J Muscle Res Cell Motil (2015) 36:71–143 DOI 10.1007/s10974-015-9407-3
ABSTRACT
Abstracts
Springer International Publishing Switzerland 2015
O0.0 Keynote lecture Wed 19:00–20:00 The control of muscle mass Sandri Marco Department of Biomedical Sciences, Padua, Italy; Venetian Institute of Molecular Medicine, Dulbecco Telethon Institute, Italy Skeletal muscle adapts its mass as consequence of physical activity, metabolism and hormones. Catabolic conditions or inactivity induce signaling pathways that regulate the process of muscle loss. Muscle atrophy in adult tissue occurs when protein degradation rates exceed protein synthesis. Two major protein degradation pathways, the ubiquitin–proteasome and the autophagy-lysosome systems, are activated during muscle atrophy and variably contribute to the loss of muscle mass. These degradation systems are controlled by a transcription dependent program that modulates the expression of ratelimiting enzymes of these proteolytic systems. The transcription factors FoxO3, which is negatively regulated by Insulin-Akt pathway, and NF-jB, which is activated by inflammatory cytokines, were the first to be identified as critical for the atrophy process. In the last years a variety of pathways and transcription factors have been found to be involved in regulation of atrophy. I will focus on the last progress in ubiquitin–proteasome and autophagy-lysosome systems, their involvement in muscle atrophy and the interplay between protein breakdown and protein synthesis. Keywords: degradation
Muscle
atrophy;
Muscle
hypertrophy;
Protein
Originally planned keynote lecture Calcium—a lifelong struggle for life or death Schaub Marcus C Institute of Pharmacology, University of Zurich, Switzerland Calcium—How and why? It fulfills a myriad of functions in multicellular organisms. Ca2+ serves as first, second and third messenger within and between cells. In combination with organic matter it provides tough structures like bones and teeth. Primordial life with self-replicating units (prokaryotes) probably originated in the ocean and was confronted
with an excess of ions from its surrounding sea water, including Na+ and Cl- and lesser Mg2+, Ca2+ and K+. Of these ions Na+ and Cl- had to be extruded from the cells by various kinds of transmembranous pumps in order to keep a balanced osmotic homeostasis. The divalent cations Ca2+ and Mg2+ are more apt to specifically bind and interact with biological compounds than the monovalent cations. Ca2+ (i) reacts 103 times faster than Mg2+ with its cognate protein binding sites, and (ii) it displays more irregular coordination geometry with 6 to 8 contacts (including association with one water molecule) than Mg2+, which binds with proteins in a rigid octahedral configuration involving 6 coordination points. Mg2+ activates a number of enzymes in the energy and nucleic acid metabolism. Due to its greater flexibility and dynamics Ca2+ gained the edge over Mg2+ in biological function. During evolution over the last billion years with the development of more complex life, Ca2+ acquired regulatory competence for almost all cellular activities. This ranges from initiation of life by fertilisation, regulation of muscle contraction, linking mitochondrial energy production by oxidative phosphorylation to the actual energy demand, right through to several pathways signaling for cellular death (apoptosis, necrosis, autophagy). To function as signaling component the millimolar concentration of Ca2+ carried over from seawater needed to be drastically reduced to reach about 10-7 M in the cytosol of a quiescent cell. After activation Ca2+ is removed from the cytosol by two ways, (i) the Ca2+ pump (SERCA) collects cytosolic Ca2+ in the sarcoplasmatic reticulum (SR), while (ii) the sarcolemmal Ca2+ pump (PMCA) extrudes Ca2+ in cooperation with the Na–K-pump and the Na–Caexchanger out of the cell. A specific Ca2+ signal with Ca2+ spikes controlled in number, intensity and frequency is only possible in front of a very low background of this cation. The extant human genome contains over 200 genes coding for Ca2+ sensing and signaling proteins. Eukaryotic cellular life pays dearly for the gain of complexity by Ca2+ signaling and function since Ca2+ has become the most poisonous substance the organism is exposed to. All cells are bathing in a 10,000 times higher outside Ca2+ concentration and every invasion of Ca2+ through a membrane defect or other pathologic mechanisms induces cell death (cardiac infarction, brain stroke etc.). Although only 10–15 g calcium presents the vital dynamic fraction essential for life, about one kg calcium resides in the bony skeletal system. Calcium is the fifth most abundant element in the Earth crust, in the ocean as well as in the human body. The most variants (35 phyla with around 8 million species) occur in the animal kingdom, yet animals spending the entire life cycle on dry land primarily come from two phyla, including insects from arthropods and vertebrates from chordata. Insects and vertebrates also are the only
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72 classes that conquered the airs. First, since all animals are heterotrophic they had to await colonisation of the land by plants in the Ordovician (around 460 million years ago). Second, the chordata, before they could follow suit the terrestrial plants, had to develop a tough enough endoskeleton to support life on land. Calcium was the most suitable element that could be carried over in sufficient amounts from sea to land. The change from cartilaginous fishes (Chondrichthyes) to bony fishes (Osteichthyes) occurred already in the sea at the transition from Silurian to Devonian 410 million years ago. Thus well equipped, the bony fishes could gradually conquer land by transformation to Amphibia, Reptilia, Aves and Mammalia. All land vertebrates carry a huge reservoir of calcium in their bones. As ionised Ca2+ functions as intracellular messenger, it is also the ionised Ca2+ in the blood that serves as intermediary between the calcium stored in the bones and the cellular requirement for it. This Ca2+ level in the blood is one of the best controlled physiological parameters by a set of hormones including calcitonin (lowering blood Ca2+) and parathyroid hormone (increasing blood Ca2+). Given the complex functions of Ca2+ and the many proteins involved in cellular Ca2+ homeostasis it is not surprising that most diseases are also affected by derangement of this system. Genetic defects in the many Ca2+ related proteins add their share in disease burden. Regulation of myocardial contraction including heart failure with reduced and with preserved systolic function as well as other pathologies involving Ca2+ will be discussed. Keywords: Calcium signaling; Evolution of calcium biology; Calcium in pathology References: Berridge MJ (2005) Unlocking the secrets of cell signalling. Annu Rev Physiol 67:1–21 Schaub MC, Heizmann CW (2008) Calcium, troponin, calmodulin, S100 proteins: from myocardial basics to new therapeutic strategies. Biochem Biophys Res Commun 369:247–264 Schaub MC, Heizmann CW (2013) Calcium in health and disease. In: Kretsinger RH, Uversky VN, Permyakov EA (eds) Encyclopedia of metalloproteins. Springer, Berlin, pp. 444–457 Williams RJP (2006) The evolution of calcium biochemistry. Biochim Biophys Acta 1763:1139–1146
O1.1 Contractile structure, Thu 09:00–09:15 Structure of myosin filaments in striated muscle: an introduction Luther Pradeep K National Heart and Lung Institute, Imperial College London, UK The myosin filaments in striated muscle are composed of myosin molecules which are packed with their tails forming the filament backbone and the heads (or crossbridges) protruding from the surface. In the last decade electron microscopy and X-ray diffraction studies have greatly aided our understanding of their fine structure. Craig and co-workers (Nature 436:1195, 2005) have applied cryo-electron microscopy on isolated tarantula thick filaments to show that the filament backbone is composed of twelve subfilaments. They showed that the crossbridges in relaxed muscle are arranged in an ‘‘interacting-heads’’ motif in which one head from each pair blocks the other. This motif has now been found in other invertebrate muscles, mammalian cardiac muscle and vertebrate smooth muscle. Studies by AL-Khayat et al. on human cardiac muscle (Proc Nat Acad Sciences 110:318, 2013) revealed tracks of density along the filament which they identified as two tracks of titin. Density was also seen which they attributed to the C-terminus of cardiac myosin binding protein C. By working with thin sections of fast-frozen/freeze-substituted skeletal muscle, Luther et al.
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J Muscle Res Cell Motil (2015) 36:71–143 (Proc Nat Acad Sciences 108:11423, 2011) obtained high preservation of the sarcomere which allowed visualisation of the whole myosin binding protein C along 43 nm spaced discs of density perpendicular to the filament and making contact with actin. Keywords: Thick filament, Electron microscopy, Striated muscle
O1.2 Contractile structure, Thu 09:15–09:30 The muscle thin filament and the cytoskeletal lattice of sarcomere: an introduction to structure, mechanics and signaling Maeda Yuichiro Structural Biology Research Center, Nagoya University Graduate School of Science, Japan We now know a near-atomic resolution structure of F-actin, and major conformation change of actin associated with polymerization. On the other hand, we do not know structural differences between ATP-F-actin and ADP-F-actin, which are distinguished by many actin binding proteins. And we have no clue how conformation of actin propagate along the actin filament. The muscle thin filament (actin/tropomyosin/troponin) plays the central roles in Ca2+-dependent regulation of muscle contraction. High resolution structures of the thin filament complex remain to be obtained. What is required is to know how the Ca2+binding to troponin induces a chain of alterations of protein–protein interactions within the complex, which change actin–actin interactions and conformations of the actin polymer. It has been anticipated that thick filament-associated titin/connectin and thin filament associated nebulin specify the lengths of thick and thin filaments, respectively. It remains an enigma how titin/connectin specifies the thick filament length. Nebulin is now viewed not as a ruler but as an actin filament stabilizer required for length maintenance. More importantly, we should know much more about how the thick and thin filaments are assembled during development, and how actin and myosin are turned over in adult muscle cells. In the Z-disk, actin filaments of opposite polarities are linked by a network of a-actinin. In the M-band, the central bare zone of the thick filament are linked together mainly by myomesin. In either structure, the force generated by muscle applies shear force to the structure. This stress is implicated to causes major conformational changes of either some alpha-actinin binding proteins in the Z-disk, or some protein kinase domains of titin in the M-band. In this way, force is converted to biochemical activities. In order to gain more insight into the mechanism of these mechanical sensors, detailed knowledge of protein–protein interactions is required. Keywords: Actin filament, Muscle thin filament, Sarcomeric lattice
O1.3 Contractile structure, Thu 09:30–09:45 Mapping of myosin heavy chain isoforms with subcellular resolution in histological sections of cardiac tissue by polarization second harmonic generation Amat-Roldan Ivan1***, Luther Pradeep K2, Torre Iratxe2 ***Candidate for Young Investigator Award
1
Advanced Microscopy, Expert Ymaging S L, Barcelona, Spain; Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, UK
2
J Muscle Res Cell Motil (2015) 36:71–143 Polarization second harmonic generation (PSHG) is a promising tool in biomedicine that enables label-free and highly specific imaging of different fibrillar structures like collagen and muscle. Origin of PSHG has been identified in muscle and there is a consensus that the contributions from Light Meromyosin (LMM) and myosin heavy chain (MHC) S2 domains are dominant. However, a small amount of PSHG signal (about 10 %) seems to arise from other parts like MHC-S1 heads. Our previous works have shown that it is possible to discriminate different MHC isoforms in histological sections of cardiac tissue by fitting PSHG signal to a well known biophysical model. This biophysical model enables to unwrap the relationship among three nonlinear coefficients and obtain the average helical pitch angle of peptides (AHPAP) of MHC for a tissue image. Computer simulations of different MHC isoforms calculated from sequences of Protein Data Base and measured 3D crystallography of subfragment MHC-S2 of human MYH7 enabled to confirm these experimental results. In this work, we show that it is possible to robustly retrieve AHPAP of MHC in unlabeled cardiac tissue sections to map expression of protein isoforms with subcellular resolution in adult rats expressing fast isoform (cMYH6) and rats fed an iodine-deficient diet which indices the expression of [95 % of slow isoform (cMYH7). Our computer simulations of the PSHG response based on 3D protein structure show that different mutations could be also distinguished with this technique. Thus, PSHG has the potential to become a tool for in situ quantification of myosin expression of different isoforms and mutations avoiding sample processing and labelling as PSHG is naturally produced by myosin. This would provide currently unavailable information for interpretation of studies in animal models expressing mutations in myosin protein and other myosin interacting proteins, like myosin binding protein C, which play critical roles in contraction. Keywords: Cardiac myosin heavy chain, Polarization second harmonic generation, Microscopy
O1.4 Contractile structure, Thu 09:45–10:00 Deleting titin’s IA junction maintains thick filament length and increases biomechanical sensing by titin’s molecular spring segments Granzier Henk1, Hutchinson Kirk1, Tonino Paola1, Methawasin Mei1, Saripalli Chandra1, Pappas Chris1, Gregorio Carol2, Smith John1 1 Department of Cellular and Molecular Medicine, University of Arizona, USA; 2University of Arizona, USA
Titin, the largest protein known, forms a giant filament in muscle where it spans the half sarcomere from Z-disk to M-band. Mutations in titin are a major cause of heart failure yet the function of large parts of titin is not understood. Titin’s IA-junction has been proposed to be crucial for thick filament length control and we made a mouse in which the IA-junction was deleted. Super-resolution optical microscopy (Structured Illumination Microscopy, SIM) and electron microscopy were used to study the thick filament length and titin’s molecular elasticity. SIM showed that the IA-junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high relative to that of I-band titin (*40-fold higher) but low relative to that of the myosin-based thick filament (*70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the thick filament backbone, and this might constitute a novel length sensing mechanism. Findings disproved the widely held view that titin at the IAjunction is crucial for thick filament length control, settling a longstanding hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin’s spring region away from the Z-disk, increasing the strain on titin’s molecular spring elements.
73 Functional studies from the cellular to ex vivo and in vivo left ventricular (LV) chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF), including hypertrophy. Thus, our work reveals titin’s important roles in diastolic function and trophic level of the heart. Keywords: Ultrastructure, Titin, Diastolic function
O1.5 Contractile structure, Thu 10:00–10:15 O-GlcNAcylation modulates sarcomeric organization and protein–protein interactions in C2C12 myotubes Lambert Matthias1***, Richard Elodie2, Duban-Deweer Sophie3, Krzewinski Frederic4, Deracinois Barbara1, Dupont Erwan1, Bastide Bruno1, Cieniewski-Bernard Caroline1 ***Candidate for Young Investigator Award
1
EA 4488 APMS, University of Lille, France; 2BioImaging Center Nord-de-France, CCMIC, University of Lille, France; 3Centre d’Analyse Prote´omique de l’Artois, University of Artois, France; 4 Structural and Functional Glycobiology Unit, UMR8576, University of Lille, France In skeletal muscle, sarcomere is characterized by an accurate structural organization through highly regulated interactions between myofilament proteins. It has been shown that many myofilament proteins are modified by O–N-acetyl-glucosaminylation (O-GlcNAcylation), an atypical glycosylation. Akin to phosphorylation, O-GlcNAcylation is involved in numerous cellular processes, including protein–protein interactions. Although it has been described as a modulator of contractile activity, the role of this glycosylation in sarcomeric organization remains to be considered. To investigate the O-GlcNAcylation role in sarcomeric structure, C2C12 myotubes were treated with Thiamet G (Thiazoline amino-ethyl gluco-configured) or DON (6-Diazo-5-Oxo-LNorleucine) to increase or decrease O-GlcNAcylation level, respectively. Both treatments were efficient to modulate O-GlcNAcylation, without alteration of cell viability or myotube maturation. Myofilament proteins were more sensitive to O-GlcNAcylation modulation than total proteome. Using immunofluorescence labeling of myosin heavy chain and confocal microscopy, we pointed out morphometric changes of the sarcomere directly correlated to O-GlcNAcylation level. We observed an increase in the dark and M-band width and a decrease in the I-band width and sarcomere length. To determine if protein–protein interactions were modulated consecutively to O-GlcNAcylation variation, we performed Red-Native PAGE and noted changes in protein complexes profiles, suggesting that protein interactions were modified. We identified through mass spectrometry proteins belonging to these modified complexes, and among them, the filamin-C, a structural Z-line protein involved in sarcomeric architecture. These results suggest that O-GlcNAcylation is involved in sarcomeric structure and protein–protein interactions. This work may provide new insights in the understanding of molecular mechanisms of diseases such as myofibrillar myopathies. Keywords: O-GlcNAcylation, Sarcomere structure, Protein–protein interactions
O1.6 Contractile structure, Thu 10:50–11:05 Mechanical and kinetic analysis of plectin-deficient skinned fibres and myofibrils from murine psoas muscle Elhamine Fatiha1, Gloistein Claas1, Winter Lilli2, Papadopoulos Symeon1, Wiche Gerhard2, Pfitzer Gabriele1, Stehle Robert1
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Institute of Vegetative Physiology, University of Cologne, Germany; Department of Biochemistry and Cell Biology, Max F Perutz Laboratories, University of Vienna, Austria
2
Mutations in the cytoskeletal linker protein plectin are linked to myofibrillar myopathy that is associated with skeletal muscle weakness. However, the impact of plectin on passive and active mechanical properties and on the kinetics of contraction and relaxation are unknown. Thus, we investigated the Ca2+-regulated contractile function of skinned muscle fibres lacking plectin (Fibre-mPle-KO). Fibres were prepared from a MCK-CreLoxP-Ple-mouse line that lacks all plectin isoforms in muscle. Additionally, kinetics of contraction-relaxation cycles were analyzed using subcellular myofibril bundles from homozygous Ple1d-KO mice (Myof-Ple1d-KO) lacking the Z-disc specific 1d-isoform of plectin. Compared to myofibrils from wildtype mice (Myof-WT), the Myof-Ple1d-KO exhibited neither significant difference in maximum active or passive tension, nor in the kinetics ofCa2+-induced or mechanically-induced force development or in relaxation kinetics. However, after the myofibrils were mechanically stressed by eccentric contractions, both phases of the biphasic relaxation process were slower in Myof-Ple1dKO compared to Myof-WT. Thus, presence of plectin-1d reduces stress-induced deceleration of relaxation. Compared to wildtype fibres (Fibre-WT), Fibre-mPle-KO required a higher Ca2+-concentration for half-maximum force pro2+ duction (higher Ca2þ 50 –value) and had a steeper Ca -force-relation (higher Hillcoefficient). The higher steepness of plectin-deficient fibers was mainly at the low [Ca2+] range, where the contraction of FibremPle-KO required much higher [Ca2+] than Fibre-WT. Presence of plectin apparently leads to a more graduated force response when [Ca2+] is increased at low [Ca2+]. Plectin might perform this graduation effect either by restricting axial cooperative coupling between Ca2+-regulated troponin-tropomyosin units along the thin filaments or by forming flexible transversal links between myofibrils that dampen contractile imbalances of collateral sarcomeres. Keywords: Cytoskeletal proteins, Relaxation kinetics, Calcium sensitivity
O1.7 Contractile structure, Thu 11:05–11:20 Insights into the regulation of ligand binding and Z-disk assembly from the structure of human muscle a-actinin De Almeida Ribeiro Euripedes1, Pinotsis Nikos1, Ghisleni Andrea2, Salmazo Anita1, Konarev Petr V3, Kostan Julius1, Schreiner Claudia1, Gkougkoulia Eirini A1, Pirker Katharina F4, Gautel Mathias5, Djinovic-Carugo Kristina1 1
Department of Structural and Computational Biology, Max F Perutz Laboratories, University of Vienna, Austria; 2King’s College London BHF Centre for Research Excellence, Randall Division for Cell and Molecular Biophysics and Cardiovascular Division, UK; 3EMBLHamburg c/o DESY, Germany; 4Division of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria; 5King’s College London BHF Centre for Research Excellence, Randall Division for Cell and Molecular Biophysics and Cardiovascular Division, London, UK a-Actinin is the major component of the Z-disk, where it cross-links actin filaments from adjacent sarcomeres. It is an antiparallel dimer of 200 kDa, containing in each subunit an N-terminal actin binding domain (ABD), a central rod domain assembled from spectrin-like repeats that mediate the antiparallel assembly, and a C-terminal calmodulinlike (CaM-like) domain with 4 EF-hand motifs. Additionally to actin filaments, a-actinin binds multiple other cytoskeletal and signalling proteins. In striated muscle, the tightly defined numbers of a-actinin
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J Muscle Res Cell Motil (2015) 36:71–143 crosslinks between the antiparallel actin filaments at the Z-disk are organized by specific binding sites on titin. These titin Z-repeats contain a short, hydrophobic, a-actinin binding motif. To achieve ordered cytoskeletal assemblies, the binding properties of a-actinin must be tightly spatiotemporally regulated. a-Actinin–titin interaction is regulated by an intramolecular mechanism, where the short sequence between the ABD and the rod interacts with the CaM-like domain in a pseudoligand complex, acting effectively as an intramolecular autoinhibitor. Here, we report the first complete high-resolution structure of the a-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides structural insight into the phosphoinositide-based regulatory mechanism controlling its interaction with other sarcomeric proteins like titin. Keywords: a-Actinin, Pseudoligand regulation, High resolution Structure
O1.8 Contractile structure, Thu 11:20–11:35 Structural dynamics of actin, tropomyosin, and troponin in the thin filament as studied by spin-labeling dipolar EPR spectroscopy Arata Toshiaki1, Ueda Keisuke2, Zhao Chenchao1, Somiya Takayasu1, Yamamoto Akie1, Aihara Tomoki1, Ueki Shoji3, Miki Masao4 1 Department of Biological Sciences, Graduate School of Science, Osaka University, Japan; 2RIKEN, Japan; 3Tokushima-Bunri University, Japan; 4University of Fukui, Japan
Using EPR spectroscopy we have determined how major protein actin and regulatory proteins troponin (Tn) C–I–T complex and tropomyosin (Tm) move in the thin filament in response to myosin and Ca2+ binding. The distance between spin label at Cys374 and Mn2+ in F-actin increased by *0.2 nm maximally at 30–60 % occupancy by myosin-S1, suggesting cooperativity. The distance also increased by *0.2 nm when F-actin was decorated with Tm-Tn with and without Ca2+ but returned to the original level upon further S1 binding at [60 % occupancy. As previously detected the switch action of skeletal TnI-TnC [1], we measured the distance between N-lobe of cTnC and regulatory region of cTnI both spin labeled, and found that cTnI binding to cTnC was loosened by PKA phosphorylation of cTnI which causes Ca2+ desensitization of heart muscle. We have determined the secondary structure of N-terminal phosphorylatable extension of cTnI by measuring the distance distribution between two spin labels attached on i and i + 4 residues [2]. PKAand PKC-phosphorylatable regions formed more stable conformations upon binding with cTnC. The mobility of a spin label on Tm showed unexpectedly no Ca2+ effect but that the wide region of Tm interacts with actin in highly cooperative manner upon S1 binding [3]. Distance distribution was successfully obtained by fitting the spectrum of spin labeled residues of 15N-based spin labeled Tm and 14N-based spin labeled Cys374 due to distinct spectral characteristics of 14N- and 15 N-labels. Ca2+ effects on the distance were found but restricted only at the N- and C-terminal regions of Tm. Interestingly, upon Ca2+ binding to the thin filament with bound S1 the mobility changes of a spin label on Tm were detected over the wide region of Tm [4]. Keywords: Tropomyosin, Troponin, Spin labeling References: [1] Aihara et al. J Biol Chem 285:10671, 2010 [2] Zhao et al. Biophys J Abstract 2012 [3] Ueda et al. Biophys J 100:2432, 2011 [4] Ueda et al. Biophys J 105:2366, 2013
J Muscle Res Cell Motil (2015) 36:71–143
O1.9 Contractile structure, Thu 11:35–11:50 Reduced right ventricular cardiomyocyte passive force due to hypophosphorylation at Ser-282 of cardiac myosin binding protein C in a rat model of post-ischemic heart failure ´ rpa´d1***, Kalasz Judit1, Pasztor T Eniko1, Kova´cs A Sanganalmath Santosh K2, Dhalla Naranjan S2, Papp Zolta´n1, Barta Judit1 ***Candidate for Young Investigator Award
1
Division of Clinical Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; 2Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, Winnipeg, Canada
Left ventricular (LV) remodeling in myocardial infarction (MI)-induced heart failure (HF) is well studied. Less is known about the concomitant right ventricular (RV) changes. In this rat model of MIinduced HF we aimed to: (1) characterize the contractile function of RV cardiomyocytes, (2) compare it with the LV, (3) investigate the underlying molecular mechanisms in the RV. Rat hearts in moderate HF were studied after 8 weeks of LV MI. Sham-operated animals served as controls. Maximal active force F(max), Ca-sensitivity (pCa50) and passive force F(pass) were measured in skinned isolated RV and LV cardiomyocytes (n = 12–15). Titin, myosin binding protein C (MyBP-C) and Troponin I (TnI) phosphorylation (P) were tested by gel-electrophoresis-based and Western blot methods. RV weight was higher in HF (0.48 ± 0.04 g, mean ± SEM) than in the Sham group (0.31 ± 0.02 g) (P B 0.05). F(max) and pCa50 of RV cardiac cells were similar in the HF and in the Sham group (18.7 ± 1.1 kN/m2; 5.88 ± 0.03 vs. 18.3 ± 2.1 kN/m2; 5.87 ± 0.03, respectively). In HF, the cross section area of RV cells was comparable to Sham (876 ± 133 lm2; 1007 ± 194 lm2). However, in HF F(pass) of RV cardiomyocytes was less (2.1 ± 0.2 kN/m2) than that in the Sham group (3.2 ± 0.4 kN/m2). LV cells had higher F(max) and pCa50 values in HF than in the Sham group (34.9 ± 2.7 kN/m2; 5.97 ± 0.03 vs. 26.9 ± 1.6 kN/m2; 5.85 ± 0.03). LV cell size in HF was not higher than in the Sham (1104 ± 151 lm2; 1029 ± 118 lm2). LV F(pass) was not altered in HF (3.37 ± 0.37 kN/m2) compared to Sham (2.83 ± 0.27 kN/m2). In HF animals relative to Sham total P of titin and MyBP-C showed no difference in the RV or in the LV, but P at Ser-282 of MyBP-C was decreased in the RV. In HF both the RV and the LV TnI P (total and Ser-22/23) was lower than in the Sham. Our data suggest distinct changes in RV cardiomyocyte structure and function in HF due to LV MI. Reduced RV F(pass) seems to be independent of titin P, rather might be attributable to hypo P of protein kinase A sites of MyBP-C. Keywords: Right ventricle, Passive force, Cardiac myosin binding protein C
O2.1 Contractile function, Thu 14:00–14:15 What do we learn by the rates of tension development k(act) and redevelopment k(TR) in activated myofibrils and muscle fibres Kawai Masataka, Wang Li Departments of Anatomy and Cell Biology, and Internal Medicine, University of Iowa, USA When the length of active muscle fibres/myofibrils are released by *20 % and restretched after *50 ms, tension redevelops with an
75 exponential time course with the rate constant k(TR) in rabbit psoas fibres [1]. A similar time course (rate const: k(act)) is observed on Ca2+ activation in single myofibrils [2]. These are generally interpreted to mean that k = f + g, where f is the attachment rate constant and g is the detachment rate constant in the two state model [1]. With this formulation, k is limited by a fast reaction. However, a problem has developed, because k has been generally 5–40/s in rabbit psoas, which is too slow for a fast cross-bridge reaction. Alternatively, we propose a model in which crossbridges cycle many times by stretching series elastic elements to develop force, hence k is limited by a slow reaction: k & (f + g)/fg (&means proportional). To set up this model, we made an assumption that the stepping rate (v) decreases linearly with force (F) (Fenn effect, [3]). The distance traveled by a cross-bridge stretches series elastic elements with stiffness q, which is registered as force. The rate constant of resulting time course is: k = qg0v0(1 - k)/F1, where k = m1/v0, g = step size, the subscript 0 indicates unloaded, and the subscript 1 indicate isometric conditions. We demonstrate that the ATP hydrolysis rate is proportionate to k(TR) with k = 0.28 as the temperature is changed. We conclude that k(TR) and k(act) are limited by the cross-bridge turnover rate; hence it represents the rate constant of the slowest reaction of the cross-bridge cycle. This model further explains why the time course of tension rise (stretch series elastic elements) is slower than that of tension decay (direct observation of cross-bridge detachment) when the Pi concentration is quickly changed with myofibril experiments [2]. Keywords: Turnover rate, Elementary steps, Cross-bridge References [1] Brenner B. Proc Nat Acad Sci (USA) 85:3265–3269, 1988 [2] Tesi C, Colomo F et al. Biophys J 78(6):3081–3092, 2000 [3] Fenn WO, J Physiol 58(2–3):175–203, 1923
O2.2 Contractile function, Thu 14:15–14:30 Mutations in the central part of a Tropomyosin molecule alter Ca2+ sensitivity and tension relaxation of skeletal muscle myofibrils after troponin–tropomyosin removal and reconstitution Scellini Beatrice1, Piroddi Nicoletta1, Ferrara Claudia1, Matyushenko Alexander M2, Levitsky Dmitrii I2, Poggesi Corrado1, Tesi Chiara1 1
Department of Experimental and Clinical Medicine, University of Florence, Italy; 2A N Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia
Tropomyosin (Tm) is a a-helical coiled-coil actin-binding protein regulating muscle contraction. Previous observations suggested that the highly conserved non canonical residues Asp-137 and Gly-126 in the central part of Tm confer instability. Replacement of these residues by canonical ones (Leu and Arg, respectively) decreases molecular flexibility and modulates the Ca2+ sensitivity of in vitro regulated actomyosin systems (Sumida et al. J Biol Chem 283:6728–6734, 2008; Nevzorov et al. J Biol Chem 286:15766–15772, 2011). Here, we investigated the functional impact of recombinant a Tm carrying one (D137L) or both (D137L/G126R) stabilizing substitutions on the mechanical behavior of skeletal myofibrils. Endogenous Tm and Tn were replaced into rabbit psoas myofibrils (Scellini et al. Adv Exp Med Biol 682:163–174, 2010) with purified Tn and recombinant a Tm (WT,D137L and D137L/G126R). Force recordings from myofibrils (15 C) showed the increase in Ca2+ sensitivity expected from in vitro studies. At saturating [Ca2+] maximal isometric tension and the rates of force activation (kACT) and redevelopment (kTR) were not significantly affected by Tm mutations. Interestingly, a clear effect was observed on force relaxation: D137L/G126R myofibrils showed
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76 prolonged duration of the slow phase of relaxation and a decreased rate of the fast phase compared to WT. This effect was smaller in D137L myofibrils. Tm mutations also strongly decreased slack sarcomere length (SL) at sub-activating [Ca2+] as well as they increased the steepness of the SL—passive tension relation. These effects were reversed by 10 mM BDM, suggesting that single and double Tm mutations compromise the full inhibition of acto-myosin interactions in the absence of Ca2+. These data support the hypothesis that flexibility of the Tm coiled–coiled structure critically modulates the turning off of the thin filament system and muscle relaxation dynamics. Supported by PRIN 2010–2011 of the Ministero Universita` e Ricerca (MIUR). Keywords: Tropomyosin, Skeletal muscle, Regulation
O2.3 Contractile function, Thu 14:30–14:45 Stiffness of the myosin head domain Brenner Bernhard1, Seebohm Benjamin1, Werkman Christoph1, Hahn Nils1, Schweda Aike1, Steffen Walter1, Kraft Theresia2 1 Molecular and Cell Physiology, Hannover Medical School, Germany; 2Molecular and Cell Physiology, Hannover Medical School, Germany
Elastic distortion of the myosin head is fundamental to the generation of contractile forces. Thus, stiffness of the myosin head, together with filament compliance, is a main determinant of forces generated by a myosin head. Differences in head stiffness among myosin isoforms, or changes in head stiffness with nucleotide would yield different forces generated by different myosin isoforms and in different states of the ATPase cycle, respectively. Studying effects of point mutations in the converter of the slow skeletal/b-cardiac myosin heavy chain that cause familial hypertrophic cardiomyopathy, we found some mutations to increase head stiffness and force generation by the myosin head. This implied that the converter is a main determinant of myosin head stiffness. These FHC-mutations were at positions in the converter sequence where fast skeletal myosin differs from the slow/b-cardiac isoform, suggesting that different heavy chain isoforms may have different head stiffness. Comparing in skinned fibers and by optical trapping fast (rabbit psoas) and slow (rabbit/human soleus) myosins we found head stiffness of the slow myosin to be B1/3rd of the fast isoform stiffness. The finding that changes in the converter strongly affect head stiffness implies that reorientation of the converter vs. the catalytic domain, e.g., during the myosin working stroke, may also change stiffness of the head domain. We tested this hypothesis by optical trapping of a single headed myosin-5a construct. We selected myosin-5a because a sub-step had been observed in optical trapping and was assigned to a reorientation of the converter in the myosin-5 working stroke. We found that after this substep, i.e., after converter reorientation, the stiffness of the myosin-5a head domain was C2-fold increased. In conclusion, stiffness and force generation of a myosin head (i) are different for fast and slow myosin-2 isoforms, and (ii) change as a myosin head progresses through its working stroke. Keywords: Force per myosin head, Stiffness of myosin isoforms, Stiffness of cross-bridge states
O2.4 Contractile function, Thu 14:45–15:00 The conformation of myosin motors in relaxed skeletal muscle Fusi Luca***, Huang Zhe, Irving Malcolm ***Candidate for Young Investigator Award
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J Muscle Res Cell Motil (2015) 36:71–143 King’s College London, UK Electron microscopy studies of isolated thick filaments from a wide range of muscle types and species are consistent with an asymmetric arrangement of the two motor domains of each myosin molecule folded back against the filament backbone and called the J motif (Woodhead et al. Nature 436:1195–1199, 2005). This conformation is associated with the OFF state of myosin, although it is unclear how the molecule is switched on during contraction. In this study we investigated the in situ conformation of myosin motors in relaxed demembranated fibres from rabbit psoas muscle using fluorescence polarisation from bifunctional rhodamine (BSR) probes at four sites on the myosin regulatory light chain (RLC). Under relaxing conditions the order parameters of the orientation distribution of each probe, P2 and P4 respectively, had a sigmoidal dependence on temperature in the range 3–33 C, with half-maximal change at 18 C. Either lattice compression by 5 % dextran or addition of 25 lM Blebbistatin decreased the transition temperature to 13 C. Maximum entropy analysis showed that at 5 C the RLC domain of myosin is almost perpendicular to the filament axis, whereas above 20 C two populations with more parallel orientations appear, and either dextran or Blebbistatin stabilize these two conformations above 16 C. The angles of these two conformations match the RLC orientations of the blocked and free heads in the J motif observed in isolated filaments, suggesting that this fraction of motors occupy the J motif in relaxed muscle in situ. Slow ramp stretches applied in relaxing solution at 25 C and at sarcomere lengths [2.6 lm produced large changes in RLC orientation towards the ON conformation seen during calcium activation, with partial reversal during force relaxation after the stretch. The correlation between RLC orientation and passive fibre tension provides evidence that the orientation of relaxed myosin motors is sensitive to thick filament strain. Supported by Wellcome Trust, UK. Keywords: myosin, skeletal muscle, muscle fibres
O2.5 Contractile function, Thu 15:00–15:15 The increase in non cross-bridge force after stretch of activated striated muscles is related to titin isoforms Cornachione Anabelle1, Leite Felipe1, Nocella Marta2, Colombini Barbara2, Bagni Maria Angela2, Rassier Dilson3 Kinesiology, McGill University, Montreal, Canada; 2Experimental and Clinical Medicine, University of Florence, Italy; 3Kinesiology, Physics, Physiology, McGill University, Montreal, Canada 1
Introduction: There is evidence that skeletal muscles present a crossbridge independent increase the sarcomere stiffness upon Ca2+ activation. It has been hypothesized that the increase in stiffness is caused by Ca2+-dependent changes in the properties of titin molecules. To test this hypothesis, we investigated the cross-bridge independent stiffness in muscles containing different titin isoforms. Methods: Permeabilized myofibrils were isolated from the psoas, soleus and heart ventricle muscles of the rabbit. Intact trabeculae were isolated from the heart of the mouse. Myofibrils were tested in relaxing and activating conditions, before and after treatment with chemicals that inhibit myosin-actin interactions. Trabeculae were electrically stimulated in Krebs-Henseleit solution. The force produced by these preparations during and after stretches of different magnitudes was measured in sarcomere lengths between 1.6 and 2.4 lm for the heart, and between 2.2 and 3.0 lm for skeletal muscles. Titin isoforms in these muscles were identified with gel electrophoresis.
J Muscle Res Cell Motil (2015) 36:71–143 Results: The cross-bridge independent stiffness was present in psoas and soleus myofibrils activated at pCa 4.5, but not in cardiac myofibrils activated supra-maximally in the presence of myosin inhibitors or in intact trabeculae. The stiffness increase with Ca2+ was higher in myofibrils isolated from the psoas than in myofibrils isolated from the soleus (54.3 and 34.6 % at SLs of 3.0 lm, respectively). In both cases, the cross-bridge independent stiffness increased at increasing sarcomere lengths. Conclusion: The increase in passive forces with Ca2+ and the crossbridge independent stiffness is directly associated with the isoforms of titin. These results suggest that a Ca2+-dependent change in titin is responsible for the increases in non cross-bridge stiffness. Keywords: Myofibrils, Myosin, Stiffness
O2.6 Contractile function, Thu 15:15–15:30 Mechanism of force potentiation after stretch in mammalian muscle fibre Nocella Marta, Cecchi Giovanni, Bagni Maria Angela, Colombini Barbara Experimental and Clinical Medicine, University of Florence and IIM, Italy It is well known that slow stretching of an active muscle induces a force enhancement (RFE) above the isometric level that persists after the stretch. RFE has been extensively studied, nevertheless its mechanism remains debated. Here, we investigated force enhancement after stretch on tetanized fibre bundles from FDB mouse muscle at 30 C. Unlikely RFE studies, stretches used here were small and fast (amplitude 3–5 % sarcomere length (sl), duration 0.6 ms) and were applied at low forces on the tetanus rise. Following our previous works, force enhancement after stretch was termed Static Tension (ST). To investigate if crossbridges (CB) affect ST, fibres were stretched at various sl up to 4.4 lm. In a group of experiments, BTS (10 lM) was added to the Tyrode solution to reduce active force. In spite of the different experimental procedure all the results indicated that ST is equivalent to RFE. Upon stimulation, ST developed faster than force. It was present 1 ms after the stimulus, when active force was zero, and reached a peak after 3 ms. ST increased with stretch amplitude and at the optimal sl of 2.7 lm for a stretch of 1 % sl, it was 3 % of tetanic force. This value was 8 times greater than the response of the passive fibre, indicating a corresponding increase of sarcomere stiffness. ST increased with sl up to a peak at 3.4 lm to decrease again at higher lengths reaching zero at 4.4 lm. At 4 lm sl active force fell to zero, but ST was still 50 % of maximum. BTS reduced force by *75 % but had almost no effect on ST. In no case ST and active force were correlated. In contrast to the current hypotheses, these results indicate that force enhancement after stretch is independent of crossbridges and is due to the stiffening of a non-crossbridge structure, very likely titin. The Ca2+ induced stiffening of the PEVK titin segment and actin-titin interaction could explain the increase of sarcomere stiffness following stimulation and the resulting force enhancement after stretch. Keywords: Force enhancement, Non-crossbridge stiffness, Titin
77 Ramp stretch of isometrically contracting muscle results in biphasic rise of tension. Initially it rises linearly and rather quickly and then the velocity of the rise decreases either smoothly or with an overshoot. Linear regressions of these two slopes intersect in a point called critical tension, Pc. We studied this phenomenon in single permeabilized fibres from rabbit psoas muscle with different stretch velocities in a wide temperature range. Blebbistatin was also used to depress actomyosin interaction. Changes in tension, stiffness and sarcomere length were monitored. Stiffness was measured with sinusoidal oscillations of 0.1 % fibre length at frequency of 2.5 kHz with subsequent detection of changes in tension. The most interesting observation was that at any experimental conditions the stiffness rise was monophasic and completed a few milliseconds before Pc was achieved. This can be explained on the base of the recent model (Ferenczi et al. PLoS One 9(1): e85739, 2014) with two extra assumptions: (1) stretching force applied to myosin molecule stereospecifically bound to actin first bends its lever arm domain and only then enables the head to detach from actin; (2) the non-stereo-specific bond is less strong than the stereo-specific one. With these assumptions the mechanism underlying the response of contracting muscle to the ramp stretch can be explained as follows: (1) the bending allows non-stereospecific attachment of the second head and this leads to an increase in both fibre stiffness and tension; (2) further stretching breaks the stereo-specific bond and both heads become attached non-stereo-specifically; (3) during the stretch force rise is slower if both heads are non-stereo-specific than if one of them is stereo-specific. The work was supported by the Program of Ural Branch of RAS, project No 12-P-4-1007. Keywords: Muscle contraction, Ramp stretch, Stiffness
O2.8 Contractile function, Thu 16:20–16:35 How actin initiates the motor activity of myosin Houdusse Anne1, Isabet Tatiana1, Llinas Paola1, Ropars Virginie1, Song Lin2, Zong Bin2, Sweeney Lee H2 Institut Curie, CNRS UMR 144, Paris, France; 2University of Pennsylvania School of Medicine, USA 1
Fundamental to cellular processes are directional movements that are driven by molecular motors, yet we lack a detailed understanding of how these motors use ATP hydrolysis to move on tracks within the cell. Myosin superfamily members are molecular motors that produce force and movement on F-actin tracks. Mechanochemical transduction by myosin on actin is coupled to unknown structural changes that result in the sequential release of the MgATP hydrolysis products, inorganic phosphate (Pi) and MgADP. We present a previously unseen myosin structure containing the hallmarks of the missing state that initiates force generation and movement. These include a new actin interface and a tunnel (back door) that has opened for Pi to escape without moving the myosin lever arm that drives its movements. We propose that in fact this state represents the beginning of the powerstroke on actin and explains how actin initiates force generation by myosin. This study brings highly novel concepts about how actin and myosin interactions control the powerstroke and how these interactions are related to the specific release of the products Pi and then ADP. Keywords: Chemo-mechanical transduction, Allosteric transition, Pi release
O2.7 Contractile function, Thu 16:05–16:20 O2.9 Contractile function, Thu 16:35–16:50 Possible mechanism of biphasic tension rise in contracting muscle fibre during ramp stretch Kochubey Pavel, Bershitsky Sergey
Electron microscopic recording of ATP-induced myosin head power stroke in hydrated mixture of actin and myosin filaments
Institute of Immunology and Physiology, Ural Branch of Russian Acad Sci, Russia
Sugi Haruo1, Chaen Shigeru2, Tanokura Masaru3, Minoda Hiroki4
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Department of Physiology, Teikyo University School of Medicine, Itabashi, Tokyo, Japan; 2Department of Integrated Sciences, Nihon University, Tokyo, Japan; 3Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan; 4Department of Applied Physics, Tokyo University of Agriculture and Technology, Tokyo, Japan It is generally believed that the myosin head (M), in the form of MADPPi, first attaches to actin (A), performs a power stroke associated with release of Pi and ADP, and detaches from A on binding with a new ATP. Up to the present time, however, the amplitude of the myosin head power stroke can only be estimated by indirect ways, and still remains to be a matter for debate and speculation. Using the gas environmental chamber, which enables us to record dynamic structural changes of hydrated biomolecules, we attempted to record the myosin head power stroke in the mixture of actin and myosin filaments, in which each myosin filament was surrounded by actin filaments, running in parallel due to rigor actin-myosin linkage formation. Individual myosin heads were position-marked with colloidal gold particles (diameter, 20 nm) at the distal or the proximal region of their catalytic domain (Sugi et al. Proc Natl Acad Sci USA 105(45):17396–401, 2008; Minoda et al. Biochem Biophys Res Commun 405(4):651–6, 2011). In the absence of ATP, the position of individual myosin heads did not change appreciably with time, indicating stability of time-averaged myosin head position. When ATP was applied iontophoretically, individual myosin heads were found to move parallel to the myosin filament axis. The amplitude of ATP-induced myosin head movement was about 3.1 nm and about 2.4 nm at the distal and the proximal region of myosin head catalytic domain, respectively. Since only a small proportion of myosin heads in a myosin filament can be activated by applied ATP, the observed myosin head movement was regarded as myosin head power stroke in the isometric condition, in which individual ATP-activated myosin heads can only move by pulling adjacent elastic structures. At low ionic strength, the amplitude of myosin head power stroke increased to 4 nm, being consistent with physiological experiments that the force exerted by individual myosin heads increases twofold at low ionic strength. Keywords: Myosin head power stroke, Muscle contraction, Myofilament sliding
O2.10 Contractile function, Thu 16:50–17:05 Effects of congenital myopathy mutations in tropomyosin on troponin binding to the thin filament Moraczewska Joanna1, Cyranka-Czaja Anna2, Robaszkiewicz Katarzyna1, Ostrowska Zofia1 1 Department of Biochemistry and Cell Biology, Kazimierz Wielki University in Bydgoszcz, Poland; 2BLIRT S A, Poland
Congenital myopathies are muscle diseases mostly caused by mutations in genes encoding proteins of the actin thin filament. Mutations in TPM3, c-tropomyosin gene expressed in type1 slow muscle fibers, were found in patients diagnosed with nemaline myopathy, congenital fiber type disproportion and cap disease. Previously we have shown that point mutations in tropomyosin (TM) reduced Ca-induced activation of the actomyosin ATPase and slowed down gliding velocity of actin filaments over myosin heads in a motility assay. However, the degree of activation loss depended on the localization and type of amino acid substitution (Robaszkiewicz et al. Biochim Biophys Acta 1822(10):1562–9, 2012). We hypothesized that the mutations changed TM structure in such a way that it affected binding of Tn complex. To verify this hypothesis in this project we compared Tn
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J Muscle Res Cell Motil (2015) 36:71–143 binding to rat recombinant wild type TM and TM mutants carrying the following substitutions: Leu100Met, Ala156Thr, Arg168Gly, Arg168Cys, Arg168His, Lys169Glu, and Arg245Gly. TM-Tn binding was analysed by Far Western Blot. It demonstrated that Tn binding to wild type and all mutant TMs was similar. Co-sedimentation of Tn with actin filaments reconstructed in the presence of all studied TM variants did not detect significant differences in Tn binding to the thin filament. However, detailed analysis of Tn-TM interactions with the use of surface plasmon resonance has shown that all mutations impaired kinetics of Tn binding to TM alone and reduced the equilibrium binding constant 1.2–2.8 fold. Mutations located in the region involved in binding of Tn core domain were the most severe. Together the analyses suggested that myopathy causing mutations affected communication between TM and Tn, which impaired the transition of the thin filament from ‘‘off’’ to ‘‘on’’ state. Keywords: Thin filament, Tropomyosin, Congenital myopathy
O2.11 Contractile function, Thu 17:05–17:20 Abnormal movement of mutant b-tropomyosin associated with congenital myopathy causes aberrant response of myosin heads and actin during the ATPase cycle Avrova Stanislava1, Rysev NikitaA1, Karpicheva Olga1, Simonyan Armen2, Chernev Alexey2, Piers Adam3, Redwood Charles3, Borovikov Yurii S1 1
Molecular basis of cell motility lab, Institute of Cytology RAS, St Petersburg, Russia; 2Department of biophysics, St Petersburg State University, Russia; 3John Radcliffe Hospital, University of Oxford, UK We have investigated how the b-TM mutations R91G, E139del, and E41K that cause congenital myopathy affect TM’s position on the thin filament and the spatial arrangement of actin monomers and myosin heads during the ATPase cycle. We labeled recombinant wild-type and mutant TMs with 5-IAF, S1 with 1.5-IAEDANS and F-actin with FITC-phalloidin, incorporated them into troponin-free ghost muscle fibres and measured polarized fluorescence at different stages of the ATPase cycle. It was found that the position of wild-type TM was correlated with the position and conformational state of the myosin heads and actin monomers at all the intermediate stages of the ATPase cycle, and each of the mutations uncoupled this relationship. The R91G mutation shifted TM strands further to the centre of the filaments, increased the proportion of the switched on actin monomers during strong binding between actin and myosin. During weakbinding, the R91G mutation, on the contrary, shifted TM strands further to the periphery of the filaments and decreased the proportion of the switched on actin monomers. In addition, the R91G mutation increased the number of myosin heads in the strong-binding states at all intermediate stages of the cycle. The E139del mutation stabilized TM strands near the filament centre during the ATPase cycle, and decreased the number of the switched on actin monomers and myosin heads in the strong-binding states. The E41K stabilized TM strands near the filament periphery during the ATPase cycle and increased the amount of the myosin heads in the strong-binding states during relaxation. It is suggested that the aberrant TM movement causes abnormal response of the contractile system, resulting in contracture and muscle weakness. This work was supported by the Russian Fund for Fundamental Research (Grants No. 14-04-00454a, 14-04-31527a), the Program of Presidium of RAS (theme No. 7) and the Muscular Dystrophy Campaign. Keywords: Myopathy, Mutant tropomyosin, Polarized fluorimetry
J Muscle Res Cell Motil (2015) 36:71–143
O2.12 Contractile function, Thu 17:20–17:35 Toward the realization of a sarcomere-like machine Melli Luca1, Bianco Pasquale1, Falorsi Giulia1***, Salvi Luca1, Coceano Giovanna2, Cojoc Dan2, Lombardi Vincenzo1,2 ***Candidate for Young Investigator Award
1
Department of Biology, University of Florence, Italy,2IOM-National Research Council, Trieste, Italy
The aim of the project is to build a sarcomere-like machine consisting of a synthetic array of myosin motor surface brought to interact with an actin filament. The ensemble of myosin motors will provide the condition for generating steady force and shortening by cyclic interactions with the actin filament. The mechanical outputs of the machine will be measured by means of a Dual Laser Optical Tweezers apparatus (DLOT, range 0.5–200 pN force, 1–10,000 nm displacement), able to control either the movement or the force of the biomachine. The motor protein chosen is the HMM fraction of myosin II from frog and rabbit skeletal muscle. Mechanical measurements are done with a simplified version of the machine, in which the number of the motors interacting with the actin filament is limited by using as a support the TMCS (Trimethylchlorosilane) coated flat tip of an etched optical fibre (diameter 3–8 lm). The position of the fibre is controlled with a three-axes piezo-stage acting as a length transducer. The correct polarity of the actin filament (5–15 lm length, purified from rabbit psoas) is controlled by attaching its barbed end to a bead that is trapped into the focus of the DLOT acting as a force transducer. The experiments (temperature 24 C, ionic strength *60 mM) include measurements of the rupture force and lifetime of the actin-HMM bound in rigor (ATP-free solution) and of the active force developed by the biomachine in 2 mM MgATP. The rupture force of the rigor bond is 12.85 ± 0.35 pN. The bond lifetime under a load of 8 pN has a bi-exponential distribution and the time constant of the major, faster component is *1 s. When the ensemble of myosin motors is perfused with 2 mM MgATP force develops to a steady value of 50 pN in *2 s. Taking into account the compliance of the trap (7 nm/pN), the sliding of the actin filament during force development amounts to 350 nm, which is accounted for by ATP driven cycles of detachment/reattachment of individual myosin motors. Supported by IIT (Genova) and PRIN 2011 (MIUR, Italy). Keywords: Dual laser optical tweezers, Single molecule mechanics, Biomachine
O3.1 Muscle plasticity, exercise, and aging, Fri 09:00–09:15 Skeletal muscle fatigue in health and disease: a functional viewpoint Karatzaferi Christina Muscle Physiology & Mechanics Group, DPESS, University of Thessaly, Greece Skeletal muscles confer movement to the human body using vast amounts of energy provided through complex metabolic pathways. Thus, whole body mobility and energy balance are dictated by muscle contraction. On the other side, muscle function reflects overall health status, as chronic conditions and/or ageing affect either or both of muscle quality (protein and fat content) and quantity (mass) as well as negatively influence the ability of muscle to resist to fatigue. In health, muscle fatigue, the inability to maintain force or power for a given task, is temporary and recovery occurs rapidly. However, in ageing and/or in chronic disease muscle fatigue may occur
79 prematurely and be persistent, endangering thus a person’s safety, and leading the sufferer in a self-perpetuating vicious cycle of inactivity to further disuse muscle atrophy/metabolic disturbance/weakness and so on, that compounds morbidity (i.e. causing metabolic syndrome, fatness, hypertension, muscle cachexia) and that eventually leads to premature death. Various investigative approaches can provide evidence that link the events taking place at the level of actomyosin interaction to whole muscle function. This talk will provide a functional perspective with regards to the various mechanisms implicated in muscle fatigue. Reference will be made to evidence from in vitro studies of muscle fatigue and from in vivo human exercise protocols. The concept of a functional threshold and the benefits of exercise will be mentioned. Then, the characteristics of muscle dysfunction in chronic kidney disease patients will be presented and new results from ongoing studies in this area will be discussed. Acknowledgments: This research has been co-financed by the European Union (European Social Fund—ESF) and Greek national funds through the Operational Program ‘‘Educational and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF)—Research Funding Program: Thales (MuscleFun Project-MIS 377260) Investing in knowledge society through the European Social Fund. Keywords: Muscle dysfunction, Chronic disease, Exercise
O3.2 Muscle plasticity, exercise, and aging, Fri 09:15–09:30 A titin based in vivo approach to sarcomere biology Fink Claudia1***, Rudolph Franziska1, Polack Christopher1, Govindarajan Thirupugal1, Radke Michael1, Granzier Henk2, Gregorio Carol2, Michael Gotthardt1 ***Candidate for Young Investigator Award
1
Max Delbruck Center for Molecular Medicine, Berlin, Germany; University of Tucson, Arizona, USA
2
Titin is a giant protein that forms the backbone of the sarcomere. Its exceptional size makes it difficult to study the holoprotein. With the availability of knockout technology, it has become feasible to not only study patient mutations in the mouse model but also to address the function of titin’s individual domains in adapting the mechanical properties of the sarcomere and regulating growth. Extending the repertoire of titin mutant mice, we have fluorescently tagged titin’s Z-disc and M-band regions to enable the analysis of titin mobility within the sarcomere as well as sarcomere assembly and remodeling. Live imaging of titin-GFP derived cardiomyocytes has provided novel insights into the role of titin as the backbone of the myofilament and its calcium dependent interaction with sarcomeric proteins. Now we have adapted a proximity proteomics approach to investigate the protein composition of the sarcomere along the titin filament. As the repertoire for genetic engineering of the mouse genome and its application to other species is improved, we will see models that more closely mimic human physiology and obtain deeper insights into sarcomere assembly, composition, disassembly, and function. Keywords: Animal models, Titin, Sarcomere
O3.4 Muscle plasticity, exercise, and aging, Fri 09:45–10:00 GDF-15 modifies the muscle microRNA profile to enhance muscle wasting chronic disease
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80 Bloch Susannah, Patel Mehul, Baz Manuel, Griffiths Mark, Polkey Michael, Kemp Paul Imperial College London, UK Muscle wasting is an important co-morbidity in many chronic diseases including chronic obstructive pulmonary disease (COPD) and intensive care unit acquired weakness (ICUAW). Muscle wasting is associated with a reduction in both the quantity and quality of life. The TGF-b family of proteins has been shown to contribute to muscle wasting. One member of this family, GDF-15, is associated with increased all cause mortality but its effects on muscle mass are not clear. We investigated the association of GDF-15 with muscle mass in COPD and in patients with ICUAW. Plasma GDF-15 was elevated in COPD and was associated with muscle mass (r = 0.49, p \ 0.001). In ICUAW, circulating GDF-15 was elevated and associated with an assessment of multiple organ failure (r = 0.64, p = 0.002). We have previously found that miR-1 and miR-499 are associated with muscle mass in COPD. Analysis of these miRs as well as miR-133 and miR181a in ICUAW showed that the miRNAs were suppressed in patients compared to controls and that miR expression was associated with plasma GDF-15 protein and muscle GDF-15 mRNA. In vitro GDF-15 suppressed the expression of miR-181a, miR-1 and miR-133, consistent with a direct role for GDF-15 in their expression. GDF-15 did not activate a SMAD reporter gene nor did it increase the expression of Cyr-16, a TGF-b target gene. Pathway analysis suggested that all the miRs analysed inhibited the TGF-b/myostatin signalling pathway. Consistent with this analysis miR-181a suppressed TGF-b signalling in vitro and previous studies have shown that miR-1 and miR-499 suppress myostatin signalling. Furthermore, nuclearSMAD2/3p and the expression of Cyr-61 and atrogin-1 were increased in ICUAW patients compared to controls. These data suggest that GDF-15 inhibits the expression of miRNAs to sensitise muscle to TGF-b/ myostatin signalling. Keywords: Atrophy, TGF beta signalling, miRNA
J Muscle Res Cell Motil (2015) 36:71–143 control patients (N = 15) were obtained. The cross sectional area (CSA) of fast- and slow-twitch muscle fibers was determined by histology. In single permeabilized diaphragm fibers, we studied maximal active tension (Fmax normalized to CSA) by exposing the fibers to saturating calcium concentration. In the same fibers we determined the myosin heavy chain (MHC) concentration and force per MHC content. Results: The CSA of fast- and slow-twitch diaphragm muscle fibers was not significantly different between groups. Diaphragm fiber Fmax was significantly lower in slow-twitch muscle fibers of CTEPH patients (p \ 0.05), but was unchanged in fast-twitch muscle fibers. MHC concentration was significantly reduced in both slow(p \ 0.02) and fast-twitch (p \ 0.001) muscle fibers of CTEPH patients. The force per MHC content was significantly increased in CTEPH patients, in both slow- (p \ 0.05) and fast-twitch (p \ 0.01) fibers. The CSA and the Fmax of slow-twitch diaphragm fibers of CTEPH patients correlated significantly with MIP, but not with 6 MWT. Conclusion: Diaphragm muscle contractility of CTEPH patients is hampered, which is most likely caused by a reduction in MHC concentration. The correlation of diaphragm muscle fiber size and contractility with MIP suggests that diaphragm weakness might contribute to the sensation of dyspnea in CTEPH patients. Keywords: Single muscle fibers, Myosin heavy chain, Pulmonary hypertension
O3.6 Muscle plasticity, exercise, and aging, Fri 10:50–11:05 The state of protein synthesis signaling pathways in rat soleus during early recovery from simulated microgravity Mirzoev Timur***, Tyganov Sergey, Shenkman Boris ***Candidate for Young Investigator Award
O3.5 Muscle plasticity, exercise, and aging, Fri 10:00–10:15 Diaphragm dysfunction in patients with pulmonary hypertension Manders Emmy1***, Bonta Peter2, Kloek Jaap3, Symersky Petr3, Lommen Wies4, Westerhof Nico5, Stienen Ger JM4, VonkNoordegraaf Anton5, De Man Frances5, Ottenheijm Coen AC4 ***Candidate for Young Investigator Award
1
Pulmonology and Physiology, VU University Medical Center, Amsterdam, Netherlands; 2Pulmonology, Amsterdam Medical Center, Netherlands; 3Cardiothoracic surgery, Amsterdam Medical Center, Netherlands; 4Physiology, VU University Medical Center, Amsterdam, Netherlands; 5Pulmonology, VU University Medical Center, Amsterdam, Netherlands Introduction: Recently, it was proposed that patients with pulmonary hypertension suffer from diaphragm dysfunction. To test this proposition, we studied the contractile strength of individual diaphragm muscle fibers of chronic-thromboembolic pulmonary hypertension patients (CTEPH). Furthermore, we determined whether diaphragm fiber contractile strength correlates with in vivo measures of diaphragm function and exercise capacity. Methods: In CTEPH-patients we assessed maximal inspiratory pressure (MIP) and exercise capacity with a 6 min walk test (6MWT). Diaphragm muscle biopsies from CTEPH patients (N = 12) and
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Myology Laboratory, Institute of Biomedical Problems RAS, Moscow, Russia The aim of our study was to measure protein synthesis rate and analyze (using inhibitors wortmannin and 1-butanol) anabolic signaling pathways in rat soleus after recovery from disuse atrophy. Wistar male rats weighing 190–210 g were divided into the following groups: (1) Control (n = 7), (2) 14HS (n = 7)—hindlimb suspension for 14 days, (3) 14HS + 3R + placebo (n = 7)—3-day readaptation with saline administration, (4) 14HS + 3R + wort (n = 7)—3-day readaptation with wortmannin administration, (5) 14HS + 3R + but (n = 7)—3-day readaptation with 1-butanol administration. Simulated microgravity was produced by HS. Protein synthesis rate was determined by SUnSET technique. Content of p-AKT, p-p70S6 K, p-p90RSK, p-GSK3-beta was assessed by Western blotting. The experiment was approved by Bioethics Committee of the RAS. Two-week HS induced a 33 % (p B 0.05) decrease in protein synthesis compared to control. However, after 3-dayreadaptation we observed an increase in protein synthesis by 40 % (p B 0.05) vs. 14-HS. In wortmannin-treated and 1-butanol-treated rats there was a decrease in protein synthesis rate vs. 14HS + 3R + placebo by 35.6 and 38.5 % (p B 0.05), respectively. Following 3-day recovery we observed increase in p-p70 content vs. 14-HS. In 14HS + 3R + but p-p70 content was lowered by 57.7 % (p B 0.05) vs. 14HS + 3R + placebo. There was 84.5 % (p B 0.05) decrease in p-p90RSK of 3R + placebo compared to control. In recovering 1-butanol-treated rats there was a significant decrease in p-AKT and
J Muscle Res Cell Motil (2015) 36:71–143 p-GSK3-beta vs. 14HS + 3R + placebo, while wortmannin administration did not induce significant changes of these parameters. Thus, the increased rate of protein synthesis during the early recovery from simulated microgravity is most likely caused by both PA-dependent activation of mTORC1 and some mTORC1- independent pathway. The work was supported by the Program No.7 of the Presidium of the RAS and RFBR Grant No. 14-04-31414. Keywords: Soleus muscle, Protein synthesis, Anabolic signaling pathways
O3.7 Muscle plasticity, exercise, and aging, Fri 11:05–11:20 Sarcoplasmic reticulum Ca2+ leak in human skeletal muscle fibres is increased with age Lamboley Cedric1, Victoria Wyckelsma1, Murphy Robyn2, McKenna Michael1, Lamb Graham2 1 Institute of Sport, Exercise and Active Living, Victoria University, PO Box 14428, Melbourne, VIC 8001, Australia; 2School of Life Sciences, La Trobe University, Melbourne, Australia
We investigated the role of sarcoplasmic reticulum (SR) Ca2+ leak as an underlying mechanism for our recent observation that SR Ca2+ content is reduced in both type I and type II muscle fibres in aged compared to young adult human subjects. Fibre segments from vastus lateralis muscle, obtained by needle biopsy in 12 healthy young (25 ± 4.8 years) and 11 old (71 ± 4.3 years) adults, were mechanically skinned and their contractile apparatus and SR Ca2+ uptake properties characterized. The relative SR Ca2+ leak through the ryanodine receptors (RyRs) in young and aged muscle fibres was gauged from the net SR Ca2+ uptake achieved in a load solution at pCa 6.7 when the cytosolic free [Mg2+] was set at 1 mM or 10 mM, the latter expected to substantially reduce any leak through the RyRs. Net SR Ca2+ uptake was ascertained from the time-integral of the force response upon releasing all SR Ca2+ with a caffeine-low [Mg2+] solution. Each fibre was classified as type I or II by western blotting. The pCa producing half maximal force (pCa50) was decreased (p \ 0.05) in type II fibres of aged subjects (5.76 ± 0.03 and 5.83 ± 0.01 pCa units in aged and young, respectively) but not in type I fibres (5.91 ± 0.02 and 5.94 ± 0.01 pCa units, respectively). In type I fibres, the maximal SR Ca2+ content reached after loading in the presence of 10 mM Mg2+, relative to that in 1 mM Mg2+, was significantly higher (p \ 0.05) in aged compared to young subjects (107 ± 6 %, n = 10, and 87 ± 2 %, n = 15, respectively), indicating greater leakage through the RyRs in the aged type I fibres. No such difference was observed in type II fibres (82 ± 4 %, n = 6 and 82 ± 3 %, n = 11, respectively). The decreased Ca2+ sensitivity of the contractile apparatus in aged type II fibres may contribute to muscle weakness observed in the aged population. Furthermore, the greater Ca2+ leakage occurring through the RyRs in type I fibres of aged compared to young subjects may have deleterious effects on Ca2+ movements and muscle function. Keywords: Skinned fibre, SR Ca2+ leak, Ageing
O3.8 Muscle plasticity, exercise, and aging, Fri 11:20–11:35 A little stress for short periods is good for skeletal muscle function Pe´rez Maria1, Mallinson David2, Mutungi Gabriel3
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School of Biological Sciences, Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ, UK; 2Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G4 0RE, UK; 3Department of Medicine, Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ, UK Glucocorticoids (GCs) are steroid hormones released from the adrenal gland in response to both physical and emotional stress. They are also released during strenuous exercise and are the only drugs that have been shown to improve skeletal muscle strength and function as well as delay the onset of disability in suffers of Duchenne muscular dystrophy (DMD) (Angelini, Muscle Nerve 36(4):424–435, 2007) suggesting that they may have beneficial effects in skeletal muscle. However, when GCs are used at high doses for long periods they have many side-effects including a myopathy whose cause is still poorly understood. Consequently, most previous studies that have investigated their effects in skeletal muscles concentrated mostly on the effects of administering high doses of GCs to laboratory animals for several weeks and their physiological actions have been largely ignored. Therefore, the primary aim of this study was to investigate the effects of treating small skeletal muscle fibre bundles with physiological doses of GCs for short periods of time. Our results show that the glucocorticoid receptor (GCR) is expressed mainly in oxidative fibres and treating muscle fibre bundles with physiological concentrations of GCs increases maximum isometric force (Po) in slowtwitch muscle fibres without significantly affecting that of the fasttwitch ones. The increase in Po occurred within 10 min and was insensitive to actinomycin D suggesting that it was non-genomic. It was blocked by the glucocorticoid receptor (GCR) inhibitor, RU486 and a monoclonal anti-GCR; suggesting that it was mediated by a membrane (m) GCR (Perez et al. J Physiol 591:5171–5185, 2013). From these findings we conclude that the rapid/non-genomic actions of GCs are mediated by a mGCR and that they are physiologically/ therapeutically beneficial in slow-twitch muscle fibres. Keywords: Glucocorticoids, Glucocorticoid receptor, Force
O3.9 Muscle plasticity, exercise, and aging, Fri 11:35–11:50 Effects of long term supplementation of anabolic androgen steroids on human skeletal muscle Malm Christer1, Yu Ji-Guo1, Bonnerud Patrik2, Eriksson Anders2, Sta˚l Per S3, Tegner Yelverton2, Nordstro¨m Anna1 1
Department of Surgery and Perioperative Science, Sports Medicine, Umea˚ University, Sweden; 2Department of Health Sciences, Lulea˚ University of Technology, Sweden; 3Department of Integrative Medical Biology, Section for Anatomy, Umea˚ University, Sweden Ten Doped (AAS) and seven Clean elite strength athletes gave muscle and blood samples, and were tested for maximal muscle strength. In addition, iDEXA scanning and blood hormones were analysed. Muscle samples were analysed using fluorescent immunohistochemistry and proteomic screening (2D DIGE/MALDI-TOF). Doped and Clean athletes had higher bone mineral density at all measured sites compared to Controls, and Doped had more lean body mass compare to Clean and Controls. The use of AAS in combination with strength training results in significantly different skeletal muscle gene expression compared to strength training without AAS. The Doped athletes produced significantly lower results in the strength measurement then the Clean athletes despite more muscle mass and similar muscle fiber size and fiber type composition. By using cluster analyses, there is a clear separation between Doped and Clean athletes based on morphological, hematological and proteomic data. Major
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82 findings were that the number of capillaries/fiber and myonuclei/fiber was higher in Doped compared to Clean athletes. The most important hormones separating the groups were, FSH, LH, 17-OH-Prog, Urea and Prolactin. The doped group had approximately one more capillary per muscle fiber compared to the clean group. Based on the proteomic analysis, major proteins separating the Doped from the Clean group were related to energy metabolism, cytoskeleton and cell signaling. We conclude that the Doped group has a lower capacity to generate force per muscle mass and fiber cross section area, possible due to the long term use of AAS. Results from the present study should be used as a base for further investigations regarding long term (possible permanent) effect of AAS. The research approach and analytical methods used in the present study can distinguish an AAS doped athlete from a Clean athlete, regardless of substance used, training regimen, age and duration of AAS supplementation. Keywords: Strength, Steroids, Proteomics
O4.1 Muscle development, differentiation, and regeneration, Fri 14:00–14:15 Mechanisms controlling protein dynamics and turnover in muscle cells
J Muscle Res Cell Motil (2015) 36:71–143 constitutively phosphorylated, serines within murine N1-line titin at S2078 and S2080 (positions according to UniProtKB identifyer, A2ASS6). In addition, by using heart tissue from the SILAC mouse mixed with wild-type (WT) or calcium/calmodulin dependent protein kinase-II (CaMKII) delta/gamma double-knockout (DKO) mouse heart, we studied CaMKII-dependent phosphorylation by quantitative in vivo phosphoproteomics. This screen revealed a conserved serine at position S1805 as a novel CaMKII-dependent phosphosite on N1line titin. Back-phosphorylation and autoradiography on recombinantly expressed human titin fragments were used to identify the phosphorylating kinases in vitro. S1805 and S2080 were both phosphorylated by CaMKII and G-protein-coupled receptor kinase-4 (GRK4). S2078 was phosphorylated by cyclin-dependent kinase-1 (CDK1), extracellular signal-regulated kinase 2 (ERK2), p38 mitogen-activated protein kinase-d (p38d), and casein kinase-2 (CK2). Protein kinase A (PKA), protein kinase C-alpha (PKCalpha), and protein kinase-G (PKG), which phosphorylate spring elements in I-band titin, did not phosphorylate any of these N1-line titin sites. Interestingly, S2080 was found to be phosphorylated in fetal rat hearts (e18) and in early stages of C2C12-cell differentiation. We conclude that phosphorylation of N1-line titin can be mediated by various protein kinases and could play a role in the integration of titin in the sarcomere during early stages of myofibrillogenesis. Keywords: Titin, Phosphorylation, N1-line
Fu¨rst Dieter O Institute for Cell Biology, University of Bonn, Germany Myofibril formation is a paradigm for macromolecular assembly, as it is based on the stepwise incorporation of numerous sarcomeric proteins into an almost crystalline arrangement. The subsequent maintenance of the contractile apparatus poses additional problems, because distinct turnover rates of the composing proteins as well as locally differing strain have to be integrated. A great deal of these tasks is executed by a plethora of cytoskeletal proteins. Of pivotal importance are stringent spatiotemporal control mechanisms regulating protein–protein interactions and/or the life time of a protein. Recent work has revealed how multiple distinct protein interactions, kinase signaling and autophagy determine both a protein’s dynamic state as well as its turnover rate.
