Abstract 3620: A769662 Protects the Heart Against Ischemia-Reperfusion Injury via the AMPK-eNOS Pathway
Intrinsic AMP-activated protein kinase (AMPK) activation has been shown to be a protective mechanism during myocardial ischemia-reperfusion (I/R). Pre-treatment with the small molecule AMPK activator, A769662, protects the murine heart against I/R injury. However, the mechanism of A769662 action is not well understood. AMPK activates endothelial nitric oxide synthase (eNOS), which has a cardioprotective effect. Here, we investigated whether the eNOS pathway is a possible mechanism for A769662-induced cardioprotection. A769662 treatment augmented AMPK activation (1.7 fold vs. vehicle control, p<0.05) and increased eNOS phosphorylation at the AMPK target site Ser1177 (1.6 fold vs. vehicle control, p=0.002) during ischemia. Ser1177 is also an Akt phosphorylation site, but A769662 did not change Akt Ser473 phosphorylation during baseline or ischemic periods. In order to determine whether A769662-induced cardioprotection is mediated by eNOS, we investigated whether the genetic deficiency in eNOS abrogates the A769662 protective effect. eNOS knockout (Nos3−/−, C57Bl/6 mice) hearts were perfused in Langendorff mode with or without A769662 (100μM) for 30 minutes (n=5–7/group) prior to no-flow ischemia for 25 minutes and subsequent reperfusion for 30 minutes. In WT C57Bl/6 hearts, A769662 pre-treatment led to notably better recovery of LV contractile function after ischemia compared to vehicle-treated hearts (55% vs. 29% of baseline rate-pressure product (RPP); p=0.03), accompanied by a 56% decrease in myocardial necrosis. In contrast, AMPK kinase dead hearts, which over-express mutated (K45R) AMPK catalytic subunit in myocytes, failed to be protected by this compound, indicating that this effect is mediated specifically via myocardial AMPK activation. Furthermore, A769662 had no significant effect to improve the recovery of cardiac function after ischemia in the eNOS knockout hearts (24+/−7% vs. 19+/−3% of baseline RPP). A769662 caused no significant reduction in myocardial necrosis in TTC-stained sections and by measurement of creatine kinase release after ischemia in the eNOS knockout hearts. Thus, the protective effect of A769662 against I/R injury in the heart is mediated in part via AMPK activation of the eNOS pathway.