COLOCALIZATION OF THE CYSTEINE PROTEASE CASPASE-3 WITH APOPTOTIC MYOCYTES AFTER IN-VIVO MYOCARDIAL-ISCHEMIA AND REPERFUSION IN THE RAT

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 distributi on of the pro-apoptotic cysteine protease caspase-3 (CPP32, apopain, Y ama) in an in vivo rat model. Male Sprague-Dawley rats (250-400 g) wer e anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the left ex ternal carotid artery was isolated to monitor arterial pressure and a left thoracotomy was performed. Regional myocardial ischemia was induc ed by occluding the left main coronary artery for 45 min. The heart wa s reperfused for 0, 60, 120 or 180 min. TUNEL staining of formalin-fix ed, paraffin-embedded left ventricle, and DNA fragmentation analysis, showed that apoptosis occurred during 45 min of ischemia alone, but fu rther developed during the 3-h reperfusion period. Immunohistochemical analysis of ischemic/reperfused left ventricle showed caspase-3 level s were substantially elevated and localized in the ischemic/reperfused region, and that caspase-3 co-localized to TUNEL positive myocytes. T herefore, regional myocardial ischemia serves as a stimulus for myocyt e apoptosis, and this form of cell death progresses time-dependently a fter the onset of reperfusion. Our studies implicate caspase-3 to be i nvolved in apoptotic cell death in ischemic/reperfused rat heart. (C) 1998 Academic Press Limited.