O4.3 Muscle development, differentiation, and regeneration, Fri 14:30–14:45
Keywords: Cytoskeleton, Protein dynamics, Autophagy
The maximal shortening velocity and ATP consumption of a muscle fibre is largely dictated by the predominant Myosin Heavy Chain (MyHC) isoform expressed and increases in the order of MyHC I-IIAIIX-IIB. Expression of the very fast contracting, MyHC IIB isoform displays species-specific expression, resulting in vastly different muscle phenotypes between species. We utilized approximately 1 kb of the MyHC IIB promoter from a domestic pig and a human (two large mammals which express and do not express MyHC IIB, respectively) to elucidate the role of the promoter sequence in regulating high and low MyHC IIB expression in large mammals. We identified a 3 bp genomic difference within the CArG and E-box region of the MyHC IIB promoter of pigs and humans that dictates the differential expression of MyHC IIB in these large mammals. Subtle species-specific genomic differences in the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MyHC IIB promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the vastly different muscle phenotypes displayed in these otherwise very physiologically similar mammals. Further, we report that both the pig and human MyHC IIB promoters can be induced by MyoD over-expression or dbcAMP treatment but the capacity to activate the human MyHC IIB promoter is severely limited by the 3 bp mutation located in the CArG-box region of the proximal MyHC IIB promoter.
O4.2 Muscle development, differentiation, and regeneration, Fri 14:15–14:30 Novel phosphorylation sites in the peripheral Z-disk region of the giant sarcomeric protein titin Breitkreuz Martin Jan1***, Voelkel Tobias1, Kru¨ger Marcus2, Hamdani Nazha1, Linke Wolfgang A1 ***Candidate for Young Investigator Award
1
Department of Cardiovascular Physiology, Ruhr University Bochum, Germany; 2Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany At the so-called N1-line of the sarcomere, the inextensible Z-disk segment of the giant protein titin adjoins the extensible I band segment. This titin region consists of four Ig domains (Z7-I1) separated by unique sequences and is encoded by titin exons 27/28. We aimed to identify novel phosphorylation sites within this titin region and the responsible protein kinases. Using multidimensional MS-based protein identification technology (MudPIT) we detected two conserved,
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Functional role of the CArG-box in species-specific expression of myosin heavy chain IIB Brown David***, Brameld John, Parr Tim1 ***Candidate for Young Investigator Award
Biosciences, University of Nottingham, UK
Keywords: Myosin heavy chain IIB, CArG-box, Myogenesis
J Muscle Res Cell Motil (2015) 36:71–143
O4.4 Muscle development, differentiation, and regeneration, Fri 14:45–15:00 The overexpression of NDRG2 promotes C2C12 myoblast proliferation and caspase activity Anderson Kimberley***, Russell Aaron P, Foletta Victoria ***Candidate for Young Investigator Award
Centre for Physical Activity and Nutrition Research (C-PAN), School of Exercise and Nutrition Sciences, Deakin University, Burwood VIC, Australia N-myc downstream-regulated gene 2 (NDRG2) is a putative tumor suppressor and stress-responsive gene that is highly expressed in skeletal muscle. In C2C12 myoblasts, the knockdown of NDRG2 slows proliferation and impairs differentiation with early cell cycle exiting. In contrast, NDRG2 overexpression is well described to decrease proliferation and increase apoptosis rates in cancer cells. Here, we investigated the effect of overexpressing both NDRG2 and a phospho-mutant version of NDRG2 (3A-NDRG2), where the amino acids Ser332, Thr348 and Ser350 have been replaced with alanines, on myoblast proliferation, differentiation and apoptosis. NDRG2 and 3A-NDRG2 both increased myoblast proliferation; however, the effect of 3A-NDRG2 on proliferation remained significantly less than NDRG2. Both gene and protein expression of positive cell cycle regulators CDK2, cyclin B and cyclin D were enhanced, while the cell cycle inhibitor p27 Kip protein expression was reduced, with NDRG2 overexpression only. During early differentiation stages, Myf5 and MyoD expression levels as well as myotube nuclei fusion index were significantly greater with NDRG2 overexpression. While 3A-NDRG2 also demonstrated an enhanced fusion index and upregulation of some positive cell cycle and early differentiation markers, they were all consistently lower than the effects found with NDRG2 overexpression. Finally, the level of apoptosis was investigated. While no differences in DNA fragmentation by TUNEL assay or levels of cleaved caspase 3 or PARP proteins were detected, caspase 3/7 activity was increased with NDRG2, but not with 3A-NDRG2. Therefore, these findings show that NDRG2 overexpression increases C2C12 myoblast proliferation and contributes to the early onset of myoblast differentiation, at least partially through enhanced caspase activity. Additionally, amino acids Ser332, Thr348 and Ser350 contribute to the function of NDRG2 in regulating myogenesis. Keywords: NDRG2, Proliferation, Myogenesis
O4.5 Muscle development, differentiation, and regeneration, Fri 15:00–15:15 Ubiquitin ligase Cbl-b iams a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading Nikawa Takeshi1, Tomoki Abe1, Shohei Kohno2, Katsuya Hirasaka3, Ayako Ohno1, Shigetada Kondo1, Ted Mills2 1
Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; 2Pharmacology/Toxicology, College of Pharmacy, University of Texas at Austin, USA; 3Graduate School of Fisheries Science and Enviromental Studies, Nagasaki University, Japan Introduction: Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth
83 factors and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Methods: In unloading experiments, rodents were subjected to spaceflight for 16 days on the space shuttle Columbia, tail suspension, and denervation, as described previously. Results: Our results indicate that the ubiquitin ligase Cbl-b plays a major role in skeletal muscle atrophy induced by unloading. The mechanism of Cbl-b-induced muscle atrophy is unique, in that it does not appear to involve the degradation of structural components of the muscle; rather, it impairs muscular trophic signals in response to unloading conditions. Recent studies of the molecular mechanisms of muscle atrophy have focused on the role of the IGF-1/PI3K/Akt-1 signaling cascade as a vital pathway in the regulation of the balance between hypertrophy and atrophy. These studies indicate that under muscle-wasting conditions, such as disuse, diabetes, and fasting, decreased IGF-1/PI3K/Akt-1 signaling augments the expression of atrogin-1, resulting in muscle atrophy. However, these studies did not address the mechanisms of the unloadinginduced impairment of growth factor signaling. In the present study, we found that under both in vitro and in vivo experimental conditions, Cbl-b ubiquitinated and induced the specific degradation of IRS-1, a key intermediate of skeletal muscle growth regulated by growth hormone, resulting in the inactivation of Akt-1. The inactivation of Akt-1 led to the upregulation of atrogin-1 through the activation of FOXO3, as well as a reduced mitogen response in skeletal muscle. Conclusion: The activation of Cbl-b may be an important mechanism underlying the failure of atrophic muscle to respond to growth factorbased treatments such as IGF-1 under unloading conditions. Keywords: Cbl-b, Muscle atrophy, Unloading
O4.6 Muscle development, differentiation, and regeneration, Fri 15:15–15:30 Non-synaptic roles of agrin in the early stages of skeletal muscle regeneration Pirkmajer Sergej1***, Gros Katarina1, Matkovicˇ Ursˇka1, Parato Giulia2, Misˇ Katarina1, Podbregar Matej3, Grubicˇ Zoran1, Marsˇ Tomazˇ1, Lorenzon Paola2 ***Candidate for Young Investigator Award
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Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Slovenia; 2Department of Life Sciences, University of Trieste, Italy; 3University Medical Centre Ljubljana and Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Slovenia Agrin is a heparan-sulphate proteoglycan that plays a major role in development and maintenance of the neuromuscular junction. Accelerated agrin degradation has recently been linked to degeneration of the neuromuscular junction and pathogenesis of the ageing-related sarcopenia. Pharmacological strategies to enhance agrin action in sarcopenic muscle may therefore alleviate muscle wasting in the elderly. However, agrin overexpression has also been linked to reduced formation of new myofibers, suggesting a role for agrin as a negative regulator of muscle regeneration. Whether and how agrin suppresses regenerative muscle formation remains unclear. We explored whether agrin modulates the early stages of muscle regeneration. Using cultured human myoblasts we found that proliferation of myoblasts from young donors remained unaltered upon acute or chronic agrin exposure. Conversely, agrin enhanced proliferation of myoblasts from aged donors. Notably, myoblast fusion and maturation of the excitation–contraction coupling in myotubes
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84 remained unaltered in the presence of agrin, indicating increased proliferation was not linked to impaired myogenic differentiation. While myoblasts expressed agrin receptor Lrp4/MuSK, agrin exposure did not stimulate phosphorylation of downstream kinases ERK1/2 and Abl. In addition, pharmacological activation of Abl markedly suppressed myoblast proliferation. These data suggest that agrin does not stimulate myoblast proliferation via the canonical agrin receptor Lrp4/MuSK. Taken together, our results demonstrate that agrin does not suppress muscle regeneration. Furthermore, we show that agrin stimulates proliferation of myoblasts from old donors, which suggests that agrin could promote the regenerative capacity of sarcopenic muscle. Collectively, our findings support the notion that agrin-based pharmacological approaches may lead to novel treatments for sarcopenia. Keywords: Agrin, Skeletal muscle regeneration, Sarcopenia
O4.7 Muscle development, differentiation, and regeneration, Fri 16:05–16:20 Elucidating the complex signaling network involved in neuromuscular junction formation Herbst Ruth1, Du¨rnberger Gerhard2, Camurdanoglu Bahar Z3, Mechtler Karl2 1
Center for Brain Research & Institute of Immunology, Medical University of Vienna, Austria; 2IMP & IMBA, Vienna, Austria; 3 Center for Brain Research, Medical University of Vienna, Austria A reciprocal exchange of signals between muscle fibers and motor neurons results in the formation of neuromuscular junctions. Muscle-specific kinase (MuSK) is a receptor tyrosine kinase, which is the key player during neuromuscular junction formation. MuSK is activated as tetrameric complex together with the low-density lipoprotein receptor Lrp4. Tetramerization is promoted by the binding of the motor neuronderived heparan proteoglycan agrin. Signal transduction events downstream of MuSK activation induce pre- as well as postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors (AChRs) at synaptic sites. Impaired MuSK signaling results in acute neuromuscular deficiencies as presented during myasthenia gravis or, even more severely, in respiratory failure and perinatal death in newborn mice lacking MuSK. Although the critical role of MuSK function as signaling molecule has been well established the signaling cascade downstream of MuSK is less well understood. We therefore used a quantitative phosphoproteomics approach to study the phosphorylation events and their temporal regulation downstream of MuSK. We identified a total of 10183 phosphopeptides of which 203 were significantly up- or downregulated. Regulated phosphopeptides were classified into four different clusters according to their temporal profiles. Within these clusters we found an overrepresentation of specific protein classes associated with different cellular functions. In particular, we found an enrichment of regulated phosphoproteins involved in posttranscriptional mechanisms and in cytoskeletal organization. These findings provide novel insights into the complex signaling network downstream of MuSK and form the basis for future mechanistic studies. Keywords: Neuromuscular junction, Receptor tyrosine kinase, Phosphorylation
O4.8 Muscle development, differentiation, and regeneration, Fri 16:20–16:35 Oxidative stress regulates titin elasticity by affecting Ig-domain stability
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J Muscle Res Cell Motil (2015) 36:71–143 Linke Wolfgang A1, Alegre-Cebollada Jorge2, Hamdani Nazha1, Breitkreuz Martin Jan1, Leichert Lars3, Fernandez Julio M2 ***Candidate for Young Investigator Award
1
Department of Cardiovascular Physiology, Ruhr University Bochum, Germany; 2Columbia University, New York, USA; 3Medical Proteome Center, Ruhr University Bochum, Germany
Background: The elasticity of titin is regulated through several mechanisms, including isoform switching and phosphorylation of unique spring elements (N2-Bus, PEVK). However, the titin springs consist mainly of immunoglobulin-like (Ig) domains, which are centrally involved in the molecular mechanism of titin elasticity. Passive force-regulating mechanisms targeting the Ig-domains of titin have not been described. Methods and Results: We have elucidated a novel oxidative stressrelated mechanism regulating muscle elasticity by altering the stability of titin-Ig domains (Alegre-Cebollada et al. Cell 156:1235–1246, 2014). Using single-molecule AFM force spectroscopy, force measurements of isolated skinned human myocytes, and redox proteomics, we show that I-band Ig-domains of titin are weakened by oxidative modification of cryptic cysteines. We demonstrate that mechanical unfolding of these Ig domains exposes hidden cysteines, which now become accessible to disulfide bonding or S-glutathionylation in the presence of millimolar concentrations of oxidized glutathione (GSSG). In the AFM experiments, the cysteines of unfolded titin-Ig domains preferentially formed mixed disulfides with glutathione, which prevented the refolding of these domains. Oxidation by GSSG substantially reduced the passive tension of stretched human myocytes, and the effect was fully reversible with the incubation of reduced glutathione. Exposing perfused mouse hearts to oxidative stress (0.1 mM H2O2) revealed that the Ig-domains from I-band titin are preferential targets of oxidation, as monitored using ICAT labeling/mass spectrometry. Conclusions: Titin elasticity in striated muscle is modulated by oxidative stress through reversible weakening of Ig-domain stability via S-glutathionylation of buried cysteines. These titin Ig domains could also represent individual mechanosensors, whose mechanical properties determine mechano-chemical signaling processes in stressed myocytes. Keywords: Redox stress, Molecular mechanics, Elasticity
O5.1 Excitation–Contraction Coupling, Sat 09:10–09:30 New approaches to answer long-standing questions in EC coupling research Flucher Bernhard E Department of Physiology and Medical Physics, Medical University Innsbruck, Austria Excitation–contraction (EC) coupling is the mechanism by which action potentials are transduced into the cytoplasmic calcium signal that regulates muscle contraction. At the heart, it is the interaction of a voltage-gated calcium channel (DHPR) and the SR calcium release channel (RyR). In cardiac muscle calcium entering through the DHPR triggers the release of further calcium by the RyR, and both channels contribute to the myoplasmic calcium increase. In skeletal muscle DHPR and RyR physically interact and SR calcium release is activated directly by a conformational change of the DHPR voltagesensor. Calcium influx through the DHPR is dispensable for skeletal muscle EC coupling. In fact, the DHPR calcium currents are small
J Muscle Res Cell Motil (2015) 36:71–143 and activate slowly at +30 mV more depolarized voltages than the activation of EC coupling. This unique signaling mechanism poses a number of problems that, even after half a century intensive research, we are only beginning to understand. For example: What is the nature of the interaction between the DHPR and the RyR in skeletal muscle EC coupling? What, if any, is the physiological significance of the slowly activating calcium current through the DHPR? How can the DHPR voltage-sensor activate two processes—opening of the RyR and gating of its own channel pore—at such greatly different voltages? This introductory lecture will portray the state of the art in EC coupling research and demonstrate examples of current experimental approaches, which shed new light on these imminent research questions. Keywords: Excitation–contraction coupling, Voltage-gated calcium channels, Skeletal muscle
O5.2 Excitation–contraction coupling, Sat 09:30–09:45 Voltage-gated ion channel dysfunction in dystrophic cardiomyopathy Koenig Xaver1, Rubi Lena1, Gawali Vaibhavkumar S1, Bittner Reginald E2, Todt Hannes1, Hilber Karlheinz1 1 Center for Physiology and Pharmacology, Medical University of Vienna, Austria; 2Center for Anatomy and Cell Biology, Medical University of Vienna, Austria
Duchenne muscular dystrophy (DMD), induced by mutations in the gene encoding for dystrophin, is an inherited disease characterized by progressive muscle weakness and premature death. Besides skeletal muscle degeneration, DMD is also associated with cardiac problems. These include cardiomyopathy development and cardiac arrhythmias. The current understanding of the pathomechanisms in the dystrophic heart is limited, but recent research indicates an involvement of dysfunctional ion channels. The aim here was to characterize abnormalities in voltage-gated ion channel function in dystrophic ventricular cardiomyocytes. By using the whole cell patch–clamp technique, the properties of currents through sodium and L-type calcium channels in cardiomyocytes isolated from the hearts of normal mice and DMD mouse models were compared. We found that sodium currents were significantly reduced in cardiomyocytes isolated from adult dystrophic mice. Calcium currents, on the other hand, were markedly increased, and calcium channel inactivation was reduced in dystrophic myocytes. Studies on cardiomyocytes derived from neonatal dystrophic animals showed that ion channel abnormalities already occur prior to cardiomyopathy development. Finally, the described ‘‘dystrophic’’ channel abnormalities entailed alterations in the electrocardiograms of dystrophic mice. We conclude that ion channel abnormalities in the dystrophic heart may cause cardiac arrhythmias. These abnormalities occur prior to pathology development and may thus be considered cardiomyopathy triggers, as well as potential new therapeutic targets to prevent cardiomyopathy in DMD patients. Supported by the Austrian Science Fund FWF (P19352, P23060). Keywords: Dystrophic cardiomyopathy, Voltage-gated ion channels, Cardiac arrhythmia
O5.3 Excitation–Contraction Coupling, Sat 09:45–10:00 Role of ClC-1 and KATP channel activation during prolonged action potential firing for action potential waveform and SR Ca2+ release
85 Holm Pedersen Thomas, Riisager Anders, Bækgaard Nielsen Ole Department of Biomedicine, Aarhus University, Denmark Repetitive firing of action potentials (APs) has previously been shown to be associated with biphasic changes in the resting membrane conductance (Gm) of rat fast-twitch muscle: Onset of activity reduces Gm through ClC-1 Cl- channel inhibition (Phase 1) while prolonged AP firing ([1500 APs) is associated with fully reversible 3–4 fold rise in Gm that reflects (re)-activation of ClC-1 and KATP channels (Phase 2). Interestingly, very similar activity loads are required to trigger Phase 2 and fatigue in single fast-twitch fibres. Since such fatigue reflects loss of SR Ca2+ release the present study explored for co-temporality between Phase 2 and loss of SR Ca2+ release. Ca2+ transients, Gm and propagating APs were recorded in the same fibres of intact EDL muscles from rat. Three electrodes were inserted in a fibre. The middle electrode ionophoretically injected Mag-Fluo-4. After loading, a peripheral electrode repeatedly triggered short AP and Ca2+ trains and Gm was determined in between these trains. As previously observed, Gm underwent a biphasic regulation during repetitive AP firing. Ca2+ transients were well maintained during Phase 1 declining no more than 15 % within the first 1500 APs (n = 4). In contrast, with onset of Phase 2 the transients fell markedly with the peak of the 2500th transient being around 20 % of first transient. The electrode farthest from the AP-triggering electrode recorded fully propagating APs. Their analysis showed that onset of Phase 2 was associated with a marked drop in AP waveform above 20 mV, a voltage comparable to half-activation voltage of the t-system voltage sensor (charge movements) in rat EDL. After firing around 4000 APs, the activity was stopped and Gm completely recovered within one-minute rest while Ca2+ transients recovered only partially. In conclusion, these preliminary data suggest that the rise in Gm during Phase 2 is associated with changes in AP waveform that may underlie loss of Ca2+ release during prolonged activity. Keywords: Skeletal muscle excitability, Chloride channels, Calcium release
O5.4 Excitation–contraction coupling, Sat 10:00–10:15 Depression of voltage-activated Ca2+ release in skeletal muscle by activation of a voltage-sensing phosphatase Jacquemond Vincent1, Berthier Christine1, Bouvard Clement1, Kutchukian Candice1, Okamura Yasushi2 1
Centre de Genetique et de Physiologie Moleculaire et Cellulaire, CNRS UMR 5534, Universite Lyon 1, France; 2Laboratory of Integrative Physiology, Osaka University, Japan Phosphoinositides (PtdInsPs) act as signaling molecules in a vast diversity of cellular transduction processes and PtdIns(4,5)P2 is known to regulate the function of several types of plasma membrane ion channels. We investigated the potential role of PtdIns(4,5)P2 in the regulation of Ca2+ homeostasis and excitation–contraction (E-C) coupling in adult mouse muscle fibres using in vivo expression of the voltage-sensing phosphatases Ci-VSP and Dr-VSP. Confocal images of EGFP-tagged Dr-VSP revealed a double-banded pattern consistent with localization of the protein within the triadic region. In both CiVSP and Dr-VSP expressing fibres, membrane current measurements from a depolarized holding potential established the presence of a gating current component consistent with the voltage-sensing domain of the VSP being active in the t-tubule membrane with a mid-activation voltage of *+60 and *+90 mV, respectively. Rhod-2 Ca2+ transients generated by voltage-clamp depolarizing pulses to levels below the range of activation of VSPs were unaffected by expression of the VSPs. However, in Ci-VSP expressing fibres challenged by 5-s
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86 long depolarizing pulses to +20 and +120 mV, the late Ca2+ level during the pulses was significantly lower at +120 mVthan at +20 mV. Furthermore, upon repeated application of 200 ms-long depolarizing pulses to +100 mV, Ci-VSP expressing fibres exhibited a depression of Ca2+ release with, in average, the peak Ca2+ transient being depressed by *30 % after ten pulses. The phenomenon was reversible and was also observed in Dr-VSP expressing fibres. Cav1.1 Ca2+ channel activity appeared unaffected by VSP activation. Results suggest that VSP-induced depletion of t-tubule PtdIns(4,5)P2 alters voltage-activated Ca2+ release. As InsP3 signaling is unlikely to play a role in this process, the effect may rather result from a regulatory interaction between PtdIns(4,5)P2 and a t-tubule membrane protein involved in the activation of the ryanodine receptor. Keywords: Sarcoplasmic reticulum Ca2+ release, Phosphoinositides, Ryanodine receptor
O5.5 Excitation–contraction coupling, Sat 10:50–11:05 Structural and binding studies of the Cav1.1 b1a subunit Casarotto Marco1, Karunasekara Yamuna1, Aditya Shouvik1, Cappello Jean1, Dulhunty Angela F1, Board Philip G1, Oakley Aaron2, Norris Nicole1 1 John Curtin School of Medical Reseach, Australian National University, Canberra, Australia; 2School of Chemistry, University of Wollongong, Australia
Excitation–contraction (EC) coupling in skeletal muscle requires a physical coupling between the voltage-gated calcium channel (Cav1.1) in the surface membrane and the skeletal ryanodine receptor (RyR1) Ca2+ release channel in the membrane of the sarcoplasmic reticulum Ca2+ store. Although the exact molecular mechanism of EC coupling is unresolved, both the a1s and b1a subunits of Cav1.1 are essential for this process. The b1a subunit has a modular structure consisting of SH3/ guanylate kinase (GK) domains separated by a variable hook region. The GK domain binds with high affinity to the I-II loop of the a1 subunit, but the functional significance of the SH3 domain remains undefined. Until now the structure of the Cav1.1 b1a subunit has not been experimentally determined, but other Cav b-isoform structures have suggested that the SH3 binding site is occluded, preventing binding to polyproline-rich partners. This prediction is at odds with our findings that show the Cav1.1 b1a subunit and the a1s subunit II-III loop interact (Kd = 3 lM). We demonstrate that this interaction takes place through the SH3 domain of the b1a subunit and a proline-rich region of the a1s II-III loop, which has previously been shown to be critical for skeletal-type EC-coupling (Kugler et al. J Biol Chem 279(6):4721–4728, 2004). Through mutational studies we demonstrate that isoform-specific differences in the SH3 RT loop enable the interaction of the b1a SH3 domain with prolinerich binding motifs. Our determination of the crystal structure of Cav1.1 b1a provides the first opportunity to examine differences between this isoform and other published structures. In light of this novel structure and binding data, we discuss the specific role of the b1a subunit in EC coupling and its relationship with the Cav1.1 a1 subunit and RyR1. Keywords: Protein-protein interaction, Dihydropyridine receptor, X-ray crystallography
O5.6 Excitation–contraction coupling, Sat 11:05–11:20 Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes: role of adenine nucleotide translocase
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J Muscle Res Cell Motil (2015) 36:71–143 Roussel Julien1, Thireau Jeroˆme1, Brenner Catherine2, Saint Nathalie1, Scheuermann Nathalie1, Lacampagne Alain1, Le Guennec Jean-Yves1, Fauconnier Je´re´my1 1 INSERMU1046, Universite´ Montpellier 1, Universite´ Montpellier 2, Montpellier, France; 2INSERM UMR-S769LabEx LERMIT, Universite´ Paris-Sud, Chaˆtenay Malabry, France
Long chain fatty acids bind to cartinine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulations are associated with severe cardiac arrhythmia such as ventricular tachycardia or fibrillation. We hypothesized that LCAC, such as palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca2+ dependentarrhythmia. In isolated cardiac mitochondria from C57/Bl6 mice, application of 10 lM PC decreases adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial ROS production, measured with MitoSOX red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongrekic acid (20 lM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation and S-nitrosylation, dissociated FKBP12.6 and increased sarcoplasmic reticulum (SR) Ca2+ leak. ANT inhibition or anti-oxidant strategy (N-acetyl cysteine) prevented SR Ca2+ leak, FKBP12.6 depletion and RyR2 oxidation induced by LCAC. Finally, both bongrekic acid and NAC significantly reduced spontaneous Ca2+ waves occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca2+ handling in a ROS-dependent pathway. In pathophysiological conditions, PC accumulation may contribute to development of ventricular arrhythmia through RyR2 dysfunction. Keywords: RyR2, ANT, Long chain acyl carnitine
O5.7 Excitation–contraction coupling, Sat 11:20–11:35 Muscle activity controls the association of mitochondria to calcium release units in skeletal fibers Pietrangelo Laura1, Ambrogini Patrizia2, Sartini Stefano2, Kern Helmut3, Boncompagni Simona1, Protasi Feliciano1 1 CeSI – Center for Research on Ageing, University G d’Annunzio, I-66013 Chieti, Italy; 2Department of Earth, Life and Environment Sciences, University of Urbino Carlo Bo, I-61029 Urbino, Italy; 3 Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, A-1010 Vienna, Austria
At the most basic level, skeletal muscle contraction requires Ca2+ and ATP and, thus, is under direct control of two major intracellular organelles: Ca2+ release unit (CRU), and mitochondria. CRUs are the sites of excitation–contraction coupling, the process responsible for triggering Ca2+ release from intracellular stores, i.e. sarcoplasmic reticulum (SR), in response to a propagating action potential in the T-tubule membrane. Mitochondria are the powerhouse of the cell, being responsible for aerobic production of ATP. CRUs and mitochondria in skeletal fibers are functionally and structurally coupled: a) Entry of Ca2+ into the mitochondrial matrix is able to stimulate the respiratory chain. b) We have recently discovered that, in adult skeletal muscle fibers, mitochondria and CRUs are structurally linked to one another by small stands, or tethers. Here we tested the following hypothesis: muscle activity improves/maintains the correct association between CRUs and mitochondria, which is challenged by ageing and inactivity. Using electron and confocal microscopy
J Muscle Res Cell Motil (2015) 36:71–143 (respectively EM and CM), we studied: a) ageing human biopsies and mouse muscle fibers and b) denervated rat muscle (by nerve crush). Our quantitative EM analysis shows that ageing (in humans and mice) and transient denervation (14 days, in rats) results in decreased association between CRU and mitochondria (2-to-3 folds decrease), whereas exercise and re-innervation either maintains or rescues the association between the two organelles (up to control levels). Functional implication of maintained/rescued proper-association between CRUs and mitochondria is potentially large: indeed, efficient Ca2+ uptake into mitochondria likely depends on their position in respect to sites of Ca2+ release. Keywords: Excitation–contraction coupling, Sarcoplasmic reticulum, ageing
O5.8 Excitation–contraction coupling, Sat 11:35–11:50 Usage of a novel microflow pipette shows that mitochondria networks are not extensive in skeletal muscle fibres Bruton Joseph1, Jefferies Gavin2, Ainla Alar2, Westerblad Ha˚kan3 1
Physiology & Pharmacology, Karolinska Institute, Stockholm, Sweden; 2Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden; 3 Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden In some tissues, mitochondria form dynamic networks that change over the minute timescale. In contrast, mitochondria in adult mammalian skeletal muscle show little motility or dynamic changes in structure over a period of several hours. Here, we use a novel three channelled microflow pipette device to test whether mitochondria in mouse skeletal muscle are coupled to each other. The central channel in the pipette delivers compounds to a restricted region, typically 30 lm in diameter. Two channels on either side of the central channel use suction to remove the compound completely from the bulk solution. Compounds were delivered to the end or side of single adult mouse muscle fibres to test whether local changes in mitochondrial membrane potential were observed in more distantly located mitochondria. Mitochondrial membrane potential was monitored with tetramethylrhodamine ethyl ester (TMRE) during the application of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 100 lM). Fluo-3 was used to monitor cytosolic calcium. When a pulse of FCCP (30 lm in diameter) was applied to the muscle fibre, there was a rapid depolarization and decrease in the mitochondrial TMRE signal to 0.38 ± 0.04 of its initial value. After washout of FCCP, the TMRE signal partially recovered as expected (0.50 ± 0.03, n = 8). At distances of 50 lm and greater away from the site of FCCP application, the mitochondrial TMRE signal did not change. Similar results were observed when two sites on the length of the fibre were pulsed sequentially with FCCP. At the end of the experiment, fibres showed no change in cytosolic calcium and twitched normally in response to electrical stimulation. The results of this study indicate that dynamic networks of mitochondria do not exist in skeletal muscle. Furthermore, the limited and reversible effects of the targeted application of FCCP with the microflow pipette highlight its advantages over bulk application of compounds to isolated cells. Keywords: Skeletal muscle, Mitochondria, Calcium
O5.9 Excitation–contraction coupling, Sat 11:50–12:05 Modulation of contractile apparatus Ca2+ sensitivity in a fibre type specific manner following repeated sprint exercise
87 Ørtenblad Niels1,2, Hvid Lars G1, Jensen Rasmus1, Andersson Erik2, Willis Sarah J2, Holmberg Hans Christer2, Gejl Kasper Degn1 1
Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Denmark, 2Swedish Winter Sports Research Centre, Mid Sweden University, Sweden
The purpose of the present study was, to examine the effects of repeated sprint skiing on the contractile apparatus of single muscle fibres. Eleven elite male cross-country skiers (VO2max 5.1 ± 0.5 L min-1) performed 4 times *4 min all-out intermittent sprint skiing on a treadmill. Muscle biopsies were obtained in arm muscle (m. biceps brachii) before and after the sprint exercises. Single muscle fibre segments were isolated, attached to a sensitive force recording transducer, and activated by Ca2+ buffered solutions to measure specific force (max force/cross sectional area) and Ca2+ sensitivity and thereafter fibre typed. A total of 150 fibres containing myosin heavy chain 1 (MHC1) and 82 MHC2 fibres were analysed. At rest, specific force was 37 % higher in MHC2 fibres than in MHC1 fibres. The repeated high-intensity exercise did not affect specific force in neither MHC1 nor MHC2 fibres. However, Ca2+ sensitivity was significantly increased in MHC2 fibres, with no effect in MHC1. The increase in Ca2+ sensitivity in MHC2 fibres, was fully reversed when the fibres were incubated with a strong reducing agent (DTT) prior to activation, indicative of an oxidative mediated effect on the contractile apparatus with high-intensity exercise in human. Further, plasma total plasma anti-oxidative capacity and thiol compound redox status (GSH/GSSG) were significantly reduced by 28 and 18 % after exercise. By using a translational approach from whole-body exercise to single fibre measurements, we supports previous in vitro findings of oxidative changes in the skeletal muscle cell with exercise, affects Ca2+ sensitivity in a fibre type dependent manner. Keywords: Contractile apparatus, Exercise, Fibre type
O6.1 Heart muscle & cardiomyopathy, Sat 14:00–14:15 An introduction on the pathomechanisms of heart failure Stienen Ger JM Department of Physiology, Institute for Cardiovascular Research VU University Medical Center, Amsterdam, Netherlands Heart failure is a multi-factorial progressive disease in which eventually the contractile performance of the heart is insufficient to meet the demands of the body even at rest. A distinction between can be made on the basis of the cause of the disease in genetic or acquired heart failure and at the functional level between systolic and diastolic heart failure. Here the basic determinants of contractile function of myocardial cells will be introduced and their role in the alterations in contractile function in heart failure will be described. The subjects covered are: myocardial structure, the tension generating capacity, passive tension and the rate of tension development of myocardial cells, the rate of ATP utilisation, calcium sensitivity of tension development, the regulatory aspects of phosphorylation of contractile proteins, length dependent activation and stretch activation. The reduction in contractile performance during systole can be attributed predominantly to a loss of cardiomyocytes (necrosis), myocyte disarray, a decrease in myofibrillar density and likely also to limited energy supply. This leads to a decline in the ejection fraction of the heart. Diastolic dysfunction can be attributed mainly to an increase in wall thickness, fibrosis and an increase in titin stiffness which result in an increase in stiffness of the ventricular wall that hampers filling of the heart with blood during diastole. A large number of post translation modifications of regulatory sarcomeric proteins influence
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88 myocardial function by altering calcium sensitivity of tension development. It is unclear whether in concert these are adaptive or maladaptive during the disease process. Keywords: Cardiomyocyte, Stiffness, Calcium sensitivity
O6.2 Heart muscle & cardiomyopathy, Sat 14:15–14:30 Experimental and computational studies of myofilament structure–function in normal and diseased muscle Regnier Michael1, Cheng Yuanhua2, Lindert Steffan3 1 Department of Bioengineering, University of Washington, Seattle, USA; 2University of Washington, Seattle, USA; 3University of California at San Diego, USA
We use a combination of site-directed mutagenesis, protein biochemistry, multi-scale mechanical analysis and computational modeling to study the regulation of normal muscle myofibril contraction and how this is altered in diseases of the sarcomere. This interdisciplinary approach allows us to do detailed structure–function analysis. Mutations in cardiac troponin C (cTnC) that increase Ca2+ binding affinity also increase its affinity for cardiac troponin I (cTnI) and when these cTnC mutants are exchanged into myofibrils or skinned trabeculae, they increase the magnitude and rate of force generation at sub-maximal, but not maximal levels of activation, and can slow the early phase of relaxation. In contrast, cTnC mutations that reduce Ca2+ binding affinity have either no effect or reduce interaction with cTnI, reduce the magnitude and rate of force generation at all levels of Ca2+ and speed relaxation. Molecular Dynamics (MD) simulations show positive correlation between Ca2+ binding affinity and stability of both (1) interaction of Ca2+ with coordinating side chains in site II and (2) the hydrophobic patch of cTnC. Together the data suggest that native cTnC may operate just at the edge of maximal effectiveness. Mutations in cTnI associated with hypertrophic cardiomyopathy increase its affinity for cTnC and also Ca2+ binding affinity of cTnI. Interestingly, they also blunt the ability of cTnI Ser 23/24 phosphorylation to reduce its affinity for cTnC and increase the rate of early phase relaxation. MD simulation studies of whole cTn suggest Ser 23/24 phosphorylation leads to the formation of the intrasubunit interaction between the N-terminus and the inhibitory peptide region of cTnI. We are studying how this, and other intra-molecular interactions may be affected by hypertrophic cardiomyopathy (HCM) associated mutations and the contraction and relaxation properties of cardiac muscle. HL65497, HL11197 (MR), 8P41GM103426 (UCSD). Keywords: Troponin, Contraction, Molecular dynamics simulation
O6.3 Heart muscle & cardiomyopathy, Sat 14:30–14:45 Structure and dynamics of the human cardiac myosin regulatory light chain: implications for heart function and regulation Pfuhl Mark King’s College London, UK The myosin regulatory light chain (RLC) plays a complex role in muscle contraction: on one hand it provides mechanical stability to the lever arm, on the other hand its phosphorylation involves it in the regulation of myosin activity. The latter is poorly understood because of its heterogeneity amongst different types of muscle: in smooth muscle, RLC phosphorylation is the main switch to activate contraction. In striated muscle this function is performed by the troponin
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J Muscle Res Cell Motil (2015) 36:71–143 complex so that the precise purpose of RLC phosphorylation is still not clear. It is certainly important in cardiac muscle where it appears to be correlated to the wringing motion of this complex muscle. The understanding of the function of RLC is further hampered by the absence of structural information for mammalian RLCs. We have therefore embarked on the solution structure determination of the human cardiac RLC. The structure is based on 3421 NOE distance constraints, 382 dihedral angle constraints and refined against 134 residual dipolar coupling constraints. The static picture of the structure is supplemented by the measurement of 15N relaxation parameters to add the dynamics to give a complete view. The most interesting features of the structure are the complex dynamics of the highly divergent N-terminus close to the phosphorylation site and the linker that connects the N- and C-terminal lobes as well as the heavy chain hook. The latter adopts an unusually obtuse angle of *126 on average with a broad spread across the structures of the family (112– 138). The N-terminus appears to make transient interactions with other parts of the RLC which are changed upon phosphorylation. The relevance of these new findings are discussed with respect to known RLC structures, their implications for the role of the RLC in myosin function, it’s regulation and the recently described interaction with domain C0 of myosin binding protein C. Keywords: cardiomyopathy, myosin binding protein C, phosphorylation
O6.4 Heart muscle & cardiomyopathy, Sat 14:45–15:00 The organisation of mitochondria near the intercalated disc in normal and DCM heart Bennett Pauline, Wilson Amanda, Ehler Elisabeth The Randall Division of Cell and Molecular Biophysics, King’s College London, UK We recently presented evidence that cardiomyocyte growth can occur by insertion of new sarcomeres at the ends of myofibrils near the intercalated disc (ID) (Wilson et al. Cell Mol Life Sci 71:165–181, 2014). Although myofibrils make up the bulk of the cell, mitochondria form columns interspersed between the fibrils which occupy some 35 % of the cell cross sectional area. To maintain the ordered structure during myocyte elongation mitochondria need to proliferate at the same rate as the myofibrils. The mitochondrial columns are frequently interrupted at the Z-disc level by t-tubules. T-tubules and junctional(j) SR both exhibit regions of close contact with the mitochondria. We have found that similar close associations occur between mitochondria and the plasma membrane and peripheral SR couplings at the ID membrane peaks. This suggests that the membrane peaks may be the basis for new t-tubule formation after sarcomere addition. In a model for dilated cardiomyopathy (DCM), the muscle Lim protein (MLP) null mouse, regions of uncontrolled sarcomere proliferation are found near the ID and are characterised by the absence of mitochondria. Published data show that in DCM, in general, mitochondria function below par and are frequently clumped. Furthermore, there is a breakdown of the t-tubule system and loss of jSR leading to reduced availability of calcium to both mitochondria and myofibrils. We have looked in more detail at the distribution of mitochondria near the ID in control and DCM hearts. We find that in diseased tissue even where the structure looks well organized there is a reduction in the number of mitochondria associated with the last sarcomere before the ID indicating that the growth of mitochondria in these hearts lags behind that of the myofibrils. We are presently investigating whether the organisation of peripheral SR at the ID is affected. Keywords: t-Tubules
Dilated
cardiomyopathy,
Sarcoplasmic
reticulum,
J Muscle Res Cell Motil (2015) 36:71–143
O6.6 Heart muscle & cardiomyopathy, Sat 15:15–15:30 Hypertrophic cardiomyopathy: unequal abundance of mutated myosin in individual cardiomyocytes causing imbalanced force generation—a possible trigger for disease development Montag Judith1, Makul Mirza1, Tripathi Snigdha1, Dunda Sebastian1, Perrot Andreas2, Francino Antonio3, Navarro-Lopez Francesco3, Van der Velden Jolanda4, Brenner Bernhard1, Kraft Theresia1 1 Molecular and Cell Physiology, Hannover Medical School, Germany; 2Cardiovascular Genetics, ECRC, ChariteUniversita¨tsmedizin Berlin, Germany; 3Hospital Clinic, University of Barcelona,Barcelona, Spain; 4Cardiovascular Research, VU University Medical Center, Amsterdam, Netherlands
Familial hypertrophic cardiomyopathy (FHC) is frequently caused by mutations in sarcomeric proteins. Among genotyped patients about 1/3 are heterozygous for missense mutations in ß-myosin (ß-MyHC). Yet, it is still unclear how numerous different mutations in different proteins lead to a quite similar phenotype but variable severity. We previously observed for slow skeletal muscle fibers of FHC patients a large functional variation among individual fibers, presumably resulting from fiber-to-fiber variation in abundance of mutated ßMyHC. We therefore hypothesized that FHC-typical features in myocardium may arise from similar variable expression of mutated myosin from cardiomyocyte to cardiomyocyte. If so, functional alterations caused by the mutated myosin, e.g., higher or lower force generation that we previously found for FHC-related ß-MyHC-mutations, would lead to imbalanced force generation among neighboring cardiomyocytes. Such imbalances could induce distortions and even disarray within the cellular network of FHC-affected myocardium. We tested this hypothesis by quantifying the relative abundance of mutated ß-MyHC-mRNA in individual cardiomyocytes of cardiac samples of FHC-patients with ß-MyHC mutations R723G and A200 V. To check for functional variation from cell-to-cell we performed biomechanical studies on cardiomyocytes from the same tissue samples. For both mutations we found the relative expression of ß-MyHC-mRNA to vary from cell to cell from almost pure mutant to almost pure wildtype mRNA. Force measurements on individual myocytes at different calcium concentrations revealed large variation in calcium sensitivity from much reduced to essentially control (wildtype) sensitivity. From this, we conclude that in FHC myocyte disarray and hypertrophy may be set off by unequal expression of mutated ß-MyHC among individual cardiomyocytes, thus causing functional imbalances that trigger alterations in morphology and cell signaling in FHC-affected myocardium. Keywords: Hypertrophic cardiomyopathy, Myosin, Single human cardiomyocytes
O6.7 Heart muscle & cardiomyopathy, Sat 16:05–16:20 Effects of the N-terminal regulatory regions of cardiac troponin I and cardiac troponin T on diastolic and systolic functions of the heart Jin Jian-Ping Wayne State University, Detroit, USA The N-terminal regions of cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are both regulatory structures. Restrictive proteolysis in vivo generates N-terminal truncations of cTnI (cTnI-ND, removing the first 30 residues) and cTnT (cTnT-ND, removing the first 71 residues). cTnI-ND and cTnT-ND remain in the cardiac myofilaments
89 with functional effects as compensatory mechanisms in cardiac adaptation and heart failure. We have demonstrated that cTnI-ND enhances diastolic function. Ex vivo working hearts showed that cTnI-ND mouse hearts exhibited faster velocity of relaxation, lower left ventricular end diastolic pressure and larger stroke volume than that of wild type hearts, demonstrating an enhanced Frank-Starling response. Slack sarcomere length in isolated cardiomyocytes and the optimal sarcomere length for maximum development of active tension in intact papillary muscle were unchanged, indicating that cTnIND extended the range of Frank-Starling response independent of sarcomere length. cTnT-ND on the other hand affects the systolic function of cardiac muscle. Transgenic mouse hearts expressing predominantly cTnT-ND showed a small but statistically significant decrease in contractile velocity. This change elongates the ventricular rapid ejection time to compensate for ventricular stroke volume against pressure load. Therefore, cTnT-ND hearts demonstrated significantly larger stroke volume with a minimal increase in energy consumption in comparison to wild type controls. This observation indicates a novel mechanism to increase the energetic efficiency of ventricular pumping. These results suggest that restrictive proteolysis to remove the N-terminal extension of cTnI or the N-terminal variable region of cTnT provides posttranslational regulations with effects selectively on the diastolic or systolic function of the ventricular muscle, which are potentially submolecular targets for the development of new treatments for diastolic and systolic heart failures. Keywords: Troponin, Posttranslational modification, Systolic and diastolic function
O6.8 Heart muscle & cardiomyopathy, Sat 16:20–16:35 Changes in cardiac titin domain phosphorylation increase passive myofilament stiffness in patients with type 2- diabetes Mu¨ller Anna-Eliane1***, Andresen Christian1, Ko¨tter Sebastian1, Ro¨ll Wilhelm2, Kru¨ger Martina1 ***Candidate for Young Investigator Award
1
Cardiovascular Physiology, University Du¨sseldorf, Germany; Cardiac Surgery, University Clinic Bonn,Germany
2
The sarcomeric protein titin is one main component that contributes to the passive stiffness of striated muscle tissue. In mammalian heart, Titinbased myofilament stiffness is an important determinant of myocardial distensibility and diastolic function. The passive mechanical properties of cardiac titin depend on the expression ratio of the isoform N2BA (*3.2–3.7 MDa, compliant) and N2B (*3.0 MDa, stiffer) and are dynamically modified by phosphorylation of its elastic I-band regions N2Bus and PEVK. Importantly, phosphorylation of N2Bus by e.g. PKG reduces titin-based myofilament stiffness, whereas phosphorylation of PEVK by PKCalpha increases it. Here, we analyzed cardiac samples from diabetic (type-2 diabetes mellitus) and non-diabetic patients that underwent cardiac surgery due to coronary artery disease and studied the influence of altered insulin homeostasis on cardiac titin phosphorylation. Western blot analyses using phosphosite-directed antibodies demonstrated a diabetes-related increase in the relative phosphorylation of the PEVK-region, and a hypo-phosphorylation of the N2Bus-region of titin. In line with the reduced N2Bus phosphorylation we observed decreased expression of soluble guanylyl cyclase, and increased levels of phosphodiesterase 5a, indicative for reduced cardiac PKG-activity in diabetic patients. As a result of the altered phosphorylation status passive stiffness of isolated cardiomyocytes from diabetic patients was significantly increased compared to non-diabetic controls. Using cultured embryonic rat
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90 cardiomyocytes we confirmed that insulin application induces rapid changes in titin phosphorylation by initiating PKG- and PKCalpha-activating pathways. Hence, the insulin deficit in diabetic patients probably results in reduced PKG and PKCalpha-mediated titin phosphorylation. Interestingly, the oral antidiabetic drug metformin also activated the PI3K/PDK1/PKCalpha pathway and may therefore account for the increased phosphorylation of the titin PEVK-region in the diabetic patients. We conclude from our findings that disturbed insulin homeostasis and oral anti-diabetic drug treatment strongly affect cardiac titin, and may therefore modify myocardial stiffness and diastolic function in patients with type-2 diabetes. Keywords: Titin, Sarcomere, Passive stiffness
O6.9 Heart muscle & cardiomyopathy, Sat 16:35–16:50 The effect of cardiac a-actin HCM mutants E99K, A230V, and A331P on force generation and cross-bridge kinetics in thin filament reconstituted cardiac muscle fibers Bai Fan, Caster Hannah M, Dawson John F, Kawai Masataka Department of Anatomy and Cell Biology, University of Iowa, USA Recombinant human cardiac actin HCM mutants E99K, A230V, A331P, and WT were expressed in baculovirus/insect cells and used to reconstitute the thin-filament of bovine cardiac fibers, together with Tm and Tn. After actin filament reconstitution, tension of WT actin reconstituted fibers at 25 C reached 0.75 ± 0.06 T0 (T0 = 26.9 ± 1.1 kPa = tension of native fibers), which was comparable to rabbit skeletal actin reconstituted fibers (0.73 ± 0.07 T0). Tension was less in E99 K (0.58 ± 0.04 T0), A230 V (0.58 ± 0.03 T0), and A331P (0.55 ± 0.03 T0). After Tm/Tn reconstitution, tension for A331P was 0.65 ± 0.02 T0, which was less than that of E99 K (0.85 ± 0.06 T0), A230V (0.89 ± 0.06 T0), and WT (0.85 ± 0.06 T0). E99K and A230V showed increased Ca2+ sensitivity (pCa50 = 5.80 ± 0.03 and 5.77 ± 0.03, respectively) but decreased cooperativity (g = 1.8 ± 0.2 and 1.9 ± 0.2, respectively) compared to WT (pCa50 = 5.59 ± 0.02, g = 2.6 ± 0.3). A331P decreased pCa50 (5.57 ± 0.03), but no change in g (2.3 ± 0.3). The kinetic constants of the cross-bridge cycle were deduced using sinusoidal analysis. E99K, A331P, and WT did not show any significant changes in these. A230V caused significant decreases in K1 (ATP association), k2 and k-2 (rate constants of crossbridge detachment step). The number of strongly-attached crossbridges were similar among WT, E99K, A230V, and A331P, indicating that force/cross-bridge is decreased in A331P, but remained the same as WT in E99K and A230V. In conclusion, our results demonstrate that the first step of HCM pathogenesis with E99K and A230V is increased pCa50 and decreased g, which result in larger tension during partial activation to cause a diastolic problem. A230V has an additional problem of decreased cross-bridge kinetics, which affects normal functions of the cross-bridge cycle and may contribute to the first step of HCM pathogenesis.A331P has a problem of decreased force/crossbridge and decreased pCa50, which result in inadequate tension generation by myocytes to cause a systolic problem. Keywords: Cross-bridge kinetics, Sinusoidal analysis, Tension transients
O6.10 Heart muscle & cardiomyopathy, Sat 16:50–17:05 TRPC3/NCX1 signalcomplexes control cardiac contractility and arrhythmogenesis
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J Muscle Res Cell Motil (2015) 36:71–143 Doleschal Bernhard1, Wolf Stefan1, Schernthaner Michaela2, Lichtenegger Michaela1, Antoons Gudrun3, Heinzel Frank3, Groschner Klaus3 University of Graz, Austria; 2Center for Advanced Bioanalysis GmbH, Linz, Austria; 3Medical University of Graz, Austria
1
TRPC3 have been suggested as a key player in cardiac maladaptive remodeling. This non-selective cation channel proteins were identified as determinants of Ca2+-transcription coupling but their impact on cardiac contractility and excitability is so far elusive. We therefore analyzed electrophysiological and contractile consequences of TRPC3 activation in the heart, using a murine model of cardiacspecific TRPC3 overexpression and GSK1702934A, a novel, selective TRPC3 agonist. GSK1702934A (1 lM) induced a non-selective cation conductance and prolonged action potentials in TRPC3overexpressing but not in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmic activity in TRPC3-overexpressing but not wild type cardiac preparations. GSK1702934A substantially promoted NCX (Na+/Ca2+ exchanger) currents in TRPC3-overexpressing myocytes, and the arrhythmogenic action of GSK1702934A was eliminated by either intracellular Ca2+ buffering (11 mM EGTA, pH 7.2) or NCX inhibiton. Immunocytochemical experiments revealed significant colocalization of TRPC3 with NCX1, which was disrupted upon activation of the channel by either GSK1702934A or receptor-mediated stimulation of phospholipase C. We conclude that cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1 thereby elevating cellular Ca2+ levels to evoke positive inotropy. Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1 and arrhythmogenesis. TRPC3-NCX micro/nanodomain communication is suggested to determine cardiac contractility and susceptibility to arrhythmogenic stimuli. Supported by the FWF (DK-MCD; W2126-B18) and the LBI for translational heart failure research. Keywords: TRPC channels, NCX1, Cardiac remodeling
O6.11 Heart muscle & cardiomyopathy, Sat 17:05–17:20 Small heat shock protein B7 (HSPB7) plays a multiple functional role in sarcomere assembly Yan Yu-Ting1, Shih Yen-Ling2, Juo Liang-Yi2, Liao Wern-Chir2, Yang Bih-Ying1 1
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; Institute of Biochemistry and Molecular Biology, National YangMing University, Taipei, Taiwan
2
HSPB7, also cardiovascular small heat shock protein (cvHSP), belongs to the small heat shock protein (sHSP) family. sHsp members have been found as the co-chaperones and play a protective role for preventing the irreversible aggregation of misfolded proteins and maintain the normal assembly and stability of cellular cytoskeleton. HSPB7 highly expresses in striated muscle cells including cardiomyocytes and skeletal muscle cells since the onset of myofibrillogenesis. Despite of the findings about the association of SNPs in HSPB7 with idiopathic and sporadic dilated cardiomyopathy and HSPB7 is a potential early biomarker of cardiac infraction, the biological function of HSPB7 still remains enigmatic. To elucidate the physiological function of HSPB7, we generated conditional gene targeting mouse lines. Conventional knockout HSPB7 causes embryonic lethality at E11.5 with sarcomere formation defects. Fragmental and disarray sarcomere structures are observed in Hspb7
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deficient heart. Abnormal location of several costamere complex and Z disk proteins is detected in the mutant cardiomyocytes. Furthermore, specific ablation of HspB7 in adult cardiomyocytes results in lethality within 10 days with a significant down-regulation of Connexin43 expression but without obvious sarcomere defect phenotypes. On the contrary, no significant sarcomere abnormality in skeletal muscle-specific knockout HspB7 mouse is identified during embryonic myogenesis, but a progressive myopathy phenotype is further developed at older stage.Taken together, our findings highlight the novel role of sHSPs affecting the sarcomere assembly and suggest the pathological role of HSPB7 in muscle related diseases.
