Cardioprotective effect of angiotensin-converting enzyme inhibition against hypoxia/reoxygenation injury in cultured rat cardiac myocytes

Background-Although ACE inhibitors can protect myocardium against ischemia/ reperfusion injury, the mechanisms of this effect have not yet been charact erized at the cellular level. The present study was designed to examine whe ther an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes aga inst hypoxia/reoxygenation (WR) injury. Methods and Results-Neonatal rat cardiac myocytes in primary culture were e xposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. M yocyte injury was determined by the release of creatine kinase (CK). Both c ilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during WR, where as CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by H oe 140 (a bradykinin B-2 receptor antagonist), N-G-monomethyl-L-arginine mo noacetate (L-NMMA) tan NO synthase inhibitor), and methylene blue (a solubl e guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine tan inhibitor of inducible NO synthase), or ind omethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguan idine. Conclusions-The present study demonstrates that cilazaprilat can directly p rotect myocytes against WR injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive N O synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modula tes guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R i njury.