ANO1 Inhibits Myocardial Fibrosis After Myocardial Infarction By Inhibiting The Phosphorylation Of Smad3

Background: Coronary Heart Disease(CHD)is the first of top 10 global cause of motality,and myocardial infarction(MI)is the main cause of death among CHD patients.Although the reperfusion therapy have reduced the motality in acute MI,the cardiac remodeling after MI causes heart failure.Cardiac fibrosis is one of the most important factors of cardiac remodeling after MI,excessive fibrosis impairs the compliance of left ventricle,results in heart failure,arrhythmia and sudden death.Therefore,inhibiting cardiac fibrosis is impotant for improvement of the prognosis of MI.ANO1,which encoding for Calcium activated Chloride Channels,plays important role in many physiopathologic processes like epithelial secretion,smooth muscle contraction,neuronal excitability,nociception,tumorigenesis,cell proliferation and so on.We have found ANO1 inhibits cell proliferation and differentiation under hypoxia,however,the effect of ANO1 on cardiac fibrosis post-MI and the underlying mechanism is still elusive.Object: The study aimed to illustrate the effect and mechanism of ANO1 on TGF-β1-induced cardiac fibroblasts(CFs)proliferation and differentiation and cardiac fibrosis after myocardial infarction(MI).Methods: 1.CFs were isolated from hearts of one-to-three-day-old neonatal mice and cultured,the second or third passage of cells was used in the following experiments.2.Whether ANO1 was expressed in the mouse CFs was examined by immunofluorescence analysis.3.CFs were treated with TGF-β1 with different concentration(0,5ng/ml,10ng/ml,20ng/ml)for 48 hours,then the proliferation of each group was measured by MTT and the protein expression of a-SMA was determined by Western blotting.4.CFs were divided into two groups: control group and TGF-β1(10ng/ml)group.The two groups of cells were treated with or without TGF-β1 10ng/ml for 48 hours,then the protein and m RNA expression of ANO1 were examined by Western blotting and q RT-PCR.5.CFs were divided into two groups: Adenovirus vector with green fluorescent protein(Ad-GFP)group and Adenovirus vector with Anoctamin1(Ad-ANO1)group.CFs were transfected with Ad-GFP or Ad-ANO1,Western blotting and q RT-PCR were used to evaluate the protein and m RNA expression of ANO1.6.CFs were divided into the following groups: Ad-GFP group,Ad-GFP+TGF-β1 group,Ad-ANO1 group and Ad-ANO1+TGF-β1 group.After transfected with Ad-GFP or Ad-ANO1,CFs were treated with or without TGF-β1 10ng/ml for 48 hours.MTT was used to measure the cell viability of each group,Western blotting and q RT-PCR were used to determine the protein and m RNA expression of a-SMA and Western blotting was used to determine the phosphorylation of Smad3.7.MI was induced by ligating the left anterior descending coronary artery(LAD)of six-to-eight-week-old C57Bl/6 mice.ECG was performed after the operation to testify whether MI model was successful.8.Two weeks after operation,mice in MI or sham group were sacrificed,Masson’s trichrome staining was used to measure the fibrotic area in the infarcted zone,bordering zone,remote zone of the infarcted heart tissue and in the heart tissue of the sham group.9.Immediately after MI,45 ul Ad-ANO1 or Ad-GFP(1*1010 pfu/ml)was injected into the left ventricular wall bordering the infarction zone at three different points.Western blotting and q RT-PCR were used to determine the protein and m RNA expression of ANO1 one week and two weeks after the injection.10.Mice were randomly divided into four groups: sham+Ad-GFP,sham+ Ad-ANO1,MI+ Ad-GFP and MI+ Ad-ANO1.Cardiac structure and function was measured by echocardiography two weeks later.Then mice were sacrificed,we weighed the body weight(BW)and heart weight(HW)of mouse in each group and calculated the ratio of HW versus BW.The fibrotic area was examined by Masson’s trichrome staining and the expression of a-SMA was determined by Western blotting and immunofluorescence staining and p-Smad3/Smad3 was measured by Western blotting.Results 1.ANO1 was expressed in neonatal mouse cardiac fibroblasts(NMCFs)as determined by immunofluorescence staining.2.Compared to the control group,the cell viability of CFs treated with different concentration of TGF-β1(5ng/ml,10ng/ml,20ng/ml)both increased significantly(P<0.001)and the protein expression of a-SMA also increased(P<0.05).After treatment with TGF-β1 10ng/ml for 48 hours,the protein and m RNA expression of ANO1 was reduced significantly(P<0.05).3.When CFs were transfected with Ad-ANO1,the protein and m RNA expression of ANO1 increased obviously(P<0.001).4.The TGF-β1-induced proliferation of CFs was inhibited by ANO1 overexpression(P<0.001).5.The protein and m RNA expression of a-SMA induced by TGF-β1 was attenuated by ANO1 overexpression(P<0.05).6.The phosphorylation of Smad3 induced by TGF-β1 was inhibited by ANO1 overexpression(P<0.05).7.The protein and m RNA expression of ANO1 in myocardium transfected with AdANO1 was increased gradually one week and two weeks after the transfection(P<0.05).8.Echocardiography showed:the ejection fraction(EF)and fraction shortening(FS)were increased significantly in MI+Ad-ANO1 group as compared with MI+AdGFP group(P<0.05).Although Left ventricular internal diameter at diastole(LVIDd)and Left ventricular internal diameter at systole(LVIDs)showed the trend to decline,the difference did not reach significance(P>0.05).9.The ratio of HW versus BW(HW/BW)showed that: HW/BW in MI+ Ad-GFP group increased significantly compared to that in sham+Ad-GFP group and sham+ Ad-ANO1 group(P<0.05),and HW/BW in MI+ Ad-ANO1 group was reduced comparing with that in MI+Ad-GFP group(P<0.05).10.Overexpression of ANO1 reduced the fibrotic area(P<0.05)in the bordering zone of the infarcted hearts and overexpression of ANO1 also reduced the number of aSMA and vimentin positive staining cells in the bordering zone of the infarcted hearts(P<0.001). 11.The phosphorylation of Smad3 after MI increased significantly and it was repressed significantly by overexpression of ANO1(P<0.001).Conclusion 1.ANO1 is expressed in NMCFs.2.ANO1 overexpression inhibits TGF-β1-induced proliferation and differentiation of CFs.3.ANO1 overexpression can improve the cardiac function and cardiac fibrosis after MI.4.ANO1 overexpression represses the phosphorylation of Smad3 induced by TGF-β1 and MI.5.ANO1 inhibits cardiac fibrosis after myocardial infarction might through repressing phosphorylation of Smad3.