Keywords: Carboxy terminal domain of glutathione transferase M2 (GSTM2C), Cardiac RyR2 channels, Inhibition of RyR2 channels
Keywords: Small heat shock protein B7, Sarcomere assembly, Gene targeting
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O6.12 Heart muscle & cardiomyopathy, Sat 17:20–17:35 The therapeutic potential of glutathione transferase M2-derived peptides to treat heart failure through cardiac ryanodine receptor Ca2+ channel inhibition Kaveenda Samarasinghe1***, Liu Dan1, Tummala Padmaja1, Arnolda Leonard2, Dulhunty Angela F1, Board Philip G1 ***Candidate for Young Investigator Award
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Molecular Bioscience Department, The John Curtin School of Medical Research, Australian National University, Canberra, Australia; 2Cardiology Department, Canberra Hospital, Canberra, Australia Introduction: Ca2+ release from sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential component of the excitation–contraction (EC) coupling process in cardiomyocytes and thus necessary for normal heart function. Excess Ca2+ release due to overactive RyR2 can cause fatal arrhythmia that can lead to cardiac arrest. The carboxy terminal domain of glutathione transferase M2 (GSTM2C) specifically inhibits RyR2 activity by binding to residues in RyR2 that are not conserved in other RyR isoforms. Our aim was to improve this inhibition and assess the therapeutic potential of GSTM2C based peptides in treating Ca2+ release-based heart failure. Methodology: Random and directed mutagenesis were used to generate numerous GSTM2C mutants and their in vitro RyR2 inhibitory capability was measured using caffeine-induced SR Ca2+ release and single channel bilayer assays. Flow cytometry and confocal microscopy with field stimulation are being used to assess the ability of these mutants to efficiently translocate and functionally improve isolated adult mouse cardiomyocytes. In vivo therapeutic effects of these mutants will be investigated using a mouse model for heart failure. Results: Several of the GSTM2C mutants showed significantly greater in vitro inhibition of single RyR2 channels as well as significantly increased reduction in Ca2+ release from isolated cardiac SR when compared to the same effects on RyR2 caused by wild type GSTM2C. So far we have confirmed that one of these mutants can significantly reduce the Ca2+ release rate and enhance Ca2+ uptake in enzymatically isolated adult mouse cardiomyocytes, thus improving Ca2+ cycling. We have also experimentally confirmed that these mutants can enter cells. Conclusion: Certain GSTM2C mutations improve its specific RyR2 inhibition and Ca2+ cycling in cardiomyocytes, thus enhancing its potential as the basis for a novel therapeutic agent for use in Ca2+ release-based cardiac disorders.
O7.1 Neuromuscular disease, Sun 09:00–09:22 Altered Ca2+ handling in murine models of malignant hyperthermia and central core disease Reggiani Carlo1, Canato Marta1, Capitanio Paola1, Michelucci Antonio2, Protasi Feliciano2 Department of Biomedical Sciences, University of Padova, Padua, Italy; 2Department of Neuroscience, Imaging and Clinical Sciences, University G d’Annunzio, Chieti, Italy Proper Ca2+ storage, release, and reuptake are essential for normal physiological functioning of muscle excitation, contraction, and relaxation. Several human skeletal muscle disorders result from dysfunction in the control and coordination of these three critical processes. Malignant hyperthermia (MH) and central core disease (CCD) are inherited human disorders of skeletal muscle Ca2+ homeostasis. Both MH and CCD are often linked to mutations and/or deletions in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the intracellular Ca2+ release channel, which is essential to excitation–contraction (EC) coupling. To understand the pathophysiology of MH and CCD, murine lines carrying mutations of RyR1 have been developed. Among them, mutation of tyrosine 522 to serine (Y522S) which is associated with human MH, has been demonstrated to make mice MH-susceptible [1], while mutation of isoleucine 4898 to threonine (I4895T) causes a progressive myopathy with formation of minicores [2]. Interestingly, also ablation of skeletal muscle calsequestrin (CASQ1), a SR Ca2+-binding protein that modulates RyR1 function, causes susceptibility to MH-like lethal episodes in response to halothane and heat stress [3] and development of central cores. Work carried out in our laboratory aims to elucidate the alterations in Ca2+ concentrations in the cytosol, sarcoplasmic reticulum and mitochondria in isolated Flexor Digitorum Brevis (FDB) fibres from these murine lines, taking advantage of Fura-2 and genetically encoded Ca2+ indicators (Cameleons). Massive alterations of sarcoplasmic reticulum free Ca2+ concentration [4] and of mitochondrial Ca2+ handling [5] have been demonstrated in CASQ1 null mice. Keywords: Sarcoplasmic reticulum, Calcium signalling, Muscle diseases References: [1] Chelu et al. FASEB J 20:329–330, 2006 [2] Zwaritch et al. PNAS 106:21813–21818, 2009 [3] Paolini et al. J Physiol 583:767–784, 2007 [4] Canato et al. PNAS 107:22326, 2010 [5] Scorzeto et al. PLoS One 8(10):e749192013, 2013
O7.2 Neuromuscular disease, Sun 09:23–09:45 A bench to bedside study on critical illness myopathy: underlying mechanisms and specific intervention strategies Larsson Lars Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden Severe muscle wasting and impaired muscle function accompany long-term immobilized and mechanically ventilated intensive care unit (ICU) patients with negative consequences for recovery from primary disease and weaning from mechanical or assisted ventilation
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92 with elderly patients being more severely affected than young. There is a growing awareness concerning the health-related quality of life (QoL) in surviving ICU patients due to the gradually increasing high costs of modern ICU care, but also to the increasing body of knowledge regarding the impact of impaired muscle function on the prolonged and more complicated weaning from mechanical or assisted ventilation, extensive rehabilitation and decreased patient QoL with consequence for long-term outcomes. Specific interest is focused on underlying mechanisms, muscle type specific differences, effects of aging and the impact of specific intervention strategies. Keywords: Myosin, Protein degradation, Muscle disease
O7.3 Neuromuscular disease, Sun 09:45–10:00 Search for causative mutations in the Austrian malignant hyperthermia (MH) population Weigl Lukas, Mann Anda, Mashaghi Maryam, Kraft Birgit, Michalek-Sauberer Andrea Clinical Department of Special Anaesthesia and Paintherapy, Medical University Vienna, Austria Aim of the study: To offer a genetic test to members of Austrian families with a known malignant hyperthermia (MH) history, we analyzed the genomic DNA of 105 members of such families which were diagnosed susceptible for MH (MHS) in the in vitro contracture test for the presence of one of 31 approved causative MH mutations. In addition, for four individuals who did not show a known mutation, the complete coding region of the RYR1 was sequenced. Methods: Of the ryanodine receptor 1 gene (RYR1) 21 exons and of the dihydropyridine receptor gene (CACNA1S) one exon were amplified with the PCR technique and sequenced. The analyzed regions thus comprised the three MH hot spot regions with 30 of the currently 31 approved MH mutations (97 %) and 74 of those 187 variants (40 %) which are listed in the European MH group register to have a possible linkage to MH. Complete sequencing of the RYR1 was done by mRNA isolation of cultured myotubes and synthesis of cDNA. Functional testing of new variants was done with Ca2+ release experiments with cultured myotubes using the specific RYR1 activators caffeine and 4-chloro-m-cresol (4-CmC). Results: In only six individuals out of the 105 tested (5.7 %), we found one of the known causative mutations and in 11 individuals a possible mutation was detected (10.5 %). In three out of four individuals with the complete RYR1 sequence checked, we found new variants, which are highly suspicious of being causative for MH. These three new variants showed increased sensitivity of Ca2+ release against caffeine, 4-chloro-m-cresol and halothane in Ca2+ release experiments of cultured and differentiated skeletal muscle cells. Conclusion: The search for approved causative MH mutations in the three hot spot regions revealed a surprising low frequency of known variants. For a genetic test of MH the list of causative mutations has to be expanded and the sequencing must not be restricted to the hot spot regions of the RYR1 gene. Supported by FWF P24922-B13 and by the Verein FWFAS Keywords: Malignant hyperthermia, Ryanodine receptor, Calcium release
O7.4 Neuromuscular disease, Sun 10:00–10:15 Involvement of adiponectin in the pathogenesis of dystrophinopathies
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J Muscle Res Cell Motil (2015) 36:71–143 Abou-Samra Michel1***, Schakman Olivier2, Lecompte Sophie1, Boursereau Raphael1, Noel Laurence1, Many Marie C1, Guiot Yves1, Gailly Philippe2, Brichard Sonia M1 ***Candidate for Young Investigator Award
1
Institute of Experimental and Clinical Research – UCL, London, UK; 2Institute Of NeuroScience – UCL, London, UK The hormone adiponectin (ApN) is decreased in the metabolic syndrome, where it plays a key pathogenetic role. We have previously shown that ApN exerts anti-inflammatory effects on the skeletal muscle in mice exposed to acute inflammation (lipopolysaccharide injection) or metabolic stress (obesogenic diet). Here we investigate whether ApN could be sufficiently potent to counteract a severe degenerative muscular disease. To this end, mdx mice (a model of Duchenne muscular dystrophy, where inflammation worsens the consequences of the genetic deficiency) were crossed with transgenic mice overexpressing ApN in order to generate mdx-ApN mice. Circulating ApN was markedly reduced in mdx mice compared to controls. Replenishment of ApN drastically reduced inflammation and oxidative stress in muscles of mdx-ApN mice and enhanced the expression of myogenic differentiation markers. Moreover, mdx-ApN mice exhibited higher global muscle force and endurance along with decreased muscle damage as quantified by curtailed extravasation of Evans Blue Dye in myofibers. Lastly, the anti-inflammatory effects of ApN were also reproduced in human myotubes in vitro. Adiponectin turns out to be an extremely potent anti-inflammatory hormone that protects the skeletal muscle against injury. These properties of ApN may be of interest in myopathies, but also in several other diseases where inflammation plays a triggering or worsening pathogenic role. Keywords: Skeletal muscle, Inflammation, Adiponectin
O7.5 Neuromuscular disease, Sun 10:50–11:05 Characterization of a Kbtbd13-deficient mouse model that recapitulates nemaline myopathy with mutations in the KBTBD13 gene De Winter Josine1***, Joureau Barbara1, Stienen Ger JM2, Granzier Henk3, Ottenheijm Coen AC4 ***Candidate for Young Investigator Award
1
Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Physics and Astronomy, VU University, Amsterdam, The Netherlands; 3 Department of Physiology, Tuscon AZ, USA; 4Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands; Department of Physiology, Tucson AZ, USA Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy and is characterized by muscle weakness and the presence of nemaline bodies in the muscle fibers. To date, nine genes are implicated in NM, of which most are associated with thin filament proteins. Recently a novel implicated gene was discovered— KBTBD13—of which the function of its protein product is unknown. NM patients with KBTBD13 mutations have muscle weakness and show a typical slowness of movement. Here, we generated a mouse model that lacks the kbtbd13 gene to obtain insight into the role of this new gene and its protein product and to elucidate the pathophysiology of this new form of NM. Methods Kbtbd13-KO mice were generated at the GEMM Core facility of the University of Arizona (Tucson, AZ,
J Muscle Res Cell Motil (2015) 36:71–143
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USA) by homologous recombination of the kbtbd13 locus with a NEO cassette. Kbtbd13-KO mice are viable and live into adulthood. Muscle weights of cardiac and skeletal muscle were collected. Intact soleus muscles of 3 month-old male kbtbd13-KO and wild-type (WT) mice were used for in vitro experiments. Muscle tendons were attached to a force transducer and a length motor. Muscles were electrically activated by field stimulation at 30 C. Results Maximal tetanic force at 150 Hz was reduced in kbtbd13-KO (n = 10). The relative force-frequency relationship in kbtbd13-KO mice was shifted to the left compared to WT mice. Both activation and relaxation kinetics were slower in kbtbdKO mice. Muscle weight data reveal atrophy of skeletal muscles, whereas the cardiac muscle shows hypertrophy of the left ventricle. Heterozygous kbtbd13-mice phenocopy WT mice. Conclusion Kbtbd13-KO mice phenocopy the normal life span and typical muscle weakness observed in NM patients with mutations in KBTBD13. Hence, we present a novel NM mouse model that is a useful tool to elucidate the role of this new gene and its protein product and to study the mechanism underlying the muscle weakness observed in patients.
change in Fo is observed in biopsies from severe TPM3 (95 ± 14). Note that the classification of severity is based on the age of diseaseonset. No shift in the force-sarcomere length relationship was observed in mild ACTA1, TPM3 and TPM2 patients. In contrast, in mildly and severely affected NEB patients the force-sarcomere length relationship was shifted leftwards, indicating shorter thin filaments. Conclusion: Our data suggest that mutations in nebulin (NEB) result in the most pronounced changes in thin filament length. Insights in the mechanisms underlying weakness in patients with thin filament mutations are necessary to improve specific treatment strategies.
Keywords: Nemaline myopathy, KBTBD13, Mouse model
Kiiski Kirsi1, Lehtokari Vilma-Lotta1, Lo¨ytynoja Ari2, Laari Liina1, Wallgren-Pettersson Carina1, Pelin Katarina3
O7.6 Neuromuscular Disease, Sun 11:05–11:20 Force-sarcomere length relations in patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2 and TPM3 Joureau Barbara1***, De Winter Josine1, Pappas Chris2, Malfatti E3, Beggs A4,Clarke Nigel F5, Romero NB3, Granzier Henk6, Gregorio Carol2, Stienen Ger JM7, Ottenheijm Coen AC1 ***Candidate for Young Investigator Award
1
VU University Medical Center, Department of Physiology, Amsterdam, The Netherlands; 2University of Arizona, Department of Cell Biology, Tucson AZ, USA; 3Unite´ de Morphologie Neuromusculaire, Institut de Myologie, Groupe Hospitalier Universitaire La Pitie´-Salpeˆtrie`re, Paris, France; 4Division of Genetics and Program in Genomics, The Manton Center for Orphan Disease Research, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA; 5Institute for Neuroscience and Muscle Research, The Children’s Hospital at Westmead, NSW, 2145; 6 University of Arizona, Department of Physiology, Tucson AZ, USA; 7 VU University, Department of Physics and Astronomy, Amsterdam, The Netherlands Background: Mutations in the nebulin gene (NEB), skeletal muscle alpha-actin1 gene (ACTA1), beta-tropomyosin 2 gene (TPM2) and alpha-tropomyosin 3 gene (TPM3) lead to thin filament myopathies, such as nemaline myopathy (NM), congenital fiber type disproportion (CFTD) and cap disease (CAP). A hallmark feature of these myopathies is muscle weakness. Here, we aimed to elucidate the effect of NEB, ACTA1, TPM2 and TPM3 mutations on thin filament length by determining the sarcomere length-dependence of force. Methods: Quadriceps biopsies from NM, CFTD, and CAP patients (n = 36) with mutations in NEB, ACTA1, TPM2 or TPM3 were compared to biopsies from controls (n = 7). Using permeabilized musclefibers, maximal active tension was determined at incremental sarcomere lengths (range 2.0–3.5 lm) to obtain the force-sarcomere length relationship. Results: The maximal active tension (Fo (in mN/mm2, mean ± SEM)) was significantly lower in biopsies from severe NEB (18 ± 5), mild NEB (55 ± 6), severe ACTA1 (53 ± 4), mild ACTA1 (81 ± 14), mild TPM2 (74 ± 2) and mild TPM3 (90 ± 8) patients compared to biopsies of controls (146 ± 7), whereas no significant
Keywords: Myopathy, Muscles weakness, Force-sarcomere length relations
O7.7 Neuromuscular disease, Sun 11:20–11:35 Large copy number variations are frequent in the nebulin gene
1 The Folkha¨lsan Institute of Genetics and the Department of Medical Genetics, University of Helsinki, Haartman Institute, Finland; 2 Institute of Biotechnology, University of Helsinki, Finland; 3 Department of Biosciences, Division of Genetics, University of Helsinki, Finland
Recessive mutations in the nebulin gene (NEB) cause nemaline myopathy (NM), distal myopathy and core-rod myopathy. NEB has 183 exons and spans 249 kb of genomic DNA in the chromosomal region 2q23. We have estimated that approximately 7 % of all variants in the coding region of NEB are pathogenic. Of the pathogenic mutations, splice site mutations are the most common ones (34 %), followed by frameshift mutations (32 %), nonsense mutations (21 %) and missense mutations (11 %). Approximately 3 % of the pathogenic mutations are large ([1 kb) deletions or duplications. The most common type of variation in NEB is, however, copy number variation (CNV) in the middle part of the gene comprising a triplication of eight exons, i.e. the block of exons 82–89, 90–97 and 98–105 are identical. One block is roughly 10 kb in size, and the whole triplicate region is about 32 kb. LINE elements located in introns 89, 97 and 105 may predispose the region for non-allelic homologous recombination causing CNV. We have analyzed NEB for CNVs using our custom-made NM-CGH microarray. To date, 250 samples from 185 NM families have been analyzed on the NM-CGH microarray. We have identified CNVs in the triplicate region of NEB in approximately 15 % of the NM families. Triplicate region duplications, i.e. addition of one to four extra copies of eight exons, were identified in 17 families, and triplicate region deletions, i.e. deletion of one block of eight exons, were identified in nine families. In one family, a triplicate region duplication, but not the deletion encountered in the same family, segregated with NM. We also analyzed DNA samples from 30 unrelated healthy controls and found triplicate region CNVs in 17 % of them. Four of the controls carried deletions and one a duplication. Interestingly, only deletion or addition of one eight-exon block were identified in the controls. Our current hypothesis is therefore that NEB alleles with five or more copies of the eight-exon block might be pathogenic. Keywords: Nebulin, Mutation, Nemaline myopathy
O7.8 Neuromuscular disease, Sun 11:35–11:50 Recessive myosin myopathy with external ophthalmoplegia associated with MYH2 mutations
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J Muscle Res Cell Motil (2015) 36:71–143
Tajsharghi Homa1, Hammans Simon2, Lindberg Christopher3, Lossos Alexander4, Clarke Nigel F5, Mazanti Ingrid6, Waddell Leigh B5, Fellig Yakov7, Foulds Nicola8, Katifi Haider2, Raheem Olayinka9 1
Department of Pathology, Institute of Biomedicine University of Gothenburg, Sweden; 2Wessex Neurological Centre, Southampton General Hospital, Southampton, UK; 3Department of Neurology, Institute of Neuroscience and Physiology, University of Gothenburg, Sahlgrenska Hospital, Sweden; 4Department of Neurology, HadassahHebrew University Medical Center, Jerusalem, Israel; 5Institute for Neuromuscular Research, The Children’s Hospital at Westmead & Discipline of Paediatrics and Child Health, University of Sydney, Australia; 6Cellular Pathology, Southampton General Hospital, Southampton, UK; 7Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 8Wessex Clinical Genetics Services, UHS NHS Foundation Trust and Department of Human Genetics and GenomicMedicine, Faculty of Medicine, University of Southampton, UK; 9Neuromuscular Research Unit, Tampere University and Hospital, Finland Myosin myopathies comprise a group of inherited diseases caused by mutations in myosin heavy chain (MyHC) genes. Homozygous or compound heterozygous truncating MYH2 mutations have been demonstrated to cause recessive myopathy with ophthalmoplegia, mild to moderate muscle weakness and complete lack of type 2A muscle fibers. In this study, we describe the clinical and morphological characteristics of recessive MYH2 missense mutations. Seven patients of five different families with a myopathy characterized by ophthalmoplegia and mild to moderate muscle weakness were investigated. Muscle biopsy was performed to study morphological changes and MyHC isoform expression. Five of the patients were homozygous for MYH2 missense mutations, one patient was compound heterozygous for a missense and a nonsense mutation and one patient was homozygous for a frame-shift MYH2 mutation. Muscle biopsy demonstrated morphologically abnormal or absent type 2A muscle fibers and reduced or absent expression of the corresponding MyHC IIa transcript and protein. We conclude that missense mutations in MYH2 may cause a recessively inherited myopathy associated with external ophthalmoplegia, similar to that of truncating MYH2 mutations, but with different type 2 fiber pathology. Keywords: myopathy
Myosin
heavy
chain,
Ophthalmoplegia,
Myosin
O7.9 Neuromuscular disease, Sun 11:50–12:05 Mutations in LINC-complex associated proteins impair mechanosensing responses and alter Yes-associated protein (YAP) signaling in human muscle precursors Fischer Martina, Arake´lian Tsole`re, Duche`ne He´le`ne, Bertrand Anne, Mamchaoui Kamel, Bigot Anne, Ziaei Simindorkh, Voit Thomas, Bonne Gise`le, Coirault Catherine INSERM U974, France The LINC (linker of the nucleoskeleton and cytoskeleton) complex enables transmission of forces between the nucleus and the extracellular matrix (ECM) via the cytoskeleton and may directly contribute to the muscle cell’s ability to probe its mechanical environment. Mutations in LINC-complex associated proteins, including lamins and nesprins cause human muscular dystrophies but disease mechanisms still remain to be elucidated. Our first aim was to determine whether human muscular dystrophies resulting from mutations in some of the LINCcomplex associated proteins (A-type lamin and nesprin1) affected the capacity of myoblasts to sense the stiffness of the ECM. Our second aim
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was to determine whether defects in YAP signaling, an important regulator of the mechano-response, were involved in the mechanosensing defects. Myoblasts with various mutations in the A-type lamins gene (LMNA), or in nesprin1 gene (SYNE1) were obtained from patients exhibiting muscular dystrophies. Control myoblasts (WT) were obtained from 2 subjects without muscular disorders. Immortalized myoblasts were cultured on 2D soft or hard surfaces. Focal adhesion (vinculin), actin cytoskeleton, and YAP signaling pathways were investigated. On 2D hard surface, there was no obvious difference in actin cytoskeleton and focal adhesion between WT, LMNA and SYNE1 myoblasts. In contrast, LMNA and SYNE1 myoblasts cultured on soft surface exhibited enlarged focal adhesions and stress fibers compared with WT. Cytoplasmic translocation of YAP observed in WT in response to reduced stiffness matrix was absent in LMNA and SYNE1 cells, suggesting a permanent activation of YAP in mutant cells. Our data indicate that cell culture stiffness is critical to reveal mechanosensing defects in dystrophic muscle cells. Deregulation of YAP could be implicated in the mechanosensing defects in human muscular dystrophies resulting from mutations in LINC-complex associated proteins. Keywords: Mechanotransduction, Cytoskeleton, LINC complex
P1.1 Contractile structure, Thu 12:00–14:00 Sensing the Z-disc: production platform for proteins and complexes comprising components of Z-disc interactome Mlynek Georg1, Leeb Sarah1, Pinotsis Nikos2, Lehner Anita3, Neuhold Jana3, Kostan Julius1, Charnagalov Alexej1, StoltBergner Peggy3, Djinovic-Carugo Kristina1 1 Department of Structural and Computational Biology, Max F Perutz Laboratories, Vienna, Austria; 2Birkbeck College, University of London, UK; 3Protein Technologies Facility, Campus Science Support Facilities GmbH, Vienna, Austria
In striated muscle cells, the Z-disc constitutes the borders of individual sarcomeres, where anti-parallel actin filaments spanning the sarcomeres are crosslinked, and represents a highly organized threedimensional structure containing a number of proteins assembled in multi-protein complexes. A major challenge in understanding the networks of protein–protein interactions involved in function and control of the Z-disc in the striated muscle is the definition and unraveling of the molecular basis of highly concerted operations providing assembling and functionality of sarcomeres. To understand substructural mechanisms that operate Z-disc a deeper view into ‘‘little talks’’ between single or multiple Z-disc components is necessary, and, thus, isolation and structural studies of native and/or structurally modified individual Z-disc proteins is required. At the centre for optimize structural studies (COSS) we have established a middle- to high-throughput protein production platform which integrates different protein production steps from bioinformatics analysis to large-scale expression and purification of proteins followed by their biophysical characterization. By using this platform we have managed to prepare several fragments of a Z-disc protein, filamin C, comprising single or multiple Ig-like domains of filamin’s C rod 2 domain, which is its hot spot for protein–protein interactions. Furthermore, we were able to characterize binary interactions between several fragments of filamin C and FATZ-1 by using of microscale thermophoresis (MST). Presented here, results on X-ray crystallography, electron microscopy and solution X-ray scattering analysis of some filamin C fragments confirm complex and dynamic nature of Z-disc functional machinery as well as suggest the importance of engineering and production of multiple variants of Z-disc proteins to study the interaction pattern within Z-disc interactome.
J Muscle Res Cell Motil (2015) 36:71–143 Keywords: Z-disc interactome, Filamin C, Protein production
P1.2 Contractile structure, Thu 12:00–14:00 Electron tomography analysis of the effect of stretch on the Zband in striated muscle Cope Sam1***, Stehle Ina2, Kraft Theresia2, Brenner Bernhard2, Luther Pradeep K3 ***Candidate for Young Investigator Award
1
National Heart and Lung Institute, Imperial College London, UK; Hannover Medical School, Germany; 3Imperial College London, UK
2
The Z-band (Z-line/Z-disc) of the sarcomere in striated muscle is home to many proteins with diverse functions. The Z-band is responsible not only for anchoring the actin filaments but for mechanotransduction and signalling pathways within the cell. The structural effectiveness of the Z-line is due to the strength of its protein interactions. The Z-band is primarily composed of a tetragonal lattice of anti-parallel actin filaments cross-linked by the actin binding protein alpha-actinin. Alpha-actinin is a spectrin-family protein composed of two actin-binding head regions and a rod domain of spectrin repeats. In transverse sections of the Z-band, electron micrographs show two distinct appearances, described as the smallsquare form typical of relaxed muscle and the basketweave form typical of active muscle. These states are thought to be due to changes in the spatial arrangement of alpha-actinin due to tension transmitted along the actin filaments. Here we have investigated the structural basis of the two forms and their transformation by electron tomography of the Z-band in rabbit psoas muscle. We have firstly investigated the structure of the Z-band in fibres put into rigor with no increase in tension. Secondly we investigated the structure of the Z-band in fibres put into rigor and then stretched to achieve tension equivalent to maximal activation tension. The fibres were chemically fixed and processed conventionally for electron microscopy. Dualaxes tomograms were generated from which we obtained mean images by sub-tomogram averaging. We present our results on the details of the structure we have observed in the two forms and we show by modelling how the transformation may occur. Keywords: Electron tomography, Electron microscopy, Z-line
P1.3 Contractile structure, Thu 12:00–14:00 Thick filament structure and calcium activation in skeletal muscle and their dependence on interfilament spacing Brunello Elisabetta1, Caremani Marco1, Fusi Luca2, Reconditi Massimo1, Linari Marco1, Narayanan Theyencheri3, Piazzesi Gabriella1, Irving Malcolm2, Lombardi Vincenzo1 Department of Biology, University of Florence, Italy; 2Randall Division, King’s College London, UK; 3European Synchrotron Radiation Facility, Grenoble, France 1
Osmotic compression of demembranated fibres from rabbit psoas muscle with 5 % Dextran T-500, to recover the in vivo interfilament spacing, increases the Ca2+ sensitivity of force without altering maximum force (Godt & Maughan, Pflugers Arch 391:334, 1981; Diamond et al. Biophys J 50:1215, 1986). We investigated the structural basis of this effect using X-ray diffraction at beam-line ID02, ESRF, Grenoble, France. Bundles of 3–5 fibres were activated
95 isometrically at different pCa by a temperature jump from 1 C to 12 C. In relaxed fibres at 12 C, addition of 5 % Dextran induced (i) a three-fold increase in the intensity of the first myosin layer line, (ii) a four-fold increase in the intensity of the so-called forbidden meridional reflections, and (iii) *0.5 % reduction in the spacings of all the myosin-based meridional reflections. Thus osmotic compression to restore the in vivo interfilament spacing induces recovery of the thick filament structure observed in resting intact muscle, in particular the systematic axial perturbation of the three layers of myosin heads within each 43 nm repeat attributed to the presence of links between thick and thin filaments mediated by Myosin Binding Protein-C (MyBP-C) (Reconditi et al. J Physiol 592:1119, 2014). The [Ca2+]-dependence of active force and the intensity of the M3 reflection from the axial periodicity of the myosin heads was described by a pCa50 of 6.22 ± 0.02 (mean ± SD) in the presence of 5 % Dextran, 0.09 ± 0.01 pCa units larger than in its absence. These results indicate that the recovery of the in vivo interfilament spacing by osmotic compression of demembranated fibres enhances the signalling pathway involved in Ca2+ activation of the contractile apparatus, probably by recovery of MyBP-C links to the thin filaments. Supported by FIRB-Futuro in Ricerca and MIUR-PRIN (Italy), MRC (UK). Keywords: Thick filament activation, Thin filament activation, Interfilament spacing
P1.4 Contractile structure, Thu 12:00–14:00 Functional studies and preliminary crystallization data of Entamoeba hystolitica alpha-actinin 2 mutants Zielinska Karolina, Gkougkoulia Eirini A, Djinovic-Carugo Kristina Department for Structural and Computational Biology, Max F Perutz Laboratories, University of Vienna, Austria Alpha-actinin is a scaffolding dimerizing protein which bundles and cross-links actin filaments. Alpha actinin is composed of actin binding domain containing 2 calponin homology domains, centrally located spectrin repeats and C-terminal calmodulin-like domain, containing 4 EF-hands [1]. In vertebrates muscle (isoform 2 and 3) and non-muscle isoforms (1 and 4) are present. The non-muscle isoforms are regulated by calcium binding to EF-hands [1]. During evolution of alpha-actinins the calcium-sensitive variants appeared earlier, the appearance of calcium-insensitive alpha-actinins is related to the development of muscle tissue. Up to date our understanding of the exact mechanism of calcium regulation of calcium-sensitive alpha-actinins is not complete. Isoform 2 of alphaactinin from Entamoeba hystolitica shows high sequence similarity with vertebrate non-muscle isoforms of alpha-actinin. Actin bundling ability of Entamoeba hystolitica alpha-actinin 2 is regulated by calcium ions and thus resembles behaviour of non-muscle vertebrate isoforms. Entamoeba hystolitica alpha-actinin 2 shows typical alphaactinin domain organization. However, in comparison with vertebrate alpha-actinins this protein contains only 2 instead of 4 spectrin repeats [3]. Thus Entamoeba hystolitica alpha-actinin 2 constitutes a good working model of ancestral homologue of vertebrate nonmuscle alpha-actinin. The insight into structure and function of this protein may shed light on regulation of calcium-dependent alphaactinin isoforms. In order to elucidate the role of calcium for the function of Entamoeba hystoliltica alpha –actinin 2 the actin bundling activity was studied upon deletion of EF-hands 3 and 4 and 1–4, respectively. In order to investigate the impact of calcium ions on the protein structure we aimed to crystallize the calcium-insensitive variant of the protein.
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P1.5 Contractile structure, Thu 12:00–14:00 Electron tomography analysis of cardiac myosin binding protein C: preservation of fine structure by high-pressure freezing and cryosections Torre Iratxe1***, De Tombe Pieter2, Luther Pradeep K1 ***Candidate for Young Investigator Award
1
Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, UK; 2Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, Illinois, USA Myosin binding protein C (MyBP-C or C-protein) is a 140 KDa accessory protein of vertebrate striated muscle thick filaments which is located within the middle third of each half A-band (C-zone) of the sarcomere. There is considerable interest in this protein as mutations in the cardiac isoform (cMyBP-C) gene (MYBPC3) are known to be a leading cause of familial hypertrophic cardiomyopathy affecting millions of people worldwide. Understanding cMyBP-C structure is important as it will enable us to understand its role in regulating cardiac function. cMyBP-C is highly labile so it is essential that the cardiac preparation is in a pristine physiological condition. We isolated thin trabeculae from the right ventricles of Sprague–Dawley male rats, mounted them in our apparatus, bathed in oxygenated Krebs-Henseleit solution and stimulated them to ensure viability. For electron microscopy studies, rapid freezing methods ensure the best preservation of biological fine structure. We used a Leica EMPACT high pressure freezing (HPF) machine; however the small size of the freezing chamber precluded mounting of live muscles. Instead we used glutaraldehyde-fixed muscles and processed them further by HPF, freeze substitution and embedding in epoxy resin, or by cutting thin cryosections. We assessed the quality of the samples from the transverse striations typical of the A-band in skeletal and cardiac muscle. Here we show the three-dimensional reconstruction of the C-zone in cardiac thick filaments by electron tomography and subtomogram averaging of the samples from these methods. Keywords: Cardiac myosin binding protein C, Electron tomography, High-pressure freezing
P1.6 Contractile structure, Thu 12:00–14:00 Involvement of active muscles force and passive energy storage into Octopus vulgaris arm performance Zullo Letizia1, Fossati Sara Maria1, Noedl Marie Therese1, Benfenati Fabio1, Hochner Binyamin2 1 Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genova, Italy; 2Department of Neurobiology, The Hebrew University of Jerusalem, Israel
Several characteristics of the octopus (Octopus vulgaris) arm muscle hydrostat make it a challenging model to study. Unlike vertebrate muscles, invertebrate cephalopods do not show significant differences in the proteins of the myofilament lattice in their appendages even if they evolved a clear functional specialization. Instead, a dramatic difference in the dimensions and arrangement of the myofilaments
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J Muscle Res Cell Motil (2015) 36:71–143 and connective tissue in each of the three main muscle layers is one of the key elements producing a difference in the limb performance. Connective tissue in muscle hydrostat is essential for providing structural reinforcement and to control shape, transmit stress and store elastic energy. In this study we aim at investigating the dynamic and elastic energy of segments of arm stiffened to different levels. To do that we used a Dual-Mode Lever Arm System on an in vitro preparation of an arm sample stimulated to create simultaneous activation of all the muscles in the segment. We show that the initial state of the arm is important for the generation of a determined force. In fact, when stimulated, arm segments under a stretch condition exert a higher force than in their resting state. We also found that the stretching force needed to elongate an arm segment to a certain length is dependent from its initial resting or contracting state. This may reflect the existence of passive elastic forces modulated by the level of the arm activation. Nonetheless, the ratio of ‘energetic’ involvement of each muscle layers during movements remains also to be elucidated. In order to depict this point we sequenced and cloned a probe for mTOR protein to evaluate its expression variation during muscle exercise. mTOR complex represents an interesting family of molecules involved in sensing of cellular nutrition and energy status. This study will open further investigations onto the exercise induced variation of mTOR signaling in invertebrate species. Keywords: Muscle hydrostat, Cephalopod, mTOR
P1.7 Contractile structure, Thu 12:00–14:00 Electron tomography of the boundary-bridge/titin kinase region in fish skeletal muscle Hunter Steve1***, Luther Pradeep K2 ***Candidate for Young Investigator Award
1
National Heart and Lung Institute, Imperial College London, UK; Imperial College London, UK
2
Titin is a 1 lm long string-like protein composed of a tandem arrangement of immunoglobulin and fibronectin domains. It spans half sarcomeres from the Z-band to the centre of the A-band and M-band. In the crossbridge part of the A-band, there is evidence for binding of titin to the thick filaments. This is supported by the coincidence of the thick filament repeat of 43 nm and the *44 nm length of the 11-domain super-repeats of titin. In the M-band and M-region, there are no super-repeats and no evidence of binding to the thick filaments. Hence titin may dislodge from the thick filament as it enters the M-region. In this C-terminus region, titin has a 150 kDa catalytic domain, titin kinase which immunolabelling shows is located near the M-band. There is great interest in titin kinase as it is believed to be involved in muscle turnover. Our recent electron microscopy investigation of striated muscle has shown a transverse ‘‘boundary-bridge’’ network between the M-band and the start of the crossbridge region. We hypothesize that the boundary-bridges network is related to titin kinase. Previously we studied this region in frog sartorius muscle, a muscle which has inherent disorder because of the superlattice arrangement of the thick filaments. Teleost fish muscle is more ordered as the thick filaments have the same orientation in each sarcomere. We have investigated this transverse network region in fish muscle by electron tomography and sub-tomogram averaging. We present our results on modelling the structure and fitting the crystal structure of titin kinase in this region. Keywords: Electron tomography, Titin kinase, M-band
J Muscle Res Cell Motil (2015) 36:71–143
P1.8 Contractile structure, Thu 12:00–14:00 Longitudinal stretching of desmin intermediate filaments with receding meniscus reveals large longitudinal tensile strength Kiss Bala´zs***, Kellermayer Miklo´s SZ ***Candidate for Young Investigator Award
Department of Biophysics and Radiation Biology, and MTA-SE Molecular Biophysics Research Group, Semmelweis University, Budapest, Hungary Desmin forms the intermediate filament system of muscle cells where it plays important role in maintaining mechanical integrity and elasticity. Although the importance of intermediate-filament elasticity in cellular mechanics is being increasingly recognized, the molecular basis of desmin’s elasticity is not fully understood. In the present work we explored the elasticity of purified desmin filaments by stretching them longitudinally with the application of molecular combing. The stretch force acting on the entire cross-sectional area of desmin was calculated to be 4 nN. The average contour length of surface-equilibrated desmin filaments increased from 0.89 to 1.38 lm when molecular combing was applied, which corresponds to a 1.6fold axial stretch on average. Molecular combing together with EDTA-treatment caused the fragmentation of desmin filament into short, 60–120 nm long and 4 nm wide structures. The surface profile of these fragments displayed periodic protrusions with an average peak-to-peak distance of 34 nm. Based on these calculations the observed fragments are hypothesized to be protofibril fragments composed of laterally attached desmin dimers. The orientation axis of the surface-constrained fragments usually deviates from the filament axis, suggesting that either the protofibrils are not aligned in parallel within the filament during axial load, or a recoil occurs during the surface adhesion behind the receding meniscus. The emergence of protofibril fragments suggests that the head or tail domains of coiledcoil desmin dimers are the load-bearing elements during axial stretch. Keywords: Desmin, Receding meniscus, Atomic force microscopy
P1.9 Contractile structure, Thu 12:00–14:00 Sarcomeric localization of the truncated Novex-3 titin isoform Beckendorf Lisa, Voelkel Tobias, Unger Andreas, Linke Wolfgang A Cardiovascular Physiology, Ruhr-University Bochum, Germany The Novex-3 titin isoform (650 kDa) is comprised of 5604 amino acids and hence is the shortest of the titin isoforms expressed in striated muscles. Novex-3 titin is integrated into the Z-disk via its N-terminal part, which is identical to the full-length titin isoforms, but due to the truncated C-terminus it does not reach beyond the I-band of the sarcomere. Unique to the Novex-3 isoform is the large C-terminus which includes six immunoglobulin-like (Ig) domains interspersed with long unique sequences, encoded by the ‘‘Novex-3’’ exon (titin exon 48). Novex-3 was found to bind to obscurin and suggested to span a distance of up to *200 nm from the Z-disk centre into the I-band (Bang et al. Circ Res 89:1065–1072, 2001). However, how the C-terminus of the protein is embedded into the sarcomeric structure remains unknown. We used antibodies against the most C-terminal Ig-domain (I23) in Novex-3 to detect the sarcomeric localization of this Ig-domain at different stretch states of rat cardiomyocytes by immunoelectron (nanogold) microscopy. We
97 also compared the relative position of Novex-3-I23 with that of alpha-actinin (Z-disk), PEVK-titin (central I-band), and titin at the Z/I-band junction (Z7-I1). We found that Novex-3-I23 localized very near the sarcomeric N1-line region adjoining the Z-disk, comigrating with the Z7-I1 antibody on stretch. At a sarcomere length of 1.81 lm the Novex-3-I23 epitope was a distance of 78 ± 12 nm away from the Z-disk centre and at a sarcomere length of 2.05 lm a distance of 94 ± 21 nm (mean ± SEM). Alpha-actinin localized closer to the Z-disk centre than Novex-3. These findings indicate that Novex-3 does not protrude significantly into the sarcomeric I-band. Instead, the C-terminus of Novex-3 remains at the N1-line, suggesting this truncated titin isoform is barely extensible. The Novex-3 isoform may instead play a role in maintaining sarcomeric stability at the N1-line and provide a scaffold for other proteins at this site. Keywords: Novex-3, Titin, Sarcomeric structure
P1.10 Contractile structure, Thu 12:00–14:00 Structural and functional studies on tropomyosin with stabilizing substitutions D137L and G126R in the middle part of its molecule Matyushenko Alexander M1***, Artemova Natalia V1, Shchepkin Daniil2, Kopylova Galina2, Bershitsky Sergey2, Tsaturyan Andrey3, Levitsky Dmitrii I1 ***Candidate for Young Investigator Award
1
Russian Academy of Sciences, A N Bach Institute of Biochemistry, Moscow, Russia; 2Russian Academy of Sciences, Institute of Immunology and Physiology, Moscow, Russia; 3Moscow State University, Institute of Mechanics, Russia Tropomyosin (Tm) is an a-helical coiled-coil protein that binds along the length of actin filament and plays a key role in the regulation of muscle contraction. There are two highly conserved non-canonical residues in the middle part of Tm, D137 and G126, which are thought to impart conformational instability (flexibility) to this region of Tm which is considered crucial for its regulatory functions. It was shown previously that G126R and D137L substitutions result in stabilization of the coiled-coil in the middle of Tm and affect its regulatory function (Sumida et al. J Biol Chem 283:6728–6734, 2008; Nevzorov et al. J Biol Chem 286, 15766–15772, 2011). We extended these studies and compared structural and functional features of Tm mutants carrying stabilizing substitutions D137L and G126R. Moreover, we analyzed the properties of Tm carrying both these substitutions within the same molecule. The results show that both substitutions similarly stabilize the Tm coiled-coil, and their combined action leads to further significant stabilization of the Tm molecule. This stabilization has no appreciable influence on the actin affinity for Tm, but it makes the Tm-actin complexes more stable as was shown by measuring the temperature dependences of thermal dissociation of these complexes monitored with light scattering. The stabilizing substitutions in the middle part of Tm increase the ATPase activity of the myosin heads upon their interaction with actin filaments containing Tm and enhance maximal sliding velocity of regulated actin filaments containing Tm and troponin in the in vitro motility assay at high Ca2+ concentrations; moreover, this stabilization increases Ca2+sensitivity of the actin-myosin interaction underlying this sliding. We propose that these effects can be explained by the influence of these stabilizing substitutions on the interactions between the middle part of actin-bound Tm and certain sites of the myosin head. Supported by RFBR Grants.
