Background: Reperfusion of ischemic myocardium causes cardiomyocyte apoptosis in concert with downregulation of Bcl-2 gene. Ischemic preconditioning (PC) mediated by cyclic episodes of short-term ischemia and reperfusion reduces apoptotic cell death. PC also triggers a signaling pathway by potentiating tyrosine kinase phosphorylation leading to the activation of p38 MAP kinase and MAPKAP kinase 2. The nuclear transcription factor, NFkappaB, plays a crucial role in this signaling process. Because NFkappaB is a target 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 presence of either dimethyl thiourea (DMTU), a hydroxyl radical scavenger, or SN50 peptide, a NFkappaB blocker. Hearts were then subjected to PC by 4 repeated episodes of 5-minute ischemia, each followed by 10 minutes reperfusion. All hearts were then made globally ischemic at normothermia for 30 minutes followed by 2 hours of normothermic reperfusion. Creatine kinase release and malonaldehyde formation were determined in the coronary effluent collected during reperfusion. At the end of each experiment, hearts were processed for infarct size determination and analyses of apoptosis, DNA fragmentation, NFkappaB, and Bcl-2. Myocardial infarction was reduced by PC. DMTU and SN50 abolished this cardioprotective effect of PC. PC resulted upregulation of Bcl-2 gene which was partially prevented by DMTU and SN50. Both ischemia/reperfusion and PC caused nuclear translocation and activation of NFkappaB, which was blocked by both DMTU and SN50. PC reduced cardiomyocyte 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 the extent of cardiomyocyte apoptosis and DMTU and SN50 reversed it only minimally.
Conclusions: The results demonstrate that reactive oxygen species play a crucial role in signal transduction mediated by PC. This signaling process appears to involve NFkappaB. NFkappaB becomes translocated and activated by both ischemia/reperfusion, which induces apoptosis and PC which reduces apoptosis. However, the amount of NFkappaB binding activity is significantly higher in the PC hearts compared with ischemic reperfused hearts. The upregulation of the antioxidant gene, Bcl-2, is inversely correlated with the reduction of cardiomyocyte apoptosis associated with PC.