| Background and Aim: Heart failure(HF)has always been a difficult problem for cardiologist in clinical practice.It has the characteristics of high incidence,high readmission,and high mortality,which seriously affects the quality of life of patients.In recent years,heart failure with preserved ejection fraction(HFpEF)has gradually gained people's attention.If HF can be controlled in the HFpEF,then we can delay the process of developing to heart failure with reduced ejection fraction(HFrEF).The mechanisms of HF is still unclear,and it is thought to be related to ventricular remodeling and myocardial fibrosis.Ivabradine is a sinus node If current selective specific inhibitor,and some studies have suggested that Ivabradine can inhibit myocardial fibrosis in HF animal models and reverse ventricular remodeling,but its mechanism has rarely been reported.This study intends to establish a transverse aortic constriction(TAC)model to simulate the progression of HF from HFpEF to HF rEF,and to elucidate the effects of Ivabradine on myocardial fibrosis and ventricular systolic and diastolic dysfunction induced by pressure overload,and to explore the molecular mechanism of Ivabradine regulation of ventricular fibroblasts proliferation and transdifferentiation.At the same time,we explored its role in miR-133 a and TGF-?/Smad signaling pathways to provide a new theoretical basis for the prevention and treatment of HF myocardial fibrosis.Methods: Twenty 8-week-old C57BL/6J male mice were randomly divided into Sham group and TAC model group.The changes of body weight,heart rate and tail artery pressure were recorded before and after operation.The changes of cardiac structure and function were analyzed by echocardiography and hemodynamics at 4,8,12 and 17 weeks after operation.The cardiac structure and fibrosis were observed by H-E,masson staining,sirius red staining and immunofluorescence staining.The expression of miR-133 a and TGF-?/Smad signal pathway related proteins was detected by qRT-PCR and Western Blotting.The plasma NT-proBNP level was measured by ELISA.Four weeks after operation,mice were randomly divided into TAC-EFp model group,IVA-L group and IVA-H group.Nine weeks after operation,mice were randomly divided into TAC-EFr model group,IVA-L group and IVA-H group.Treatment for 8 weeks with IVA.Echocardiography and hemodynamics were used to analyze the changes of cardiac structure and function in different groups of mice.H-E,Masson staining,sirius red staining and immunofluorescence staining were used to observe the cardiac structure and the degree of myocardial fibrosis.The expression of miR-133 a and TGF-?/Smad signaling pathway related proteins was detected by qRT-PCR and Western Blotting.The plasma NT-proBNP level was measured by ELISA.Mapping lab and Langendorff were used to detect conduction velocity,conduction direction,conduction heterogeneity and ventricular tachycardia induction rate.Extract and culture ventricular fibroblasts(vFBs)of neonatal SD rat,and establish fibroblast transdifferentiation model by Ang ?.IVA was given at low and high dose intervention respectively.The expression of TGF-?/Smad signaling pathway related proteins and vFBs proliferation rate were measured by qRT-PCR,Western Blotting and CCK-8.MiR-133 a was overexpressed and silenced by lipofection transfection,miR-133 a was detected by qRT-PCR,CTGF,Collagen ? and Collagen ? was observed by qRT-PCR and Western Blotting,and vFBs proliferation was detected by CCK-8.After overexpression and/or silence of miR-133 a in vFBs by lipofection transfection,the fibroblast transdifferentiation model was established by Ang ?,and then IVA was given at low and high dose intervention respectively.The expression of CTGF,Collagen ? and Collagen ? was observed by qRT-PCR and Western Blotting,Results: DBP,IVSTd,LVPWd,IVRT,EDT,LVEDP and Tau increased,and LVIDs,E/A and-dp/dtmax decreased significantly at 4 weeks after TAC(p<0.05).HR,SBP increased(p<0.05),the significant difference of LVIDs disappeared(p>0.05),and LVEF,LVFS,+dp/dtmax had no significant changes at 8 weeks after TAC(p>0.05).At 9 weeks after TAC,LVIDs,LVIDd began to increase,LVEF,LVFS,+dp/dtmax began to decrease,NT-proBNP increased significantly(p<0.05),and it was positively correlated with the time after TAC.Pathological results showed that the CVF of TAC mice increased significantly(p<0.05),which was positively correlated with the time after TAC.With the lapse of time after TAC,the expression of miR-133 a decreased,and the expression of target genes CTGF and TGF-?