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Journal of Muscle Research and Cell Motility (2005) 26:57–89 DOI 10.1007/s10974-005-9006-9

Springer 2005

Abstracts SESSION I. AUXILIARY TRIADIC PROTEINS, SUNDAY, 18TH OF SEPTEMBER 2005, 8:30–10:30 The role of auxiliary dihydropyridine receptor subunits in muscle B.E. FLUCHER1, G.J. OBERMAIR1, P. TULUC1, J. SCHREDELSEKER2, G. KERN1 and M. GRABNER2 1 Department of Physiology & Medical Physics and 2 Department of Medical Genetics, Clinical & Molecular Pharmacology, Innsbruck Medical University, Innsbruck, Austria The skeletal muscle dihydropyridine receptor is a slowly activating calcium channel that functions as the voltage sensor in excitation– contraction coupling. In addition to the pore-forming a1S subunit it contains the transmembrane a2d-1 and c1 subunits and the cytoplasmic b1a subunit. Although the roles of the auxiliary subunits in calcium channel function have been extensively studied in heterologous expression systems, their functions in excitation–contraction coupling have only recently been elucidated in null-mutant muscle cells. The b1a subunit is essential for skeletal muscle function. Results from a novel zebra ﬁsh mutant indicate that its role is in the coordinated organization of the DHPR in tetrads opposite the ryanodine receptor. The a2d-1 and c1 subunits are not necessary for ec-coupling but function to limit the activity of the calcium channel in skeletal muscle. siRNA depletion of a2d-1 in muscle cells demonstrated that a2d1 determines the slow speed of L-type calcium currents. Analysis of muscle ﬁbres from c knock-out mice showed that c1 limits calcium inﬂux and calcium release by increasing the voltage-sensitivity of inactivation.

Initial characterization of a skeletal calsequestrin knock-out mouse C. PAOLINI1, C. REGGIANI2, P.D. ALLEN3 and F. PROTASI1 1 Univ. G. d¢Annunzio – Ce.S.I., Chieti, Italy 2 University of Padova, Department of Anat. and Physiol., Padova, Italy 3 Department of Anesth. Res., BWH, Boston, USA Calsequestrin (CSQ) is a Ca2+-binding protein located at the junctional face of the SR terminal cisternae (jSR) near the sites of Ca2+ release, the ryanodine receptors. Two isoforms, skCSQ and caCSQ, are diﬀerentially expressed in adult skeletal muscle ﬁbres: exclusively skCSQ in fast twitch ﬁbres, while in slow twitch about 25% of the total amount is caCSQ. To better deﬁne the role of CSQ in the excitation–contraction coupling apparatus, we have created a knock-out model, null for the skCSQ isoform. Western blot analysis of skCSQnull limb muscles conﬁrms the absence of skCSQ, while caCSQ is still present. Mice are usually smaller in size and preliminary hand grip strength test values are signiﬁcantly lower compared to control mice values (30–40%). We have analyzed skCSQ-null calcium release units (CRUs) in the EDL and SOLEUS using electron microscopy and our results show striking ultra structural eﬀect of the mutation that are more evident in the EDL muscle than in the SOLEUS. In both muscles the overall shape of the jSR is ﬂatter than normal and the caCSQ still expressed seems to be condensed into dense bands,

similarly to cardiac muscle. Our results suggest that skCSQ is an important determinant of spatial orientation and ultrastructure of skeletal muscle CRUs.

Developmental expression of ank1.5 and obscurin in developing mouse skeletal muscle E. GIACOMELLO, V. CUSIMANO, E. ASSENZA and V. SORRENTINO Molecular Medicine Section, Department of Neuroscience, University of Siena, Siena, Italy In skeletal muscle cells, the sarcoplasmic reticulum (SR) is organized around each individual myoﬁbril in a regulated way which aligns speciﬁc domains like terminal cisternae and longitudinal tubules with deﬁned regions of the sarcomere. Proteins responsible of the spatial arrangement of the SR with the respect of myoﬁbrils are not known. Recently, we reported that ank1.5, a small muscle speciﬁc isoform of the ankyrin1 gene, localized on the SR, is capable of interacting with obscurin, a protein localized at the periphery of the myoﬁbrils. It is interesting to consider that the interaction between ank1.5 and obscurin may have a potential role in anchoring these two compartments. During skeletal muscle development, the SR starts to be organized around embryonic day 14, and it appears to undergo a series of modiﬁcations to reach an adult-like organization around birth. In parallel, SR speciﬁc proteins acquire the ﬁnal localization at speciﬁc sites. We have performed studies on the expression of ank1.5 and obscurin in order to follow their localization during development of the SR in muscle skeletal cells. Our results indicate that the localization of both ank1.5 and obscurin is developmentally regulated. The localization of ank1.5 during development with respect to that of other proteins of the SR will be discussed.

Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) needs sarcoplasmic reticulum Ca2+ to become activated P. KARCZEWSKI, S. BARTEL, B. HOCH and I. MORANO Max Delbru¨ck Centre for Molecular Medicine, Berlin, Germany CaMKII was established to function as a molecular switch in cardiac EC coupling. However, the origin of Ca2+ coupled to CaMKII activation has not been deﬁned. We studied cytoplasmic CaMKII activation with respect to speciﬁc phosphorylation at Thr17 of its wellcharacterized substrate phospholamban (PLB), the inhibitor protein of the cardiac sarcoplasmic reticulum (SR) Ca2+ pump. In neonatal cardiomyocytes the ß-adrenergic agonist isoproterenol (ISO) and the Ltype Ca2+ channel agonist Bay K8644 induced CaMKII catalysed Thr17 phosphorylation of PLB. When the SR Ca2+ release was blocked by ryanodine or thapsigargin, Thr17 phosphorylation of PLB was abolished. In H9c2 cardiomyoblasts, which do not contain a functional SR comparable to cardiomyocytes, stably transfected with PLB ISO produced the cAMP-dependent phosphorylation of PLB but was unable to induce Thr17 phosphorylation. However, the massive elevation of intracellular Ca2+ by ionomycin resulted in Thr17 phosphorylation indicating that CaMKII was activated. From these data

58 we conclude that activation of CaMKII involved in cardiac EC coupling essentially depends on Ca2+-induced Ca2+ release from the SR.

Presence of SERCA and Calcineurin during fetal development of porcine skeletal muscle H.W. DE JONGE, C.W. VAN DER WIEL, K. EIZEMA and M.E. EVERTS Fac. of Vet. Med., Dept. of Pathobiology, Div. of Anatomy and Physiology, Utrecht, The Netherlands Mechanisms of skeletal myoﬁber diﬀerentiation in large animals during gestation are poorly understood. Studies in rodents suggest that the calcineurin (Cn) pathway is involved. Neural activity is a prerequisite for Cn activity. To study a role of Cn in fetal myoﬁber differentiation, we monitored the temporal and spatial distribution of myosin heavy chain (MyHC) isoforms and Cn subunits A and B in porcine m. semitendinosus at 55 and 75 days and at the end of gestation. Sarcoplasmic reticulum Ca2+ ATPase (SERCA) and phospholamban (PLB) were studied in relation to the presence of nerves. Immunoﬂuorescence analysis revealed no speciﬁc distribution of MyHC in relation to neuromuscular contacts. At 75 days of gestation, SERCA2 expression was accompanied by PLB expression in primary ﬁbers. SERCA1 was expressed in all ﬁbers at all stages. Cn subunit expression was not ﬁber type speciﬁc but became more prominent at term. From these results we speculate that in porcine m. semitendinosus diﬀerential SERCA2 expression precedes Cn expression and that the Cn pathway is not involved in prenatal myoﬁber diﬀerentiation.

The auxiliary a2d-1 subunit modulates current properties of CaV1.2 calcium channels, cardiac action potentials and calcium dynamics P. TULUC, G.J. OBERMAIR, G. KERN, G. KUGLER and B.E. FLUCHER Dept. Physiology & Medical Physics, Innsbruck Medical University, Innsbruck, Austria The L-type calcium channel (CaV1.2) is important for excitation and contraction of the heart. Heterologous expression of the pore-forming a1C with and without the auxiliary a2d-1 subunit in non-muscle cells indicated functions of a2d-1 in membrane expression and current modulation. However, the role of a2d-1 in cardiac function was not known. Coexpression of a1C and a2d-1 siRNA in dysgenic myotubes demonstrated that depletion of the a2d-1 subunit does not affect membrane expression and triad targeting of a1C. The voltage dependence of activation was shifted by +12 mV, causing a 20% reduction of current density. Activation kinetics were slowed down by shifting the ratio from fast to slowly activating channels. Applying these altered current properties to an established mathematical model, predicted that depletion of a2d-1 prolongs the action potential and leads to a substantial increase of calcium load. These results demonstrate that the eﬀects of a2d-1 depletion on CaV1.2 are suﬃciently large to cause defects in cardiac function. Support: Austrian Science Fund P16532-B05, P17806-B05, P17807-B05; EU HPRN-CT-2002-00331.

59 SESSION II. EC AND ET COUPLING, SUNDAY, 18TH OF SEPTEMBER 2005, 10:45–12:45 Excitation–transcription coupling in adult skeletal muscle: Signalling pathways in activity-dependent ﬁbre type plasticity Y. LIU1, T. SHEN1, W.R. RANDALL2 and M.F. SCHNEIDER1 1 Departments of Biochemistry and Molecular Biology and 2Departments of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, USA Adult fast- and slow-twitch skeletal muscle ﬁbres exhibit characteristic diﬀerences in functional properties due to diﬀerences in the isoforms and quantities of expression of many muscle proteins. These diﬀerences may be reversed by chronic electrical stimulation using an activity pattern characteristic of the other ﬁbre type. Here we consider two putative intracellular signalling pathways that may contribute to fast to slow ﬁbre type transformation. The ﬁrst pathway involves cytosolic activation of the Ca2+ sensitive phosphatase calcineurin (CaN) due to elevated cytosolic [Ca2+] during muscle activity. CaN activation results in dephosphorylation of cytoplasmic NFATc, translocation of dephosphorylated NFATc from cytoplasm into the nucleus and activation of slow ﬁbre gene expression by NFATc in the nucleus. The second pathway involves the elevation of intranuclear [Ca2+], likely by diﬀusion of elevated cytosolic Ca2+ into the nucleus. The Ca2+-dependent activation of nuclear calmodulin-dependent protein kinase then causes phosphorylation of HDAC within the nucleus and thereby permits nuclear eﬄux of HDAC. The resulting decline of nuclear HDAC decreases HDAC suppression of MEF2 activation of slow ﬁbre gene expression. Kinetic properties of NFAT and HDAC intracellular movement in electrically stimulated individual adult mouse FDB muscle ﬁbres will be presented.

The scorpion toxin maurocalcine; a tool to study the excitation–contraction coupling in skeletal muscle X. ALTAFAJ1, K. MABROUK2, I. JONA3, L. CSERNOCH3, M. DE WAARD1 and M. RONJAT1 1 Inserm U607/DRDC, Grenoble, France 2 CNRS FRE 2738, Faculte´ de Me´decine Nord, Marseille, France 3 Dept. of Physiol., RCMM, MHCS, Univ. of Debrecen, Debrecen, Hungary Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa binds directly to the type 1 RyR on the same binding site as the domain A of the II–III loop of the alpha1 subunit of the dihydropyridine receptor (DHPR). MCa induces a large increase in ryanodine binding on RyR1 (also observed on RyR3 but not on RyR2) and the appearance of long-lasting channel openings in a mode of smaller conductance. MCa is also able to cross biological membranes. A biotinylated derivative of MCa was produced (MCab) and complexed with a ﬂuorescent streptavidine-cyanine 3 in order to follow the cell penetration of the toxin. The toxin complex eﬃciently penetrated in various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called Cell-Penetrating Peptides (CPP). We used MCa to study the coupling of DHPR and RyR in mammalian skeletal cells. External application of MCa to cultured myotubes produces Ca2+ release from intracellular stores without changing depolarisation-induced Ca2+ release. In adult cells, MCa does not induce Ca2+ release by itself but strongly modiﬁes the frequency and amplitude of calcium release events without modifying their spatial distribution. We propose that functional coupling of RyR and DHPR in Ca2+ release unit prevents MCa from either reaching its binding site or from being able to modify the RyR gating.

Activation of calcium release depends on recent history of calcium inﬂux E. POLA´KOVA´, A. ZAHRADNI´KOVA´ Jr., A. ZAHRADNI´KOVA´ and I. ZAHRADNI´K UMFG SAV, Bratislava, Slovakia Eﬃciency of calcium current (ICa) to activate Ca2+ release is important for reliable function of cardiac muscle cells. We have studied the relationship between ICa activation and activation of Ca2+ release in rat ventricular myocytes using whole-cell patch clamp and confocal microscopy. Depolarization of cells to the ICa reversal potential was used to eliminate Ca2+ inﬂux during speciﬁc time periods. When Ca2+ inﬂux was present both, during a brief (5 ms) depolarizing pulse and after subsequent repolarization (tail ICa), the probability of Ca2+ release activation was signiﬁcantly larger than in the absence of tail ICa. Brief depolarization to voltages that evoked only small Ca2+ inﬂux that by itself activated little Ca2+ release dramatically shortened the latency of the subsequent tail ICa-induced Ca2+ release events, while it did not signiﬁcantly aﬀect probability of tail ICa-induced Ca2+ release. Less negative repolarization potentials increased the probability of tail ICa-induced Ca2+ release. These observations directly demonstrate the potentiating eﬀect of subthreshold local Ca2+ inﬂux on the Ca2+ release machinery, supposedly achieved by relieving the ryanodine receptors of the pre-bound Mg2+ ions.

Expression of Calcineurin and SR Ca2+ handling proteins in equine muscle ﬁbres during early postnatal growth K. EIZEMA, M. VAN DEN BURG, D. VAN DER WAL, H.W. DE JONGE and M.E. EVERTS Dept. of Pathobiology, Utrecht University, Utrecht, The Netherlands The MyHC expression pattern changes dramatically in the m. gluteus medius in the ﬁrst year of life. A calcineurin (Cn) Ca2+-dependent pathway is believed to play a role in activating and maintaining a slow MyHC phenotype. Other Ca2+ handling proteins such as SERCA1 and SERCA2 and its inhibitor phospholamban (PL) are also important for the contractile properties of the ﬁbres. We studied the expression patterns using immunoﬂuorescence in biopsies taken at 0, 2, 4, 22 and 48 weeks of age. At all time points both Cn A and B proteins show expression in all ﬁbres, with higher expression in MyHC type I. From 4 weeks onwards expression of CnA was also higher in MyHC type 2a and 2ad ﬁbres. CnB remained only high in type I ﬁbres. At birth the SERCA isoforms and PL expression were not yet restricted to either fast or slow ﬁbres. At 4 weeks of age, restriction of SERCA1 to fast MyHC ﬁbres and SERCA2 and PL to slow ﬁbres was eminent. We conclude that the high plasticity of MyHC in young foals is also reﬂected in the SERCA1, 2 and PL expression. The CnA and B relative expression however changes within the ﬁbre types.

The complex regulation of slow muscle type genes E. ZA´DOR1, R. FENYVESI1, G. RA´CZ1 and F. WUYTACK2 1 Institute of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary 2 Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Leuven, Belgium The formation of slow muscle phenotype is entirely dependent on the slow type innervation. The main marker of slow muscle, the myosin heavy chain (MyHCI) is also expressed in a strictly nerve dependent manner. Other genes, like the slow type sarco/endoplas-

60 mic reticulum Ca2+ ATPase (SERCA2a) correlates with MyHCI in the ﬁbres in many adaptation models, suggesting that the regulation of slow muscle type genes is common. However, we found that SERCA2a is not regulated directly by slow type innervation and neither depends on the calcineurine-NFAT or the Ras pathways which are each essential for the high expression level of slow myosin. Similarly, the expression of SERCA2a is not regulated mainly at the transcription level, like the expression of slow myosin, but it depends on the levels of splicing and translation. This shows that at least two important slow muscle type genes are regulated in a diﬀerent manner suggesting that there is no common regulatory mechanism that applies to all genes typical for slow skeletal muscle. However, there is a complex adaptation of diﬀerent levels of regulations on the long term forming the slow type muscle.

Intracellular Ca2+ signals and whole-cell and unitary activity of plasma membrane channels in voltageclamped mouse skeletal muscle ﬁbres under conditions of SR Ca2+ release and depletion B. ALLARD, H. COUCHOUX, S. POUVREAU and V. JACQUEMOND University C. Bernard Lyon 1, UMR CNRS 5123, Physiologie Inte´grative Cellulaire et Mole´culaire, Villeurbanne, France Voltage-independent cationic channels in skeletal muscle are suspected to play a role in Duchene dystrophy but the conditions and/or parameters that control their activity remain controversial. Here we used simultaneously single channel- and whole-cell-voltage clamp techniques and ﬂuo-3-Ca2+ imaging on single enzymatically isolated skeletal muscle ﬁbres to unravel any possible change in membrane conductance that would depend upon SR Ca2+ release and/or SR Ca2+ depletion. Delayed rectiﬁer K+ single channel activity was routinely detected during whole-cell depolarizing pulses. Activity of channels carrying inward currents of 1.5 pA at )80 mV was detected in 15 out of 117 and in 21 out of 59 patches in control and in mdx ﬁbres, respectively. In control and mdx muscles, series of whole-cell depolarizing pulses did not reproducibly increase this channel activity even when they led to a decrease in Ca2+ transients giving evidence for SR depletion. Inhibitors of the SR Ca2+ pump produced a slowing of the decay phase of the voltageevoked Ca2+ transients and a progressive increase in the resting Ca2+ level. Still, under these conditions, there was no sign of increase in both the resting whole-cell conductance and in the inward single channel activity. Overall results add no support to the possibility that these channels are regulated by the intracellular Ca2+ store content.

Recovery from voltage-dependent inactivation of Ca release and force in adult muscle ﬁbres of a DHP receptor c1-subunit knockout mouse Z. ANDRONACHE1, D. URSU1, R.P. SCHUHMEIER1, F. KRETZ1, M. FREICHEL2, V. FLOCKERZI2 and W. MELZER1 1 University of Ulm, Germany 2 University of Homburg, Germany Adult ﬁbres of mice lacking the DHP receptor c1 subunit exhibit an altered voltage dependence of inactivation of both Ca conductance and Ca release [Ursu et al. (2004) JGP 124: 605]. Here we studied recovery from inactivation in single voltage-clamped ﬁbres of the M. interosseus and in ﬁbre bundles of the EDL. Ca release ﬂux and tetanic force were recorded. Recovery of Ca release at )80 mV after long-lasting (60 s) depolarization to +10 mV was similar in c)/) cells compared to controls (c+/+). In contrast, tetanic force recovery in normal Krebs–Ringer solution after a period of depolarization by 120 mM K was incomplete. A similar partial restoration could be observed in c+/+ muscle at slightly elevated K concentration (15 mM)

raising the question whether an altered resting potential might explain the observation. In fact, microelectrode measurements of resting potentials in ﬁbres subjected to the recovery protocol revealed a persistent depolarization by about 15 mV in c)/) EDL. This ﬁnding indicates a role of the DHPR c1 subunit in stabilizing the resting potential and maintaining excitation.

NFATc1 and slow-to-fast shift in MyHC composition under gravitational unloading A.V. ARDABIEVSKAYA1, B.S. SHENKMAN2, A.M. MOUKHINA2 and T.L. NEMIROVSKAYA1,2 1 Lomonosov Moscow State University, Moscow, Russia 2 Institute of Biomedical Problems, RAS, Moscow, Russia We have tested the hypothesis, supposing that increased ﬂexor activities in the course of unloading exerted suppressive inﬂuence on postural extensors (e.g. m.soleus) and thus brought about changes in their NFATc1 and MyHC expression. Experiment was performed on 21 male Wistar rats divided into 3 groups: cage control (C, n=7), 14 day hindlimb suspended group (HS, n=7) and 14 day hindlimb suspension with tenotomy of m.tibialis anterior (HST, n=7). NFATc1 content in nuclear extract was analyzed as optical density of 3 bands (110, 82 and 76 kDa) on WB. We found signiﬁcant increase (p<0.05) in HS group compared to C group of NFATc1 content in nuclear extract m.soleus (110 kDa+140%; 82 kDa+87.5%; 76 kDa+78.2%) and of the percentage of MyHCIIa type ﬁber m.soleus (+69.7%). None of these parameters diﬀers signiﬁcantly from C group in HST group. We have concluded that the tenotomy of ﬂexors prevents NFATc1 changes and MyHC transformation in m.soleus under gravitational unloading. The increase of NFATc1 content in nuclear extract associated with the increase MyHCIIa. The work was supported by Grants RFBR 05-04-49255a, 04-0449044.‘

Calcium handling in skeletal muscles from transgenic mice over-expressing Junctate, a novel sarco(endo)plasmic reticulum protein A. DIVET1, S. PAESANTE1, C. GRASSO1, C. TIVERON2, C. HUCHET-CADIOU3, S. TREVES4 and F. ZORZATO1,4 1 Dept. of Exp. and Diag. Med., Sect. of Gen. Pathol., Univ. of Ferrara, Ferrara, Italy 2 Transgenic Mice Service Centre, Exp. Res. Centre, Regina Elena Inst., Roma, Italy 3 UMR CNRS U3B, Fac. des Sci. et Tech., Univ. de Nantes, France 4 Dept. of Anaesthesia and Res., Basel Univ. Hosp., Basel, Switzerland The sarcoplasmic reticulum (SR) regulates the intracellular Ca2+ concentration and plays an important role in excitation–contraction coupling (ECC). The portion of SR terminal cisternae facing the transverse tubules, referred as junctional face membrane, is endowed with numerous proteins for which the function still remains unclear. The aim of this study is to determine the functional role of junctate, a newly identiﬁed SR/ER membrane protein, in Ca2+ homeostasis and ECC mechanism. Experiments were performed on a transgenic mouse model over-expressing junctate in skeletal muscle. Studies of Ca2+ ﬂux on SR microsomes, using a spectrophotometric determination with Antipyrylazo-III, showed that maximal Ca2+ loading capacity was signiﬁcantly increased in transgenic mice compared to their wild type counterpart. Contractile protein properties of fast- (EDL) and slowtwitch (soleus) muscles were investigated in Triton X-100 skinned ﬁbres. The results indicated that maximal Ca2+-activated tension and Ca2+ sensitivity were not aﬀected in transgenic mice. In addition, Sr2+

61 sensitivity was similar between transgenic and wild type mice, excluding a fast to slow transition of transgenic muscle ﬁbres. These ﬁndings demonstrate that over-expression of junctate in skeletal muscle aﬀects the Ca2+ loading characteristics of SR membranes without aﬀecting contractile protein properties.

Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its beta-adrenergic regulation H. HAASE1, J.L. ALVAREZ2, D. PETZHOLD1, J. BEHLKE1, G. VASSORT2 and I. MORANO1 1 Max Delbru¨ck Centre for Molecular Medicine, Berlin, Germany 2 INSERM U-637, Montpellier, France The cardiac L-type Ca channel (ICaL) is activated by protein kinase Amediated phosphorylation of the a1C- and b2-channel subunits and ahnak, an associated 5643-amino acid protein. We examined the role of a naturally occurring, genetic variant I5236T-ahnak on ICaL elicited under whole-cell patch-clamp conditions on rat ventricular myocytes. Intracellular perfusion with a synthetic ahnak peptide (10 lM) carrying the I5236T mutation increased ICaL amplitude from 11.9±0.8 to 18.9±1.4 pA/pF (p<0.05; n=18, 20) at 0 mV depolarization. The ICaL enhanced by this peptide was not further stimulated by isoprenaline. The wild-type peptide had no eﬀect on ICaL. Binding experiments with recombinant b2 subunit and the ahnak-C1 domain (aa 4646– 5288) revealed a dissociation constant for the ahnak-C1/b2 subunit complex of 155±31 nM (n=14). The binding aﬃnity of this complex decreased by 50% by either PKA phosphorylation or the presence of 10 lM mutated peptide (but not wild-type peptide). Hence, we suggest that tuning the ahnak/b2 subunit interaction is an important determinant for controlling ICaL.

