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The aim of our study was to characterize the temporal relationship of apoptosis to regional myocardial ischemia and reperfusion and we aimed to determine the effect of ischemia and reperfusion on the distribution of the pro-apoptotic cysteine protease caspase-3 (CPP 32, apopain, Yama) in an in vivo rat model. Male Sprague-Dawley rats (250-400 g) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the left external carotid artery was isolated to monitor arterial pressure and a left thoracotomy was performed. Regional myocardial ischemia was induced by occluding the left main coronary artery for 45 min. The heart was reperfused for 0, 60, 120 or 180 min. TUNEL staining of formalin-fixed, paraffin-embedded left ventricle, and DNA fragmentation analysis, showed that apoptosis occurred during 45 min of ischemia alone, but further developed during the 3-h reperfusion period. Immunohistochemical analysis of ischemic/reperfused left ventricle showed caspase-3 levels were substantially elevated and localized in the ischemic/reperfused region, and that caspase-3 co-localized to TUNEL positive myocytes. Therefore, regional myocardial ischemia serves as a stimulus for myocyte apoptosis, and this form of cell death progresses time-dependently after the onset of reperfusion. Our studies implicate caspase-3 to be involved in apoptotic cell death in ischemic/reperfused rat heart.

Using a paced Lagendorff-perfused rabbit heart paradigm, we investigated the role of protein kinase C (PKC) in the development of ventricular fibrillation (VF) in hearts subjected to hypoxia (12 min) and re-oxygenation (40 min). We studied the effect of putative activators and inhibitors of PKC on the incidence of VF. Hearts exposed to 4 beta-phorbol,12,13-dibutyrate (PDBu), isophorbol or the membrane permeant diacylglycerol analog, 1-oleoyl-2-acetyl-rac-glycerol (OAG), during the prehypoxic phase had an increased incidence of VF during the hypoxic and reoxygenation periods. The incidence of VF was 90%, 83% and 75% in hearts exposed to PDBu, isophorbol and OAG, respectively (P < 0.05 vs control). Perfusion of hearts with PDBu was associated with a significant increase in the membrane fraction of cardiac PKC activity. In the presence of the inactive phorbol ester 4 alpha-phorbol didecanoate, the incidence of VF was 17% (P > 0.05 vs control). PKC activators were profibrillatory at concentrations that did not affect cardiac function: neither left ventricular developed pressure nor coronary perfusion pressure were affected. The effect of PDBu was antagonized by staurosporine: the incidence of VF was 17% in PDBu+staurosporine treated hearts (P < 0.05 vs control). To further study the profibrillatory effect of PDBu, hearts were exposed to PDBu in the presence of the ATP-dependent potassium channel antagonist glibenclamide. The latter prevented PDBu-induced VF. The results show that under the conditions employed, PDBu-induced activation of PKC induces redistribution of PKC activity and is associated with the development of VF.