Apoptotic cell death during ischemia/reperfusion and its attenuation by antioxidant therapy

Abstract

Apoptosis is a form of programmed cell death that can be induced in susceptible cells by a wide variety of normal physiological stimuli as well as by deleterious environmental conditions and cytotoxic agents. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca(2+) which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine how free radicals are directly involved in apoptosis, rats were divided into three groups. The first group of rat hearts were perfused for 15 min with KHB buffer, the second group of rat hearts were perfused with superoxide dismutase plus catalase, and the hearts were subjected to 30 min of ischemia and 120 min of reperfusion. The third group of rat hearts, served as control which were subjected to 165 min of perfusion with KHB buffer (where n=6 rats in each group). At the end of each experiment, hearts were saved (at -70 degrees C) and analysed for apoptosis, DNA laddening and MDA production. During the reperfusion continuous cardiac pressure measurements were recorded in real time with a data acquisition and analysis system (CORDAT II, Triton Technologies). Direct measurements of heart rate, developed pressure and the first derivative of the developed pressure were recorded before the intervention and during the reperfusion. Coronary flow was measured by timed collection of coronary effluent. The results of our study revealed apoptotic cells after 120 min of reperfusion as demonstrated by the intense fluorescence of the immunostained digoxigeninlabeled genomic DNA when observed under fluroscence microscopy. Evaluation of DNA fragmentation showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA, about 180 bp. The presence of apoptotic cells and DNA fragmentation in the myocardium were abolished by preperfusing the hearts in the presence of SOD and catalase, which also reduced the oxidative stress as evidenced by the MDA production. In concert, myocardial function was significantly better when compared with the ischemic control. Taken together, these results clearly demonstrate that oxidative stress developed in the ischemic reperfused myocardium induces apoptosis and free radical scavengers can play a crucial role in apoptotic cell death associated with ischemia/reperfusion.