Oxidative stress developed during open heart surgery induces apoptosis: Reduction of apoptotic cell death by ebselen, a glutathione peroxidase mimic

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this also is true for other animals, a surgically relevant model was established using an isolated in situ swine heart. Hearts were subjected to 15 min of normothermic regional ischemia by left anterior descending artery (LAD) occlusion followed by 30 min of normothermic cardioplegic arrest and 3 h of reperfusion. Oxygen free radicals have been shown to be the inducers of apoptosis and because reperfusion of ischemic myocardium is associated with the generation of free radicals, an additional group of hearts was preperfused with three different doses (5, 10, and 25 nM) ebselen, a glutathione peroxidase mimic, for 15 min before 15 min of LAD occlusion. Hearts were then subjected to 30 min of normothermic cardioplegic arrest followed by 3 h of reperfusion at normothermia. Control experiments were performed by perfusing the hearts for 4 h at normothermia. Two other groups of hearts were subjected to either 30 or 60 min of LAD occlusion followed by 30 min of cardioplegic arrest without subjecting them to reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end-labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The apoptotic cells appeared only after 90 min of reperfusion, as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts. The presence of apoptotic cells and DNA fragmentation in the myocardium was abolished by preperfusing the hearts in the presence of 10 nM ebselen, which also moderated the oxidative stress developed in the heart. Apoptotic cells and DNA ladders were completely absent in the hearts subjected to either 30 or 60 min of LAD occlusion. The results demonstrate that reperfusion of the ischemic heart induces apoptosis, which can be reduced with ebselen by reducing the oxidative stress associated with ischemia/reperfusion.