Eﬀects of natural derivatives of phenol on calcium transport of sarcoplasmic reticulum B. LUKACS, G. NAGY, N. DOBROSI, I. GHERASIM and I. JONA Department of Physiology, Medical and Science Centre, Univ. Debrecen, Debrecen, Hungary The eﬀect of natural derivatives of phenol was investigated on sarcoplasmic reticulum calcium pump (SERCA) isolated from rabbit and calcium release channel (RyR1) isolated from normal and malignant hyperthermia susceptible pig (MHS) skeletal muscle. SERCA activity was determined using ‘coupled enzyme method’, the decrease of NADH concentration was followed photometrically. The eﬀects of some of these agents on the calcium release and on the 3H ryanodine binding were investigated too. Thymol and trans-anethole inhibited the Ca2+ATPase with a Kd of 224±10 lM and 168±10 lM, respectively. The vanillin, orthovanillin, 4-methyl-2-nitrophenol, meta-anisaldehyde and cineole exerted only unspeciﬁc inhibition. In calcium release measurements, the thymol and the carvacrol activated the channel with a Kd of 158±16 lM and 211±55 lM, respectively. In binding assay experiments the thymol activated both the MHS and wild type RyR1. The MHS type RyR1 showed lower Kd to ryanodine and could bind more ryanodine than the normal type RyR1 both in control and in the presence of thymol. Supported by the EU (HPRT-CT-2002-331) and OTKA (037727).

Nicotinic acetylcholine receptors as a physiological target of the illicit drug ecstasy W. KLINGLER1,2, J.J.A. HEFFRON4, K. JURKATROTT2, G. O’SULLIVAN4, A. ALT3, F. SCHLESINGER5, J. BUFLER5 and F. LEHMANN-HORN2 1 Dept. of Anaesthesiology 2Applied Physiology and 3 Forensic Medicine, Ulm Univ., Germany

4

Dept. of Biochem., Univ. College Cork, Ireland Dept. of Neurology, Medizinische Hochschule Hannover, Germany 5

3,4-Methylendioxymethamphetamine (MDMA, ecstasy) is a drug which is abused because of its psychodelic eﬀects in the central nervous system. However, clinical features as fasciculations, muscle cramps, trismus are frequent and severe complications as hyperthermia, hyperkalemia and rhabdomyolysis were linked to the pharmacogenetic trait of malignant hyperthermia (MH). Hence, we tested the hypothesis of a direct muscle eﬀect of MDMA on surgically dissected muscle strips, cultivated myotubes and isolated sarcoplasmic reticulum (SR) vesicles. Mechanographic registrations showed that MDMA enhanced the sensitivity to the Ca2+-releasing agents halothane and caﬀeine. MDMA increased cytosolic [Ca2+] and cellular metabolism from myotubes but did not release Ca2+ from the SR. Alpha-bungarotoxin, a speciﬁc antagonist of the nicotinic acetylcholine receptor (nAChR) abolished the MDMA eﬀects. The nAChR agonistic action of MDMA was conﬁrmed by current measurements on HEK-cells expressing nAChR. We were surprised that MDMA did not aﬀect EC-coupling and we conclude that an activation of the nAChR contributes to the muscle-related symptoms of MDMA.

Reduced calcium available for release during skeletal muscle fatigue B.R. MACINTOSH1,2, J.J. CAMPBELL1, S. ESAU1 and M.B. MACNAUGHTON2 1 Faculty of Kinesiology and 2Faculty of Medicine University of Calgary, Calgary, Alberta, Canada Muscle fatigue results from reduced Ca2+ release from the sarcoplasmic reticulum due to either impaired release mechanism, or decreased Ca2+ in the terminal cisternae. We have shown that the extra force per activation is not decreased by impaired Ca2+ release, but this would be decreased with reduced Ca2+ availability, possibly due to precipitation with inorganic phosphate (Pi). This should resolve within 5 min of stopping repetitive stimulation, as phosphocreatine (PCr) resynthesis occurs. The medial gastrocnemius muscle of the anesthetized rat was prepared for isometric contractions near optimal length in situ. The force per activation late in a train was determined by subtraction of the force transient with 10 pulses at 50 Hz from the transient with 11 pulses: prior to, during, and following 6 min of repetitive stimulation (50 Hz, 300 ms, 1Æs)1). The force per activation was 2.1±0.2 (mean±SD) times the twitch amplitude prior to and 0.82±0.2 at 3 min of repetitive stimulation. By 5 min of recovery, the extra force was still signiﬁcantly diﬀerent (1.24±0.2) from control (no repetitive stimulation) values (1.7±.01), indicating that recovery did not occur within the time that PCr resynthesis would be complete. Ca2+ availability appears to decrease during repetitive stimulation, but factors other than precipitation with Pi may be involved. Supported by NSERC and the Markin-Flanagan award.

Stretch of in-series structures cannot explain length dependence of fatigue M.B. MACNAUGHTON1,2 and B.R. MACINTOSH1,2 Faculty of Kinesiology1and Faculty of Medicine2, University of Calgary, Calgary, Alberta, Canada Relative force depression associated with muscle fatigue is greater when assessed at short vs. long muscle lengths. A rightward shift of the length-dependence of force may be caused by stretch of in-series structures, making sarcomere lengths shorter at any muscle length. Such a rightward shift would cause proportionately greater force depression at short vs. long muscle lengths. Submaximal force–length

62 relations (double-pulse) were evaluated before and after repetitive contractions (50 Hz, 300 ms, 1 s)1) in an in situ preparation of the rat medial gastrocnemius muscle. Fascicle lengths were measured with sonomicrometry. Prior to repetitive stimulation, fascicle lengths were 11.3±0.8, 12.8±0.9 and 14.4±1.2 mm at lengths corresponding to )3.6, 0 and 3.6 mm where 0 is a reference length with maximal active force. After repetitive stimulation, these lengths were 11.4±0.8, 12.6±0.9 and 14.2±1.2 mm. There was no signiﬁcant change in fascicle lengths. The length-dependence of fatigue was, therefore, not due to a stretch in the SEC. Interestingly, the rightward shift was more evident when active force was calculated in the traditional method. When passive force associated with the peak of contraction was subtracted from peak force, the length-dependence of fatigue was still present, however, postfatigue absolute active force depression was similar at all lengths. Supported by NSERC.

Splice variants of the CACNA1S calcium channel gene N. MOLENDA, N. MAO, F. LEHMANN-HORN and K. JURKAT-ROTT Abteilung Angewandte Physiologie, Universita¨t Ulm, Ulm, Germany Alternative splicing is likely to be the primary source of human proteomic diversity. Our aim was to investigate the splice variants of the human CACNA1S gene. We extracted RNA from human muscle and human myoblast cell culture, performed reverse-transcriptase PCR and direct sequencing of all variants found. We identiﬁed 10 diﬀerent splice variants: 6 generating premature truncations and 4 generating putatively functional channels. The splice variant excluding exon 29 is comparable to the variant of cardiac channel gene, CACNA1C, excluding exon 33. Exclusion of exon 29 is also known for mouse cacna1s indicating a high conservation of this variant. It is highly expressed in myotubes (80% of transcripts) but relatively rare in adult muscle (20% of transcripts). For this reason, we want to undertake functional studies of this variant.

Wetware innovative model of artiﬁcial siliconneuromuscular junction for EC coupling stimulation of ﬁbre and myotubes on metal-oxide-semiconductor (MOS) devices and MicroCHIPs M. QUARTA1, M. CANATO1, S. VASSANELLI2 and C. REGGIANI1 1 Muscle Biophysics Lab, Dept. of Anatomy and Physiology, Univ. of Padua, Italy 2 NaChip Lab, Dept. of Anatomy and Physiology, Univ. of Padua, Italy The aim of this study was to develop an artiﬁcial silicon-muscle junction to allow a ﬁne control of in vitro long-term contraction patterns, based on software and instrumental to muscular tissue engineering, new experimental approaches, and muscular regeneration models. Two distinct biological models were employed: (1) Satellite primary mouse cells were cultured on diﬀerent silicon chip devices and induced to diﬀerentiate to myotubes developing a muscle-silicon junction stimulation spots integrated in the chip wire. This was used to elicit single myotubes excitation, EC coupling and contraction and (2) Fully diﬀerentiated muscle ﬁbres were dissociated from one month mice and cultured on the same chip devices for the same purposes. The development of this model has allowed us to replace the diﬀuse ﬁeld stimulation of a cell population with the control of single cells with the high spatial resolution given by capacitive stimulation. Single cells can be stimulated and monitored in a precise and targeted way and cells exposed to diﬀerent conditions can be compared.

Eﬀects of a calcium chelator TPEN on sarcoplasmic reticulum calcium release ryanodine receptor channel S. SARKOZI, J. ALMASSY, C. SZEGEDI and I. JONA Department of Physiology, Medical and Science Centre, University of Debrecen, Debrecen, Hungary The aim of the study was to determine the eﬀects of a widely used calcium and zinc chelator TPEN (N,N,N¢,N¢-Tetrakis (2-pyridylmethyl)ethylendiamine) on skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel (RyR). Investigations of TPEN-buﬀered glioblastoma cells resulted in controversial consequences raised the question if TPEN has any inﬂuence on the Ca2+ transport mechanisms of the SR. To clarify if RyRs are concerned in the putative action of TPEN isolated Ca2+ release channels were reconstituted into planar lipid bilayers and the gating behaviour of incorporated channels was studied. TPEN altered the RyR gating in a dose-, polarity-, and voltage-dependent manners. These eﬀects were proved to be dual: if the applied holding potential induced current was opposite of the direction occurring physiologically through the RyR TPEN closed the channel in potential dependent way. This result suggests that positively charged TPEN works as a plug and acting so directly blocks RyR’s pore. Oppositely, when the direction of the current ﬂowing through the incorporated RyR was of the same during Ca2+ release from SR TPEN slightly activated RyR by increasing the open probability of the channel. Supported by the EU (HPRT-CT-2002-331) and OTKA (037727).

Blot overlay analysis of the skeletal ryanodine receptor D.N. SCHREIBER and K. OHLENDIECK Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland The RyR1 isoform of the ryanodine receptor Ca2+-release channel was isolated from rabbit skeletal muscle by immunoprecipitation. Following conjugation to activated horseradish peroxidase, the protein was used as bait in blot overlay assays. Triads were separated in a 1D denaturing polyacrylamide gel and in a 2D native/denaturing gel system. Detection of protein–protein interactions using enhanced chemiluminescence revealed three bands which probably represent RyR1 itself and triadin. Crosslinking experiments showed several additional interaction-partners of lower molecular mass which might be involved in the formation, regulation and maintenance of excitation–contraction coupling. Corresponding proteins will be identiﬁed by immunoadsorption, chemical crosslinking and MALDI-ToF mass spectrometry. Research was funded by project Grant HPRN-CT-2002-00331 from the European Commission.

Down-regulation of resting calcium sparks activity in dystrophin deﬁcient myotubes with mini-dystrophin introduction H. BALGHI, S. SEBILLE, B. CONSTANTIN, G. RAYMOND and C. COGNARD Institut de Physiologie et Biologie Cellulaires, CNRS UMR 6187, University of Poitiers, France In Duchenne muscular dystrophy (DMD), cytosolic calcium levels are known to be increased, leading to cell necrosis. We studied resting local calcium release activities in myotubes from a dystrophin-deﬁcient skeletal muscle cell line (SolC1) and from SolD (a derivated clone expressing mini-dystrophin). These spark-like events were found to be more numerous in SolC1 than in SolD myotubes as demonstrated with the sparks ‘site density’ determination. Exposure of ryanodine drastically reduced Ca2+ sparks site density in SolD myotubes but not in SolC1 while IP3 pathway inhibitors signiﬁcantly reduced Ca2+ sparks site density in SolC1 myotubes. Immunocytochemistry of

63 IP3R-1 and IP3R-2 revealed localization of a possible IP3-dependent release apparatus in both cell types. Increases in duration and spread of calcium sparks suggest the involvement of IP3R in resting calcium release activity in dystrophin-deﬁcient myotubes. Down-regulation of this pathway by introduction of BMD-minidystrophin illustrates the regulatory role of dystrophin on Ca2+ homeostasis.

Comparison of the eﬀect of ischiadic and selective denervation on gene expression in rat soleus muscles A. SZABO and E. ZADOR Institute of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary Denervation is a frequently used method to study the eﬀect of motoric innervation on skeletal muscle. For hindlimb muscles this is usually carried out by dissecting a part of the ischiadic nerve. However, it is less frequently applied method when a piece from the soleus nerve is dissected therefore the muscle is selective denervated. In the later case, the soleus is passively moved by the other hindlimb muscles while in ischiadic denervation it is practically in paralyzed condition. This diﬀerence in motility aﬀects the fresh weight, the RNA and protein content of the denervated and the denervated-regenerated muscle and apparently inﬂuences gene expression. In this work we investigate the mRNA and protein levels of myogenic transcription factors, myosins, sarco/endoplasmic reticulum Ca2+ pumps (SERCAs). Our results will compare the eﬀects of selective and ischiadic denervation on gene expression of developed and regenerating soleus muscles.

The novel suramin analogue NF676 is a potent inhibitor of the calcium release channel – ryanodine receptor C. SZEGEDI and M. HOHENEGGER Institute of Pharmacology, Centre for Bio-Molecular Medicine and Pharmacology, Medical University of Vienna, Vienna, Austria The trypanocidal drug, suramin is potent activator of the ryanodine receptor via the calmodulin binding site. As calmodulin is an activator and inhibitor of the ryanodine receptor, depending on the presence of Ca2+, respectively, we screened a panel of suramin analogues for a possible inhibitory action. Using high-aﬃnity [3H]ryanodine binding to sarcoplasmic reticulum membranes, we have found that NF676 is capable to inhibit the ryanodine receptor. This assumption is corroborated by [3H]ryanodine binding to the puriﬁed ryanodine receptor and single channel recordings. In saturation isotherms, NF676 decreased the [3H]ryanodine binding to the puriﬁed ryanodine receptor and reduced the aﬃnity for [3H]ryanodine. In single-channel recordings NF676 conﬁrmed its inhibitory potency. NF676 induced a concentration dependent closure of the channel with no eﬀect on current amplitude. The NF676-induced inhibition was preceded by a strong ﬂuctuation of the channel activity and the closed channel could be fully reactivated upon ATP administration. Taken together these data suggest that NF676 is a novel potent inhibitor of the ryanodine receptor.

Travelling elementary calcium release events in the presence of a low aﬃnity calcium buﬀer in skeletal muscle ﬁbres P. SZENTESI1, C. SIMUT1, T. DELI1 and L. CSERNOCH1,2 1 Cell Physiol. Res. Group, RCMM and 2Dept. of Physiol., Univ. Debrecen, Debrecen, Hungary The calcium content of sarcoplasmic reticulum (SR) plays important role in the electro-mechanical coupling of skeletal muscles. With a low aﬃnity Ca2+ buﬀer (TPEN) we modiﬁed the free calcium concentration in the SR ([Ca2+]SR) in frog and rat skeletal muscle ﬁbres.

TPEN in high concentration (>100 lM) decreased the probability of spontaneous elementary Ca2+ releases (sparks) in permeabilized ﬁbres, while in low concentration in low intracellular magnesium concentration ([Mg2+]i) caused travelling sparks, with a speed of 365±23 lm/s. In intact ﬁbres TPEN in high concentration did not modify the parameters of depolarization evoked Ca2+ release. In low concentration the drug slowed down the inactivation of SR Ca2+ release channel (RyR) from 5±1 to 10±3 ms. Our ﬁndings suggest that TPEN in high concentration decreases the Ca2+ release from the SR by inhibiting the Ca2+ reuptake. In low concentration in the absence of Mg2+ inhibits the inactivation of RyRs by binding the released Ca2+. These results suggest that in physiological conditions the high [Mg2+]i inhibits the spreading of Ca2+-induced Ca2+ release in skeletal muscle. Furthermore, the [Ca2+]SR must be suﬃciently high for developing spontaneous elementary Ca2+ release events. Supported by the EU (HPRT-CT-2002-331) and OTKA (T0 49151).

Statistical properties of automated detection methods for elementary calcium release events F.V. WEGNER and R.H.A. FINK Medical Biophysics, Institut f. Physiologie und Pathophys., Heidelberg University, Germany We analyzed the statistical properties of two algorithms for the detection of elementary Ca2+ release events (ECRE) in line scan images using synthetic images with variable noise levels and amplitude scaled, synthetic ECRE. Two statistics representing (i) detection eﬃciency (sensitivity) and (ii) reliability (positive predictive value) were determined for diﬀerent ECRE amplitudes. The amount of amplitude distortion was evaluated and event histograms for amplitude, rise time, FDHM and FWHM were computed from artiﬁcial images containing ECRE scaled according to random scan line positioning. We developed a new wavelet transform-based algorithm and compared it with the commonly used one originally proposed by Cheng et al. (1999). The wavelet based algorithm allows for highly eﬃcient noise reduction without excessive amplitude reduction. As the spectral information is highly preserved, ECRE were detected on all wavelet levels. The novel algorithm shows considerably higher sensitivities and lower false-positive counts. Repetitive events are easily detected due to the multiresolution properties of our algorithm.

Allosteric coupling between calcium binding and channel opening in the ryanodine receptors A. ZAHRADNI´KOVA´1, S. GYO¨RKE2 and I. ZAHRADNI´K1 1 UMFG SAV, Bratislava, Slovakia 2 DHLRI, OSU, Columbus, OH, USA Numerous RyR point mutants, implicated in disease phenotypes of skeletal and cardiac muscle, have defective regulation by calcium even in heterozygous state. We have developed a method for adaptation of RyR models to account for the presence of wild-type and mutant monomers. The adapted models were used to analyze the eﬀects of allosteric regulation, modal gating, and structure of the Ca2+ binding site on activity of RyRs containing monomers deﬁcient in calcium-dependent activation [Li and Chen (2001) J Gen Physiol 118: 33]. To describe the eﬀect of mutation on RyR activity, RyR models needed (i) four independent Ca2+ binding sites; (ii) allosteric coupling between Ca2+ binding and channel opening; and (iii) the presence of low-activity gating modes. Our results give a quantitative basis to the previously suggested relationships between conformational changes and gating transitions of the RyR. These results open new insights into the molecular mechanisms of RyR activation by calcium and provide important clues for understanding RyR regulation in health and disease. Supported by VEGA 2/4150/04 and APVT-51-31104.

64 SESSION III. CROSS BRIDGE STRUCTURE AND MOVEMENTS, SUNDAY, 18TH OF SEPTEMBER 2005, 17:45–19:45 Temperature-jump induced force generation in muscle is inhibited during lengthening and enhanced during shortening K.W. RANATUNGA, M.E. COUPLAND and G.J. PINNIGER Department of Physiology, School of Medical Sciences, University of Bristol, Bristol, UK A small rapid temperature-jump (T-jump) induces an increase of active force in isometric muscle representing the conformational change in the acto-myosin power-stroke that leads to the muscle force generation [Goldman et al. (1987) J Physiol 392: 71; Davis and Harrington (1987) Proc Natl Acad Sci 84: 975; Bershitsky and Tsaturyan (1992) J Physiol 447: 425; Ranatunga (1999) Proc Roy Soc B 266: 1381]. When an active muscle is shortening, the force is decreased below the isometric force, whereas the force is increased above the isometric level when muscle is lengthening. From four experiments on maximally Ca-activated (skinned rabbit psoas) muscle ﬁbres at 10 C, we ﬁnd that the relative amplitude and the rate of force generation induced by a 3 C laser T-jump (in 0.2 ms) are increased as the shortening velocity is increased (n>30). Conversely, the amplitude decreases as the ﬁlament sliding velocity is decreased from shortening towards isometric and is near zero during lengthening; a T-jump does not produce a net increment in tension in lengthening muscle. This is direct evidence for the molecular basis of the well known Fenn eﬀect that energy liberation in muscle is enhanced during shortening (We thank the Wellcome Trust for the support).

The number of the N-terminal negative charges of actin modiﬁes the amount of force generated per cross-bridge in reconstituted bovine cardiac muscle ﬁbres M. KAWAI1, X. LU1 and P.A. RUBENSTEIN2 1 Department of Anatomy and Cell Biology and 2Department of Biochemistry, University of Iowa, Iowa City, USA Mutant yeast actins were used to determine the role of actin’s Nterminal negative charges in force generation. The thin ﬁlament was selectively removed from skinned cardiac muscle ﬁbres by gelsolin, and the actin ﬁlament was reconstituted from puriﬁed Gactin. For reconstitution, yeast wild type actin (2Ac: 2 N-terminal negative charges), yeast mutant actins (3Ac and 4Ac), and rabbit skeletal muscle actin (MAc) were used. The eﬀects of phosphate, ATP, and ADP on force development were studied at 25 C. With MAc, isometric tension was 77% of the initial tension owing to the lack of a regulatory system. With 2Ac, isometric tension was 10%; with 3Ac, isometric tension was 23%; and with 4Ac, isometric tension was 44%. Stiﬀness followed a similar pattern (2Ac<3Ac<4Ac
Muscle mechanics and uncoupling protein 3 in mice D.C. BICKHAM and N.A. CURTIN Division of Biomedical Sciences, Imperial College London, London, UK Uncoupling protein 3 (UCP3) is a mitochondrial transporter believed to waste energy as heat without ATP synthesis. Transgenic mice overexpressing UCP3 (UCP3-oe) are lean, though hyperphagic with a normal level of locomotion [Clapham et al. (2000) Nature 406: 415–418]. Knock-outs lacking the gene (UCP3-ko) are physically similar to wild type (WT) mice, but have metabolic diﬀerences, not fully elucidated. Muscle ﬁbre types are characterized by their myosin isoforms and metabolic proﬁle. Disruption of the matching of contractile proteins and metabolism may result in altered contractile performance. We have reported that UCP3-oe and WT muscles are similar [Bickham and Curtin (2005) J Physiol 565P: C97]. Here we report results for soleus (slow twitch) and edl (fast twitch) muscles isolated from UCP3ko mice (25 C). The force–velocity relationship was investigated by the ‘step and ramp’ method [Curtin et al. (1998) J Exp Biol 201: 103– 114] and the slack test method [Edman (1979) J Physiol 291: 143– 159]. We found no signiﬁcant diﬀerences in the maximum shortening velocities or maximum power outputs between WT, UCP3-oe and UCP3-ko, when tested in brief contractions.

Decreased muscle function in uremia in human and rat is ﬁbre type dependent E.M. BARTELS1, M. TOFTDAL2, S. PIERZYNOWSKI3, A.H. NIELSEN4, I. EIDEMAK5, S. MOELSTED5 and A.P. HARRISON2 1 The Nat. Libr. of Sci. and Med., Copenhagen 2 The Royal Vet. and Agr. Univ. KVL, Frederiksberg, Denmark 3 Animal Physiol. Lab., Lund, Sweden 4 Copenhagen Gen. Hosp., KAS, Herlev, Denmark 5 Frederiksborg Gen. Hosp., Hillerød, Denmark Uremia is known to cause muscle weakness and early fatigue. In an extended study of 25 uremia patients, surface EMG was measured before, and immediately after, dialysis during voluntary movement of muscles. m. vastus lateralis and m. interosseus dorsalis II, using a sampling rate of 40,000 s)1 with a MacLab 8s and a G4Powerbook. An improvement of action-potential frequency was found in both muscles after dialysis, but most clearly for the m. interosseus dorsalis II muscle, which is mainly a fast muscle. In vitro experiments measuring muscle strength from m. soleus (mainly slow ﬁbres) and the EDL muscle (mainly fast ﬁbres) of rats, in an uremic and a normal Ringer’s solution showed that, during tetanus, the slow muscle was hardly aﬀected, while the fast muscle was more fatigued, when the uremic condition was compared with controls. Our results indicate that uremia particularly aﬀects the fast muscle ﬁbres, as seen from both action potential and mechanical measurements.

