Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found t o play a role in ischemic reperfusion injury in several animal species incl uding rats and rabbits. To examine whether this is also true for other anim als, an isolated perfused mouse heart was subjected to 30 min of ischemia f ollowed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. A t the end of each experiment, hearts were processed for the evaluation of a poptosis and DNA laddering. The in situ end labeling (ISEL) technique was u sed to detect apoptotic cardiomyocyte nuclei while DNA laddering was evalua ted by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The re sults of our study revealed that apoptotic cells appear only after 60 min o f reperfusion as demonstrated by the intense fluorescence of the immunostai ned genomic DNA when observed under fluorescence microscopy. None of the is chemic hearts showed any evidence of apoptosis. These results were corrobor ated with the findings of DNA fragmentation showing increased ladders of DN A bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies sho wed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we pe rformed identical experiments using isolated hearts from GSHPx-1 knockout m ice and transgenic mice overexpressing GSHPx-1. GSHPx-1 knockout mice showe d evidence of apoptotic cell death even after 30 min of reperfusion. Signif icant number of apoptotic cells were found in the cardiomyocytes as compare d to non-transgenic control animals. To the contrary, very few apoptotic ce lls were found in the hearts of the transgenic mice overexpressing GSHPx-1. Hearts of GSHPx-1 knockout mice were more susceptible to ischemia/reperfus ion injury while transgenic mice overexpressing GSHPx-1 were less susceptib le to ischemia reperfusion injury compared to non-transgenic control animal s. The results of this study clearly demonstrate a role of GSHPx in ischemi a/reperfusion-induced apoptosis in mouse heart.