This study was designed to evaluate the role of ULK1 in AMPK-mediated myocardial autophagy and contractile dysfunction
following acute alcohol challenge. Wild-type and AMPK knockout mice were challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial
function was evaluated using echocardiography and edge-detection. Western blot analysis was employed to evaluate the levels of
AMPK, Raptor, mTOR, the AMPK downstream signal ULK1 and autophagy markers Beclin-1 and LC3-II. siRNA was used to knockdown
ULK1 in H9C2 myoblasts. GFP-LC3 puncta was used to evaluate autophagosome formation. Alcohol challenge compromised cardiac
function as evidenced by decreased fractional shortening, peak shortening and intracellular Ca2+ rise, prolonged relengthening and
intracellular Ca2+ decay in WT mice, the effects of which were mitigated by AMPK knockout. Ethanol exposure facilitated myocardial
autophagy as evidenced by enhanced LC3-II level, as well as phosphorylation of AMPK, Raptor, and dephosphorylation of mTOR and
ULKI in WT hearts, which were alleviated by AMPK knockout. Pharmacological inhibition of AMPK using compound C attenuated
ethanol-induced autophagosome formation, AMPK phosphorylation, ULK1 dephosphorylation and apoptosis. Ethanol exposure-induced
cardiomyocyte contractile defects and autophagosome accumulation were reversed by the autophagy inhibitor 3-MA. Similarly, knockdown
of ULK1 using siRNA in H9C2 cells ablated ethanol-induced autophagosome accumulation, LC3-II expression and cell death.
Lysosomal inhibition using bafilomycin, E64-D and pepstatin A potentiated ethanol-induced increase in autophagosome formation.
Taken together, our results suggest that ULK1 may play a critical role in AMPK-mediated myocardial autophagy, apoptosis and contractile
dysfunction following acute alcohol challenge.