The Role And Mechanism Of Methyltransferase EZH2 In Diabetic Myocardial Fibrosis

Background: Diabetes is a metabolic disease characterized by chronic elevated blood glucose,which seriously endangers human health.Diabetic cardiomyopathy will eventually lead to heart failure,which is one of the important causes of death from diabetes.Studies have shown that diabetic fibrosis promotes the development of diabetic cardiomyopathy.The differentiation of cardiac fibroblasts transfer into cardiac myofibroblasts plays an important role in cardiac fibrosis.Therefore,exploring the mechanism of diabetic myocardial fibrosis and the mechanism of myocardial fibroblast differentiation may provide new ideas for the understanding and management of diabetic cardiovascular complications.Enhancer of zeste homolog 2(EZH2)is a methyltransferase that can catalyze the trimethylation of lysine 27(H3K27me3)of histone H3.In recent years,studies have reported that EZH2 is involved in the occurrence and development of organ fibrosis,but whether EZH2 is involved in diabetic myocardial fibrosis is still unclarified.Objective: The purpose of this study was to investigate the role and mechanism of methyltransferase EZH2 in diabetic fibrosis and differentiation of cardiac fibroblasts.Methods:(1)Establishment of type I rat and mouse diabetes model;(2)Echocardiography was used to detect the cardiac function of model animals.(3)Masson staining was used to detect the degree of myocardial fibrosis in rats.(4)Immunohistochemical staining was used to measure the protein expression of animal myocardium tissue.(5)Western blot was used to detect the protein expression of animal myocardium tissue.(6)Real-time quantitative PCR(q-PCR)was used to measure the gene expression in rat myocardial tissue.(7)Isolation and culture of cardiac fibroblasts,stimulation with high glucose;EZH2 inhibitor(GSK126),PPARγ agonist(Rosiglitazone)and AMPK agonist(A769662)were added before high glucose stimulation;(8)Immunofluorescence was used to detect the α-SMA protein expression of cardiac fibroblasts.(9)Western blot was used to detect the proteins expression of cardiac fibroblast.(10)Transwell and scratch test was used to measure the migration ability of myocardial fibroblasts.(11)CCK8 assay was used to detect the proliferation ability of myocardial fibroblasts.Results:(1)The percentage of collagen fiber area in the total myocardial area in the control group and diabetic rats was 2.29±1.35%,8.48±1.03%(P < 0.05,n = 4).The area of collagen fibers in diabetic rats was more than that in the control group.The relative expressions of fibrotic proteins α-SMA,Collagen I,Collagen III and TGF-β1 protein increased in the myocardial tissue of diabetic rats.(2)The relative protein expressions of H3K27me3 in ventricular muscle tissue of rats in the control group and the diabetic group were 1.02±0.23,2.21±0.34(P < 0.05,n = 3).The relative protein expressions of H3K27me3 in ventricular muscle tissue of mice in the control group and diabetes group were 1.00±0.52,3.83±0.85(P < 0.05,n = 6).The level of H3K27me3 in myocardial tissues of diabetic rats and mice was higher than that of control rats and mice.The protein and m RNA levels of EZH2 did not change significantly in myocardial tissues between control and diabetic groups.(3)In cultured ventricular fibroblasts in vitro,the relative protein expressions of Collagen I,TGF-β1,and α-SMA protein in the normal glucose group and the high glucose group were 1.00±0.27,3.21±0.54(P < 0.05,n = 3),1.00±0.10,1.41±0.22(P < 0.05,n = 3),1.00±0.31,2.04±0.30(P < 0.05,n = 3).The relative expressions of fibrotic proteins α-SMA,Collagen I and TGF-β1 protein and the migration ability of cardiac fibroblasts increased in ventricular fibroblasts treated with high glucose in vitro.