Mol Cell Biochem (2013) 379:43–49 DOI 10.1007/s11010-013-1625-7
Pharmacological postconditioning by bolus injection of phosphodiesterase-5 inhibitors vardenafil and sildenafil Bernd Ebner • Annette Ebner • Anna Reetz • Stefanie Bo¨hme • Antje Schauer • Ruth H. Strasser Christof Weinbrenner
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Received: 6 October 2012 / Accepted: 21 March 2013 / Published online: 27 March 2013 Ó Springer Science+Business Media New York 2013
Abstract Postconditioning enables cardioprotection against ischemia/reperfusion injury either by application of short, repetitive ischemic periods or by pharmacological intervention prior to reperfusion. Pharmacological postconditioning has been described for phosphodiesterase-5 inhibitors when the substances were applied as a permanent infusion. For clinical purposes, application of a bolus is more convenient. In a rat heart in situ model of ischemia reperfusion vardenafil or sildenafil were applied as a bolus prior to reperfusion. Cardioprotective effects were found over a broad dosage range. In accordance with current hypotheses on pharmacological postconditioning signaling, the protective effect was mediated by extracellular signal-regulated kinase and protein kinase C pathway. Interestingly, the extent of protection was independent of the concentration applied for both substances. Full protection comparable to ischemic postconditioning was reached with half-maximal human equivalence dose. In contrast, mean arterial pressure dropped upon bolus application in a dose-dependent manner. Taken together, the current study extends previous findings obtained in a permanent infusion model to bolus application. This is an important step toward clinical application of pharmacological postconditioning with sildenafil and vardenafil, especially because the beneficial effects were proven for concentrations with reduced
B. Ebner (&) A. Ebner A. Reetz S. Bo¨hme A. Schauer R. H. Strasser C. Weinbrenner Department of Medicine/Cardiology, Heart Center Dresden, University Hospital, University of Technology Dresden, Fetscherstr. 76, 01307 Dresden, Germany e-mail:
[email protected] Present Address: C. Weinbrenner Medizinische Klinik I, Klinikum Hanau, Leimenstr. 20, 63450 Hanau, Germany
hemodynamic side effects compared to the dosage applied for erectile dysfunction treatment. Keywords Postconditioning PDE5 Bolus application Ischemia Myocardial infarction Cardioprotection Abbreviations ANOVA Analysis of variance Akt Protein kinase B BW Body weight cGMP Cyclic guanosine monophosphate ELISA Enzyme-linked immunosorbent assay ERK Extracellular-regulated kinase GSK-3b Glycogen synthase kinase 3b HPLC High performance liquid chromatography KATP ATP-dependent potassium channels MAP Mean arterial pressure mPTP Mitochondrial permeability transition pore PDE5 Phosphodiesterase 5 PKC Protein kinase C PKG Protein kinase G RISK Reperfusion injury salvage kinase RIVA Ramus interventricularis anterior SEM Standard error of mean TBAS Tetrabutylammoniumhydrogensulfate
Introduction The intracellular homeostasis of cAMP and cGMP is regulated by phosphodiesterases (PDEs) [1]. Phosphodiesterase-5 (PDE5) regulates adrenergic stimulation by an eNOSdependent mechanism. Therefore, its inhibitors sildenafil and vardenafil are clinically approved for treatment of erectile
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dysfunction. Both substances have been extensively studied and their effects on adrenergic signaling and nitric oxide production have been proven in other organs, e.g., the healthy heart [2–7]. In the diseased heart, inhibition of PDE5 signaling can be protective against anthracycline toxicity, myocardial hypertrophy, dysfunction secondary to pressure overload, and ischemia–reperfusion [8, 9]. Modified ischemia–reperfusion or ‘‘postconditioning’’ is the rescue of heart tissue by protective interventions applied after the incident of a myocardial infarction. Protective interventions can either be repetitive short periods of ischemia (ischemic postconditioning) or the application of a pharmacological stimulus (pharmacologic postconditioning) [10]. As of date various signaling pathways have been identified to play a role in postconditioning processes. Due to the numerous signal transduction pathways potentially involved in postconditioning, the mechanisms need to be clarified for each pharmacological postconditioning agent. In addition, the effects may differ between a permanent infusion and bolus application due to wash out effects. For clinical purposes, application of a bolus is more convenient compared to permanent infusion during the procedure of re-opening of the occluded vessels. In previous studies [11] our group could demonstrate that the calcium sensitizer and PDE III inhibitor levosimendan that rescues rat myocardial tissue by a RISK pathway when given as a bolus prior to reperfusion. Although many substances for successful application of pharmacological postconditioning have been described, bolus applications have not been tested thoroughly. For the PDE5inhibitors sildenafil and vardenafil pharmacological postconditioning has been described for a permanent infusion [12, 13]. This study was designed to test, whether these findings can be extended to bolus application. A special focus is set on the doses applied for estimation of hemodynamic side effects. In additions, this study aimed to further clarify the underlying signal transduction mechanism of cardioprotection following bolus application of PDE5-inhibitors during ischemia.