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98 Keywords: Tropomyosin, Protein–protein interactions, Regulation of muscle contraction
P1.12 Contractile structure, Thu 12:00–14:00 Effect of sarcomere length on calcium diffusion in a 3-D sarcomere model Holash Robert, MacIntosh Brian Kinesiology, University of Calgary, Canada Skeletal muscle behaves in an isovolumic manner, maintaining volume as the muscle shortens and lengthens. This behaviour is carried through to the level of the sarcomere. As volume is maintained, the sarcomere diameter changes with sarcomere length (SL). Subsequently, the myofilament lattice spacing also changes with muscle length, compressing as the muscle is lengthened, and expanding as it is shortened. To examine how calcium ion (Ca2+) diffusion, binding, and uptake is affected by changes in SL, we developed a realistic 3D mesh model of a sarcomere and myofilament lattice, and then compared the diffusion, binding and uptake of Ca2+ at SL of 1.8, 2.3 and 2.8 lm using the software program Mcell. The model was a 1/8th slice of a half sarcomere, with 120 actin and 71 myosin filaments. The model was divided into fifteen sub compartments (SC), by making three radial and five lengthwise divisions. Eight different seed values were run for each of the three experimental conditions (sarcomere length). Only SL and subsequent lattice spacing were changed between trials. 300 lM of Ca2+ were released during a simulated action potential. The concentration of free calcium (DCa2+) and percentage of doubly-bound troponin (DBT) was measured in each compartment and within the sarcomere as a whole, average values for each simulation condition were calculated and recorded. In all three experimental conditions, DCa2+ in the sarcomere peaked at a value of *9 lM at *0.003 s, however, the percentage of DBT in the sub compartments varied greatly between the conditions. Within the most central SC the percentage of DBT varied between 24 % at a SL of 1.8 lm and 54 % at a SL of 2.8 lm. Also in last SC towards the m-band, the percentage of DBT varied between 15 % at SL 2.3 lm and 0.7 % at SL 2.8 lm. These results suggest that while the overall free DCa2+ is not affected by changes in SL, changes in SL can greatly affect the distribution of Ca2+ within the sarcomere. Keywords: Sarcomere, Structure, Calcium diffusion
P1.13 Contractile structure, Thu 12:00 Inhibition of myostatin activation prevented dexamethasonemediated muscle atrophy in C2C12 myotubes Abe Tomoki1, Kenro Utsunomiya1, Yuushi Okumura2, Ayako Ohno1, Shigetada Kondo1, Takeshi Nikawa1 1 Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Japan; 2 Graduate School of Nutritional Sciences, Sagami Women’s University, Japan
Introduction: Muscle atrophy causes by both decreased protein synthesis and increased proteolysis. Recent studies reported that glucocorticoid treatment causes muscle atrophy via increased expression and activation of myostatin. Myostatin is a negative regulator of muscle growth, resulting in muscle atrophy. Processing of myostatin is necessary for its activation. Although the inhibition of myostatin activation is a potent therapeutic strategy for muscle
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J Muscle Res Cell Motil (2015) 36:71–143 atrophy, there is little known about mechanisms for glucocorticoidinduced myostatin activation. Method: To elucidate roles of proteases associated with myostatin activation in dexamethasone-mediated muscle atrophy, we evaluated effects of an inhibitor of furin in C2C12 myotubes. We also checked effects of an inhibitor of furin on dexamethasone-induced expr4ession of atrogin-1/MAFbx and MuRF1 in C2C12 myotubes. Results: We found that the treatment with an inhibitor of furin inhibitor prevented dexamethasone-mediated muscle atrophy without decreased expression of myostatin in C2C12 myotubes. An inhibitor of furin also reduced atrogin-1/MAFbx and MuRF1 expression induced by dexamethasone. The processing of myostatin mediated by dexamethasone was suppressed in C2C12 myotubes treated with an inhibitor of furin. Conclusion: The treatment with an inhibitor of furin prevented dexamethasone-mediated muscle atrophy through suppression of myostatin activation. These results indicate that proteases associated with myostatin activation can be therapeutic targets for muscle atrophy. Keywords: Glucocorticoid, Muscle atrophy, Myostatin
P1.14 Contractile structure, Thu 12:00–14:00 Characterizing and simulating skeletal muscle fatigue. Experimental and 3D simulation methodology Sierra Marta1***, Grasa Jorge2, Miana-Mena Francisco Javier1, Mun˜oz Marı´a Jesu´s1, Calvo Begon˜a2 ***Candidate for Young Investigator Award
1
Department of Physiology and Pharmacology, University of Zaragoza, Spain; 2Department of Mechanical Engineering, University of Zaragoza, Spain
Skeletal muscle fatigue manifests as a result of repetitive or sustained muscle contraction and can be defined as a temporary reduction in the capacity of the tissue to generate force. In this work, a formulation of a thermodynamically consistent model in three dimensions is proposed for simulating skeletal muscle behaviour under fatigue conditions. Moreover, this model includes one biochemical parameter related to the current fatigue state of the tissue: glycogen concentration. To obtain the muscle force evolution, the study was conducted on isolated rabbit Extensor Digitorum Longus muscles. These muscles were divided in two groups (n = 5) subjected to ‘‘in vitro’’ stimulations of 100 Hz, 100 V and 200 ms, inducing tetanic contractions, with an interval of 10 s between each burst, for 25 and 60 min, respectively. Force evolution was recorded by means of an electro-mechanic universal testing machine. Furthermore, glycogen inside the tissue was determined at the end of each experiment. The force evolution was not uniform through time, showing a strong drop for the first 25 min of stimulation and reaching forces around 16 % of its initial value, in the second part of the stimulation, the force dropped until 4,76 %. Furthermore, glycogen concentration analysis showed a significant decrease (36.99 %, p B 0,05) for the first 25 min. From this point to the end, there was no significant difference in glycogen concentration. Force evolution and glycogen concentration exhibited a determination coefficient (r2) of 84 %. The computational model is based on that proposed by Herna´ndez-Gasco´n et al. (Theor Biol. 335:108–118, 2013) where active and passive responses were implemented by means of a decoupled strain energy function. Active stress was obtained as the maximum tetanic stress penalized by three functions related to the stimulus filaments overlapping and the
J Muscle Res Cell Motil (2015) 36:71–143 fatigue. The implementation of the model into a finite element code reproduces the isometric contractions in a three-dimensional reconstruction of the muscle. Keywords: Skeletal muscle contraction, Fatigue simulation, Glycogen
P1.15 Contractile structure, Thu 12:00–14:00 How the binding of CP (capping protein) to the B-end of actin filament is regulated Takeda Shuichi1, Koike Ryotaro2, Ota Motonori2, Maeda Yuichiro1 1 Structural Biology Research Center, Nagoya University Graduate School of Science, Japan; 2Department of Complex Systems Science, Nagoya University Graduate School of Information Science, Japan
CP, the hetero-dimeric capping protein, binds to the B-end of the actin filament, thereby reduces the number of free B-end and increase the elongation rate there. CP is one of the essential proteins for the actin treadmilling, which drives cell locomotion. The normal mode analysis and crystal structures of CP in complex with CP binding peptides (Takeda et al. PLoS Biol 8(7):e1000416, 2010; Takeda et al. Phys Biol (3):035005, 2011) have indicated that CP is divided into two domains, CP-L and CP-S, each of which reciprocally rotates relative to each other. Both the CARMIL protein and twinfilin, a B-end binding protein, bind CP and are co-localized with CP underneath the membrane in the area where the actin filaments elongates rapidly. Upon binding to CP, the CARMIL-peptide dissociates CP from B-end, while TwfC36 peptide, a C-terminal segment of twinfilin, does not. Crystal structures (Takeda et al. PLoS Biol 8(7):e1000416, 2010 and unpublished) have revealed that both CARMIL- and TwfC36 peptides bind CP along the common groove away from the interface CP/B-end. Although binding sites are largely overlap, the former peptide crosses the boundary between the two CP domains, while the latter does not. The normal mode analyses indicate that the reciprocal rotational fluctuation between the two domains is drastically dumped by the binding of CARMIL peptide, but not by TwfC36 peptide (unpublished). Finally, the two peptides compete for CP binding. Based upon these findings, we hypothesize that, CARMIL proteins un-cap CP, keeping the actin treadmilling going, while twinfilin protects CP against the uncapping, removing the actin filament from the treadmilling. The structural fluctuations, not the structure itself, would play crucial roles in altering the CP affinity to B-end. CARMIL peptides, but not TwfC36 peptide, dump the fluctuation between two domains, detaching CP from B-end. This implies the reverse would also be true: reduction in structural flexibility of the actin filament should detach CP from B-end. Keywords: Capping protein, Actin filament, Protein–protein interactions
P1.16 Contractile structure, Thu 12:00–14:00 Force enhancement modeled by three filament model Cibera Vaclav1***, Herzog Walter2 ***Candidate for Young Investigator Award
1
New Technologies – Research Centre, University of West Bohemia, Czech Republic; 2Faculty of Kinesiology, University of Calgary, Canada
99 The purpose of this study was to model the unexplained increase in force during and after active muscle stretching. Specifically, the aim was to theoretically simulate force during and after active muscle stretching at the single sarcomere and half sarcomere level. The simulations are based on a three filament sarcomere model consisting of the contractile protein actin and myosin, and the structural protein titin. The mathematical description of the three filament model is comprised of two main parts. The first part is based on the classical, two-state Huxley (1957) cross-bridge model, which captures the active, actin-myosin based cross-bridge forces. The second component of the model describes the passive forces produced in a sarcomere, which can be associated virtually exclusively with titin. When a sarcomere is stretched, titin acts as a molecular spring. The elastic properties of titin’s spring segments (the proximal and distal Immunoglobulin, Ig domains, the N2A and the PEVK regions) have been well described and they have been used here for simulating titin’s force-extension properties. Our simulations can explain the extra force observed in eccentric contractions, the reduced energy cost of eccentric contractions and the long lasting increase in force (force enhancement) following active sarcomere stretching. Keywords: Force enhancement, Three filament model, Eccentric contraction
P1.17 Contractile structure, Thu 12:00–14:00 Force-dependent kinetics of domain unfolding contribute to an accelerometer function of titin Ma´rtonfalvi Zsolt***, Bianco Pasquale, Naftz Katalin, K} oszegi Dorina, Kellermayer Miklo´s SZ ***Candidate for Young Investigator Award
Department of Biophysics and Radiation Biology, Semmelweis University, Hungary Titin, a giant filamentous intrasarcomeric protein, is a serial chain of approximately 300 globular (Ig or FN) domains and a few unique sequences. Although force-dependent unfolding has been extensively investigated in recombinant homopolymeric constructs of titin domains, neither the global kinetics, nor the spatial pattern of mechanically-driven domain unfolding is known within the complexity of the full-length molecule. In the present work we stretched individual titin molecules isolated from rabbit m. longissimus dorsi using force- and velocity-clamp optical tweezers protocols. When clamped at high forces, the molecule extended in discrete steps via unfolding of its constituent globular domains. In an apparent violation of mechanically-driven activation kinetics, however, neither the global domain unfolding rate, nor the folded-state lifetime distributions of titin were sensitive to force. The contradiction can be reconciled by assuming a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of force increases. To explore whether there is a spatial pattern in this gradient of domain unfolding, we carried out a topographical screening of individual titin molecules stretched to varying degrees with receding meniscus. We found that unfolded domains were distributed homogenously along the entire length of the overstretched titin molecule. The spatially randomized domain stability ensures that titin is a quasi Hookean expander across a wide range of stretch and loading rates, thereby behaving as an apparently linear sensor of the mechanical environment. Because the unfolding-based apparent stiffness varies with stretch rate, titin may function as an accelerometer within the muscle sarcomere.
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100 Keywords: Titin, Optical tweezers, Unfolding kinetics
P1.18 Contractile structure, Thu 12:00–14:00
J Muscle Res Cell Motil (2015) 36:71–143 a 5 nm 3D reconstruction of the Z-band that shows clear detail in actin and alpha actinin. We present our results on the fitting of the two proteins in the 3D map and discuss the implications of our findings. Keywords: Nemaline myopathy, Z-line, Electron microscopy
Calcium-calmodulin competes with actin for binding to the Mdomain of cardiac myosin binding protein-C Azimi Vahid1, Hoye Elaine2, Harris Samantha P1 University of Arizona, USA; 2University of California, Davis, USA
P2.1 Contractile function, Thu 12:00–14:00
1
The regulatory M-domain of cardiac myosin binding protein-C (cMyBP-C) binds to myosin, actin, and to calmodulin when calcium is present (i.e., calcium-calmodulin), but it is unclear whether binding of all three ligands is independent or if binding interactions are competitive. Here we investigated whether calcium-calmodulin (CaCam) binding to the M-domain affected the ability of the M-domain to bind to actin using cosedimentation binding and calmodulinsepaharose pull-down assays. Results of actin cosedimentation binding assays showed that Ca-Cam significantly reduced specific binding of a recombinant protein containing three N-terminal domains of cMyBP-C (i.e., C1-M-C2, referred to as C1C2) when 10 lM calmodulin was present in the presence of calcium (pCa 3.0). In the absence of calcium (at pCa 10.0) calmodulin had no effect on C1C2 binding to actin. Increasing Ca-Cam concentrations to achieve higher molar ratios with respect to C1C2 further reduced the amount of C1C2 that bound to actin. Conversely, in calmodulin-Sepharose pulldown experiments, binding of C1C2 to calmodulin was only modestly reduced in the presence of increasing concentrations of F-actin. Taken together, these results indicate that binding of Ca-Cam can compete with actin for binding to the M-domain. These results suggest a potential mechanism whereby the functional effects of cMyBP-C binding to actin can be regulated by calcium. This work supported by NIH HL080367. Keywords: Myosin binding protein-C, Calmodulin, Actin
P1.19 Contractile structure, Thu 12:00–14:00
High-resolution 3D cryo-EM reconstructions of actin-myosin V in the rigor and ADP-bound state Wulf Sarah F1, Fujita-Becker Setsuko1, Trabuco Leonardo G1, Hofhaus Go¨tz1, Oster Marco1, Safer Daniel2, Sweeney Lee H2, Houdusse Anne M3, Schro¨der Rasmus R1 1
CellNetworks, BioQuant, University Heidelberg, Germany; Department of Physiology, University Pennsylvania, USA; 3Institut Curie, Paris, France
2
We report two new cryo-EM reconstructions of chicken myosin V decorated F-actin in the ADP-bound state and nucleotide-free Rigor, ˚ resolution. These are the first ‘‘decorated actin’’ both at about 7–8 A reconstructions of myosin V comparing two important nucleotide states of myosin V when bound to actin. We obtained corresponding quasi-molecular models by Molecular Dynamics flexed-fitting of known myosin V crystal structures into our reconstructed 3D densities. The analysis of the resulting high-resolution models confirms the strong binding of myosin V to actin for both nucleotide states, observing the 50 kDa domain cleft-closure for both states. In addition, the high-resolution reconstructions allow a detailed analysis of changes in the secondary structure of myosin. Preliminary findings are subtle changes of the actin-myosin interface between the two different nucleotide states and—at the same time—a significant change of the central beta-sheet (transducer domain) coupled to a change of the lever arm orientation. Comparing the two flexed macromolecular structures we find that the lever arm swings by *13 upon ADP release in the direction of the actin barbed-end. Keywords: Actin-myosin V, Cryo-EM, Rigor ADP
Unravelling the structure of alpha-actinin/actin interaction in Zbands Lacey Samuel1, Pinotsis Nikos2, Djinovic-Carugo Kristina3, Squire John M4, Morris Edward P5, Luther Pradeep K6 ***Candidate for Young Investigator Award
1
National Heart and Lung Institute, Imperial College London, UK; Birkbeck College, University of London, UK; 3Max F Perutz Laboratories, Vienna, Austria; 4Department of Physiology and Phamacology, University of Bristol, UK; 5Chester Beatty Laboratories, Institute of Cancer Research, London, UK; 6National Heart and Lung Institute, Imperial College London,UK 2
Alpha actinin and actin are highly conserved proteins which are ubiquitous in eukaryotic cells. Crosslinking of actin filaments by alpha actinin is of fundamental importance in cell locomotion and structure of muscle. Alpha actinin is a primary component of striated muscle Z-bands. The recent crystal structure of the complete alpha actinin molecule allows analysis of its interaction with the known atomic structure of actin. Nemaline myopathy is a skeletal muscle disease characterised by nemaline rod inclusions composed of large crystalline arrays of Z-bands. By electron microscopy of negatively stained cryosections of nemaline rods, we have obtained
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P2.2 Contractile function, Thu 12:00–14:00 Myocardial titin: an important modifier of cardiac stiffness Hamdani Nazha1, Sahin Senem1, Backs Johannes2, Linke Wolfgang A1 1
Department of Cardiovascular Physiology, Ruhr University Bochum, Germany; 2Department of Cardiology, Angiology and Pulmology University of Heidelberg, German A well-established function of titin is the determination of passive tension, F(passive), in myocardium. Modifications to the elastic titin region have been suggested to contribute to left ventricular diastolic dysfunction in heart failure (HF). Titin-based stiffness can be modulated by isoform switch or phosphorylation. We find that titin-isoform switch accounts for a significant amount of myocardial stiffness modulation, giving rise to increased or reduced F(passive) in different types of heart failure. In addition, both acute and chronic modulations of cardiomyocyte F(passive) occur via altered titin phosphorylation. Cyclic AMP-dependent protein kinase-A, cGMP-dependent protein kinase-G, and extracellular signal-regulated kinase-2 phosphorylate titin at a cardiac-specific domain, the N2Bus; this phosphorylation results in a reduction in
J Muscle Res Cell Motil (2015) 36:71–143 cardiomyocyte F(passive) in various species. PKC phosphorylates the PEVK-domain of titin, which increases F(passive) of normal mouse cardiomyocytes, but does not significantly alter F(passive) of cardiomyocytes obtained from a dog HF model. Calcium/calmodulin-dependent protein kinase-II (CaMKII) is the first kinase found to phosphorylate both the N2Bus and the PEVKdomain. This phosphorylation reduces cardiomyocyte F(passive), as demonstrated in skinned mouse cardiomyocytes incubated with recombinant CaMKII. Moreover, F(passive) is elevated in cardiomyocytes of CaMKII double knockout mice and reduced in those of CaMKII-overexpressing transgenic mice. In both human and experimental HF, a global titin phosphorylation deficit is observed, but site-specific titin phosphorylation can be increased or decreased in HF, presumably depending on the activity and expression level of the relevant kinases. Titin phosphorylation may have beneficial effects in the heart via reducing myocardial diastolic stiffness and improving ventricular filling. Altered titin phosphorylation in HF may severely affect F(passive) and compromise cardiac function. Keywords: Titin, Phosphorylation, Cardiac stiffenss
P2.3 Contractile function, Thu 12:00–14:00 X-ray diffraction studies of the cross-bridge structure during shortening or stretch of contracting rabbit muscle fibres Koubassova Natalia1, Bershitsky Sergey2, Ferenczi Michael3,4, Kopylova Galina2, Fernandez Manuel5, Narayanan Theyencheri5, Tsaturyan Andrey1 1
Institute of Mechanics, Moscow State University, Moscow, Russia; Institute of Immunology and Physiology, Ural Branch of RAS, Yekaterinburg, Russia; 3National Heart and Lung Institute, Imperial College London, UK; 4Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore; 5European Synchrotron Radiation Facility, Grenoble, France 2
Upon stretch contracting skeletal muscle effectively absorbs energy and protects joints from damage. During shortening it produces mechanical work. Our goal was to characterize the cross-bridge structure in these states and to understand how it corresponds to the observed mechanical function of muscle. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch and ramp shortening compared to those during isometric contraction at physiological temperature using synchrotron radiation at ESRF (Grenoble, France). The intensities of the off-meridional layer lines and the fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that during shortening at low velocity the fraction of myosin heads stereo-specifically bound to actin is approximately the same as that during isometric contraction; however their level arms are tilted towards the Z-line with respect to their motor domains. At high shortening velocity the fraction of actin-bound heads decreases, so that the same power is delivered by fewer actin-bound myosin heads as a result of their higher turnover. Upon stretch the total number of cross-bridges increases in accordance with Brunello et al. (Proc Natl Acad Sci USA 104(50):20114–20119, 2007), while a significant fraction of myosin heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. This explains high stiffness, low ATPase and effective energy absorption. Supported by Grants from RFBR 13-04-40100-N, 13-04-40101-N, the Program of Ural Branch of RAS, project No 12-P-4-1007, EMBO, the Royal Society, EBSA and MRC. Keywords: X-ray diffraction, Interference, Modelling
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P2.4 Contractile function, Thu 12:00–14:00 Analysis of the motor domain of mutant and wild type human embryonic myosin: changes induced by Freeman Sheldon Syndrome mutations at R672 Walklate Jonathan1***, Velazquez Carlos Vera2, Leinwand Leslie A2, Geeves Michael A1 ***Candidate for Young Investigator Award
1
School of Biosciences, University of Kent, UK; 2Department of Molecular Cellular and Developmental Biology, University of Colorado, USA
Human embryonic myosin (Emb) is expressed during development and is lost shortly after birth, re-expression occurring during muscle repair. We have expressed the WT motor domain with the light chain binding sites in mouse C2C12 muscle cells. We have also expressed R672H and R672C two point mutations in Emb myosin linked to Freeman–Sheldon syndrome or muscle distal arthrogryposis. Analysis of the rate and equilibrium constants of the cross bridge cycle show the WT myosin to be distinct from the adult human isoforms previously characterised (Bloemink et al. J Biol Chem 288(38):27469– 27479, 2013). The Emb isoform is expected to be a slow isoform and is compared to the slow/cardiac b muscle isoform. The rate of ATP binding to both the motor domain alone (M, K1k+2) and to actin.M 0 0 (A.M, K1 K2 ) is halved for b compared to Emb. The maximum rate of ATP binding to the myosin motor is increased by 30 % while the apparent affinity of the motor for ATP is tightened (15–10 lM). For 0 A.M the maximum rate of actin dissociation Kþ2 is decreased from -1 1200 to 780 s while 1/K1 is decreased from 274 to 84 lM. R672C: The rate of ATP binding to Emb R672C is reduced 10 fold with a 5 fold reduction in the maximum rate constant of binding (k+2). ATP binding to A.M is reduced 4 fold again mainly due to a 3 fold re0 duction in Kþ2 . ADP binding appears unaffected. R672H: For this mutation the ATP binding induces a very small change in Tryptophan fluorescence (DFl = 1.5 % compared to 7–8 % for WT) making the measurement of nucleotide binding difficult. The rate of ATP binding is reduced 30 fold with the k+2 reducing 9-fold and the 1/K1 increasing 3 fold. The reduction in DFl suggests a disruption of conformational change associated with the recovery stroke. ATP 0 binding to AM is relatively normal with a similar 1/K1 to WT, both 0 0 0 Kþ2 and K1 Kþ2 are reduced 2-+fold. The affinity of ADP for AM is 3-fold tighter the rate constant for ADP release being reduced 3-fold. Actin affinity is reduced 20-fold. Keywords: Myosin, Embryonic, Freeman–Sheldon
P2.5 Contractile function, Thu 12:00–14:00 Functional comparison of cardiac and skeletal acto-myosin from zebrafish and other species via in vitro motility assay Kaschel Lisa-Mareike***, Weber Cornelia, Mosqueira Matias, Fink Rainer HA ***Candidate for Young Investigator Award
University of Heidelberg, Institute for Physiology and Pathophysiology, Medical Biophysics Unit, Germany The in vitro motility assay (IVMA) is a useful tool to investigate interactions and mechanical properties of the acto-myosin motor.
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102 Determination of maximum sliding speeds of labeled actin filaments moved by working strokes of the heads of immobilized myosin molecules under optimal ATP concentration allows conclusions regarding the unloaded shortening velocity of muscle fibers. Thus IVMA is a versatile method to investigate functional outcomes of e.g. mutations of motor or regulatory muscle proteins leading to cardiac insufficiency and skeletal muscle deficiency. Since the first IVMA were conducted with myosin II from rabbit skeletal muscle and Dictyostelium discoideum, myosins of several classes and organisms were investigated. Furthermore, actin and reconstituted thin filaments were studied in IVMA, enabling also Ca2+ regulation in vitro. Taking into account the increasing importance of zebrafish as a model organism in muscle research, we established isolation protocols for skeletal and cardiac myosin and polymerizable actin from zebrafish. Cardiac and skeletal motor and regulatory muscle proteins from adult wild type zebrafish extracts were confirmed by SDS-PAGE and western blot and their activity by IVMA. Functional cardiac myosin was extracted from hearts of wildtype zebrafish directly into the IVMA flow cells. The presence of regulatory proteins in these extracts allows a comparison of Ca2+ regulation of acto-myosin from zebrafish hearts to those of other origin. IVMA with both actin and myosin from zebrafish origin were compared to acto-myosin interactions using actin and myosin combinations from different species, thus creating heterospecific systems. The analysis of instantaneous speeds and travelled distances over time of actin filaments and myosins suggest differences in the acto-myosin interaction of homospecific and heterospecific acto-myosin systems. Keywords: In vitro motility assay, Zebrafish, Heterospecific actomyosin
P2.6 Contractile function, Thu 12:00–14:00 Chemical modification of protein thiols mimics contractile dysfunction observed at the single fiber and molecular level with muscle disuse Callahan Damien M1, Miller Mark S1, Maughan David W1, Toth Michael J2 1
Departmentof Molecular Physiology & Biophysics, University of Vermont, USA; 2Departmentof Medicine, College of Medicine, University of Vermont, USA Contractile dysfunction accompanies muscle disuse and various diseases and may hasten the development of physical disability. Oxidative modification of proteins is commonly thought to be one mechanism whereby these physiological and pathological conditions alter muscle contractility. Our studies explored the potential link between chemical modification of myofilament proteins and contractile dysfunction in human skeletal muscle. We evaluated contractile performance at the molecular, cellular and whole muscle levels in patients with end-stage, symptomatic knee osteoarthritis (OA; n = 16), a clinical model of muscle disuse, and healthy, active controls (n = 15). The most prominent functional adaptation in myofilament proteins with disuse was a slowing of cross-bridge kinetics in myosin heavy chain (MHC) I fibers. We have previously shown this functional phenotype with aging, chronic heart failure and cancer. To test the possibility that such alterations in molecular muscle function can be mediated by oxidative modification, we investigated the impact of treatment with the thiol-specific alkylating agent N-ethylmaleimide (NEM), which irreversibly modifies protein thiol groups, on cross-bridge kinetics. We found a concentration dependent reduction in cross-bridge kinetics, as reflected by an increase in myosin attachment time (to), with NEM. In fact, at 12.5 lM NEM, we
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J Muscle Res Cell Motil (2015) 36:71–143 found a 17 % increase in to with no change in tension, a result that bears striking similarity to the differences observed with disuse. Considering that protein thiol groups are particularly sensitive to oxidative modification and that NEM has been shown to mimic the functional effects of biologically-relevant oxidants in pre-clinical models, our results provide preliminary evidence to support the notion that post-translational modification of protein thiols may partially explain the alterations in myofilament protein function observed with muscle disuse, aging and chronic disease in older adults. Keywords: Muscle disuse, Contractile dysfunction, Oxidative modification
P2.7 Contractile function, Thu 12:00–14:00 Study of lifetime of actomyosin complex with fast skeletal myosin Nabiev Salavat1, Ovsyannikov Denis1, Tsaturyan Andrey2, Bershitsky Sergey1 1
Institute of Immunology and Physiology, Ural Branch of RAS, Yekaterinburg, 620049, Russia; 2Institute of Mechanics, Moscow State University, Moscow, 119992, Russia Lifetime dependence of the complex of myosin from rabbit psoas muscle with filamentous actin on the load and ATP concentration was studied with two-beam optical trap (Finer et al. Biophys J 68(4): 291S–297S, 1995) operating in the force clamp feedback mode (Takagi et al. Biophys J 90(4): 1295–1307, 2006). Range of used loads included those exceeding isometric level thus reproducing conditions of eccentric contraction of muscle at single molecule level; ATP concentration was 3 lM or 6 lM. Attachment of a single myosin molecule to trapped actin filament and its force generating event triggered experimental protocol of loading the molecule. The load rose to a set level and then was left constant until detachment of myosin from actin. The actomyosin lifetime was measured from the moment of reaching the set force level to the detachment. Lifetime frequency distribution was biphasic: it quickly rose and then decreased more slowly. The distribution was fitted with two exponentials. The rate constants of both components increased with the load and the faster component showed much load higher sensitivity than the slower one. The increase in ATP concentration accelerated the slower component in direct proportion but did not affect the faster component. As observed by Veigel et al. (Nat Cell Biol 5:980–986, 2003) in similar optical trap experiments, the rate constants of lifetime distribution of smooth muscle myosin well corresponds to those of the kinetics of two phases of its working stroke. Taking this we can suppose that the distribution found in our experiments indicates similar two-step mechanism of the working stroke in the fast skeletal myosin. Supported by RFBR Grants 13-0440100-N and 13-04-40101-N and the Program of Ural Branch of RAS, project No 12-P-4-1007. Keywords: Actomyosin lifetime load dependence, Actomyosin lifetime ATP dependence, Optical trap
P2.8 Contractile function, Thu 12:00–14:00 The structural state of actomyosin in the presence of Gln147Pro mutant tropomyosin evaluated by polarized fluorimetry Karpicheva Olga1***, Simonyan Armen2, Piers Adam3, Borovikov Yurii S1, Redwood Charles3 ***Candidate for Young Investigator Award
J Muscle Res Cell Motil (2015) 36:71–143
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1
Institute of Cytology RAS, Russia; 2Institute of Cytology RAS, Saint Petersburg State University, Russia; 3University of Oxford, John Radcliffe Hospital, UK
Keywords: Sarcomere dynamics, New model, Invertebrate muscle
The molecular mechanisms of skeletal muscle dysfunction in congenital myopathies remain unclear. The present work examines the effect of a myopathy-causing mutation (Q147P) in beta-tropomyosin on the structural state of the myosin head and F-actin during different steady states of the ATP hydrolysis cycle observed by polarized fluorescence technique. In three series of experiments, F-actin, myosin S1 and recombinant tropomyosins (wild-type and with Q147P mutation) were modified by FITC-phalloidin, 1,5-IAEDANS and 5-IAF, respectively, with further incorporation into single ghost muscle fibers predominantly containing actin filaments free of troponin and tropomyosin. The Q147P tropomyosin binds to actin in muscle fibers despite its reduced affinity for actin in cosedimentation assay. However, as compared to the wild-type tropomyosin, it occupies a different position on F-actin, according to the steric model of regulation closer to the centre of the filament. The mutant tropomyosin increases the proportion of strong-binding actomyosin cross-bridges at some stages of the ATPase cycle and prevents the formation of the weak-binding state. The incomplete relaxation and excessive activation in the presence of the Q147P tropomyosin is likely to underlie the contractile abnormalities caused by this mutation. This work was supported by the FEBS Collaboration Scholarship, the Russian Foundation for Basic Research (Grants No. 14-04-00454a, 14-04-31527a) and the Muscular Dystrophy Campaign.
P2.10 Contractile function, Thu 12:00–14:00
Keywords: Mutation in tropomyosin, Conformational changes, ATP hydrolysis cycle
P2.9 Contractile function, Thu 12:00–14:00 Lancelets—a prototype model for vertebrate sarcomere dynamic research? A look on potential and technical feasibilities Dettwiler Fabian1, Denoth Jachen2, Agarkova Irina 1
Institute for Biomechanics, ETH Zurich, Switzerland; 2InSphero AG Switzerland
The locomotive muscle from the marine invertebrate branchiostoma lanceolatum (lancelet), an evolutionary prototype for skeletal muscle, is introduced as a model for sarcomere dynamic research. It is investigated whether isolated sarcomere networks and muscle fibers from lancelet can be used as an experimental system that is principally similar but structurally less complex than classic vertebrate fiber models and more intact than classic myofibrils models. Despite the increasing popularity of lancelets in biological science, no methods exist for mechanical studies on isolated fibers and myofibrils. Some of the main reasons for that are the extremely small cell size (some have only a diameter of 1 lm and a length of 200 lm) and fragility, compared to those from frog and mouse and the low level of knowledge on muscle cell physiology. Therefore methods for mechanical experiments on isolated muscle fibers and myofibrils were developed and first data on sarcomere dynamics were collected. Mononucleated muscle fibers with length of 0.1–0.8 mm that contain only one or a few myofibrils and single and small bundles of myofibrils with sarcomeres of various cross-sections (1 9 1 up to 1 9 10 lm2) were isolated. Myofibrils were isometrically activated at 5 C. Samples from mice were tested under the same conditions. Our findings provide a novel model to study elementary sarcomere dynamics and the origin of vertebrate muscle function. Isolated myofibrils from lancelet offer unique possibilities to reveal more details on inter- and intra-sarcomere dynamics. Isolated muscle fibers from lancelet might soon allow studying myofibril dynamics within an autonomous cell.
Aldosterone-inhibition exhibits repolarizing effects in osmotic muscle Breitenbach Simon, Fan Chunxiang, Jurkat-Rott Karin, Lehmann-Horn Frank Division of Neurophysiology, University Ulm, Germany Pseudohypertrophy, a swelling of especially the lower leg muscles associated with a paradoxical lack of muscle force, is the term for a major symptom of Duchenne muscular dystrophy. Manifesting before the onset of the muscle’s catastrophic transformation into fatty and connecting tissue, the underlying mechanism has remained inexplicable for two centuries. With a modern MRI protocol developed in collaboration with the DKFZ in Heidelberg we were able to show increased intracellular sodium in the muscle. It was proposed that an increased sodium conductance diminishes the membrane potential und thus the ability to contract. The osmotic effect of sodium is the cause of the pseudohypertrophic swelling. A sodium overload with corresponding oedema was already known from patients with hypokalemic periodic paralysis. Treatment with eplerenone, a potassium-sparing diuretic was able to reduce this overload and reduce the oedema. In diaphragm muscle fibers of rat we were able to show a fast hyperpolarizing effect after administration of eplerenone. Further, a depolarization caused by a cationophore was reversed. Eplerenone is a very specific blocker of the mineralocorticoid receptor (MR) in the muscle. The MR with its bound ligand aldosterone is a functional transcription factor but has also fast, non-genotropic interactions. Aldosterone is described as an inhibitor of insulin signaling, thus an inhibitor of PI3 K/Akt/mTOR pathway and a regulator of serum and glucocorticoid-induced protein kinase. This makes the MR as an upstream regulator of the activity of an array of ionchannels and transporters by control of their localization and modification of phosphorylation. Besides the sodium–potassium pump this includes the sodium-chloride co-transporter, sodium– potassium-chloride co-transporter and sodium-proton exchanger. In this study we investigate the contribution of said transporters to the eplerenone effect. Keywords: Mineralocorticoid receptor, Eplerenone, Membrane potential
P2.11 Contractile function, Thu 12:00–14:00 Functional and biochemical characterization of extensor digitorum longus (EDL) muscle from S1P3null mice Germinario Elena1,2, Bondı` Michela1, Cencetti Francesca2,4, Donati Chiara2,4, Nocella Marta2,5, Colombini Barbara2,5, Betto Romeo2,3, Bruni Paola2,4, Bagni Maria Angela2,5, Danieli-Betto Daniela1,2 1
Department of Biomedical Sciences, University of Padova, Italy; IIM, Interuniversity Institute of Myology, Italy; 3CNR-Institute for Neuroscience, CNR, Padova, Italy; 4Department of Biomedical, Experimental and Clinical Sciences, Mario Serio, University of Florence, Italy; 5Department of Experimental and Clinical Medicine, University of Florence, Italy
2
The bioactive sphingolipid sphingosine 1-phosphate (S1P) is an important regulator of skeletal muscle functions. S1P elicits most of its
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104 biological responses through the activation of five specific surface G-protein coupled receptors (S1P1–5) that activate distinct signalling pathways, whose consequences depend on the set of receptors expressed by the target cell. S1P3 receptor seems to regulate satellite cells proliferation, muscle regeneration and muscle trophism. Since S1P3 is involved in cell calcium homeostasis, it is possible that it can regulate skeletal muscle contractility. This study explored the possible role of S1P3 receptor in the extensor digitorum longus (EDL) muscle from 3-month old S1P3-null mice (KO). To this end, KO and wildtype (WT) EDL muscle contractile properties and fatigue responses were investigated in isolated intact fiber bundles, with or without the addition of 1 lM S1P. Force and stiffness were measured during a fatiguing protocol (105 consecutive tetani every 3 s) and during recovery (15 tetani every 120 s). Force decline was smaller in fibers treated with S1P (*50 % of the maximum tetanic tension) respect to untreated bundles (*80 %) in KO, but not in WT. The beneficial effect of S1P on KO was also present in the recovery phase. It is worth reporting that the KO EDL was morphologically similar to the WT muscle, with comparable mean fiber CSA (840 ± 67 vs 879 ± 95 lm2, respectively) and mitochondria content. SDS-PAGE analysis revealed that the KO EDL contains more type 1 fibers than the WT muscle (6.0 ± 1.0 vs 0.5 ± 0.5, respectively, P \ 0.05). Moreover, the mRNA quantification by Real-Time PCR showed that the expression of S1P1 and S1P2 receptors were significantly enhanced in KO compared to WT EDL muscle. On the whole this study demonstrates that S1P3 ablation appreciably affects the physiological properties of EDL muscle.
J Muscle Res Cell Motil (2015) 36:71–143 pCa values were fitted in the Hill equation. At pH 7, pCa50 values (± SEM), a reflection of calcium sensitivity, were 6.18 ± 0.02 for SHAM, and 6.15 ± 0.02, for UREM fibres. Exposure to pH 6.2 lowered calcium sensitivity for both groups but more so for uremic fibres (SHAM by 12 %, UREM by 16 %). Consistent results transpired with analysis of individual fibre data. It appears that low pH affects more the force-pCa relationship of UREM than SHAM fibres. Acknowledgments. This research has been co-financed by the European Union (European Social Fund—ESF) and Greek national funds through the Operational Program ‘‘Educational and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF)—Research Funding Program: Thales (MuscleFun ProjectMIS 377260) Investing in knowledge society through the European Social Fund. Keywords: Muscle dysfunction, Chronic disease, Animal model
P2.13 Contractile function, Thu 12:00–14:00 Cation influx via TRPC3 and TRPC6 plays a central role in the slowly increasing force during sustained stretch Yamaguchi Yohei1***, Toshiyuki Kaneko2, Keiji Naruse1, Gentaro Iribe1 ***Candidate for Young Investigator Award
Keywords: S1P receptors, EDL muscle, Fatigue 1
P2.12 Contractile function, Thu 12:00–14:00 Effect of uremia on force-pCa relationship of permeabilized single fibers Mitrou Georgia I1, Sakkas Georgios K1, Karioti Aggeliki1, Poulianiti Konstantina P1, Stefanidis Ioannis2, Tepetes Konstantinos3, Christodoulidis Gregory3, Koutedakis Yiannis4, Geeves Michael A5, Karatzaferi Christina1 1 Muscle Physiology & Mechanics Group, DPESS, University of Thessaly, Greece; 2Dept Nephrology, Faculty of Medicine, University of Thessaly, Greece; 3Department of Surgery, Faculty of Medicine, University of Thessaly, Greece; 4Human Performance Group, DPESS, University of Thessaly, Greece; 5School of Biosciences, University of Kent, UK
Chronic renal failure (CRF) presents with muscle weakness and premature fatigue, leading to reduced functional capacity, poor quality of life and premature death. The causes of muscle dysfunction in CRF have not been yet established. We aimed to evaluate the force-pCa relationship of permeabilized single psoas fibres from healthy (sham-operated, SHAM) and uremic (via partial nephrectomy, UREM) NZ rabbits. Surgery and euthanasia protocols were approved by the University of Thessaly ethics committee. At 3 months, psoas muscle samples were harvested and, after a 24-hour permeabilisation treatment, were stored in 50 % glycerol solution at -20 C until assessment. Isometric tension was assessed at 10 C, pH 7, using an SI Heidelberg micro dynamometer, in various concentrations of calcium chloride (total N = 120). A subset of fibres (N = 12) were also assessed at 10 C, pH 6.2. Basic rigor buffer (in mM): 120 KAc, 5 MgAc2, 1 EGTA, 50 MOPS, pH 7, or MES pH 6.2; Relaxing: with 5 mM ATP; Activating: with various concentrations of CaCl2 by mixing. Relative force data, averaged per free calcium condition (N = 3–8 fibres), and free calcium expressed in
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Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan; 2Department of Physiology, Asahikawa Medical University, Japan
When cardiac muscle is held in a stretched position, its intracellular calcium transient and twitch force slowly increase over several minutes probably due to stretch activated ion channels. Several members of the transient receptor potential canonical (TRPC) channel family are known as mechano-sensitive, nonselective cation channels. However, their involvement in the phenomenon remains unclear. In this study, we hypothesized that the activation of TRPC channels in isolated ventricular myocytes contributes to stretch-induced increase in cation influx during the slowly increasing forces. To test our hypothesis, mouse ventricular myocytes were enzymatically isolated, and a pair of computercontrolled piezo-positioned carbon fibers was attached to each cell end to apply stretch. Myocytes were electrically stimulated at 1 Hz and perfused in normal Tyrode solution at room temperature. Passive and active forces were calculated from carbon fibers bending. Myocytes were stretched by moving carbon fibers and the stretch was maintained for 200–300 s to record the slowly increasing force. The stretch caused an immediate increase in twitch force by Frank-Starling mechanism. Then, the force slowly increased by 111.4 ± 2.1 % of the force immediately after the stretch (n = 27). Inhibition of TRP channels with 2-Aminoethyldiphenylborinate (10 lM) significantly reduced the slowly increasing force (93.2 ± 4.7 %, n = 5). To clarify the subtypes contributed to the phenomenon, we tested the effect of TRPC3 and TRPC6 specific inhibitor, Rox-4560, on the slowly increasing force. Rox-4560 (5 lM) also significantly reduced this response (97.8 ± 2.4 %, n = 5). These results suggest that the cation influx via TRPC3 and TRPC6 plays a central role in the slowly increasing force during the stretch. Keywords: Transient receptor potential canonical 3, Transient receptor potential canonical 6, Stretch
J Muscle Res Cell Motil (2015) 36:71–143
P2.14 Contractile function, Thu 12:00–14:00 Titin–actin interaction observed in labeled skeletal myofibrils DuVall Mike1, Jinha Azim2, Leonard Tim2, Herzog Walter2 1
Biomedical Engineering, University of Calgary, Canada; 2Human Performance Laboratory, University of Calgary, Canada Within skeletal and cardiac muscle exists a molecular spring called titin, responsible for preventing stretch related damage and contributing to the development of muscle force. Titin is primarily thought to be associated with the passive (elastic) force arising when muscles are stretched, however more recent work has suggested there may be a dynamic nature to titin that can be used to understand active stretches in muscle as well. Upon activation, we believe this spring has the capability of binding to neighboring actin rendering the elastic region of titin shortened. To test this, we tracked the location of fluorescently conjugated antibodies directed at the PEVK and M-line regions of rabbit psoas myofibrils. The labeled myofibrils were mounted on our new force transducer system, and the preparations were gradually stretched to different lengths both passively (low calcium) and actively (high calcium) while recording the real-time force and translocation of fluorescent labels. With passive stretch, the labels elongate relatively linearly with sarcomere length. Interestingly, when muscles are activated at the plateau region (*2.2 lm) and then stretched, we see that some segments do not elongate initially while others are forced to elongate greatly. With continued stretch, the pattern reverses. Careful label tracking suggests titin becomes entangled in the end of the myosin thick filament briefly upon activation, before it is released and becomes anchored firmly to actin for the remainder of the active stretch. This behaviour is not seen in the passive case at all, and lends support to the previously evaluated notion of titin-actin interaction (Fukushima et al. J Biomed Biotechnol 2010:727239, 2010). In light of recent experiments in our lab, we speculate that the molecular spring titin has the ability to modulate its stiffness in an actin dependent manner (Leonard & Herzog, Am J Physiol Cell Physiol 299(1):C14–C20, 2010) thereby affecting both passive and active stretch forces. Keywords: Titin, Actin, Sarcomere
P2.15 Contractile function, Thu 12:00–14:00 D137L and G126R mutations affect the changes in position of a-tropomyosin during the ATPase cycle Rysev Nikita A1***, Matyushenko Alexander M2, Artemova Natalia V2, Levitsky Dmitrii I2, Borovikov Yuri S1 ***Candidate for Young Investigator Award
1
Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia; 2A N Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia The effect of G126R and D137L substitutions within skeletal atropomyosin (TM) on spatial arrangement of TM and actin subunits in troponin-free actin filaments of ghost muscle fibres were studied at different stages of the ATPase cycle. The wild-type recombinant TM, G126R, D137L and G126R/D137L mutant TMs were labelled with 5-IAF at Cys36 residue introduced into TM by C190A/S36C mutation. F-actin was labelled with FITC-phalloidin. The ghost fibres containing the labelled proteins were studied by polarized fluorimetry technique. It is believed that TM shift to the centre of the filament
105 during the ATPase cycle correlates with a clockwise rotation of actin subunits around the filament axis. These changes in conformation switch the actin subunits from the off to the on state, imparting them the ability to promote phosphate release from the myosin active centre, and thereby to activate the ATPase reaction. Consistent with this view we observed that, during the ATPase cycle, the control TM moved from the filament periphery to the centre and actin subunits switched on. As compared to the control TM, the mutant TMs were found to be shifted further to the periphery of actin filament at mimicking the weak-binding stages of actomyosin interaction, which resulted in a pronounced increase in the number of the switched off actin subunits. At mimicking the strong-binding stages, the mutant TMs moved further to the centre of the filament and the amount of the switched on actin subunits extremely increased. The effect reached its maximum with the G126R/D137L mutant. The abnormal pattern of mutant TM movement during the ATPase cycle may be responsible for the earlier observed increase in Ca2+-sensitivity of actin-myosin interaction and maximal sliding velocity of actin filaments in the in vitro motility assay induced by D137L and G126R substitutions in TM. This work was supported by RFBR (Grants 14-04-00454-a, 12-04-00411-a, 13-04-40099-H). Keywords: D137L and G126R mutations, Tropomyosin, Polarized fluorescence
P2.16 Contractile function, Thu 12:00–14:00 Functional implications of the protein complex formed by histidine-proline-rich glycoprotein and AMP deaminase in fast skeletal muscle Ranieri-Raggi Maria1, Martini Daniela1, Ronca Francesca1, Sabbatini Antonietta RM1, Moir Arthur JG2, Raggi Antonio1 1 Laboratory of Biochemistry, Department of Pathology University of Pisa, Italy; 2Department of Molecular Biology and Biotechnology, University of Sheffield, UK
Histidine-proline-rich glycoprotein (HPRG) is a plasma protein synthesized by parenchymal liver cells. Muscle cells do not synthesize muscle HPRG but instead actively internalize it from plasma. We demonstrated that rabbit skeletal muscle AMP deaminase (AMPD) associates with a HPRG-like protein and that an antibody against human plasma HPRG selectively binds to type IIB fibers that are known to contain the highest level of AMPD. The role of HPRG as zinc chaperone for skeletal muscle AMPD has recently been reviewed. We now propose that two correlated observations suggest a role for the AMPD-HPRG complex in the regulation of muscle contraction via the interaction of AMPD with the N-terminal region of troponin T (TnT). Firstly, in rabbit fast skeletal muscle, that has the highest level of AMPD and HPRG, the hypervariable N-terminal region of TnT (fTnT) has the sequence 29-HEPAPEVHVPEEVHEDALEDMR-50 containing 2 potential zinc binding sites (HEXXXE) that are compatible with the HEXXXH sequence that has been shown to bind zinc in rabbit skeletal muscle AMPD N-terminus. Secondly, the inhibitory effect by ATP on AMPD that is removed by limited proteolysis by trypsin in vitro and by a calpain-like proteinase in vivo is restored by interaction with stoichiometric amounts of rabbit fTnT or the phosphorylated 50-residue N-terminal peptide of fTnT. Therefore, TnT mimics the regulatory action exerted in AMPD by the 95-residue N-terminal fragment. We propose the observed interaction of AMPD with TnT is related to the physiological role of the enzyme during muscle contraction, by which AMPD displaces the myokinase equilibrium to generate from ADP the ATP required by myosin ATPase. Thus, the otherwise unrestrained AMPD activity that follows
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106 the proteolytic cleavage of the enzyme in strenuously exercised muscle can be counteracted by the binding of AMPD to TnT. Keywords: AMP deaminase, Histidine-proline-rich glycoprotein, Fast skeletal muscle troponin T
P2.17 Contractile function, Thu 12:00–14:00 Effect of cMyBP-C on the calcium regulation of interaction of myosin from fast and slow skeletal muscles with the regulated thin filament Shchepkin Daniil, Kopylova Galina, Nabiev Salavat, Nikitina Larissa Institute of Immunology and Physiology, Ural Branch of RAS, Yekaterinburg, Russia cMyBP-C is a thick filament-associated protein that consists of 8 immunoglobulin I-like and three fibronectin 3-like domains called C0–C10 (from N- to C-terminus). Numerous data report cMyBP-C contribution not only to the thick filament structure but also to the calcium regulation of contractility of the heart. Skeletal muscles are divided into fast and slow ones consisting of type II and type I fibers, respectively. In muscle tissue of adult animals four genes are expressed which encode isoforms of myosin heavy chains. One isoform is expressed in slow muscles (type I) and three isoforms are expressed in fast muscles (IIA, IIX, and IIB). Fast and slow skeletal muscle myosins were obtained from m. psoas and m. soleus of the rabbit, respectively. Troponin, tropomyosin and skeletal actin were extracted from fast skeletal muscle of the rabbit. Whole cMyBP-C was obtained from chicken hearts. We assessed dependences of the sliding velocities of regulated thin filaments on calcium concentration over skeletal isomyosins in the presence and absence of cMyBP-C in the in vitro motility assay. The effects of cMyBP-C on the interaction of single myosin molecule with F-actin were studied using the optical trap. Addition of cMyBP-C to myosin at physiological proportion (1:5 molar ratio of cMyBP-C/myosin) did not affect ‘pCa–velocity’ relationships for fast skeletal myosin. Presence of cMyBP-C increased the sliding velocity of regulated thin filaments at any pCa over slow myosin. cMyBP-C also increased calcium sensitivity from 6.8 to 7.13. We suggest that cMyBP-C effects on calcium regulation of actinmyosin interaction depend on the isoforms of skeletal muscle myosin. Supported by the Program of Ural Branch of RAS (projects 12-P-41007 and 12-P-4-1042), RBRF Grant 13-04-96027-Ural and by the Government of Sverdlovsk Region. Keywords: Myosin, cMyBP-C, In vitro motility assay
P2.18 Contractile function, Thu 12:00–14:00 Effect of botulinum neurotoxin A on neuromuscular transmission during doublet stimulation in rat skeletal muscle Gjessing Sofie L1, Rahbek Ole1, Møller-Madsen Bjarne1, Bækgaard Nielsen Ole2 1
Department of Children’s Orthopaedics, Aarhus University Hospital, Denmark; 2Department of Biomedicine, Aarhus University, Denmark Intramuscular injections of botulinum A (BoNT-A) is a widespread treatment of focal spasticity in children with cerebral palsy. Often the aim is to improve gait by reducing hypertonicity while preserving motor function. An important functional aspect of locomotion is fast force generation at the beginning of contractions, which in human
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J Muscle Res Cell Motil (2015) 36:71–143 often is achieved by initiating trains of activating action potentials by double pulses with inter-pulse intervals as short as 1.4 ms. Here, we hypothesized that BoNT-A reduces the ability of the neuromuscular junction to transmit doublets and, therefore, also reduces the potentiating of the doublets on force development. Juvenile Wistar rats received under anesthesia (hypnorm/midazolam) injection with BoNT-A (6 U/kg bw) in one hind leg leaving the other as a control. After 1–4 days, soleus muscles and part of their motor nerve were isolated and incubated in Krebs–Ringer buffer. Isometric contractions were elicited via electrical stimulation of the nerve. In control muscles, contractions elicited by doublet with 2 or 4 ms inter-pulse interval potentiated force production by 96 ± 16 and 132 ± 18 % (N = 16), respectively. This potentiation was, however, attenuated by BoNT-A in a manner that depended on the reduction in nerve stimulated force and the interpulse interval. Thus, in muscles where the maximal tetanic force was reduced by respectively 25–50 % or by more than 60 %, the force potentiation by doublets was reduced to 154 ± 8 % (N = 11, P \ 0.05) and 144 ± 6 % (N = 13, P \ 0.01), respectively. This effect of BoNT was seen at an inter-pulse interval of 2 ms but not at 4 ms or longer. In conclusion, BoNT-A reduced the ability of the neuromuscular junction to transmit doublets in rat muscles. This suggests that BoNT-A can interfere with motor function during dynamic contraction, which, depending on the dose used, could compromise gait in children treated with the compound. Keywords: Botulinum toxin A, Spasticity, Neuromuscular transmission
P2.19 Contractile function, Thu 12:00–14:00 Effects of disulfide formation on sarcoplasmic reticulum Ca2+ uptake during recovery from sustained muscle contractions in rat slow-twitch muscles Hirano Katsutoshi, Watanabe Daiki, Wada Masanobu Graduate School of Integrated Arts and Sciences, Hiroshima University, Japan It has previously been shown that vigorous muscle contractions, in many instances, result in decreases in the ability of sarcoplasmic reticulum (SR) to sequester Ca2+ in fast-twitch muscles, whereas this is not necessarily the case in slow-twitch muscles. The purpose of this study was to examine a cause of changes in SR Ca2+ uptake in slowtwitch muscles fatigued by low-frequency stimulation. Intact rat soleus muscles were exposed to continuous 10-Hz stimulation via the sciatic nerve for 60 min and excised immediately (R0) after the cessation of stimulation, or after 0.5 h (R0.5) or 48 h (R48) of recovery. The maximal activity of sarco/endoplasmic reticulum Ca2+ATPase (SERCA) was decreased by 20 % at R0, increased by 32 % at R0.5 and unchanged at R48. Application of dithiothreitol, a disulfide reducing reagent, returned the altered activities observed at R0 and R0.5 to control levels. The amount of phosphorylated phospholamban was increased, but only at R0.5. The content of SERCA2, a slow isoform of SERCA, was unaltered at any time points investigated. These results suggest that during recovery after sustained muscle contractions, 1) the disulfide formation that occurs in SERCA may be involved both in decreases and increases in the maximal capacity of SR Ca2+ uptake and 2) phosphorylation of phospholamban may contribute to increases in the submaximal capacity. In view of the previously suggested effect of reduced glutathione and oxidation on SERCA activity, it may be speculated that S-glutathionylation of SERCA is associated with the increased SR Ca2+ uptake. Keywords: Muscle fatigue, Oxidative stress, Enthusing activity
J Muscle Res Cell Motil (2015) 36:71–143
P2.20 Contractile function, Thu 12:00–14:00 Direct myosin activation represents a novel therapeutic approach for acute heart failure ´ rpa´d, Bo´di Bea´ta, E´des Nagy La´szlo´***, To´th Attila, Kova´cs A Istva´n, Papp Zolta´n ***Candidate for Young Investigator Award
Division of Clinical Physiology, Faculty of Medicine, University of Debrecen, Hungary Decreased cardiac contractility is a central feature of acute heart failure, and omecamtiv mecarbil (OM), a novel cardiac myosin activator drug may provide inotropic support for weakened cardiac contractions. The aim of this study was to characterize a hypothetical Ca2+-sensitizing effect of OM and to determine the tissue selectivity of its action. Direct force measurements were performed in skinned myocyte-sized preparations from left ventricular and diaphragmatic tissue samples of Wistar rats, exposed to test solutions with increasing concentrations of OM (between 10nM and 10 lM) to test its concentration dependent effects on Ca2+-regulated force production and on its Ca2+-sensitivity. OM treatment resulted in significant increases (P B 0.05) of the Ca2+-sensitivity of force production in cardiomyocytes (pCa50 in drug free controls: 5.88 ± 0.02; at 0.1 lM and 1 lM drug concentrations: 5.98 ± 0.02 and 6.452 ± 0.05, respectively; n = 8–10; mean ± SEM) as well as in diaphragmatic myofibers (pCa50 in drug free controls: 5.61 ± 0.02; and at 1 lM drug concentration: 5.85 ± 0.02; n = 8). Moreover, OM significantly increased passive forces (Fpassive) at 1 lM concentration and higher in isolated cardiomyocytes (Fpassive drug free: 0.62 ± 0.05 kN/m2 and at 1 lM drug concentration: 2.12 ± 0.12 kN/m2; n = 10) and in diaphragmatic fibers (Fpassive in drug free controls: 0.68 ± 0.09 kN/ m2, and at 1 lM drug concentration: 0.93 ± 0.08 kN/m2; n = 10). OM slowed down the kinetics of force development significantly from 30nM OM concentration (arbitrary; drug free: 1.57 ± 0.24 and at 30nM OM concentration: 1.09 ± 0.12; n = 8). Our data illustrate OM as a potent positive inotropic agent, exerting its cardiotonic effect via a Ca2+-sensitizing mechanism. However, OM appears to be a nonselective myosin activator drug also enhancing the contractility of the rat diaphragm. Moreover, the drug mayimpair diastolic function due to Ca2+-sensitization at diastolic Ca2+ concentration and by increasing cardiomyocyte passive force.
