Effect Of FTO On H9C2 Cardiomyocyte Apoptosis Induced By High Glucose

Objectives:Diabetic Cardiomyopathy（DCM）is a serious complication of diabetes mellitus.It is a specific cardiomyopathy that is independent of hypertension,coronary heart disease,vascular heart disease,and other heart diseases.DCM has a high incidence and great harm,and is closely related to the high incidence and mortality of cardiovascular disease in diabetic patients.Cardiomyocyte apoptosis plays an important role in the pathogenesis of DCM.Fat mass and obesity-associated protein（FTO）is a gene that is closely related to polygenic obesity,and it is also the first m6 A demethylase discovered.FTO is closely related to apoptosis.Studies have shown that the expression of FTO is decreased in cardiomyocytes of failing heart for humans and mice,and the expression of FTO is closely related to the function of the heart.Therefore,whether FTO is involved in the occurrence of DCM by regulating cardiomyocyte apoptosis is worth exploring.In this study,the relationship between the expression of FTO and apoptosis in H9C2 cells induced by high glucose was investigated to explore the possible mechanism of FTO in diabetic cardiomyopathy,so as to provide a new target for the treatment of diabetic cardiomyopathy.Methods:H9C2 cardiomyocytes cultured in low glucose（5.5mmol/L）were randomly selected for the logarithmic growth phase with the highest growth rate,and the following experimental scheme was carried out:1.H9C2 cardiomyocytes were cultured with low glucose（5.5 mmol/L）as blank control,2.H9C2 cardiomyocyte were cultured with high glucose（35mmol/L）for 72 h,3.H9C2 cells cultured with high glucose（35mmol/L）were treated with Lipofectamine^TM 3000 reagent transfected lentivirus stably knocking down FTO,and we also used small interfering RNA（si RNA）to knock down FTO instantaneously for H9C2 cells cultured with high glucose（35mmol/L）.Overexpression of FTO in H9C2 cells cultured with high glucose（35mmol/L）.RNA and total protein were extracted from H9C2 cardiomyocytes of the above groups after high glucose stimulation or cell transfection,and the changes of FTO content.the changes of apoptosis-related proteins,Cleaved-Caspase-3 and Bax,and the changes of apoptosis-inhibiting protein Bcl-2 were detected by q-PCR and Western Blot methods respectively.The changes of myocardial cell apoptosis were detected by flow cytometry.4.Whole genome RNA sequencing was performed in the high glucose group and the high glucose group transfected with lentivirus to stably knock down FTO.According to the sequencing results and literature review,the differential genes were analyzed.And the relationship between differential genes and FTO was analyzed.Effect of possible interaction between differential genes and FTO on apoptosis of H9C2 myocardial cells stimulated by high glucose was investigated.Results:1.Compared with the low glucose（5.5mmol/L）group,the high glucose（35mmol/L）group showed decreased FTO expression,increased Cleaved caspase3 and Bax expression,decreased Bcl-2 expression,and increased myocardial cell apoptosis after 72 h.2.After stable and transient FTO knockdown,pro-apoptotic Cleaved caspase3 and Bax expression increased,and anti-apoptotic Bcl-2 expression decreased.Overexpression of FTO decreased Cleaved caspase3 and Bax,and increased Bcl-2expression.3.The common high glycemic group and lentivirus transfected with low FTO high glycemic group were sequenced by whole genome RNA,and the differential genes were analyzed by GO and KEGG enrichment.According to the analysis,Ptgs2 was one of the differential genes closely related to iron death,and the m6 A prediction website showed that Ptgs2 gene had m6 A modification site.4.m RNA and protein expression of Ptgs2 were increased in high glucose cells with low FTO expression.Conclusion:1.FTO expression was decreased in cardiomyocytes stimulated by high glucose.2.The change of FTO affects the apoptosis of cardiomyocytes,the expression of FTO decreases,the apoptosis level of cardiomyocytes increases,the expression of FTO increases,the apoptosis level of cardiomyocytes decreases.3.Ptgs2 is one of the target genes regulated by FTO,and FTO may be involved in the apoptosis of cardiomyocytes stimulated by high glucose by regulating Ptgs2.