Materials and methods All procedures were in conformance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the local Animal Care and Use Committee. Study groups and experimental protocols Male Wistar rats (Charles River) of 250–300 g body weight were used for in situ heart ischemia–reperfusion experiments. All hearts experienced 30 min of regional ischemia followed by reperfusion. Animals were randomly assigned to one of the following study groups:
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Control rats received neither pharmacologic nor ischemic postconditioning (n = 22). Ischemic postconditioning as a positive control was applied for three cycles of reperfusion (30 s) followed by regional ischemia (30 s) before the onset of reperfusion (n = 23). PDE5 inhibitors were applied as a bolus 5 min prior to reperfusion. Here, chosen concentrations were adapted from human doses of equivalence: 6.5, 3.3, 0.7, and 0.007 times of human equivalence dose were applied as published earlier in animal studies by Salloum and co-workers [12, 14]. For sildenafil the respective concentrations were 1400, 700, 70, and 0.7 lg/kg body weight, for vardenafil the respective concentrations were 140, 77, 14, and 0.14 lg/kg body weight. Application of ERK1/2 inhibitor PD 98059 (0.3 mg/kg body weight) or PKC inhibitor chelerythrine (5 mg/kg body weight) as a bolus was performed. These inhibitors were tested in the absence and presence of high (140 lg/kg BW vardenafil) and low (70 lg/kg BW sildenafil) concentrations of PDE5 inhibitors. In-situ myocardial infarction Myocardial infarction was induced in situ as described previously [11, 15]. Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). They were tracheotomized and ventilated with room air supplemented with oxygen at 65 Hz (rodent ventilator, TSE, Bad Homburg, Germany). Atelectasis was prevented by maintaining a positive endexpiratory pressure of 5–10 cm H2O. Arterial pH, pO2, and pCO2 were monitored by a blood gas analyzer (AVL 990) and maintained within physiological range to exclude postconditioning effects by acidosis. Body temperature was kept at 36.8 to 37.2 °C with a heating pad. The left axillary artery was cannulated to monitor arterial blood pressure and heart rate was recorded by dedicated analysis software (ADInstruments Chart V 5.5.4) via a pressure transducer (MLT 844, ADInstruments, Spechbach, Germany). The right axillary vein was cannulated in order to infuse drugs or saline. A left thoracotomy was performed at the fourth intercostal space to expose the heart. The pericardium was removed and a 5-0 Prolene suture (Ethibond Excel, Ethicon, Germany) was passed next to the middle of R. interventricularis anterior (RIVA). The ends of the ligature were threaded through a short-propylene tube which was used as a snare to allow later reversible occlusion of the RIVA. Heart rate and blood pressure were allowed to stabilize for 10 min before induction of index ischemia. Coronary artery occlusion was verified by epicardial cyanosis and subsequent decrease in blood pressure. Regional ischemia was induced for 30 min and PDE5 inhibitors were infused as a bolus application 5 min prior to reperfusion. Reperfusion was confirmed by epicardial hyperemia.
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Determination of infarct size
Statistical analysis
Following 30 min of reperfusion a bolus of propidium iodide (10 mg/kg body weight diluted to 1 mg/mL) was given in order to determine the infarct size [16, 17]. The heart was excised after an additional reperfusion time of 15 min, quickly mounted on a Langendorff apparatus, and perfused with 0.9 % saline and 0.2 % heparin to drain the blood. The coronary artery was re-occluded and zinc/ cadmium–sulfide fluorescent particles (1–10 lm, Duke Scientific Corp.) were infused to demarcate the risk zone as the area without fluorescence. The heart was then weighed, frozen and cut into 2 mm transverse slices. The heart slices were photographed under ultraviolet light and the areas of infarct (bright red fluorescent) and the risk zones (nonfluorescent) were determined by planimetry. The infarct size was expressed as percentage of the risk zone.
