| BackgroundDiabetic cardiomyopathy(DCM)is the main fatal complication of diabetic patients.In the process of occurrence and development,the clinical manifestation of diabetic cardiomyopathy is asymptomatic diastolic dysfunction in the early stage.With the progress of the disease,abnormal ventricular systolic function gradually appears and finally aggravates to symptomatic congestive heart failure.The main pathological manifestations are myocardial hypertrophy,myocardial cell death,myocardial inter-stitium and perivascular fibrosis.PurposeThis study discussed the role and molecular mechanism of Wnt/β-catenin/TCF7L2 signaling pathway in DCM,and provided new targets for the prevention and treatment of DCM.Methods1.A 3-4-week-old C57BL/6J male mouse model of type 2 diabetes was established by high fat diet for 2 weeks and intraperitoneal injection of Streptozotocin(STZ,85 mg/kg).The measurement of random blood glucose ≥ 16.7mmol/L was considered to be a successful model establishment,which could be used for subsequent experiments.The experiment was divided into control group(Control,CON)and diabetes mellitus group(DM).After 20 weeks of diabetes,the8 expressions of β-catenin and TCF7L2 in the heart of type 2 diabetic mice were detected by Western blot(WB),RT-PCR,and Immunohistochemical(IHC).The above studies were conducted to clarify the expression changes of Wnt/β-catenin/TCF7L2 signaling pathway in diabetic cardiomyopathy.2.The Wnt/β-catenin/TCF7L2 signaling pathway inhibitor i CRT14 and the positive control Rosiglitazone(RSG)were administered in type 2 diabetic mouse models.The experimental groups were control group(CON),diabetic group(DM),diabetic group + i CRT14 2.5 mg/kg dose group(DM + i CRT14 2.5),diabetic group + i CRT14 5 mg/kg dose group(DM + i CRT14 5),diabetic group +i CRT1410 mg/kg group(DM + i CRT14 10),diabetes + RSG 10 mg/kg group(DM + RSG10).The changes of blood glucose in mice were measured at each time point.Cardiac function of mice in each group was detected by small animal ultrasound imaging system,and morphological changes of myocardial tissue were observed by HE and Masson staining.Changes of Wnt/β-catenin/TCF7L2 signaling pathway related proteins and cardiac hypertrophy indexes were detected by WB.To investigate the effect of inhibition of Wnt/β-catenin/TCF7L2 signaling pathway on myocardial remodeling in diabetic mice.3.The primary cardiomyocytes were isolated from the heart of neonatal SD rats,and treated with high glucose(33 mmol/L)and high insulin(100 nmol/L)for 48 hours to detect the changes of Wnt/β-catenin/TCF7L2 signaling pathway.Then,on the basis of high glucose and high insulin treated cardiomyocytes,Wnt/β-catenin/TCF7L2 signaling pathway agonist SKL2001 and inhibitor i CRT14 were administered.The experimental groups were control group(CON),high glucose and high insulin group(GLU + INS),high glucose and high insulin +SKL2001 40 μmol/L concentration group(GLU + INS + SKL),high glucose and high insulin + i CRT14 2.5 μmol/L concentration group(GLU + INS + i CRT142.5),high glucose and insulin + i CRT14 5 μmol/L concentration group(GLU +INS + i CRT14 5),high glucose and insulin + i CRT14 10 μmol/L concentration group(GLU + INS + i CRT14 10),the expression of Wnt/β-catenin/TCF7L2 signaling pathway related proteins and genes in each group were detected by WB9 and RT-PCR.Cell morphology of each group was detected by Immunofluorescence(IF)and the surface area of myocardial cells was measured.4.Lenti-Tcf7l2-sh RNA and Lenti-Tcf7l2-WT were constructed to regulate the expression of Tcf7l2 in primary cardiomyocytes.RNA was extracted from each group.The analysis of differential gene expression was performed by RNA-sequence technology,and potential target genes were screened and verified by WB.5.According to the results of transcriptome analysis,the protein and RNA of the myocardial tissue of diabetic mice and the protein and RNA at each time point of cardiomyocytes treated with high glucose and high insulin were extracted,and the carbonic anhydrases gene family of different target genes screened by transcriptome were analyzed by RT-PCR and WB.It indicated that carbonic anhydrase 2(CA2),a member of the carbonic anhydrase family gene,might be a new target gene regulated by β-catenin/TCF7L2 pathway.6.According to the screening data of RNA sequencing,the protein and m RNA levels of CA2 in hearts of diabetic mouse and high glucose and high insulin treated cardiomyocytes were detected by WB,RT-PCR and IF to determine the expression and distribution of CA2 in diabetic cardiomyopathy.7.SKL2001 and Lenti-Tcf7l2-sh RNA were administered to cardiomyocytes treated with high glucose and high insulin.The experimental