ULK1 Plays a Critical Role in AMPK-Mediated Myocardial Autophagy and Contractile Dysfunction following Acute Alcohol Challenge

Machender   R.   Kandadi; Nan      Hu; Jun      Ren

Abstract

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.

Keywords: Alcohol, cardiac function, autophagy, AMPK, ULK1.

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