107 capacity. The mechanism underlying PH-induced diaphragm dysfunction is unclear, but may be due in part to the reduced myofibrillar proteins. Recently, heat shock protein (HSP) 72 has been shown to inhibit the degradation of myofibrillar proteins in skeletal muscle. Here we examined the effect of heat stress on PHinduced diaphragm dysfunction. Male Wistar rats were randomly assigned to control (C), control with heat stress (CH), PH (M), PH with heat stress (MH) group. PH was induced by a single injection of monocrotaline. The rats in the CH and MH group were exposed to heated water at 42 C for 30 min every other day. After 4 weeks of injection, diaphragm muscle was removed and analyzed for force production and immunoblotting. Compared with C group, the maximum tetanic force per cross-sectional area was markedly decreased in the M group. The levels of HSP72 in whole muscle lysates were elevated in both the CH and MH groups, while the levels of HSP25 and aB-crystallin were unchanged. Moreover, the expression of HSP72, but not the HSP25 and aB-crystallin, in the myofibrillar fraction was increased in the M, CH, and MH groups. In conclusion, increased levels of HSP72 by intermittent heat stress may attenuate force loss in diaphragm of PH rats through preventing myofibrillar dysfunction. Keywords: Pulmonary hypertension, Diaphragm, Heat shock protein
P2.22 Contractile function, Thu 12:00–14:00 Two types of stretch activation responses in fast human skeletal muscle fibres at different step length Kro¨ss Markus***, Galler Stefan ***Candidate for Young Investigator Award
Department of Cell Biology, University of Salzburg, Austria
Health Sciences, Sapporo Medical University Graduate School, Hokkaido, Japan; 2Health Sciences, Sapporo Medical University, Hokkaido, Japan
When muscles fibres, activated under isometric condition, are stepwise stretched by 0.1–0.5 % of fibre length, characteristic force responses appear: A rise in force simultaneously with the stretch, followed by a rapid force decay and a subsequent delayed force increase (stretch activation). The kinetics of stretch activation is conspicuously correlated with the isoforms of the myosin heavy chain (Galler et al. J Physiol 478: 513–521, 1994). On skinned fast fibres of frog and rat we previously found two types of stretch activation: a fast type at high levels of Ca2+ activation and an up to 30 times slower type at low levels of activation (Galler et al. BBRC 385: 44–48, 2009). At intermediate Ca2+ activation levels both types were simultaneously visible in one stretch experiment. In the present study we extended our investigation to human skeletal muscle fibres. Human fast fibres did not show the two types of stretch activation in a single stretch experiment. However, when the step length was increased to 2–4 %, the usual fast type of stretch activation disappeared and a slow type appeared. This slow response was up to 9 times slower than the fast one observed at small step lengths (0.1–0.5 %). With increasing Ca2+ activation level increasing step lengths were necessary to observe the slow type of stretch activation. At maximal Ca2+ activation no slow type of stretch activation was found in stretches up to 7 %. Like frog and rat slow fibres, also human slow fibres displayed only one type of stretch activation over the whole Ca2+ activation range. The kinetics of this response increased about 3 times when the Ca2+ activation level increased from near threshold to maximum.
Patients with pulmonary hypertension (PH) suffer from diaphragm muscle weakness, along with dyspnea and reduced exercise
Keywords: Stretch activation, Skinned muscle fibres, Human skeletal muscle
Keywords: Omecamtiv mecarbil, Acute heart failure, Cardiac myosin activator
P2.21 Contractile function, Thu 12:00–14:00 Effects of heat stress on contractile function in the diaphragm from rat with pulmonary hypertension Masami Abe1***, Jaesik Lee1, Daisuke Tatebayashi1, Kouichi Himori1, Takashi Yamada2 ***Candidate for Young Investigator Award
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P2.23 Contractile function, Thu 12:00–14:00
P2.25 Contractile function, Thu 12:00–14:00
Ventilator-induced diaphragm contractile dysfunction: emerging role of the Small Ubiquitin-related Modifier (SUMO) pathway
Dystrophin deficiency impairs upper airway dilator muscle function in the mdx mouse
Gastaldello Stefano***, Cacciani Nicola, Kalamgi Rebeca Corpeno, Hedstro¨m Yvette, Larsson Lars
Burns David***, O’Halloran Ken D ***Candidate for Young Investigator Award
***Candidate for Young Investigator Award Department of Physiology, University College Cork, Ireland Department of Neuroscience, Uppsala University, Sweden Mechanical ventilation is often an essential intervention in patients suffering from sepsis, neuromuscular diseases and during surgery along with postsurgical recovery. Mechanical ventilation can be a life-saving intervention and is used clinically to maintain gas exchange in patients unable to provide adequate alveolar ventilation on their own. On contrary, mechanical ventilation can promote ventilator-induced diaphragmatic dysfunctions (VIDD). A decrease in force-generating capacity is a primary diaphragm muscle dysfunction, while atrophy has been observed after a long period treatment. Taken together, these effects result to be a major contributor to problems in weaning patients from the ventilator. Reversible post-translational modification by covalent conjugation of the Small Ubiquitin-related Modifier (SUMO) is emerging as an important means to regulate cellular responses to endogenous and exogenous stressors both cellular and organismic levels. Given the central role of the SUMO network, we present a new study that carries important results within the complexity of the signal mechanisms responsible for VIDD in young rat mechanical ventilated diaphragm muscles. Keywords: Diaphragm, Mechanical ventilation, SUMO
P2.24 Contractile function, Thu 12:00–14:00
Duchenne muscular dystrophy (DMD) is a fatal genetic disorder associated with multi-system dysfunction. Limb and respiratory muscle is adversely affected. The sternohyoid (SH) muscles are representative pharyngeal dilators involved in the maintenance of airway patency. We sought to examine the effects of dystrophin deficiency on SH muscle function in the mdx mouse model of DMD. 8 week old male wild type (WT) control (C57BL10, n = 8) and mdx (C57BL10mdx, n = 8) mice were studied. Isolated SH muscle was examined ex vivo under control conditions (95 % O2/5 % CO2) at 35 C. Isometric and isotonic functional properties were measured. Data were compared using Student’s t-tests and two-way ANOVA as appropriate. SH muscle functional capacity was significantly decreased in mdx animals compared to WT. There was a significant reduction in peak twitch force (p = 0.0021) and peak tetanic force (p = 0.004) and maximum work (p = 0.0098) and power (p = 0.0349) production in mdx SH compared to WT muscles. Furthermore, there was a significant decrease in mechanical work (p \ 0.0001) and power (p \ 0.0001) production and shortening velocity (p = 0.020) across the load continuum in mdx compared to WT animals. In conclusion, SH muscle is adversely affected in the mdx mouse. Pharyngeal dilator muscle dysfunction may impair the control of airway patency in patients with DMD, increasing the risk of airway obstructive events and blood gas disturbances. Keywords: DMD, Sternohyoid muscle, Upper airway
Different effects of isoforms of cardiac contractile and regulatory proteins on calcium regulation of actin–myosin interaction Kopylova Galina, Shchepkin Daniil, Nikitina Larissa Laboratory of Biological Motility, Institute of Immunology and Physiology, Ural Branch of RAS, Yekaterinburg, 620049, Russia Contractile function of the heart depends on the pattern of contractile and regulatory proteins in cardiomyocytes. Change the conditions of the heart functioning leads to a change in composition of sarcomere proteins in a cardiomyocyte. The aim of this work was to investigate the role of b-tropomyosin (TM) in functioning of myocardium. For this Ca2+-regulatory effect of tropomyosin isoforms with different content of a- and b-chains on actin-myosin interaction was studied in the in vitro motility assay with cardiac myosin and a-actin isoforms. V1 and V3 isomyosins were obtained from left ventricles of hyper- and hypothyroid rabbits, respectively. Troponin, cardiac a-actin and aa-Tm were extracted from left ventricles of euthyroid rabbits. ab-TM was obtained from left ventricles of bovine heart. The maximal sliding velocities of the regulated thin filaments over V1 isomyosin were higher for the filaments contained aa-TM. Ratio of a/b-chains of TM did not affect maximal velocity over V3 isomyosin. The Hill coefficient of ‘pCavelocity’ relationship of cardiac isomyosins did not depend on the ratio of a/b-chains of TM with both cardiac and skeletal actin. The Hill coefficient was higher for V1 as compare V3 isomyosin. Supported by Grants of the Program of Ural Branch of RAS (project No 12-P-4-1007 and 12-P-4-1042), RBRF Grants 13-04-40101-N and 13-04-96027Ural and by the Government of Sverdlovsk Region. Keywords: Cardiac myosin, Calcium regulation, In vitro motility assay
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P2.26 Contractile function, Thu 12:00–14:00 Intermittent heat stress fails to ameliorate contractile dysfunction in extensor digitorum longus muscle from adjuvant-induced arthritis rats Lee Jaesik***, Abe Masami, Tatebayashi Daisuke, Himori Kouichi, Yamada Takashi ***Candidate for Young Investigator Award
Health sciences, Sapporo Medical University Graduate School, Japan Muscle wasting and weakness are prominent features in patients with rheumatoid arthritis. We suggested previously that NO-derived radicalinduced modifications in myofibrillar proteins contribute to the arthritis-induced muscle dysfunction (Yamada et al. Arthritis Rheum 60:3280-9, 2009). On the other hand, heat shock protein (HSP) 72 has been shown to reduce redox stress in skeletal muscle. In this study, we examined the effects of heat stress on contractile properties of extensor digitorum longus (EDL) muscle from adjuvant-induced arthritis (AIA) rats. Rats were divided into four groups (n = 6/group): a control (C), heat stress (H), AIA (A), and AIA with heat stress (AH) group. AIA was induced by an injection of complete Freund’s adjuvant to the knee joint cavity. Rats in the CH and AH groups were exposed to heated water at 42 C for 30 min every other day. After 21 days of injection, EDL muscles were removed and analyzed for force production and
J Muscle Res Cell Motil (2015) 36:71–143 immunobloting. Compared with C group, the maximum specific force was decreased in the EDL muscles from the A and AH groups. Moreover, the reduction in force production was accompanied by increased expression of tumor necrosis factor-a and 3-nitrotyrosine content in both groups. The levels of HSP72 were markedly increased in the CH and AH groups. These results demonstrate that the increased HSP72 expression by intermittent heat stress does not ameliorate muscle dysfunction and inflammatory redox stress in AIA EDL muscles. Keywords: Adjuvant-induced arthritis, Heat shock protein 72, Contractile dysfunction
P2.27 Contractile function, Thu 12:00–14:00 The effect of MHC isoform expression on the history-dependence of force production
109 Comparative Biomedical Sciences, Royal Veterinary College, UK Skinned psoas fibres from laboratory rabbits (LR) are an established model for investigating cross-bridge mechanics. We have begun tests on a range of muscle types from wild rabbits (WR), partly with the aim of evaluating muscle power capabilities in free-moving animals. Here we compare our findings to date for the mechanics of psoas fibres from WR and LR, determined using temperature-jump activation to 20 or 25 C, and using force-control approaches. When adjusted to a temperature of 25 C, assuming Q10 of 2.25, power was [40 % greater (P \ 0.001) in WR (142 ± 1.5 W/l, n = 21) compared to LR (100 ± 1.5 W/l, n = 12). Maximum shortening speed (Vmax) was [2-fold higher (P \ 0.001) in WR (8.9 ± 0.15) compared to LR (4.4 ± 0.04), and the shortening speed at peak power was [60 % faster (P \ 0.001) in WR. The higher power and shortening speed of WR compared to LR are likely to be due to both differences in myosin heavy chain isoform and to ‘training effects’. Keywords: Power, Psoas, Rabbit
Power Geoffrey A***, Joumaa Venus, Hisey Brandon, Caicedo Andres, Stutz Jan, Herzog Walter ***Candidate for Young Investigator Award
P2.29 Contractile function, Thu 12:00–14:00
Human Performance Lab, University of Calgary, Canada
High troughput transducers to measure in vitro muscle contraction
A fundamental property of skeletal muscle, unaccounted for by the current framework of the cross-bridge theory, is the history-dependence of force production. There is a greater isometric steady-state force following active lengthening and a lower isometric steady-state force following shortening compared with force achieved in a purely isometric contraction at the same final muscle length. These welldocumented properties of skeletal muscle are termed residual force enhancement (RFE) and force depression (FD), respectively. A distinct difference exists in force characteristics between Type II and Type I muscle fibres during both active lengthening and shortening. However, we do not expect fibre type differences during lengthening or shortening to influence the isometric steady-state following the dynamic action. Thus, we do not expect fibre type differences to influence the level of RFE or FD. Glycerinated and isolated muscle fibre segments from the psoas and soleus of New Zealand white rabbits were tested to determine: RFE, stretched from 2.6–3.2 lm sarcomere length (SL); and FD, shortened from 3.2 to 2.6 lm SL. Following the experiments, single fibre MHC isoforms were identified via SDS-PAGE and fibres were divided into Type II (expressing MHC IId) and Type I (expressing MHC I).The main finding was that RFE was not different across fibre types, while FD was greater in the Type II compared with Type I fibres. The work performed during shortening was greater in the Type II compared with Type I fibres, which may have contributed to the greater FD in Type II fibres. Despite there being distinct differences in the transient aspects of active lengthening, there does not appear to be a difference in the steady-state RFE of permeabilized Type II and I muscle fibre segments. However, FD was higher in the Type II compared with Type I fibres, owing to the ability to perform more work during shortening. AIHS, CIHR, NSERC, CRC Programme and The Killam Foundation. Keywords: Force enhancement, Force depression, Single fibre
P2.28 Contractile function, Thu 12:00–14:00 Comparison of power output in single skinned psoas fibres from wild rabbit (Oryctolagus cuniculus) and laboratory rabbit (New Zealand White) West Timothy, Diack Rebecca, Goodwin David, Curtin Nancy, Woledge Roger, Wilson Alan
Giazzon Marta1, Favre Melanie1, Araromi Oluwaseun2, Poulin Alexandre2, Shea Herbert2, Liley Martha1 1 CSEM Centre Suisse d’Electronique et de Microtechnique SA, Switzerland; 2EPFL, Lausanne, Swizerland
Asthma is a chronic disease in which contraction of bronchial smooth muscle causes breathing difficulties. Existing in vitro models of smooth muscle tissue contraction are complex, cannot be scaled to allow large numbers of measurement in parallel and do not directly measure muscle contraction. In the project ‘Breathe’ we are going to develop a highly parallel in vitro platform for direct measurement of the force smooth muscle cells generate when they contract, and thus for study of the effect of different therapeutic drugs. Human Bronchial Smooth Muscle Cells (hBSMCs) are used to create the muscle tissue. Sub–micrometre surface topography and PLL-PEG/fibronectin coatings are used to obtain a pattern monolayer of aligned, interconnected and synchronous myoblasts. A strain transducer device will be developed to sense the very small forces generated by a single cell layer. The transducer will use extremely soft elastomer electrodes whose electrical properties change as function of strain. A culture and measurement platform in 24-well plate format will be developed. It will allow the contractile force of 24 cell cultures to be studied simultaneously. Keywords: Smooth muscle, Contraction, Cell force transducer
P3.1 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Changes in size and function of single muscle fibres of young and elderly subjects in response to two weeks of bed rest and subsequent rehabilitation Cancellara Lina1, Canato Marta1, Moro Tatiana1, Katja Koren2, Rado Pisot2, Paoli Antonio1, Reggiani Carlo1, Toniolo Luana1 1
Department of Biomedical Sciences, University of Padova, Italy; Primorska University, Slovenia
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Muscle unloading is known to induce muscle atrophy and, in this study, we aimed to assess whether such effect is more pronounced in elderly than
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110 in young people. Muscle biopsies were obtained from the vastus lateralis before and after 15 days of bed-rest from seven young (age 20–30) and sixteen elderly (age 60–65) male subjects. A third biopsy was taken after 15 days of rehabilitation based on three sessions/week of exercise. All subjects were healthy, normally active sedentary people. Single fibres were dissected, cross sectional area (CSA) and isometric force (Fo) during maximal activation were measured and fibres were classified as slow, fast 2A, 29and hybrid in relation to their content in myosin isoforms determined with SDS-PAGE. Before bed rest, significant differences were present between young and elderly subjects as the proportion of slow fibres and slow myosin isoforms was greater in the elderly, while the average CSA and isometric force of single muscle fibres were lower in the elderly than in the young subjects. After two weeks of bed rest, average fibre CSA showed a decrease in both groups, which was followed, during rehabilitation by a partial recovery towards the initial values in the young but not in the elderly group. Average isometric force underwent to a decrease during the bed rest period without any increase during the rehabilitation period. Taken together the results point to a high sensitivity to disuse of muscle fibre size and contractile performance in the elderly and to a slow and incomplete recovery during the rehabilitation period. Keywords: Muscle single fibres, Bed rest, Contractile properties
P3.2 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Protein kinase A (PKA) activation suppress the ubiquitin– proteasome system (UPS) activity and MuRF1 mRNA expression by phosphorylating Foxo1 in rat skeletal muscle Silveira WilianA1, Gonc¸alves Dawit AP1, Grac¸a Fla´via A1, Zanon Neusa M1, Kettelhut Isis C2, Navegantes Luiz CC1 1
Physiology, Ribeira˜o Preto Medical School, USP, Brazil; Biochemistry/Immunology and Physiology, Ribeira˜o Preto Medical School, USP, Brazil
2
It has been shown that cAMP signaling activation prevents skeletal muscle atrophy, however the molecular mechanisms remain unclear. This study tested the hypothesis that the ubiquitin–proteasome system (UPS) could be regulated by cAMP/PKA signaling. The effects of 6-Bnz-cAMPS (a specific PKA activator) on PKA/CREB and Akt/Foxo signaling, as well as on rates of protein degradation and on mRNA levels of Atrogin-1 and MuRF1 were determined in isolated extensor digitorum longus (EDL) muscles from normal rats. Additionally, the effects of forskolin (FSK; a adenylyl cyclase activator) and H89 (a PKA inhibitor) on cAMP/PKA and Akt/Foxo signaling were examined in C2C12 myotubes. In EDL muscles, 6-Bnz-cAMPS reduced the UPS activity (*45 %), and mRNA levels of MuRF1 (40 %), and increased the phosphorylation levels of CREB (64 %) and Foxo1 (*39). In serum-deprived C2C12 myotubes, the activation of cAMP/PKA signaling induced by FSK (10 lM) increased the phosphorylation levels of CREB and Foxo1, an effect that was prevented by H89 pre-incubation. The phosphorylation levels of Akt were not altered by FSK, 6-BNZ-cAMP or H89. These data suggest that PKA inhibits UPS activity through a novel mechanism that involves the phosphorylation and inhibition of Foxo1, independently of Akt. Keywords: cAMP, CREB, C2C12 myotubes
J Muscle Res Cell Motil (2015) 36:71–143 Sultana Nasreen1***, Flucher Bernhard E1, Benedetti Ariane1, Obermair Gerald J1, Tuluc Petronel2, Schwarzer Christoph3, Szentesi Pe´ter4, Geyer Nikolett4, Dienes Beatrix4, Csernoch La´szlo´4 ***Candidate for Young Investigator Award
1
Division of Physiology, Medical University Innsbruck, Austria; Department of Pharmacology, University of Innsbruck, Austria; 3 Division of Pharmacology and Toxicology, Medical University Innsbruck, Austria; 4Department of Physiology, Faculty of Medicine, University of Debrecen, Hungary 2
CaV1.1e is the calcium channel splice variant of embryonic skeletal muscle. It functions as voltage sensor in EC coupling, but in contrast to the adult CaV1.1a variant it also supports sizeable calcium currents activating at the same membrane potential as SR calcium release. Splicing defects resulting in elevated expression levels of the CaV1.1e variant in mature muscle correlate with the degree of muscle weakness in patients suffering from dystrophic myotonia. In order to study the physiological importance of the developmental switch from a well conducting to a poorly conducting channel and to examine its putative involvement in dystrophic muscle weakness, we generated a mouse model in which exon 29 of the Cacna1 s gene is permanently deleted. Quantitative RT-PCR analysis demonstrates that exon 29 knockouts express exclusively the CaV1.1e splice variant at levels comparable to total CaV1.1 in wildtype muscle; although Western blot analysis showed a 20–40 % reduction of CaV1.1 protein. A battery of behavioral tests (home-cage activity, voluntary and forced running, grip strength and rotarod tests) revealed no signs of muscle weakness or altered motor performance compared to wildtype siblings. Combined voltage clamp and fluorescence calcium recordings of isolated muscle fibers from exon 29 knockout mice revealed a substantial influx-dependent component of the calcium transient. Histological staining for succinate dehydrogenase activity and immunofluorescence analysis of myosin heavy chain isoforms revealed a reduction in oxidative metabolism and a change of fiber type composition, respectively, in knockout soleus and EDL muscles. However, fatigue resistance of both muscles was increased. Together these findings suggest that expression of L-type calcium currents in embryonic muscles and their perinatal down-regulation are important for the determination and maintenance of the specific fiber type composition in skeletal muscles. Grants: FWF P23479, W1101, OTKA-NN107765. Keywords: Calcium determination
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muscle,
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P3.4 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Epidermal growth factor (EGF) promotes the fast skeletal muscle fiber phenotype in chronic disease Natanek S Amanda1, Ciano Margherita1, Willis-Owen Saffron1, Thompson Anna1, Polkey Michael2, Cookson William1, Moffatt Miriam1, Kemp Paul1
P3.3 Muscle plasticity, exercise, and aging, Fri 12:00–14:00
2
Increased calcium influx through CaV1.1e in mature skeletal muscle disrupts the coordinated expression of fiber type properties
A preponderance of fast-twitch (FT), compared to slow-twitch (ST), fibres commonly occurs in the locomotor muscles of patients with chronic disease. This ST to FT shift (FS) is associated with reduced
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National Heart and Lung Institute, Imperial College London, UK; NIHR Biomedical Research Unit of the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London, UK
J Muscle Res Cell Motil (2015) 36:71–143 exercise capacity in patients with Chronic Obstructive Pulmonary Disease, COPD, and type II diabetes. This association may be causal since reversal of FS increases exercise capacity in animals. Targetable mechanisms driving FS in adult muscle are limited. Gene array coupled with network analysis using muscle from 98 elderly COPD patients and 19 age-matched controls identified EGF close to the hub of a network map strongly and inversely correlated with quadriceps ST fibre proportion in patients. Serum EGF was higher in patients with a low ST fibre proportion than controls. Consistent with EGF regulating fibre type, EGF is reported as more highly expressed in mouse FT type IIb fibres than ST fibres. To determine whether EGF promotes ST fibres in vitro, Day 8 C2C12 myotubes were treated with a neutralizing EGF antibody. EGF removal increased MHC I (10-fold p \ 0.001) and reduced MHC IIb expression (2-fold, p \ 0.001). Similarly, Day 8 myotubes treated with a selective EGF receptor inhibitor, tyrphostin 1478, had increased transcripts of MHC I and mitochondrial genes UCP3 and citrate synthase, compared to control myotubes. Previous studies have shown that inhibition of mitogenactivated protein kinases, ERKs, promote ST fibre type. Indeed, inhibition of ERK1/2 signaling with PD98,059 in C2C12 myotubes increased MHC I expression 5-fold (p \ 0.001) but did not suppress MHC IIb expression. Together our data suggest that EGF contributes to suppression of ST gene, and promotion of FT gene, expression, partly through ERKs. EGF may contribute to FS in chronic disease and loss of exercise capacity. Inhibition of EGF signaling may have therapeutic potential; EGF receptor inhibitors are already used clinically for cancer. Keywords: Slow-twitch, Fast-twitch, Muscle plasticity
P3.5 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Focal adhesion proteins are regulated in dependence of the exercise intensity in humans Andresen Bernhard, Bloch Wilhelm, Suhr Frank Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany Mechanical stimuli critically determine structural and functional adaptations of human skeletal muscle tissues. Focal adhesions (FAs) constitute protein assemblies that mediate transformations of mechanical stimuli into biochemical signals allowing cells and tissues to adapt. Among FA proteins, integrin-linked kinase (Ilk) was found to regulate skeletal muscle integrity in mice under sedentary conditions. However, it remains unknown whether FAs are regulated by physiological loading, e.g. exercise, in human muscle, why we aimed to study Ilk and its partners a-/b-parvin (Parva/Parvb) and Pinch-1/-2 as well as related signaling pathways after exercise conditions. 2 cycling intervention groups: 1. Moderate (Mod, 9 sessions in total, each at 45 % of VO2max, 16.50 h total exercise time) and intensive interval (Int, 9 sessions in total, each at 95 % of VO2max, 3.71 h total exercise time); 3 weeks of exercise; 6 biopsies from Vastus lateralis (pre, 4 h/24 h after 3rd session, 4 h/24 h/72 h after 9th session). FA components and signaling pathways were analyzed by western blot (WB), immunofluorescence (IF) and qPCR. FA components were significantly (p \ 0.05) increased at 4 h post 3rd and 9th sessions, while the general FA components’ expression patterns diverge between Mod and Int groups. IF analyses showed that FA components have sarcolemmal localizations and Ilk was also observed in myonuclei. Parvb and Pinch-1 are more prominently expressed in human skeletal muscle than Parva and Pinch-2. Furthermore, we observed signaling pathway patterns, e.g. pAktS473, homologous to FA component expression patterns in Mod and Int groups. Our data show that
111 FA components, such as Ilk, Parvs, and Pinchs, are regulated by physiological loading. Importantly, FA components obviously discriminate between moderate and intensive as well as the durations of exercise modes. Therefore, FA components are mechanically sensitive sensors that are regulated by physiological loading in human skeletal muscle. Keywords: Human skeletal muscle, Integrin-linked kinase, Focal adhesions
P3.6 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Muscle stem cell differentiation; the role of age oxidative environment and muscle origin Loughna Paul1, Redshaw Zoe2 1 School of Veterinary Medicine and Science, University of Nottingham, UK; 2University of Nottingham, UK
The resident stem cell population within skeletal muscles contributes to significant periods of hypertrophy, particularly during the postnatal period, and also to remodelling and regeneration in response to physiological stimuli, disease and traumatic insults. In both rodent models and humans it has shown that age reduces the capacity of these stem cell populations to contribute to such processes. The ability of these cells to trans-differentiate into other cell types has also been recognised. It has been shown that with age there is a progressive loss of muscle mass (sarcopenia) and that in humans and large animals this is associated with infiltration of adipose tissue into the muscle. In these studies we examine the properties of stem cells derived from different muscles of a large animal (porcine) model. It was shown that cells derived from different skeletal muscles exhibited different differentiation properties in vitro. We have examined signalling pathways involved in differentiation and shown that activation of the canonical Wnt pathway prevents adipogenic differentiation. In contrast an increased oxidative environment encourages adipogenesis but inhibits myogenic differentiation. We have shown that this acts via MAPKs and through a protein kinase C pathway. Stem cells were isolated from both young and old animals. With age there is a decreased capacity of muscle stem cells to proliferate and undergo myogenic differentiation. We have examined the role of a number of muscle specific micro-RNAs in this process. It was observed that certain micro-RNAs with specific roles in differentiation were down-regulated whilst others were up-regulated. Keywords: MiRNAs, Stem cells, age
P3.7 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Dihydrotestosterone (DHT) rescues the age-dependent decline in protein synthesis in mouse fast-twitch skeletal muscle fibres Wendowski Oskar***, Mutungi Gabriel ***Candidate for Young Investigator Award
University of East Anglia, Norwich, UK Sarcopenia (muscle wasting due to old age) is a debilitating condition characterised by the gradual loss of skeletal muscle mass and function that manifests itself predominantly in the elderly. It leads to reduced activity, increased susceptibility to falls and eventually to loss of
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112 independence. However, despite its physical and socioeconomic importance, the causes and mechanisms underlying sarcopenia are still poorly understood. Previously, we have published data which suggests that DHT may have therapeutic potential in the management of sarcopenia (Hamdi & Mutungi, J Physiol 589:3623-40, 2011). However, no studies have investigated the effects of DHT on protein synthesis in muscles isolated from old mice. The primary aim of this study was to investigate the effects of treating small skeletal muscle fibre bundles isolated from young (*100 days) and old ([600 days) female mice with physiological concentrations of DHT on protein synthesis. All the experiments were performed at *20 C using small fibre bundles isolated from the extensor digitorium longus and the soleus of adult mice. Two types of experiments were performed. In the first experiment, proteins isolated from the EDL and soleus of young and old mice were probed for the expression of the sodiumcoupled neutral amino acid transporter (SNAT) 2 and the L-type amino acid transporter (LAT2). In the second experiment, the effects of DHT on protein synthesis was determined using the trichloroacetic acid protein precipitation assay followed by liquid scintillation counting. Our results show that the expression of both amino acid transporters increases with age in both the EDL and soleus. They also show the presence of a second smaller protein band of * 56 kDa that was present in muscles of mice [500 days old. Ageing led to a decline in protein synthesis in the fast-twitch fibres and this was reversed by treating the fibre bundles with DHT. We suggest sarcopenia is caused by the reduction in bioavailable DHT. Keywords: Dihydrotestosterone, Sarcopenia, Ageing
P3.8 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 b2-Adrenergic agonist suppresses proteasome activity, autophagic flux and atrogenes expression in skeletal muscle from fasted mice Gonc¸alves Dawit AP1, Manfredi Leandro H1, Grac¸a Fla´via A1, Silveira Wilian A1, Heck Lilian C1, Bertaggia Enrico2, Sandri Marco3, Kettelhut Isis C4, Navegantes Luiz CC1 1
Physiology, Ribeira˜o Preto Medical School, USP, Brazil; 2Venetian Institute of Molecular Medicine- Padua, Italy; 3Venetian Institute of Molecular Medicine, Padua, Italy; 4Biochemistry/Immunology and Physiology, Ribeira˜o Preto Medical School, USP, Brazil It has been shown that b2-adrenergic agonists (BAA) are able to promote muscle growth and mitigate muscle wasting in several atrophic conditions. However, the intracellular mechanisms of action of BAA are yet unclear. Therefore, the aim of this work was to investigate whether Formoterol (FOR), a highly selective BAA, could regulate proteasomal (UPS) and autophagic/lysosomal proteolysis, the two major proteolytic systems involved in muscle atrophy. For that, 2-day fasted mice received a single injection of FOR (300 mg.kg-1; sc) or saline and then were euthanized after 1, 2 and 4 h. FOR suppressed (*40 %) the mRNA levels of the atrophy-related Ub-ligases Atrogin-1 and MuRF1 and the autophagy gene Gabarapl1 4 h after injection, as measured by qPCR. For the evaluation of proteasome activity, tibialis anterior muscles were transfected with the mutant ubiquitin UbG76 VGFP and, 7 days later, mice were treated with FOR or saline. UbG76VGFP is targeted to proteasomal degradation, and the GFP fluorescence is detected by microscopy in a very low level in transfected myofibers from saline-treated fasted mice, but is highly expressed when proteasome function is inhibited by FOR peaking at 2 h after injection. Autophagosome formation, in turn, was measured by the conversion of LC3-I to LC3-II by immunoblot. At 1 h, FOR reduced by 80 % LC3-II/ LC3-I ratio in muscles from fasted mice. In order to measure autophagic flux, mice were concomitantly fasted and treated for 2 days with
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J Muscle Res Cell Motil (2015) 36:71–143 colchicine (COL; 0.4 mg.kg-1; ip), an agent that inhibits autophagosome-lysosome fusion. Again, FOR reduced LC3-II/LC3-I ratio in COL-treated mice indicating that BAA indeed suppressed the delivery of substrate to lysosome. Taken together, our data show that BAA counteracts muscle protein catabolism induced by food deprivation, which might alleviate muscle atrophy, by down-regulating UPS and autophagy/lysosome proteolytic systems. Supported by FAPESP (2012/18861-0 and 2012/24524-6) Keywords: Formoterol, Atrogin-1 and Murf1, Lysosome
P3.9 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Phosphorylation/O-GlcNAcylation interplay in the modulation of protein synthesis: focus on the IGF-1 pathway in differentiated C2C12 myotubes Deracinois Barbara1, Dupont Erwan1, Richard Elodie2, Lambert Matthias1, Bastide Bruno1, Cieniewski-Bernard Caroline1 EA4488 – University of Lille 1, France; 2BioImaging Center of Lille – University of Lille 1, France 1
Muscle atrophy corresponds to a decrease in skeletal muscle mass resulting from the dysregulation of the equilibrium between protein synthesis and degradation. As described in many other acquired disorders, a dysregulation of cellular processes is implicated in skeletal muscle atrophy. Among them, the PI3 K/Akt/mTOR pathway, activated through IGF-1, is largely involved in the development of muscle atrophy. Since phosphorylation has been recognized as a major player in cell signalling, several actual research programs concern the study of the deregulation of phosphorylation process on intracellular pathways. However, the characterization of the impact of O-linked-N-acetyl-D-glucosaminylation (termed O-GlcNAcylation) dramatically lacked in these research strategies. Indeed, this atypical glycosylation, highly dynamic and reversible such as phosphorylation, is involved in the modulation of several intracellular signalling pathways. Nevertheless, its role on the modulation of protein neosynthesis through the IGF-1 pathway was never considered to date. The aim of our project is to characterize the role of the interplay between phosphorylation and O-GlcNAcylation in the cellular regulation of protein synthesis in a skeletal muscle cell line. In particular, O-GlcNAcylation level was increased on C2C12 myotubes using Thiamet G (a specific inhibitor of O-GlcNAcase the enzyme responsible of O-GlcNAc removal) alone or concomitantly with IGF1. We evaluated the impact of IGF-1 and Thiamet G, alone or together, on protein neosynthesis through fluorescent labelling of nascent proteins. We identified several key signalling proteins bearing an O-GlcNAc moiety, and quantified the interplay between phosphorylation and O-GlcNAcylation by immunoblot on key signalling proteins. Interestingly, we showed that O-GlcNAcylation can influence the phosphorylation pattern. Data obtained from this project will provide new insights in the knowledge of muscle atrophy syndrome. Keywords: O-GlcNAcylation, Protein synthesis, IGF1 pathway
P3.10 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Whey protein supplementation attenuates the reduction in muscle oxidative capacity during 21 days bed rest Bosutti Alessandra1,2, Blottner Dieter3,4, Salanova Michele4, Rittweger Joern5, Degens Hans2
J Muscle Res Cell Motil (2015) 36:71–143 1
Department of Scienze Mediche, Chirurgiche e della Salute, University of Trieste, Italy; 2School of Healthcare Science, Manchester Metropolitan University, UK; 3Charite´ Universita¨tsmedizin Berlin, Vegetative Anatomy, Germany; 4Center of Space Medicine Berlin (ZWMB), Germany; 5Institute of Aerospace Medicine, DLR-German Aerospace Center, Cologne, Germany Muscle unloading, as encountered during space flight and disuse, induces skeletal muscle atrophy and weakness, switch of fibre types and loss of capillaries. Less attention has been given to disuse-induced changes in oxidative capacity, which may affect endurance. Here we investigated the impact of 21 days, 60 head-down bed rest on muscle fibre size, fibre type composition, capillarisation and muscle oxidative capacity and the efficacy of milk-based proteins (whey protein) to counteract disuse-induced muscle changes. Muscle biopsies were taken from the soleus (SOL) and vastus lateralis (VL) muscles before and after bed rest from 10 healthy men (aged 31 ± 6 years). Subjects were assigned to two groups and studied twice in two follow-up campaigns, in once receiving whey protein and once receiving a standardized isocaloric diet. Fibre types and capillaries were identified by immunohistochemical staining of muscle section with anti-myosin type1 and lectin. Analysis was done with the method of capillary domains. Oxidative capacity of individual fibres was determined as the optical density (OD) at 660 nm of succinate dehydrogenase (SDH) stained histological sections. The fibre cross-sectional area times the SDH-OD of that fibre (integrated SDH) was index of maximal oxygen consumption of that fibre (VO2max). Statistical analysis was done by repeated measures ANOVA. Bed rest did not induce significant changes in fibre type composition, fibre size or capillarisation, but decreased SDH activity (p \ 0.001) and fibre VO2max (p \ 0.01) in both muscles. Whey protein partially counteracted the decrease in oxidative capacity. Our results suggest that decrements in oxidative capacity may be an early hallmark of disuse-induced muscle adaptations and occurs even in the absence of significant fibre atrophy and capillary rarefaction. The Whey diet attenuated these effects and may thus be an effective supplement to sustain oxidative capacity during space flight and long-term bed rest. Keywords: Bed rest, Muscle disuse, Muslce oxidative capacity
P3.11 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Exercise training promote a decrease in catabolic changes but did not change anabolic factors in plantaris muscle of old rats after immobilization-induced atrophy ***Candidate for Young Investigator Award
Vechetti-Junior Ivan J***, Bertaglia RS, Paula TG, Dal-Pai-Silva M Department of Morphology, Sao Paulo State University, Brazil Growth and maintenance of skeletal muscle mass is critical for longterm health and quality of life. Furthermore, the mechanisms involved in the control of muscle mass in aging has heightened interest in trying to understand the mechanisms responsible for prevent the loss of muscle mass. In addition, the exercise represent an appropriate therapeutic intervention in the muscle recovery process in aging. The goal of this study was to examine the morphological and molecular effects of immobilization (7 days) followed of muscle re-growth with exercise training in old rats (18 months). The animals underwent to the regrowth process with (aerobic or resistance training) or without exercise
113 during 3 or 7 days. At the end of the experiment, the animals were sacrificed and plantaris muscle removed and subsequently subjected to morphological, histochemical, biochemical and molecular analyzes. The animals were kept in the same condition throughout the experiment (body weight and body fat) and showed a muscle atrophy after 7 days of immobilization (15 % by muscle weight, 9 % muscle-to-tibia length ratio, 31 % by total protein concentration and 19 % muscle fiber cross sectional area). After 3 days of muscle re-growth, none of the animals undergoing the atrophy process regained muscle mass in any of the parameters analyzed. After 7 days of recovery, only the groups submitted to both exercise regained muscle mass. In addition, rats submitted to immobilization-induced atrophy presented an increase in mRNA of catabolic factors MAFbx, MuRF1 and FoxO1 with no changes in anabolic factors (AKT, mTOR, p70s6k). After 7 days of regrowth, all animals submitted to both exercise showed a decrease in the catabolic factors expression. Interestingly animals that were not subjected to exercise remained a high level of catabolic factors expression at the end of the experimental period. Our results showed that both aerobic or resistance training enhances muscle recovery during muscle atrophy in old rats. Keywords: Exercise training, Muscle atrophy, Muscle re-growth
P3.12 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Fragmentation of the aged NMJ is associated with alterations in the agrin-MuSK signalling pathway Elkrief Daren1, Sally Spendiff1, Gilles Gouspillou2, Madhusadanrao Vuda1, Russell Hepple1 1
Kinesiology and Physical Education, McGill University, Montre´al, Canada; 2Kinanthropologie, Universite´ du Que´bec a` Montre´al, Canada The loss of muscle mass and strength with aging is a major contributor to loss of health in old age. Muscle denervation is particularly important at clinically relevant ages when muscle atrophy is most severe (Rowan et al. PLoS One 7(1):e29082, 2012), but the mechanisms remain unknown. The Agrin/MuSK pathway is essential for maintenance of the adult neuromuscular junction (NMJ) and itsdysfunction contributes to diseases like myasthenia gravis (Ruegg et al. 2012). In particular, agrin binds to the LRP4 receptor to activate (phosphorylate) MuSK, subsequently recruiting rapsyn, allowing the formation of the rapsyn/acetylcholine receptor (AChR) complex on the cytoskeleton to maintain AChRs in a ‘‘pretzel-like’’ configuration. Although mice which transgenically over-express the endogenous protease of agrin, neurotrypsin, recapitulate many key features seen in normally aging muscle (Butikofer et al. FASEB J (12):4378-93, 2011), changes in this pathway with normal aging are unknown. As such, we examined this issue in immunolabeled cross-sections of vastus lateralis (VL) muscle from young adult (YA) and very old (VO) Fisher 3449 Brown Norway F1-hybrid rats. We also extracted RNA from VL muscle to examine transcripts in this pathway. We observed characteristic fragmentation of the AChR clusters in VO animals (1.33 ± 0.11 versus 1.69 ± 0.15 [mean ± SD] fragments in YA vs VO, respectively), mirrored by fragmentation of the Musk and Rapsyn localization at the NMJ. In addition, we found lower levels of MuSK and Rapsyn at the NMJ in VO muscle, consistent with alterations in this pathway contributing to AChR cluster fragmentation in aging muscle. Interestingly, transcripts of MuSK, Rapsyn, and AChR subunits a, b, and the fetal isoform c were up-regulated in very old muscle, likely as an attempted compensation for unstable NMJs in aging muscle. Collectively, our data show that fragmentation of the
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114 aged NMJ is associated with a loss of key proteins that regulate the stability of the AChR cluster. Keywords: Sarcopenia, Agrin/MuSK pathway, Denervation
P3.13 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Metabolic alterations in the hypermuscular Compact mice Kocsis Tama´s, Julia Baan, Mendler Luca, Dux La´szlo´, KellerPinte´r Aniko´ Department of Biochemistry, Faculty of General Medicine, University of Szeged, Hungary The Compact mice carry a naturally occurring 12-bp deletion in the propeptide region of the myostatin precursor and additional modifier genes are responsible to determine the hypermuscular phenotype. Myostatin as a potent negative regulator of the skeletal muscle mass influences Smad2/3 and PI3 K/Akt signaling. The PI3K/Akt pathway plays a central role in integrating of catabolic and anabolic responses; therefore, we aimed to investigate the metabolic effects of the Compact mutation. We showed that the absolute weight of the investigated hindlimb muscles and liver significantly increased in Compact animals compared to wild-type; however, the muscle weight/body weight ratio significantly increased and the liver weight/body weight ratio significantly decreased in Compacts. The total glycogen and protein amount increased in Compact M. tibialis anterior, M. quadriceps femoris, M. gastrocnemius and liver samples. Western blot analysis revealed that the mutation resulted in increased level of phosphoAkt(Ser473) in both muscle and liver samples despite the presence of Compact myostatin, indicating the absence/reduced effect of myostatin. The glucose tolerance test revealed increased glucose uptake in peripheral tissues of Compacts, and the hepatic gluconeogenesis decreased determined by pyruvate tolerance test. To further assess liver function the activity of alanine transaminase (ALT) was measured in vitro, and the total liver ALT activity to body mass ratio significantly decreased in Compact animals. In conclusion, the Compact mutation results in systemic metabolic alterations; it has opposite effect on the relative weight and glycogen concentration of skeletal muscle and liver, influences liver function and increases the peripheral glucose uptake. ´ MOP-4.2.2.A-11/1/KONV-2012This research was supported by TA ´ MOP-4.2.4.A/2-11/1-2012-0001. 0035 and TA Keywords: Compact, Myostatin, Glycogen
P3.14 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 MicroRNA-23a has minimal effect on endurance exercise-induced adaptation of mouse skeletal muscle Wada Shogo1, Kato Yoshio2, Sawada Shuji3,6, Aizawa Katsuji1, Park Jong-Hoon1, Russell Aaron P4, Ushida Takashi1, Akimoto Takayuki5 1 Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Japan; 2National Institute of Advanced Industrial Science and Technology (AIST), Japan; 3Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology, Japan; 4Center for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Australia; 5Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Japan; 6Waseda University, Japan
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J Muscle Res Cell Motil (2015) 36:71–143 Skeletal muscles contain several subtypes of myofibers that differ in contractile and metabolic properties. Transcriptional control of fiber type specification and adaptation has been intensively investigated over the past several decades. Recently, microRNA (miRNA) mediated posttranscriptional gene regulation has attracted increasing attention. MiR23a targets key molecules regulating contractile and metabolic properties of skeletal muscle, such as myosin heavy chains and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1a). In the present study, we analyzed the skeletal muscle phenotype of miR23a transgenic (miR-23a Tg) mice to explore whether forced expression of miR-23a affects markers of mitochondrial content, muscle fiber composition and muscle adaptations induced by 4-weeks of voluntary wheel running. When compared with wild type mice, protein markers of mitochondrial content, including PGC-1a, and cytochrome c oxidase complex IV (COX IV), were significantly decreased in the slow soleus muscle, but not the fast plantaris muscle of miR-23a Tg mice. There was a decrease in type IId/x fibers only in the soleus muscle of the Tg mice. Following 4 weeks of voluntary wheel running there was no difference in the endurance exercise capacity as well as in several muscle adaptive responses including an increase in muscle mass, capillary density or the protein content of myosin heavy chain IIa, PGC-1a, COX IV and cytochrome c. These results show that miR-23a targets PGC-1a and regulates basal metabolic properties of slow, but not fast twitch muscles. Elevated levels of miR-23a did not impact on whole-body endurance capacity or exercise-induced muscle adaptations in the fast plantaris muscle. Keywords: microRNA, Exercise performance, Muscle fiber-type
P3.15 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Slow-to-fast myosin shift in soleus muscle during mechanical unloading. Involvement of nuclear MuRFs and calsarcin expression Shenkman Boris, Lomonosova Yulia Laboratory of Myology, SSC RF Institute of Bio-Medical Problems, RAS, Moscow, Russia Exposure to actual and simulated microgravity is known to induce decrease in expression of slow MyHC mRNA and increase in expression of fast MyHC mRNAs. We supposed that altered expression of the calsarcin (CS) I and II (specific for type I and type II fibers respectively) may provide the control over myosin phenotype during unloading. We found that after 3 days of hindlimb unloading (HU) the content of CSII mRNA increases two-fold in rat soleus as compared to the cage controls. This level was maintained till the 7th day of the exposure and increased by more than 5-fold (as compared to controls) after two weeks of HU. In contrast to CSII, CSI mRNA expression didn’t change after 3 days of HU, but decreased more than 2-fold by the 7th and 14th day of HU. The increase of CSII RNA (in type II fibers) may be explained as the mechanism of stabilization of fast phenotype in all, but more important, in newly transformed type II fibers. At the same time, the decrease of CSI mRNA (in type I fibers) may be understood as counteracting the slow-to-fast transformation. Morriscot et al. (J Struct Biol 170(2):344–353, 2010) demonstrated that calsarcin II expression decreased only in the double knockouts MuRF1-/MuRF2-. So, we hypothesized that increase of CSII mRNA content in unloaded soleus muscle might be associated with the cytoplasm-nucleus translocation of MuRF1 and MuRF2. We observed more than 4-fold accumulation of MuRF1 and 80 % accumulation of MuRF2 in the nuclear fraction (western blotting) after 3 days of HU. Thus the accumulation of MuRFs in myonuclei may
J Muscle Res Cell Motil (2015) 36:71–143 promote the expression of CSII, necessary for stabilization of fast phenotype in the course of slow-to-fast shift in unloaded soleus muscle. We express our gratitude to Prof. S. Labeit (Mannheim) for kind presenting the best antibodies against MuRF1 and MuRF2. Keywords: Unloading, MyHC, MuRf E3-ligases
P3.16 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Effects of functional focused strength training on muscle function and strength in the elderly Danter Daniel***, Ko¨sters Alexander ***Candidate for Young Investigator Award
Department of Sport Science and Kinesiology, University Salzburg, Austria With a rapidly growing older population, there has been great interest in developing approaches to counteract the effects of sarcopenia, and thereby reduce widespread clinical implications. It has turned out that strength training is indeed an effective method to increase muscle mass, strength and functional performance in frail elderly individuals, but unfortunately this population with the greatest need of training machines and equipment have the least opportunity to use such tools. Therefore, methods that are not costly and adapted for older people’s needs are required. The aim of this study was to investigate the effects of a functional focused strength training (FFST) orientated at the activities of the daily living on the mobility and strength of senior citizens over 70 years. 18 seniors participants, aged 70 + years, were randomly assigned to either training group (TG, N = 10, age: 77.0 ± 7.04) or control group (CG, N = 8, age: 79.38 ± 6.63). The training was held 2 times per week, 45 min per session, for the period of 10 weeks. The CG had no training. The mobility was assessed with the Short Physical Performance Test Battery (SPPB) and the Timed Up and Go (TUG) test. The maximum strength was determined with the one repetition maximum (1RM) during the free squat. A two way ANOVA with repeated measurements revealed that the TG improved highly significant (p = 0.004) compared to the CG in the SPPB. In the TUG was no such improvement (p = 0.093). At the 1RM the TG also improved significantly (p = 0.016) compared to the CG. The results suggest that a FFST can improve the mobility and the maximum strength of seniors and is therefore an effective and elderly friendly method to counteract the effects of sarcopenia. Keywords: Aging, Sarcopenia, Strength training
P3.17 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Phospho-GlcNAc modulation of MLC2 is different in slowand fast twitch muscle during treadmill running Hortemo Kristin Halvorsen1,2***, Aronsen Jan Magnus1,2,3, Sjaastad Ivar1,2, Lunde Per Kristian1,2, Sejersted Ole M1,2 ***Candidate for Young Investigator Award
1
Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Norway; 2KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Norway; 3Bjørknes College, Oslo, Norway
115 Myosin light chain 2 (MLC2) is a small regulatory protein located at the neck of the myosin complex. We have previously reported dephosphorylation of slow isoform of MLC2 (sMLC2) in soleus muscle during in situ shortening contractions. Here, we investigate the effects of in vivo treadmill running on phosphorylation of sMLC2 in soleus and fast isoform of MLC2 (fMLC2) in extensor digitorum longus (EDL), and if there are reciprocal effects on O-GlcNAcylation of MLC2. Healthy rats performed a fatiguing exercise protocol with incremental running speed, and after 30 min running soleus and EDL were immediately harvested and snap-frozen. Analysis of myofilament proteins revealed a striking dephosphorylation of sMLC2 in soleus after running compared to resting control, while phosphorylation of fMLC2 in EDL remained high. Enzymes regulating phosphorylation of MLC2 was quantified, and Myosin protein phosphatase regulatory subunit (MYPT2) was abundantly expressed in soleus but not in EDL, while Myosin light chain kinase 2 (MLCK2) was highly expressed in EDL compared to soleus, fitting with the higher phosphorylation level of MLC2 in EDL. O-GlcNAcylation of MLC2 was also higher in EDL compared to soleus, accompanied by lower expression of O-GlcNAcase, the enzyme reversibly removing GlcNAc. We did not detect changes in O-GlcNAcylation of MLC2 after running, this could be due to low stoichiometry of O-GlcNAc especially in soleus, but suggests that sMLC2 can be dephosphorylated without a reciprocal increase in O-GlcNAcylation. In contrast to fMLC2 in EDL, sMLC2 in soleus is profoundly dephosphorylated during exercise, but we did not detect significant changes in O-GlcNAcylation of either isoform. Keywords: Myosin light chain 2 (MLC2), Protein phosphorylation, Protein O-glcnacylation, Treadmill running
P3.18 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Skeletal muscle function and serum thiobarbituric acid reactive substances level are improved by the combination treatment of catechin-ingestion with heat stress in healthy elderly women Yoshioka Toshitada1, Oda Hideshi2, Ota Noriyasu3, Watanabe Atsuya4, Kitazawa Hidefumi3, Igaki Michihito2, Suzuki Atsushi2, Shimotoyodome Akira3, Hase Tadashi3, Sugiura Takao5, Goto Katsumasa6 1
Hirosaki Gakuin University, Japan; 2Personal Health Care Products Research Laboratories, KAO Corporation, Japan; 3Biological Science Research Laboratories, KAO Corporation, Japan; 4Department of Orthopeadic Surgery, Teikyo University Chiba Medical Center, Japan; 5Department of Exercise and Health Sciences, Yamaguchi University, Japan; 6Department of Physiology, Graduate School of Health Sciences, Toyohashi SOZO University, Japan
Catechin has not only antioxidant activity but also anti-atrophic effects on unloading-induced muscle atrophy. On the other hand, heat stress, one of hypertrophic stimuli for skeletal muscle, induces skeletal muscle hypertrophy in aged animal. However, there was no evidence showing that effects of catechin-ingestion and/or heat stress on skeletal muscle in elderly people. In this study, we investigated the effects of catechin and/or heat treatment on the volume in quadriceps muscles and isometric force of knee extension in healthy elderly women. Subjects (71.0 ± 0.7 years old, n = 27) were divided into 3 groups; 1) catechin-ingested (CAT), 2) heat-stressed (Heat), and heat stress with catechin-ingested groups (CAT + Heat). Subjects in both CAT and CAT + Heat groups daily ingested 540 mg of green tea catechin in one hour for 10 weeks. Heat stress was applied for the quadriceps muscles for 8 h a day and 4 days a week by using two
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116 heat- and steam-generating sheets. In CAT + Heat group, maximal isometric force and muscle volume were significantly increased by 10-week of treatment, but not in CAT and Heat groups. Time of TUG in both CAT and CAT + Heat groups was significantly improved by the treatment. Following 10-week of the treatment, the level of serum thiobarbituric acid reactive substances (TBARS), an oxidative stress marker, in both CAT and CAT + Heat groups was significantly decreased, but not in Heat group. Evidences suggest that cathechin ingestion with heat stress might improve impaired muscle function of elderly women. This study was supported, in part, by KAKENHI (22240071, 24650407, 24650411, 26350818, 26560372) from Japan Society for the Promotion of Science, the Science Research Promotion Fund from The Promotion and Mutual Aid Corporation for Private Schools of Japan, and KAO Corporation. Keywords: Sarcopenia, Oxidative stress, Countermeasure
P3.19 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Prolonged exhaustive exercise induces diastolic dysfunction with preserved systolic function mediated by oxidative stress Kleindienst Adrien1, Boissie`re Julien2, Nottin Ste´phane1, Gayrard Sandrine1, Lacampagne Alain2, Reboul Cyril1, Cazorla Olivier2 1 Universite´ d’Avignon, France; 2INSERM U1046 Montpellier, France
Intense physical practices like marathon or triathlon result in transient left ventricular (LV) dysfunction, characterized by transient reduction of LV diastolic relaxation without changes in systolic function by unknown mechanisms. Our study aimed to investigate in vivo and in vitro the involvement of known regulators of diastolic function such as eNOS and oxidative stress signalling pathways in a rat model. Wistar rats experienced prolonged exhaustive exercise (PEE) (3 h; 65 % of maximal aerobic velocity), in association or not with an antioxidant treatment (Nacetylcysteine, 50 mg.kg-1, i.p.). Systolic cardiac function evaluated in vivo by echocardiography and ex vivo in Langendorff isolated heart was unchanged by PEE, while parameters of diastolic function were severely altered. At the cellular level, PEE had no effect on myofilament calcium sensitivity, TnI expression and phosphorylation. However, PEE was associated with marked alteration of calcium handling during contraction. The amplitude of calcium transient was reduced in PEE hearts when compared to Ctrl ones and both Tau and time to baseline50 (TTBl50), two indexes reflecting the kinetic of Ca2+ reuptake by the SR were increased by PEE. Finally, an interesting point is that antioxidant treatment with NAC during PEE totally prevents the alteration of the diastolic function. To conclude, the present results show that PEE induced oxidative stress that targeted preferentially calcium handling rather than myofilaments, and may be responsible for transient diastolic dysfunction.