Data are presented as mean values ± SEM. To assess effects ANOVA data analysis with post hoc tests (pressure measurement and infarct size) or Student’s t test was used (one-tailed, cGMP measurements). A value of p \ 0.05 was considered to indicate a statistically significant difference. Statistics were performed using SPSS for Windows (SPSS Inc., version 11.5).
Determination of cGMP Determination of cGMP in heart tissue homogenate was performed by HPLC measurement with ultraviolet detection at 255 nm following derivatisation with isatoic anhydride to 2-o-anthraniloyl-cGMP for better sensitivity and peak resolution. Derivatisation procedure was adapted from [18] and modified to fit small scale reactions. Whole hearts were homogenized in liquid nitrogen. Proteins were removed by acid precipitation (5 % perchloric acid), and potassium perchlorate was precipitated by sample neutralization with KOH. Samples or standard (100 lL) were added with 40 lL freshly prepared isatoic anhydrid solution (1.6 mg/mL in water adjusted to pH 9.6). Following incubation with continuous shaking at 37 °C for 2 h formic acid (10 lL) was added to stop the reaction. HPLC-analysis was performed on an Alliance 2695 pump module (Waters, Germany) equipped with a UV detector (W2487, Waters, Germany) on a Waters Symmetry C18 column (75 9 4.6 mm, particle size 3.5 lm) at a flow rate of 0.8 mL/min. Gradient elution (Eluent 1: 3.5 mmol/L TBAS and 5 % acetonitrile in 30.5 mmol/L KH2PO4 adjusted to pH 4.1; eluent 2: two parts eluent 1 plus three parts acetonitrile) was performed as follows: Starting conditions were 100 % eluent 1. Over 7.5 min composition was changed in a linear manner to 0 % eluent 1 (100 % eluent 2). This condition was maintained for 2.5 min. Initial conditions were re-established within 1 min and the column was equilibrated for 4 min prior to the next run. Retention times were 3.66 min for cGMP and 4.25 min for cAMP. Data were confirmed using a commercially available Immunoassay Kit (Sigma, Germany) according to manufacturer’s instructions.
Results Infarct size reduction by bolus application of sildenafil Infarct size was determined as described in the methods section in control animals without postconditioning (negative control) or with ischemic postconditioning (positive control) and bolus application of rising sildenafil doses (Fig. 1a). Application of very low concentrations of sildenafil (0.7 lg/kg BW) did not alter the infarct size significantly. In contrast, the higher concentrations of sildenafil resulted in a significant infarct size reduction versus control conditions (all p \ 0.001) comparable to that achieved by ischemic postconditioning. However, there was no linear dose–response relationship for infarct size reduction by sildenafil. This is remarkable, because the mean arterial blood pressure (MAP) was influenced by sildenafil in a dose-dependent manner. Approximately 2–3 min after sildenafil bolus application a drop in MAP was observed and this pressure drop was significantly more pronounced when higher concentrations of sildenafil were applied (Fig. 1b). For all following experiments a sildenafil concentration of 70 lg/kg BW was applied because this concentrations reduced the infarct size and had the slightest effects on the blood pressure. Infarct size reduction by bolus application of vardenafil Infarct size was determined in control animals without postconditioning, in an ischemic postconditioning group and in four further groups with bolus application of rising vardenafil doses (Fig. 2a). Ischemic postconditioning significantly reduced the infarct size from 48 ± 2 % to 32 ± 3 % (p \ 0.001). Apart from the lowest dose, application of various doses of vardenafil as a bolus resulted in a significant reduction versus control conditions (all p \ 0.001) comparable to that achieved by ischemic postconditioning. Again, there was no linear dose–response relationship for infarct size reduction by vardenafil. In contrast to this, the applied concentrations of vardenafil had a dose-dependent effect on the mean arterial blood pressure (Fig. 2b).