groups were divided into high glucose and high insulin group(GLU + INS),high glucose and high insulin +Lenti-GFP group(GLU + INS + GFP),high glucose and high insulin +Lenti-Tcf7l2-sh RNA group(GLU + INS + sh RNA),and high glucose and high insulin +Lenti-GFP + SKL2001 group(GLU + INS + GFP + SKL),high glucose and high insulin + SKL2001 + Lenti-Tcf7l2-sh RNA group(GLU + INS + SKL +sh RNA).WB and PCR were used to detect CA2 in each group,and it was confirmed that TCF7L2 was the key nuclear transcription factor forβ-catenin-induced up-regulation of CA2 expression.8.To further verify that Car2 is the downstream target gene of β-catenin/TCF7L2,the binding changes of β-catenin and TCF7L2 in the CA2 promoter region were10 studied by Chromatin immunoprecipitation(Chip)with myocardial tissue from the mice in each group.9.The expression of CA2 was inhibited by CA2-si RNA in primary cultured cardiomyocytes and then SKL2001 was administered.The experimental groups were divided into blank control group(NC),CA2-si RNA group(CA2-si RNA),blank control + SKL2001 group(NC+SKL),and CA2-si RNA + SKL2001 group(CA2-si RNA +SKL).The role of CA2 in β-catenin/TCF7L2 activation induced diabetic cardiac hypertrophy was determined by WB,RT-PCR and IF to detect related indexes of cardiac hypertrophy.Results1.With the prolongation of diabetic time,random blood glucose and fasting blood glucose in DM group increased gradually,and random blood glucose ≥ 16.7mmol/L.The blood glucose of glucose tolerance test mice did not recover after 2h.Randomized blood glucose and fasting blood glucose were stable in CON group.Western blot results showed that compared with CON group,the protein expression of Active β-catenin,β-catenin,TCF7L2 and c-Myc in heart of mice in type 2 diabetes group was increased.RT-PCR results showed that compared with CON group,m RNA expression of β-catenin and Tcf7l2 in heart of diabetic mice were up-regulated.Immunohistochemical results showed that the expression ofβ-catenin was increased,and the expression of TCF7L2 was increased in the nucleus of diabetic mice.These results indicate activation of the Wnt/β-catenin/TCF7L2 signaling pathway in myocardial tissue of type 2 diabetic mice.2.After 16 weeks of continuous administration of β-catenin/TCF7L2 binding inhibitors i CRT14 and positive control drug RSG,there was no significant change in random blood glucose,and fasting blood glucose decreased significantly after12 weeks of administration,glucose tolerance tests also did not return to pre-glucose levels.The results of echocardiography showed that in DM group,the thickness of left ventricular anteroposterior wall and anteroposterior wall at the11 end of diastole and systolic became thinner,the ejection fraction decreased and ratio of E peak(MVE)/A peak(MVA)and mitral valve flow peak area(MVE /A VTI)decreased.In each group treated with i CRT14,the anterior and posterior wall thickness at end diastolic and end systolic of the left heart increased,the ejection fraction increases and ratio of mitral valve flow peak E(MVE)/A(MVA)and mitral valve flow peak area(MVE /A VTI)increased.After RSG administration,the anterior and posterior wall thickness at the end of diastolic end of the left ventricular wall and the end of systolic end of the left ventricular wall increased in all groups,the ejection fraction increases and ratio of mitral valve flow peak E(MVE)/A peak(MVA)and mitral valve flow peak area(MVE /A VTI)increased.The results of HE staining showed that the cross-sectional area of myocardial cells in the DM group increased,and the cross-sectional area of myocardial cells in the i CRT14 group decreased to varying degrees.Masson staining results showed that the area of myocardial fibrosis was increased in the DM group,and the area of myocardial fibrosis in the i CRT14 dose groups was reduced to varying degrees,while the area of myocardial fibrosis in the RSG group was reduced to a certain extent.WB results showed that the expression of Active β-catenin β-catenin c-Myc Atrial natriuretic peptide(ANP)was increased in the DM group,and the expression of these proteins was significantly down-regulated in the i CRT14 group and RSG group.These results suggest that i CRT14 and RSG can significantly improve cardiac remodeling in diabetes mouse.3.Cardiomyocytes of primary neonatal rat were treated with high glucose and insulin for 48 hours.WB results showed that compared with CON group,the expression of Active β-catenin,β-catenin and ANP increased in(GLU + INS)group;Compared with GLU + INS group,the expression of Active β-catenin,β-catenin and ANP increased in(GLU + INS + SKL)group.The expressions of Active β-catenin,β-catenin and ANP were all