J Muscle Res Cell Motil (2015) 36:71–143 1
Muscle Physiology & Mechanics Group, DPESS, University of Thessaly, Greece; 2DPESS, University of Thessaly, Greece; 3 Department of Nephrology, Faculty of Medicine, University of Thessaly, Greece; 4Department of Surgery, Faculty of Medicine, University of Thessaly, Greece Uremia affects skeletal muscle structure and function leading to muscle atrophy, muscle weakness, metabolic disorders, diminished exercise capacity and fatigue. Complex mechanisms that stimulate muscle dysfunction have been proposed, and oxidative stress may be implicated. The aim of this study was to evaluate the effects of uremia on muscle redox status in rabbit model of renal insufficiency. Psoas and soleus muscle samples from nephrectomised NZ rabbits were harvested and homogenized according to institutional ethics approval. Samples were stored at -80 C until analyzed for Glutathione Reduced (GSH), Glutathione Oxidized (GSSG), GSH:GSSG ratio, Catalase Activity (CAT), Protein Carbonyls (PC), Thiobarbituric Acid Reactive (TBARS) and Total Antioxidant Capacity (TAC). Uremic (UREM) psoas and soleus had reduced levels of GSH by 31.1 and 8 % vs sham-operated control (CON). Compared to CON, CAT activity was decreased by 24.5 and 5.23 % in UREM psoas and soleus respectively. In other indices responses were not in the same direction for both UREM muscle types, e.g. compared to CON, PC levels were lower in uremic psoas by 45 % and higher in soleus by 5.94 %, as with TBARS (74 % lower in psoas, 89 % higher in soleus). In conclusion, there is evidence for muscle redox status changes in uremia. Further work is underway to clarify the meaning of these changes and the possible contribution of oxidative stress on muscle functionality. Acknowledgments. This research has been co-financed by the European Union (European Social Fund—ESF) and Greek national funds through the Operational Program ‘‘Educational and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF)— Research Funding Program: Thales (MuscleFun Project-MIS 377260) Investing in knowledge society through the European Social Fund. Keywords: Oxidative stress, Chronic disease, Animal model
P3.21 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Single muscle fibre analysis of proteins with roles in glycogen metabolism in skeletal muscle from trained cyclists following varying bouts of exercise Frankish Barnaby1***, Lane Stephen2, Areta Jose2, Stapleton David3, Hawley John2, Murphy Robyn1 ***Candidate for Young Investigator Award
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Department of Zoolgy, La Trobe University, Melbourne, Australia; Exercise & Nutrition Group, School of Medical Sciences, RMIT University, Melbourne, Australia; 3Department of Physiology, University of Melbourne, Australia
Keywords: Diastolic dysfunction, Exercise, Oxidative stress
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P3.20 Muscle plasticity, exercise, and aging, Fri 12:00–14:00
Background: Glycogen is an important fuel source, providing energy for contracting skeletal muscle. Skeletal muscle is heterogeneous in nature, comprised of slow-twitch (Type I) and fast-twitch (Type II) fibres, which are distinct in their metabolic and contractile properties. This study investigated the affect of varying glycogen availability upon specific glycogen related proteins, in response to different bouts of exercise. Methods: Eight endurance-trained cyclists performed two experimental trials (Low and High glycogen), where glycogen concentration was manipulated via exercise-diet interventions. Each experimental trial lasted 15 h including overnight sleep, consisting of
The effects of uremia on rabbit skeletal muscle redox status Poulianiti Konstantina P1***, Karioti Aggeliki1, Kaltsatou Antonia1, Jamurtas Athanasios2, Mitrou Georgia I1, Stefanidis Ioannis3, Tepetes Konstantinos4, Christodoulidis Gregory4, Koutedakis Yiannis2, Sakkas Georgios K1, Karatzaferi Christina1 ***Candidate for Young Investigator Award
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a high-intensity interval training (HIT, evening of day 1) and aerobicintensity training (AIT, morning of day 2) bout of exercise. Muscle biopsies from the vastus lateralis were collected Pre- and Post- each exercise bout using the Bergstrom biopsy technique. Individual muscle fibres were collected under paraffin oil and prepared for western blotting analysis. Results: Following HIT bout of exercise, there was a fibre type- and trial- specific decrease in glycogen synthase and glycogen phosphorylase, with concomitant increases in their phosphorylated forms. No changes in glycogen branching or glycogen debranching enzymes were seen following HIT in either Low or High glycogen. AIT did not result in any changes seen for those proteins in either the Low or High glycogen trials. Conclusion: Glycogen availability influences the expression of various glycogen related proteins, and does so in a fibre specific manner. These findings highlight the complexity of glycogen metabolism and that fibre type needs to be considered when understanding its regulation. Analyses conducted in whole muscle should therefore be interpreted with caution.
type fiber diameter was observed either in ES or LP trained subjects (p \ 0.001). Altogether these results demonstrate the effectiveness of ES as a safe home-based method to counteract fast type fiber atrophy associated with ageing and to improve the performances of ageing muscles.
Keywords: Exercise, Glycogen, Metabolism
1
Keywords: Elderlies, Electrical Stimulation, Mobility
P3.23 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Exercise-induced fibre type transformation is impaired in humans with a single nucleotide polymorphism in the PGC1a gene Steinbacher Peter1, Scho¨nauer Anna-Lena1, Reinhardt Sandra1, Stoiber Walter1, Sa¨nger Alexandra Maria1, Fo¨rster Holger2, Feichtinger Rene´ G3, Paulweber Bernhard4, Ring-Dimitriou Susanne5 Department of Cell Biology, University of Salzburg, Austria; Physician, Salzburg, Austria; 3Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria; 4Department of Internal Medicine, Paracelsus Medical University, Salzburg, Austria; 5 Department of Sport Science and Kinesiology, University of Salzburg, Austria 2
P3.22 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Physical exercise in Aging: results of 9 weeks of electrical stimulation or leg press trainings in 70 years old sedentary elderlies Zampieri Sandra1,3, Mosole Simone2, Lo¨fler Stefan3, Fruhmann Hannah3, Musaro` Antonio4, Sandri Marco5; Cvecˇka Ja´n6, Sedliak Milan7, Sˇarabon Nejc7, Carraro Ugo2, Kern Helmut3 1
Department of Biomedical Sciences, Padova, Italy; 2Department of Biomedical Sciences, Padova, Italy; 3Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Vienna, Austria; 4 DAHFMO-Unit of Histology and Medical Embryology, IIM, Sapienza University of Rome, Italy; 5Department of Biomedical Sciences, Padova, Italy; Venetian Institute of Molecular Medicine, Dulbecco Telethon Institute, Italy; 6Faculty of Physical Education and Sport, Comenius University, Bratislava, Slovakia; 7University of Primorska, Science and Research Centre, Institute for Kinesiological Research, Koper, Slovenia A greater understanding of the mechanisms leading to impaired muscle function during ageing is of central importance for prevention of disability and for optimization of independence of the elderlies. Our recent results from a peculiar group of well-trained seniors that exercised regularly in their previous 30–40 years, suggest that lifelong physical exercise have beneficial effects on age related decay not only affecting muscle trophism and phenotype, but also counteracting denervation atrophy by promoting reinnervation. Based on this findings, 70 years old seniors with normal life style were recruited (n = 26) and trained for 9 weeks either with electrical stimulation (ES) or leg press (LP). Before and after training, all the subjects were submitted to mobility functional tests and muscle biopsies from Vastus Lateralis of both legs. No signs of degeneration, and/or of inflammation were observed in muscle biopsies after the training. Functional tests show that ES was able to significantly improve muscle torque and functional performances of seniors and morphometrical and immunofluorescent analyses performed on muscle biopsies show that ES significantly increased the size of fast type muscle fibers (p \ 0.001), together with a significant increase of the number of Pax7 and NCAM positive satellite cells (p \ 0.005). Signaling pathways controlling muscle mass and satellite cells activation were accordingly modulated. A significant decrease of slow
PGC1a (peroxisome proliferator-activated receptor c co-activator 1a) is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP) in the PGC1a gene affects insulin sensitivity, blood lipid metabolism, and muscle parameters such as myofibrillar structure and mitochondrial oxidative capacity. Individuals carrying this SNP were shown to have a reduced physical fitness and a higher risk for type 2 diabetes. Following the hypothesis that such persons should also have a reduced exercise trainability, we investigated the responses of sedentary men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 9 60 min/week, HR@70–90 % VO2peak). Results show that persons carrying the Gly482Ser SNP had a diminished exercise effect at the sub-maximal performance level compared to the control group. Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a traininginduced increase in content of slow contracting oxidative fibres. Capillary supply and mitochondrial density increased similarly in both groups. Keywords: Endurance exercise, PGC1alpha, Mitochondria
P3.24 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Unacylated Ghrelin inhibits dexamethasone-induced skeletal muscle atrophy in mice Agoni Valentina1, Minetto Marco2, Bottinelli Roberto1, Canepari Monica1 1
Department of Molecular Medicine, University of Pavia, Italy; Department of Internal Medicine, Division of Endocrinology, Diabetology and Metabolism, Molinette Hospital, University of Turin, Italy
2
To understand the triggering mechanisms of steroid myopathy we studied the adaptations to dexamethasone (DEX) administration
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118 (5 mg/Kg day) in vastus lateralis (VL) and soleus (Sol) muscles of adult mice at different times (1, 3 h and 3, 7 and 15 days). It has been recently demonstrated that the unacylated form of ghrelin, a peptide hormone that stimulates GH release, is able to counteract dexamethasone effects on cultured myoblasts. Therefore, the effects of administration of unacylated ghrelin (UAG, 100ug/Kg day) during the onset of steroid myopathy in mice was also evaluated. Significant fibers atrophy in VL was found after 7 (13 %) and 15 days (31 %) of DEX treatment, whereas in Sol atrophy (33 %) was found only after 15 days of DEX treatment. UAG administration blunted the atrophy observed after 15 days of DEX treatment: 11 and 23 % lower atrophy in VL and SOL respectively. Significant up-regulation of MuRF-1 was found after 1, 3 and 10 h of DEX treatment in VL and after 1 h in Sol. A significant up-regulation of myostatin was found after 1, 3 and 10 h of DEX treatment in both VL and Sol. In both VL and Sol UAG prevented MuRF-1 and myostatin up-regulation after 1 and 3 h of DEX treatment, but failed to prevent MuRF-1 and myostatin induction after 10 h of DEX treatment. Results suggest that: (a) in the early phases of steroid myopathy atrophy is supported by the activation of the ubiquitin–proteasome system and by the induction of myostatin; (b) UAG exhibits anti-catabolic effects capable to counteract steroid atrophy; (c) UAG has short-term effects. Keywords: Steroid myopathy, Skeletal muscle, Atrophy
P3.25 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Effect of colon carcinoma, swimming and high-fat mixed lipid diet on the muscle fibre type transitions in skeletal muscles of rats Smerdu Vika1, Persˇe Martina2 1 Institute of Anatomy, Faculty of Medicine, University in Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia; 2Medical Experimental Centre, Institute of Pathology, Faculty of Medicine, University in Ljubljana, Zalosˇka 4, 1000 Ljubljana, Slovenia
The functional properties of skeletal muscle fibres are substantially determined by myosin heavy chain (MyHC) isoforms, whose expression can be modulated by altered physiological and pathological conditions. We studied the effect of experimentally induced colon carcinoma with 1,2-dimethylhydrazine (DMH), 21-week swimming and high-fat mixed lipid diet (HFD) on fibre type transitions by immunohistochemical analysis of MyHC expression in soleus (SOL), extensor digitorum longus (EDL), deep-red and superficial-white portion of gastrocnemius medialis (GMr, GMw) and lateralis (GLr, GLw) muscles of rat swimmers and non-swimmers, fed either with HFD or low-fat corn oil diet (LFD). The effect of DMH treatment was studied by comparison of MyHC expression in DMH-treated and untreated non-swimmers, fed with LFD. Although all three factors mostly induced non-significant changes in MyHC expression, the tendency of fibre type transitions was evaluated. DMH treatment induced moderate transitions from slow to fast fibre types in all muscles except SOL and GLr, in which transitions proceeded in the opposite direction. Further, we demonstrated that the paradigm about fast to slow fibre type transition after endurance exercise obviously does not hold true for all muscles. Namely, the transitions from fast to slow fibre types were found in some fast muscles (GMr, GMw, GLw) except EDL and GLr in which bi-directional transitions with elevated number of hybrid fibres were noticed, but in slow SOL the transitions from slow towards fast fibre types were induced. The HFD diet resulted in transitions either partly (EDL, GMw, GLw) or completely opposite to those induced by swimming (SOL, GMr), but in GLr the effect was the same as that of swimming. However, the extent of fibre
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J Muscle Res Cell Motil (2015) 36:71–143 type transitions differed among the muscles and was probably related to the original, i.e. genetically determined muscle fibre type composition and to the muscle functional role in swimming as well. Keywords: Skeletal muscle, MyHC, Rat
P3.26 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Effect of nifedipin administration on proteolytical events in rat soleus muscle under 3-day of hindlimb suspension Belova Svetlana1, Lomonosova Yulia1, Shenkman Boris1, Nemirovskaya Tatiana2 1
Lab Myology, Institute for Bio-Medical Problems, RAS, Moscow, Russia; 2Faculty of Basic Medicine, Lomonosov Moscow State University, Russia We tested the hypothesis that prevention of cytoplasmic calcium influx via L-calcium channels during 3 days of rat hindlimb suspension could lead to decrease of l-calpain level, and calcium leakage to cytoplasm could serve a triggering mechanism, which activated the work of ubiquitin-proteosomic pathway in rat soleus. It have been demonstrated earlier that L-channels blocking diminished the calcium level in muscle fibers during hindlimb suspension (Mukhina et al. Neurosci Behav Physiol 38(2):181–188, 2008). 21 male Wistar rats were divided into 3 groups: Control group (C, n = 7), Hindlimb suspended rats during 3 days (HS, n = 7), HS + nifedipin administration (in m.soleus twice a day (7 mg/kg in 0.3 % DMSO, HSN, n = 7)). M.soleus weight and MyHC protein content haven’t been changed after HS. We neither reveal l-calpain level increase, nor desmin destruction in HSN group in contrast to HS group. However MuRF-1 and MAFbx mRNA levels were both increased (p \ 0.05) and pAkt content was decreased (p \ 0.05) in both hidlimb suspended groups. In conclusion: l-calpain content under soleus unloading (but not its mRNA level) could be regulated by calcium level modulation, destruction of the cytoskeletal protein desmin occurs up to reduce the contractile proteins and noticeable muscle atrophy. L-channel blocking (calcium level modulation in the cytoplasm of muscle fibers) doesn’t prevent decrease of pAkt content and MuRF-1 and MAFbx elevated level. Grants RFBR 11-04-00787-a, 14-04-01632. Keywords: Hindlimb suspension, l-Calpain level, MuRF-1 and MAFbx levels
P3.27 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Changes in MHC isoforms relative content of atrophied and recovering rat plantaris muscle Aru Maire1, Alev Karin1, Kaasik Priit1, Puhke Raivo1, Pehme Ando2, Seene Teet3 1
Institute of Exercise Biology and Physiotherapy, University of Tartu, Estonia; 2Institute of Sport Pedagogy and Physiotherapy, University of Tartu, Estonia; 3Institute of Exercise Biology, University of Tartu, Estonia Glucocorticoids (GCs) are the most important and frequently used anti-inflammatory and immunosupressive drugs in the treatment of patients with a variety of disorders. The catabolic effects of GCs have been well known for many years. GC-induced muscle atrophy is
J Muscle Res Cell Motil (2015) 36:71–143 characterized by fast-twitch, glycolytic muscles atrophy illustrated by decreased fiber cross-sectional area and reduced myofibrillar protein content. Muscle mass and muscle phenotype are determinants of contractile performance. The contractile phenotype is mainly determined by the expression of myosin heavy chain (MHC) isoforms in skeletal muscle. MHC composition is an indicator of regenerative events and adaptation to changing demands in skeletal muscle. The aim of the study was to assess the effect on the myosin heavy chain (MHC) isoforms composition of dexamethasone (DEX) treatment and recovery of atrophied skeletal muscle using chronic overload of plantaris (PLA) muscle. Adult male rats of the Wistar strain were used. Muscle atrophy was induced by intraperitoneal injection of dexamethasone daily during 7 days. The control animals received appropriate amounts of 0.15 M NaCl. After 7-days administration of DEX, compensatory hypertrophy was applied by synergist tenotomy, which lasted 10 days. MHC content of muscle samples was analyzed electrophoretically. Results showed changes in MHC isoform composition in response to both DEX administration and compensatory hypertrophy (CH). Results demonstrate qualitative remodelling of the isoform profile during overloading and recovery of the atrophied muscle. The combined treatment compensates the effects of DEX treatment. The expression of fast MHC isoforms was stimulated by the combined treatment. DEX + CH induced a significant shift towards fast MHC isoforms with increase in MHC IIb and MHC IId with a reduction of MHC IIa in comparison with control group. Keywords: Skeletal muscle atrophy, Glucocorticoids, MHC
P3.28 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Use it or lose it. A life-long-style of recreational sport activities increases reinnervation of aging muscle Mosole Simone1, Carraro Ugo1, Kern Helmut2, Lo¨fler Stefan2, Fruhmann Hannah2, Vogelauer Michael3, Cvecˇka Ja´n4, Sedliak Milan4, Tirpakova Veronika4, Sˇarabon Nejc5, Zampieri Sandra6 1
Department of Biomedical Sciences, University of Padova, Italy; 2 Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Vienna, Austria; 3Department of Physical Medicine and Rehabilitation, Wilhelminenspital, Vienna, Austria; 4Faculty of Physical Education and Sport, Comenius University, Bratislava, Slovakia; 5University of Primorska, Science and Research Centre, Institute for Kinesiological Research, Koper, Slovenia; 6Department of Biomedical Sciences, University of Padova, Italy; Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Vienna, Austria Age-related changes in skeletal muscle are known to contribute to the decline in quality of life often reported in elderly; however, these changes and the mechanisms by which they occur are not clear. We examined the effects of lifelong high-level physical activity on a cohort of sportsmen and collect evidence that aging atrophy is a result of progressive denervation that can be counteracted by lifelong highlevel exercise. We analyzed the fiber type composition of muscle biopsies from 3 groups (1) senior sportsmen *70 years old, who exercised regularly at high levels for 30 years; (2) healthy, sedentary seniors, age-matched subjects; and (3) active young sportsmen (age *26 years). Our results demonstrate that: 1. biopsies from young men seldom contain denervated (0.2 ± 0.5 %), reinnervated or transformed muscle fibers (0.5 ± 0.6 %); 2. biopsies from sedentary seniors contain both denervated (2.6 ± 1.9 %), coexpressing myofibers (1.8 ± 1.7 %) and a few reinnervated clustered myofibers of the fast type (3.0 ± 4.7 %); 3. senior sportsmen show a larger
119 percentage of healthy, slow myofibers (68.5 ± 14.1 %,) that appear mainly clustered in slow fiber-type groupings (7.9 ± 7.4 %). Data reveal that there was no difference between athletic and sedentary senior groups in percentages of muscle fibers co-expressing fast and slow MHCs (0.6 ± 0.6 %), suggesting that lifelong exercise doesn’t induce motor unit transformation. However, sportsmen had both considerably higher percentages of slow-type myofibers and greater numbers of slow fiber-type groupings than the sedentary group. It appears thatlifelong exercise allows the body to adapt to the consequences of aging and to preserve muscle function by saving lost muscle fibers through reinnervation by different, mainly slow, motor axons. These observations suggest that long-term physical activity promotes reinnervation of muscle fibers undergoing age-related denervation and these one can be summarized in the phrase ‘‘Use It or Lose It…’’. Keywords: Elderlies, Life-long exercise, Reinnervation
P3.29 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Molecular adaptation of sarcoplasmic reticulum of human skeletal muscle to functional electrical stimulation and leg-press training in aging Mosole Simone1, Carraro Ugo1, Kern Helmut2, Lo¨fler Stefan2, Fruhmann Hannah2, Vogelauer Michael3, Cvecˇka Ja´n4, Sedliak Milan4, Tirpakova Veronika4, Sˇarabon Nejc5, Zampieri Sandra1,2 1
Department of Biomedical Sciences, University of Padova, Italy; Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, Vienna, Austria; 3Department of Physical Medicine and Rehabilitation, Wilhelminenspital, Vienna, Austria; 4Faculty of Physical Education and Sport, Comenius University, Bratislava, Slovakia; 5University of Primorska, Science and Research Centre, Institute for Kinesiological Research, Koper, Slovenia
2
Physical activity plays an important role in preventing chronic disease and muscle degeneration in adults and the elderly. Voluntary physical exercise, such as leg press (LP) training, is not always feasible and other therapies should be applied such as Functional Electrical Stimulation (FES). The key process of Ca2+ storage uptake and release is essential in muscle adaptation. This study shows the effects of physical training and FES on expression of calsequestrin (CASQ) the main Ca2+ binding protein of the sarcoplasmic reticulum (SR) and SR Ca2+ ATPase (SERCA1, 2) in human vastus lateralis (VL) muscle after FES or LP training. Muscle total homogenates were obtained from biopsies performed before and after completing a nine weeks FES treatment on a group of volunteers (n = 17), mean age 71. CASQ, SERCA, sarcalumenin, phospholamban protein expression was determined by western blot. In 5 out of 17 samples, FES induced increase in CASQ1 concentration (p \ 0.05) whereas the CASQ2 isoform did not change; SERCA1 and 2 expression did not show a univocal trend. Comparison with leg press training on a group of volunteers (n = 9), mean age 72, was performed. Additionally, myonuclear translocation and increase of NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1) expression was observed after functional electrical stimulation. These results indicate that FES potentiates Ca2+ storage in skeletal muscle via up-regulation of CASQ1 and suggest that NFATc1 translocates from the cytoplasm into the nuclei of human skeletal muscles during FES simulating a slowtype firing pattern. Keywords: Aging, Electrical stimulation, Calcium binding proteins
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P3.30 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Respiratory muscle gene expression in a mouse model of sustained hypoxia O’Leary Andrew James***, Ken D O’Halloran ***Candidate for Young Investigator Award
Department of Physiology, University College Cork, Ireland Chronic obstructive pulmonary disease (COPD) is a pathological disorder characterized by sustained hypoxia (SH). SH causes apparently unique adaptations in the principal muscle of breathing—the diaphragm. There remains however a general paucity of information concerning the molecular mechanisms underpinning respiratory muscle remodelling in hypoxia. We assessed if there are early changes in expression of genes encoding transcription factors/transcriptional co-activators and Ca2+ handling proteins of the sarcoplasmic reticulum (SR) in the diaphragm of mice exposed to acute SH. Adult male C57BI6 J mice (n = 8 per group) were exposed to 1, 4 and 8 h of SH (FiO2 = 0.10) or normoxia (FiO2 = 0.21). Diaphragm muscles were excised post-mortem. A qRT-PCR approach was used to assess relative changes in gene expression. Data were statistically compared by one-way ANOVA. Statistically significant changes in expression were observed in PGC-1a, NF-jB and NRF1 mRNA in the diaphragm after acute exposure to SH. PGC-1a expression was increased significantly in the 1 h SH group vs. control (P \ 0.05), but this increase was no longer significant after 4 h of SH and returned to levels equivalent to the control level in the 8 h group (P \ 0.05 for 1 vs. 8 h groups). NF-jB was significantly decreased after 4 h of SH (P \ 0.05 vs. control and vs. 1 h group); a similar result was observed for NRF1 (P \ 0.05, 4 vs. 1 h groups). Both were still decreased in the 8 h group, but the decrease was no longer statistically significant. There were no statistically significant changes in the expression of genes encoding Ca2+ handling proteins of the SR (SERCA 1 & 2, Junctophilin 1 & 2, Calsequestrin 1). The effect of acute SH on gene expression in the mouse diaphragm is time-dependent and dynamic. The next step of this study is to determine if there are differences in the expression profiles of these genes comparing diaphragm to sternohyoid (upper airway dilator muscle) and limb muscle.
J Muscle Res Cell Motil (2015) 36:71–143 spinal stenosis (lss) patient. After completing body perception and core stability physiotherapy (PT), a 70-year-old man with lss started extensive endurance training on ergometers to increase reduced physical condition. At this time walking distance was limited to 250 m because of spreading low back pain. Therefore, functional AlterG training was initiated two times per week for an eight month period. Besides the AlterG training, a Nordic Walking technique instruction in the department was held, to ensure transfer into daily routine. Early in AlterG therapy, visual analog scale (VAS) for his lower back was set at 5.5 (0 = no pain, 10 = worst pain), reducing to 0 after eight months of training. At the beginning of AlterG training, body weight was decreased by 40 % up to 5 %. As walking time increased from 13 to 35 min, walking distance increased equally from 870 m to 2.4 km. The walking capacity of 2.4 km and the alleviation of pain should allow the patient to perform normal daily activities, and thus participate in social life and preserve independence. The case will be discussed in the context of application within a multistage rehabilitation program. Keywords: Exercise, Rehabilitation References: [1] Pfeifer, 2004: http://www.bertelsmann-stiftung.de/cps/rde/xbcr/ SID-37D37FB0-115060FF/bst/Bewegungsinterventionen_2004.pdf [2] Wessinghage & Morsch, Public Health Forum, 21(2):21.e1-21.e3, 2013 [3] www.alterg.com [4] Frobo¨se et al. (Eds): Training in der Therapie, 2010 [5] Chun & Mishra in www.alterg.com/clinical-summary
P3.32 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Post mortal changes in structure and function of skeletal muscle proteases Pfisterer Alexander1***, Pittner Stefan1, Sa¨nger Alexandra Maria1, Stoiber Walter1, Monticelli Fabio C2, Steinbacher Peter1 ***Candidate for Young Investigator Award
1
Keywords: COPD, Diaphragm, Hypoxia
P3.31 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Physical active rehabilitation of spinal stenosis using AlterG treadmills Landkammer Yvonne, Bernecker Robert, Wicker Anton Physikalische Medizin und Rehabilitation, SALK, Salzburg, Austria Regained physical condition increases physical activity, showing positive consequences for lower back pain [1, 2]. AlterG treadmills, operating with a differential air pressure technology reducing body weight up to 80 %, are commonly used as a training device for medical complaints of lower extremities [3]. One aim of functional rehabilitation is to decrease pain [4]. One goal of using AlterG in rehabilitation is to restore normal walking mechanics [5]. The following case report refers to an experimental application with a lumbal
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Department of Cell Biology, University of Salzburg, Austria; Department of Forensic Medicine and Forensic Neuropsychiatry, University of Salzburg, Austria
2
Improvement of methods to determine time of death is crucial to modern forensic science. Exact methods to date are only able to cover the first 36 h post mortem (pm). The aim of this study is to search for regular decay processes that allow for precise time-of-death estimation also beyond this present limit. For this purpose, we used casein zymography to determine the function of l- and m-calpains, the two main proteases of muscle degradation, in porcine biceps femoris muscle at different times pm. We found that in all samples investigated, the native l-calpain band disintegrates into two bands until 36 h pm while it takes until 72 h pm that the same occurs for m-calpain. A similar difference became evident for the final disappearance of the l- and m-calpain bands which lasts until 120 and 240 h pm, respectively. These results show clearly that calpains degrade in a predictable fashion with a time course extending clearly beyond 36 h pm, thus making them promising molecular candidates for use in time-of-death determination. Keywords: Calpain, Muscle degradation, Post mortem
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Pittner Stefan1***, Monticelli Fabio C2, Sa¨nger Alexandra Maria1, Steinbacher Peter1
results showed that during short-term of feed deprivation (4 days), gene expression of Murf1a, Murf1b, MAFBx and fbox25 were strongly activated, and the expression level decreased significantly after refeeding. Our results showed that fasting followed by refeeding periods can alter the molecular mechanisms involved in muscle growth control in juvenile of pacu, and this fact could influence the muscle fiber recruitment rate and hypertrophy in this specie.
***Candidate for Young Investigator Award
Keywords: Muscle growth, Fasting, Refeeding
1
P4.1 Muscle development, differentiation, and regeneration, Fri 12:00–14:00
P3.33 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Estimation of the time since death: degradation of skeletal muscle
Cell Biology, University of Salzburg, Austria; 2Forensic Medicine, University of Salzburg, Austria Estimating the time since death is a very important aspect in forensic sciences that is pursued by a variety of methods. The most precise method to determine the post mortem interval (PMI) is the temperature method which is based on the deviation of the body core temperature from environmental temperature. However, this method is only useful in the early post mortem phase (0–36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used SDS-PAGE and Western blotting to analyze the time course of degradation of selected proteins in porcine biceps femoris muscle until 240 h post mortem. Results demonstrate that desmin, titin and SERCA1 degraded in a predictable fashion in all samples investigated, with degradation intervals being confined to 18–48 h, 36–72 h and 168–240 h, respectively. This degradation behavior identifies these proteins as promising substrates for future molecular-based PMI determination technologies. Keywords: Post mortem interval, Cytoskeletal proteins, Muscle degradation
P3.34 Muscle plasticity, exercise, and aging, Fri 12:00–14:00 Nutritional status regulate the expression levels of genes related with catabolic signaling pathways in skeletal muscle of fish pacu (Piaractus mesopotamicus) Paula TG***, Mareco EA, Salomao R, Santos VB, Dal-Pai-Silva M ***Candidate for Young Investigator Award
Department of Morphology, Sao Paulo State University, Brazil In fish, fasting can affect physiological systems, influencing directly on skeletal muscle, which may alter the rate of myogenesis, the number and diameter of muscle fibers and the expression patterns of genes related to muscle development. Feed deprivation can increase the expression levels of genes involved in the catabolic pathways resulting in a decrease of muscle growth. The aim of this study was to evaluate the influence of fasting followed by refeeding in gene expression of Murf1a, Murf1b, MAFBx and fbox25 in juvenile pacu. Fish (15 g weight) were distributed in tanks containing water under distinct recirculation systems (six replicates for each group), during the experiment. Fish were fed to satiation for 1 week, then feed deprived for 4 days (-4, -3, -2 and -1/fasting condition), followed by 24 h of refeeding period (0, 6 h, 12 h and 24 h/refeeding state). Skeletal muscle samples were obtained in all periods of the experiment and the quantitative gene expression analysis of Murf1a, Murf1b, MAFBx and fbox25 was performed by RT-qPCR. Our
The M-line protein, obscurin, in the development and symmetry of insect flight muscle Katzemich Anja1, Leonard Kevin2, Sweeney Sean1, Sparrow John1, Bullard Belinda1 1
Department of Biology, University of York, YO10 5DD, UK; EMBL-EBI-Wellcome Trust Genome Campus, Cambridge, CB10 1SD, UK
2
The M-line protein, obscurin, is made up of 21 Ig domains, 3 Fn3 domains, a Rho-GEF domain near the N-terminus and two kinase domains near the C-terminus. We have investigated the effect of down-regulating obscurin on the development and structure of Drosophila flight muscle (IFM). Obscurin assembles into periodic striations in the pupal IFM at 30 h after puparium formation, at which time the Z-disc protein, kettin, and myosin are in amorphous strands. Thus, M-line assembly precedes the formation of the Z-disc and A-band. Obscurin expression was reduced by RNAi techniques. The IFM of these flies had sarcomeres of normal length but there was no M-line and the H-zone was irregular. Isolated thick filaments were asymmetrical, with bare zones shifted towards one end of the filaments. In the myofibril, the position of the bare zone determined the length and polarity of adjacent thin filaments. Therefore, early expression of obscurin nucleates assembly of symmetrical thick filaments and controls the length of thin filaments. We have used tandem affinity purification (TAP) to identify ligands of the two kinase domains of obscurin. Constructs of the domains, with tags, were injected into embryos, and the kinases expressed in IFM were isolated using the tags. Ball (another kinase) was bound to kinase1, and MASK (an ankyrin-repeat protein) was bound to kinase2; additional ligands were identified for both kinases. RNAi lines of ball and MASK produced phenotypes in IFM that were similar to that of obscurin RNAi flies. However, expression of obscurin in RNAi lines of both ligands was normal, suggesting ball and MASK act downstream of obscuirn. Both ligands are genetically linked to signalling pathways in the development of Drosophila muscle. Keywords: Obscurin, M-line, Insect flight muscle
P4.2 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 NDRG2 suppression in C2C12 myoblasts causes mitochondrial dysfunction and altered metabolism Foletta Victoria1, McGee Sean2, Russell Aaron P1 1 Centre for Physical Activity and Nutrition Research (C-PAN), School of Exercise and Nutrition Sciences, Deakin University, Melbourne, Australia; 2Centre for Molecular and Medical Research
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122 (MMR), School of Medicine, Deakin University, Melbourne, Australia The N-myc downstream-regulated gene 2 (Ndrg2) gene is linked to the control of cell proliferation and differentiation and is responsive to stress. In cancer cells, NDRG2 is considered a tumor suppressor as its increased expression blocks cancer cell growth and enhances apoptosis. In contrast, in skeletal muscle cells, NDRG2 suppression impairs myoblast growth and differentiation. Recently, we identified that NDRG2 is a transcriptional target of the PGC-1a and ERRa proteins, key controllers of energy metabolism and mitochondrial biogenesis in skeletal muscle. Hence, we investigated whether NDRG2 suppression in myoblasts also impairs metabolic processes and mitochondrial function in addition to the reduced proliferation observed. Initial morphological and quantitative analyses of mitochondria in NDRG2-deficient myoblasts indicated the cells to have a higher mitochondrial content with a more elongated mitochondrial network; however, no increase in mitochondrial biogenesis activity was evident based on Pgc-1a, Nrf-1 and Tfam gene expression levels. Analyses of mitochondrial membrane properties revealed NDRG2deficient cells to display a higher membrane potential enhanced further by the addition of oligomycin. Mitochondrial respiration was lowered significantly in the basal and maximal respiration states and greater levels of reactive oxygen species were also measured in the NDRG2-deficient myoblasts. While citrate synthase activity was not altered, decreased oxidation of a variety of fuel substrates including glucose and palmitate was found. In addition, genes involved in fatty acid transport and metabolism were significantly altered. Together, these findings indicate that NDRG2 plays a novel role in mitochondrial-based function and cell metabolism in proliferating muscle cells. These findings have implications for both metabolic functioning in muscle and cancer cells via the control of NDRG2. Keywords: NDRG2, Mitochondria, Metabolism
P4.3 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 In vitro engineered muscle: identification and characterization of murine dermal precursor cells with myogenic potential Garcı´a-Parra P1, Naldaiz-Gastezi N1, Lacalle J2, Goicoechea M3, Alonso-Martin S4, Aiastui A3, Garcı´a-Belda P5, Garcı´a-Verdugo JM5, Carvajal JJ6, Lo´pez de Munain A7, Izeta A8 1 Instituto Biodonostia, Spain; 2School of Nursing, University of the Basque Country UPV/EHU, Spain; 3Neuroscience Area, Instituto Biodonostia, Spain; 4Development and Stem Cells, Institut de Myologie, Paris, France; 5Laboratorio de Neurobiologı´a Comparada, Instituto Canavilles, Valencia, Spain; 6Molecular Embryology Team, Centro Andaluz de Biologı´a del desarrollo, Seville, Spain; 7 Department of Neurology, Hospital Universitario Donostia, San Sebastia´n, Spain; 8Tissue Engineering Laboratory, Instituto Biodonostia, Spain
Our group has recently derived skeletal muscle from dermis-derived cells, by using an ECM that recreates the myogenic niche. After one week of differentiation, we observed isolated, twitching myotubes followed by spontaneous contractions of the entire tissue-engineered muscle construct. In vitro engineered myofibers expressed canonical markers, ultrastructure and electrophysiological characteristics of skeletal muscle. Interestingly, after one-month engineered muscle constructs showed progressive degradation of the myofibers concomitant with fatty infiltration, paralleling the natural course of muscular degeneration. However, we don’t yet know how dermisresident precursors are related with our isolated-myogenic precursors,
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J Muscle Res Cell Motil (2015) 36:71–143 a critical point for extrapolating our results to the human system. Knowing that dermal cells and muscle cells share a common embryonic origin from somite-derived dermomyotome and taking into account that there are different types of cells within the skin that have myogenic potential, our main objective is to identify and characterize the origin of murine dermal subpopulation with a myogenic potential, hypothesizing that may be (i) satellite cells from the Panniculus carnosus, (ii) dermomyotome-derived adult stem cells, (iii) perivascular cells and/or (iv) neural crest-derived precursor cells. For this end, lineage tracing experiments [(i) Pax3-GFP, (ii) Pax7CE, (iii) Myf5-Cre, (iv) CSPG4-Cre, and (v) Sox10-Cre] combined with FACS strategies and cellular differentiation assays have been developed. Our results show a high contribution of Myf5+ cells, a low contribution of Cspg4+ cells, and no contribution of Sox10+ cells to dermis-derived myogenic precursor cell subset. In principle, that means that dermomyotome-derived but not neural crest-derived cells are involved in subsequent engineered muscle differentiation. Keywords: Muscle differentiation, Dermal precursor cells, Engineered muscle
P4.4 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Sarcophagy—the ubiquitin-mediated autophagic turnover of sarcomeric proteins Masiero Eva1, Kho Ay Lin1, Jenniches Katharina1, Waters Sarah1, Kru¨ger Marcus2, Whitehouse Caroline1, Sandri Marco3, Gautel Mathias1 1
Cardiovascular Division and Randall Division for Cell and Molecular Biophysics, King’s College London, UK; 2Max-PlanckInstitute for Heart and Lung Research, Bad Nauheim, Germany; 3 Department of Biomedical Science, University of Padova and Dulbecco Telethon Institute at Venetian Institute of Molecular Medicine, Padova, Italy Dynamic turnover of muscle sarcomeres is regulated by two major pathways, the ubiquitin-proteasomal (UPS) and autophagy-lysosomal (AL) systems. Muscle-specific ubiquitin E3 ligases target proteins for degradation by the UPS and can cooperate with proteases like calpains. Several UPS and AL-associated proteins and proteases associate directly with sarcomeres, including the giant sarcomeric ruler protein titin. Titin contains multiple signalling domains, including a C-terminal kinase domain (TK) at the M-band. TK shows unusual sequence features, raising the question whether its main function is catalytic, that of a scaffold, or both. Lange et al. (Science 308(5728):1599–1603, 2005) reported a signalling complex where TK interacts with the autophagy adaptor proteins nbr1 and p62/ SQSTM1, and ubiquitin E3 ligases MURF-1 and -2. To understand the role of this complex in load-dependent muscle protein turnover, we generated genetically altered mice carrying truncated versions of Nbr1. These animals show distinct muscle phenotypes. Unloading and loading experiments in skeletal and cardiac muscle show that loaddependent muscle protein turnover depends on full-length Nbr1, whereas complete knockout of p62 is compensated. Proteomic analysis reveals altered turnover of metabolic enzymes, sarcomeric proteins, signalling proteins and components of the endosomal pathway. Surprisingly, sarcomeric components whose turnover depends on Nbr1 include M-band titin and myomesin. Previously identified cytoskeletal targets for autophagosomal turnover were restricted to the peri-myofibrillar protein filamin-C. We show that Nbr1 recruits M-band components including C-terminal titin to LC3- and ubiquitinpositive autophagosomes in both skeletal muscle and cardiomyocytes.
J Muscle Res Cell Motil (2015) 36:71–143 The sarcomere thus interacts as a mechano-sensitive scaffold with autophagy adaptors that are critical and non-redundant for cardiac and skeletal muscle maintenance and turnover. We call this new form of autophagy of sarcomeres ‘‘sarcophagy’’. Keywords: Sarcomere, Titin, Autophagy
P4.5 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 The L-type Ca2+ channel auxiliary subunit a2d is involved in structural muscle organization—a novel developmental role Reuveny Adriana, Talila Volk Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Muscle contraction is triggered by excitation of the muscle cell membrane, a mechanism mediated by increasing levels of intracellular Ca2+. Ca2+ channels are present in muscle membrane indentations, the t-tubules, where they facilitate the contraction of highly ordered structures defined by two neighboring z-discs (also known as z-lines) and thin (actin) and thick (myosin) filaments in between them. The L-type Ca2+ channel auxiliary subunit a2d represents a family of evolutionary conserved proteins that are expressed in many tissues, and are mainly known for their function in membrane anchoring of the channel complex. In this work, we identified and studied the muscular a2d in Drosophila and we reveal a novel developmental role for a2d. We find that a2d is required for proper organization of the z-disc KASH domain protein, MSP-300 (an evolutionarily conserved Nesprin family member protein). We found that a2d colocalizes with MSP-300 to the nuclear membrane and z-discs of the larva muscles. Knock down of a2d induces severe aggregation of the muscle nuclei and disruption of the Z-disc anchored protein MSP-300. Genetic interaction experiments reveal that muscles of double heterozygous mutants for a2d and MSP-300 exhibit severe alterations in the localization of these proteins, together with nuclei disorganization and disruption of the t-tubular structure. Consistently, a2d mutant flies do not develop to adulthood. This work identifies a novel developmental role for the muscle a2d protein. Our findings indicate that a2d interacts with MSP-300, and that this interaction is mutually required for their correct subcellular localization. We suggest that this interaction promotes proper structural organization of the muscle nuclei and t-tubular network, via anchoring to the nuclear membrane and muscle z-disc and, two functions important for proper muscle function. Keywords: Drosophila, MSP300, Nesprin
P4.7 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Ultrastructural localisation of titin during sarcomere assembly in regenerating vertebrate skeletal muscle after intoxication with notexin Unger Andreas, Siebrecht Sebastian, Linke Wolfgang A Cardiovascular Physiology, Ruhr-University Bochum, Germany The giant filamentous protein titin spans from the Z-disk to the M-line in the sarcomere of striated muscle cells, where it functions as a molecular spring during stretching and relaxation. During myofibrillogenesis, titin is thought to act as a molecular template for the
123 assembly of A-bands, because super-repeats of titin domains in the C-zone of the A-band show a *43 nm periodicity coinciding with the *43-nm spacing of myosin heads and MyBP-C. In this study we used immunogold electron microscopy and three-dimensional reconstruction of electron micrographs to localise titin, a-actinin, myosin, myosin binding protein-C and myomesin at different stages of myofibrillogenesis in regenerating rat soleus muscle after notexininduced myofibre breakdown. Two days after intoxication with notexin we observed premyofibrils containing titin, myosin and Z-bodies; three days after intoxication we found Z-bodies fusing to Z-disks and I- and A-Bands beginning to show typical striation patterns. Three dimensional reconstructions revealed that extrasarcomeric titin colocalises with thick filaments in sarcomeres and is also nearby the assembling sarcomeres. Our results support a model in which titin acts as a molecular scaffold protein for the assembly and the integration of the thick filaments into the sarcomere during skeletal muscle regeneration. Keywords: Sarcomere assembly, Muscle regeneration, Titin
P4.8 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Leucine supplementation accelerates connective tissue repair and functional recovery during skeletal muscle regeneration Pereira Marcelo1***, Silva Meiricris1, Carlassara Eduardo1, Abrahamshon Paulo2, Moriscot Anselmo1, Aoki Marcelo3, Miyabara Elen1 ***Candidate for Young Investigator Award
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Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Brazil; 2Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil; 3School of Arts, Sciences and Humanities, University of Sao Paulo, Brazil Although the regenerative process has been investigated in detail, studies on the strategies to improve it are still on going. We investigated the effects of leucine supplementation on the skeletal muscle regenerative process, focusing on the remodeling of connective tissue (CT) and functional recovery in the phenotypically distinct muscles. Male Wistar rats were subjected or not to leucine supplementation (1.35 g/kg), which started 3 days prior to cryolesion of soleus and tibialis anterior (TA) muscles from left hind limb, and continued receiving leucine until 10 days later. Muscles from right hind limb were used as control. Muscle cross-sections were used to reveal the overall morphology, and determine myofibre cross-sectional area (CSA). Quantification of the CT area density was analyzed under light and polarized microscopes. Neonatal myosin heavy chain (MyHC-n) and the content of MyHC-I and II were analyzed by immunostaining and Western Blot, respectively. Phosphorylation of transforming growth factor-b receptor type I (TbR-I), and the positive nuclei for Smad2/3, were assessed by immunostaining. Muscle function was assessed by in vivo electrostimulation. Leucine supplementation promoted an increase in the CSA of regenerating muscles, which was more pronounced in the soleus (p \ 0.05) than in the TA. Leucine also reduced the amount of CT and the activation of TbR-I and Smad2/3 on both regenerating muscles (p \ 0.05); and accelerates the shift of MyHC-n to MyHC-I and II on soleus and TA, respectively (p \ 0.05). In addition, prevented the development of fatigue in regenerating soleus and the decrease in the tetanic strength in regenerating TA (p \ 0.05). Leucine supplementation accelerates CT
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124 repair and consequent functional recovery of regenerating soleus and TA through the attenuation of TbR-I and Smad2/3 activation. These results suggest that leucine supplementation can be used as a nutritional strategy to prevent or attenuate several events in muscle disease. Keywords: Leucine, Fibrosis, Functional recovery
P4.9 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Lysosomal V-type ATPase inhibitors acidify undifferentiated skeletal muscle cells and trigger apoptosis Hiess Florian1, Wanke Lorenz1, Weigl Lukas2, Hohenegger Martin1 1
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria; 2Department of Anesthesia and Pain Managment, Medical University of Vienna, Austria Skeletal muscle cells tolerate acidification better than other cells. Bafilomycin A1 is a widely used V-type ATPase inhibitor to trigger autophagy and to selectively prevent nicotinic acid adenine dinucleotide phosphate (NAADP) induced calcium transients from acidic lysosomes, mediated by two pore channels. Here we investigated the role of bafilomycin A1 and the related compound concanamycin A1 in human and murine undifferentiated skeletal muscle cells at concentrations usually used to inhibit NAADP induced calcium transients. Application of 100 nM V-type ATPase inhibitors evoked a transient raise in cytosolic calcium concentration. This calcium transient was accompanied by cellular shrinkage and a significant decline of lysosomal staining with an organelle specific fluorescence dye. Expectedly, bafilomycin A1 and concanamycin A1 triggered acidification of undifferentiated skeletal muscle cells within the same time frame. The morphological changes in these undifferentiated skeletal muscle cells are reminiscent of apoptosis, which was confirmed by FACS analysis and specific measurement of caspase 3 activity with a fluorescent substrate. Taken together these data show that V-type ATPase inhibitors should be carefully evaluated when used to inhibit acidification of lysosomes or NAADP mediated calcium signals. This work was supported by Herzfeldersche Familienstifung and the Austria Science Funds (P22385 to M.H.). Keywords: Skeletal muscle cells, V-type ATPase inhibitor, Lysosomes
P4.10 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Adaptive response of soleus muscle after crush denervation and during re-innervation Bardi Eleonora1, Sartini Stefano2, Cuppini Riccardo2, Bottinelli Roberto1, Pellegrino Maria Antonietta1 1
Department of Molecular Medicine, University of Pavia, Italy; Department of Earth, Life and Environmental Sciences, University of Urbino, Italy
2
Peripheral nerve injury causes skeletal muscle inactivity, by destroying nerve–muscle communication, and muscle atrophy. Reinnervation is known to promote muscle mass recovery, but few data are available about the pathway supporting such process. The aim of this study was to analyze the relation among skeletal muscle mass,
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J Muscle Res Cell Motil (2015) 36:71–143 intracellular signaling pathways involved in its control and neurotrophins expression. Particular attention has been paid to the potential role of neurotrophins in the maintenance of skeletal muscle mass. Fibers cross sectional area, ubiquitin proteasome and autophagy systems, anabolic pathway, GDNF, IGF-1 and NT-4 neurotrophins expression have been analyzed in denervated rat soleus and during reinnervation, which starts on day 4, with and without exercise. Rat soleus muscle was analyzed after denervation and during re-innervation at 3, 7 and 10 days from nerve crush. A significant decrease of fibers CSA was found both following denervation and during reinnervation. Phosphorylation level of Akt, S6 and 4E-BP1 was significantly lower in denervated soleus compared to un-denervated control; reduction of Phospho-4E-BP1 persisted during reinnervation. As regard catabolic pathways, a significant decrease of beclin-1 mRNA and increase of Chatepsin-L mRNA and LC3-B protein level was found in denervated soleus and during re-innervation with respect to un-denervated control. No significant changes of neurotrophic factors were found after denervation and re-innervation. Our preliminary results suggest that muscle atrophy found in denervated muscle and during reinnervation depends on a decrease of protein synthesis and autophagy activation and show the basal time course of neurotrophins mRNA expression. The next step of the research will be the study of the same adaptation during re-innervation with exercise. Keywords: Denervation, Skeletal muscle, Neurotrophins
P4.11 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Molecular mechanisms of TEAD transcription factor function in skeletal muscle differentiation Joshi Shilpy***, Davidson Irwin ***Candidate for Young Investigator Award
Department of Functional Genomics and Cancer, IGBMC, Strasbourg, France The TEAD transcriptional factors comprise a highly conserved TEA DNA binding domain that binds a consensus MCAT (50 -CATTCCA/ T-30 ) element present in the promoters of many muscle-expressed genes. We have shown that expression of the TEA DNA binding domain acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts and inhibits their differentiation, while selective shRNA knockdown of TEAD4 results in abnormal differentiation characterized by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridization (ChIP-chip) or DNA sequencing (ChIPseq) shows that TEAD4 occupies the promoters of genes critical for differentiation and that TEAD4 directly induces Myogenin, CDKN1A, Caveolin 3 and myogenic miRNAs to promote myoblast differentiation. In contrast, TEAD4 represses expression of the growth factor CTGF and cyclin D1 to promote differentiation. TEAD4 therefore appears interact with both co-activators and co-repressors to differentially regulate gene expression at the transition from proliferation to differentiation. We performed tandem affinity purification coupled to Mass spectrometry (TAP-MS) in differentiated C2C12 cells engineered to express Flag-HA-TEAD4 and identified several novel and some already well-known TEAD4 interaction partners such as YAP1 and VGLL proteins. This led us to investigate their role in C2C12 and primary myoblast differentiation. We performed gain or loss-of-function studies in C2C12 myoblast cell line and mouse primary myoblasts in order to identify their role in
J Muscle Res Cell Motil (2015) 36:71–143 TEAD4 transcriptional activity. We compared the gene expression profiles in C2C12 and primary myoblasts following loss-of function of TEAD4 or its co-factors. This study investigates the molecular function of TEAD4 and its transcriptional co-factors in two model systems, which will help us further understand how TEADs regulate specific sets of genes during proliferation or differentiation. Benhaddou et al. Cell Death Differ. 2012. Keywords: TEAD4, Transcription co-factors, Interactome
P4.12 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Lack of HO-1 influences microRNAs expression after cardiotoxininduced injury Gese Anna***, Czypicki Ryszard, Cies´la Maciej, Kozakowska Magdalena, Jo´zkowicz Alicja, Dulak Jo´zef ***Candidate for Young Investigator Award
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology,Jagiellonian University, Cracow, Poland Heme oxygenase (HO-1) is cytoprotective, anti-inflammatory and antioxidant enzyme degrading heme to billiverdin, carbon monoxide and ferrous ion. Since HO-1 was shown previously by us to strongly influence differentiation of myoblasts we hypothesized that it can be also crucial for regeneration of skeletal muscles after cardiotoxin injection. Cohort included 60 C57BL/69 FVB mice. Half of them were HO-1 knockout (KO) while the rest (wild type—WT) had functional Hmox1 gene encoding HO-1 protein. 25 KO and 25 WT mice were intramuscularly injected with cardiotoxin (CTX) solution in saline and 5 mice per genotype were sacrificed 1, 3, 7, 14 and 28 days after CTX injury. Gastrocnemius muscles were collected and expression of mRNAs and microRNAs was analyzed. H&E staining was also performed. Remaining 10 mice that served as control groups were sacrificed one day after intramuscular saline injection. In the early inflammatory phase, 1 day after CTX injury the expression of IL-1b and IL-6 was higher in HO-1 KO than WT mice. Induction of MyoD appears to be delayed and expression of Myf5 and Myf6 was lower in HO-1 KO than in WT mice. HO-1 deficient animals displayed also higher levels of Pax7. The latter is the target of miR-206 and interestingly, the expression of miR-206, which steadily increased in later phases of muscle regeneration, was significantly higher in WT than in KO mice. Also expression of microRNAs-1, 133b, 206, 146a and 378 was higher in WT than in KO mice at the end of the experiment. In sum, the data indicate that the lack of HO-1 may influence regeneration of muscles after injury through regulation of microRNAs expression. Supported by the MAESTRO Grant from the National Science Center (NCN 2012/06/A/NZ1/00004). Keywords: Muscle regeneration, Cardiotoxin injury, Heme oxygenase-1
P4.13 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Overexpression of Gasp-1 leads to a deregulation of adiposity and glucose homeostasis in adult mice Perie Luce, Brun Caroline, Baraige Fabienne, Forestier Lionel, Blanquet Veronique
125 UMR INRA 1061, Animal Molecular Genetics, Limoges, France Myostatin (Mstn) is a powerful negative regulator of muscle mass. Knock-out myostatin mice present an increase of muscle mass with hypertrophy and hyperplasia of myofibres. Recent studies have shown that myostatin can also have an impact on the metabolism. The Mstn inactivation leads to an increase of insulin sensitivity, a decrease of adiposity and a resistance to obesity (McPherron & Lee, J Clin Invest 109(5):595–601, 2002). GASP-1 (Growth Associated Growth and Differentiation Factor Associated Serum Protein) is known to inhibit myostatin. Recently, we have shown that overexpressing Gasp-1 mice present an increase of muscle mass associated with hypertrophy without hyperplasia (Monestier et al. BMC Genomics 13:541, 2012). Compared to knock-out myostatin mice, no reduced adiposity was observed. However, old mutant Gasp-1 mice ([1 year) develop a distinct group of metabolic phenotypes, including an increase of fat mass, an ectopic fat deposition, and a higher glucose rate in serum. They also present a hepatic steatosis. Gasp-1 mice do not seem to be resistant to obesity. Molecular mechanisms involved in this deregulation will be presented. Keywords: Aging, Skeletal muscle lipid deposition, Insulin resistance
P4.14 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 The compact mutation of myostatin influences the expression of skeletal muscle growth regulators Baa´n Julia Aliz1***, Kocsis Tama´s1, Mu¨ller Ge´za2, Keller-Pinte´r Aniko´1, Dux La´szlo´1, Mendler Luca1 ***Candidate for Young Investigator Award
1
Department of Biochemistry, Faculty of General Medicine, University of Szeged, Hungary; 2Egis Pharmaceuticals, 1475 Budapest, Hungary Introduction: Myostatin (Mstn) is an important negative regulator of skeletal muscle growth. Compact (Cmpt) mice, similar to Mstn knockouts, are characterized by hypermuscularity. Cmpt mutation comprises a 12-bp deletion in the propeptide region of Mstn, however, modifier genes are also involved in the Cmpt phenotype. Previously we performed morphological analysis of Cmpt muscles. By now we have created an appropriate genetic control for the Cmpt line and aim to analyze the molecular mechanisms of the Cmpt hypermuscularity in comparison with both BALB/c and genetic control lines. Materials and Methods: Hindlimb muscles of 10–12-week-old male Cmpt and control lines (n = 3–5) were removed and frozen in liquid nitrogen. Total RNA isolation was carried out from the tibialis anterior (TA) muscles using TRI reagent followed by qRT-PCR analysis. Transcript levels of Mstn, its receptor activin receptor IIB (ActRIIB), its potential inhibitor follistatin (Fstn), as well as positive regulators of muscle growth such as insulin-like growth factor 1 (IGF1) and androgen receptor (AR) were analyzed using hypoxantine guanine phosphoribosyltransferase (HPRT) as an internal control. Results: Mstn transcript levels in TA muscle were significantly increased in Cmpt mice compared to both types of controls. The expression of ActRIIB was significantly decreased in the genetic control as well as in Cmpt mice in comparison with the BALB/c ones. Fstn transcript levels showed striking decrease in the genetic controls compared to BALB/c line, however, a further reduction was revealed in Cmpts as well. No differences were detected in the expression of
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126 IGF-1, while a decreasing tendency in AR transcript levels were measured in Cmpt mice compared to both control lines. Conclusion: The modified gene expression of the examined muscle growth regulators may contribute to the hypermuscular phenotype of the Cmpt mice as part of a complex regulatory network. Grant sup´ MOP-4.2.2.A-11-1-KONV-2012-0035 port: TA Keywords: Myostatin, Compact mice, Expression levels of skeletal muscle regulators
P4.15 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Development of a 3-D biomimetic scaffold for human craniofacial skeletal muscle regeneration Shah Rishma1, Knowles Jonathan1, Hunt Nigel1, Lewis Mark2 1 UCL Eastman Dental Institute, London, UK; 2School of Sport, Exercise and Health Sciences, Loughborough University, UK
Background: Current methods used to manage craniofacial skeletal muscle defects are associated with many limitations. Engineered skeletal muscle tissue has the potential to provide an alternative approach with fewer disadvantages. Aim: To develop a 3-D biomimetic scaffold to support craniofacial skeletal muscle regeneration. Materials and Methods: 3-D scaffolds were produced by encasing degradable phosphate glass fibres within a collagen matrix. Following seeding with human craniofacial skeletal muscle-derived cells at 106 cells/ml, scaffolds were incubated for 21 days. Cells were also seeded on aligned degradable glass fibre scaffolds. Macroscopic images were taken with a Canon 300D digital camera, and modulation contrast images were obtained with an inverted microscope used together with a COHU CCD camera and Leica FW4000TZ imaging software. The alamarBlue metabolic assay assessed cell survival. RT-PCR quantified levels of the MyoD1, myogenin and MYH genes. Results: Construct size was approximately 30 mm long and microscopic examination confirmed unidirectional cell alignment. Good cell survival was noted throughout the experimental period and RT-PCR demonstrated upregulation of the myogenic regulatory factors and developmental and adult MYH genes. Compared to the glass fibre scaffolds, the glass fibre-collagen scaffolds had later gene upregulation. Conclusions: Glass fibres and fibres embedded within a collagen matrix can support skeletal muscle formation and maturation and may be used for different applications. The glass fibres may be used as a ‘fast’ cell delivery system providing an extra cell source for muscle fibre regeneration. The glass fibre-collagen scaffolds provided for a sizeable construct with easier handling and the potential for implantation into areas of muscle defect. Further development will focus on the formation of vascular channels within the scaffold as the fibres degrade and utilisation of the fibre ends to support tendon/bone formation. Keywords: Craniofacial skeletal muscle, Phosphate glass fibres, Collagen
P4.16 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Muscle regeneration in cardiotoxin-treated Nrf2 knockout mice Czypicki Ryszard***, Gese Anna, Cies´la Maciej, Kozakowska Magdalena, Jo´zkowicz Alicja, Dulak Jo´zef ***Candidate for Young Investigator Award
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J Muscle Res Cell Motil (2015) 36:71–143 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), encoded by Nfe2l2 gene is a transcription factor regulating expression of several cytoprotective genes with antioxidant properties. Hmox-1, encoding HO1, is a target of Nrf2, and recently we demonstrated the strong effect of HO-1 on myoblasts differentiation. Therefore, here we aimed to investigate the effect of Nrf2 deficiency on skeletal muscle injury and repair after cardiotoxin (CTX) injection. 25 WT and 26 KO mice were injected into gastrocnemius muscles with CTX from Naja mossambica mossambica on day 0. 5 animals per group were sacrificed on 1, 3, 7, 14 and 28 days post treatment. Additionally, 5 WT and 5 KO control mice were sacrificed one day after saline injection. Samples for real-time PCR, western-blot, serum, blood and histological analyses were collected. Shortly after injury, Nrf2 deficient animals exhibited lover level of Hmox1. These changes were accompanied with higher mRNA levels of IL-1b and IL-6, indicating ongoing inflammation. However, in later phases of regeneration higher levels of myogenic factors such as MyoD, Myf5, Myf6 and myogenin were observed in Nrf2 deficient animals than WT mice. Overall, the expression of miR-1, miR-133a, miR-133b, miR-146, miR-206 and miR-378, which are involved in muscle regeneration, do not differ significantly between the genotypes. However, the increase in miR-206 and miR-1 expression in comparison to basal level was higher in WT than Nrf2-/- mice, while miR-378 increase stronger in Nrf2 KO mice. Results obtained to date suggest a role of Nrf2 in the regulation of proliferation and differentiation of muscle cells. Supported by the MAESTRO Grant from the National Science Center (NCN 2012/06/A/NZ1/00004). Keywords: Muscle regeneration, Cardiotoxin injury, Nrf2
P4.17 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Influence of incubation temperature on body and muscle growth in two ecotypes of whitefish Coregonus lavaretus Brandauer Magdalena1***, Stoiber Walter1, Wanzenbo¨ck Josef2, Steinbacher Peter1 ***Candidate for Young Investigator Award
1
Department of Cell Biology, University of Salzburg, Austria; Research Institute for Limnology, University of Innsbruck, Austria
2
Several lakes around the world are inhabited by ecotype pairs of teleost fish that differ in habitat utilisation, spawning behaviour and maximum size (dwarf type and regular type). Such ecotype pairs provide well-suited model systems for study of intraspecific phenotypical diversification. Recent research has demonstrated that the thermal experience of a teleost embryo is likely to become ‘imprinted’, thus having continued effects on the growth of the body and its dominant tissue mass, the axial muscle. However, it is as yet unknown to which extent thermal imprinting due to different spawning site conditions may aid the segregation of small and large forms within the ecotype pairs. This work contributes to clarify this issue by investigating muscle growth in an ecotype pair of Coregonus lavaretus from a lake at the northern rim of the Alps in Austria. Batches of fish of each form were kept at 2 and 6 until hatching and subjected to similar thermal treatment afterwards. We present results from digital assessment of trunk muscle cross section (a reliable
J Muscle Res Cell Motil (2015) 36:71–143 measure of body mass development) undertaken as the initial step of a larger programme also focusing at stem cell behaviour. These results show clearly that fish of the regular form are much smaller when imprinted at thermal conditions typical for the spawning sites of the dwarf form (6 C) than when imprinted at the conditions usually experienced at their own spawning sites (2 C). Surprisingly, the fish of the dwarf form exhibit a similar response pattern to thermal history (2-fish much larger than 6-fish), indicating that in their case, normal spawning site temperature (6 C) is indeed likely to act as a growth limiting factor. This is of major significance to aspects of ecological developmental biology, fisheries biology and from the evolutionary perspective. Keywords: Phenotypic plasticity, Muscle growth, Fish muscle
P4.18 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Developing a protocol for the immunoelectron microscopic localisation of Mef2 in the myotomes of zebrafish embryos Platzl Christian***, Stoiber Walter, Steinbacher Peter
127 broad clinical application, little is known about the effects of ESWT at the cellular and molecular level. Only recently, it has been shown that low energy ESW up-regulates the expression of several growth factors, leading to proliferation and differentiation of various stem/ progenitor cells and to increased blood supply. ESWT may be beneficial for muscle injuries as well. Following the observations described above, we hypothesize that ESWT induces myogenic stem/ progenitor cell activation and together with increased blood supply promotes muscle regeneration processes. The present study investigates, whether ESWT improves the regenerative process of skeletal muscle tissue. For this purpose, M. quadriceps femoris of male Sprague–Dawley rats was injured by injection of cardiotoxin and regeneration processes in ESW-treated and ESW-untreated muscle were compared. Specifically, IHC and western blotting were used to analyze expression of Pax7, MyoD, Myogenin, VEGF and CD31. Results are discussed in the context of ESW effects on regeneration processes in muscle tissue. Keywords: ESWT; Skeletal muscle; Regeneration
P4.20 Muscle development, differentiation, and regeneration, Fri 12:00–14:00
***Candidate for Young Investigator Award Regeneration of skeletal muscle: therapeutic effects of extracorporeal shockwaves Cell Biology, University of Salzburg, Austria
Bergmann Fiona***, Sa¨nger Alexandra Maria, Steinbacher Peter
Over the last decades, the zebrafish Danio rerio has become one of the most important model organisms for the study of vertebrate embryonic development and cell fate decision. In this context, the processes governing the formation of muscle cells were most intensely studied, thereby contributing to the identification of the key molecular regulators of vertebrate myogenesis. However, related immunolabelling work to date is almost entirely based on light microscopy while attempts to localize the regulatory proteins at the ultrastructural level have remained scarce and unsatisfactory. The present work aims to improve upon this situation by testing immunoelectron microscopy procedures for suitability to trace myocyte enhancer factor-2 (Mef2), one of the important transcription factors regulating myogenic differentiation, in zebrafish muscle precursor cells. A set of experiments varying pre-embedding and post-embedding procedures is performed to optimize fixation, infiltration and signal enhancement regimes.
