title: ULK1 plays a critical role in AMPK-mediated myocardial autophagy and contractile dysfunction following acute alcohol challenge.
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.
authors: Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, Wyoming 82071, USA.
authors: Machender R
authors: MR
authors: Kandadi
authors: Nan
authors: N
authors: Hu
authors: Jun
authors: J
authors: Ren
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D000071189:Autophagy-Related Protein-1 Homolog; D020013:Calcium Signaling; D002310:Cardiomyopathy, Alcoholic / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D002460:Cell Line; D004195:Disease Models, Animal; D020868:Gene Silencing; D006321:Heart / Q000503:physiopathology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D032386:Myoblasts, Cardiac / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D009200:Myocardial Contraction; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D010588:Phagosomes / Q000378:metabolism / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D051381:Rats
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; C586255:ULK1 protein, rat; C112444:Ulk1 protein, mouse; D055372:AMP-Activated Protein Kinases