(4)The ratio of p EZH2/EZH2 protein and the relative protein expressions of H3K27me3 in ventricular fibroblasts of normal glucose group and high glucose group were 1.00±0.15,0.49±0.70(P < 0.05,n = 3)and 1.00±0.33,3.40±0.48(P < 0.05,n = 3).The level of H3K27me3 is higher in cardiac fibroblasts treated with high glucose than that of cardiac fibroblasts cultured with normal glucose,and the phosphorylation of EZH2 protein was reduced.(5)The expressions of H3K27me3 in NG + DMSO,NG + GSK126,HG + DMSO,and HG + GSK126 were 1.00±0.04,0.84±0.26,1.78±0.11,0.94±0.35(P < 0.05,n = 3).The expressions of Collagen I,TGF-β1,and α-SMA in the four groups were: 1.00±0.01,1.07±0.15,1.38±0.13,1.06±0.15(P < 0.05,n = 3);1.00±0.01,1.29±0.11,1.80±0.15,1.07±0.26(P < 0.05,n = 3);1.00±0.01,1.00±0.18,1.32±0.63,0.97±0.25(P < 0.05,n = 3).Inhibition of EZH2 reduces the relative level of H3K27me3,fibrotic related protein α-SMA,Collagen I and TGF-β1 protein in cardiac fibroblasts stimulated by high glucose.Inhibition of EZH2 reduces the proliferation and migration of cardiac fibroblasts under high glucose stimulation.(6)The m RNA expression of PPARγ in the ventricular muscle tissue of the control group and the diabetic group was 0.96±0.06,0.45±0.03(P < 0.05,n = 4).Down-regulation of PPARγ m RNA expression was found in myocardial tissue of diabetic rats.(7)The expressions of PPARγ in NG + DMSO,NG + GSK126,HG + DMSO,HG + GSK126 were 1.10±0.13,1.04±0.24,0.35±0.13,0.83±0.40(P < 0.05,n = 3).Inhibition of EZH2 upregulated the m RNA expression of PPARγ in cardiac fibroblasts under high glucose stimulation.(8)The expressions of Collagen I,TGF-β1,and α-SMA in the four groups of NG + DMSO,NG + Rosiglitazone,HG + DMSO,and HG + Rosiglitazone were 1.00±0.01,1.08±0.12,1.46±0.05,1.02±0.51(P < 0.05,n = 3);1.00±0.06,0.97±0.19,1.62±0.25,1.00±0.06(P < 0.05,n = 3);1.01±0.13,1.10±0.25,1.68±0.15,1.11±0.13(P < 0.05,n = 3).Activation of PPARγ reduced the relative expression of myocardial fibrosis-related proteins α-SMA,Collagen I and TGF-β1 under high glucose stimulation.Activation of PPARγ reduced the migration ability of myocardial fibroblasts under high glucose stimulation.(9)The relative protein expressions of p AMPK in the ventricular muscle tissue of the control group and the diabetic group were 1.00±0.07 and 0.42±0.12(P < 0.05,n = 3).The phosphorylation level of AMPK in ventricular myofibroblasts was 1.00±0.05 and 0.64±0.11 in normal glucose and high glucose groups(P < 0.05,n = 3).The phosphorylation of AMPK decreased diabetic rat myocardial tissue cardiac fibroblasts treated with high glucose.(10)The relative protein expressions of H3K27me3 and p EZH2/EZH2 in the four groups of NG + DMSO,NG + A769662,HG + DMSO,and HG + A769662 were: 1.00±0.03,1.33±0.10,2.03±0.29,1.11±0.43(P <0.05,n = 3);1.00±0.09,1.11±0.26,0.73±0.80,1.08±0.14(P <0.05,n = 3).Activation of AMPK reduced the expression of H3K27me3 protein in high glucose induced cardiac fibroblasts and up-regulated the phosphorylation of EZH2.Conclusion:(1)The expression of EZH2 is unchanged in the myocardial tissues of diabetic rats and mice.The level of H3K27me3 in myocardial tissues is up-regulated in diabetic rats and mice than the control group.(2)The phosphorylation of EZH2 is down-regulated and the level of H3K27me3 is up-regulated in cardiac fibroblasts treated with high glucose.(3)EZH2 promotes fibrotic protein expression,the differentiation,migration and proliferation of cardiac fibroblasts.Inhibition of EZH2 reduces the expression of fibrotic protein,differentiation,migration and proliferation ability of cardiac fibroblasts under high glucose stimulation.(4)Rosiglitazone stimulates PPARγ to reduce the expression of myocardial fibroblasts fibrosis-related protein,the differentiation and migration ability of myocardial fibroblasts under high glucose stimulation,and improve myocardial fibrosis.