***Candidate for Young Investigator Award
Keywords: Immune electron microscopy, Myogenesis, Zebrafish
P4.19 Muscle development, differentiation, and regeneration, Fri 12:00–14:00
Animal Structure and Function, Cell Biology, University Salzburg, Austria Extracorporeal shockwave therapy (ESWT) is a non-invasive procedure and since some decades used for therapeutic treatment of a variety of different medical issues. This is based on its effects on wound healing supported by proliferative events and neovascularization. Nevertheless, little is known about the effect of ESWT on skeletal muscle tissue, particularly in terms of molecular and cellular. The aim of the study is to investigate the effects of ESW on damaged skeletal muscle tissue and its impact on regeneration processes. Muscle injury is set by cardiotoxin (Naja naja) injection into rat hindlimb muscle (M. quadriceps femoris) and proliferative events are assessed by means of intraperitoneal BrdUapplication. Specifically, the appropriate response of endothelial cells, fibroblasts as well as myogenic progenitor cells is studied via immunohistochemistry. Keywords: ESWT, BrdU, Skeletal muscle
Low energy shock wave therapy of injured skeletal muscle tissue Zissler Angela1***, Zimmermann Reinhold2, Steinbacher Peter1, Sa¨nger Alexandra Maria1 ***Candidate for Young Investigator Award
1
Animal Structure and Function, Cell Biology, University Salzburg, Austria; 2Department of Urology and Andrology, Paracelsus Medical University, Salzburg General Hospital, Austria In the last decade, numerous clinical experiments have shown the beneficial role of low energy extracorporeal shockwave therapy (LEESWT) in the regeneration of various tissues and organs. Despite this
P4.21 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Application of knee braces and socks during postural tasks Landkammer Yvonne1***, Bernecker Robert1, Herfert Ju¨rgen1, Edtinger Sebastian2, Moder Angelika3, Wicker Anton1 1
University Clinic of PM&R SALK, Salzburg, Austria; Gesundheitszentrum Johannesbad, Salzburg, Austria; 3Institut of Diseases PMU, Salzburg, Austria
2
Introduction: Braces are used very often during sport activities because of subjective increase of joint stability. They don’t need to be
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128 seen as a passive tool only with mechanical effects, literature reports influence on sensomotoric function [1–4]. Aim of the study is to evaluate the effect of two different stability devices during postural tasks. Methods: 24 healthy probands complete three valid measurements on each side for three different test situations (without braces, with genumedi knee braces, with CEP socks) standing on the Posturomed platform running with Microswing software in order to stabilize the platform as quick as possible after slackening a lever. The more points achieved, the better the result. Friedman ANOVA and Kendall Coeff.of Concordance were used for statistical analysis. Results: The results show that postural control increases by using braces and socks. On the left leg, there is a higher score between Knee brace and no brace, socks and no brace and even socks and knee braces. On the right leg we can find the same results, but there is no difference between knee brace and socks. Discussion: As described in the literature [1–4] we could find an increase in postural control by using knee braces and cep socks. This effect seems to result from reflex change of sensomotoric function induced by the braces and socks. The best results were achieved, when the participants wore the cep socks. Increasing postural control and injury prophylaxis could be new fields for the use of cep socks, which should be investigated by further research. Keywords: Postural control, Braces, Cep socks References: [1] Birmingham et al. Med Sci Sports Exerc 32(2):304–308, 2000 [2] Cawley et al.Am J Sports Med 19(3):226-233, 1991 [3] Kramer et al. Clin J Sport Med 7(1):32-39, 1997 [4] Jerosch et al. Dtsch Z Sportmed 49:263-269, 1998
P4.22 Muscle development, differentiation, and regeneration, Fri 12:00–14:00 Effect of resistance training on capillary density at slow muscle fibers in diabetic and normal rats Shekarchizadeh Parivash, Karimian Jahangir Isfahan University of Medical Sciences, Iran Introduction: It is well accepted that skeletal muscle conforms to exercise stimulus by increasing capillary density and/or capillary/fiber ratio, but there is less evidence of the effect of resistance training on capillary density in Flexor Hallucis Longus (FHL) and soleus muscle. In this study, we evaluated the effect of resistance training on capillary density in soleus muscles in type 1 diabetic rats. Materials and methods: Thirty-six male rats were divided into four groups: (1) control; (2) diabetic; (3) diabetic trained and (4) control trained (n = 9 each). A single intraperitoneal injection of streptozotocin at a dose of 55 mg/kg was used for induction of diabetes. The rats in the trained group undertook one training session per day for 3 days/week. Training was done with the use of a 1 metre high ladder inclined at 80. After 4 weeks, the plasma nitrite concentrations were measured. Capillary/fiber ratio was determined in soleus muscles by immunohistochemistry. Results: Capillary/fiber ratio in soleus muscle of diabetic group was more than control rats. Resistance training did not alter capillary/fiber ratioin diabetic animals (1.00 ± 0.6 vs. 1.07 ± 0.07, respectively). There was a positive correlation between plasma nitrite and capillary density in soleus muscle (R2 = 0.65). Conclusion: Although resistance training increased plasma NO level, however, it could not improve capillary/fiber ratio in soleus muscle of diabetic animals. Keywords: Capillary density, Diabetes, Exercise
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P5.1 Excitation–contraction coupling, Sat 12:00–14:00 Lethal exertional-strokes in susceptible mice are prevented by drugs used to treat Malignant Hyperthermia in humans Michelucci Antonio1***, De Marco Alessandro1, Pietrangelo Laura1, Paolini Cecilia1, Hamilton L Susan2, Protasi Feliciano1 ***Candidate for Young Investigator Award
1
CeSI – Center for Research on Ageing, UniversityG d’Annunzio, 66013 Chieti, Italy; 2Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
In humans, lethal over-heating episodes are triggered by administration of halogenated anesthetics (a disorder known as Malignant Hyperthermia, MH) and by high temperature and/or strenuous exercise (crises identified as Environmental/Exertional Heat Strokes, EHSs). Whereas MH crises are successfully treated by intravenous administration of dantrolene, to date no pharmacological interventions are available to prevent or reverse EHS. We previously demonstrated that mice carrying a mutation linked to MH in humans (ryanodine receptor type-1 YS mice) and Calsequestrin-1 knockout (CASQ1-null) mice trigger lethal crises when exposed to both halothane and heat. Here we tested the following hypotheses: a) strenuous exercise in challenging environmental conditions is a stress capable of triggering EHS-lethal episodes; b) drugs used to treat MH crises in humans (dantrolene or its analog azumolene) can prevent such exertion-induced strokes. When RYR1-YS (n = 10) and CASQ1-null (n = 14) mice were subjected to an exertional-stress protocol (executed on a treadmill at 34 C, 30–40 % of humidity) which was tolerated by WT animals (n = 10), their mortality was dramatically increased (80 and 79 %, respectively). During the exertional protocol RYR1-YS and CASQ1-null mice showed: a) rise in core temperature (respectively 5.3 ± 0.2 C and 4.9 ± 0.3 C) significantly higher than WT mice (2.6 ± 0.2 C); b) high percentage of skeletal fibers presenting severe structural damage (respectively 99 % and * 64 %); and c) elevated blood levels of creatine-kinase, K+, and Ca2+ (suggestive of rhabdomyolysis). Interestingly, pre-treatment of animals with dantrolene or azumolene prevented completely exertional-strokes in both RYR1-YS (n = 7) and CASQ1-null (n = 8) mice, strongly suggesting that: a) exertional-crises may share common molecular mechanisms with anesthetic-induced MH; b) drugs used in the operating room to treat acutely MH crises should be considered for treatment of environmental/ exertional heat strokes. Keywords: Ryanodine receptors, Calsequestrin, Dantrolene
P5.2 Excitation–contraction coupling, Sat 12:00–14:00 Contractile apparatus uses glycogen from specific subcellular locations: Evidence of cytosolic compartmentalization between glycogen metabolism and energy consumption in skeletal muscle Nielsen Joachim, Christensen Peter T, Ørtenblad Niels Department of Sports Science and Clinical Biomechanics, University of Southern Denmark The link between glycogen and fatigue in skeletal muscle has been suggested to originate from a specific subcellular location. However, factors controlling the localization-dependent regulation of glycogen during muscle work remain unknown. Here we used electron microscopy to quantify glycogen content of intermyofibrillar (IMF), intramyofibrillar (Intra) and subsarcolemmal (SS) localizations within
J Muscle Res Cell Motil (2015) 36:71–143 muscle fibres from rat soleus muscles, which were stimulated electrically in vitro with or without pre-incubation with the myosin ATPase inhibitors BTS and blebbistatin. IMF glycogen (medians and IQR) was higher in muscles stimulated with inhibited myosin ATPases (Inhib) (6.01 (4.63:8.01) lm3 lm-3 103, n = 39 fibres from 13 muscles) compared to normally contracting stimulated muscles (Norm) (5.13 (2.45:6.17), n = 36 (12), P = 0.001), but still lower than resting control muscles (Con) (8.53 (6.66:10.79), n = 27 (9), P = 0.003). While Intra glycogen also was higher in Inhib muscles (4.68 (2.17:5.67) lm3 lm-3 103) compared to Norm muscles (0.69 (0.25:5.79), P = 0.010), it was not different from Con (3.89 (2.49:4.71), P = 0.685). In contrast, inhibition of the myosin ATPases during stimulation did not affect the utilization of SS glycogen, which was comparable lower than Con muscles (5.91 (4.07:9.54) lm3 lm-2 103) in both Norm muscles (4.97 (3.09:6.22), P = 0.026) and Inhib muscles (4.57 (3.61:6.54), P = 0.042). These findings demonstrate the existence of a cytosolic compartmentalization of glycogen metabolism within skeletal muscle fibres and indicate that energy delivery from glycogen breakdown to the myosin ATPases derives mainly from Intra glycogen, to a less extent from IMF glycogen, and not from SS glycogen. Keywords: Skeletal muscle, Myosin ATPases, Glycogen metabolism
P5.3 Excitation–contraction coupling, Sat 12:00–14:00 CB1 cannabinoid receptors are involved in the regulation of excitation–contraction coupling in mammalian skeletal muscle Ola´h Tama´s1***, Bodna´r Do´ra1, To´th Adrienn1, Fodor Ja´nos1, Kova´cs Adrienn1, Farkas Anna1, Na´dro´ Bı´borka2, Szentesi Pe´ter2, Csernoch La´szlo´ ***Candidate for Young Investigator Award
1
Faculty of Medicine, Department of Physiology, University of Debrecen, Hungary; 2University of Debrecen, Faculty of Medicine, Department of Physiology, Debrecen, Hungary The presence of CB1 cannabinoid receptors (CB1R) has been shown in skeletal muscle, but it is yet to be cleared whether they have any significance in the regulation of contractions. CB1-knockout (CB1KO) mice showed hypoactivity, however, it is questionable whether this was solely due to effects on the central nervous system or impairment of muscle function also contributes. Our aim was to investigate the role of CB1R in mammalian skeletal muscle, and the effects of cannabinoid drugs on Ca2+ transients. Running ability of control and CB1-KO mice was studied by activity-wheel-tests while in vivo muscle force of the animals was measured by grip-tests and hang-tests. Ca2+ transients evoked by KCl-depolarization in the presence and absence of cannabinoid agonists were investigated on flexor digitorum brevis (FDB) fibers of control and CB1-KO mice. CB1-KO mice performed worse as compared to control in all behavior tests applied. In contrast, depolarization-evoked Ca2+ transients were significantly higher in FDB fibers isolated from CB1KO mice (848 ± 98 nM, n = 47) compared to control (376 ± 60 nM, n = 32, p \ 0.01). When KCl-evoked Ca2+ transients were repeated on control FDB fibers in the absence and presence of the CB1 agonist WIN55,212 (WIN), the transients after WIN treatment were significantly smaller (44 ± 7 % of the first transient, n = 27) than in untreated (79 ± 5 % of the first transient, n = 32, p \ 0.01) fibers. Our observations suggest that CB1R-mediated signaling contributes to the regulation of excitation–contraction coupling and skeletal muscle contractions. Nevertheless, the effects mediated by the absence of CB1R in the central nervous system on the inferior muscle
129 performance of CB1-KO mice in vivo cannot be ruled out. These results can contribute to the identification of the side effects of medically used cannabinoid drugs on skeletal muscle. Grants: ´ MOP 4.2.4. A/2-11-1-2012-0001 National Excellence Program. TA Keywords: Skeletal muscle, CB1 cannabinoid receptor, Excitation– contraction coupling
P5.4 Excitation–contraction coupling, Sat 12:00–14:00 On the interdependency of a1s and b1a for trafficking and excitation–contraction (EC-) coupling in skeletal muscle Polster Alexander1,2, Linde Nina F2, Beam Kurt G3, Papadopoulos Symeon2 1
Department of Physiology and Biophysics, University of Colorado – Anschutz Medical Campus, USA; 2Institute of Vegetative Physiology, Medical Faculty of the University of Cologne, Germany; 3Department of Physiology and Biophysics, University of Colorado – Anschutz Medical Campus, USA The functional expression of ec-coupling proteins involves general and skeletal muscle-specific mechanisms, which remain to be elucidated. Both, the b1a subunit and the pore forming a1s subunit (Cav1.1) are required for focal targeting of the two proteins to the junctional membrane and for proper skeletal muscle ec-coupling. What is the contribution of either subunit? b1a expressed in myotubes lacking a1s (dysgenic) is cytoplasmic, and a1s is retained in the ER when the b subunit is absent or when a b interacting region (AID) within the cytoplasmic I-II linker of a1s is deleted (a1del). The mere linkage between b1a and a1s is not sufficient: We covalently attached b1a to the N-terminus of either a1s or a1sdel and expressed the respective plasmid in dysgenic myotubes. While b1a-a1s was fully functional, b1a-a1sdel was still retained in the ER. However, co-expression of b1a-a1sdel with I-II loop peptide or with intact a1s had a dramatic effect, where fluorescently tagged b1a-a1sdel displayed a stripe and dot-like appearance close to the cell surface, though still not functional. Dot-like b1a translocation towards the surface membrane was also observed in the absence of any a1s, when b1a was either co-expressed with I-II loop peptide or as b1a–I–II loop fusion protein. Thus, interaction with the I-II loop seems to unveil a b1a driving force towards the membrane, which can even include the otherwise trafficking deficient, membrane-bound a1sdel. Interestingly, crystal structure studies on the b– I-II loop peptide interaction reported no significant changes in the b structure. Currently, we are investigating the impact of residue exchanges within the I-II loop, at positions reported to be critical for the b–I–II loop interaction. So far, single residue exchanges had no effect on a1s trafficking or ec-coupling. Supported by a DFG Grant to SP (PA801/6-1). Keywords: Excitation–contraction coupling, Skeletal muscle dihydropyridine receptor, Calcium channel
P5.5 Excitation–contraction coupling, Sat 12:00–14:00 Glycolytic derived ATP is essential for muscle fibre excitability and Na,K-ATPase activity in the transverse tubular system of skeletal muscle fibers Jensen Rasmus***, Nielsen Joachim, Ørtenblad Niels ***Candidate for Young Investigator Award
Department of Sports Science and Clinical Biomechanics, University of Southern Denmark
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130 This study examined whether Na,K-ATPase function in the T-system is dependent on ATP derived via the glycogenolytic-glycolytic pathway under conditions with normal cytoplasmic ATP concentration. Single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the T-system permitting action potential triggered twitch and tetanic force (F) responses. The T-system can then be depolarized and/or the glycogen phosphorylase (GP) activity inhibited by DAB (2 mM) or CP-316,819 (10 lM). Tetanic F decreased by 28 ± 3 % when depolarized to *-67 mV. In the fully polarized fibres, inhibition of GP, decreased tetanic F by 33 ± 3 % (CP, P \ 0.001) and 12 ± 4 % (DAB, P = 0.004), which was further pronounced in the depolarized fibre (CP 86 ± 3 % and DAB 61 ± 10 %, P \ 0.001). Similar results were obtained with twitch responses. To evaluate the ability of the T-system to repolarize following a depolarization, double pulses were applied, with various time spacings (1–20 ms). This repriming period (RP) before the second pulse elicits a larger F response is dependent on the Na,KATPase activity. In a fully polarized fibre, the RP was 4 ± 0.2 ms (20 fibres), which was increased to 6 ± 0.5 ms (19 fibres, P \ 0.001) in the depolarized fibre, indicative of a longer time for the Na,K-ATPase to repolarize the fibre. There was no effect of GP inhibition on RP in the fully polarized fibre. However, when the T-system was depolarized and GP inhibited, the RP increased to 8 ± 1.2 ms (P \ 0.001) (13 fibres). These findings demonstrate that the triad junction is a highly restricted microenvironment, where glycolytic resynthesis of ATP is critical to meet the high demand of the Na,K-ATPase and maintain muscle excitability. Keywords: T-system excitability, Glycogen metabolism, Single muscle fibers
P5.6 Excitation–contraction coupling, Sat 12:00–14:00 Mechanisms underlying prolonged low-frequency force depression Daiki Watanabe1***, Keita Kanzaki2, Satoshi Matsunaga3, Masanobu Wada1 ***Candidate for Young Investigator Award
1
Graduate School of Integrated Arts and Sciences, Hiroshima University, Japan; 2Faculty of Food Culture, Kurashiki Sakuyo University, Japan; 3Faculty of Education and Culture, Miyazaki University, Japan One of the form of muscle fatigue is characterized by greater loss of force at low compared to high stimulation frequency and prolonged recovery, which is called ‘‘prolonged low-frequency force depression (PLFFD)’’. The purpose of this study was to investigate the mechanisms of PLFFD with focus on the functional behavior of myofibril and ryanodine receptor 1 (RyR1; a Ca2+ release channel of sarcoplasmic reticulum). Intact rat gastrocnemius (GS) muscles were electrically stimulated via the sciatic nerve until force was reduced to * 50 % of the initial. The superficial region of GS muscles was excised 30 min after the end of fatiguing stimulation and used for skinned fiber and biochemical experiments. Skinned fibers were activated in heavily buffered Ca2+ solutions and in caffeine solutions to assess myofibrillar Ca2+ sensitivity and RyR1 caffeine sensitivity, respectively. A Ca2+ concentration at half-maximum force was unaltered, whereas a caffeine concentration at 10 % of maximum force (caffeine threshold) was increased in stimulated fibers. Biochemical analyses on whole GS muscles indicated that fatiguing stimulation elicited appreciable increases in the level of S-glutathionylation of troponin I (TnI) and in the amounts of non-
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J Muscle Res Cell Motil (2015) 36:71–143 phosphorylated RyR1. Only when skinned fibers dissected from quiescent muscles were treated with protein phosphatase 1 and muscle homogenate, the caffeine threshold was increased. These findings suggests that, at least in an in situ model for muscle fatigue, i) PLFFD is caused by impaired RyR1 Ca2+ release, but not by decreased myofibrillar Ca2+ sensitivity, ii) S-gluathionylation that occurs in TnI may offset the depressive effects on myofibrillar Ca2+ sensitivity, and iii) impaired RyR1 function may relate to dephosphorylation of RyR1. Keywords: Muscle fatigue, Ryanodine receptor phosphorylation, Skinned fibre
P5.7 Excitation–contraction coupling, Sat 12:00–14:00 An anti-oxidant treatment prevents/reduces formation of cores in a mouse model of Central Core Disease De Marco Alessandro1, Michelucci Antonio1, Hamilton L Susan1, Boncompagni Simona2, Protasi Feliciano1 1 CeSI – Center for Research on Ageing, UniversityG d’Annunzio, 66013 Chieti, Italy; 2Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
Central core disease (CCD) and Malignant Hyperthermia (MH) are related skeletal muscle diseases often linked to mutations in the ryanodine receptor type-1 (RYR1) gene, encoding for the sarcoplasmic reticulum (SR) Ca2+ release channel. CCD is characterized by hypotonia, proximal muscle weakness and presence of amorphous regions of myofibrillar disorganization (cores) lacking mitochondrial activity. In humans, the Y522S (YS) mutation in RYR1 is associated with MH susceptibility with cores. Knock-in heterozygous RYR1-YS mice are viable, suffers of MH, and develop structural cores: previous studies indicated that oxidative stress likely represent a key event in the myopathic cascade. We tested the hypothesis that reducing oxidative stress prevents/reduces structural damage and consequent formation of cores. We treated RYR1-YS mice from 2-to-4 months of age with a potent anti-oxidant, N-acetylcysteine (NAC) provided ad libitum in drinking water (1 % w/v), and analyzed skeletal muscle preparations by histology, electron and confocal microscopy. Analysis of extensor digitorum longus (EDL) muscles of 4-month-old un-treated mice confirmed the presence of severe structural alterations—previously defined as un-structured and contracture cores—in a significant percentage of fibers: respectively 28 % and 17 % presented un-structured and contracture cores (3 muscles; 149 fibers). On the other hand, in agematched NAC-treated mice frequency of fibers presenting the two types of alterations was greatly reduced: only 2 and 5 %, respectively (3 muscles; 335 fibers). Being mitochondria the main source of oxidative stress, we evaluated the grade of mitochondrial damage in muscle fibers of RYR1-YS mice. After NAC treatment, we found a reduction in the frequency of highly damaged mitochondria (from 23 % to 13 %) and in mitochondrial swelling of about 24 %. Taken together our data suggest that oxidative stress plays an important role in the development of cores in RYR1-YS mice. Keywords: Ryanodine Mitochondria
receptors,
Sarcoplasmic
reticulum,
P5.8 Excitation–contraction coupling, Sat 12:00–14:00 Does cooperativity underlie mechano-calcium feedback in myocardium or does it not? Examination by means of mathematical modeling
J Muscle Res Cell Motil (2015) 36:71–143 Katsnelson Leonid B, Shikhaleva Elena Lab of Mathematical Physiology, Institute of Immunology and Physiology of the RAS, Ekaterinburg, Russia Mechano–calcium feedbacks (MCFs) are important regulatory links for the excitation–contraction Coupling (ECC) in the myocardium. They reveal themselves e.g. in load-dependent relaxation in isotonic twitches, in inactivating effects of quick deformations during isometric twitches, and in the specific responses of calcium transients and action potentials on the mechanical conditions of these twitches. In general, MCFs provide a fine tuning of electrical and calcium activation to the mechanical conditions of myocardium contractions. There is much experimental and theoretical evidence that cooperative dependence of CaTnC kinetics on the cross-bridge concentration ([Xb]) is a key mechanism underlying MCFs in the intact cardiomyocytes. Nevertheless, a discrepancy seems to arise between this role of cooperativity in the intact myocardium behavior and experimental data obtained on skinned heart muscles showing that mechanical conditions significantly affect only calcium sensitivity of the ‘pCa-force’ relationship but practically do not affect its Hill coefficient of cooperativity (see e.g. Konhilas et al. J Physiol 544(Pt 1):225–236, 2002). These findings prompted many authors to doubt that cooperativity may be a mechanism contributing to the mechano-calcium feedbacks. Trying to overcome this discrepancy we suggest now a refined concept of XbCaTnC cooperativity that seems to explain both mechano-dependence of calcium activation in the intact myocardium and the above mentioned data obtained on the skinned muscles. Briefly speaking, the concept allows the cooperativity to reveal itself differently in steady-state and transitional processes. Equations formalizing this concept are incorporated now in our mathematical model used many times before to analyze ECC in the intact myocardium (see e.g. Katsnelson et al., Prog Biophys Mol Biol 107(1):81–89, 2011). The adjusted cooperativity does enable us to overcome the described above discrepancy at least in the framework of the model. Supported by RFBR Grant 13-04-00365. Keywords: Myocardium, Calcium activation, Mathematical model
P5.9 Excitation–contraction coupling, Sat 12:00–14:00 Effects of ranolazine and nicardipine on the relaxation of precontracted isolated rabbit aortic rings Malavaki Christina1***, Aidonidis I2, Stefanidis Ioannis1, Karatzaferi Christina3, Hatziefthimiou A2 ***Candidate for Young Investigator Award
1
Division of Nephrology Medical School, University of Thessaly, Larissa, Greece; 2Laboratory of Physiology, Faculty of Medicine, University of Thessaly, Larissa, Greece; 3Muscle Physiology & Mechanics Group, DPESS, University of Thessaly, Trikala, Greece
Patients with vasospastic angina are prescribed slow-release dihydropyridine-type Ca2+ antagonists to prevent coronary artery spasm, however these don’t protect against spasm-accompanied arrhythmias. Ranolazine (RAN), has been found to exert antiarrhythmic actions in addition to vasorelaxation. We hypothesized that there may be additional benefits from a combination of RAN with a nifedipine-like Ca2+ antagonist (nicardipine, NIC). We investigated the effect of 10-9 M to10-4 M RAN, 10-9 M to 105 M NIC and their combination (3 lL RAN and 3 lM NIC) on aortic rings of adult NZ rabbits pre-contracted with 2 lL of phenylephrine (Phe). The study received ethics approval (IEC, Medical School, UTh). Treatment with RAN or NIC for 25 min induced dose-dependent relaxation. The EC50 values for RAN or NIC were 6.5 9 10-6 M and 1.4 9 10-5 M respectively (N = 4 animals). Treatment with 3 lL RAN or NIC up to 65 min revealed that both agents had a biphasic relaxant effect. At 40 min
131 of incubation (earlier phase), the effect of RAN or NIC on relaxation was 26.4 ± 5 % and 21.0 ± 5.3 % respectively, and 38.8 ± 3.6 % for their combination (N = 5; P \ 0.05, t test compared to the effect of NIC alone). At 50–65 min the effect of NIC prevailed and tended to overlap with values of the combination treatment. In conclusion, the combination of ranolazine and nicardipine resulted in enhanced relaxation in Phe-precontracted rabbit aortic ring preparations, supporting a possible synergistic effect. Acknowledgements: We thank I. Makantasis for technical support. Christina Malavaki was supported by ‘‘IKY Fellowships of Excellence for Postgraduate Studies in Greece-Siemens Program’’. Keywords: Ranolazine, Nicardipine, Contractility
P5.10 Excitation–contraction coupling, Sat 12:00–14:00 The effect of follistatin on the calcium homeostasis and differentiation of the C2C12 skeletal muscle cells Fodor Ja´nos1, To´th Adrienn1, Vincze Ja´nos1, Ola´h Tama´s1, o2, Csernoch La´szlo´1 Dienes Beatrix1, Za´dor Ern} 1 Faculty of Medicine, Department of Physiology, University of Debrecen, Hungary; 2Faculty of Medicine, Department of Biochemistry, University of Szeged, Hungary
Myostatin (MSTN), a member of the transforming growth factor b superfamily has emerged as a negative regulator of skeletal muscle growth. During embryogenesis, myostatin is exclusively expressed in skeletal muscle to control the differentiation and proliferation of the myoblasts. It mediates the cell signaling cascade through activin receptors in the muscle. Follistatin (FS) is a high affinity activin-binding protein that can act as an activin antagonist. In our experiments we applied FS and the effect of the elimination of activin A signaling pathway on the Ca2+homeostasis and the expression pattern of the key proteins involved in the differentiation of C2C12 skeletal muscle cells was studied. Functional experiments were performed on differentiated C2C12 myotubes by measuring the changes in [Ca2+]i following the stimulation by KCl or caffeine. The amplitude of the caffeine-induced Ca2+-transients were significantly higher in FS-treated cells (378 ± 32 nM) as compared to control (241 ± 30; p = 0.004;) in the presence of normal [Ca2+]e. While the application of KCl did not modify the amplitude of the Ca2+-transients, the Ca2+-uptake capacity of the sarcoplasmic reticulum assessed as the uptake rate of the calcium pumps (SERCA) significantly increased (340 ± 16 lM/s vs. 280 ± 11 lM/s; p = 0.007 in FS-treated and control cells, respectively). Following FS treatment changes in expression of SERCA were also detected. Additionally, expression of MSTN, Akt and P-Akt were significantly altered during differentiation. Our results suggest that the application of FS increased the Ca2+-influx via store operated calcium entry when RyRs were activated with caffeine. Furthermore, the altered MSTN and P-Akt expression could be responsible for the increased muscle differentiation. This work was supported by ´ MOP-4.2.1/B-09/1/KONV-2010-0007 Grants: OTKA NN-107765, TA ´ MOP-4.2.2/B-10/1-2010-0024, Bo´lyai Research Scholarship of and TA the Hungarian Academy of Sciences to J.F. Keywords: Myostatin, C2C12 cell, Follistatin
P5.11 Excitation–contraction coupling, Sat 12:00–14:00 JP-45 variants as functional modifiers of the MH phenotype Sztretye Mo´nika1,2***, Rokach Ori1, Vincze Ja´nos2, Csernoch La´szlo´2, Treves Susan1, Zorzato Francesco1 ***Candidate for Young Investigator Award
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Departments of Anesthesia and Biomedizin, Universita¨tsspital Basel, Switzerland; 2Department of Physiology, University of Debrecen, Hungary
Skeletal muscle excitation–contraction coupling (ECC) is operated by a macromolecular complex comprising the voltage-dependent L-type Ca2+ channel (DHPR), the ryanodine receptor (RyR), calsequestrin (Casq) and other auxiliary proteins in the contact region between the T-system and the SR membrane, such as junctophilin, triadin, junctin and JP45. JP45 (encoded by JSRP1) is an integral protein constituent of the skeletal muscle SR interacting with the DHPR and Casq. Several JSRP1 variants have been found in the human population and two of these (G150A and P108L) have been shown to downregulate DHPR activity (Yasuda et al. Hum Mutat 34(1):184–190, 2012). Alteration of JP45 signaling may influence the phenotype of malignant myperthermia (MH) individuals, the latter being a pharmacogenetic potentially lethal reaction occurring in predisposed individuals during anaesthesia. Our working hypothesis is that the presence of JP45 polymorphic variants may explain the variable phenotype seen in MH patients carrying the same mutation. In addition JP45 variants may counteract the hypersensitivity of ECC caused by RyR1 mutations (Andronache et al. Proc Natl Acad Sci USA 106(11):4531–4536, 2009). To test our hypothesis we generated RyR1Y522S knock-in mice on JP45 null background. FDB fibres from the RYRY522S knock-in mice on JP45 KO background, were reconstituted via in vivo electroporation with pDsRed tagged wild type cDNA or the cDNA carrying either variant of JP45. Intact fibers isolated from the FDB muscles were loaded with the calcium-sensitive fluorescent dye mag-fluo-4 AM, stimulated with brief suprathreshold pulses (5 ms) to initiate action potentials, and the accompanying changes in mag-fluo-4 fluorescence were detected on a confocal microscope. Preliminary data suggest that JP45 variants affect the calcium transients of the FDBs. We propose that the presence of JP45 polymorphic variants fine tune skeletal muscle ECC and we hope to unveil a signaling pathway which could be targeted by new drugs to be used prophylactically in MH patients.
J Muscle Res Cell Motil (2015) 36:71–143 as controls. Of the main regulatory proteins, expressions of phospho protein kinase B (P-Akt), myogenic differentiation factor (MyoD), stromal interaction molecule 1 (STIM1), calsequestrin (CSQ), and calcineurin were confirmed at the protein level, while myostatin and myocyte-enriched calcineurin-interacting protein (MCIP1.4) were identified at mRNA level. Quantitative analysis of the Western blots normalized to actin also confirmed the significant alterations in CSQ, STIM1, P-Akt, and calcineurin expression detected in the clones as compared to control. Furthermore, to examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+transients were evoked by the applications of 120 mM KCl. Significantly higher [Ca2+]i was measured in the 20th and 40th seconds after the beginning of KCl application (112.3 ± 3.2 nM, and 110.4 ± 3.1 vs. 150.3 ± 6.5 nM and 134.9 ± 4.7, in control and in the clone C1 respectively) indicating decreased Ca2+ uptake capability of the SERCA pumps. This maximal pump rate was 453.6 ± 41.01 vs. 143.9 ± 24.01 lM/s, in control and in the clone C1. These results suggest that SERCA1b plays an essential role in the regulation of [Ca2+]i and ab ovo gene silencing results in decreased skeletal muscle differentiation. This work was supported by Grants: ´ MOP-4.2.1/B-09/1/KONV-2010-0007 and OTKA NN-107765, TA ´ MOP-4.2.2/B-10/1-2010-0024, Bo´lyai Research Scholarship of TA the Hungarian Academy of Sciences to J.F. Keywords: SERCA1b, C2C12 cell, Calcium homeostasis
P5.14 Excitation–contraction coupling, Sat 12:00–14:00 Characterisation of Junctophilin 1 (JPH1) expression in rabbit skeletal muscle. English Kathleen K***, Mackrill John J ***Candidate for Young Investigator Award
Keywords: Skeletal excitation–contraction coupling, Malignant hyperthermia, JP45 Department of Physiology, University College Cork, Ireland
P5.12 Excitation–contraction coupling, Sat 12:00–14:00 Investigation of the role of SERCA1b in the calcium homeostasis of C2C12 skeletal muscle cells To´th Adrienn1***, Fodor Ja´nos1, Vincze Ja´nos1, Ola´h Tama´s1, o3, Csernoch La´szlo´1 Juha´sz Tama´s2, Za´dor Ern} ***Candidate for Young Investigator Award
1
Faculty of Medicine, Department of Physiology, University of Debrecen, Hungary; 2Faculty of Medicine, Department of Anatomy, Histology and Embryology, University of Debrecen, Hungary; 3 Faculty of Medicine, Department of Biochemistry, University of Szeged, Hungary The neonatal isoform of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1b) is a dominant Ca2+ pump in fibers of regenerating muscle. Our aim was to study the role of SERCA1b during skeletal muscle differentiation and to provide a detailed understanding of role in Ca2+ homeostasis. SERCA1b protein synthesis has been suppressed using a specific shRNA sequence. Clones with decreased SERCA 1b expression were selected for additional experiments. Scrambled shRNA transfected and parental cells were used
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Junctophilins (JPH) are transmembrane proteins expressed in skeletal muscle, heart and neuronal tissue. In skeletal muscle, the predominant isoform is JPH1, while JPH2 is found in both skeletal and cardiac muscles. JPHs function in maintaining the close apposition of the plasma membrane and endoplasmic or sarcoplasmic reticulum. In skeletal muscle, JPH1 interacts with the type 1 ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) while helping to maintain triad structure, and thus play a crucial role in regulating calcium ion release during the process of excitation– contraction coupling (EC coupling) (Golini et al. J Biol Chem 286(51):43717–43725, 2011). To characterise JPH1 levels in diverse skeletal muscles, western blotting analyses of latissimus dorsi, diaphragm, intercostal, extensor digitorum longus (EDL) soleus, gastrocnemius, vastus lateralis (VL), sternomastoid and sternohyoid muscle from rabbit was performed using the monoclonal antibody, previously shown by our group to recognise JPH1 (English et al. Cal Sig 1:1–6, 2014). The results show that the highest amount of JPH1 was found in the latissimus dorsi, followed by the VL and EDL muscles, while the diaphragm had much lower quantities when normalised to total protein per lane. This indicates that JPH1 expression might be differentially regulated depending on muscle function, and has implications for the regulation of EC coupling in different muscle types, as well as the future study of JPH1 in skeletal muscle. Keywords: Junctophilin 1, EC coupling, Skeletal muscle
J Muscle Res Cell Motil (2015) 36:71–143
P6.1 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Ahnak deficiency causes sarcomeric defects and mitochondrial abnormalities in murine heart and skeletal muscle Suhr Frank1***, Bloch Wilhelm2, Morano Ingo1 ***Candidate for Young Investigator Award
1
Institute of Molecular Muscle Physiology, Max Delbruck Center for Molecular Medicine, Berlin-Buch, Germany; 2Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Cologne, Germany
Ahnak is localized in cardiac and skeletal muscle fibers at costameric sites, interacts with dysferlin, shows co-localizations with vinculin, but not with myomesin in skeletal muscles and is involved in cardiac Ca2+ handling. Ahnak is implicated in skeletal muscle dystrophy LGMD2B, is not involved in sarcolemmal repair processes and might function as an elastic bridge between costameres and Z-lines. Thus, Ahnak is critical for cardiac and skeletal muscle integrity. To shed further light on Ahnak’s roles, we used an Ahnak-deficient (Ahnak-/-) mouse strain to study cardiac and skeletal muscle integrities. Ahnak-/- mice were generated and proven by PCR and western blot. We studied cardiac and skeletal muscle integrity at ultrastructural levels by transmission electron microscopy (TEM). Succinat dehydrogenase (SDH) staining and OXPHOS western were performed to evaluate mitochondrial phenotypes. We also performed functional contraction analyses of isolated cardiac and skeletal muscle fibers. TEM revealed that subsarcolemmal Z-lines were perpendicularly aligned to neighboring sarcomeres suggesting loss of proper Z-line linkage to the sarcolemma. Compared to WT, Ahnak-/- quadriceps (Quf) muscles showed stretched sarcomeres (sarcomere length WT range: 2.2 to 2.8 lm, sarcomere length Ahnak-/- range: 3.4 to 4.1 lm) as well as highly disorganized Z-lines and M-bands. Gastrocnemius (Gas) muscles showed abnormal accumulations of mitochondria beneath sarcolemmas and almost 4-fold higher SDH intensities. In Ahnak-/- cardiac muscle, mitochondria showed significantly increased numbers, heterogeneous sizes, and abnormally shaped structures. Interestingly, isolated Ahnak-/- skeletal muscle fibers showed increased transverse stiffness. Our data show that Ahnak determines cardiac and skeletal muscle structural integrity. We also show that Ahnak is involved in mitochondrial regulation leading to the hypothesis that Ahnak has much more profound functions than currently known. Keywords: Ahnak, Skeletal muscle, Cardiac muscle
P6.2 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Biochemical kinetic analysis of the hypertrophic cardiomyopathycausing R403Q mutation in the human b-cardiac myosin motor domain Ujfalusi Zoltan1***, Velazquez Carlos Vera2, Geeves Michael A1, Leinwand Leslie A2 ***Candidate for Young Investigator Award
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School of Biosciences, University of Kent, UK; 2Department of Molecular Cellular and Developmental Biology, University of Colorado, USA Inherited cardiomyopathies are a serious health problem in all age groups, and are the leading cause of sudden death in young people.
133 They affect 1 in 500 individuals and approximately 40 percent of such cardiomyopathy-causing mutations are found in the motor protein b-cardiac-myosin. R403Q was the first human HCM mutation identified 20 years ago, yet we still have little molecular explanation of how this mutation causes the disease, despite numerous attempts to study its impact on the protein. Part of the problem is access to human b-cardiac myosin with the R403Q mutation. Here we report biochemical kinetics of recombinant human b-cardiac R403Q myosin motor-domain produced in mouse C2C12 muscle cells. We compared the R403Q mutation with our previous data on the wild-type (WT) b-myosin motor (Deacon et al. Cell Mol Life Sci 69:4239–42355, 2012). This is the first time the effectof the mutation on biochemical kinetics has been examined in the relevant human myosin background. Surprisingly, few kinetic parameters are altered by the mutation. ATP and ADP binding to both the motor domain and the actin motor domain complex are changed by less than 20 %. The one parameter showing a major alteration is the affinity of the motor for actin in the rigor-state which was reduced 5–10 fold. This may not be unexpected since the R403 is the actin binding site on myosin. Keywords: Human b-cardiac myosin, R403Q, Cardiomyopathy
P6.3 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Myocyte force measurements and cardiac hemodynamics in transgenic mice with a gain-of-function mutation in myosin binding protein-C Van Dijk Sabine J1, Bezold Kristina L2, Harris Samantha P1 1
University of Arizona, USA; 2Stanford University, USA
Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Effects of cMyBP-C on contraction are mediated in part by limiting the interactions of actin and myosin to slow myocyte shortening velocity and power output. Although interactions with myosin S2 on the thick filament have been proposed as a way in which cMyBP-C could limit shortening velocity (e.g., by creating a drag force on myosin heads), interactions of cMyBP-C with actin could also account for slowed shortening velocity. For instance, cMyBP-C could create a drag that opposes filament sliding by transiently linking thick and thin filaments together. To explore this possibility we created transgenic mice that express a mutant cMyBP-C with a point mutation (L348P) located in a conserved sequence within the regulatory M-domain that increases cMyBP-C binding to actin in vitro (Bezold et al. J Biol Chem 288(30):21496–505, 2013). We reasoned that if the mutation also enhanced binding to actin in sarcomeres then shortening velocity would be slowed in myocytes from L348P mice and that L348P hearts would show diastolic function due to increased myocardial stiffness. Results show that transgenic mice expressing the L348P mutation are viable and that L348P cMyBP-C is expressed in sarcomeres. However, hearts from L348P have significantly enlarged left atria consistent with diastolic dysfunction. Permeabilized myocytes from transgenic mice showed altered force production including reduced maximal force and enhanced Ca2+ sensitivity of tension. Shortening velocity and power output were significantly reduced whereas passive stiffness and myocyte visco-elasticity were significantly increased. Together these data are consistent with the idea that cMyBP-C creates an internal load in the sarcomere by binding to actin. This work supported by NIH R01 HL080367 (SPH) and AHA pre-doctoral (KLB) and postdoctoral (SJvD) fellowships. Keywords: Myosin binding protein-C, Transgenic mice, Myocyte
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P6.4 Heart muscle & cardiomyopathy, Sat 12:00–14:00 TPK1 deficiency is a novel deficiency in thiamine metabolism with neurologic and rarely cardiac manifestation Matakovic´ Lavinija1***, Feichtinger Rene´ G1, Zimmermann Franz A1, Banka Siddharth2, Freisinger Peter3, Prokisch Holger4, Sperl Wolfgang1, Mayr Johannes A1 ***Candidate for Young Investigator Award
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Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria; 2Manchester Academic Health Science Centre, University of Manchester, UK; 3Department of Pediatrics, Klinikum Reutlingen, Germany; 4Institute of Human Genetics, Helmholtz Center Munich, Neuherberg, Germany The deficiency of thiamine pyrophosphokinase (TPK, EC 2.7.6.2.) results from mutations in the TPK1 gene, which leads to an inability to convert thiamine into the active cofactor, thiamine pyrophosphate (TPP). Lack of TPP affects the mitochondrial energy metabolism at the level of pyruvate dehydrogenase and a-ketoglutarate dehydrogenase (aKGDH, EC 1.2.4.2), the latter is necessary for carbohydrate and fat oxidation. We identified TPK1 deficiency in 5 families with different genetic cause. Clinically they presented with episodic encephalopathy and progressive neurologic dysfunction manifesting as gait disturbances, ataxia, dystonia, and spasticity. Elevated levels of alpha-ketoglutaric acids were found in urine of most TPK1 patients reflecting aKGDH deficiency. Left ventricular hypertrophic cardiomyopathy was found in one. Recently two novel homozygous mutations were found c.664G [ C (p.Asp222His) leading to decreased TPK1 protein stability, but a high residual enzymatic activity and c.479C [ T (p.Ser160Leu) that interferes with TPK dimerization, leading to drastically decreased enzymatic activity. Recombinant mutant or wild type TPK was investigated concerning substrate and Mg2+concentrations. A clear dependence of TPK activity on thiamine and Mg2+ was found in both mutant and the wild type TPK. A difference between wild type and mutants was detectable at all investigated concentrations but it was highest at low substrate or Mg2+ concentrations. Remarkably, two of the five living patients showed clear improvement after starting a therapy with high dose thiamine. Deficiency of TPK1 causes a neurologic disease in most patients but can also cause cardiomyopathy. Beriberi is an alimentary thiamine deficiency and can manifest with a neurologic (dry beriberi) or cardiologic (wet beriberi) phenotype. Therefore, we think that TPK1 deficiency should also be considered in patients with cardiomyopathy. Keywords: TPK1, Thiamine, Cardiomyopathy
P6.5 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Phosphodiesterase-5 is not detectable in mouse, rat, dog or human myocardium Degen Chantal1***, Kalkidan G Bishu2, Zakeri Rosita2, Ogut Ozgur2, Redfield Margaret M2, Brozovich Frank V2 ***Candidate for Young Investigator Award
1
Paracelsus Medical University, 5020 Salzburg, Austria; Cardiovascular Diseases, Mayo Medical School, MN 55905, USA
2
Phosphodiesterase-5 (PDE5) is highly expressed in the pulmonary vasculature, but its expression in the myocardium is controversial. Cyclic guanosine monophosphate (cGMP) activates protein kinase G
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J Muscle Res Cell Motil (2015) 36:71–143 (PKG), which has been hypothesized to blunt cardiac hypertrophy and negative remodeling in heart failure. Although PDE5 has been suggested to play a significant role in the breakdown of cardiac cGMP and hence PKG regulation in the myocardium, the RELAX trial, which tested effect of PDE5 inhibition on exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF), failed to show a beneficial effect. These results highlight the controversy regarding the role and expression of PDE5 in the healthy and failing heart. This study used 1- and 2-dimensional electrophoresis and Western blotting to examine PDE5 expression in mouse (before and after trans-aortic constriction), rat, dog (control and HFpEF) as well as human (healthy and failing) heart. We were unable to detect PDE5 in any of the cardiac tissue lysates, whereas PDE5 was present in the bovine lung samples used as positive controls. These results indicate that if PDE5 is expressed in cardiac tissue, it is present in very low quantities. Further, there is no detectable increase in PDE5 expression in heart failure. Therefore in cardiac muscle, PDE5 cannot be important for the regulation of the cGMP-PKG signaling, and is not a suitable drug target for the treatment of cardiac hypertrophy. These results highlight the importance of basic science investigation prior to clinical trial design. Keywords: PDE5, Heart failure, Hypertrophy
P6.6 Heart muscle & cardiomyopathy, Sat 12:00–14:00 The CNS-Heart axis is a source of cardiac dysfunction in mouse models of Huntington’s Disease Mielcarek Michal1, Inuabasi Linda1, Bondulich Marie K1, Muller Thomas1, Osborne Georgina F1, Franklin Sophie A1, Smith Donna L1, Neuder Andreas1, Rosinski Jim2, Rattray Ivan1, Protti Andrea3, Bates Gillian P1 1 Department of Medical and Molecular Genetics, King’s College London, UK; 2CHDI Management Inc/CHDI Foundation Inc, Los Angeles, California, 90045 USA; 3King’s College London British Heart Foundation Centre of Excellence, Cardiovascular Division and Division of Imaging Sciences and Biomedical Engineering, King’s College London, UK
Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer’s, Parkinson’s and Huntington’s disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HDrelated dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However themechanism leading to cardiac dysfunction in the clinical and preclinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the
J Muscle Res Cell Motil (2015) 36:71–143 HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor. Keywords: Huntington’s disease, Cardiomyopathy, Misfolded proteins
P6.7 Heart Muscle & Cardiomyopathy, Sat 12:00–14:00 A novel role for PP5 in regulating titin phosphorylation and function in the heart Krysiak Judith Cardiovascular Physiology, Ruhr University Bochum, Germany Within the I-band region of the giant protein titin, a cardiac-specific N2B-unique sequence (N2Bus) can be phosphorylated by various protein kinases, including PKA, PKG, ERK2, and CaMKII, which reduces titin-based passive stiffness. Specific phosphatases acting on this titin region are not known. We carried out a yeast-2-hybrid screen using human N2Bus (‘‘bait’’) and a human cardiac cDNA library (‘‘prey’’) and detected the catalytic domain of protein phosphatase-5 (PP5c) as a binding partner of N2Bus. PP5 is a unique member of the PPP family of serine/threonine specific protein phosphatases, based on the presence of tetratricopeptide repeats (TPR) at the molecule’s N-terminus. The low basal activity of PP5 compared to other phosphatases is due to the inhibitory interaction between the TPR domain and the C-terminus. PP5 is activated by proteolytic cleavage of the TPR region, upon binding of the TPR domain by polyunsaturated fatty acids such as arachidonic acid, or through binding of heat-shock protein-90 (HSP90). The N2BusPP5 interaction was verified by GST-pulldown assays and PP5 bound more strongly to (PKA- or PKG-) phosphorylated N2Bus than to unphosphorylated N2Bus. Co-localization of N2Bus and PP5 in the sarcomeric I-band was suggested by confocal immunofluorescence and immunoelectron microscopy of PP5-overexpressing transgenic (TG) mouse heart. Autoradiography/back-phosphorylation experiments showed that recombinant PP5 dephosphorylates the phosphorylated recombinant N2Bus. Phosphorylation of titin isoforms, N2B and N2BA, in human heart tissue could also be reduced by ex vivo application of PP5. Additionally, PP5 TG mouse hearts showed lower N2Bus phosphorylation than wildtype (WT) hearts. Force measurements on permeabilized single cardiomyocytes revealed increased passive stiffness in PP5 TG vs. WT hearts. We conclude that PP5 binds to titin N2Bus and acts as an antagonist to the protein kinase-mediated effects on titin stiffness.
