HDAC3 Promotes Hypoxia-induced Autophagy In H9C2 Cardiac Cells Through Negatively Regulating MTOR Protein

Background Heart failure(HF)is a group of clinical syndrome caused by structural or functional abnormalities of the heart that impair the ability of the ventricle to contract or relax,it’s an important disease endangering human health.The prevalence of HF in adults is 1-2%,with the increase of age,the prevalence of elderly people over 70 years old exceeds10% and it’s now in a stage of continuous increase year by year.In recent years,although great progress and development have been made in the diagnosis and treatment of HF,the mortality rate of HF patients during hospitalization was as high as5.3%,it caused a huge economic and social burden.Therefore,studying the pathological molecular mechanism of the occurrence and development of HF,exploring new targets and new drugs for the prevention and treatment of HF,increasing the success rate of clinical treatment of HF and improving its prognosis are important medical problems that need to be solved urgently.Autophagy is a process of cell catabolism,it plays an important role in the occurrence and development of many diseases.Cardiomyocyte autophagy is an important homeostasis pathway,which is essential for adapting to altered myocardial metabolic needs.On the one hand,autophagy`in cell homeostasis and protect cardiomyocytes by degrading misfolded proteins and damaged cardiomyocytes,on the other hand,under pathological conditions,cardiomyocyte autophagy can induce cardiomyocyte hypertrophy and promote myocardial remodeling,and this is one of the important causes of HF.However,the current research on the molecular mechanism of cardiomyocyte autophagy is still unclear.Histone deacetylase is one of the 11 HDAC isoforms of the HDAC superfamily,and is an important molecule for epigenetic regulation.Studies have found that HDAC3 can regulate a variety of cell growth and apoptosis processes,and can promote cardiomyocyte hypertrophy and induce cardiac insufficiency,which is closely related to congenital heart disease.However,the function and mechanism of HDAC3 in regulating autophagy in cardiomyocytes is still unclear.ObjectiveThis study attempted to establish a hypoxia model of H9C2 cardiac cells,simulate the hypoxia damage of cells in vivo,and explore the function and mechanism of HDAC3 regulating autophagy of H9C2 cells.MethodsThe hypoxia model of rat H9C2 myocardial cell line was established in vitro,the experimental cells were divided into control group,hypoxia 1 h group,hypoxia 6 h group and hypoxia 12 h group,and cell viability was detected by tetramethylazolium salt(MTT).The expression and fluorescence activity of HDAC3 and autophagy-related molecules LC3 B,P62 Beclin-1 and m TOR were detected by immunofluore-scence staining and Western blot.H9C2 cardiomyocytes were screened to establish a cell model of HDAC3 knockdown and overexpression,the transfection effect of HDAC3 plasmid and interfering RNA were detected by Real-time fluorescence quantitative PCR,and the expression and fluorescence activity of m TOR and autophagy-related molecules after down-regulation or up-regulation of HDAC3 were detected by immunofluorescence staining and Western blot.Results(I)With the increase of hypoxia time,hypoxia induced the decrease of H9C2 cells activity.After hypoxia for 1 h,6 h and 12 h,the mean viability of H9C2 cardiomyocytes were 81.41 %,65.06 % and 43.78 %,respectively.Compared with the mean of 99.67 % in the normal control group,the survival rate of cells in each hypoxia model group decreased(P<0.05).(II)Compared with the control group,the expression of autophagy-related proteins LC3B-II and Beclin-1 in H9C2 cardiac cells increased after hypoxia for 1 h,6 h,and 12h(P<0.05),and the expression of P62 protein increased first at 1 h and then decreased at6 h(P<0.05).After hypoxia for 1 h and 6 h,the expression of HDAC3 and m TOR increased(P<0.05).After hypoxia for 12 h,the expression of HDAC3 and m TOR decreased(P<0.05).(III)Overexpression of HDAC3 can reduce the protein expression and fluorescence activity of m TOR,and it can increase the protein expression and fluorescence activity of LC3B(P<0.05);Down-regulation of HDAC3 can increase protein expression and fluorescence activity of m TOR,and reduce protein expression and fluorescence activity of LC3 B.ConclusionHypoxia induced the decrease of H9C2 cells activity and the increase of autophagy level,and there was a time-dependent relationship.HDAC3 negatively regulates m TOR protein and promotes hypoxia-induced autophagy in H9C2 cardiac cells.