ISCHEMIC PRECONDITIONING ATTENUATES APOPTOTIC CELL-DEATH ASSOCIATED WITH ISCHEMIA REPERFUSION/

Apoptosis or programmed cell death is a genetically controlled respons e for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free ra dicals/oxidative stress and Ca2+ which are also implicated in the path ogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isol ated perfused rat hearts were subjected to 15, 30 or 60 min of ischemi a as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of r eperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence det ection of digoxigenin-labeled genomic DNA using APOPTAG(R) in situ apo ptosis detection kit. DNA laddering was evaluated by subjecting the DN A obtained from the hearts to 1.8% agarose gel electrophoresis and pho tographed under UV illumination. The results of our study revealed apo ptotic cells only in the 90 and 120 min reperfused hearts as demonstra ted by the intense fluorescence of the immunostained digoxigenin-label ed genomic DNA when observed under fluorescence microscopy. None of th e ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed incr eased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). T he presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated sh ort-term ischemia and reperfusion which also reduced the ischemic repe rfusion injury as evidenced by better recovery of left ventricular per formance in the preconditioned myocardium. The results of this study i ndicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by m yocardial adaptation to ischemia.