Structural and mechanical behaviour of non-forcegenerating cross-bridges S.Y. BERHITSKY1, M.A. FERENCZI2, N. KOUBASSOVA3, V. SITHTHANANDAN2 and A.K. TSATURYAN3 1 Institute of Immunology and Physiology, Russian Academy of Sciences, Yekaterinburg, Russia 2 Imperial College, London, UK 3 Institute of Mechanics, Moscow University, Moscow, Russia

65 Properties of acto-myosin cross-bridges in pre-force-generating state were studied in bundles of permeabilized muscle ﬁbres. Structural changes were monitored by low-angle time-resolved X-ray diﬀraction at SRS station 16.1 (Daresbury Laboratory, UK). Low ionic strength relaxing solution (LISRS) or an addition of AlF4 or BeFx to activating solution was used as factors populating the pre-forcegenerating state(s). Muscle tension was suppressed to 25% while instantaneous stiﬀness left 90% of those in actively contracting muscle. The ﬁrst actin layer line, an indicator of cross-bridge stereospeciﬁc binding, was not seen in the diﬀraction pattern. Cyclic stretches-releases caused diﬀerent responses of the intensity of M3 meridional reﬂection (IM3) that depends on the shape of cross-bridges. A stretch led to a 30% transient increase in IM3 in LISRS, produced no changes in IM3 in the presence of BeFx and initiated a transient decrease in IM3 with AlF4 like that in active contraction. Supported by HHMI, NATO, RFBR, MRC.

Diﬀerent activity of HIF-1 in rat slow and fast skeletal muscle ﬁbres under systemic hypoxia N. CACCIANI1, A. MATSAKAS2, M. MURGIA2, L. TONIOLO1, S. SCHIAFFINO2 and C. REGGIANI1 1 Department of Anatomy and Physiology, University of Padova, Italy 2 Department of Biomedical Sciences, University of Padova, Italy Recent studies have shown that cellular responses to hypoxia are mediated by the hypoxia inducible factor 1 (HIF-1), a transcription factor active on many genes relevant to oxygen homeostasis. The purpose of this study was to assess whether the activity of HIF-1 is diﬀerent in slow oxidative muscles, as soleus, and in fast glycolytic muscles as EDL. Rat soleus and EDL were transfected in vivo with plasmids carrying luciferase as a gene reporter under control of a HIF-1 responsive promoter. Transcriptional activity of the HIF-1 was measured with luciferase assay in homogenates of soleus and EDL of rats breathing spontaneously in normoxia (21% oxygen) or hypoxia (10% oxygen). In normoxic conditions HIF-1 activity in the soleus was nearly double than in the EDL. The diﬀerence between soleus and EDL disappeared after 24 h of denervation suggesting a role of muscle contractile activity in determining the higher HIF-1 activity in soleus. In hypoxic conditions, HIF-1 transcriptional activity increased both in EDL and soleus. Whereas in soleus activity remained high, in EDL activity returned to the initial values after approximately 24 h.

Viscosity and protein osmotic pressure are the indissoluble partners of muscle contraction E. GRAZI and C. DI BONA Dipartimento di Biochimica e Biologia Molecolare, Universita` di Ferrara, Italy A simple model is presented where, by an iterative procedure, the forces delivered by the power strokes are summed up to overcome the load. The system is moderated by the viscous hindrance. The model reproduces the features of muscle contraction as deﬁned by the data of He et al. on rabbit psoas muscle ﬁbres. The model recalls the concept of impulsive force ﬁrst introduced by Wortington and elaborated by Wortington and Elliot. The force generated by the power stroke is transmitted to the zeta disk and promotes contraction. To raise a load force summation is required. This occurs when the persistence of the force generated by the power stroke is longer than the time elapsing between two power strokes. Both cross-bridge attachment and stretching decrease water activity thus increasing the elastic energy of the cross-bridges and the viscosity of the medium. From one side, the decrease of water activity allows the attached cross-bridge to withstand to and to deliver larger forces, from the other side the decrease of

water activity increases viscosity, thus promotes heat dissipation, hinders the energetic yield and decrease the velocity of the contraction.

Interpretation of the X-ray interference in muscle with a multi-state structural model N. KOUBASSOVA Institute of Mechanics, Moscow University, Moscow, Russia Temperature jump from 5 C to 30 C in contracting permeabilized muscle ﬁbres from the rabbit leads to a 2.5-fold increase in active tension. M3 myosin meridional X-ray reﬂection arising from 14.5 nm axial repeat of myosin heads along myosin backbone is split into two peaks due to interference of the X-rays on the symmetrical halves of a sarcomere. Interference ﬁne splitting of M3 was recorded at steady state contraction at low and high temperatures [Tsaturyan AK et al. (2003) J Muscle Res Cell Motil 24: 332]. Fivestate kinetic-structural model has been proposed to explain observed changes in the intensities of M3 and the ﬁrst actin layer line, A1, accompanying tension rise [Ferenczi M.A. et al. (2005) Structure 13: 131–141]. Analysis of the model showed that the movement of the centre of electron density of myosin heads in the 5-state model, Dc, is diﬀerent from that estimated from ﬁne axial M3 proﬁle by generally used point diﬀractor model. Modiﬁed model is able to explain both cited sets of data. The changes in the M3 interference proﬁle in response to applied axial displacement of cross-bridges predicted by the multi-state model are discussed. Supported by RFBR, INTAS, NATO, HHMI.

Interaction of signalling pathways that regulate muscle atrophy and hypertrophy E. LATRES, A.R. AMINI, A.A. AMINI and D. GLASS Regeneron Pharmaceuticals, Inc., Tarrytown, New York, USA Adult skeletal muscle regulates mass and ﬁbre size in response to changes in workload, pathological conditions, or treatment by pharmacologic agents. Insulin-growth-factor-I is a crucial signalling mechanism underlying muscle hypertrophy, while increase in protein degradation by the ubiquitin pathway is a key mechanism during atrophy. We identiﬁed and characterized components of both hypertrophy and atrophy, including two genes up-regulated in multiple models of muscle atrophy; MAFbx, and MuRF1 encode E3 ubiquitin ligases, required to conjugate ubiquitin to speciﬁc substrates. To investigate the interaction of the hypertrophy and atrophy processes, we investigated global changes in gene expression in response to muscle hypertrophy and atrophy, directly comparing these inverse processes. Using DNA-microarray analyses we identiﬁed ﬁve genes which are signiﬁcantly and inversely regulated during IGF-induced hypertrophy and dexamethasone-induced atrophy in C2C12 myotubes. Most importantly, regulation of these genes in response to dexamethasone could be prevented with IGF-I, demonstrating that this pathway can block the cellular changes that underlie muscle atrophy.

Eﬀects of EGTA administration for MyHC and SERCA isoform transformation in m. soleus under hindlimb unloading in rats A. MOUKHINA, K. LITVINOVA, N. GASNIKOVA, T. NEMIROVSKAYA and B. SHENKMAN Institute for Bio-Medical Problems RAS, Moscow, Russia Hindlimb unloading (HU) lead to increase of intracellular Ca2+ level and MyHC slow-to-fast shift in m. soleus. Study was aimed to investigate the role of intracellular Ca2+ level for MyHC and SERCA shift in muscle ﬁbres under HU. Thirty-six male Wistar rats were divided in 3 groups: cage control (C, n=12), 14 days HU (HU,

66 n=12), 14 days HU with 0.3 ml 10% EGTA i.p./day (HUE, n=12). Level of intracellular Ca2+ increased in HU group. % muscle ﬁbres with MyHCI decreased in HU group (68, 6+2.0 vs. 78.0±1.1% in C group, p<0.05) and % muscle ﬁbres with MyHCII increased (28.7+2.1 vs. 19.1±0.7 % in C, p<0.05) respectively. % of ﬁbres with SERCA 1 and SERCA II increased in HU group (47.4±4 and 80.2±2) vs. those in C group (32.3±3 and 70.3±3, p<0.05, respectively). EGTA injection in group HUE decreased level of intracellular Ca2+, prevented MyHC slow-to-fast shift and prevented increase % of muscle ﬁbres with SERCA I, but not SERCA II (78+2 vs. 70+3 in C, p<0.05). Conclusion: Increase of intracellular Ca2+ level under HU lead to MHC slow-to-fast shift, increase % of muscle ﬁbres with SERCA I, but not SERCA II. Supported by grants RFBR N05-04-49255a, 04-04-49044.

Eﬀects of contractions on MGF expression in isolated mouse skeletal muscle M. OTTENS, M.E. EVERTS and P.W.J. VAN DEN WIJNGAARD Department of Pathobiology, Utrecht University, The Netherlands The insulin-like growth factor (IGF-1) gene is involved in exercisedinduced hypertrophy. IGF-1 can undergo alternative splicing to generate diﬀerent products, like the mechano-growth factor (MGF) expressed in mechanically overloaded muscle. The aim of this study was to determine the correlation between diﬀerent modes of contractions and MGF expression in time. Therefore, mouse EDL muscles were isolated and lengthening and isometric muscle contractions were performed. Expression levels were determined for MGF and IGF-1 and also for speciﬁc markers of activated satellite cells like Proliferating Cell Nuclear Antigen (PCNA) and MyoD. 15¢ of lengthening contractions induced MGF expression after 4 h and IGF-1 expression after 6 h. Isometric contractions did not induce MGF and only a small amount of IGF-1. PCNA and MyoD are expressed during lengthening contractions, indicating that satellite cells (SC) start to proliferate. These results suggest that MGF expression is induced in muscles which are subjected to lengthening contractions and MGF plays a role in the activation of SC.

Eﬀect of diﬀerent types of mechanical loading on the MHC synthesis rate and composition in skeletal muscle A. PEHME, K. ALEV, P. KAASIK, R. PUHKE, M. ARU, K. REISBERG, M. KIPRI and T. SEENE Department of Functional Morphology, University of Tartu, Tartu, Estonia The aim of the study was to investigate the response of protein synthesis rate, particularly myosin heavy chain (MHC) isoforms synthesis and the magnitude of its isoform transformation in rat fast-twitch plantaris muscle to diﬀerent modes of prolonged mechanical loading. Diﬀerent protocols of mechanical loading were used: resistance training (RT), compensatory hypertrophy (CH) of m. plantaris after tenotomy of m. gastrocnemius and the combination of the two previous loadings (RT + CH). MHC I and IID isoform synthesis rate increased in all experimental groups, as well as their relative content. MHC IIA relative content decreased during RT and RT+CH and increased during CH. MHC IIB isoform relative content decreased in all experimental groups, but in comparison with CH in CH+RT MHC IIB isoform content increased in plantaris muscle. These results demonstrate diﬀerent modes of mechanical loading resulted in the selective up- and down-regulation of MHC isoforms in fasttwitch skeletal muscle. The synthesis rate and relative content of two fastest isoforms of MHC IIB and IID are regulated to diﬀerent directions during mechanical loading.

Relation between contractile proteins turnover rate, nuclear domain size and skeletal muscle functional capacity T. SEENE1, K. ALEV1, P. KAASIK1, A. PEHME1, M. UMNOVA2, R. PUHKE1, E.-M. RISO1, M. ARU1 and K. REISBERG1 1 Department of Functional Morphology, University of Tartu, Estonia 2 Institute of Ecology and Evolution, RAS, Moscow, Russia The purpose of this study was to investigate the eﬀect of muscle hypertrophy and atrophy on the functional capacity of slow-twitch (ST) and fast-twitch (FT) skeletal muscle, changes in myosin heavy (MyHC) and light chain (MyLC) isoforms pattern, turnover rate and nuclear domain size. The animals used were adult male rats of Wistar strain. Muscle atrophy was caused by infusion of dexamethasone and by chronic exhaustive exercise, muscle hypertrophy by resistance training. Screening of the amount of ambulatory and total movements carried out with Opto-Varimex-Mini and expressed as movements/h, the hindlimb grip strength was measured with Grip Strength Meter and expressed as N/100 g bw. Turnover rate of myosin isoforms was measured by double isotope method (3H/14C), nuclear domain size was expressed as protein/DNA ratio. Atrophy of skeletal muscles starts with destruction of myoﬁbrils, accompanied by disarray of thick myoﬁlaments and spreads over the contractile apparatus in FT muscles. In both types of atrophy MyHC IIb isoform relative content and turnover rate decreases and DNA unit size increases, demonstrating the increase of the amount of protein per nucleus and simultaneous decrease of muscle functional capacity. Muscle hypertrophy was accompanied by increase of turnover rate of MyHC, decrease of relative content of MyHC IIb isoform in FT muscles without any signiﬁcant changes in DNA unit size. There exist good relations between changes in muscle strength, muscle and DNA unit size and turnover rate of contractile proteins. MyHC and MyLC isoforms turnover rate is related with skeletal muscle functional capacity.

Eﬀects of 30-day low-frequency electrostimulation on human stretched m. vastus lateralis P.P. TARAKIN, B.S. SHENKMAN, E.V. LYUBAEVA, Y.S. LEMESHEVA, D.V. POPOV, Y.R. BRAVYI and O.L. VINOGRADOVA State Scientiﬁc Centre of RF Institute for Biomedical Problems of Russian Academy of Sciences, Moscow, Russia Twelve male volunteers took part in the study. They were divided into two groups. The knee extensors of both legs of six men were under chronic low-frequency electrostimulation. The second group of 6 men was under stimulation combined with stretching of m. quadriceps femoris. In the ‘pure stimulation’ group we found 10% decrease of cross-sectional area (CSA) of fast-twitch (FT) ﬁbres. CSA of slow-twitch (ST) ﬁbres didn’t change. Muscle electrostimulation in combination with stretch induced ﬁbre CSA increase of 11% for FT and 13% for ST ﬁbres. After the electrostimulation percentage of ﬁbres containing slow myosin heavy chain (MHC) isoforms increased by 10% independently of the experimental group involved and also percentage of ﬁbres stained with antibodies against fast MHC decreased by 12%. Activity of succinate dehydrogenase (SDH), in FT ﬁbres was increased by 55–57% without any diﬀerences between groups. The increase of SDH activity in ST ﬁbres was 38% in ‘pure stimulation’ group and 27% in subjects involved in electrostimulation combined with muscle stretching.

67 Eﬀects of overtraining on Na,K-ATP-ase and ﬁbre type expression in equine vastus lateralis muscle M.M.M. VAN DEN BURG, B.J.L.J. JOOSTEN, E. VAN BREDA, I.D. WIJNBERG, J.H. VAN DER KOLK, H.A. KEIZER, K. EIZEMA and M.E. EVERTS Dept. of Pathobiology, Utrecht University, Utrecht, The Netherlands The capacity for active Na,K transport (i.e. the Na,K pump) of skeletal muscle is essential for the maintenance of muscle excitability. MyHC composition of skeletal muscle is related to resistance to fatigue. We investigated the eﬀects of overtraining on the Na,K-ATPase concentration by measurement of [3H]ouabain binding and the MyHC isoform expression by immunohistochemistry in the m. vastus lateralis of standard reds. Six horses (age 2 years) were trained for 18 weeks (TT2). Subsequently, they were divided into two groups during 6 weeks (TT3). Training group (T) trained at the same level as the ﬁrst 18 weeks. Overtraining group (OT) trained intensively 7 days a week. Biopsies were taken at TT2 and TT3. No diﬀerences in MyHC isoform expression were detected. Na,K-ATPase content increased by 20% (p>0.05, n=3) in T group, while the rise in OT group was 65% (p<0.05, n=3). Up-regulation of Na,K-ATPase is strongly related on the speciﬁc workload. Despite clear overtraining symptoms skeletal muscle is still able to adapt to exhaustive workload, without changing its ﬁbre type composition.

68 SESSION IV. SMOOTH MUSCLE – CONTRACTILE PROCESS AND REGULATION, MONDAY, 19TH OF SEPTEMBER 2005, 8:30–10:30 Mechanism for controlling Ca2+ sensitivity of smooth muscle contraction M. ETO1, F. MATSUZAWA2, S. AIKAWA2 and S. OHKI3 1 Centre for Cell Signalling, Dept. of Mol. Physiol. and Biol. Phys., Univ. Virginia Sch. Med., USA 2 Dept. of Clin. Gen., Tokyo Metropolitan Inst. Med. Res., Japan 3 Centre for Nanomaterials, JAIST, Japan Agonist-induced contraction of smooth muscle is involved in regulation of vascular tone. Agonist stimulation triggers cellular Ca2+ release and phosphorylation of myosin by Ca2+/calmodulin dependent-myosin light chain kinase. The receptor activation also increases Ca2+ sensitivity of smooth muscle contraction. This Ca2+ sensitization is attributed to the agonist-induced inhibition of myosin phosphatase, enhancing myosin phosphorylation independently of Ca2+ signals. The agonist-induced inhibition of myosin phosphatase is mediated by at least two kinases, PKC and ROCK. Both PKC and ROCK phosphorylate CPI-17 at Thr38 that converts this protein into a potent inhibitor for myosin phosphatase. Thus, the phosphorylation of CPI-17 at Thr38 is a determinant of the Ca2+ sensitivity of smooth muscle contraction through controlling myosin phosphatase activity. CPI-17 consists of a four-helix v-shape bundle plus a loop structure around Thr38 (P-loop). In unphospho-protein, the P-loop is placed between two helices. Upon phosphorylation, Thr38 moves 9 A˚ away from the helices that exposes the P-loop into the solvent. The exposed phospho-Thr38 directly recognizes and docks at the active site of myosin phosphatase. This speciﬁc interaction of phospho-CPI-17 with myosin phosphatase triggers the agonist-induced Ca2+ sensitization of smooth muscle contraction.

Regulation of contraction in smooth muscle from new-born mice A. ARNER1,2, M. EKMAN2, K. STAKEBERG2 and K-E. ANDERSSON3 1 Dept. Physiology and Pharmacology, Karolinska Instituitet, Stockholm, Sweden 2 Dept. Experimental Medical Research and 3Dept. Clinical Pharmacology, Lund Univ., Lund, Sweden Developmental regulation of smooth muscle contraction was examined in urinary bladders from new-born and adult mice. In the newborn, non-muscle myosin contributes to contraction and potentially also to slow contractile kinetics [Lo¨fgren et al. (2003) J Gen Physiol 121: 301]. Activation of bladder muscle from newborn mice had a signiﬁcantly higher sensitivity to Ca2+ [Ekman et al. (2005) J Gen Physiol 125: 187), associated with a lowered expression of myosin phosphatase (MYPT-1). Nerve-induced contractions of new-born tissue were less inﬂuenced by purinoceptor desensitisation, suggesting a lower contribution of the transmitter ATP. This is most likely due to properties of the nerves, since the sensitivity of newborn bladder tissue to alpha-beta-methylene-ATP was not altered. The phasic component of cholinergic contractions was pronounced and initiated at low carbachol concentrations in the new-born tissue. The carbachol contractions of both new-born and adult urinary bladder tissue were inhibited by Y27632 and 8-Br-cGMP. However, the sustained phase of carbachol contraction in new-born tissue was less sensitive to these compounds, suggesting that the receptor-mediated calcium sensitisation mechanism is less prominent in new-born compared with adult mice and that the contractions of new-born bladders are less inﬂuenced by the NO-cGMP inhibitory pathway.

PHI-1 induced enhancement of MLC20 phosphorylation in chicken smooth muscle A.M.R. EL-TOUKHY1, A.M. GIVEN1, A. 1 1,2 COCHARD and F.V. BROZOVICH 1 Departments of Physiology and Biophysics and Medicine (2Cardiology), Case Western Reserve University, Cleveland, USA The regulation of force in smooth muscle can occur through Ca2+dependent or independent pathways. The Ca2+-independent pathway has been well characterized in many tissues, however the relative contribution of multiple regulatory pathways makes comparison between tissues and species diﬃcult. In this study, chicken smooth muscle was used to demonstrate that both agonist activation of intact tissues and G-protein stimulation of skinned preparations leads to an increase in the phosphorylation of MLC20 through a Rho-kinase dependent pathway. However, unlike many mammalian systems, G-protein stimulation did not cause an increase in phosphorylation at Thr695 or Thr850 of the myosin targeting subunit (MYPT1) of the myosin phosphatase. In addition, the smooth muscle speciﬁc MLC phosphatase inhibitor protein, CPI-17, was not expressed, while an analogous protein, PHI-1, was present in chicken smooth muscle tissues. Both agonist and GTPcS stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.

Deletion of functional domains from the C-terminal region of Caldesmon by homologous recombination in mice S. CHACKO, A.K. CHANGOLKAR, Y. ZHENG, E. POLYAK, J.A. HYPOLITE, S. CHANG, M.E. DISANTO, S.A. ZDERIC and A.J. WEIN Department of Pathobiology and Division of Urology, University of Pennsylvania and Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA A 14.8 kb genomic fragment encompassing exons 6–11 of the caldesmon (CaD) gene was cloned from a 129/sv mouse genomic library. We used targeted gene disruption to ablate the CaD C-terminal region containing the functional domains important for actin–myosin interaction and actomyosin ATPase. The insertion of Neo (carrying a stop codon) into exon 7 was conﬁrmed by Southern, PCR and DNA sequence analyses. Deletion of amino acid sequence 626–793 had no obvious eﬀect on reproduction or survival. Homozygous ()) mice revealed urinary bladder smooth muscle hypertrophy. Smooth muscle strips from )/) generated less force/gm of muscle in response to 125 mM KCl; however, the rate (dp/dt) of force generation by whole bladder in response to KCl was higher for the )/) compared to )/). Electron microscopy demonstrated normal thick myosin and thin actin ﬁlaments in )/) mouse bladder. However, confocal microscopy of myocytes from )/) mouse bladder revealed fewer cytoplasmic ﬁbrils reacting with antibody against alpha smooth muscle actin, compared to that of +/+. These ﬁbrils also react with antibody against an epitope in the un-deleted region in the CaD. Our results show phenotypic changes in smooth muscle upon deletion of the C-terminal functional domains of CaD and suggest this knockout mouse to be a valuable tool to elucidate the still unclear physiological function of CaD.

69 Age-dependence of cholinergic and purinergic receptors in urinary bladder G.P. SZIGETI1, G.T. SOMOGYI3, L. CSERNOCH1 and E. SZELL2 1 Dept. of Physiology, Univ. Debrecen, Debrecen, Hungary 2 Dept. of Paediatrics, Univ. Debrecen, Debrecen, Hungary 3 Dept. of Urology, Baylor College of Medicine, Houston, TX, USA

Increased level of hydrogen peroxide enhances myogenic tone in arterioles of type 2 diabetic mice Z. BAGI3, N. ERDEI3, A. TOTH3, I. EDES3, G. KALEY1 and A. KOLLER1,2 1 Dept. of Physiol., New York Med. Coll., Valhalla, New York, USA 2 Dept. of Pathophysiol., Semmelweis Univ., Budapest, Hungary, 3 Dept. of Clin. Physiol., Inst. of Card., Univ. of Debrecen, Debrecen, Hungary

In neonatal animals during the early postnatal period, micturition is mediated by a somatobladder spinal reﬂex pathway. During postnatal development this primitive reﬂex is replaced by supraspinal mechanisms. In the rat, this developmental change in the central neural control of voiding occurs in concert with changes in peripheral neurotransmission and intrinsic properties of the bladder smooth muscle. The neurally evoked bladder contractions which are mediated entirely by cholinergic mechanisms in the bladder strips from 1week-old rats become primarily purinergic in later stage. In our experiments the age dependence of the nicotinic and purinergic neurotransmission was studied on smooth muscle strips and isolated smooth muscle cells. Atropine (1 lM) reduced the amplitude of spontaneous contractions and reduced the basal tone in muscle strips from 2- to 3-week-old rats while it was less eﬀective in 4- to 5-week-old bladders. The muscarinic receptor agonist carbachol (1 lM) increased the amplitude of the spontaneous contractions. This eﬀect was more prominent on 2- to 3-week-old than on 4- to 5-week-old bladders. In contrast, the purinergic receptor agonist a, b-methyl ATP stimulated the contractions mainly in older animals. The carbachol- and a, b-methyl ATP-induced currents in isolated smooth muscle cells showed the same age-dependent changes as the data from muscle strips. Supported by Hungarian Research Grant (OTKA T0 49151).

