Akt‐ and PKA‐mediated endothelial nitric oxide synthase activation triggers early ischemic preconditioning in isolated rat hearts

Ischemic preconditioning (IPC) is a powerful cardioprotective phenomenon, however, the role of endothelial nitric oxide synthase (eNOS) and mechanisms of its activation are unclear in early IPC. While eNOS is an important downstream target of Akt and/or protein kinase A (PKA), questions remain regarding the role of Akt or PKA in eNOS activation and cardioprotection. Hearts were subjected to 30 min global ischemia and 120 min reperfusion (I/R) with or without IPC cycles (3 cycles of 5 min I and 5 min R). To address signaling proteins in the trigger phase and early R, myocardial tissues were collected following IPC cycles or inhibitors (PI3K inhibitor, LY294002/LY; PKA inhibitor, H89) plus IPC cycles or IPC cycles plus 30 min global I and 15 min R and Western blot analysis was performed. IPC significantly improved postischemic LVDP and reduced myocardial infarction compared to I/R group, and IPC effects were abrogated by L-NAME. I/R caused significant loss of total myocardial eNOS, and this loss was prevented by IPC. Phosphorylation of eNOS, Akt, and PKA was significantly increased following IPC. Pretreatment with LY or H89 either abolished or decreased phosphorylation of Akt or PKA, respectively. Importantly, LY or H89 abolished IPC-induced increase in eNOS activation and abrogated the cardioprotective effect of IPC. Thus, Akt- and PKA-mediated eNOS activation is critical for early IPC-induced cardioprotection.