| BackgroundHeart failure(HF)is a major public health problem.In heart failure,energy metabolism is disordered,and the level of Smad2 acetylation increases,which further promotes the development of myocardial fibrosis and HF.Therefore,Smad2 acetylation is important to improve myocardia l fibrosis in HF.The literature shows that by increasing the silent information regulator 2-related enzymes 1(Sirtuin 1,Sirt1),regulating the expression of Smad2 acetylation in a mouse model of pressure overload improves myocardial fibrosis and prevents the development of HF.At the same time,aerobic exercise can regulate Sirt1 to improve myocardial fibrosis in HF.Therefore,we speculate that aerobic exercise can effectively improve HF myocardial fibrosis by mediating Smad2 acetylation modification by regulating Sirt1 expression.Objectives1.To explore whether aerobic exercise improves cardiac function and myocardial fibrosis in HF through Sirt1/acetylated Smad2.2.Determine whether Sirt1 regulates cardiac fibroblast phenotype transformation(the expression of α-smooth muscle actin(α-SMA)is a marker)through Smad2,acetylated Smad2 pathway.3.Understand the biological functions,pathways and key molecules of myocardial fibrosis from bioinformatics analysis,and the roles of Sirt1 and Smad2 in it.Methods1.Using the mouse HF model,aerobic exercise for 4 weeks,combined with Smad2 interference,Sirt1 agonist and inhibitor.Measure body weight,heart weight,and echocardiographic indicators.Heart tissue section staining(HE,Masson,α-SMA immunohistochemistry).Western blot was used to detect the protein expression of Sirt1,acetylated Smad2,Bax,Caspase3 and collagen type III alpha 1 chain(Col3a1)in cardiac tissue.2.The cells used primary mouse cardiac fibroblast cell line.Resveratrol mimics the effect of aerobic exercise(increases Sirt1 expression),and cell experiments explore the molecular mechanism of Sirt1,Smad2 and myocardial fibrosis: CF was treated with normoxia or hypoxia for 24 hours,combined with Smad2 knockout,and treated with resveratrol.Western blot detection of Sirt1,Smad2,acetylated Smad2,α-SMA protein expression in each group of CF.3.On March 29,2022,myocardial fibrosis-related genes were collected from the NCBI-Gene database,and gene ontology and K yoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed to explore biological functions and signaling pathways,and to assemble protein interaction networks to identify central genes associated with myocardial fibrosis mechanisms.Results1.Aerobic exercise improves myocardial fibrosis in HF through Sirt1 and acetylated Smad2 signaling pathways.Compared with wild-type sham-operated group(WT-sham),the wild-type HF group(WT-HF)showed left ventricular posterior wall in systole(LVPWs),inter-ventricular septum thickness in systole(IVSs),fractional shortening(FS)and ejection fraction(EF)decreased(p<0.05)and left ventricular internal diameter in systole(LVIDs),left ventricular internal diameter in diastole(LVIDd),systolic left ventricular volume(LV Vols),and diastolic left ventricular volume(LV Vold)increased(p<0.05).In WT-HF group(compared with WT-sham group),HE staining showed that myocardial cells were disordered,Masson staining showed increased collagen fibers,and immunohistochemical staining of α-SMA showed increased positive staining of α-SMA.In WT-HF group(compared with WT-sham group),Sirt1 protein expression decreased(p<0.05),and acetylated Smad2,Col3a1,Bax,and Caspase3 protein expressions increased(p<0.05).Compared with the WT-HF group,the wild-type HF exercise group(WT-HE)showed LVPWs,FS and EF increased(p<0.05).In WT-HE group(compared with WT-HF group),HE staining showed that cardiomyocytes were arranged regularly,Masson staining showed that collagen fibers were reduced,and immunohistochemical staining of α-SMA showed that the positive staining of α-SMA was reduced.In WT-HE group(compared with WT-HF group),the expression of Sirt1 protein was increased(p<0.05),and the protein expression of acetylated Smad2,Col3a1,Ba x and Caspase3 was decreased(p<0.05).The results suggest that aerobic exercise can improve cardiac function and myocardial fibrosis while up-regulating Sirt1 and inhibiting the expression of acetylated Smad2.Compared with the Smad2 targeted interference sham operation group(S-sham),the Smad2 targeted interference HF group(S-HF)showed LVPWs,FS,and EF decreased(p<0.05).In S-HF group(compared with S-sham group),HE staining showed that myocardial cells were disordered,Masson staining showed increased collagen fibers,and immunohistochemical staining of α-SMA showed increased positive staining of α-SMA.In S-HF group(compared with S-sham group),Sirt1 protein expression decreased(p<0.05)and acetylation Smad2,Bax,Caspase3,Col3a1 protein expression increased(p<0.05).Compared with S-HF,Smad2 targeting interfered HF exercise group(S-HE)showed FS,and EF increased(p<0.05).In S-HE group(compared with S-HF group),HE staining showed no difference in the arrangement of cardiomyocytes,Masson staining showed no difference in collagen fiber content,and α-SMA immunohistochemical staining showed no difference in the degree of α-SMA positive staining.In S-HE group(compared with S-HF group),the expression level of Sirt1 increased(p<0.05),the expression of acetylated Smad2 and Col3a1 proteins did not change significantly,and the protein expressions of Bax and Caspase3 decreased(p<0.05).The results suggest that interfering with Smad2 and affecting acetylated Smad2 will block the protective effect of aerobic exercise,so aerobic exercise may improve myocardial fibrosis in HF