In type 2 diabetes mellitus (T2-DM) reactive oxygen species play important roles in the development of vascular dysfunction. Among them, hydrogen peroxide (H2O2) has been proposed to interfere with several vasoregulatory pathways, although its mechanism of action in T2-DM has not yet been elucidated. We hypothesized that in T2-DM vascular production of H2O2 is enhanced, which affects the synthesis of prostaglandins. We found that in isolated carotid arteries the level of dichloroﬂuorescein-detectable H2O2 was signiﬁcantly greater in mice with T2-DM (db/db), as compared to controls. In isolated, pressurized gracilis muscle arterioles (80 lm) of db/db mice step increases in intraluminal pressure (20– 120 mmHg) elicited greater constrictions than in control vessels, which were reduced either by catalase or PGH2/TXA2 receptor antagonist, SQ29548. Administration of H2O2 (10)8–10)5 M) elicited substantial dilations in control vessels, but caused constrictions in db/db arterioles, which was then inhibited by SQ,29548. These ﬁndings suggest that in mice with T2-DM vascular production of H2O2 is enhanced, which by increasing production of constrictor prostaglandins enhances myogenic tone. Hence, these H2O2-mediated alterations may contribute to the disturbed regulation of tissue blood ﬂow in type 2 diabetes (NIH HL-43023, HL-46813 and OTKA M045186, 048376, Zs-711/84289, F-048837).

Anandamide as an antagonist of the vanilloid receptor-1 (TRPV1) in rat skeletal muscle arterioles E. LIZANECZ1, Z. BAGI1, Z. PAPP1, I. E´DES1, N. KEDEI2, P.M. BLUMBERG2 and A. TO´TH1,2 1 Division of Clinical Physiology, Institute of Cardiology, University of Debrecen, Debrecen, Hungary 2 Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA It has been proposed that the activation of TRPV1 aﬀects the vasotone in resistance arterioles. Here we tested the eﬀects of anandamide on the diameter of isolated, pressured (80 Hgmm) skeletal muscle (m. gracilis) arterioles and aimed to elucidate the anandamide-induced, TRPV1-dependent receptor mechanisms. Capsaicin (1 lM), a well-known agonist of TRPV1, elicited a substantial constriction in isolated arterioles (to 51±12% of initial diameter). In contrast, anandamide (1 nM– 100 lM) did not aﬀect the arteriolar diameter signiﬁcantly. To test if anandamide desensitized TRPV1, isolated vessels were preincubated with anandamide (30 lM, for 20 min). This approach revealed that anandamide indeed desensitized TRPV1 for capsaicin (arteriolar constriction by 1±1% following capsaicin administration). However, subsequent cyclosporine-A treatment (phosphatase 2B inhibitor) reverted this desensitization (arteriolar constriction by 31±1% following capsaicin). These results suggest that the interaction between anandamide and TRPV1 is modulated by protein phosphorylation and this might have an impact on TRPV1-dependent vasoregulation. Support by OTKA F 048873.

Calcium trans-membrane inﬂux and gene expression activation in satellite cells M. CANATO2, V. DE ARCANGELIS1, M. ALVISI1, F. NARO1 and C. REGGIANI2* 1 Dipartimento di Anatomia e Fisiologia, Universita` di Padova, Italia 2 Dipartimento di Istologia ed Embriologia Medica, Universita` di Roma ‘‘La Sapienza’’, Italia Modulation of intracellular calcium concentration ([Ca2+]i) is an important regulative mechanism for the establishment of the muscular phenotype and for myoﬁber function. The variety of the biological responses regulated by Ca2+ suggests that the cell is capable to discriminate between diﬀerent Ca2+ signals but how it does it is still matter to debate. Exposure of L6 myogenic cells to high extracellular Ca2+ ([Ca2+]o) concentrations transiently increases [Ca2+]i through a selective increase of the conductance of membrane channels (DHPR) and ion exchangers (Na+/Ca2+) [Naro et al. (2003) AJP]. The increase of [Ca2+]i due to the plasma membrane inﬂux, induces a hypertrophic phenotype and an increased expression of slow-ﬁbre genes but not fast-ﬁbre genes which, instead, are stimulated by release of Ca2+ from intracellular stores [DeArcangelis et al. (2004) JCP]. Preliminary data on primary mouse culture of satellite cells show induction of a hypertrophic phenotype similar to that observed in myogenic L6 cells suggesting a possible physiological role of [Ca2+]o in the control of muscle skeletal function and gene expression.

Eﬀect of lysenin on smooth muscle actomyosin ATPase activity E. CZURYŁO and N. KULIKOVA Nencki Institute of Experimental Biology, Warsaw, Poland

70 Lysenin is a 33.5-kDa protein isolated from coelomic ﬂuid of an earthworm Eisenia foetida. It interacts with lipids, speciﬁcally recognizing sphingomyelin. It also causes long-lasting (more than 30 min) contraction of isolated rat aorta strips. The mechanism underlying induction of contraction as well as the biological action of lysenin in animals has not been clariﬁed. We studied the eﬀect of lysenin on the ATPase activity of regulated hybrid actomyosin system reconstituted from skeletal muscle actin and myosin and smooth muscle regulatory proteins, tropomyosin and caldesmon. Under conditions where the ATPase activity was maximally inhibited by caldesmon, addition of lysenin resulted in the abolishment of inhibition and further activation of the ATPase activity up to the level of its activation by tropomyosin. It suggests that in the presence of lysenin the contraction force should reach its maximum. The eﬀect was observed at molar ratios of actin to lysenin of 1000:1 and lower. Electron microscopic observations have shown that addition of lysenin to F-actin results in bundling. Possible eﬀect of lysenin at the parameters of Factin double helix is discussed. Supported by Grant 2 P04A 046 26 of Polish State Committee for Scientiﬁc Research.

Protein kinase C modulates myoﬁlaments Ca2+sensitivity in vascular smooth muscle: possible role in vasospasm development I.V. KIZUB, A.A. PAVLOVA and A.I. SOLOVIEV Department of Experimental Therapeutics, Institute of Pharmacology and Toxicology, Academy of Medical Sciences of Ukraine, Kiev, Ukraine We investigated protein kinase C (PKC)-dependent myoﬁlaments Ca2+-sensitivity in vascular smooth muscles (VSM) under hypoxia (pulmonary artery), ionising radiation and genetically determined hypertension (aorta). Contractile recordings experiments were made using chemically permeabilised (b-escin) smooth muscle. It was shown that vascular smooth muscles in all above-mentioned experimental conditions demonstrated an increasing of myoﬁlaments Ca2+-sensitivity. A potent PKC inhibitors, chelerythrine (10)6 M) and staurosporine (10)7 M), abolished or signiﬁcantly decreased Ca2+-sensitizer eﬀect of hypoxia, ionising radiation and normalise myoﬁlaments Ca2+-sensitivity at hypertension. To summarise, we have shown the key role of the PKC activity in the increase of the Ca2+-sensitivity of VSM myoﬁlaments. We conclude that the PKCmediated myoﬁlaments Ca2+-sensitisations in VSM may be one of the common mechanisms for vasospasm development of diﬀerent genesis.

The contractile properties of the bladder and the proximal urethra in female pig J.J.M. PEL, E. VAN ASSELT and R. VAN MASTRIGT Department of Urology, sector Furore, Erasmus MC, 3000 DR, Rotterdam, The Netherlands To study the mechanism of urinary continence, the contractile properties (force–length and force–velocity) of small bundles from bladder and proximal urethral muscle of the female pig were measured using the stop test. The proximal urethra was divided in two sections (I and II) of 10 mm in length. The rate of force development (time constant) was derived from phase plots. Immunohistochemical staining of urethral cross sections was used to identify smooth and striated muscle ﬁbres. In urethral segments I and II, spontaneous contractions were measured with a frequency of 0.4 Hz. On average, isometric force was highest in the bladder. The length range over which force was produced was smallest in urethral segment II, followed by segment I and ﬁnally bladder. The unloaded shortening velocity was respectively 0.15, 0.25 and 0.35 l/s. Isometrically, the

urethral muscle bundles revealed a fast (0.5 s) and a slow (2.1 s) exponential force development, whereas in bladder only a slow ( 2.3 s) development was measured. Histological preparations showed that smooth as well as striated muscle was presented in urethral segments I and II. The spontaneous contractions may be part of the continence mechanism in the female pig urinary tract. Diﬀerences in contractility between urethra and bladder may be ascribed to the presence of striated muscle in the proximal urethra.

Regulation of soluble guanylate cyclase activity by direct interaction with heat shock protein Hsp90 AB. POSTNIKOV1, VL. BETIN1, F. MURAD2 and AY. KOTS1,2 1 Dept. of Bioorganic Chemistry, School of Biology, Lomonosov Moscow State Univ., Moscow, Russia 2 Dept. of Integrative Biology and Pharmacology, Univ. of Texas at Houston Med. School, Houston, TX, USA Soluble guanylate cyclase (sGC) is the main receptor of nitric oxide (NO). sGC is inhibited by various oxidants which either convert ferrous heme to ferric suppressing NO binding or modify essential thiol groups of sGC. Hence, we suggested that Hsp can block oxidant-induced inhibition of sGC. According to surface plasmon resonance binding experiments, Hsp90 can directly interact with sGC. The resulting KD value of 23.1 nM suggests that the binding is characterized by high aﬃnity. Hsp90 (0.1 mg/ml) protected NOdependent activity of sGC during preincubation. However, other Hsps (70, 60, 40 and 25) failed to protect sGC. Hsp90 protected sGC from inhibition by Mn2+ and Cd2+ known to be potent blockers of thiol groups. The complex of Hsp90 and sGC is formed slowly but once formed, should remain relatively stable for a long period of time. Hsp90 protects essential functional groups of the enzyme. The interaction of Hsp90 with sGC can be important for maintenance of NO-dependent cellular cGMP synthesis in damaged tissues.

Caldesmon inhibits myosin-induced movement of smooth muscle tropomyosin during the actomyosin ATPase cycle D.YU. SAVKIN1, G.A. CHUDAKOVA1, G.V. MIKHAILOV1, O.E. PRONINA1, N. KULIKOVA2, A. WRZOSEK2, R. DABROWSKA2 and YU.S. BOROVIKOV1 1 Institute of Cytology RAS, Russia 2 Nencki Institute of Experimental Biology, PAS, Poland We have studied the eﬀect of caldesmon on polarized ﬂuorescence from 1.5-IAEDANS or 5-IAF attached to Cys-36 and/or Cys-190 of smooth muscle tropomyosin (TM) induced by myosin subfragment-1 in ghost muscle ﬁbres in the absence and presence of MgADP, MgAMP-PNP, MgATPyS or MgATP. The probes were rigidly immobilized on TM, making the absorption and emission dipoles of the dyes directly sensitive to tropomyosin movements. The changes in the orientation of 5-IAF and 1.5-IAEDANS dipoles under the inﬂuence of nucleotides indicated the multistep alterations of TM rotation during the actomyosin ATPase cycle. The maximal TM rotation by 2–3 relative to the ﬁbre axis was observed at transition between the A**Æ M**ÆADPÆPi and AÆMÆADP states. In the presence of caldesmon the tropomyosin rotation decreased by 50%. It is suggested that caldesmon alters the position of TM in a reconstituted actin thin ﬁlament thereby limiting the ability of myosin heads to move tropomyosin. Supported by RFFR (05-04-48812) and the grant of Polish State Committee for Scientiﬁc Research to the Nencki Institute.

71 The importance of L-type channels for the endotheliumindependent relaxation induced by estradiol in male rat aorta and small mesenteric arteries D. N. SERBAN, I. L. SERBAN and C. OPRISA Department of Physiology, University of Medicine and Pharmacy ‘‘Gr. T. Popa’’, Iasi, Romania Mechanisms of the non-genomic vascular eﬀects of steroids are still under investigation. We observed that in male rat aorta and small mesenteric arteries relaxation by 17-beta estradiol shows an important endothelium-independent component, which is stronger with phenylephrine vs. high K. Others have found that this estradiol effect on smooth muscle relies on inhibition of calcium inﬂux and that estradiol inhibits L-type channels. To evaluate the relevance of L-type channel inhibition for estradiol relaxation, we examined the eﬀect of estradiol upon the D600-insensitive component of aortic contraction induced by phenylephrine This contraction is estradiolresistant, even at doses able to completely relax phenylephrine contraction in the absence of D600. Half-eﬀective concentrations of D600 induce a shift to the right of the dose–relaxation curve for estradiol in both aorta and small mesenteric arteries. These data show that relaxation by estradiol of the contraction induced by alpha-1 adrenoceptor activation requires unblocked L-type channels. Alternative relaxing mechanisms may be of little relevance.

The relative contribution of endothelium-dependent relaxing mechanisms in rat small mesenteric arteries depends on the contracting agent I.L. SERBAN and D.N. SERBAN Department of Physiology, University of Medicine and Pharmacy ‘‘Gr. T. Popa’’, Iasi, Romania Mechanisms of endothelium-dependent relaxation vary with species, age, sex, hormonal status, vascular territory and calibre, the mechanical or chemical vasodilator, pre-existing vascular tone and its determinants. There is little data available on the latter. We noticed that in rat small mesenteric arteries the EDHF component of carbachol-induced relaxation is more important with precontraction by prostaglandin F2a (PGF) vs. phenylephrine (PHE). Glibenclamide (GLI) aﬀects the carbachol response stronger when NO involvement proved to be more important (PHE), while tetraethylammonium (TEA) visibly reduces the carbachol response only with PGF precontraction. In both cases the EDHF response is inhibited by TEA, not by GLI. In the case of angiotensin the phasic component of contraction is aﬀected similarly to the PHE response, and the tonic one similarly to the PGF response. K+ channel inhibitors indicate the involvement of KATP in the NO eﬀect, but not in the EDHF one, mediated by KCa. The relative importance of NO and EDHF depends upon the contracting agent, with an interesting correlation at the level of target K+ channels.

72 SESSION V. MOLECULAR MECHANISMS OF MYOSIN MOTORS, MONDAY, 19TH OF SEPTEMBER 2005, 10:45–12:40 Ca2+ inhibits the activity of a myosin II by binding to an EF-hand of a calmodulin-like light chain and altering the dynamics of the neck region L. FARKAS1, J.E. DEBRECZENI2, V. HARMAT2, CS. HETE´NYI1, I. HAJDU´4, P. ZA´VODSZKY4, K. KOHAMA3 and L. NYITRAY1 1 Dept. of Biochem. and 2Protein Modelling Group, Hungarian Academy of Sci., Eo¨tvo¨s Lora´nd Univ., Budapest, Hungary 3 Dept. of Pharmac., Gunma Univ., Maebashi, Japan 4 Inst. of Enzymol., Biol. Res. Centre, Hungarian Academy of Sci., Budapest, Hungary Ca2+ binding to light chains or calmodulins (CaM), associated with the neck region, regulates the activity of several classes of myosin. We have previously identiﬁed an inhibitory Ca2+-binding site in the essential light chain (ELC) of Physarum myosin II, and here we report the high resolution structure of its regulatory domain (RD, the neck region). The structure reveals a Ca2+-bound canonical EFhand in a closed state, similar to the Ca2+-activated scallop myosin RD which has an unusual Ca2+-binding EF-hand, and unlike CaM in which Ca2+-binding generally induces a closed-to-open transition. The large change is prevented in the Physarum ELC by interactions with the heavy chain and lack of a Ca2+-site in the second EF-hand. Diﬀerences between the Physarum and scallop RD at the interface of the two light chains and at bends of the heavy chain helix may contribute to the opposite functional eﬀect of Ca2+ on these myosins. Hydrogen–deuterium exchange experiments by Fourier-transformed infrared spectroscopy and molecular dynamic simulations show that removal of Ca2+ increases the global internal mobility of RD. Based on these results we propose that Ca2+-induced alteration in the dynamics of RD is a major factor in suppressing the activity of myosin in Physarum plasmodium. Similar mechanism may contribute to Ca2+-inhibition of certain unconventional myosins. Supported by OTKA.

Coupling of conformational changes of myosin II catalytic domain functional regions by using switch I intrinsic ﬂuorescence sensors B. KINTSES1, M. GYIMESI1, B. JELINEK1, W. ZENG2, C.R. BAGSHAW2 and A. MA´LNA´SICSIZMADIA1 1 Department of Biochemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary 2 Department of Biochemistry, University of Leicester, Leicester, UK We are investigating conformational changes of the switch I region of myosin during ATPase cycle by using single trytophan residues introduced to the positions of 239 and 242 of Dictyostelium myosin II motor domain. These tryptophans sense diﬀerent events compared with the relay loop tryptophan W501 that reports on switch 2 movements. W239 and W242 respond to MgATP binding with a 40% ﬂuorescence quench, as does W242 to MgADP binding. The response of W239 on MgADP binding is slightly diﬀerent (biphasic and changed amplitude). Interestingly with the W239 construct, two states can be distinguished in the presence of MgADP which are perturbed by a temperature jump, under conditions where only a single species is sensed in the presence of ADP (no Mg2+). Single predominant species are also detected by temperature jump in the apo state and under conditions where the c-phosphate site is occupied. The W239 and

W242 responses to actin binding diﬀer from that of W501, indicating that actin aﬀects the conformation of switch 1. Actin also shifts the equilibrium of the bound MgADP towards a single state. Based on these results we are testing models of actomyosin cycle to clarify the relationship between switch 1, switch 2, the actin binding cleft and the relay regions.

The working stroke of skeletal muscle myosin is performed in two steps M. CAPITANIO1, M. CANEPARI2, P. CACCIAFESTA1, V. LOMBARDI3, R. CICCHI1, M. MAFFEI2, F.S. PAVONE1,4 and R. BOTTINELLI2 1 European Laboratory for Non-linear Spectroscopy, Univ. of Florence, Firenze, Italy 2 Dept. of Exp. Med., Human Physiol. unit, Univ. of Pavia, Pavia, Italy 3 Lab. of Physiol., DBAG, Univ. of Florence, Firenze, Italy 4 Dept. of Physics, Univ. of Florence, Firenze, Italy Shortening velocity of skeletal muscle ﬁbres and its dependence on the load vary widely among ﬁbres expressing diﬀerent myosin isoforms, but the underlying mechanisms remain poorly understood. Here we apply a novel single molecule approach to rapidly (300 ls) and precisely (0.1 nm) detect acto–myosin interactions of two myosin isoforms (type 1 from rat and type 2B from mice) having large diﬀerences in shortening velocity. Thanks to the high spatial and temporal resolution we show for the ﬁrst time that skeletal myosin propels actin ﬁlaments performing its conformational change (working stroke) in two steps. The ﬁrst step (3–5 nm) occurs immediately after myosin binding and is followed by a smaller step (1.0–1.5 nm), and then by dissociation of the acto–myosin complex. The two mechanical conformations of myosin when attached to actin deﬁne two phases within bound state with independent durations ton1 and ton2. The rate of the ﬁrst phase (1/ton1) is much higher in the fast (2B) myosin isoform (1156±396 s)1) than in the slow (1) one (42.8±5.9 s)1), independently of ATP concentration. On the other hand, the rate of the second phase (1/ton2) depends linearly on ATP concentration and is similar in the two isoforms. Our ﬁndings provide a simple model for regulation of shortening velocity among skeletal myosin isoforms and for the load sensitivity of the cross-bridge cycle.

A fresh interpretation of muscle transient experiments with demanding impact on how muscles contract G.F. ELLIOTT1 and C.R. WORTHINGTON2 1 Vision Sciences and Optometry, Cardiﬀ Univ., Cardiﬀ, UK 2 Physics and Biology, Carnegie-Mellon Univ., Pittsburgh, USA Piazzesi et al. [(2002) J Physiol 545: 145–151] studied the length transients due to fast step changes in force, with high time resolution. They recorded ﬁlament movement and shortening velocities in the 4 phases, highlighting Phase 2 and Phase 4 (working muscle). Siding with the swinging-cross-bridge model they record, however, that the ‘power stroke’ (usually regarded as a constant) in fact decreases with load ( 11 nm at zero load). The experimental result conforms to our impulsive model [W and E (1996) Int J Biol Macromol 18: 123– 131]. This has a smaller load-dependent step-size distance z ( 2 nm at zero load), the distance moved by one actin past one myosin ﬁlament for the consumption of one ATP in average time frame t (the ‘interval time’; £1 ms).

73 From their data we calculate precise interval times and ﬁlament movements in Phase 2 and in working muscle [W and E (2005) IJBM 35: 265–268]. An important result is that the time frame in Phase 2 is the same as in working muscle. The data veriﬁes the correctness of our z values. There are 8 active ATP events on each actin ﬁlament in Phase 2 in time frame t compared to 1 in working muscle. The numbers follow directly from their experimental observation that the shortening velocity in Phase 2 is eight times that in working muscle; they do not comment on this. For the ﬁrst time this gives precise numbers for contractile events and supports the impulsive model with a smaller ﬁlament (zero-load) motion, 2 nm per ATP split, proposed previously on energetic grounds.

Fluorescence lifetime imaging applied to muscle ﬁbres D.I. GARCI´A1, P. LANIGAN2, J. ECCLESTON3, M. WEBB3, J. REQUEJO2, M. NEIL2, P. FRENCH2 and M.A. FERENCZI1 1 Biomedical Science Division and 2Photonics group, Physics Department, Imperial College London, London, UK 3 NIMR, Mill Hill, London, UK The ﬂuorescence life time imaging (FLIM) of suitable ﬂuorophores provides information about the nano-environment of the molecules in a concentration-independent way. We are investigating the use of FLIM to detect changes in the environment of nucleotide binding site in the myosin cross-bridge by imaging sarcomeres in permeabilized muscle ﬁbres, incubated with low concentrations (10 lM) of ﬂuorescent analogues of ATP. Using analogues labelled at the 3¢ ribose position with a variety of ﬂuorophores, the lifetime of the nucleotide in the A band is found to diﬀer from than in the I-band, where there is no myosin to bind the nucleotide. The eﬀect of changing the sarcomere length was also investigated. It was found that in muscle ﬁbre stretched to partial overlap (2.7–3.2 lm) the ﬂuorescence lifetime was diﬀerent in the actin–myosin overlap region of the A-band, compared to the H-zone where there is no overlap. These ﬁndings suggest that the environment around the nucleotide pocket is sensitive to actomyosin interactions. The changes in life-time might reveal subtle structural changes in the myosin molecule and may provide information about the kinetics of ATP hydrolysis and its strain dependence.

