Regulation Mechanism Of Acetyl-FoxO1-mediated Autophagy Pathway In Resistance Training Alleviates Hypoxia-induced Muscle Atrophy

Objective:Skeletal muscle atrophy caused by hypoxia,not only affects the effects of athletes'altitude training,but also reduces the quality of production and life of the people living on the plains for generations to the plateau.The purpose of this study was to investigate the effects of resistance training on hypoxia-induced muscle atrophy and the role of acetyl-forkhead box protein O1(Ac-FoxO1)-mediated autophagy pathway in this process.Methods:Forty male SD rats were randomly divided into normoxic control group(C),normoxic resistance training group(R),hypoxic control group(H),and hypoxic resistance training group(HR).The R and HR groups were trained with incremental weight-bearing ladder training every other day;the H and HR groups were lived in a hypoxic room with an oxygen concentration of 12.4%(24 h/d).After4 weeks,the lean body mass was tested by dual-energy X-ray(DEXA);and the wet weights of the soleus(SOL),gastrocnemius(GAS),extensor digitorum longus(EDL),and musculus biceps brachii(MBB)were weighed;muscle fiber morphology were observed HE staining;Laminin was stained by immunofluorescent(IF),and muscle fiber cross-sectional area(FCSA)were calculated;the expression of[Myosin,muscle-specific ring finger protein 1(Mu RF1)and muscle atrophy F-box protein(Atrogin1)and other atrophy-related proteins,as well as Ras-related protein 7(Rab7),autophagy related gene 7(Atg7),sequestosome 1(SQSTM1/p62),Coiled-coil myosin-like BCL2-interacting protein 1(Beclin1),microtubule-associated protein light chain(LC3,LC3?/?)and autophagy-related proteins were tested by Western blot(WB);the functions and the interaction with FoxO1 of differentially expressed autophagy genes were tested by PCR array;the expression and activity of the deacetylase sirtuin-2(SIRT2),as well as the protein expression and localization of FoxO1 and Ac-FoxO1 were tested by WB and IF.Furthermore,the expression of FoxO1,Ac-FoxO1,SIRT2,Rab7,and Atg7 in the nucleus and cytoplasm of L6myotubes in hypoxia were tested by WB;then the myotubes were intervened by3-MA(the autophagy inhibitor)in hypoxia,and the expression of muscle atrophy and autophagy were tested by WB and IF;then the plasmids of r FoxO1(FoxO1overexpression),K?Q(mimicked acetyl-FoxO1)and K?R(mimicked deacetyl-FoxO1)were transfected to myotubes,and the expression of the muscle atrophy and autophagy proteins were tested in normoxia and hypoxia.Results:1)Compared with group C,the lean body weight,the wet weight of EDL,FCSA and Myosin protein expressions in group H were significantly decreased,and the protein expressions of Mu RF1 and Atrogin1 of EDL were increased(P<0.05);compared with group H,the lean body weight and the FCSA of EDL of group HR were increased,and the proteins expression of Mu RF1 and Atrogin1 were decreased(P<0.05);2)Compared with group C,the expression of autophagy-regulating proteins Rab7,Atg7,and key proteins Beclin1 and LC3?/?increased,and the expression of p62 decreased(P<0.05).The expression of autophagy differential genes upregulated in group H/C,and the function is mainly concentrated in the process of autophagy vesicle formation;compared with group H,the expression of Atg7 and LC3?/?in the group HR were decreased,and the expression of p62 was increased(P<0.05).The expression of autophagy differential genes upregulated in group HR/H,and the function were enriched in the co-regulation process of autophagy and apoptosis;FoxO1 and differential autophagy genes were interacted in each group;the autophagy genes interacting with FoxO1 in group HR/H were enriched in the early stage of autophagy;3)Compared with group C,the expression of FoxO1 and Ac-FoxO1,as well as the nuclear localization of FoxO1 and cytoplasmic localization of Ac-FoxO1 increased in group H(P<0.05).Compared with group H,the expression of FoxO1,the nuclear localization of FoxO1 and cytoplasmic localization of Ac-FoxO1decreased in group HR(P<0.05);4)The diameter of L6 myotubes were reduced and the expression of atrophy proteins were increased under 1%O₂ hypoxia exposure for 6 h(P<0.05);5)The expression of FoxO1 and Rab7 in nucleus and Ac-FoxO1 and Atg7 in cytoplasm of myotube were increased by hypoxia(P<0.05);6)The autophagy levels were increased and the myotube diameter were decreased by hypoxia,but effectively relieved by the early stage of autophagy inhibitor 3-MA(P<0.05);7)The autophagy levels were increased and the myotube diameter were decreased with transfected K?Q plasmids,more significantly than transfected with r FoxO1 in normoxia;but the autophagy levels were decreased and the myotube diameter were increased by transfected K?R plasmids in hypoxia(P<0.05).Conclusion:1)Skeletal muscle atrophy caused by hypoxia in rats could be effectively alleviated by 4-week resistance training,especially the EDL;2)Autophagy-lysosome pathway mediated by nuclear FoxO1 and cytoplasmic Ac-FoxO1 is an important way to regulate skeletal muscle mass in hypoxia;3)Hypoxia-induced muscle atrophy could alleviate by resistance training through inhibiting cytoplasmic acetyl-FoxO1-mediated the early stage of autophagy.