135 handling and membrane electrophysiology, leading to a pro-arrhythmogenic phenotype and alteration of relaxation. The relative contribution of primary vs. secondary alterations is still unknown. Methods: We aim to study these changes in intact trabeculae, single cardiomyocytes and skinned preparations from the ventricles of transgenic mouse models carrying HCM-related mutations of cTnT (R92Q, E163R). Results: Compared to WT, R92Q trabeculae and cells showed: (i) preserved peak isometric twitch tension at low inotropic level with reduced contractile reserve; (ii) slower Ca2+ transient kinetics, elevated diastolic [Ca2+] and prolonged relaxation, associated with reduced SERCA function; (iii) frequent Ca2+ waves, after-contractions or spontaneous beats during pauses, which increased in response to isoproterenol. In E163R vs. WT trabeculae and cells, force and Ca2+ transient amplitude were preserved in all conditions. Interestingly, the kinetics of force development and relaxation was prolonged, despite Ca2+ transient kinetics was faster and SERCA function unchanged. Nonetheless, E163R myocardium showed increased arrhythmogenic activity. Further, E163R myofibrils showed a prolongation of the overall relaxation, with incomplete inactivation in the absence of Ca2+. Energy cost of contraction, measured with an enzyme-based assay, as well as myofilaments Ca2+ sensitivity, were increased in E163R vs. WT skinned trabeculae. Conclusions: In R92Q hearts, secondary changes of EC-coupling and membrane electrophysiology appear to be the major contributors to the observed mechanical dysfunction and arrhythmogenicity. In E163R hearts instead, in the absence of major secondary cellular remodelling, mutation-driven impairment of myofilament function is likely to be the leading element determining mechanical and electrical abnormalities. Keywords: Hypertrophic cardiomyopathies, Troponin T, Transgenic mouse
P6.9 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Phenotypic analysis of human iPS-derived cardiomyocytes cultured under diabetic conditions Jeworutzki Elena1***, Delobel Frederic2, Bailly Jacques2, Iacone Roberto2, Zorzato Francesco1 ***Candidate for Young Investigator Award
1
Keywords: PP5, Titin, Phosphatase
P6.8 Heart Muscle & Cardiomyopathy, Sat 12:00–14:00 Myocardial dysfunction in hypertrophic cardiomyopathy: primary effects of sarcomeric mutations versus secondary cardiomyocyte remodeling Coppini Raffaele1, Gentile Francesca2, Mazzoni Luca1, Ferrantini Cecilia2, Tosi Benedetta2, Ferrara Claudia2, Tardiff Jil3, Tesi Chiara2, Cerbai Elisabetta1, Poggesi Corrado 1
Department NeuroFarBa, University of Florence, Italy; 2Department of Clinical and Experimental Medicine, University of Florence, Italy; 3 University of Arizona, Tucson, USA Introduction: In HCM human tissue, primary alterations at the level of the sarcomeres are associated with secondary changes of Ca2+
Unispital Basel, Department Biomedizin, ZLF, Hebelstr 20, 4031 Basel, Switzerland; 2Pharma Research and Early Development, F Hoffman-La Roche Ltd, 4070 Basel, Switzerland Diabetes is a multifactorial disease that can also perturb proper calcium cycling in the heart resulting in a diabetic cardiomyopathy independent of pathogenic effects on the vascular system. Numerous studies in various in vitro animal models indicate that cardiac myopathy can be induced either by mutations in calcium transporting or storing proteins or by malfunction due to phosporylation or glycosylation events in these proteins or direct interaction partners. Since most calcium transporting proteins are also directly regulated by calcium, the spatial distribution and precise amount of local calcium is critical for proper calcium cycling. Hyperglycaemic cardiomyocytes (CMs) were shown to increase SR calcium leak and calcium sparks. In this study we investigate calcium signalling in human iPS-derived cardiomyocytes which were cultured under condition which mimic insulin resistance and glucose toxicity. To assess the calcium signalling tool kit components of the hiPS derived CMs, we performed extensive western blot analysis and high resolution immunohistochemistry. The hiPS CMs that were kept
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136 under diabetic conditions exhibit a strong hypertrophic phenotype, disturbed sarcomeric integrity and changes in calcium transients and contractility. The use of voltage sensitive dyes in functional experiments will allow us to identify ventricular cardiomyocytes to perform calcium measurements specifically in this cell type. Preliminary data of this study will be presented. Keywords: hiPS cardiomyocytes, Diabetes, Calcium
P6.10 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Structural dynamics of N-terminal extension of cardiac troponin I by site directed spin labeling-EPR Arata Toshiaki1, Zhao Chenchao1, Takai Shinji1, Somiya Takayasu1, Ueki Shoji2 1 Department of Biological Sciences, Graduate School of Science, Osaka University, Japan; 2Tokushima-Bunri University, Japan
Different from skeletal muscle, troponin I in cardiac muscle (cTnI) has a unique N-terminal extension, that is firmly essential for modulating Ca sensitivity of troponin C (cTnC) and force tension by phosphorylation of itself causing cardiotonic action. The secondary structure of N-extension of cTnI was determined by measuring the distance distribution between spin labels attached on i and i + 4 residues. All the EPR spectra of them had broadened and reduced amplitude to two-third of single spin labeled one and showed a very broad residual distance distribution over 0.8–2.5 nm by best-fit simulation. At lower ionic strength, the amplitude of their spectra decreased to over half of single spin labeled one and the first two peaks became convergent to some extent. Correspondingly, the distance distribution of them became narrower, mainly with 1.5–2 nm in the full width of distance distribution. In particular, 23/27 and 43/47 showed more broadened spectra in cTnI-cTnC complex than in monomer. Distance analysis showed that 23/27 where had PKA phosphorylation sites Ser23/24, transmitted from flexible in monomer to relatively stable and extended conformation in cTnI-C complex with center and width of 1.18 nm and 1.15 nm, respectively. In addition, similar to 23/27 region, 43/47, located on PKC phosphorylation sites Ser42/44, demonstrated a short distance with much narrower distribution (center and width of 0.8 and 0.78 nm) and represented a rather stable alpha-helix conformation in cTnI-C complex than that in monomer state. The interspin distances between TnC and the N-extension have been tried to explore which residues are interacting. Keywords: Troponin, Cardiac muscle, Spin labeling
P6.11 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Study of ‘force–velocity’ and ‘power-load’ relations for rabbit cardiac myosin isoforms V1 and V3 in the in vitro motility assay Nikitina Larissa1, Kopylova Galina1, Shchepkin Daniil1, Katsnelson Leonid B2 1
Lab of Biological Motility, Institute of Immunology and Physiology of the RAS, Ekaterinburg, Russia;2Lab of Mathematical Physiology, Institute of Immunology and Physiology of the RAS, Ekaterinburg, Russia Mammalian ventricular myocardium contains two types of myosin heavy chains —a and b. The amino acid sequence identity between
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J Muscle Res Cell Motil (2015) 36:71–143 a and b MHC is 93 %, with nonidentical residues situated in functionally important domains associated with actin binding and ATPase activity. Myosin isoforms V1 and V3 are homodimers of aand b-heavy chains respectively. Expression of isomyosins V1 and V3 is different in cardiomyocytes from various layers of the ventricle wall, depends upon species, age, and hormonal state of the animal. Changes in the expression of isoforms are associated with cardiac pathologies. The aim of this work is to investigate ‘force– velocity’ and ‘power-load’ relations for rabbit cardiac myosin isoforms V1 and V3 in the in vitro motility assay. Using the actinbinding protein a-actinin as a load, we recorded the ‘force–velocity’ and ‘power-load’ relationships (plotting a-actinin concentrations vs. respective regulated thin filaments movement velocities) for both cardiac myosin isoforms at two calcium concentrations in solution, pCa 6.5 and 7.0. We found ‘force–velocity’ relationships for both myosin isoforms V1 and V3 in the in vitro motility assay corresponded to hyperbolic Hill’s equation under low loads and deviated from it under high loads. Increase in calcium concentration caused more pronounced change in the curvature of the ‘force–velocity’ line for the V3 isoform. This result suggests that V3 seems to be more sensitive to changes in calcium concentration. We conclude myosin heavy chains composition determines shape of ‘force–velocity’ curves in myocardium. Supported by RAS (12-G-4-1042 and 12-G-4-1042), RBRF (13-04-00365, 13-04-96027-Ural) and the Government of Sverdlovsk Region. Keywords: Myocardium, Regulation, Isomyosins
P6.12 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Mitochondrial phosphate carrier deficiency presenting with cardiomyopathy and generalized skeletal myopathy Feichtinger Rene´ G1, Zimmermann Franz A1, Konstanopoulou Vassiliki2, Haack Tobias B3, Prokisch Holger3, Bhoj Elizabeth J4, Ficicioglu Can4, Yudkoff Marc4, Mayr Johannes A1, Sperl Wolfgang1 1 Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria; 2Department of Pediatrics, Medical University Vienna, Austria; 3Institute of Human Genetics, Helmholtz Zentrum, Munich, Germany; 4Department of Pediatrics, University of Pennsylvania and CHOP, Philadelphia, USA
Recently, we described for the first time defects of the mitochondrial phosphate carrier in 5 patients with muscular hypotonia, hypertrophic cardiomyopathy and lactic acidosis. This carrier transports anorganic phosphate necessary for ATP synthesis in mitochondria. Here we add patients from 3 further families. Patient 1 is an adult female with reduced exercise capacity since early childhood with muscular hypotonia, hypertrophic cardiomyopathy and 3-methylglutaconic aciduria. Histological investigations of a muscle biopsy showed increased lipid content and high respiratory chain activities. One sibling died at age of 9 months. Patient 2 and 3, two boys both with an age of one year, also manifested with hypertrophic cardiomyopathy and generalized skeletal myopathy already from the neonatal period on. Exome sequencing revealed pathogenic mutations in SLC25A3, affecting the isoform expressed in skeletal and heart muscle in patient 1 and 2. Patient 3 had compound heterozygous mutations in parts of the protein that are not spliced in a tissue-specific way. We describe three further patients with a phosphate carrier deficiency, with novel mutations. The cardiomyopathy was critical in all patients during the neonatal period, but a favorable outcome with normal intellectual development was found in longer surviving individuals.
J Muscle Res Cell Motil (2015) 36:71–143 Keywords: Cardiomyopathy, 3-Methylglutaconic aciduria
Phosphate
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deficiency,
P6.13 Heart muscle & cardiomyopathy, Sat 12:00–14:00 Regional differences in contractility of isolated subepicardial and subendocardial ventricular myocytes Vasilyeva Anastasia1, Iribe Gentaro2, Naruse Keiji2, Solovyova Olga1 1
Institute of Immunology and Physiology of the RAS, Ural Federal University, Yekaterinburg, Russia; 2Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Previously, several studies have reported on the transmural heterogeneity in the mechanics of ventricular cardiomyocytes. It has been shown that subendocardial (ENDO) cells are significantly stiffer (steeper end-diastolic force–length relationship, EDFLR) and reveal more effective pre-load-dependent force activation (steeper end-systolic FLR, ESFLR) than subepicardial (EPI) cells. In the present study, we investigate mechanisms underlying the distinctions in FLR between EPI and ENDO cells in a simulation study combined with wet experiments in isolated mouse cardiomyocytes. We utilized our Ekaterinburg-Oxford mathematical model accounting for the ionic and myofilament mechanisms of excitation–contraction coupling in cardiomyocytes to develop models of EPI and ENDO myocytes. We used the models to simulate FLR in isometric mode of contractions. In combination with modeling study, we performed wet experiments on cellular mechanics in EPI and ENDO single cells of mouse left ventricle with using carbon fiber technique. In consistence with the experimental data, ENDO model produced steeper ESFLR versus EPI model. Modeling results suggested that distinguishing features of the cooperativity mechanisms of calcium activation of myofilaments (i.e. inter-relations between the kinetics of cross bridges and calcium– troponin C complexes) assumed in EPI and ENDO models may essentially contribute to the distinctions in ESFLR in the cells. As the model predicted, wet experiments demonstrated that EPI cells under free shortening (n = 22) have shorter time to peak contraction and higher rate constant of relaxation compared to the ENDO cells (n = 22). However, preliminary single cell stretch experiments showed no statistically significant differences in the slopes of either EDFLR or ESFLR between EPI and ENDO due to wide variability. Supported by UB RAS (Program #1, 12-M-14-2009, 12-G-4-1067), RBRF (14-01-00885, 14-01-31134), by UrFU (Act 211 #02.A03.21.0006), and JSPS KAKENHI 2628212. Keywords: Transmural heterogeneity, Cell mechanics, Modeling
P7.1 Neuromuscular disease, Sat 12:00–14:00 Phospholamban overexpression impairs SERCA activity and leads to mixed skeletal muscle myopathy Fajardo Val Andrew***, Bombardier Eric, Bellissimo Catherine, McMillan Elliott, Wadsworth Brennan, Quadrilatero Joe, Russell Tupling A ***Candidate for Young Investigator Award
Kinesiology, University of Waterloo, Canada
The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump catalyzes the active transport of Ca2+ into the sarcoplasmic reticulum and is largely responsible for muscle relaxation as well as regulation of intracellular Ca2+ levels. In this study we characterized a mouse model of myopathy caused by reduced SERCA function. Specifically, we found that mice overexpressing the well-described SERCA inhibitor, phospholamban (PlnOE), in their slow-twitch type I fibres had reduced maximal rates of SERCA ATPase activity (477.1 ± 24.5 vs. 558.5 ± 29.1, p = 0.05) and Ca2+-uptake (19.2 ± 1.03 vs. 25.1 ± 1.5, p = 0.009) compared to wild-type (WT) mice in homogenates obtained from their gluteus minimus muscles. Enhanced fibrosis, inflammation, and necrosis were observed upon histological analysis of the gluteus minimus and soleus muscles. In addition, there were a greater number of fibres with centrally located nuclei (partly due to greater regeneration cycles) and when combined with the slight elevation in serum creatine kinase levels (118 ± 22 vs 325 ± 93, p = 0.05) our results suggest that PlnOE mice exhibit dystrophic-like features. Interestingly, the greater levels of centrally located nuclei may also be associated with centronuclear myopathy as we also observed type I fibre predominance and hypotrophy as well as central aggregation of oxidative activity. On occasion, core-like lesions reminiscent of multi-minicores disease were also present. Muscular dystrophy, centronuclear myopathy and multi-minicores disease all lead to generalized muscle weakness and not surprisingly force-frequency curves generated using soleus muscles revealed that PlnOE mice had lower force at sub-maximal and maximal frequencies compared to their WT littermates (p \ 0.05). These results demonstrate that PlnOE mice present with mixed skeletal muscle myopathy and illustrates clearly the importance of SERCA function in overall muscle health. Keywords: Myopathy, SERCA, Phospholamban
P7.2 Neuromuscular disease, Sat 12:00–14:00 Enhanced oxidative stress in soleus muscles from phospholamban overexpressing mice may contribute to impairments in SERCA activity Fajardo Val Andrew, Tran Khanh, McMillan Elliott, Bombardier Eric, Quadrilatero Joe, Russell Tupling A Kinesiology, University of Waterloo, Canada Functional impairment of the sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) pump is a common secondary defect contributing to Ca2+ dysregulation and disease pathology in a variety of myopathies. Enhanced oxidative stress is often observed in many muscle diseases and can contribute to reduced SERCA function by altering SERCA’s structure through mechanisms including S-nitrosylation. Here we questioned whether oxidative stress and S-nitrosylation are contributing factors to the impaired SERCA function in a mouse model of mixed skeletal muscle myopathy used in our laboratory. To this end, soleus muscles from mice overexpressing phospholamban (PlnOE) were extracted and used to assess SERCA function, calpain activity, oxidative stress, and S-nitrosylation. Soleus muscles displayed muscle atrophy with soleus:body weight ratios being significantly lower in PlnOE mice compared to their wild-type (WT) littermates (0.12 ± 0.01 vs. 0.18 ± 0.01, p \ 0.0001). Both SERCA Ca2+- ATPase activity (181.6 ± 23.8 vs. 248.4 ± 11.5, p = 0.04) and uptake (4.1 ± 1.04 vs. 15.3 ± 0.6 p \ 0.001) were reduced in thesoleus homogenates obtained from PlnOE mice compared to WT. Reduced SERCA activity was associated with increased calpain activity in soleus muscles from PlnOE mice compared to WT mice (1.60 ± 0.2 vs. 1.0 ± 0.1, p = 0.02). Finally, results from a dichlorofluorescein assay (1.6 ± 0.1 vs. 1.0 ± 0.2, p = 0.03) and Western blot analysis of SERCA S-nitrosylation (2.4 ± 0.2 vs 1.1 ± 0.2, p = 0.05) indicate greater levels of oxidative stress and SERCA
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138 S-nitrosylation in the soleus homogenates of PlnOE mice compared to WT mice. Our results suggest that impaired SERCA activity resulting from phospholamban overexpression leads to increased oxidative stress that may further attenuate SERCA’s catalytic activity. In conclusion, protecting SERCA from oxidative damage may aid in combatting the myopathy seen in these transgenic animals. Keywords: Myopathy, Calcium overload, Oxidative stress
P7.4 Neuromuscular disease, Sat 12:00–14:00 Impact of sporadic denervation on muscle mitochondrial function in neurotrypsin over-expressing mice and its implications for aging muscle Vuda Madhusudanarao1***, Aare Sudhakar Reddy1, Perez Anna1, Spendiff Sally1, Taivassalo Tanja2, Hettwer Stefan3, Hepple Russell1 ***Candidate for Young Investigator Award
1
Critical Care Division, McGill University Health Center, Montre´al, Canada; 2Department of Kinesiology, McGill University, Montre´al, Canada; 3Neurotune AG, Schlieren, Zurich, Switzerland Although there is evidence of altered mitochondrial function in aging muscle, surgical denervation also modifies mitochondrial function and advanced age is associated with a marked accumulation of denervated myofibers. Thus, determining the extent to which mitochondrial alterations in aging muscle are a primary defect versus secondary to sporadic denervation is important in evaluating the therapeutic potential of targeting mitochondrial dysfunction in aging muscle. To address the effect of sporadic denervation on muscle mitochondrial function, we examined male neurotrypsin over-expressing (Sarco) mice (8 months old), a model that exhibits unstable neuromuscular junctions and many hallmark features of aging muscle. Mitochondrial respiration, reactive oxygen species (ROS) emission and calcium retention capacity were assessed using permeabilized myofibres from gastrocnemius muscle. In addition, to isolate the impact of sporadic denervation on mitochondrial function in Sarco mice we used an approach previously shown to block the denervation-induced mitochondrial ROS signal: cytosolic phospholipase A2 (cPLA2) inhibition using arachidonyl trifluoromethyl ketone (AACOCF3). Sporadic denervation did not affect indices of mitochondrial content nor the function at the whole tissue level or in permeabilized myofibers, respectively. However, myofibers treated with cPLA2 inhibitor reduced basal ROS emission to 51 % compared to the vehicle treated myofibers in Sarco mice (vehicle, 1.67 ± 0.48 vs. AACOCF3, 0.81 ± 0.33, p = 0.03) but not in wild type mice, suggesting that sporadic denervation is significantly modulating mitochondrial ROS emission in Sarco mice. We conclude that since sporadic denervation can modulate muscle mitochondrial function (as revealed through the impact of cPLA2 inhibition on ROS emission in Sarco mice), alterations in muscle mitochondrial function with aging are likely to be at least in part secondary to the accumulation of denervated myofibers in advanced age. Keywords: Sporadic denervation, Muscle mitochondria, Reactive oxygen species
P7.5 Neuromuscular disease, Sat 12:00–14:00 Functional study of a Cav1.1 R528G mutation causing hypokalaemic periodic paralysis type 1
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J Muscle Res Cell Motil (2015) 36:71–143 Bednarz Marcin1, Da Yu-Wei2, Jurkat-Rott Karin1, Fan Chunxiang1, Lehmann-Horn Frank1 1
Division of Neurophysiology, Ulm University, Germany; Deparment of Neurology, China Medical University, Taiwan, China
2
Familial Hypokalemic periodic paralysis (HypoPP) is the most common periodic paralysis. It is a genetic skeletal muscle channelopathy of autosomal dominant inheritance, which is characterized by episodic attacks of muscle weakness associated with a decrease in blood potassium level. In a large HypoPP family of 19 affected individuals we found a missense mutation, R528G of Cav1.1 (DII/S4). Until now there have been no functional studies of R528G mutant Cav1.1 channels. Therefore the purpose of this study was to investigate: i) the alteration in gating caused by the mutation R528G of skeletal muscle L-type Ca2+ channels and ii) identify omega currents, that may contribute to the pathogenesis of HypoPP. To investigate the gating properties of wild type (WT) and mutant (R528G) channels, we performed whole-cell patch clamp experiments on transiently transfected GLT myotubes, expressing either WT or R528G mutant channels. Based on GFP fluorescence, 13-16 days old myotubes were selected. The mutation didn’t affect the voltage dependence of activation and inactivation, but slowed the kinetics of activation and fastened the kinetics of inactivation. It shifted the steady state inactivation curve to the left by slowing the recovery from fast inactivation. Additionally, our data shows an increased amplitude of hyperpolarization- induced inward currents in the R528G mutation compared to untransfected and WT-expressing GLT cells, indicating a potential omega current. Based on the current–voltage relationship in the untransfected and WT-expressing GLT cells, this inward current might reflect currents passing through the inwardly rectifying Kir-channels. To illustrate omega currents in R528G more clearly, we are currently blocking the inward rectifying Kir-channel. Keywords: Calcium channel, Periodic paralysis, Omega current
P7.6 Neuromuscular disease, Sat 12:00–14:00 Archvillin: a new player in skeletal muscle mechanical signal transduction Spinazzola Janelle1***, Liu Min2, Barton Elisabeth R3 ***Candidate for Young Investigator Award
1
Department of Anatomy and Cell Biology, and Pennsylvania Muscle Institute, University of Pennsylvania, USA; 2Department of Physiology, University of Pennsylvania, USA; 3Department of Anatomy and Cell Biology, University of Pennsylvania, USA Deficiency in the gamma-sarcoglycan subunit (gSG) of the DGC is a feature of muscular dystrophies, and is sufficient to induce muscle degeneration and signaling defects in adaptation to mechanical load. Mechanisms of gSG mediated signaling functions and the proteins involved are poorly understood. We investigated gSG as a component of the load-sensing machinery of muscle cells, and through a yeasttwo-hybrid screen, identified the muscle-specific protein archvillin (AV) as a gSG interacting protein. AV is an attractive candidate because it promotes ERK signaling and interacts with dystrophin through its N-terminus. AV expression and localization were analyzed by IB, qRT-PCR and IHC in hindlimb muscles of C57Bl/6 (WT), gSG -null (gsg-/-), and mdx mice. AV protein and mRNA expression was significantly upregulated in gsg-/- muscle, but was similar to WT levels in mdx muscle. In cross-sections, AV was upregulated at the sarcolemma in gsg-/- muscle, but absent in mdx
J Muscle Res Cell Motil (2015) 36:71–143 muscle. Reintroduction of gSG in gsg-/- muscle by rAAV injection returned AV levels toward that of WT. Mechanical perturbation of TA muscles with an in situ eccentric contraction protocol revealed that AV associates with P-ERK in a stimulus-dependent manner, but this interaction was lost in gsg-/- and mdx muscle. Nuclear/cytoplasmic fractionation studies showed that nuclear localization of P-ERK1/2 increased in gsg-/- and mdx muscle after eccentric contraction. These results suggest that AV expression is dependent on the SG complex and that AV may impact ERK signaling and contribute to the aberrant signaling observed in dystrophic muscle. Mechanical fragility may not be the only deficit contributing to the dystrophic process. Analyzing potential components of gSG-mediated mechanotransduction will be an important step in understanding the pathways leading to dystrophic pathology, and may contribute to the development of therapies for muscular dystrophies. NIH T32AR053461. Keywords: Muscular dystrophy, Extracellular signal-regulated kinase 1/2, Sarcoglycan
P7.7 Neuromuscular disease, Sat 12:00–14:00 Impaired myosin ATPase activity due to actin oxidation underlies skeletal muscle dysfunction in adjuvant-induced arthritis rat Yamada Takashi, Lee Jaesik, Abe Masami, Tatebayashi Daisuke, Chikazoe Sachiko, Hirota Kento Department of Physical Therapy, Sapporo Medical University, Hokkaido¯, Japan In addition to the primary symptoms arising from inflamed joint, muscle weakness is prominent in patients with rheumatoid arthritis (RA). Here, we investigated the mechanisms of arthritis-induced muscle dysfunction in the fast-twitch extensor digitorum longus (EDL) muscle of rat with adjuvant-induced arthritis (AIA). AIA was induced in rat knees by Freund’s adjuvant injection and was allowed to develop for 21 days. Muscle contractility was assessed in whole EDL muscle. To further assess mechanisms underlying contractile dysfunction, we investigated alterations in expression and activity of excitation–contraction coupling proteins, the expression levels of inflammatory mediators, and changes in redox protein expression and metabolism. There was a decreased tetanic force per cross-sectional area and an increased twitch contraction and relaxation time in AIA EDL muscles. These contractile dysfunctions were accompanied by a marked decrease in myosin ATPase and sarcoplasmic reticulum Ca2+ATPase activity, whereas myosin heavy chain content and isoforms were not changed in AIA muscle. There was a significant increase in 3-nitrotyrosine and malondialdehyde protein adducts, as well as in NADPH oxidase 2, neuronal nitric oxide synthase, TNF-a, and HMGB-1 protein expression from the muscles of rat with AIA. We here show contractile dysfunction in EDL muscle of AIA rat, which can be explained by redox modification of actin due to TNF-a-induced peroxynitrite overproduction. Keywords: Rheumatoid arthritis, Specific force, Redox stress
P7.8 Neuromuscular Disease, Sat 12:00–14:00 Cold shock domain protein A—a novel nemaline myopathycausing gene? Laitila Jenni1***, Lehtokari Vilma-Lotta1, Kiiski Kirsi1, Wallgren-Pettersson Carina1, Pelin Katarina2 ***Candidate for Young Investigator Award
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The Folkha¨lsan Institute of Genetics and Department of Medical Genetics, University of Helsinki, Finland; 2Department of Biosciences, Division of Genetics, University of Helsinki, Finland Mutation analysis of the known nemaline myopathy (NM)-causing genes in a Finnish patient with an unusual form of NM, with legs clearly stronger than arms, did not reveal the cause of the disease. Aheterozygous TPM3 splice site mutation, delTAGG, in intron 1 was identified, inherited from the healthy father. The mutation was predicted to be recessive, but no TPM3 mutation was found on the maternal allele. Large copy number variations affecting the known nine NM genes were excluded by NM-CGH array analysis. Wholeexome sequencing revealed a heterozygous RYR1 missense mutation, p.Leu2031Phe, predicted to be probably damaging by PolyPhen-2, but the healthy father also carried this mutation. No other putative pathogenic mutation in RYR1 was found in the patient, and in myoblasts of the patient, epigenetic silencing of the maternal allele was ruled out. Exome sequencing did, however, disclose two putative pathogenic mutations in the cold shock domain protein A encoding gene, CSDA. The CSDA gene has 11 exons and encodes three different protein isoforms: 303 aa, 372 aa and 200 aa. CSDA is highly expressed in skeletal muscle and heart. Our patient was compound heterozygous for two missense mutations, p.Ser34Arg and p.Arg129Trp, in CSDA, each inherited from one of the healthy parents. The mutations are not listed in the EVS database (http://evs.gs.washington.edu/EVS/). Ser34 and Arg129 are highly conserved between species, and reside in the DNA-binding domain (cold shock domain) of CSDA. Furthermore, Ser34 is phosphorylated by ERK2 and GSK3b, and the phosphorylation is thought to be important for the formation of nuclear CSDA complexes and binding of CSDA to single-stranded DNA. Western blot analysis of cultured myotubes from the patient confirms CSDA expression on the protein level. Further analyses are ongoing in order to quantify the expression levels and determine the intracellular location of CSDA in myoblasts from the patient and healthy controls. Keywords: Nemaline myopathy, CSDA, Cold shock domain protein A
P7.9 Neuromuscular disease, Sat 12:00–14:00 Proteomic profiling of skeletal muscle from the wobbler mouse model of motor neuron disease using label-free mass spectrometry Holland Ashling1***, Ohlendieck Kay2 ***Candidate for Young Investigator Award
1
Department of Biology, National University of Ireland Maynooth; Department of Biology, NationalUniversity of Ireland Maynooth
2
Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease in adult patients. While the long-term physiological adaptation of the neuromuscular system to changed functional demands is usually reflected by unilateral skeletal muscle transitions, the progressive degeneration of distinct motor neuron populations is often associated with more complex changes in the abundance and/or isoform expression pattern of contractile proteins and metabolic enzymes. The wobbler (WR) mouse is an established animal model of human motor neuron disease and is characterized by a large variety of cellular abnormalities including muscular atrophy. In order to evaluate the intricate effects of primary motor neuronopathy on the skeletal muscle proteome of the wobbler mouse, label-free mass spectrometry was employed to study the global protein alterations. Proteomic analysis verified the complexity of neurodegeneration-
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140 related changes in skeletal muscle from the WR mouse model of ALS and identified novel changes in proteins. Our proteomic profiling confirmed pathobiochemical complexity of disease-induced changes and showed distinct alterations in 72 protein species including a variety of fibre type-specific isoforms of contractile proteins, metabolic enzymes, metabolite transporters and ion-regulatory proteins, as well as changes in molecular chaperones and various structural proteins. The establishment of novel disease-specific biomarkers may be advantageous in the design of improved diagnostic tools and the identification of novel therapeutic targets of motor neuron disease. Keywords: Muscle proteomics; Motor neuron disease; Wobbler mouse
P7.10 Neuromuscular Disease, Sat 12:00–14:00 Extracellular calcium and magnesium reduce myotonia in ClC-1 inhibited rat and human muscle Skov Martin1***, Riisager Anders1, Fraser, James A2, Bækgaard Nielsen Ole1, De Paoli Frank V1, Holm Pedersen Thomas1 ***Candidate for Young Investigator Award
1
Biomedicine, Aarhus University, Denmark; 2Physiological Laboratory, University of Cambridge, UK
Myotonia congenita is a genetic disorder caused by loss-of-function mutations in skeletal muscle ClC-1 chloride channels. The symptoms in myotonia congenita are spontaneous excitations and delayed relaxation of muscle. Characteristically, these symptoms vary markedly between patients with identical ClC-1 mutations and on a day-to-day basis in individual patients. Certain conditions that increase the risk of Mg2+ and Ca2+ wasting (pregnancy and use of diuretics) have been associated with worsening of myotonia. Prompted by this, we investigated whether physiological variations in extracellular concentration of Mg2+ and/or Ca2+ affect myotonic contractions in isolated rat (EDL) and human (abdominal rectus) muscle. To induce experimental myotonia, muscles were exposed to ClC-1 inhibitor 9-AC (100 lM). In all experiments, muscles were field-stimulated (5 Hz for 2 s) every 10 min and contractile force was quantified from force integrals (AUC). In both rat and human muscles, ClC-1 inhibition markedly increased AUC reflecting the appearance of myotonia. This was confirmed by intracellular recordings of trains of action potentials before and after 9-AC addition. When extracellular [Mg2+] or [Ca2+] were subsequently lowered from 1.2 and 1.27 to 0.4 and 0.8 mM, the AUC rose 3.4 and 3.0 fold, respectively. With readdition of divalent cations in steps of 0.1 mM, the AUC gradually recovered. When elevated above control levels, the myotonia could be largely abolished. Further experiments showed that the two ions can substitute one another suggesting a common mechanism of the two ions on myotonia. Electrophysiological experiments and mathematical modelling strongly implicate a depolarizing shift in activation of voltage gated Na+ channels as a cellular mechanism underlying the effects of Mg2+ and Ca2+ on myotonia. These findings suggest that physiological variation in extracellular Mg2+ and Ca2+ may contribute to variation in symptoms in myotonia congenita. Keywords: Myotonia, Phenotypic variability, Extracelluar divalent cations
P7.11 Neuromuscular Disease, Sat 12:00–14:00 A novel desmin mutation (Q348P) associated with myofibrillar myopathy in a polish family
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J Muscle Res Cell Motil (2015) 36:71–143 Redowicz Maria Jolanta1, Fichna Jakub Piotr2, Karolczak Justyna1, Potulska-Chromik Anna3, Miszta Przemyslaw4, Berdynski Mariusz2, Sikorska Agata1, Filipek Slawomir4, Kaminska Anna M3, Zekanowski Cezary2 1 Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St, 02-093 Warsaw, Poland; 2Department of Neurodegenerative Disorders, Mossakowski Medical Research Centre, 5 Pawin´ski St, 02-106 Warsaw, Poland; 3Department of Neurology, Medical University of Warsaw, 1 Banach St, 02-097 Warsaw, Poland; 4Faculty of Chemistry & Biological and Chemical Research Centre, University of Warsaw, 1 Pasteur St, 02-093 Warsaw, Poland
Desmin is a muscle-specific intermediate filament protein, which forms a network connecting sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin cause myopathies, cardiomyopathies and related phenotypes. Molecular mechanisms of the changes leading to the disease remain ambiguous. Here, we describe a novel mutation (Q348P) found in a Polish family, which is predicted as pathogenic using a bioinformatic approach. To test its impact on desmin expression and its intrafibre distribution in vitro analyses of quadriceps muscle biopsy were performed. An elevated desmin level in the patient’s muscle as well as atypical desmin localization within the fibers were observed. Additional staining for M-cadherin, a-actinin and MHCs confirmed severe disruption of myofibrillar organization. Also, the aberrant myofibers displayed numerous centrally localized nuclei. The structure of desmin 2B fragment (residues 333–411) built by homology modeling tools (SwissProt server) based on the known structure of human vimentin coiled coil 2B fragment (residues 328–411) revealed that the mutation introduced a large distortion in the desmin coiled coil structure. This could cause a strong bent at a mutation site,most probably leading to the protein aberrant folding. It could be thus speculated that the observed aggregates formed by misfolded desmin trigger the myofibrils disruption and, in consequence, severe myopathy symptoms. Keywords: Desmin, Myopathy, Disease
P7.12 Neuromuscular Disease, Sat 12:00–14:00 Taurine supplementation can improve tibialis anterior (TA) force production and muscle architecture: A comparison between the 28 and 70 day old mdx mouse model of Duchenne muscular dystrophy (DMD) Barker Robert1***, Van der Poel Chris2, Murphy Robyn1, Horvath Deanna2 ***Candidate for Young Investigator Award
1
Zoology, La Trobe University, Melbourne, Australia; 2Human Biosciences, La Trobe University,Melbourne, Australia Introduction: Due to its role in mediating a number of proposed pathways associated with the pathophysiology of DMD, the amino acid taurine is a desirable candidate as a therapeutic supplement. Here we investigated the effect of taurine supplementation on the dystrophic mdx mouse at two time points 1) 28 d, peak damage and 2) 70 d, where a chronic but low level of active damage persists. Methods: Offspring from C57BL/10ScSn (WT), mdx and mdx taurine (mdx Tau) supplemented mice were utilised at ages 28 days and 70 d (± 1 day). Mdx Tau mice received taurine (dose 3 % v:v in water) from birth until experimentation. Tibialis anterior (TA) force
J Muscle Res Cell Motil (2015) 36:71–143 characteristics were measured in situ and histological parameters assessed. Protein expression was investigated by western blot analysis and TA taurine content measured using HPLC. Results: Taurine supplementation increased both specific and peak twitch force in 28 days mdx Tau mice but no differences were observed in the complimentary 70 days groups. Histological analysis of the TA identified extensive damage in 28 days mdx mice evident by fibres both highly variant in size and centrally nucleated. Both were less prominent in the mdx Tau mice which more closely emulated the WT. This improvement was not as apparent in 70 days mdx Tau mice. Preliminary muscle taurine content analysis indicates an increase in the 28 days mdx mice with supplementation that was not observed in the 70 d group. Largely, neither proteins important for excitation–contraction coupling (DHPR, SERCA) nor the taurine transporter protein were different between groups. Conclusion: Taurine supplementation is effective at reducing muscle pathology and improving muscle function at the peak damage period of 28 d in the mdx mouse. However, it is evidently less effective when only low level but chronic muscle pathology is present (70 d). These findings suggest that age is a very important consideration when investigating potential therapeutic interventions for DMD. Keywords: Taurine, Dystrophy, Skeletal muscle
P7.13 Neuromuscular disease, Sat 12:00–14:00 Proteomic analysis shows that low intensity training reduces the carbonylation level and increases the expression of energy metabolism and muscle contraction proteins in mdx skeletal muscle Hyzewicz Janek, Tanihata Jun, Kuraoka Mutsuki, Ito Naoki, Miyagoe-Suzuki Yuko, Shin’ichi Takeda Department of Molecular Therapy, National Institute of Neuroscience, Tokyo, Japan Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene and results in muscle degeneration. High intensity training injuries muscles of mdx mice, an animal model for DMD, however, low intensity training rescues the mdx phenotypes and even reduces the oxidative stress level. We performed a 2D Electrophoresis analysis to assess the effect of low intensity swimming on protein oxidation, expression and protein–protein interactions at single protein level in 8 weeks mdx gastrocnemius. We observed an overcarbonylation and a down-regulation of mitochondria, muscle contraction and glycogenolysis proteins in mdx muscle. Protein–protein interaction analysis revealed an absence of complete ATP synthase complex in mdx muscle. Here we showed that low intensity training had a mild effect on reduction of carbonylation level, but restored expression and protein–protein interaction of proteins involved in energy metabolism and muscle contraction in mdx muscle. Among muscle contraction proteins, we paid a special attention to troponin T and myosin-binding protein C slow and fast isoforms. We showed that low intensity training not only increased the expression of slow isoforms in wild type and mdx muscle, but restored the expression of fast isoforms in mdx gastrocnemius too. Keywords: Proteomic, Exercise, Duchenne muscular dystrophy
P7.14 Neuromuscular disease, Sat 12:00–14:00 Comparative study of dystrophy progression and TNF-a and TGF-b levels in different skeletal muscles of the mdx mice
141 Maranha˜o Juliana, Moreira Drielen de Oilveira, Mauricio Adriana Fogagnolo, De Carvalho Samara Camac¸ari, Marques Maria Julia, Santo Neto Humberto Structural and Functional Biology, University of Campinas, Brazil In Duchenne muscular dystrophy (DMD), lack of dystrophin causes progressive skeletal muscle degeneration. Tumor necrosis factor-alpha (TNF-a) and transforming growth factor-beta (TGF-b) contribute to inflammation and fibrosis deposition and may serve as markers of dystrophy progression. Skeletal muscles are affected under different intensities, with the respiratory muscles being the most affected and some muscles, such as the intrinsic laryngeal, spared. We performed a comparative evaluation of dystrophy progression in the dystrophic diaphragm (DIA) and quadriceps (QDR) muscles in the mdx mice model of DMD. We used C57BL/10 as control. We assessed their histopathological features (morphometric analysis) and molecular composition (TNF-a and TGF-b levels by Western blot) at ages 1, 4 and 9 months. Fibrosis was the main histopathological feature in mdx DIA, which increased over time (1 month: 1.8 %, 4 months: 18.2 %, 9 months: 32 % of fibrosis), being significantly elevated in mdx DIA compared to mdx QDR (9 months: 9 % of fibrosis). Conversely, muscle regeneration, as demonstrated by areas with centrally nucleated fibers, was the main feature of the mdx QDR (1 month: 10 %, 4 months: 79 %, 9 months: 90 % of centrally nucleated fibers areas) and significantly elevated in comparison to mdx DIA (9 months: 66 % of central nucleated areas). Inflammation-regeneration area was higher in QDR at 1 month (50 % in QDR vs. 6.0 % in DIA), reaching similar levels (about 3 %) in both muscles, at 9 months. Mdx DIA presented higher levels of TNF-a and TGF-b compared to mdx QDR, from 4 months on. In mdx QDR, the levels of TNF-a and TGF-b were increased (compared to control) during dystrophy progression. Elevated levels of TNF-a and TGF-b correlated with dystrophy progression mainly in mdx DIA. We draw attention to the extensive regeneration in mdx QDR concomitantly to elevated levels of proinflammatory and profibrotic cytokines. Keywords: Dystrophy, TGF-b, Diaphragm
P7.15 Neuromuscular disease, Sat 12:00–14:00 The R6/2 mouse model of Huntington’s disease displays a denervation-like phenotype Mielcarek Michal1, Jolinon Nelly1, Ahmed Mhoriam2, Smith Donna L1, Dick James R2, Greensmith Linda2, Bates Gillian P3 1 Department of Medical and Molecular Genetics, King’s College London, UK; 2Sobell Department of Motor Neuroscience and Movement Disorders and MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, London, UK; 3Department of Medical and Molecular Genetics, King’s College London, UK
Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by the expansion of a polyglutamine stretch within the huntingtin protein (HTT). The neurological symptoms, that involve motor, cognitive and psychiatric disturbances, are caused by neurodegeneration that is particularly widespread in the basal ganglia and cereberal cortex. HTT is ubiquitously expressed and in recent years it has become apparent that HD patients experience a wide array of peripheral organ dysfunction including skeletal muscle wasting. To test the hypothesis that mutant HTT leads to skeletal muscle denervation we used the well-established R6/2 mouse model that is transgenic for a mutated N-terminal exon 1 HTT fragment and develops the majority of HD-related symptoms by 3 months of age. Functional in vivo assessment of the force and contractile characteristics of the hind limb muscles tibialis anterior (TA) and extensor
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142 digitorum longus (EDL) revealed a progressive impairment at 12 and 14 weeks of age. This was accompanied by a reduced number of motor units innervating the EDL muscles as determined by estimating the number of functional motor units, by stimulating the nerve with stimuli of increasing intensity. We conclude that the HD-related muscle wasting phenotype is accompanied by progressive denervation. Keywords: Huntington’s disease, Skeletal muscle wasting, Denervation-like phenotype
P7.16 Neuromuscular disease, Sat 12:00–14:00 Diaphragm muscle fibre function in response to mechanical ventilation and ageing. Effects of a chaperone co-inducer Cacciani Nicola, Ogilvie Hannah, Akkad Hazem, Larsson Lars Neuroscience, Clinical Neurophysiology, Uppsala University, Sweden We have studied the combined effects of controlled mechanical ventilation (CMV) and ageing on diaphragm muscle single fibres using a unique rat model simulating Intensive Care Unit (ICU) conditions and the effects of chaperone co-inducer (BGP-15). The effects of 5 days CMV on diaphragm muscle fibre size and function were investigated in young and old F344 Brown Norway hybrid rats. The experimental groups were deeply sedated, pharmacologically paralyzed and extensively monitored 24 h per day. BGP-15 was given in systemically by intravenous infusion. After 5 days, the mid-costal parts of the diaphragm were dissected, chemically skinned and prepared for contractile measurements. Cross sectional area (CSA) absolute force (P0) and specific force (absolute force/CSA) were measured at the single muscle fibre level. Results: An ageing-related hypertrophy and decline in specific force were observed in control fibres. A compensatory hypertrophy was observed in response to 5 days CMV in both young and old animals. Systemic administration BGP-15 had a significant positive effect on diaphragm muscle fibre specific force in the young, restoring it to control values, but not in the old. The strong effect of BGP-15 on muscle fibre function in the young was paralleled by a significant up-regulation of HSP 70, but this effect was absent in the old.
J Muscle Res Cell Motil (2015) 36:71–143 properties that allow them to perform extremely rapid twitch contractions over extended periods and to escape from damage in the muscular dystrophies, such as Duchenne muscular dystrophy (DMD). A better ability to handle intracellular calcium (Ca2+) has been suggested to explain these properties, through mechanisms poorly understood. We investigated whether ILM are endowed with a superior Ca2+ homeostasis in rodents (rats and mice) that enables them to remain protected from damage in dystrophic mdx mice. Quantitative PCR (qPCR) indicated that mRNA of 7 (Casq1, Casq2, Pln, Pvalb, CaM, Pmca1 and Ncx) out of the 12 genes quantified were expressed at significantly higher levels in rat ILM vs. tibialis anterior (TA). The highest fold-change was detected for the Pmca1 and Ncx (up to 30-fold increase in ILM vs. TA). CaM presented a 17-fold increase in ILM vs. TA, followed by Pln and Casq2 (about 6.5 fold increase). In dystrophic mdx mice, mRNA of Casq1, Casq2, Serca1 and Serca2 were expressed significantly higher in ILM vs. TA. The relative expression levels of selected proteins involved in Ca2+ homeostasis were examined using western blot. Rat ILM showed higher levels of CASQ1, SERCA1, SERCA2 and CaM compared to TA. CASQ2 was higher in rat TA than in ILM. Protein levels of CASQ1 and SERCA1 were higher in ILM compared to TA of normal and mdx mice. In mdx ILM CASQ2 levels were higher compared to normal. However, mdx TA showed decreased CASQ2 levels compared with normal TA. Rodent ILM (rats and mice) have comparable expression of Ca2+ handling proteins. These results indicate that ILM have a differential Ca2+ regulation system profile which suggestive of a better ability to handle Ca2+ changes in comparison to limb muscles, and this might be provide a mechanistic insight for their unique pathophysiological properties. Keywords: mdx mice, Calcium homeostasis, Sparing from myonecrosis
P7.18 Neuromuscular disease, Sat 12:00–14:00 Effects of acute sustained hypoxia on diaphragm muscle function in the mdx mouse Burns David, O’Halloran Ken D ***Candidate for Young Investigator Award
Keywords: Diaphragm, Myosin, Mechanical ventilation Department of Physiology, University College Cork, Ireland
P7.17 Neuromuscular disease, Sat 12:00–14:00 Differential expression of genes involved in the calcium homeostasis in intrinsic laryngeal muscles of mdx mice Ferretti Renato1***, Selingardi Roberta1, Dos Santos Veridiana Carvalho1, Marques Maria Julia2, Khurana Tejvir S3, Santo Neto Humberto2 ***Candidate for Young Investigator Award
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Departament of Anatomy, Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Brazil; 2Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas, Brazil; 3Department of Physiology, Perelman School of Medicine and Pennsylvania Muscle Institute, University of Pennsylvania, USA Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection with unique physiological
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Duchenne muscular dystrophy (DMD) is a genetic disease caused by defects in the dystrophin gene resulting in respiratory or cardiac failure. Patients have reduced diaphragm functional capacity due to muscle weakness caused by the accumulation of connective and adipose tissue. DMD is characterised by hypoxaemic periods caused by hypoventilation and sleep disordered breathing. We examined the effects of acute (6 h) sustained hypoxia (10 % O2) on diaphragm muscle function in the mdx mouse model of DMD. 8 week old male wild type (WT; C57BL10) and mdx (C57BL10mdx) animals were exposed to hypoxia or room air (control); animals were divided into four groups: WT control (n = 8), WT hypoxia (n = 8), mdx control (n = 8) and mdx hypoxia (n = 8). Isometric and isotonic functional properties of the diaphragm were assessed ex vivo under control conditions (95 % O2/5 % CO2) at 35 C. Data were compared using Student’s t-tests and two-way ANOVA as appropriate. Diaphragm dysfunction was observed in mdx mice, with significant reductions in peak twitch (p = 0.0008) and peak tetanic (p \ 0.001) force compared to WT. Hypoxia significantly reduced peak tetanic force in WT (p = 0.0209) but not mdx (p = 0.1549) diaphragm preparations. Maximum work and power production were unaffected by hypoxia in
J Muscle Res Cell Motil (2015) 36:71–143 WT and mdx animals. Mechanical work (p = 0.0014) and power (p \ 0.0001) production across the load continuum were both significantly reduced by hypoxia in WT animals. Conversely, hypoxia did not significantly alter work and power production in mdx animals. Hypoxia significantly reduced shortening velocity over the load continuum in WT (p = 0.0209) and mdx (p = 0.0187) animals. Acute sustained hypoxia impairs WT, but not mdx, diaphragm function. The resistance to the acute hypoxic stress in mdx diaphragm is suggestive of adaptation to hypoxia. Preliminary findings indicate that hypoxic tolerance is not maintained in mdx diaphragm at 14 weeks. Keywords: DMD, Diaphragm, Hypoxia
P7.19 Neuromuscular disease, Sat 12:00–14:00 Potential roles of M2 macrophages in fibrosis deposition in heart and skeletal muscles of the mdx mice, during later stages of disease De Carvalho Samara Camac¸ari, Santo Neto Humberto, Marques Maria Julia Structural and Functional Biology, University of Campinas, Brazil Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration and cardiorespiratory failure. The inflammatory infiltrate in dystrophic muscles is composed of M1 macrophages that
143 damage dystrophic muscles and of M2 macrophages, which promote muscle regeneration but can also lead to fibrosis. We assessed whether a predominance of M2 macrophages could increase fibrosis in skeletal and cardiac muscles, in the mdx mice model of DMD. We used omega-3 to promote M2 macrophages in the diaphragm (DIA), quadriceps (QDR) and heart. Mdx mice (3 months old) received omega-3 oil (commercially available fish oil; FDC Vitamins; Omega3), for 5 months. Control mdx mice (untreated) received mineral oil. M2 macrophages predominated in the omega-3-treated heart and DIA, as indicated by Western blot of CD206 (heart omega-3: 0.4 ± 0.1; untreated heart: 0.2 ± 0.1; DIA omega-3: 1.1 ± 0.1; untreated DIA: 0.7 ± 0.1; p \ 0.05, ANOVA) and serum IL-10 (untreated: 60 ± 24; omega-3: 120 ± 43). Fibrosis (morphometric analyses) was significantly increased in the omega-3 group (omega-3 heart: 14 ± 0.3; untreated heart: 1.1 ± 0.2; omega-3 DIA: 64 ± 2.6; untreated DIA: 41 ± 3.6; p \ 0.05, ANOVA). TGF-b, a marker of fibrosis, was decreased by omega-3 in heart (56 %) and DIA (33 %). Serum levels (ELISA) of TGF-b and IL-13 were decreased by omega3 (TGF-b: untreated, 1130 ± 128; omega-3, 461 ± 132; IL-13: untreated, 117 ± 13; omega-3, 41 ± 2.5). QDR muscle did not show significant increases in M2 macrophages by omega-3, presenting a slight increase in fibrosis (untreated: no fibrosis; omega-3: 1.3 ± 0.8) and no changes in TGF-b (untreated: 1.3 ± 0.2; omega-3: 1.4 ± 0.2). These results suggest that while the presence of M2 macrophages may be relevant to muscle regeneration, M2 macrophages can also lead to fibrosis which is negative to dystrophinopathies. Keywords: mdx, M2 macrophages, Fibrosis
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