Alternatively spliced exon B of myosin Va is essential for binding of the tail light chain shared by dynein ZS. HO´DI1, A. NE´METH1, CS. HETE´NYI1, A. BODOR2, A. PERCZEL2 and L. NYITRAY1 1 Department of Biochemistry and 2Department of Organic Chemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary A 10-kDa dynein light chain (DLC) has been previously identiﬁed as a tail domain light chain of mammalian myosin-Va (myo5a). DLC may function as a cargo-binding and/or regulatory subunit of both motor proteins. We have identiﬁed and characterized the binding site of DLC on myo5a. Various fragments of human myo5a tail and DLC2 were expressed and their complex formation was analyzed by pull-down assays, gel ﬁltration, native PAGE, and spectroscopic methods. DLC was found to bind as a homodimer to a 12 residues segment (Pro1282–Thr1293) located between the medial and distal coiled coil domains. The binding region contains three residues, coded by the alternatively spliced exon B that is essential for DLC binding. Using CD spectroscopy and limited proteolysis, we demonstrate that binding of DLC to the intrinsically unstructured DLC binding domain (DBD) stabilizes both of this and the neighbouring coiled-coil domains. NMR spectroscopy and molecular docking simulations show that a short synthetic peptide of DBD binds to the surface grooves on DLC,

formed at the sides of the dimerization interface. We assume that in vivo the second binding site of the DLC dimer is either occupied by the DBD of the other myo5a heavy chain, or by a cargo, or interacts with other regulatory proteins. Supported by OTKA and GVOP.

The DCM-causing Glu40Lys mutation in a-tropomyosin inhibits myosin-induced movement of tropomyosin in ghost ﬁbres S.S. KHAIMINA1, D.YU. SAVKIN1, G.V. MICHAILOV1, C. REDWOOD2 and YU.S. BOROVIKOV1 1 Institute of Cytology RAS, St. Petersburg, Russia 2 University of Oxford, Oxford, UK A missense mutation (Glu40Lys) in a-tropomyosin (a-TM) is known to cause dilated cardiomyopathy (DCM). To determine how this mutation might alter thin ﬁlament regulation, we analysed its eﬀect on myosin-induced movement of TM. The Cys-190 residue of both wild-type and Glu40Lys mutant recombinant human a-TM was labelled with iodoacetamide 5-ﬂuorescein and TM and myosin subfragment-1 were incorporated into ghost muscle ﬁbres. The orientation and mobility of TM were studied in the absence or presence of MgADP, MgAMPPNP, MgATPcS and MgATP by polarized ﬂuorimetry. As the ﬂuorescent probe was covalently bound to TM, the dipoles of its absorption and emission were sensitive to changes in the orientation and mobility of TM. At diﬀerent intermediate states of the ATPase cycle, the orientation and mobility of the probe oscillators were shown to change discretely indicating multistep alterations of these characteristics of TM during ATP hydrolysis. The DCM mutation inhibited these movements. We suggest that the mutation ‘freezes’ TM in the ‘OFF’ position on the thin ﬁlament and hence may reduce force generation.

Organization of dog myosin heavy chain gene clusters L. MACCATROZZO1, M. PATRUNO1, F. CALIARO1, L. TONIOLO2, C. REGGIANI2 and F. MASCARELLO1 1 Department of Experimental Veterinary Sciences and 2 Anatomy and Physiology, University of Padua, Italy Myosin heavy chain (MHCs) are actin-based motor proteins that play a key role in determining muscle contraction speed. MHC genes superfamily present a highly conserved gene clusters organization in genome of mammals. In this work we used computer–based programs to identify and characterize MHCs sequences in dog genome. The dog MHC sequences were assembled by NCBI Dog Genome Resources web site. BLAST searches suggest that the genes of dog encoding isoforms expressed in skeletal muscles (embryonic, 2A, 2X, 2B, perinatal and extraocular) have been located in a cluster on chromosomes 5; the two ‘‘cardiac’’ genes, coding for MHC-b (slow) and MHC-a (cardiac), are arranged tandemly on chromosomes 8; moreover, the gene for masticatory isoform (2 M MHC) has been located on chromosomes 6. Dog MHC isoforms show high similarity of aminoacid sequence with orthologous isoforms in other species (94% for 2B MHC, 96% for 2X and between 96–98% for 2A MHC). Multiple alignments on predicted protein sequences were used to create a phylogenetic tree. The neighbour-joining tree conﬁrmed the position of the dog orthologous isoforms within each speciﬁc MHC group.

Dog myosin heavy chain isoform composition: comparison between limb and specialised muscles F. MASCARELLO1, L. MACCATROZZO1, F. CALIARO1, M. PATRUNO1, L. TONIOLO2 and C. REGGIANI2 1 Department of Experimental Veterinary Sciences 2 Anatomy and Physiology, University of Padua, Italy

74 Four MHCs can be expressed in adult skeletal muscles of mammals. The slow, 2A and 2X isoforms have been found expressed in all species examined, whereas the 2B isoform has been so far detected only in laboratory mammals and marsupials. Recent works (Chang et al., 1997; Toniolo et al., 2004) showed the expression of 2B MHC also in pig skeletal muscles as well as in specialized eye muscles as retractor bulbi and rectus lateralis. In the cow, MHC-2B isoform is expressed only in those specialized muscles (Maccatrozzo et al., 2004). The issue about MHC isoforms expressed in fast or type 2 dog ﬁbres is still open (Snow et al., 1982; Strbenc et al., 2004). Immunohistochemistry, protein electrophoresis and RT-PCR showed that type 2 dog ﬁbres contain the MHC-2X isoform both in ‘‘hybrid’’ 2A/X and in ‘‘pure’’ 2X ﬁbres. The limb and trunk muscles express only 1, 2A and 2X-MHC isoforms, whereas the MHC-2B isoform was present only in specialized eye muscles and probably in thyroarytenoideus muscle (Wu et al., 2000). The 2B isoform of dog and cow is a muscle speciﬁc protein (allotype), as for 2 M and 2EO MHCs?

Mechanical and kinetic properties of rabbit cardiac isomyosins V1 and V3 compared in an in vitro motility assay and optical tweezers L.V. NIKITINA1, G.V. KOPYLOVA1 and J.E. MOLLOY2 1 IIP, RAS, Yekaterinburg, Russia 2 MRC NIMR, London, UK We measured the unloaded actin ﬁlament sliding velocity in an in vitro motility assay and found that rabbit cardiac V1 isoform was faster than V3 (0.42±0.08 l m s)1 vs. 0.17±0.03 (mean±sd)). To simulate higher loading we applied a-actinin (200 l g ml)1) to the ﬂow cell surface and this reduced the number of moving ﬁlaments greater for V1 than V3 (40% vs. 20%). We also measured the bound lifetimes and power stroke size for V1 and V3 isoforms using an optical trapping apparatus over a range of Mg.ATP concentrations. We found a smaller working stroke for V1 than V3 (4.4±1 nm vs. 7.4±0.98 (mean±sem) at 5 lM ATP). The actin-attached lifetimes for both isoforms were inversely dependent upon ATP concentration and detachment rate was faster for V1 than V3 (3.8 lM s)1 vs. 2.2). We conclude that rabbit cardiac V1 isoform is a faster but less forceful motor than the V3 and this might be achieved by taking slightly shorter power strokes but at a high rate. (All experiments were performed at 23 C). Supported by NATO15155/02B/TB, RFBR 05-04-48353a, HHMI 55000300.

Amoeba proteus myosin VI-immunoanalog is a dimer involved in cell migration and pinocytosis M. DOMINIK1, W. KLOPOCKA2, P. POMORSKI2 and M.J. REDOWICZ1 Departments of 1Muscle Biochemistry and 2Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland A search for myosins involved in the actin-based-only motility in Amoeba proteus revealed a 130-kDa protein reacting with four different anti-myosin VI antibodies. This protein bound to actin ﬁlaments (F-A) in an ATP-dependent manner and immunoprecipitated with the antibodies. It was crosslinked by EDC to a 260-kDa product, presumably a dimer. Analysis of the tryptic-derived peptides revealed several sequences with a high degree of identity to myosins VI. Myosin VI immunoanalog (MVI) localized to various vesicular structures and colocalized with dynamin II (DII) and F-A; in pinocytotic cells these proteins concentrated within pseudopodia. MVI and DII occupied even the same isolated vesicles. Blocking endogenous MVI with antibodies inhib-

ited amoebae locomotion but not aﬀected their morphology and manner of migration. In contrast, blocking DII with antibodies caused inhibition of only uroidal locomotion and aﬀected directionality of amoebae migration. Amoebae treated with anti-MVI or anti-DII antibodies exhibited defects in pinocytosis, suggesting that both proteins are also involved in pinocytosis.

Enzyme kinetics above denaturation temperature Z. SIMON1, M. GYIMESI1, C.R. BAGSHAW2 and A. MA´LNA´SI-CSIZMADIA1 1 Department of Biochemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary 2 Department of Biochemistry, University of Leicester, Leicester, UK A wide range of temperature dependence of enzyme kinetic measurements enables a more precise determination of reaction energetics and accurate separation of reaction steps. We have developed a novel stopped ﬂow/temperature jump method to measure enzyme transient kinetics at high temperatures where the enzyme would normally denature within seconds. This method enables us to increase the temperature over a millisecond time scale in parallel with mixing of the reactants so that the overall dead time does not increase. This is unique among combined transient kinetic techniques. Since heat denaturation normally occurs in a second time scale, essentially all events can be followed that are faster than heat denaturation reaction. We have tested the new method on a single tryptophan containing recombinant Dictyostelium myosin II catalytic domain. This single tryptophan located in the relay loop (W501) responds to the open-closed conformational change coupled to the hydrolysis step with an 80% ﬂuorescence intensity increase. Because of the large signal change this reaction is a good candidate for a test reaction of the stopped ﬂow/T-jump method. We present the calibration of the system and the results of the energetic characterisation of this reaction measured over a 70C temperature range.

Helical tail fragments of myosin VI exist as monomeric, highly ﬂexible structures D. SU¨VEGES1, A. NE´METH1, Z. GA´SPA´RI2, G. TO´TH3 and L. NYITRAY1 1 Department of Biochemistry and 2Department of Organic Chemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary 3 Agricultural Biotechnology Centre, Go¨do¨llo¨, Hungary Myosin VI is a unique motor that moves toward the minus end of actin ﬁlaments. Its tail consists of a region that was predicted to form a coiled coil dimer followed by a globular domain involved in cargo binding. Previous studies were carried out on dimer constructs, stabilized by strong coiled coil sequences, and showed that myosin VI is a processive motor. However, a recent report using full-length myosin VI revealed that it is monomeric and nonprocessive. We characterized myosin VI tail fragments by limited proteolysis, cross-linking and spectroscopic studies. Three subdomains of the tail separately or in longer constructs are all monomeric unless stabilized by a Leu-zipper, and they exist as ﬂexible sequences with high, but transient helical content. The most striking subdomain (E916-R981) has a very high charged density (0.86); modelling studies indicate that it is unlikely to form a stable coiled coil. Database search exposed only a few dozens of protein domains with similar density of charged residues (including the tail of myosin X) and none of them is known to have well-deﬁned structure, i.e. they likely exist as intrinsically unstructured domains. In myosin VI, interaction of the helical tail with other domains or interacting cargo proteins may stabilize the

75 structure and would allow this motor to operate both in a monomeric and dimeric form. (Supported by OTKA).

Molecular mechanism of motility: early results and new problems A.G. SZENT-GYO¨RGYI Brandeis University, Rosenstiel Basic Medical Sciences Research Center, Waltham, Massachusetts, USA The experiments of Albert Szent-Gyo¨rgyi, Ilona Banga, and Bruno Straub published in Studies from the Institute of Medical Chemistry University Szeged (1941–1943), but not available to Western scientists due to the war, will be summarized. These included the discoveries of pure myosin, actin, the demonstration that contraction requires actomyosin. The complex of these two proteins. The impact of these ﬁndings on the development of the sliding ﬁlament mechanism, the cross bridge cycle, single molecule studies will be discussed. Some of the present unsolved problems will also be analysed.

76 SESSION VI. CYTOSKELETON AND ACTIN, TUESDAY, 20TH OF SEPTEMBER 2005, 8:30–10:30 A Drosophila model of human ACTA1 nemaline rod myopathies? J. SPARROW1, S. HAIGH1, V. KUMAR1,2, U. NONGTHOMBA1,3 and M. PECKHAM2 1 Department of Biology, Univ. York, York, UK 2 School of Biomedical Sciences, Univ. Leeds, Leeds, UK 3 MRDG, Indian Institute of Science, Bangalore, India More than 105 human skeletal muscle actin, ACTA1, gene mutations produce mostly dominant, usually lethal, congenital myopathies. Four phenotypes – actin myopathy (AM), nemaline (NEM) and intranuclear (IRM) rod myopathies (Sparrow et al., 2003) and congenital ﬁbre type disproportion (CFTD) are found. Nemaline rods are the most common and contain Z-disc proteins. Nemaline myopathies are known in other human striated muscle thin ﬁlament protein genes. Extant and new transgenic Drosophila ﬂight muscle speciﬁc Act88F actin gene mutations corresponding to ACTA1 myopathy mutations have been examined. The G15R, A138V, V163M, V163L, R256C, G268D and R372H mutations disrupt ﬂight muscle sarcomeres, causing Z-disc disruption and serial Z-disc stacks, called zebra-tails. The structures have striking similarities to nemaline rods. To date the rarer phenotypes, AM, CFTD and IRM, have not yet been found in ﬂy muscles. Studies of ﬂight muscle development show that 3 mutants (A138V, R256C, G268D) exhibit aberrant myoﬁbrillogenesis from the outset, while young adult R327H ﬂies contain normal muscle and ﬂy but become ﬂightless and exhibit nemaline rods by day-3, reﬂecting late onset ACTA1 gene alleles. These studies suggest that nemaline myopathies develop from either an inability to assemble normal sarcomeres or an inability to maintain structural during muscle function. We also ﬁnd the nemaline phenotype in Drosophila troponin T alleles. The ﬂy is an amenable model with which to study the molecular details of this group of human myopathies.

Human gamma-actin deafness mutations in yeast actin cause a range of yeast mitochondrial and vascular phenotypes M. MCKANE1, K.K. WEN1, L. TRANEBJAERG2, K. FRIDERICI3 and P.A. RUBENSTEIN1 1 Dept. of Biochemistry, Univ. of Iowa, USA 2 Dept. of Medical Genetics, University of Tromso, Norway 3 Dept. of Microbiology, Michigan State University, USA Gamma non-muscle actin mutations cause progressive hearing loss. We have cloned 6 of these mutations into yeast actin and expressed them. The mutant actins support cell growth on glucosecontaining medium but exhibit speciﬁc growth deﬁcits in liquid culture. Mitochondria of four of the six mutant cell types have abnormal morphology but retain their DNA. All of the cells exhibit vacuole defects including hypervesiculation, hypovesiculation and inheritance defects. Five of the six actins polymerize relatively normally in vitro. The remaining actin polymerized more rapidly than normal and accelerated polymerization of WT actin indicating it may have enhanced ﬁlament nucleation ability. Two of the actins had faster monomeric nucleotide exchange rates and decreased melting temperatures as assessed by CD spectroscopy. Another mutant actually exhibited a slower rate of nucleotide exchange than WT actin but melted at the same temperature as WT actin. The association of speciﬁc organelle phenotypes with speciﬁc actin alleles indicates this series of mutations may be very helpful in delineating the role of actin in vacuole and mitochondrial function in yeast.

Synergistic eﬀect of fesselin and calponin on actin ﬁlament bundles formation and their sensitivity to calmodulin A. WRZOSEK, J. KOŁAKOWSKI and R. DA ˛ BROWSKA Nencki Institute of Experimental Biology, PAS, Poland Actin cytoskeleton of smooth muscle cells is composed of isoforms of actin and many of actin-binding proteins including those, like calponin, ﬁlamin, a-actinin and fesselin, which are capable to crosslink actin ﬁlaments into networks or bundles. We have demonstrated joint action of fesselin and calponin in bundling of actin ﬁlaments. Micromolar concentration of fesselin signiﬁcantly decreases the threshold concentration of calponin required for actin ﬁlament bundling formation. The macroscopic structure of the bundles induced by these two proteins visualized by confocal laser scanning microscopy varies from those formed by either fesselin or calponin alone. The bundles are formed at physiological-like conditions of ionic strength and pH, although maximum bundling is observed at pH 6.5. The bundling ability, taken as a cumulative eﬀect of fesselin and calponin, disappear upon interaction of these proteins with Ca2+/calmodulin. It is well documented that fesselin and calponin are localized to the dense bodies in smooth muscle cells in vivo and the results obtained in vitro may point to a potential pathway of actin cytoskeleton reorganization synchronized with smooth muscle contraction.

A junctional complex connects the terminal sarcomere with the intercalated disc in cardiac muscle P.M. BENNETT, A.M. MAGGS, E. EHLER, M. GAUTEL, A.J. BAINES and J.C. PINDER The Randall Division of Cell and Molecular Biophysics, GKT School of Biomedical Sciences, New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, UK Cardiac myocytes are linked at their distal ends by a complicated folded structure known as the intercalated disc (ICD). This is the site where both electrical and mechanical signals are transmitted between adjacent cells. The regular sarcomeric structure continues to the edge of the ICD, but the ﬁnal sarcomere does not terminate with a Z-line structure, rather, the thin ﬁlaments from the last I-band continue into the ICD and are tethered to the tension transmitting adherens junctions at the plasma membrane. This results in thin ﬁlaments with varying lengths. However, the ﬁnal A-band is usually well ordered. The Aband position within a sarcomere is normally maintained by the elastic behaviour of I-band titin which is anchored into the Z-line. In the absence of a Z-line at the junction of the ultimate sarcomere and the ICD, we have sought to determine if titin is present in the ﬁnal half sarcomere and which other Z-line proteins are associated with it. Using immuno-ﬂuorescence and immunogold electron microscopy on cardiac myoﬁbrils and cryosections of cardiac muscle we have found that an unusual junctional complex occurs at the position equivalent to that of the Z-line. This complex contains at least titin, alpha-actinin and ZASP. The question arises – how is such a complex anchored and correctly positioned on the terminal thin ﬁlament?

Characterization of a new member of the myomesin family R. SCHOENAUER, A. HIRSCHY, J.-C. PERRIARD and I. AGARKOVA Institute of Cell Biology, ETH Zurich-Hoenggerberg, CH-8093 Zurich, Switzerland Myomesin and M-protein are structural components of the sarcomeric M-band, which is thought to stabilize the thick ﬁlament lattice

77 during contraction. These related proteins consist mainly of Ig-like and Fn type III domains and belong to the same family according to sequence comparison and domain arrangement. Myomesin is constitutively expressed in the M-bands of all kinds of vertebrate crossstriated muscles, whereas M-protein is found in fast skeletal muscle and adult heart. Using comparative sequence analysis we now have identiﬁed a novel gene which is closely related to both M-protein and myomesin and shares the same intron-exon and domain arrangement. RT-PCR analysis of mouse tissues shows that this new member of the myomesin family (myomesin 3) is expressed mainly in skeletal muscle with the highest expression level in slow muscle. It is not expressed in mouse heart under normal conditions, while it is up-regulated in the heart of some mouse models of dilated cardiomyopathy. Variable levels of myomesin 3 are detected in the striated muscles of several mammalian species. We propose that myomesin 3 is a novel component of the sarcomeric cytoskeleton whose diﬀerential expression pattern, in combination with myomesin and M-protein, provides clues for the pathological development in the heart and skeletal muscle.

Relationship of cross-bridge arrangement to the backbone structure of myosin ﬁlaments of asynchronous insect ﬂight muscles (aIFM) G. ADER and G. BEINBRECH Institute of Zoophysiology, Univ. Muenster, Germany On the surface of the thick ﬁlaments of aIFM 4 cross-bridges per level (= 1 crown) repeat in distances of 14.5 nm along the longitudinal axis of the ﬁlaments. The cross-bridges are located at 4 helical tracks with axial spacing of 38.7 nm and a true axial repeat of 116 nm. To analyse the self assembly properties of myosin we expressed peptide fragments representing the C-terminal two thirds of Drosophila light meromyosin in E. coli (Ader et al., (1996) J Muscle Res Cell Motil 18: 246). Negatively stained aggregates of these fragments display 116 nm repeats with substructural elements of 43.5-29-14.5-29 nm in width. Optical diffraction patterns of the aggregates show layer line characteristics that resemble those of negatively stained, native thick ﬁlaments (Morris et al., (1991) J Struct Biol 107: 237). The results suggest that, indeed, the aIFM speciﬁc cross-bridge pattern result from the aggregation properties of insect myosin. Filament models based on these properties are consistent with electron microscopy and X-ray diﬀraction data. They include several assumptions: (a) the end by end aggregation of the fragments, (b) the coiling of 2 fragments strands to form ‘subﬁlaments’ and (c) the coiling of coiled subﬁlaments to form subﬁlament pairs.

M-band function: safety-belts for contracting sarcomeres I. AGARKOVA1, R. SCHOENAUER1, P. BERTONCINI2, E. EHLER3, S. LANGE1,3, M. HEGNER2 and J.-C. PERRIARD1 1 Cell Biology, ETH Zurich-Hoenggerberg, Zurich, Switzerland 2 Institute of Physics, University of Basel, Basel, Switzerland 3 Cardiovascular Division, King’s College London, London, UK The sarcomeric M-band is associated with dark transverse stripes in the centre of the thick ﬁlament zone on electron micrographs but its role remains obscure. Recent experiments led to a new view on M-band structure and function. The prominent M-band component myomesin was found to form dimers by antiparallel interaction of the C-terminal domains, which might cross-link the neighboring thick ﬁlaments by binding to myosin with N-terminal domains. Thus, myomesin assumes a role similar to a-actinin in the Z-discs. Using atomic force microscopy we have shown that myomesin molecules are molecular springs with complex viscoelastic properties. The elasticity of myomesin is modulated by an

alternatively spliced EH-segment, which has an intrinsically disordered conformation and works like ‘entropic spring’. In case of very strong and/or long lasting stretches the modular (Ig or Fn) domains of myomesin might unfold and function as reversible ‘shock absorbers’ preventing M-band rupture. We suggest that the M-band is an elastic web, which impedes the lateral misalignments of thick ﬁlaments and notably facilitates the titin job in restoring the central position of the thick ﬁlaments at the end of each contraction cycle.

Time-course of collagen matrix and desmin cytoskeleton alterations in rat soleus at 3, 7, 14 and 30 days of gravitational unloading I.N. CHISTYAKOV SRC Institute for Biomedical Problems of RAS, Moscow, Russia The purpose of the work was to study a time-course of desmin and collagen structures alterations under conditions of gravitational unloading (30 days hindlimb suspension (HS) rat). Soleus muscle samples were analyzed after 3, 7 and 14 day of HS. For revealing collagen frozen sections were stained with picrosirius red. The crosssectional area (CSA) of muscle, occupied with the total collagen as well as a percentage of isoform ratio didn’t increase signiﬁcantly after 3 days of HS in comparison with the control (C). After 7 day of unloading the CSA of muscle sample, occupied with the total collagen increased by 30% in comparison with the control, without the shift of isoform ratio. Fourteen days of HS resulted in 1.5-fold increase of the CSA of muscle sample, occupied with the total collagen in comparison with the control. Thus the percentage contribution of type I collagen ﬁbrils reached 70.8% (vs. 79.8% in C animals), and type III collagen ﬁbrils – 29.2% (vs. 20.2% in C). After 30-day exposure the CSA of muscle sample, occupied with the total collagen in comparison with the C, increased 3-fold, thus the contribution type I ﬁbrils reached 63.2%. Three day HS was followed with the signiﬁcant (P<0.05) decrease of the desmin level (118.2±3.4 arbitrary units of ﬂuorescence intensity vs. 137.4±2.5 in C). The 7 day exposure resulted in 104.2±6.2 ﬂuorescence intensity that signiﬁcantly diﬀered from a C level and level of the 3 day unloading (P< 0.05). The study was supported by the special program of the Russian Ministry of Education and Science.

