Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity

OBJECTIVE: Ischemia/reperfusion in the heart causes myocardial apoptosis and increase nitric oxide (NO) production. We have reported that myocardial apoptosis is related to activation of cell cycle regulatory proteins. However, the role of nitric oxide (NO) in ischemia/reperfusion-induced apoptosis is still unclear. This study was designated to elucidate novel apoptosis mechanisms induced by ischemia/reperfusion, especially the interaction between NO and cell cycle regulators. METHODS AND RESULTS: Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were subjected to 1-h ischemia and then to reperfusion. The rate of cardiomyocyte apoptosis increased significantly after 24 h of reperfusion as evaluated by TUNEL analysis. NO increased 1.8-fold after 15 min of reperfusion in cardiomyocytes. After 36 h of reperfusion, the apoptosis rate was greatly increased by the NO synthetase inhibitor, Nitro-L-arginine methyl ester (L-NAME), and decreased by the NO donor of S-nitroso-N-acetylpenicillamine (SNAP). Immunoblot analysis showed that the protein levels of cyclin A accumulated in a time-dependent manner in response to ischemia/reperfusion, and L-NAME inhibited this response. Ischemia/reperfusion also increased the activity of cyclin A-associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus. To clarify the involvement of p21(cip1/waf1) protein, which is the suppressor of cyclin A-associated kinase, we performed immunoblot analysis and examined its kinase activity. Treatment of cardiomyocytes with L-NAME suppressed the p21(cip1/waf1) protein level and increased the cyclin A-associated kinase activity. The addition of SNAP showed inverse results. CONCLUSION: Our data indicates that NO released from cardiomyocytes under condition of ischemia/reperfusion exerts an antiapoptotic effect by modulating cyclin A-associated kinase activity via p21(cip1/waf1) accumulation.