Ischemic preconditioning reduces apoptosis by upregulating anti-death geneBcl-2

Background - Reperfusion of ischemic myocardium causes cardiomyocyte apopto sis in concert with downregulation of Bcl-2 gene. Ischemic preconditioning (PC) mediated by cyclic episodes of short-term ischemia and reperfusion red uces apoptotic cell death. PC also triggers a signaling pathway by potentia ting tyrosine kinase phosphorylation leading to the activation of p38 MAP k inase and MAPKAP kinase 2. The nuclear transcription factor, NF kappa B, pl ays a crucial role in this signaling process. Because NF kappa B is a targe t of oxygen free radicals and Bcl-2 is an antioxidant gene, we hypothesized that reactive oxygen species might play a role in the signaling process. Methods and Results - Isolated rat hearts were perfused in the absence or p resence of either dimethyl thiourea (DMTU), a hydroxyl radical scavenger, o r SN50 peptide, a NF kappa B blocker. Hearts were then subjected to PC by 4 repeated episodes of 5-minute ischemia, each followed by 10 minutes reperf usion. All hearts were then made globally ischemic at normothermia for 30 m inutes followed by 2 hours of normothermic reperfusion. Creatine kinase rel ease and malonaldehyde formation were determined in the coronary effluent c ollected during reperfusion. At the end of each experiment, hearts were pro cessed for infarct size determination and analyses of apoptosis, DNA fragme ntation, NF kappa B, and Bcl-2. Myocardial infarction was reduced by PC. DM TU and SN50 abolished this cardioprotective effect of PC. PC resulted upreg ulation of Bcl-2 gene which was partially prevented by DMTU and SN50. Both ischemia/reperfusion and PC caused nuclear translocation and activation of NF kappa B, which was blocked by both DMTU and SN50. PC reduced cardiomyocy te apoptosis which was partially inhibited by DMTU and SN50. A substantial number of apoptotic cardiomyocytes were identified in the hearts subjected to 30 minutes ischemia and 2-hour reperfusion. PC significantly inhibited t he extent of cardiomyocyte apoptosis and DMTU and SN50 reversed it only min imally. Conclusions - The results demonstrate that reactive oxygen species play a c rucial role in signal transduction mediated by PC. This signaling process a ppears to involve NF kappa B. NF kappa B becomes translocated and activated by both ischemia/reperfusion, which induces apoptosis and PC which reduces apoptosis. However, the amount of NF kappa B binding activity is significa ntly higher in the PC hearts compared with ischemic reperfused hearts. The upregulation of the antioxidant gene, Bcl-2, is inversely correlated with t he reduction of cardiomyocyte apoptosis associated with PC.