Ankyrin-B relationships with calcium signalling apparatus in developing and mature skeletal muscle cells K. LEGAGNEUX, C. MAGAUD, C. COGNARD and B. CONSTANTIN Institut de Physiologie et Biologie Cellulaires, CNRS, UMR-6187, University of Poitiers, Poitiers, France Ankyrins are ubiquitously intracellular adaptor proteins involved in targeting diverse transmembrane proteins to specialized domains in the plasma membrane and endoplasmic reticulum. Several studies from the group of Dr Bennett have shown that cardiomyocytes from ankyrin-B ()/)) or (+/)) mice display mis-localization of diverse calcium transporters such as ryanodine receptors (RYR) and inositol 1,4,5-triphosphate receptors (IP3R) along with altered calcium release and contraction properties. This study addresses the question of relationships between ankyrin-B and calcium transporters in mature and developing skeletal muscle cells. Ankyrin-B ()/)) or (+/)) mice (gift from Dr Bennett) are also studied in order to determine involvement of ankyrin-B in sorting of various calcium channels and properties of calcium signalling in these conditions. Preliminary results show a developmental regulation of ankyrin-B distribution in skeletal muscle cells, analysed by means of ﬂuorescent confocal microscopy. Co-distribution of ankyrin-B with RYR anf IP3R type II were also observed in ﬁbres.

78 Myoﬁbrillogenesis in cardiomyocytes: exploring the ‘‘sarcomeroskeleton’’ E. EHLER1, J.-C. PERRIARD2 and S. LANGE1,2 1 The Randall Division of Cell and Molecular Biophysics and the Cardiovascular Division, King’s College London, Guy’s Campus, London, UK 2 Institute of Cell Biology, ETH Zurich-Hoenggerberg, Zurich, Switzerland We propose the existence of a basic framework in the sarcomere, which might act as a kind of ‘‘sarcomeroskeleton’’ and help to integrate all the other components properly during the process of myoﬁbrillogenesis. This seems to consist of alpha-actinin in the Z-disk, myomesin in the M-band and the titin ﬁlaments spanning in-between. The role of full-length titin during myoﬁbrillogenesis has already been demonstrated. However, no data exist at present to demonstrate the essential role of alpha-actinin and myomesin, respectively. We have performed RNAi experiments on primary cultures of neonatal rat cardiomyocytes to study this question. While the turnover of integral sarcomeric proteins seems to be too slow to see dramatic eﬀects on the myoﬁbrils in short term cultures, we can observe alterations in the organisation of myoﬁbrillogenesis regions in the cellular periphery in the case of alpha-actinin interference. RNAi against myomesin can also aﬀect the mature myoﬁbrils, depending on the particular cellular state. These results suggest that there might indeed be a kind of ‘‘sarcomeroskeleton’’, which is required for myoﬁbril assembly, but that Z-disc and M-bands are affected in a distinct way by interference.

Structure of ﬁlaments of modiﬁed actin using electron microscopy and single particle image processing J. GRUSZCZYNSKA-BIEGALA1,2, S.A. BURGESS1, H. STRZELECKA-GOLASZEWSKA2 and J. TRINICK1 1 School of Biomedical Sciences, University of Leeds, Leeds, UK 2 Nencki Institute of Experimental Biology, Warsaw, Poland

Actin is one of the main components in the eukaryote cells that play signiﬁcant role in many cellular processes. The ﬁlamentous form of actin (F-actin) is the principal component of the contractile and motility systems. Diﬀerent studies indicate that actin ﬁlaments are ﬂexible, and the dynamics of the ﬁlaments may have signiﬁcant role in the contractile process. One way to modify the ﬂexibility and perturb inter-monomer motions in F-actin is the chemical cross-linking of adjacent monomers. Site speciﬁc inter-monomer cross-links were prepared: between Lys-191 and Cys-374; between Gln-41 and Lys113; between Gln-41 and Cys 374. In vitro motility assay showed that constrain imposed on actin ﬁlaments by cross-linking inhibited the sliding motion of actin ﬁlaments on myosin and CD experiments revealed signiﬁcant diﬀerence in melting temperatures: 63C for uncross-linked and 74C for cross-linked (90%). In this study the eﬀect of intrastrand cross-linking of Gln 41 and Cys 374 on the F-actin was studied by DSC technique. The main transition of untreated F-actin at 63C was shifted to 70C after cross-linking and aﬀected the cooperative interaction between monomers.

Electron tomography of the C-zone in relaxed vertebrate skeletal muscle P. LUTHER1, J. LIU2, H. WINKLER2, R. PADRON3, R. CRAIG4, C. KNUPP5, K. TAYLOR2 and J. SQUIRE1 1 Imperial College London, UK 2 Florida State University, Tallahassee, USA 3 IVIC, Venezuela 4 University of Massachusetts, Worcester, MA, USA 5 University of Cardiﬀ, UK Vertebrate striated muscle comprises within each half A-band a striking set of 7–11 stripes of periodicity 43 nm that form the Czone. These stripes are attributed to myosin binding protein-C (MyBP-C), a 130 kD protein composed of repeating immunoglobulin (Ig) and ﬁbronectin domains. The cardiac and skeletal isoforms are similar, but the cardiac isoform is phosphorylated and has an extra Ig domain. There is great interest in this protein as mutations in the cardiac isoform lead to familial hypertrophic cardiomyopathy, one of the leading causes of sudden death in athletes. Our knowledge of the structural arrangement of this protein and of the C-zone is greatly lacking. In this study the ultrastructure of the C-zone has been investigated in frog sartorius muscle that was fast-frozen in a liquid helium slam-freezer and freeze-substituted. From the plastic sections we obtained the ﬁrst ever electron tomograms of the vertebrate A-band. We present results from these insightful tomograms on the ultrastructure of the thick ﬁlaments and of the C-zone.

Modiﬁcation of the N-terminal part of the DNase-I binding loop in subdomain 2 of actin by proteolytic cleavage with a protease from E. coli (ECP) inﬂuences the conformation of a putative tropomyosin (TM) binding site in subdomain 4 of actin (Moraczewska et al., (2004) J Biol Chem. 279: 31197–31204). We have previously presented 3D reconstructions of unmodiﬁed F-actin and its complex with TM based on a single particle processing method (Burgess et al., (2004) J Mol Biol. 341: 1161–1173). In this study, we applied the same method to negatively stained actin ﬁlaments modiﬁed by ECP in the presence and absence of TM. Filament segments were processed in the SPIDER software suite to create a family of helical segments with varying twist (angular rise per subunit). EM 3D reconstructions were used to examine the eﬀects of ECP modiﬁcation on the actin ﬁlament structure, as well as reveal possible shifts in TM position on the ﬁlament induced by this modiﬁcation, as suggested by our biochemical studies. Supported by the EU Marie Curie Training Site scheme.

Amyloidogenic properties of X-, C- and H-proteins of sarcomeric cytoskeleton L.G. MARSAGISHVILI1 and Z.A. PODLUBNAYA1,2 1 Institute of Theoretical and Experimental Biophysics RAS 2 Pushchino State University, Pushchino, Moscow Region, Russia

Eﬀect of inter-monomer cross-linking on the thermal transitions of F-actin D. LO¨RINCZY1, GY. HEGYI3 and J. BELAGYI2 1 Dept. of Biophysics 2Inst. of Bioanalysis University Pe´cs; Faculty of Medicine, Pe´cs 3 Dept. of Biochemistry, Eo¨tvo¨s University, Budapest, Hungary

Deposits of amyloid ﬁbrils are revealed in myositis, myocarditis, cardiomyopathies and many others diseases. Amyloid ﬁbrils have a number of common properties: beta-pleated sheet structure with individual beta-sheets oriented parallel to the main axis; insolubility in vivo; speciﬁc luminescence in the presence of Congo red and of thyoﬂavin-T. We demonstrate that like X-protein, C- and Hproteins of rabbit and ground squirrels skeletal muscles are capable to form amyloid ﬁbrils in vitro. Their amyloid nature was conﬁrmed by electron, polarization and luminescence microscopy and

79 by spectral methods. It is known that X-, C-, H-proteins already contain 90% of beta-sheet structure necessary for formation of amyloids. This in turn, creates a danger of fast growth of their amyloid deposits in vivo. Further studies on dynamics of amyloid formation by these proteins, can clear the ways for conducting by amyloidogenesis in human organs and tissues. This work is supported by grants RFBR 03-04-48487, ‘‘Universities of Russia’’ 11.01.462 and Program of the Presidium RAS ‘‘Fundamental Sciences for Medicine’’.

A new insight into ‘‘dissociative’’ mechanism of the thermal unfolding of F-actin by using small heat shock proteins V. MIKHAILOVA1, A. PIVOVAROVA1, I. CHERNIK2, N. CHEBOTAREVA1, N. GUSEV2 and D. LEVITSKY1,3 1 Bach Inst. Biochemistry RAS, Moscow 2 Dept Biochemistry, School Biol. and 3Belozersky Inst. Physico-Chem. Biol., Moscow State Univ., Moscow, Russia Earlier we proposed a ‘‘dissociative’’ mechanism for the thermal unfolding of F-actin ﬁlaments (Mikhailova et al., (2003) J Muscle Res Cell Motil 24: 329). According to this mechanism, irreversible thermal denaturation of F-actin is preceded by dissociation of short oligomers from the ﬁlament, with following denaturation and aggregation of these oligomers. More recently we have found that small heat shock proteins (sHSP) do not protect F-actin from thermal denaturation, but eﬀectively prevent aggregation of heat-denatured protein (Mikhailova et al., (2004) J Muscle Res Cell Motil 25: 251). At present we show that sHSP form stable complexes with denatured F-actin. The size of these highly soluble complexes was much smaller than that of intact F-actin, as was shown by analytical centrifugation. These data conﬁrm earlier proposed dissociative mechanism of the thermal denaturation of F-actin. Supported by grants from RFBR, Program for Support of Leading Scientiﬁc Schools in Russia, and Program ‘‘Molecular and Cell Biology’’ RAS.

Ectopic expression of KRP/telokin augments 3T3 ﬁbroblast motility at lower level of myosin II light chain phosphorylation D.V. SEREBRYANAYA, I.M. KULIKOVA, V.P. SHIRINSKY and A.V. VOROTNIKOV Institute of Experimental Cardiology, Cardiology Research Centre, Moscow 121552, Russia MLCK related protein (KRP/telokin) is abundantly expressed in smooth muscle cells and cardiomyocytes. It binds, primarily through the C-terminus, to unphosphorylated smooth/non-muscle myosin II and protects its regulatory light chain (RLC) from phosphorylation in vitro. It also unbends myosin molecules and promotes their polymerization into inactive ﬁlaments. To explore the in vivo function of KRP, we generated 3t3 ﬁbroblast cells that stably express either of human wild-type, N-terminally (DN), or C-terminally truncated (DC) KRP at levels close to that of myosin II. The fraction of myosin RLC phosphorylated at Ser19 (P-RLC) was found 60% less in the w/ t-KRP expressing cells, and 40 and 20% less in the DN- and DCKRP expressing cells, respectively, than in the mock controls. Yet, w/t-KRP cells contained more triton-insoluble myosin ﬁlaments and displayed 30% better adhesion and 50% better migration on colla-

gen than the mock controls. These data suggest that KRP enhances the cell motility by recruiting myosin II into ﬁlaments and increasing the cytoskeleton assembly, thus bypassing the need for a cell to phosphorylate myosin ﬁrst. It also appears to be a rate limiting step in cell migration, which, by itself, likely requires less level of P-RLC. Indeed, Rho-kinase inhibition resulted in substantial loss of P-RLC in all cells, but ceased the migration only of in the w/t-KRP, and not in the mock cells. Supported by FIRCA, RFBR and HHMI.

The structural mechanism of the high elasticity of giant titin molecule A.A. VAZINA1, P.M. SERGIENKO1, N.P. GORBUNOVA1, N.P.DESHCHEREVSKAYA1, N.F. LANINA1, W. BRAS1 and I.P. DOLBNYA1 1 Inst. Theor. Exp. Bioph. RAS, Pushchino, Russia 2 ESRF, B.P.220, F-38043 Grenoble, France 3 G.I. Budker’s Inst. Nucl. Phys. SB RAS, Novosibirsk, Russia The X-ray pattern of oriented titin gels shows a set of small-angle reﬂections in the form of diﬀuse spots or wide arcs in the region of the equator or nearby. In addition, two wide-angle reﬂections in the form of diﬀuse haloes at approximately 9.8 and 4.6 A˚ are registered, which are probably due to the packing of b-strands to a b-barrel domain. A characteristic feature of titin gel extension is an abrupt narrowing of the sample (the long ‘‘neck’’) with the full optical anisotropy is formed, the small-angle reﬂections on the X-ray patterns disappearing, but the wide-angle reﬂections persisting. A morphologic model of a giant ﬁbrillar titin molecule consisting of successively joined anisotropic b-barrel domains is proposed. The angles of tilting of individual domains relative to the molecule axis are diﬀerent, each domains having a rotational degree of freedom. The structural mechanism of the high elasticity of titin molecule on stretching in terms of the physics of crystalline polymers is discussed. Supported by RFBR Grants 03-02-17409, 04-02-97260

Changes in cardiac titin of ground squirrels upon hibernation: comparison with human cardiomyopathies I.M.VIKHLYANTSEV1 and Z.A. PODLUBNAYA1,2 1 Institute of Theoretical and Experimental Biophysics RAS 2 Pushchino State University; Pushchino, Moscow Region, 142290, Russia It is known that the increase of N2BA titin isoform content in myocardium leads to the decrease of its stiﬀness, the increase of extensibility and the decrease of heart rate. We have revealed the 15%increase of N2BA titin isoform content relative to that of N2B titin isoform in left ventricle and atrium of hibernating ground squirrels. This contributes to a suppression of heart contractile activity upon hibernation. The 12%-increase of N2BA titin isoform content in left ventricle of human hearts was revealed at the end stage of dilated cardiomyopathy (DCM) (Makarenko et al., 2004). The authors suppose that these changes decrease titin-based passive stiﬀness and may beneﬁt myocardial diastolic function. However, the authors suppose that this can impair the contractile performance in systole. Our data on titin-isoform changes upon hibernation conﬁrm the later supposition. In such a case, the increase in N2BA titin content in failing heart can be an indicator of the end stage of DCM. The work is supported by RFBR grants 03-04-48487, 04-04-48599, ‘‘UR’’ no. 11.01.462 and Program ‘‘Fundamental sciences for medicine’’.

80 SESSION VII. THIN FILAMENT REGULATION OF CONTRACTION IN MUSCLE, TUESDAY, 20TH OF SEPTEMBER 2005, 10:45–12:45 In vivo models of altered troponin I phosphorylation: implications for heart failure A.M. MURPHY Department of Paediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA Troponin I is the inhibitory component of the troponin complex which serves at the calcium switch for striated muscle contraction. Cardiac troponin I is dynamically phosphorylated by several kinases targeted to at least 5 sites. Previous in vitro work has demonstrated that site-speciﬁc phosphorylation of troponin I regulates the steadystate calcium sensitivity of muscle activation, and well as the kinetic properties of the contractile process. In heart failure signalling pathways are disturbed, and alterations of site-speciﬁc troponin I phosphorylation have been noted. Speciﬁcally, decreased phosphorylation at the protein kinase A (PKA) sites on troponin I has been demonstrated, and increased phosphorylation at protein kinase C sites has been inferred. In order to study the in vivo eﬀects of altered troponin I phosphorylation we created a mouse model with constitutive pseudophosphorylation of troponin I at the PKA sites. These mice demonstrated mildly increased basal systolic and diastolic left ventricular function. More strikingly, an increased force frequency response was noted in these mice. Another characteristic of the mice was an altered response to imposition of afterload. While non-transgenic mice demonstrated an increase in the relaxation constant (s) with imposition of afterload, in the PKA pseudophosphorylated mice, s au increased minimally in response to increased afterload. These results implied that the decreased phosphorylation of troponin I at PKA sites in human heart failure could impair the response to increased heart rates and altered afterload. Our recent work has more directly addressed this hypothesis in mice in which troponin I mimicking partial dephosphorylation at PKA sites and pseudophosphorylation at PKC sites was expressed. Indeed these mice demonstrate the opposite phenotype to the PKA pseudophosphorylated mice in regards to the force-frequency and afterload response. In conclusion, altered phosphorylation of troponin I in heart failure is likely to be a contributor to the pathophysiology of the common clinical condition.

Eﬀects of PKA- and PKC-mediated troponin phosphorylation on myoﬁlament contractile function in human myocardium J. VAN DER VELDEN1, N.A. NAROLSKA1, N.M. BOONTJE1, A. BORBE´LY1,3, R. ZAREMBA1, J.G.F. BRONZWAER2, Z. PAPP3, K. JAQUET4, W.J. PAULUS1 and G.J.M. STIENEN1 1 Lab. for Physiol. and 2Dept. of Cardiol., Inst. for Cardiovasc. Res., VU Univ. Med. Centre, Amsterdam, The Netherlands 3 UDMHSC, Division of Clin. Physiol., Inst. of Cardiol., Debrecen, Hungary 4 Forschungslabor Molekulare Kardiologie, St. Josef Hospital, Klinik der Ruhr-Universita¨t, Bochum, Germany In human heart failure b-adrenergic mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. This study was designed to reveal the eﬀects of PKC on myoﬁlament function in human myocardium under basal conditions, and upon modulation of protein phosphorylation by PKA and phosphatases. Isometric force was measured at diﬀerent [Ca2+] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myoﬁlament proteins and the inﬂuence of PKC, PKA and phosphatase

treatment were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting and ELISA. Troponin I (TnI) phosphorylation at the PKA-sites was decreased in failing compared to non-failing hearts and correlated well with myoﬁlament Ca2+-sensitivity (pCa50). Incubation with the catalytic domain of PKC did not alter maximal force under basal conditions nor following PKA and phosphatase pre-treatment. PKC reduced Ca2+-sensitivity to a larger extent in failing (D pCa50=0.19±0.03) than in non-failing (D pCa50=0.08±0.01) cardiomyocytes. This shift was reduced, though still signiﬁcant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa50. Protein analysis indicated that PKC phosphorylates PKA-sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. The PKC-mediated decrease in Ca2+-sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased.

Myoﬁlament function in heart failure: role of troponin R.J. BELIN, M.P. SUMANDEA, T. KOBAYASHI, V.L. RUNDELL, D.L. GEENEN, R.J. SOLARO and P.P. DE TOMBE Centre for Cardiovascular Research, University of Illinois, Chicago, USA Heart failure (HF) is associated with altered cardiac myoﬁlament function. The underlying mechanisms are largely unknown. We induced HF in rats either by chronic: left ventricular (LV) infarction (MI group; systolic dysfunction), or by aortic banding induced hypertrophy (LVH group; diastolic dysfunction). Myoﬁlament function was characterized in LV skinned myocytes by the active force-[Ca2+] relationship. Both HF groups displayed altered myoﬁlament function: Maximum force was depressed 35 and 42%; EC50 was increased 36 and 30% in LVH and MI, respectively. Replacement of endogenous cardiac troponin (cTn) with recombinant cTn reversed the depressed EC50 in both HF groups, but was without an eﬀect in non-diseased skinned myocytes. Maximum force, however, did not recover signiﬁcantly. Likewise, cTn puriﬁed from either HF or control rat hearts appropriately restored or depressed EC50 in either failing or control skinned myocytes (full cross-over design). We conclude (1) end-stage HF is associated with depressed myoﬁlament function; (2) increased EC50 in HF is due to an altered structural state of troponin, which may involve altered levels of contractile protein phosphorylation.

Eﬀects of PKA stimulation on the length-dependent of cardiac myosin binding protein C deﬁcient cardiomyocytes O. CAZORLA1, S SZILA´GYI1, N. VIGNIER2, GUY VASSORT1, L. CARRIER2 and A. LACAMPAGNE1 1 INSERM U-582, Montpellier 2 INSERM U-582, Paris, France b-adrenergic stimulation modulates cardiac contractility through protein kinase A (PKA), which phosphorylates cellular proteins, such as cardiac troponin I (cTnI) and cardiac C-protein (cMyBPC). The relative contributions of cTnI and cMyBP-C to the regulation of myoﬁlaments Ca2+ sensitivity are still controversial. In the present work we studied the PKA eﬀect on myoﬁlament Ca2+ sensitivity of left ventricular skinned myocytes isolated from young (5-weeks old) and old (55-weeks old) wild type mice (WT) and cMyBP-C deﬁcient mice (KO) at two sarcomere lengths (SL: 1.9 and 2.3 lm). Without PKA stimulation and at the shorter SL, Ca2+ sensitivity was higher in KO than in WT. The diﬀerence disappeared at the longer SL. No diﬀerence in passive tension or

81 maximal active tension was observed. PKA stimulation induced a desensitization of WT myoﬁlaments at both SL but had almost no eﬀect on young and old KO myoﬁlaments. The results suggest that cMyBP-C contributes to the regulation of cardiac contraction and that TnI phosphorylation alone by PKA was not suﬃcient to induce myoﬁlament desensitization.

Changes in contractile protein composition and function in patients with atrial dilatation and atrial ﬁbrillation S. EIRAS1, N.A. NAROLSKA1, R.B. VAN LOON1, C.R. JIMENEZ2, F.C. VISSER1, W. STOOKER3, J. VAN DER VELDEN1 and G.J.M. STIENEN1 1 Institute for Cardiovascular Research, VU University Medical Centre 2 Department of Molecular and Cellular Neurobiology, VU 3 Department of Cardiology, OLVG, Amsterdam, The Netherlands Force development was studied in mechanically isolated single skinned cardiomyocytes from atrial appendages from patients with sinus rhythm without (SR), or with atrial dilation (SR+AD) or atrial ﬁbrillation (AF). Atrial dimensions were determined by echocardiography. Protein composition was determined by 1- and 2-dimensional gel electrophoresis in conjunction with Western Immunoblotting and mass spectrometry. Compared to SR and SR+AD patients, patients with AF showed: a reduction (20%) in force normalized to cross-sectional area in isolated atrial cardiomyocytes, no change in Ca2+-sensitivity of force development, a reduction in its kinetics (Ktr), enhanced protein expression of the slow myosin heavy chain isoform (b-MHC), an increase in troponin T (TnT) phosphorylation and a marked loss of the regulatory light chain 2 (ALC-2). Our ﬁndings indicate that atrial dilatation per se does not predispose nor cause contractile alterations seen in AF.

Binding of extrinsic toponin I to rigor myoﬁbrils D.R. SWARTZ1 and S.S. LEHRER2 1 Indiana University School of Medicine, Dept. of Anatomy and Cell Biology, Indianapolis, IN, USA 2 Boston Biomedical Research Institute Boston, MA, USA Binding of troponin I (TnI) to actin, actin-tropomyosin (Tm) and actin-Tm-Tn in the presence of Ca2+, inhibits myosin ATPase activity (Geeves et al., 1995) when 1 TnI binds tightly to 7 actins/1Tm or to 7 actin/1Tn/1Tm +Ca2+, suggesting that TnI binds to a composite actin-Tm site. These studies were extended to the binding and localization of ﬂuorescent TnI (TnI*) into myoﬁbrils. The binding of TnI tin the presence of Ca2+ inhibited ATPase activity to 90% with an apparent Kd(TnI)=1 lM. For TnT-substituted myoﬁbrils, half maximal inhibition occurred at 30 nM TnI to >90%. Myoﬁbrils were incubated with TnI* and its sarcomeric distribution was determined by ﬂuorescence microscopy. For native myoﬁbrils at pCa 4, TnI* bound preferentially to the non-overlap regions (Closed State) rather than the overlap regions (Open State), consistent with the suggestion of a TnI binding site on actinTm in the Open State. At pCa 9 TnI* bound preferentially to overlap regions, suggesting that the extrinsic TnI* could preferentially exchange with the intrinsic TnI in the Open State where the TnI-actinTm interaction is weakened. For TnT-substituted myoﬁbrils, TnI* bound preferentially to non-overlap regions, suggesting that the TnI binding site is still available in the presence of TnT. These studies indicate that the binding of TnI to thin ﬁlaments involves a joint actinTm site. The presence of TnT on actinTm, enhances TnI binding, suggesting that actinTmTnT is in the Closed State.

