| Objective:1.To investigate the effect of autophagy inhibition on skeletal muscle of obese rats induced by high-fat diet(HFD).2.To clarify the role of autophagy in regulating palmitic acid(PA)-induced myotube atrophy.3.To investigate the effect of P300 on autophagy and myofiber in PA-treated C2C12 myotube and db/db mice under high lipids condition.Methods1.Male SD rats were randomly divided into normal group(Control),high-fat diet group(HFD)and high-fat diet+chloroquine group(HFD+CQ).To establish the obesity model,rats were continuously fed with high-fat diet for 16 weeks.Then,rats in the HFD+CQ group were intraperitoneally injected with the autophagy inhibitor chloroquine(chloroquine,CQ,50mg/kg/d)for 2 weeks.Meanwhile,rats in Control group and HFD group were treated with the same amount of normal saline.(1)Oil red staining was used to demonstrate the lipid deposition in muscle fibers.(2)Western Blot(WB)and immunofluorescence were used to detect the expressions of autophagy-related proteins(LC3 and p62).(3)The maximum grip strength of rates was measured by electronic grip strength meter,and the weight of gastrocnemius muscle of rats was measured by analytical balance.(4)Muscles were stained with hematoxylin-eosin(HE),and the cross-sectional area(CSA)of muscle fibers was assessed.(5)The expressions of muscle atrophy gene Atrogin-1 were detected by WB.2.(1)C2C12 myoblasts were cultured in proliferation medium.After myoblasts differentiated into myotubes,the myotubes were treated with palmitic acid(PA)to establish in vitro models of obesity.(1)Expressions of markers associated with myotube atrophy,including myosin heavy chain(MHC)and muscle atrophy genes(Atrogin-1 and MuRF1),were detected by WB,RT-qPCR and immunofluorescence.(2)Changes in autophagic flux were evaluated by WB,transmission electron microscopy(TEM)and mRFP-GFP-LC3 lentivirus infection.(3)Myotubes were treated with 0.4mM PA in the presence or absence of autophagy inhibitor 3-Methyladenine(3-Methyladenine,3-MA,2 mM)for 36 hours,and the expressions of autophagy protein(LC3 and p62)were detected by WB to investigate the effect of PA on autophagosome formation.(2)Myotubes were treated with the autophagy inhibitor chloroquine(CQ,1 ?M)or the autophagy activator rapamycin(Rapamycin,RAPA,50nM)with or without PA(0.4 mM)for 36 hours.(1)The effects of CQ and RAPA on autophagy were confirmed by WB and MDC immunofluorescence staining.(2)The expressions of markers related to myotube atrophy,including myosin heavy chain(MHC)and muscle atrophy genes(Atrogin-1,MuRF1),were detected by WB and RT-qPCR.The diameter of myotube was measured under light microscope.3.(1)The myotubes were treated with PA,and the expressions of P300 related markers,including P300,phosphorylated P300(P-P300)and acetylated histone H3(Ac-H3),were detected by Western Blot.(2)Myotubes were treated with PA in the presence or absence of P300 specific inhibitor c646.(1)To verify the inhibitory effect of c646 on P300,the expressions of Ac-H3 were detected by WB.(2)The expressions of markers associated with myotube atrophy were detected by WB and RT-qPCR.(3)Changes in autophagic flux were evaluated by WB,immunofluorescence and mRFP-GFP-LC3 lentivirus infection.(3)Myotubes were co-treated with autophagy inhibitors CQ,c646 and PA.(1)To confirm the inhibitory effect of CQ on autophagy,the expressions of autophagy-related proteins(LC3 and p62)were detected by WB.(2)To evaluate whether blocking autophagy could reverse the effect of P300 inhibition on myotube atrophy,the expressions of markers related to myotube atrophy were detected by WB and RT-qPCR,and the diameter ofmyotube was measured under light microscope.(4)Male db/db mice were divided into db/db group and db/db+c646group group,and m/m mice with the same genetic background were divided into m/m group and m/m+c646 group group.Mice in the m/m+c646group and db/db+c646 group received intraperitoneal injection of c646(30nmol/g/d)for 2 weeks,while mice in the normal m/m group and db/db group were treated with the same amount of c646 solvent.(1)Oil red staining was used to demonstrate the lipid deposition in muscle fibers.(2)WB and immunohistochemistry staining were used to detect the expressions of P300 related proteins,including P300,P-P300 and Ac-H3.(3)WB and immunofluorescence were used to detect the expressions of autophagy-related proteins(LC3,p62).(4)The maximum grip strength of the mice was measured by electronic grip strength meter,and the weight of gastrocnemius muscle of mice was measured by analytical balance.(5)Muscles were stained with hematoxylin-eosin(HE),and the cross-sectional area(CSA)of muscle fibers was determined.(6)The mRNA expressions of atrophy gene Atrogin-1 and MuRF1 were detected by RT-qPCR.Results1.Lipid heterotopic deposition was observed in skeletal muscle of HFD rats.Meanwhile,the expressions of LC3-II/I and p62 were increased,suggesting that autophagic flux was impaired.Additionlly,the muscle mass,grip strength and myofiber size in the HFD rats were significantly lower than those in the Control group,accompanied by increased expressions of Atrogin-1 protein.Treatment with the autophagy inhibitor CQ aggravated the lipid-induced autophagic flux inhibition(the expression levels of LC3-II/I and p62 were further increased)and the aforementioned characteristic changes associated with muscle atrophy.2.Treatment with PA significantly reduced the expression of MHC protein in muscle cells in a time-and concentration-dependent manner.Simultaneously,PA increased the mRNA and protein levels of atrophy genes(i.e.Atrogin-1 and MuRF1)and reduced the diameter of myotubes.In addition,PA suppressed autophagic flux,as evidenced by elevated levels of autophagy proteins LC3-II/I and p62,blocked fusion of autophagosomes and lysosomes and accumulated autophagosomes.Induction of autophagy by RAPA could alleviate PA-induced MHC protein degradation,muscle atrophy gene activation and myotube diameter reduction,whereas treatment with the autophagy inhibitor CQ produced the opposite effect.3.(1)Exposure of C2C12 myotubes to PA resulted in a significant increase in phosphorylated P300 and acetylated histone H3,a downstream substrate of P300,although the expressions of P300 total protein were not significantly changed,indicating that PA induced excessive activation of P300.Inhibition of P300 by a specific inhibitor c646 significantlypromoted the fusion of autophagosomes and lysosomes,reduce the accumulation of autophagosomes,and inhibited the expression of autophagy-related proteins(LC3-II/I and p62).Moreover,PA-induced MHC protein degradation,muscle atrophy gene activation and myotube diameter reduction were also ameliorated by P300 inhibition.However,CQ-mediated autophagy inhibition could reverse the protective effects of P300 inhibition on myoutubes.(2)In vivo results revealed that the expressions of P-P300 and Ac-H3 were significantly increased,while the autophagic flux was decreased in gastrocnemius muscle of db/db mice.Selective inhibition of P300 by c646 reactivated autophagic flux,suppressed the transcription of atrophy genes(Atrogin-1 and MuRF1),and partially restored grip strength and myofiber size.ConclusionOur research demonstrated that lipid-induced excessive activation of acetyltransferase P300 can block the autophagic flux,leading to skeletal muscle atrophy.Inhibiting P300 activity or activating autophagic flux may be the strategy for the treatment of lipid-induced muscle atrophy. |
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