through acetylated Smad2 signaling pathway.Compared with the WT-HF,the wild-type Sirt1 agonist HF group(WT-HF-R)showed LVPWs,left ventricular posterior wall in diastole(LVPWd),IVSs,inter-ventricular septum thickness in diastole(IVSd),left ventricular mass(LV Mass),FS,and EF increased(p<0.05)and LVIDs decreased(p<0.05).In WT-HF-R group(compared with WT-HF group),HE staining showed that cardiomyocytes were arranged regularly,Masson staining showed that collagen fibers were reduced,and immunohistochemical staining of α-SMA showed that the positive staining of α-SMA was reduced.In WT-HF-R group(compared with WT-HF group),Sirt1 protein expression increased(p<0.05),and acetylated Smad2,Col3a1,Ba x,and Caspase3 protein expressions decreased(p<0.05).Compared with S-HF group,Smad2 targeted interference with Sirt1 agonist HF group(S-HF-R)echocardiographic indicators(LVPWs,LVPWd,LVIDs,LVIDd,IVSs,IVSd,LV Vols,LV Vold,LV Mass,FS,EF)had no significant difference.In S-HF-R group(compared with S-HF group),HE staining showed no difference in the arrangement of cardiomyocytes,Masson staining showed no difference in collagen fiber content,and α-SMA immunohistochemical staining showed no difference in the degree of α-SMA positive staining.In S-HF-R group(compared with S-HF group),the protein expression of Sirt1 was increased(p<0.05),the expression levels of acetylated Smad2,Ba x,and Col3a1 were not different,and the protein expression of Caspase3 was decreased(p<0.05).The results showed that Sirt1 could improve cardiac function and myocardial fibrosis in HF by inhibiting acetylated Smad2.Compared with WT-HE,the wild-type Sirt1 inhibitor HF exercise group(WT-HE-527)showed the LVIDs,LVIDd,LV Vols,LV Vold were significantly increased(p<0.05),while the FS and EF were significantly decreased(p<0.05).In WT-HE-527 group(compared with WT-HE group),HE staining showed that myocardial cells were disordered,Masson staining showed increased collagen fibers,and immunohistochemical staining of α-SMA showed increased positive staining ofα-SMA.In WT-HE-527 group(compared with WT-HE group),the protein expression of acetylated S mad2,Col3a1,Bax,Caspase3 is increased(p<0.05).Compared with S-HE group,Smad2 targeting interferes with Sirt1 inhibitor HF exercise group(S-HE-527)showed the cardiac ultrasound indicators(LVPWs,LVPWd,LVIDs,LVIDd,IVSs,IVSd,LV Vols,LV Vold,LV Mass,FS,EF)had no difference.In S-HE-527 group(compared with S-HE group),HE staining showed no difference in the arrangement of cardiomyocytes,Masson staining showed no difference in collagen fiber content,and α-SMA immunohistochemical staining showed no difference in the degree of α-SMA positive staining.In S-HE-527 group(compared with S-HE group),the protein expression of Sirt1 was decreased(p<0.05),the protein expression of acetylated Smad2 and Col3a1 was not different,and the protein expression of Bax and Caspase3 was increased(p<0.05).The results suggest that aerobic exercise may improve cardiac function and myocardial fibrosis in HF through Sirt1/acetylated Smad2.Based on the above results,aerobic exercise up-regulates Sirt1,and Sirt1 can improve HF myocardial fibrosis by inhibiting acetylated Smad2.Therefore,aerobic exercise may improve cardiac fibrosis by inhibiting Smad2 acetylation signaling pathway by Sirt1.2.The role of Sirt1 and acetylated Smad2 in CF phenotype transformation.Compared with the V2 N group(normoxia treatment),the cardiac fibroblasts in the V2 H group(hypoxia treatment)had increased protein expression of Smad2,acetylated Smad2 and α-SMA(p<0.05),and decreased the expression of Sirt1protein(p<0.05).Compared with the V2 H group,the cardiac fibroblasts in the V2H+R group(hypoxia treatment + resveratrol)had decreased protein expression of Smad2,acetylated Smad2,α-SMA(p<0.05),and increased protein expression of Sirt1(p<0.05).Compared with the V2 H group,the V2 gs H group(Smad2CRISPR/Cas9 mouse CF treated with hypoxia)decreased the expression of Smad2 and acetylated Smad2 protein(p<0.05),but had no significant change in the expression of Sirt1 and α-SMA proteins.Compared with the V2 gs H group,the V2 gs H+R group(Smad2 C RISPR/Cas9 mouse CF hypoxia treatment + resveratrol)increased the protein expression of Sirt1(p<0.05),but had no change in Smad2,and decreased the protein expression of acetylated Smad2 and α-SMA(p<0.05).These results suggest that hypoxia promotes CF phenotype transformation,while resveratrol increases Sirt1 expression and inhibits acetylated Smad2,preventing CF phenotype transformation.3.The underlying mechanism of myocardial fibrosis and the role of Sirt1 and Smad2 in it.A total of 1418 myocardial fibrosis-related genes(MFRG)were obtained from the NCBI-GENE database.The biological functions of myocardial fibrosis were confirmed,including regulation of inflammatory response and positive regulation of cytokine production.At the same time,it was confirmed that the biological function regulation of Sirt1 and Smad2 in myocardial fibrosis is mainly concentrated on the transforming growth factor pathway.Conclusions1.Aerobic exercise improves HF myocardial fibrosis through Sirt1 /acetylated Smad2 signaling pathway.2.Sirt1 inhibits acetylated Smad2 and affects CF phenotypic transformation.3.The biological functions of myocardial fibrosis mainly include regulation of inflammatory response and positive regulation of cytokine production.The biological function regulation of Sirt1 and Smad2 in myocardial fibrosis is mainly concentrated on the transforming growth factor pathway. |
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