Contractile alterations in isolated human cardiomyocytes following oxidative protein modiﬁcations A. BORBE´LY1, Z. HERTELENDI1, A.TO´TH1, S. SZILA´GYI1, I. E´DES1, A.VARRO´2, J.GY. PAPP2 and Z. PAPP1 1 UDMHSC, Institute of Cardiology, Division of Clinical Physiology, Debrecen 2 University of Szeged, Department of Pharmacology and Pharmacotherapy, Szeged, Hungary We investigated the mechanical consequences of those contractile protein alterations that develop following the applications of oxidative agents in mechanically isolated, Triton X 100-permeabilized human left ventricular myocytes at a sarcomere length of 2.2 lm (T=15C). Ca2+-activated force production (Factive), its Ca2+-sensitivity (pCa50) and the steepness of the Ca2+-force relationship (nHill) were monitored before and after exposures to hydrogen peroxide (H2O2) or 2,2¢ dithiodipyridine (DTDP). H2O2 (10 mM for 30 min) decreased Factive at saturating [Ca2+] by 27±5% (P<0.05, mean±S.E.M., n=9 myocytes). DTDP (1 mM for 2 min, n=9) induced a reduction in Factive by 28±4% (31±3 kN/m2 vs. 20±2 kN/m2, before and after DTDP, respectively P<0.05). This reduction in Factive was partially reverted by incubations in the presence of the antioxidant dithiotreitol (DTT, 10 mM for 30 min, Factive= 25±2 kN/ m2, P<0.05). Moreover, DTDP also induced a signiﬁcant decrease in Ca2+-sensitivity and in nHill (pCa50:5.9±0.02 and 5.69±0.04; nHill: 2.17±0.16 and 1.70±0.07; before and after DTDP, respectively, P<0.05). These results suggest that oxidative contractile protein alterations may diminish Ca2+-activated force production via alterations in the Ca2+-regulation process of the contractile machinery in the human myocardium. Supported by the ETT 239/2003 grant.

Troponin I degradation products released into the bloodstream during myocardial ischemic injury Z. HERTELENDI1, A. TO´TH1, CS. JENEI1, I. E´DES1, K. JAQUET2 and Z. PAPP1 1 Division of Clinical Physiology, Institute of Cardiology, Debrecen, Hungary 2 St. Joseph-Hospital, Ruhr-Universita¨t Bochum, Germany We aimed to characterize the proteolytic troponin I (TnI) degradation during myocardial infarction in men. TnI and its fragments were isolated from the sera of patients with acute myocardial infarction (approximately 24 h after the onset of chest pain; n=3 patients) by immunprecipitation, and subsequently subjected to SDS-polyacrylamide gel electrophoresis and Western immunoblotting. We found that considerable part of TnI is present (37.1±5.9%) as the full length, intact molecule (28 kDa) in the serum, however more than half of the TnI immunreactivity is the consequence of TnI fragments. Four degradation products (at around 25, 20, 16, and 15 kDa) with variable densities could be clearly separated. Puriﬁed recombinant TnI spiked into control (without myocardial infarction) human serum also degraded partially. However, this degradation resulted a diﬀerent pattern of TnI fragments than that of patients’ sera. When recombinant TnI was digested in vitro by l-Calpain and the resultant degradation pattern was compared to that of the patients, striking similarities were found in the molecular weight distribution of the TnI fragments. These results imply that lCalpain can be responsible for the in vivo degradation of TnI in the ischemic myocardium. Supported by the ETT 239/2003 grant.

Degradation of human cardiac troponin I by active matrix metalloproteinase-2 in vitro M.H.M. HESSEL and A. VAN DER LAARSE Department of Cardiology, Leiden University Medical Centre, Leiden, The Netherlands Matrix metalloproteinase-2 (MMP-2) is responsible for collagen degradation and increased myocardial MMP-2 activity is associated with

82 ischemia-reperfusion induced injury and left ventricular remodelling in heart failure. It has been shown that MMP-2 also acts intracellularly and therefore may be involved in degradation of myoﬁlament proteins, leading to impaired contractility. The purpose of this study was (I) to test whether active MMP-2 is able to degrade cardiac troponin-I (cTnI) in vitro and (II) to investigate the cTnI fragments formed. cTnI was incubated with active MMP-2 at 37C for 0, 60 and 120 min (n=4). In an additional set of experiments, MMP-2 was preincubated with an inhibitor, o-phenanthroline, for 15 min (n=3). As a control, cTnI was incubated without MMP-2 (n=4). Intact cTnI and cTnI fragments were determined by Western blotting. Incubation with active MMP-2 resulted in a signiﬁcant reduction of intact cTnI (29 kDa) by 40±10% (P<0.05) after 60 min and 67±11% (P<0.01) after 120 min. Three cTnI fragments of 26, 14 and 8 kDa were formed. oPhenanthroline completely inhibited degradation of cTnI after 60 and 120 min of incubation. In the absence of active MMP-2, cTnI fragments were not observed. This study demonstrated that MMP-2 is able to proteolytically cleave cTnI in vitro, thereby forming at least three cTnI fragments. Inhibition of active MMP-2 by o-phenanthroline prevented degradation of cTnI. These results suggest that in vivo activation of MMP-2 during heart failure may result in cTnI degradation and may contribute to contractile dysfunction.

PKC alpha in the human myocardium: expression, translocation and possible functions A. MOLNA´R1, S. SZILA´GYI1, A. BORBE´LY1, M. VASZILY1, A. VARRO´2, J.GY. PAPP2, I. E´DES1, Z. PAPP1 and A. TO´TH1 1 University of Debrecen, Institute of Cardiology, Division of Clinical Physiology, Debrecen, Hungary 2 Department of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary Function, expression, translocation and substrate speciﬁcity of diﬀerent PKC isoenzymes were investigated in human myocardial preparations. PKC inhibition decreased the relative isometric force production following Ca2+ incubations (1 mM Ca2+ for 10 min; control: )4.8±1.4%; with PKC inhibition: )21.1±5.9% when compared to control maximal Ca2+-activated force). PKC-alpha expression was signiﬁcantly lower in the failing than in the donor samples, but no diﬀerence in the intracellular localization of PKC-alpha was detected by immunohistochemistry. Biochemical assays revealed that PKC-alpha translocates to the contractile machinery upon stimulations with 10 lM PMA or 5 mM Ca2+ (EC50: 172 lM Ca2+). Phosphorylation of human myocardial contractile proteins was followed by autoradiography using diﬀerent recombinant PKC isozymes, and major diﬀerences in substrate speciﬁcities were observed among the diﬀerent PKC isoforms. Our results suggest PKC-mediated modulation and related functional alterations of the contractile protein machinery in the human myocardium. Supported by the OTKA F 048873 grant.

83 SESSION VIII. MUSCLE DYSFUNCTION, TUESDAY, 20TH OF SEPTEMBER 2005, 16:00–18:00 Myotonias and periodic paralyses F. LEHMANN-HORN Applied Physiology, Ulm University, Ulm, Germany Mutations in the genes encoding voltage-gated ion channels expressed in skeletal muscle cause muscle disorders characterized by over- or hypoexcitability of the surface membrane: (i) loss-of-function mutations in the chloride channel gene, CLCN1, reduce the inhibitory chloride current through the muscle ﬁbre membrane, cause action potential bursts and lead to Thomsen myotonia or the much more frequent Becker myotonia; (ii) gain-of-function mutations in the SCN4A gene encoding the muscle sodium channel cause a persistent sodium current which is pronounced in response to physiological membrane depolarization, e.g. due to elevated serum potassium (potassium-aggravated myotonia, hyperkalemic periodic paralysis) or cold environment (paramyotonia congenita); iii) loss-offunction SCN4A mutations and mutations in the CACNA1S gene coding for the muscle L-type calcium channel show enhanced channel inactivation and cause episodes of muscle inexcitability (i.e. weakness), particularly in response to lowered serum potassium (hypokalemic periodic paralysis). Another type of periodic paralysis is Andersen syndrome, a disorder potentially associated with cardiac arrhythmia and dysmorphic features. It is caused by mutations in KCNJ2 encoding a potassium channel that is essential for the resting membrane potential. Generally, reliable information for determining the range of diagnoses may be gained by examination of additional relatives because of the intra- ad interpersonal variability. Next to clinical features, also electromyography and provocative testing combined with neuro-physiological tests are helpful for diagnosing. Genetic studies focus on conﬁrming clinical diagnosis but may be quite useful for exclusion of dystrophy-related myotonias (DM1 and DM2), an important factor for prognosis.

Myasthenia gravis – functional interaction of speciﬁc antibodies with nicotinic receptor channels J. BUFLER Neurological Departement, Medical School Hannover, Hannover, Germany Myasthenia gravis (MG) is a prototypic autoimmune disease and caused by a chronic attack of speciﬁc antibodies against muscle nicotinic acetylcholine receptor (nAChR) channels. Clinically the disease is characterized by used dependent paresis of skeletal muscles. The most important neurophysiological sign is the pathological decrement after repetitive supramaximal nerve stimulation. Anti-nicotinic antibodies are detected in around 90% of MG patients. Beside an increased rate of receptor degradation and a complement-mediated destruction of the postsynaptic membrane, there is a direct blockade of nAChR channels by speciﬁc IgG antibodies. The patch-clamp technique in combination with a system for ultrafast solution exchange allows the analysis of the function of ligand-activated channels within the range of milliseconds. For the data presented here we used cultured mouse myotubes or recombinant nAChR channels expressed in HEK293 cells. It was shown that monoclonal antibodies directed against the binding site of the receptor as well as puriﬁed IgG fractions of patients suﬀering from MG blocks embryonic- and adult-type nAChR channels reversibly and in a dose dependent manner. The blockade was observed with puriﬁed IgG from antibody-positive as well as antibody-negative patients. With increasing time of incubation of the channels with antibodies, the block becomes irreversible. As described for the action of a-bungarotoxin at nAChR channels, the binding of speciﬁc antibodies proceeds in several stages, the ﬁrst one showing a low-aﬃnity reversible block (resembling a competitive block of nAChR channel currents by drugs like d-tubocurarine) proceeding to higher-aﬃnity binding

with irreversible blockade of the channels. The ﬁndings are clinically relevant because they might explain the rapid recovery of muscle strength after plasmapheresis or ﬂuctuations of muscle weakness during the day by the pharmacological interactions of myasthenic antibodies with nAChR channels.

The functional eﬀect of a DCM-causing mutation in TnC (G159D) in skinned muscle ﬁbres and its modulation by troponin phosphorylation L. PRESTON1,2, P. ROBINSON1, C. ASHLEY2 and C. REDWOOD1 1 Department of Cardiovascular Medicine and 2Department of Physiology, University of Oxford, UK The functional eﬀect of the DCM-causing mutation in cardiac TnC (G159D) was assessed in skinned rabbit psoas ﬁbres. Whole human cardiac troponin (Tn) was reconstituted from individual wild type (WT) or mutant recombinant subunits and used to displace the endogenous Tn. Tn containing G159D TnC was found to regulate force generation in a similar way to WT Tn with no signiﬁcant diﬀerences in pCa50, nH or force/cross-sectional area. However, in kinetic analyses, when activated with NP-EGTA the mutant caused a 50% decrease in the rate of activation. Tn phosphorylation is known to be important in vivo, with both protein kinases A and C causing a reduction in Ca2+-sensitivity. We are now using the pseudophosphorylation mimics S23,24D TnI and T203E TnT to investigate the eﬀect of the DCM TnC mutant on phosphorylated troponin function. Our preliminary data shows that Tn containing G159D TnC and S23,24D TnI regulates force production with a similar pCa50 and nH to Tn containing WT TnC and S23,24D TnI, but causes a reduction in the force/crosssectional area. We will report on the eﬀect of the G159D mutation on the behaviour of T203E TnT complex and also compare the regulation given by the various mutants to reconstituted actomyosin ATPase activity.

Malignant Hyperthermia: genetic screening of the entire RYR1 gene and functional expression of mutated proteins L. GALLI1, D. ROSSI1, P. DE SMEDT1, A. ORRICO1, D. FRANCI1, F. PETRIOLI1, S. LORENZINI1, V. TEGAZZIN2 and V. SORRENTINO1 1 Molecular Medicine Section, Dept. of Neuroscience, Univ. of Siena, Siena, Italy 2 MH Laboratory, Dept. of Anaesthesia, S. Antonio Univ. Hosp., Padova, Italy Mutations in the RyR1 gene are linked to Malignant Hyperthermia (MH), an autosomal-dominant inherited disorder that causes muscle spasm and hyperthermia in susceptible patients when exposed to volatile anaesthetics and muscle relaxants. Mutations in the RYR1 gene are found in approximately 30–45% of the referred cases and they are located in three hot spot regions: MH domain 1 (aa 35–614), MH domain 2 (aa 2092–2481) and MH domain 3 (aa 4095–4990). In order to assess a reliable estimate of the characteristics and frequency of RYR1 mutations in MH susceptible individuals, we performed a systematic screening by DHPLC analysis of the entire coding region of the RYR1 gene in 50 unrelated MH probands. The results indicate that more than 85% of MHS individuals present mutations in the RYR1 gene, proving that RYR1 is associated with the large majority of MH cases. Sequence of the entire RYR1 coding region of patients negative at the DHPLC analysis is being per-

84 formed to complete the study. Many of the novel mutations detected have been found outside of the three hot-spot regions. Mutated RYR1 channels have been expressed in HEK293 cells in order to study their regulatory properties and to verify their eﬀect on intracellular calcium homeostasis.

Pharmacological treatments improve sarcoplasmic reticulum function and contractile performance of diaphragm muscle from mdx mice A. LAFOUX, A. DIVET, P. GERVIER and C. HUCHET-CADIOU UMR CNRS 6204 Laboratoire de Biotechnologie, Biocatalyse et Biore´gulation, University of Nantes, France This study investigated the potential therapeutic beneﬁts of pharmacological tools in reversing the Ca2+ sequestration function of the sarcoplasmic reticulum and improving the functional capacity of dystrophic skeletal muscle from mdx mice. Creatine (2.15 mg/ mL/day) and L-arginine (3.75 mg/mL/day) were administered per os to male mdx mice (12 weeks) during 4 weeks. Then, the eﬀectiveness of the treatment was investigated on the contractile protein properties, the sarcoplasmic reticulum Ca2+ uptake and the expression of SERCA1 and calsequestrin of diaphragm muscles from mdx mice. The data demonstrate that after treatment, the force developed by Triton-skinned ﬁbres was increased approximately by 25%. The study of sarcoplasmic reticulum function, assessed in saponin-skinned ﬁbres and vesicle preparations, shows that the Ca2+ uptake was improved. Furthermore, the analysis of the locomotor activity, evaluated using diﬀerent tests (wiretest, rotarod, and grip test), shows that mdx mice were more resistant to fatigue. In conclusion, the data show that both treatments signiﬁcantly normalized many functional and biochemical parameters by acting on events that are related to Ca2+ homeostasis.

+

Antimyotonic eﬀects of elevated K concentration and osmolarity S. CHAIKLIENG1, F. LEHMANN-HORN1 and W. KLINGLER2 1 Department of Applied Physiology, Univ. Ulm, Ulm, Germany 2 Department of Anaesthesiology, Univ. Ulm, Ulm, Germany Mutations in the chloride channel (CLCN1) are causative for Thomsen and Becker’s myotonia. Repetitive contractions reduce the myotonic stiﬀness by unknown mechanisms. The objective of this study was to investigate changes of microenvironmental conditions on this so called warm-up phenomenon. Force registrations were performed on freshly dissected mouse gastrocnemius muscle. Experimental myotonia was induced by Cl)-free solution, blocking CLCN1 with 9-antracene carboxylic acid, and the use of myotonic mice (ADR-mouse). K+ increase prevented the relaxation deﬁcit in a concentration dependent manner. At 7 mM K+ the relaxation time reached control levels. Elevated osmolarity promoted the warm-up phenomenon. This eﬀect was partially antagonized by bumetanide, an inhibitor of the Na+K+Cl) cotransporter (NKCC2). Therefore, the antimyotonic eﬀects of increased osmolarity may be explained by electrical stabilisation via NKCC2. We also conclude that the eﬄux of K+ from muscle ﬁbres after repetitive voluntary contractions reduces myotonic activity. We speculate that K+ induced depolarization leads to inactivation of voltage gated Na+channels (i.e. reduction of action potential frequency). Clinicians

should aim at elevating K+ levels in the high normal range in these patients.

Unloaded shortening in intact single wt, mdx and minidystrophin (MinD) expressing murine skeletal muscle ﬁbres under voltage-clamp conditions O. FRIEDRICH1, J.S. CHAMBERLAIN2 and R.H.A. FINK1 1 Med. Biophysics, Inst. Physiology & Pathophysiology, Univ. Heidelberg, Germany 2 Dept. Neurology, Univ. Washington, Seattle, USA Ca2+ signalling and DHPR channel function are impaired in muscle from mdx mice missing dystrophin (Friedrich et al., 2004) but can be recovered by expression of mini-/micro-dystrophins (Gregorevic et al., 2004). Whether the kinetics of depolarisation induced contraction is aﬀected is not clear. We performed 2-electrode voltage clamp experiments in single Mm. interossei wt, mdx and MinD ﬁbres (isotonic condition) with maximum Ca2+ activation by maintained depolarisation to 0 mV (15C, 25C). The time course of ﬁbre length l(t) was recorded via a CCD line-scan (500 Hz). lmin was 50% of resting length L0 throughout. Timeto-minimum (TTM) of shortening was 600 ms in wt ﬁbres (15C) and 800 ms in mdx and MinD ﬁbres. TTM decreased 20% at 25C. Time constants of the shortening phase (sfast, sslow) were larger in mdx- (100 ms, 350 ms) and MinD(70 ms, 450 ms) vs. wt-ﬁbres (60 ms, 200 ms) at 15C and decreased 50% at 25C. Max. shortening speed (vm 9L0s)1, 15C, 14L0s)1, 25C) and acceleration were similar. In mdx muscle, shortening activation kinetics seems to be slightly slowed down with almost unaltered vm.

Similarity between exercise and corticosteroid myopathies P. KAASIK1, A. PEHME1, T. SEENE1, K. ALEV1, M. UMNOVA2, R. PUHKE1, E.-M. RISO1, M. ARU1, M. KIPRI1 and K. REISBERG1 1 Department of Functional Morphology, University of Tartu 2Institute of Ecology and Evolution RAS, Russia It seems that there is some similarity between the structural changes in FT-G and FT-OG muscle ﬁbres in case of exercise myopathy, although destructive changes in the G muscle ﬁbres seem to be of smaller scope. G ﬁbres do not participate actively in the low-intensity exercise, but large range of destructive processes in these ﬁbres shows that G muscle ﬁbres participate in long-term exercise, performing the junction of the skeletal muscle in the role of an organ. FT muscle ﬁbres are more sensitive to the action of corticosteroids. It is probable that the increase of endogenous corticosterone level in endurance-type exhausted rats and cortisol level in overtrained athletes may be the important factor in the pathogenesis of the exercise myopathy. ST muscle ﬁbres with high oxidative potential are much less sensitive to the destruction of myoﬁbrils. FT muscle ﬁbres are more sensitive to the catabolic action on the level of myoﬁbrils. In case of exercise myopathy, in FT skeletal muscles with high oxidative potential the turnover rate of MyHC is decreasing. In case of corticosteroid myopathy the turnover of MyHC is decreasing in FT muscles with low oxidative potential. In conclusion: although there are similarities between development of exercise and corticosteroid myopathy there is no ground to conclude that exercise myopathy is the mild form of corticosteroid myopathy.

85 The muscle type of the Compact mouse: myostatin or androgen eﬀect? E. ZA´DOR and L. MENDLER Institute of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary The selection for high protein content and body weight in the Technical University of Berlin resulted in a mouse strain, which in its hypermuscular character was very similar to the null mutant of myostatin, a muscle growth inhibitory factor. The strain was named Compact and the genetic mapping of its Hungarian subpopulation showed that the phenotype is not monogenic. A mutation is found in the myostatin pro-region and also in the region of the androgen receptor gene. According to the previous tissue analysis in the muscle of the Compact mice both hypertrophy and hyperplasia are observed like in those of the myostatin KO mouse. In this work we aimed to investigate the hindlimb muscles of the Compact mice. Our results show that the ﬁbre type composition and ﬁbre size of the Compact is diﬀerent from those of the myostatin KO mouse or the other hypermuscular strains, and from the control Castaneus strain. The most signiﬁcant diﬀerence was present in the ﬁbre size of fast-glycolytic ﬁbres. A similar change in ﬁbre size was found in response to testosterone treatment of the androgen-dependent levator ani muscle of castrated rats, which consists of almost entirely glycolytic ﬁbres. This shows that parallel with the myostatin mutation the androgen eﬀects could also inﬂuence the formation of skeletal muscles of Compact mice.

The Interaction of Phenylglyoxal with the Calcium release channel (Ryanodine Receptor) B. MOLONEY, J. MACKRILL, T.V. MCCARTHY and J.J.A. HEFFRON Department of Biochemistry, University College Cork, Ireland Arginine (Arg) is an amino acid, which plays an important role in the function of the ryanodine receptor. This is evident from the number of arg residues conserved across species and also the number of arginines, which are involved in functionally important mutations in the protein. Phenylglyoxal (PG) is a reagent, which has been shown to react speciﬁcally with arginine residues in proteins. Sarcoplasmic reticulum (SR) isolated from rat skeletal muscle was used to investigate the eﬀect of PG on calcium release using the calcium sensitive dye Antipyrylazo III. PG induced calcium release from actively loaded sarcoplasmic reticulum in a dose dependent manner. This release was completely blocked by ruthenium red, an agent that specifically inhibits calcium release via RYR indicating that the PG induced release was occurring via this channel. Radioligand binding studies using [3H]Ryanodine show that PG causes a decrease in binding of [3H]Ryanodine to SR by decreasing the Bmax value while the Kd remains unaltered. PG also aﬀects the radioligand binding in various conditions including ranges of free calcium, pH & KCl concentration. PG also decreases the binding of BODIPY-FLX-Ryanodine (a ﬂuorescent ryanodine derivative) in diﬀerentiated mouse C2C12 cells. This work aims to identify functionally important Arg residues & to elucidate the role of these residues.

Changes of myosin light chains upon hibernation: comparison with human muscle diseases D.A. OSIPOVA1 and Z.A. PODLUBNAYA1,2 1 Institute of Theoretical and Experimental Biophysics RAS 2 Pushchino State University, Pushchino, Moscow Region, 142290, Russia Earlier we registered 30–70% appearance of ALC1 in human ventricles at early stages of DCM but not at terminal stage. (Akopova

et al., 1998; Khalina et al., 2004). The data emphasize functional role of ALC1 appearance. Studying the hibernation as an adaptive model, in ventricles of arousing ground squirrels (GS) we revealed up to 30% of ALC1 necessary for restoring the normal heart activity. In atria of hibernating GS we found from 30 to 60% of VLC1, which disappear gradually as the animal approaches to active state. It is known that cardiomyopathies can go with skeletal myopathies and exhibit the decrease in myocardial LC2 content. In skeletal myosin of hibernating and arousing GS we revealed the increase in slow LC1 and LC2 isoforms with respect to their fast isoforms. According to the hibernation strategy, the decrease of fast LCs isoforms in psoas muscle ﬁbres upon hibernation and arousal also took place. Comparative study on hibernation and myopathies helps to understand their adaptive strategy and to ﬁnd ways for their diagnostics and correction. Supported by RFBR grants 04-04-48599, 04-04-97305 and grant ‘‘UR’’ 11.01.462.

