Study Of TGF-β1 Regulated By Endothelin-1 On Atrial Fibrosis In Atrial Fibrillation

Background and Objective Atrial fibrillation(AF)is the most encountered arrhythmia in clinical practice and a serious threat to human life and health.The fundamental mechanisms of AF is complex,so it have long been debated.Electrical and structural remodeling of atrial are important contributors to the AF occurrence and persistance.The electrical remodeling of atrial is easy to reverse,however,the atrial structural remodeling is difficult to reverse.Atrial fibrosis is a hallmark of structural remodeling.Therefore,the exploration of the mechanism of atrial fibrosis and find effective ways of blocking atrial fibrosis may provide a new theory to prevent atrial structural remodeling of AF.Recent studies shown that the Endothelin-1(ET-1)may be involved in atrial fibrosis of AF by regulating fibrosis related factors.Transforming growth factor-β1(TGF-β1)is the most important profibrotic molecule which participated in atrial fibrosis and plays an important role in the pathogenesis of AF.It had demonstrated that ET-1 is an upstream regulator of TGF-β1,P38mitogen-activated protein kinase(p38MAPK)is an important downstream signaling pathway of ET-1 in the cardiomyocyte.Whether ET-1 regulates the expression of TGF-β1 in cardiomyocyte through the p38 MAPK signaling pathway and promoting atrial fibrosis of AF remains to be studied.In this study,we detected the changes of fibrosis,ET-1 and TGF-β1 in atrium of patients and canines with AF.Furthmore,ET-1 combined with ET-1 receptor antagonist or p38 MAPK signaling pathway antagonist was used to intervene the H9c2 cell.In order to explore the mechanisms of TGF-β1 regulated by ET-1 on atrial fibrosis in AF.Method 74 patients with valvular heart disease undergoing thoracotomy surgery were included in our study,40 patients with AF and 34 patients with sinus rhythm(SR).The right atrial appendage tissue(RAA)was collected before cardiopulmonary bypass.Sixteen male canines were randomized into 4 groups:Sham,AF lasting 1 week(1W),AF lasting 2 weeks(2W)and AF lasting 4weeks(4W)(n=4,each).A canine model of AF was made by rapid left atrial pacing.The tissues in left atrial(LA)were obtained from canines in each group.Collagen fiber deposit and collagen volume fraction(CVF)was assessed by Masson’s staining.Real-time quantitative polymerase chain reaction(RT-q PCR)was applied to detect the m RNA expression levels of ET-1 and TGF-β1.Western blot(WB)was used to detect the protein expression levels of ET-1 and TGF-β1.Then compared the m RNA and protein expression of TGF-β1 in H9c2 cell which was stimulated with ET-1 in different concentrations and different time,and which was cultured with ET-1 combined ET-1 receptor antagonist sulfisoxazole(SIZ)or p38 MAPK signaling pathway antagonist SB203580.ELISA(enzyme-linked immune-sorbent assay,ELISA)was applied to measured p38 MAPK and Phospho-p38 MAPK expression in H9c2 cell intervened by ET-1with or without SIZ.Results (1)The CVF of RAA in patients with AF(29.66%±4.50%)was significantly higher than in patients with SR(7.59%±2.41%)(P <0.01).(2)The ET-1 m RNA expression levels in AF group(2.3918±1.4856)was significantly higher than in SR group(1.1379±0.7944)(P <0.01);the protein expression levels of ET-1 in AF group(0.5601±0.1417)was significantly higher than in SR group(0.2432±0.0328)(P <0.01).(3)The TGF-β1 m RNA expression levels in AF group(3.0448±2.8028)was significantly higher than in SR group(1.2283±0.7480)(P <0.01);the protein expression levels of TGF-β1 in AF group(0.6420±0.1976)was significantly higher than in SR group(0.2510±0.0386)(P<0.01).(4)In the RAA of two groups,there is a significantly positive correlation between the protein expression of ET-1 and CVF(r=0.757,P<0.01),there is also a significantly positive correlation between the protein expression of TGF-β1 and CVF(r=0.734,P<0.01);meanwhile,there is a significantly positive correlation between the protein expression of ET-1 and TGF-β1(r=0.686,P<0.01).(5)With prolonged AF duration,the m RNA and protein expression of ET-1 and TGF-β1 in LA of canines gradually increased,besides CVF(P <0.05).(6)ET-1 can stimulate H9c2 cells to secrete TGF-β1,while SIZ and SB203580 can block this function of ET-1 independent.(7)ET-1 can activate p38 MAPK in H9c2 cells,while p38 MAPK activation by ET-1 was inhibited by treatment with SIZ.Conclusion ET-1 may regulate TGF-β1 by p38 MAPK signal pathway involved in atrial fibrillation.