decreased in the high glucose and high insulin + i CRT14 concentration groups.The IF results showed that the surface area of myocardial cells increased in the GLU + INS group compared with12 the con group.Compared with GLU + INS group,the surface area of myocardial cells increased in GLU + INS + SKL group,but decreased in high glucose and insulin + i CRT14 5 μmol/L group.These results indicate that high glucose and High insulin can induce cardiac hypertrophy,and i CRT14 administration can reduce the hypertrophy caused by high glucose and High insulin.4.RNA-seq results showed that lentiviral vectors Lenti-Tcf7l2-sh RNA and LentiTcf7l2-WT could significantly knock down or overexpress Tcf7l2 in cardiac cells.The expression of carbonic anhydrase gene family(CAs)was significantly changed by differential genes were obtained by screening.Further verification by WB results showed that the expression of proteins of CA2 and CA4 was increased in the Lenti-Tcf7l2-WT group,and significantly decreased in the Lenti-Tcf7l2-sh RNA group.These results suggest that Car2 and Car4 may be new potential target genes of β-catenin/TCF7L2 pathway.5.Subsequently,transcriptome sequencing results were further investigated in myocardial tissue of diabetic mice and myocardial cells treated with high glucose and high insulin.WB and RT-PCR results showed that m RNA levels of CA2 and CA4 proteins were increased in cardiomyocytes treated with high glucose and high insulin.m RNA expression of CA2 protein was increased in type 2 diabetic mice.However,only m RNA expression of CA4 was increased,while protein expression was not significantly changed.These results suggest that Car2 may be a new target gene regulated by β-catenin/TCF7L2 in diabetic cardiomyopathy.6.WB and RT-PCR results showed that the expression of CA2 protein and m RNA in diabetic mice treated with i CRT14 decreased in a dose-dependent manner compared with that in diabetic mice treated with i CRT14.The IF results showed that CA2 was mainly expressed in cardiomyocytes cytoplasm,and the expression of CA2 was increased in the DM group,but decreased after i CRT14 administration.The results of in vitro experiments were basically consistent with those of the above in vivo experiments.WB RT-PCR IF results showed that the m RNA level of CA2 protein was increased in the myocardial cells of GLU + INS group.Compared with GLU + INS group,CA2 protein m RNA expression was further up-regulated in GLU + INS + SKL group.However,the m RNA expression of CA2 protein was decreased in GLU + INS + i CRT14 groups.The results of the above models of diabetic cardiomyopathy in vitro and in vivo showed that the expression trend of CA2 was consistent with that of β-catenin.These results suggest that the β-catenin pathway may induce cardiac hypertrophy through the activation of CA2 in diabetic cardiomyopathy.7.SKL2001 and Lenti-Tcf7l2-sh RNA was used to treat cardiomyocytes induced by high glucose and insulin.WB results show compared with Glu +INS+GFP group,CA2 expression were decreased in GLU + INS + sh RNA group.Compared with GLU + INS + GFP + SKL + sh RNA group,CA2 expression was also significantly decreased in GLU + INS + SKL + sh RNA group.These results suggested thatβ-catenin entry into the nucleus in diabetes mainly up-regulated CA2 expression through TCF7L2.8.The results of Chip-q PCR showed that TCF7L2 binding site existed in the promoter region of CA2 gene.In diabetic cardiomyopathy,the binding ofβ-catenin TCF7L2 to the CA2 promoter region was enhanced.The binding ofβ-catenin TCF7L2 to the CA2 promoter region was weakened after i CRT14 administration.These results suggest that CA2 is the downstream target gene ofβ-catenin/TCF7L2 pathway.9.Inhibition of CA2 expression by si RNA further confirmed the role of CA2 inβ-catenin/TCF7L2 activation leading to cardiac hypertrophy.The results showed that the inhibition of CA2 expression by si RNA(CA2-si RNA)in cardiac hypertrophy induced by β-catenin agonist SKL2001 significantly inhibited the expression of ANP protein,Nppa m RNA,and reduced the surface area of cardiomyocytes.Conclusion1.In type 2 diabetes,the canonical Wnt/β-catenin/TCF7L2 signaling pathway to activate,effective suppression β-catenin/TCF7L2 signaling pathway can improve diabetic cardiomyopathy.2.RNA-seq screening and in vivo studies confirmed that CA2 is a downstream target gene of the canonical Wnt/β-catenin/TCF7L2 signaling pathway,β-catenin/TCF7L2 directly induces CA2 gene transcription and induces diabetic cardiac hypertrophy.3.Inhibition of CA2 expression can partially reverse the myocardial hypertrophy induced by β-catenin/TCF7L2 pathway activation. |
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