The eﬀect of oxidative stress upon aging rat heart physiology C.R. REVNIC1, F. REVNIC2 and S. BOTEA3 1 U.M.F. ‘‘Carol Davila’’, 2NIGG ’’Ana Aslan’’, 3V. Babes Institute, Bucharest, Romania Heart muscle is largely dependent upon uninterrupted blood ﬂow, which guarantees delivery of substrates and washout of harmful products of metabolism. During aging, a lot of degenerative changes occur and heart is not an exception. Ischemia is a frequent phenomenon associated with aging and this means the decrease or cessation of myocardial blood ﬂow, which leads to changes in myocardium metabolism. The aim of our study was to point out how highly interconnected network of intracellular signaling reactions behaves in stress conditions imposed by 45 min ischemia followed by 60 min reperfusion of isolated rat heart of diﬀerent ages and to what extent physiological parameters of cardiac contractility such as: heart rate (H.R.),coronary ﬂow (C.F.) and left ventricle systolic pressure (L.V.S.P.) are inﬂuenced, as well as to assay ventricular cells for apoptosis. Isolated rat hearts of 6 and 37 months old have been mounted and perfused with Krebs Hanseleit buﬀer at 37C in Langendorﬀ retrograde perfusion system at a constant pressure over 60 min (ie. at 10¢, 20¢, 30¢, 40, 50¢ and 60¢) intervals have been determined: H.R.,C.F. and L.V.S.P TACS Apoptotic DNA Laddering Etd.Br. kit (R&D System England) has been used to assay ventricular myocytes for apoptosis. Our data have pointed out that, in old rats H.R. exhibits higher values than in young controls. C.F. is variable in time in aging rats versus young ones where it has a slow decrease during the experiment. LVSP is net elevated in aging rats, but with ﬂuctuations in time. DNA ladder formation on agarose gel electrophoresis was noted in ageing left verticular cardiomyocites.

1H NMR studies of aging rat heart with experimental atherosclerosis F. REVNIC1, M. GEORGESCU1, S. BOTEA2, C. REVNIC3 and A.M. GEORGESCU1 1 ‘‘Ana Aslan’’ N.I.G.G, 2V. Babes Institute, 3UMF ‘‘Carol Davila’’, Bucharest, Romania The aim of our study was related with investigation of biophysical aspects of heart membrane permeability in old rats feed on cholesterol reach diet and on rats which received also Aslavital and Procaine treatment. Twenty-four rats aged 32 month-old have been divided in 4 groups of 6 rats each: group A control, group B feed for 6 weeks with cholesterol reach diet only, group C feed on cholesterol reach diet and treated for 6 weeks with Aslavital (4 mg/kg body weight Procaine) and group D feed on cholesterol reach diet treated with Procaine(4 mg/kg

86 body weight) for 6 weeks.1H RMN permeability studies of plasmalema from control and treated rats hearts have been done with an Aremi’78 Spectrometer at 25 MHz frequency by measuring T21 and T22 proton transverse relaxation times. There is a decrease in proton transverse relaxation time in plasmalema of cardiomyocite of rats fed on cholesterol reach diet which suggest an accelerated proton exchange. Membrane permeability to water can be taken into consideration as an index of cardiovascular system recovery, important in maintaining a dynamic equilibrium with vascular destruction phenomenon due to high blood pressure. Our results pointed out that Aslavital and Procaine treatment in rats feed on reach cholesterol diet brought about the stabilization of water exchange. Aslavital behaved as a buﬀered Procaine with a moderate eﬀect upon water permeability.

Depression of force production and ATPase activity in diﬀerent types of human skeletal muscle ﬁbres from patients with chronic heart failure P. SZENTESI4, M.A. BEKEDAM2, B.J. VAN BEEK-HARMSEN1, W.J. VAN DER LAARSE1, R. ZAREMBA1, A. BOONSTRA3, F.C. VISSER2 and G.J.M. STIENEN1 1 Dept. of Physiol. 2Cardiol. and 3Pulmonol., Inst. for Cardiovasc. Res., VUmc, Amsterdam, The Netherlands 4 Dept. of Physiol., Med. and Health Sci. Centre, Univ. of Debrecen, Debrecen, Hungary Isometric force production and ATPase activity were determined simultaneously in single human skeletal muscle ﬁbres from 5 healthy volunteers and 9 patients with chronic heart failure (CHF). ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. Calcium-activated actomyosin (AM) ATPase activity was obtained by subtracting the activity measured in relaxing (pCa=9) solutions from that obtained in maximally activating (pCa=4.4) solutions. Fibre type was determined on the basis of myosin heavy chain isoform composition by polyacrylamide SDS gel electrophoresis. Our results indicate that in CHF patients signiﬁcant reductions occur in isometric force and AM ATPase activity, but that tension cost for each ﬁbre type remains the same. Isometric force and AM ATPase activity correlated well with the maximum rate of oxygen uptake determined during a bicycle ergometer test. These results indicate that in skeletal muscle from CHF patients a decline in density of contractile proteins takes place and/or a reduction in the rate of crossbridge attachment of approximately 30%, which exacerbates skeletal muscle weakness due to muscle atrophy. Supported by NWO, the Netherlands Heart Foundation (97.118) and Hungarian Research Grant (OTKA T0 49151).

87 SESSION IX. GENE TRANSFER AND CELL THERAPY, TUESDAY, 20TH OF SEPTEMBER 2005, 18:15–20:15 Gene therapies for muscle diseases: progress towards clinical trials D.J. WELLS Department of Cellular and Molecular Neuroscience, Faculty of Medicine, Imperial College London, UK Gene therapy oﬀers the possibility of correcting the genetic defects underlying many muscle diseases. Although a number of diﬀerent systems can deliver genetic material to muscle cells both in vitro and in vivo, the eﬃciency has been limited by a number of constraints. Major stumbling blocks have included host immune responses to the transgene and/or the vector, limited diﬀusion from the site of delivery, decreasing eﬃciency with increasing muscle maturity and species diﬀerences. Recent major improvements in the delivery of genetic material to muscle are ﬁnally oﬀering the real possibility of clinically eﬀective treatments. Adeno-associated viral vectors are emerging as the viral vector of choice for muscle and the use of a range of novel serotypes has increased the eﬃciency of both local and, more importantly, systemic delivery. The use of physical methods such as pressure, electrical ﬁelds and ultrasound have dramatically increased the eﬃciency of non-viral gene transfer and have opened the prospect of eﬀective regional gene delivery. Finally, recent work has shown that mRNA modiﬁcation oﬀers real potential for eﬀective clinical treatment of muscle diseases.

Autologous transplantation of AC133+ stem cells in Duchenne Muscular Dystrophy: preclinical and clinical evidences Y. TORRENTE1, M. BELICCHI1, C. MARCHESI1, G. D’ANTONA2, F. PISATI1, M. GAVINA1, F. COJAMAGNAN1, R. TONLORENZI3, G. FAGIOLARI1, C. LAMPERTI1, R. GIORDANO4, L. PORRETTI4, R. LOPA4, M. SAMPAOLESI3, L. VICENTINI1, N. GRIMOLDI5, F. TIBERIO5, V. SONGA6, P. BARATTA6, A. PRELLE1, L. 7 1 FORSENIGO , G.P. COMI , P. BIONDETTI7, M. MOGGIO1, N. STOCCHETTI6, A. PRIORI1, P. REBULLA4, MG. D’ANGELO8, R. BOTTINELLI2, G. COSSU3 and N. BRESOLIN1,8 1 Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Dept. of Neurol. Sci., Stem Cell Lab., Dino Ferrari Centre, Univ. of Milan, Italy 2 Dept of Exp. Med., Univ. of Pavia, Human Physiol. Unit, Pavia, Italy 3 Stem Cell Res. Inst., San Raﬀaele Hosp., Milan, Italy 4 Centro Trasfusionale e di Immunologia dei Trapianti, Ospedale Maggiore Policlinico 5 Inst. of Neurosurgery, Univ. of Milan, Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Italy 6 Dept. of Anestesia and Critical Care Med., Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Univ. of Milan, Italy 7 Radiology Unit, Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Italy 8 IRCCS Eugenio Medea, Bosisio Parini, Italy Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to an defect on the gene encoding dystrophin.

The lack of a functional dystrophin in muscles results in the loss of DAPs and fragility of the muscle ﬁber membrane. This, in turn, leads to muscle ﬁber necrosis and progressive muscle weakness. Transplantation of normal satellite cells into dystrophic muscles represnts a potential approach for genetic disease such DMD but this approach dysplays various limitations including poor cell survival rates, limited dissemination of the injected cells and immune response. We recently reported that human circulating AC133+ progenitor cells can be induced to diﬀerentiate into skeletal muscle in vitro or when injected into the muscle tissue of scid-mdx dystrophic mice. Partial regeneration and consequent functional improvement in the muscles of DMD patients may be important in delaying the most severe symptoms of the disease. We speculate that transplantation of circulating AC133+ stem cells could represent a future treatment for primary myopathies and so investigations of the potential of these cells are absolutely necessary. We characterize the muscle homing properties of circulating AC133+ stem cells from normal and dystrophic after intra-arterial transplantation and identiﬁed the expression of adhesion molecules such E, P, and L-selectin on the circulating AC133+ stem cells that may are involved in the muscle homing. These data were obtained in dystrophic animal models (scid/mdx mice and Golden retriever muscular dystrophy (GRMD) dog). Successful completion of this part of project would produce the needed information for an optimisation of the intra-arterial injections of AC133+ stem cells in DMD patient. So far, we have treated 8 patients by intramuscular transplantation of autologous AC133+ stem cells and observed no side eﬀects in all patients. By this approach, we hope to limit the delay between results obtained in the dystrophic animal models and the clinical application of a controlled phase II that could incorporate new results from ongoing experimental work.

Systemic delivery of AAV vector expressing chimeric antisense-U1 snRNA partly restores dystrophin expression and ameliorates ﬁbres function in skeletal muscles of mdx mice G. D’ANTONA1, M. DENTI2, A. ROSA2, O. PANSARASA1, F. DE ANGELIS2, V. PARENTE1, O. STHANDIER2, A. AURICCHIO3, R. BOTTINELLI1 and I. BOZZONI2 1 Dept. of Exp. Med., Univ. of Pavia, Pavia, Italy 2 Dept. of Gen. and Mol. Biol. Univ. of Rome ‘‘La Sapienza’’, Rome, Italy 3 Telethon Inst. of Gen. and Med. (TIGEM), Naples, Italy Mutations in the dystrophin gene can be corrected at the post-transcriptional level by skipping the mutated exon during the splicing reaction. This can be achieved by the expression of antisense sequences against the splice junctions of the exon to be skipped. In the mdx mice, skipping of the mutated exon 23 would restore an inframe functional mRNA. We analysed the eﬀects of systemic delivery of AAV vector expressing antisense-U1 snRNAs against exon 23 on dystrophin expression and ﬁbre function of mdx mice. A group of 4 mice (2 mo) received AAV-GFP-U1 vector through a single intravenous injection into the caudal vein. One month after injection CK serum levels appeared to be intermediate between control C57/ Bl6 and untreated mdx. WB and immunostaining analysis indicated a characteristic distribution of GFP-dystrophin expression that prevailed in the gluteus, gastrocnemius and vastus. A diﬀuse GFP-dystrophin expression was detected in other muscles including the diaphragm. The analysis of a and b sarcoglycans in the same muscles enlighted a partial reconstitution of the dystrophin-sarcoglycan complex. The mechanical properties of the muscles were analysed by

88 measuring speciﬁc force (Po/CSA) of single ﬁbres from the vastus of control, untreated, mdx and GFP positive and negative bundles from treated mdx mice. Results showed a signiﬁcant lower force in mdx and GFP negative ﬁbres in comparison with GFP positive ﬁbres. The functional recovery of treated mice was conﬁrmed by treadmill exhaustion tests which revealed a higher time to exhaustion in treated mice in comparison with untreated. Our results suggest that systemic delivery of AAV/antisense-U1 induces exon skipping of the mutated dystrophin exon 23 followed by dystrophin rescue and functional recovery in mdx mice.

Transgenic rats harbouring minigenes coding N-terminal peptides of cardiac MLC-1 isoforms improve heart function H. HAASE, G. DOBBERNACK, D. PETZHOLD, W.-P. SCHLEGEL, U. GANTEN, J. BEHLKE and I. MORANO Max-Delbru¨ck-Centre for Molecular Medicine, Molecular Muscle Physiology, Berlin, Germany Actin-binding of the N-terminus of essential myosin light chains (MLC-1) slows down myosin motor function. Weakening the MLC-1-actin interaction in vitro by N-terminal MLC-1 peptides increased motor activity. We generated transgenic rats (TGR) on WKY background which overexpressed N-terminal peptides 1-15 of human atrial (TGR/hALC-1/1-15) or ventricular MLC-1 (TGR/ hVLC-1/1-15) in the heart. We calculated ca. 30 ng transgene/ 100 lg SDS-soluble protein in all TGR groups. Maximal contraction rate (mmHg/s) of Langendorﬀ hearts rose from 1444±99 in WKY to 2260±93 (P<0.001) and 2639±173 (P<0.001) in TGR/ hALC-1/1-15 and TGR/hVLC-1/1-15, respectively. Isovolumetric pressure development (mmHg/mg heart weight) rose from 22.4±1.6 in WKY to 43.1±2.1 (P<0.001) and 39.7±2.1 (P<0.001) in TGR/hALC-1/1-15 and TGR/hVLC-1/1-15, respectively. Maximal relaxation rate (mmHg/s) rose from )908±40 in WKY to )1354±38 (P<0.001) and )1450±58 (P<0.001) in TGR/hALC-1/1-15 and TGR/hVLC-1/1-15, respectively. Thus, minigenes of the N-terminus of MLC-1 isoforms may represent a tool for the treatment of the failing heart.

Cx43 gap junction communication is involved in myoblast proliferation and diﬀerentiation A. GO¨RBE1, D.L. BECKER2, L. DUX1 and T. KRENA´CS3 1 Inst. of Biochem., Fac. of Med., Univ. of Szeged, Szeged, Hungary 2 Dept. of Anat. & Develop.l Biol., UCL, London, UK 3 Bay Zoltan Found. for Appl. Res. Inst. for Biotech., Szeged, Hungary Gap junction communication (GJC) plays an important role in direct information exchange between neighboring cells. Connexins (Cx) form membrane associated GJ channels, which allows quick pathway for small molecules. GJs found in almost all tissues with the notable exception of adult skeletal muscle. The aim of this study was to investigate the possible involvement of gap junction communication in muscle development and its inﬂuence for myoblast proliferation and diﬀerentiation. In primary myoblast cultures we could detect an overexpression of Cx43 just before myoblast fusion. Overexpression and downregulation of gap junction expression resulted changes in myoblast proliferation and diﬀerentiation. Cells, transfected with wild type Cx43 showed bit smaller proliferation than the control ones and were more involved in myotube formation. Dominant negative Cx43 expression caused a signiﬁcant increase in proliferation. Manipulation of myoblast fusion also modiﬁed the Cx expression of myoblasts.

In summary, we observed the upregulation of Cx43 gap junctions expression at an early stage of skeletal muscle diﬀerentiation preceding myoblast fusion proposing that gap junctional communication is involved in early muscle diﬀerentiation. Modiﬁcation of gap junction communication resulted changes during early phase of myogenesis.

The eﬀect of a TNF-a inhibiting drug on skeletal muscle regeneration E. KOVA´CS and E. ZA´DOR Institute of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary Tumor necrosis factor a (TNF-a) is preinﬂamatory cytokine playing a key role in the formation of systemic inﬂamation and paracrine/ autocrine interactions. The level of TNF-a is increased by a magnitude after muscle injuries, but it has not been shown whether it inﬂuences muscle regeneration or not. We aimed to reveal the eﬀect of TNF-a through its inhibition by Remicade, a drug containing chimeric TNF-a antibody used in the medical practice to treat chronic inﬂamatory diseases like rheumatoid arthritis. We induced muscle regeneration by intramuscular injection of a snake venom containing notexin. Remicade was administered subcutaneously immediately after the toxin injection. The remicade treatment increased ﬁbre size and the intensity of desmin speciﬁc immunostaining of muscle compared to those of the control. Our preliminary results suggest that Remicade stimulates and TNF-a inhibits muscle regeneration.

NFATc1 regulates the establishment and maintenance of fatigue resistant ﬁbres in rat fast and slow muscles G. MUTUNGI School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, UK Although adult rat skeletal muscles are composed of a mixture of ﬁbre types, they all consist of neonatal ﬁbres at birth. Then within the ﬁrst 3 weeks of life, they change to the adult ﬁbre types. What triggers this change and the physiological mechanisms regulating it, however, remain uncertain. In this study, the expression and localization of NFATc1 in fast and slow muscles isolated from both adult and neonatal rats were investigated. In rats < 5 days old, all the ﬁbres express NFATc1 irrespective of their muscle of origin. Then, from day 7 onwards, the number of ﬁbres expressing NFATc1 progressively decreases with age in the fast muscle; but remains relatively unchanged in the slow one. Thus, by day 21, 39.8±2.6% (n=5 rats) of all the ﬁbres in the fast muscle and 98.9±3% (n=7 rats) of those in the slow one express NFATc1. A qualitatively and quantitatively similar pattern of NFATc1 expression was observed in adult rats where 41.6±3.4% (n=7 rats) of all the ﬁbres in the fast and 99±2% (n=7 rats) of those in the slow muscle express NFATc1. Further investigations revealed that all the ﬁbres expressing NFATc1 were either type 1 or type 2A/2X ﬁbres but never type 2B. NFATc1 was mostly localized in the cytoplasm in the fast and in the nucleus in the slow muscle. From these results I suggest that NFATc1 is responsible for the establishment of fatigue resistant ﬁbres in the neonate and for their maintenance in the adult.

Response to diﬀerent methods of sacriﬁce of atrial natriuretine (ANP) and sarcoplasmic Ca-ATP-ase in rat myocardium E. REMSEY-SEMMELWEIS2, J.G. KARL2.; J.R.B. WAIT2 and M.A.Q. SIDDIQUI1 Dept. of 1Cell Biology and 2Cardiac Surgery, SUNYBrooklyn, New York, USA Aim: The sacriﬁce situation is stressful associated with considerable gene expression(Exp) changes. In our study diﬀerent methods of

89 sacriﬁce and its temporal relationship were revealed to have eﬀects on the atrial (A) and ventricular (V) mRNA gene expression of ANP and Ca-ATP-ase. After earlier in vitro data we hypothetised diﬀerent response in gene expression in A and in V myocardium in vivo. Methods: Inbred male Lewis rats (350–450 gr) were sacriﬁced in three separate groups: Decapitation (Decap) (0–1 min; n=5), CO2 thank (CO2) (3–4 min; n=5), and intraperit. Pentobarb. Anesthesia (Pento) (35 mg/kg; 12–14 min; n=8). All sacriﬁces were followed by rapid heart excision and freezing in N2O. ANP and Ca-ATP-ase were analysed separately in A and V myocardium by RT-PCR. Results: Decap. represents baseline and time control (0 min). Compare Decap. to CO2 there was a signiﬁcant upregulation in ANP gene expression both in A and V (2288.8 vs. 2825.8 ; P<0.01; 507 vs. 747,2; P<0.01) and also compare Decap. to Pento (2288.8 vs. 2721; P<0.01; 507,0 vs. 1930,5, P<0.01) Between CO2 and Pento there was a highly signiﬁcant upregulation 8747.2 vs. 1930.5, P<0.01) in V ANP mRNA gene expression but not in A. In A CaATP-ase there was a signiﬁcant upregulation between Decap and Pento (1757.0 vs. 1998.0; P<0.01) and signiﬁcant downregulation in V between Decap. and Pento (2698.0 vs. 2133.3; P<0.01) and also in V between CO2 and Pento (2894.2 vs. 2133.3; P<0.01). Conclusion: Time might be a signiﬁcant factor in diﬀerent sacriﬁce methods which is reﬂected in myocardial gene expression of ANP and Ca-ATP-ase and they can have a signiﬁcant inﬂuence to the endpoint results of each experiment. V gene expression of ANP has a diﬀerent sensitivity (13 fold higher) to Pento than A. There is an inverse relationship between ANP and Ca-ATP-ase mRNA gene expression in V to Pento. The A Ca-ATP-ase gene expression is signiﬁcantly diﬀerent than V compare Pento to Decap.

The eﬀects of thyroid hormones on the diﬀerentiation of C2C12 cells do not occur through increased mRNA expression of the IGF system S. SLOB, P.W.A. CORNELISSEN, B. DRAGT, M.E. EVERTS and P.W.J VAN DEN WIJNGAARD Dept. of Pathobiology, Utrecht University, The Netherlands Triiodothyronine (T3) and thyroxin (T4) play an important role in skeletal muscle growth and diﬀerentiation. Previously, we found that T3 and T4 increased diﬀerentiation of C2C12 cells through a MyoDindependent increase in p21 expression. Autocrine expressed insulinlike growth factor-I (IGF-I) directs skeletal muscle diﬀerentiation. We therefore hypothesized that the eﬀects of thyroid hormones on the myogenic diﬀerentiation of C2C12 cells might be mediated through an increase in mRNA expression of IGF. MGF mRNA expression was determined as well. As expected expression of IGF-I and IGF-IR increased during the course of diﬀerentiation. Exposure to T3 or T4 however did not eﬀect the mRNA expression levels of IGF-I and its receptor, IGF-IR. IGF-IIR mRNA was also present in the C2C12 cells, but was unaﬀected by diﬀerentiation or thyroid hormones. MGF expression did not change after treatment with T3 or T4 either. We therefore conclude that the eﬀects of thyroid hormones on diﬀerentiation of C2C12 cells do not occur through increased mRNA expression of the IGF system.

Fibroblast Growth Factor-2 can inhibit and stimulate myogenic diﬀerentiation P.W.J. VAN DEN WIJNGAARD, P.W.A. CORNELISSEN, A.A.M. VAN OORSCHOT and M.E. EVERTS Dept. of Pathobiology, Utrecht University, The Netherlands Fibroblast growth factor-2 (FGF-2) is known to inhibit myogenic diﬀerentiation by activation of the mitogenic ERK1/2 signalling pathway. We investigated the concentration dependence of the inhib-

iting eﬀect of FGF-2 on the diﬀerentiation of C2C12 cells, since the activation of ERK1/2 occurs only at high FGF-2 concentrations. Surprisingly at concentrations below 30 pM addition of FGF-2 to diﬀerentiating C2C12 cultures stimulates the expression of myogenin after 48 h of diﬀerentiation. Addition of 6 pM FGF-2 caused a 2.3fold increase in myogenin mRNA expression as compared to control. The stimulation of myogenin expression at 48 h of diﬀerentiation is preceded by an induction of p27KIP1 protein at 24 h of diﬀerentiation. This induction is again induced by FGF-2 concentrations below 30 pM, while higher concentrations did not aﬀect p27KIP1 expression. Immunoblot analysis revealed that at 24 h of diﬀerentiation low concentrations of FGF-2 considerably inhibit ERK1/2 activation. These results suggest that the stimulation of myogenin and p27KIP1 expression is caused by inhibition of the ERK1/ 2 MAPK activation.

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