The signaling pathways that control the hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis have not been fully defined. In this study, we investigated whether extracellular signal-regulated kinase1/2 (ERK1/2) plays a role in NO's anti-apoptotic effect against H/R injury. Primary cultures of adult rat ventricular myocytes (ARVMs) were exposed to 3 h of hypoxia followed by 30, 60, 90 and 120 mm of reoxygenation in presence of a vehicle, NO donor (GSNO, 50 mu mol/L) and inhibitors of ERK1/2 (PD98059, 10 mu mol/L). GSNO protected the cardiomyocyte from reoxygenation injury, as evidenced by decreased apoptosis, and this protective effect was inhibited by co-treatment with PD98059 during reoxygenation. Consistent with this, when administered with adenoviral vector encoding dominant negative ERK (Ad-dnERK), GSNO's effect was also blocked. Western blotting revealed that GSNO increased the ERK phosphorylation during reoxygenation. Furthermore, H/R-induced activation of caspase-3 and -9 were attenuated by GSNO. Interestingly, X-linked inhibitor of apoptosis protein (XIAP) protein levels decreased in myocytes subjected to reoxygenation, and ERK phosphorylation can improve XIAP expression, which involved inhibiting caspase-3, -7 and -9 activities. Overexpression experiment with adenoviral vector containing constitutively active ERK (Ad-caERK) alone acquired protection against apoptosis triggered by H/R injury and positively regulated XIAP expression compared with control adenovirus (Ad-LacZ). Our data demonstrated that, GSNO's antiapoptotic effect against reoxygenation injury involves ERK signaling pathway. The activation of ERR increased XIAP expression and led to decreased caspase activation. (C) 2012 Elsevier Inc. All rights reserved.