ANO1 Inhibits Cardiac Fibrosis After Myocardial Infraction Via TGF-?/smad3 Pathway

BackgroundMyocardial fibrosis after myocardial infarction leads to reduced oxygen delivery,poor electrical signal conduction,with subsequent reduced cardiac diastolic and systolic function,et al.So it is very important to prevent and improve myocardial fibrosis after myocardial infarction.However,despite decades of intense research,effective treatment for myocardial fibrosis remains elusive.Until 2008,the molecular basis of classical calcium calcium activated chloride channel(CaCC)was identified,Anoctamin 1(ANO1),which has led to a worldwide upsurge on ANOl research.Numerous studies have shown that ANO1 is involved in many physiological processes,such as cell proliferation,migration and differentiation,which support for our hypothesis that ANO1 may be involved in myocardial fibrosis after myocardial infarction.ObjectiveThis study investigated the effects of ANO1 on myocardial fibrosis after myocardial infarction and the mechanisms involved.Methods1.Cardiac fibroblast isolation and culture Neonatal rat cardiac fibroblasts were isolated from 1-3-day-old Sprague-Dawley rats.The second to third passage cardiac fibroblasts were used in the following experiments.2.construction of recombinant adenovirus vector encoding ANO1 and cardiac fibroblasts infection in rats ANO1 recombinant adenovirus vector was constructed by Shanghai Ji Kai gene Chemical Technology Co.,Ltd.The overexpression of ANO1 was verified by immunoblotting for ANO1 24 hours after infection.3.Cardiac fibroblasts hypoxia The second to third passage cardiac fibroblasts were incubated in a GENbag anaer at 37 ? with 5%CO2,1%02 and 94%N2 for 6,8,10,16,and 24 hours,after serum starvation.Then the cell proliferation was detected by MTT;the expression of ANO1 mRNA was detected by qRT-PCR;and the expression of ANO1 protein was detected by western blotting.4.Animal model of MI and adenovirus transfer in vivo C57B1/6J male mice,age of 8-10 weeks,were used for the experiments.Mice were divided into four groups randomly::sham,MI,Ad-GFP+MI and Ad-ANO1+MI.The left anterior descending coronary artery(LAD)was ligated to induce MI.Briefly,mice were anesthetized with 1%sodium pentobarbital and then intubated and ventilated during the operative process.The LAD was ligated about 2mm from the tip of the left auricle with 7-0 silk suture in MI group.The same procedure was performed in sham group without the ligation of the suture under the LAD.MI was confirmed by the elevation of S-T segment on an electrocardiogram.Mice in Ad-GFP +MI and Ad-ANO1+MI groups were given intramyocardial injection of 45ul Ad-GFP or Ad-ANOl(1*1010PFU/ml)into the left ventricular wall bordering the infarction zone via a 30-gauge Hamilton needle,while mice in the sham and MI groups received the same amount of saline.The animals were sacrificed 1 week after surgery for further analysis.The research was approved by the ethical committee of Nanjing Medical University and all animal experiments were performed in compliance with the guidelines on humane use and care of laboratory animals for biomedical research published by National Institutes of Health(No.85-23,revised 1996).5.The effect of ANO1 overexpression on myocardial fibrosis after hypoxia Overexpressed ANO1 in myocardial fibroblasts,then treated with hypoxia.We tested cell proliferation by MTT,ANO1 mRNA expression by qRT-PCR,and fibrosis related proteins by Western blot.6.The effect of ANO1 overexpression on myocardial fibrosis after myocardial infarction After constructing ANO1 overexpression mouse model,we ligated the anterior descending branch of the coronary artery to establish the myocardial infarction model.7.To verify the mechanisms of ANO1 on cardiac fibrosis after MI The expression of TGF-beta,p-smad3 and related fibrosis proteins after hypoxia or/and ANO1 overexpression were detected by Western blot.8.Statistical analysis Statistics were performed using GraphPad Prism software(GraphPad Software,Inc.,CA,USA).All data was analyzed by SPSS 17.0 software(SPSS Inc,Chicago,Illinois,2008).All experiments were repeated with at least three batches of cardiac fibroblasts,and qualitatively similar data were obtained in all repetitions.Data were tested with Analysis of Variance(ANOVA)or t-test,with P<0.05 considered significantResults1.Evidence of ANO1 in the neonatal rat cardiac fibroblasts Cardiac fibroblasts and myocardial cells of neonatal rats were isolated to detect ANO1 mRNA and protein expression.The expression of ANO1 mRNA in cardiac