The Expression And Correlation Of Siat7A,TAK1 And KlF4 In The Progression Of Hypertensive Myocardial Hypertrophy

Research background: Cardiovascular disease is a serious concern and threat to human health.One of the significant risk factors for cardiovascular disease is chronic hypertension.Persistent hypertension leads to arterial wall-thickening and hardening,which eventually give rise to ventricular hypertrophy.Heart failure then occurs as a result of an increase in systolic pressure.A previous screening study that examined nearly 20,000 genes in the hypertrophic cardiac tissue of genetically diverse hypertensive rats found that Sialyltransferase7a(Siat7A),also known as CMPNeu5Ac: Galnac-Rα2,6-sialic acid transferase,uniquely displayed a sustained increase in its expression level.Another study has shown that TGF-β activated kinase 1(TAK1),a member of the mitogen-activated protein kinase(MAPK),was upregulated in the cardiac tissue of adult mice 7 days after the occurrence of transverse aortic constriction(TAC).It was also found that the overexpression of TAK1 led to the TAK1-mediated activation of p38 pathway,which promoted the expression of embryonic genes and ultimately led to cardiac hypertrophy and interstitial fibrosis.Yet another research has discovered that Kruppel like factor 4(Klf4),a member of the Klf family,was upregulated in hypertrophic myocardium of mice and cultured hypertrophic cardiomyocytes.In the current study,we have demonstrated that under hypoxia,Klf4 expression in human ventricular myocyte AC16 cells is increased and that Siat7 A expression is increased through the trans-activation of the promoter region of Siat7 A,thereby increasing the production of Sialyl-Tn(Neu5ACα2-6Gal NAC-O-Ser/Thr),the substrate antigen of Siat7 A.However,we observed an increased level of sialylation as well as a higher expression level of hypoxiainducible factor-1α(HIF-1α)in cardiomyocytes that have received hypertrophy-inducing angiotensin Ⅱ(AngⅡ)treatment.HIF-1α activates TAK1 transcription by binding to the 1285-1274 bp region of the TAK1 promoter.The increased activity of TAK1 subsequently activates the nuclear factor kappa-B(NF-κB)signaling pathway,leading to cardiac hypertrophy.Objectives:Based on the above-mentioned prior results,the problems to be solved in this study are as follows:(1)Does Klf4 expression change following AngⅡ-induced hypertrophy of myocardium?(2)Does Klf4 expression affect the expression of Siat7 A in AngⅡ-induced hypertrophy of myocardium?(3)Is there an interrelation between Siat7 A,TAK1,and Klf4 in hypertensive cardiac hypertrophy?Through the study of the above problems,we hope to provide new ideas for the clinical treatment of hypertensive cardiac hypertrophy and find a new therapeutic target for the prevention of hypertensive cardiac hypertrophy.Materials and methods: Myocardial tissue of patients with cardiac hypertrophy and patients without any background of heart disease were collected from autopsy specimens for preparation of tissue sections.Preparation of paraffin slice was made using cardiac tissue of hypertensive rats.Pathological changes of cardiac tissue were observed using hematoxylin-eosin staining(HE stain).Immunohistochemical staining was performed to detect the expressions of Siat7 A,TAK1,and Klf4 in cardiomyocytes during the occurrence of cardiac hypertrophy.Human ventricular myocyte line AC16 cells were cultured and transfected with plasmid lentivirus with knockdown or overexpression of Siat7 A gene.Cell lines with stable knockdown or overexpression of the Siat7 A gene were selected.After 24 h of AngⅡ(1 μM)stimulation,protein and m RNA samples were collected.Western Blot was used to detect the expressions of Atrial Natriuretic Peptide(ANP),α-actinin,Siat7 A,TAK1,and Klf4.Quantitative real-time PCR was used to detect m RNA expression levels of ANP,Brain Natriuretic Peptide(BNP),β-myosin heavy chain(β-MHC),Siat7 A,TAK1,and Klf4.To further clarify the correlations of Siat7 A,TAK1 and Klf4 in hypertrophic myocardial cells,AngⅡ(1 μM)were administrated to the cells for 0.5 h,1.5 h,3 h,6 h and 12 h,after which protein sample collection was performed.Western blotting was applied to test the expressions of Siat7 A,TAK1 and Klf4.Results:The protein levels of Siat7 A,TAK1,and Klf4 were all increased in both the myocardial tissue of patients with hypertension and in rat myocardium with AngⅡinduced hypertrophy.In vitro treatment of AC16 cells with different concentrations of AngⅡ(0 μM,0.1 μM,1 μM)for 24 hours resulted in the increased expression of ANP,β-MHC,α-actinin,as well as Siat7 A,TAK1,and Klf4,with a higher concentration of AngⅡ producing greater increase.After infection with knockdown-Siat7A-carrying lentivirus,AC16 cells showed significantly decreased expression of Siat7 A.When these cells were stimulated with AngⅡ(0 μM,1 μM),the expression of TAK1,Klf4,ANP,BNP,β-MHC and α-actinin decreased accordingly.Similarly,after infection with lentivirus carrying overexpressed Siat7 A gene,AC16 cells displayed increased expression of Siat7 A.The expression of TAK1,Klf4,ANP,BNP,β-MHC,and α-actinin also increased following treatment with AngⅡ(0 μ M,1 μ M).After TAK1 was knocked down using si RNA,TAK1 expression showed a marked decrease in AC16 cells.,and stimulation with AngⅡ resulted in decreased expression of Siat7 A,Klf4,ANP,BNP,β-MHC,and α-actinin.Likewise,after si RNA-mediated knockdown of Klf4,the expression of Klf4 in AC16 cells decreased significantly,while subsequent stimulation with AngⅡ also reduced the expression of Siat7 A,TAK1,ANP,BNP,β-MHC and α-actinin.Through stimulating AC16 cells with AngⅡ for different durations,we found that the expression of Klf4,Siat7 A,and TAK1 protein started to increase after 0.5 h,1.5h,and 3h respectively.Meanwhile the expression of α-actinin and ANP protein increased after 1.5 h and 3 h of stimulation respectively.Conclusion :(1)In hypertrophic myocardium of hypertensive patients,the protein expressions of Siat7 A,TAK1 and Klf4 increased uniformly.(2)During AngⅡ-induced hypertension and cardiac hypertrophy in rats,the expressions of Siat7 A,TAK1 and Klf4 in myocardium were increased.Similarly,during in vitro experiment,hypertrophy of AC16 cells could be induced by exposure to AngⅡ at certain concentrations and durations,which also caused the increased expressions of Siat7 A,TAK1 and Klf4.(3)During the development of AngⅡ-induced cardiac hypertrophy,Klf4 is likely the first gene to be upregulated,followed by Siat7 A and subsequently TAK1.The three genes were mutually reinforcing,and they jointly promoted the development of cardiac hypertrophy by forming a closed loop.