PKA-mediated eNOS phosphorylation in the protection of ischemic preconditioning against no-reflow

Objective: To investigate whether ischemic preconditioning (IP) can reduce myocardial no-reflow by activating endothelial (e-) nitric oxide synthase (NOS) via the protein kinase A (PKA) pathway. Methods and Results: In a 90-min ischemia and 3-h reperfusion model, minipigs were assigned into sham, ischemia-reperfusion (IR), IR + IP, IR + IP + L-NNA (an eNOS inhibitor, 10 mg . kg(-1)), IR + IP + H-89 (a PKA inhibitor, 1.0 mu g . kg(-1) . min(-1)), IR + L-NNA, and IR + H-89 groups. IP pretreatment improved cardiac function and coronary blood flow, decreased the activities of creatine kinase by 36.6% after 90 min of ischemia and by 32.8% after 3 h of reperfusion (P < 0.05), reduced the no-reflow areas from 49.9% to 11.0% (P < 0.01), and attenuated the infarct size from 78.2% to 35.4% (P < 0.01). IP stimulated myocardial PKA activities and the expression of PKA and Ser(133) phosphorylated (p-) cAMP response element-binding protein (CREB) in the reflow and no-reflow myocardium, and enhanced the activities of constitutive NOS and the phosphorylation of eNOS at Ser(1179) and Ser(635) in the no-reflow myocardium. IP suppressed the expression of tumor necrosis factor-alpha and P-selectin, and attenuated cardiomyocytes apoptosis by regulating the expression of Bcl-2 and caspase-3 in the reflow and no-reflow myocardium. The eNOS inhibitor L-NNA completely canceled these beneficial effects of IP without any influence on PKA activity, whereas the PKA inhibitor H-89 partially blocked the IP cardioprotective effects and eNOS phosphorylation at the same time. Conclusion: IP attenuates myocardial no-reflow and infarction after ischemia and reperfusion by activating the phosphorylation of eNOS at Ser(1179) and Ser(635) in a partly PICA-dependent manner. (C) 2012 Elsevier Inc. All rights reserved.