Inhibition of endogenous nitric oxide synthase potentiates ischemia–reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway

Objective: Apoptosisof cardiomyocytes may contribute to ischemia–reperfusion injury. The role of nitric oxide (NO) in apoptosisis controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosisof cardiomyocytes during ischemiaand reperfusion and elucidated the underlying mechanisms. Methods and results: Isolated perfused rat hearts ( n =6/group) were subjected to ischemia(30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-l-arginine. Reperfusion induced cardiomyocyte apoptosisas assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosisduring reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemiaand reperfusion-induced apoptosisof cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspasesin ischemia–reperfusion. Conclusion: The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemiaand reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspasecascade, whereas the Bcl-2/Bax protein levels remained unchanged.