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Fig. 1 Effects of sildenafil bolus application on infarct size and blood pressure rat hearts were subjected in situ to 30 min regional ischemia followed by 30 min reperfusion. Bolus application of 0.7 lg/kg BW sildenafil (n = 8), 70 lg/kg BW sildenafil (n = 7), 700 lg/kg BW sildenafil (n = 8), and 1400 lg/kg BW sildenafil (n = 7) were compared to non-postconditioned controls (n = 10) and ischemic postconditioning (iPC; n = 14). Asterisk indicates significant (p \ 0.05) changes compared to control group, hash indicates significant (p \ 0.05) differences between the various concentrations of sildenafil. (a) Bolus application of sildenafil reduces infarct size in the same magnitude as ischemic postconditioning. (b) Mean arterial blood pressure is reduced dose-dependently by sildenafil
ERK in PDE5-inhibitor-mediated cardioprotection PDE5 inhibitor sildenafil is known to enhance phosphorylation of ERK1/2 in isolated perfused mouse hearts, and this mechanism is crucial in preconditioning with sildenafil [19]. As demonstrated before, inhibition of ERK1/2 in sildenafil-treated animals showed larger infarct size compared to sildenafil treatment alone (Fig. 3). Application of ERK1/2 inhibitor PD 98059 alone did not significantly influence the infarct size. To test if ERK1/2 is also involved in postconditioning with the PDE5 inhibitor vardenafil (Fig. 3) was tested in combination with PD 98059. The combination of both substances resulted in an infarct
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Fig. 2 Effects of vardenafil bolus application on the infarct size and blood pressure rat hearts were subjected in situ to 30 min regional ischemia followed by 30 min reperfusion. Asterisk indicates significant (p \ 0.05) changes compared to control group, hash indicates significant (p \ 0.05) differences between the various concentrations of vardenafil. (a) Bolus application of 14 lg/kg BW vardenafil (n = 10), 77 lg/kg BW vardenafil (n = 6) and 140 lg/kg BW vardenafil (n = 10) reduces infarct size compared to non-postconditioned controls (n = 12) in the same magnitude as ischemic postconditioning (iPC; n = 9). (b) Mean arterial blood pressure is reduced dose-dependently by vardenafil (n = 10 for 14 and 140 lg/kg BW; n = 6 for 77 lg/kg BW vardenafil treatment)
size comparable to that of control hearts, which is significantly larger compared to modified reperfusion with vardenafil alone (p = 0.002). cGMP in PDE5-inhibitor-dependent cardioprotection Sildenafil and vardenafil are PDE5 inhibitors and should therefore elevate the bioavailability of cGMP. Measurements of cGMP tissue content by HPLC in total heart homogenates revealed a significant rise in cGMP tissue levels in vardenafil-treated animals (Fig. 4). Sildenafil treatment failed to elevate tissue total cGMP levels. ELISA measurements confirmed these results (data not shown).
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Fig. 3 Influence of ERK1/2 inhibition on the infarct size reduction rat hearts were subjected in situ to 30 min regional ischemia followed by 30 min reperfusion. ERK1/2 was inhibited with 0.3 mg/kg BW PD 98059. For the vardenafil groups 140 lg/kg BW vardenafil was used and for the sildenafil groups 70 lg/kg BW sildenafil was used. PD 98059 alone did not influence the infarct size. ERK Inhibition blunts the protective effect both PDE5 inhibitors. Controls (n = 12); PD 98059 (PD n = 6); vardenafil (Vard n = 10); vardenafil ? PD 98059 (V ? PD; n = 9); sildenafil (Sild n = 7); sildenafil ? PD 98059 (S ? PD; n = 7)
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Fig. 5 Influence of PKC inhibition on the infarct size reduction rat hearts were subjected in situ to 30 min regional ischemia followed by 30 min reperfusion. PKC was inhibited with 5 mg/kg BW chelerythrine. For the vardenafil groups 140 lg/kg BW vardenafil was used and for the sildenafil groups 70 lg/kg BW sildenafil was used. PKC inhibition alone did not influence the infarct size. Co-infusion of chelerythrine blunts the protective effect both PDE5 inhibitors. Controls (n = 10); chelerythrine (Chel: n = 7); vardenafil (Vard n = 10); vardenafil ? chelerythrine (V ? C; n = 7); sildenafil (Sild n = 7); sildenafil ? chelerythrine (S ? C; n = 9)
kinase C (PKC) is involved in the signal transduction of PDE5-inhibitor dependent infarct size reduction experiments under co-administration of chelerythrine bolus application and sildenafil/vardenafil bolus application were performed. Chelerythrine treatment alone had no effect on infarct size (Fig. 5). Combined treatment of chelerythrine and sildenafil or chelerythrine and vardenafil abolished the protective effect of sildenafil or vardenafil alone.