fibroblasts was detected by qRT-PCR analysis and ANO1 protein by western blotting analysis,which were significantly higher than those in myocardial cells.Furthermore we stained cardiac fibroblasts(red),ANOl protein(green),and nucleus(blue)by immunofluorescent confocal microscopic analysis.These results demonstrated ANO1 protein existed in the cardiac fibroblasts of neonatal rats and the expression was higher than that in myocardial cells.2.ANO1 expression increased cardiac fibrosis after hypoxia MTT analysis verified that cell viability at the point of 6,8,10,16,and 24 hour increased gradually after hypoxia.According to western blotting analysis,compared to untreated group,the level of a-SMA(marker of myofibroblasts)at the point of 6,8,10,16,and 24 hour increased gradually after hypoxia and peaked at the point of 24 hour.These results demonstrated that cardiac fibroblast activated after hypoxia(e.g.fibroblasts proliferation)developed into myofibroblasts and set off cardiac fibrosis.Then we detected ANO1 mRNA and protein expression in cardiac fibroblasts 6,8,10,16,and 24 hours after hypoxia by qRT-PCR,and western blotting.The expression of ANO1 mRNA and protein increased as hypoxia went on in the first 24 hours,and peaked 16 hours after hypoxia.These results suggest that ANO1 participates in cardiac fibrosis after cardiac fibroblasts hypoxia.3.Expression of ANO1 increased in myocardial infraction(MI)mice Western Blot was used to evaluate the expression of ANO1 in MI mice.ANO1 increased more significantly in MI group compared with the sham group.4.Overexpression of ANO1 reduced the indicators of cardiac fibrosis after hypoxia To investigate the function of ANO1 in cardiac fibroblasts,we transduced adenovirus-encoding ANO1(Ad-GFP-ANO1)or GFP(Ad-GFP)into cardiac fibroblasts.According to qRT-PCR and western blotting,ANO1 mRNA and protein expression increased dramatically in ANO1 over-expression group compared with control group,respectively.These results proved that ANO1 expression model was well constructed.Secondly,cell viability elevated in overexpression ANO1 + hypoxia 16 hours(overexpression H)group compared with overexpression ANO1(over H)group and negative control(NC)+ hypoxia 16 hours(NC H)group.This result demonstrated that ANO1 over-expression inhibited the proliferation of cardiac fibroblasts after hypoxia.Thirdly,according to Western blotting,a-SMA and collagen I protein levels were reduced in over-expression H group compared with over-expression group and NC H group.We summarized that the over-expression of ANOl inhibited cardiac fibrosis after hypoxia.5.ANOl alleviated fibrosis after MI To reveal the role of TMEM16A in fibrosis after MI,Ad-GFP or Ad-ANO1 was injected into the left ventricular(LV)wall immediately after MI.Western blot showed the protein of ANO1 increased significantly in mice with Ad-ANO1 compared to mice with Ad-GFP,which confirms the successful gene transfer.We examined myocardial fibrosis using Masson's Trichrome staining.Obvious fibrosis was observed in the infarcted margin.Fibrosis decreased significantly in the Ad-ANO1 group compared with MI and Ad-GFP group.Myofibroblast is responsible for interstitial matrix and consequent left ventricle(LV)deformation caused with fibrosis.We evaluated the effect of ANO1 on cardiac myofibroblast differentiation in MI mode.Western blot showed ANO1 reduced the expression of a-SMA significantly.Immunofluorescence analysis also revealed that ANOl reduced the number of a-SMA and vimentin positive staining cells in the bordering zone.6.ANO1 inhibits cardiac fibrosis via TGF-?/smad3 pathway To understand how ANO1 regulated cardiac fibrosis in cardiac fibroblasts,we detected the expression of fibrosis in cardiac fibroblasts infected with Ad-GFP-ANO1.According to western blotting analysis,protein levels at the point of 6,8,10,16,and 24 hours after hypoxia,TGF-? and p-smad3 increased gradually and peaked at 16 and 24 hour compared to untreated group.The protein expression of TGF-? and p-smad3 were consistent with ANO1 and fibrosis indicators expression after hypoxia above.Moreover,after the transfection of cardiac fibroblasts with Ad-GFP-ANO1 and their 16-h exposure to hypoxia,we found that TGF-? and p-samd3 protein levels decreased in group of over-expression H group compared with over-expression group or negative control(NC)H group by western blotting.These data indicate that ANO1 inhibits cardiac fibrosis via deregulating TGF-?/smad3 pathway.ConclusionsANO1 inhibits cardiac fibrosis after myocardial infraction via TGF-?/smad3 pathway.