/Smad signaling pathway related proteins increased significantly(p<0.05).After treatment with IVA in TAC-EFp mice,HR slowed down and BP remained unchanged in IVA-L mice and IVA-H mice;IVSTd,LVPWd,IVRT,EDT,LVEDP decreased and EF,FS,-dp/dtmax and + dp/dtmax increased significantly in IVA-H mice(p<0.05),but there was no significant difference in IVA-L mice.NT-proBNP,CVF decreased,while the expression of miR-133 a was up-regulated,and the expression of target genes CTGF and TGF-?/Smad signaling pathway was down-regulated significantly in IVA-L mice and IVA-H mice(p<0.05).There was no significant difference between IVA-L mice and IVA-H mice.After treatment with IVA in TAC-EFr mice,HR,NT-proBNP,CVF were decreased,and miR-133 a expression was increased in IVA-L mice and IVA-H mice,while CTGF and TGF-?/Smad signaling pathway related protein expression was decreased(p<0.05).But there was no change in BP,IVSTd,LVPWd,LVIDd,LVIDs,EF and FS.IVRT,EDT,LVEDP and Tau were decreased,and-dp/dtmax and + dp/dtmax were accelerated in IVA-H mice(p<0.05).IVRT,EDT and CVF in IVA-H mice,were lower than those in IVA-L mice(p<0.05).Electrophysiology showed,In TAC-EFr mice,LV conduction pattern was disordered,conduction heterogeneity and VT induction rate were increased(p<0.05).In IVA-H mice,LV conduction pattern tended to be normal,and conduction heterogeneity and VT induction rate were decreased(p<0.05).While there was no significant difference in RV conduction direction between groups.In Ang ? group,vFBs proliferation rate was increased,the expression of miR-133 a was decreased and the expression of TGF-?/Smad signaling pathway related protein was increased(p<0.05).After IVA treatment,the proliferation rate was decreased,the expression of miR-133 a was increased and the expression of TGF-?/Smad signaling pathway related protein was decreased(p<0.05).After overexpression of miR-133 a,the vFBs proliferation rate was decreased and the expression of CTGF,Collagen ? and Collagen ? was decreased(p<0.05).After overexpression of miR-133 a and stimulation within Ang ?,the expression of CTGF,Collagen ? and Collagen ? was down-regulated compared with Ang ? group and up-regulated compared with Ad-miR-133 a group(p<0.05).After silencing miR-133 a,the vFBs proliferation rate was increased and the expression of CTGF,Collagen ? and Collagen ? was up-regulated(p<0.05).After silence of miR-133 a with Ang ? stimulation and IVA intervention,the expression of CTGF,Collagen ? and Collagen ? in Si-miR-133a+Ang ?+IVA group was significantly up-regulated compared with Si-NC+Ang ?+IVA group,and significantly down-regulated compared withSi-miR-133a+Ang ? group(p<0.05).There was no significant difference between Si-NC+Ang ? group and Si-miR-133a+Ang ?+IVA group.Conclusions: Four weeks after TAC,TAC mice showed left ventricular hypertrophy,left ventricular diastolic dysfunction and myocardial fibrosis,which could simulate the pathological process of HFpEF myocardial fibrosis.Nine weeks after TAC,TAC mice showed left ventricular hypertrophy,left ventricular enlargement,left ventricular diastolic and systolic dysfunction and myocardial fibrosis,which could simulate the pathological process of HFrEF myocardial fibrosis.IVA can inhibit myocardial fibrosis by regulating miR-133 a and TGF-?/Smad pathways,thereby improving ventricular remodeling and reducing the inducing rate of VT.The improvement of cardiac function in HFpEF mice is more significant than that in HFrEF mice,suggesting the importance of early intervention in HF. |
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