Discussion
Fig. 4 cGMP tissue content Bolus treatment with vardenafil (0.14 mg/kg BW, n = 4) but not with sildenafil (0.07 mg/kg BW, n = 4) enhances total cGMP tissue content compared to untreated control hearts (n = 4). Asterisk indicates significant (p \ 0.05) changes compared to control group
Protein kinase C in PDE5-inhibitor-dependent cardioprotection Pharmacological postconditioning with 70 lg/kg BW sildenafil was effective to reduce the infarct size, but the cGMP tissue content was not elevated by application of this concentration of sildenafil. Therefore, the involvement of alternative pathways was evaluated. To test if protein
Rise in myocardial cGMP content has revealed protection from myocardial cell death which has been investigated in vitro [20] and in vivo [21]. For sildenafil and vardenafil cardioprotective effects have been shown in animal models when given as a permanent infusion during reperfusion [12, 13]. In addition, in vitro experiments with sildenafil have demonstrated, that protective effects on cardiomyocytes are independent of the hemodynamic effects [22]. This study extends these findings to bolus application, which is more convenient for clinical application. In the present in situ bolus application model both substances reduced the infarct size in a dose-independent manner. However, regarding mean arterial pressure a dose dependency was observed, which insures application of the correct concentrations. This is in contrast to previous in vitro studies, where cardioprotective effects for vardenafil were only demonstrated within a narrow concentration range [13]. In
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this study, chosen concentrations were adapted from human doses of equivalence: 6.5, 3.3, 0.7, and 0.07 times of human equivalence dose of sildenafil or vardenafil were applied. The three higher concentrations showed no dose dependency of the beneficial effects of PDE5-inhibitors on infarct size when given as a bolus. Therefore, it is possible to use concentrations with minimal effects on blood pressure. This is a great advantage for clinical application. Vardenafil and sildenafil were chosen as two examples for PDE5-inhibitors. In the current study only vardenafil, but not sildenafil did enhance the total tissue cGMP content. This is unexpected, but we used twofold higher concentrations of vardenafil than sildenafil for the cGMP measurements. This may explain the lack of effect given the 32-fold higher IC50 values for guanylate cyclase of sildenafil versus vardenafil [23]. However, both substances target intracellular PDE5 and the cell permeability and kinetics of both substances are similar [24]. Interestingly, the concentration of sildenafil we used for cGMP measurements was effective in protecting the hearts from ischemia–reperfusion injury. For sildenafil a direct PDE5independent protective effect on mitochondria has been described [25]. Recent evidence suggests that cGMP is highly compartmentalized within the cell [26, 27]. Therefore, elevation of cGMP within a certain cellular compartment, e.g., in the perimitochondrial region might be sufficient for the beneficial effects of sildenafil and no elevation of total cGMP tissue content is necessary. In addition, the signal transduction of the cardioprotective effects was analyzed. One prominent pathway is RISK (reperfusion injury salvage kinase) signaling via protein kinase B (Akt) or ERK [28]. In our system, inhibition of ERK1/2 completely abolished the beneficial effects of PDE5 inhibitors. In general, this pathway leads to an improved mitochondrial protection by preventing permanent opening of the mitochondrial permeability transition pore (mPTP) in the mitochondrial membrane [29, 30]. Previous studies have demonstrated involvement of cGMP/guanylate cyclase, PKG, Akt/GSK3b, mitochondrial KATP-channel and a mPTP [12, 13, 20, 31]. We extended these findings by the demonstration that specific PKC inhibitors are able to blunt the protective effect of PDE5inhibitors as pharmacological postconditioning agents completely. It has been shown that activation of PKCe1 can activate mitochondrial ATP-dependent potassium channels (KATP). KATP opening results in net potassium influx with subsequent activation of PKCe2. PKCe2 is a different PKC subset which can directly inhibit mPTP [30]. Taken together, this study provides an important step toward clinical application of pharmacological postconditioning with sildenafil and vardenafil. Both substances are effective when applied as a bolus prior to reperfusion. This
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may enable treatment during myocardial infarction with minimal hemodynamic side effects. Acknowledgments We thank Janet Lehmann for technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (We 1955/2-2). Part of this study was sponsored the Roland Ernst Stiftung fu¨r Gesundheitswesen.
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