Role Of Activin A-follistatin System In Left Ventricular Remodeling In Heart Failure

Activin A is a member of the transforming growth factor-βsuperfamily. It is involved in the acute-phase response and fibrosis . Follistatin is an activin-binding protein and is measurable in extracellular matrix and serum . Activins trapped by follistatin are then internalized by endocytosis and are subsequently degraded by proteolysis.It has been reported that the expressions of activin A and its receptors in ischemic and nonischemic regions markedly increased after myocardial infarction (MI) and that activin A increased gene expression of atrial natriuretic peptide(ANP), brain natriuretic peptide (BNP), and matrix metalloproteinase-9(MMP-9), the factors known to play an important role in myocardial remodeling and interstitial fibrosis. The coexpression of activin A and follistatin is vital in tissue remodeling and repair. The imbalance of activin A-follistatin system may induce pathological changes in tissues. Overexpression of activin in the skin of transgenic mice caused dermal fibrosis and epidermal hyperthickening, and in the epidermis of transgenic mice in which the activin antagonist follistatin was overexpressed, the skin was characterized by a mild dermal and epidermal atrophy, significantly reduced granulation tissue formation, and smaller scar area. These studies implicate an important function of endogenous activin in wound repair and scar formation. Follistatin, the activin antagonist, may prevent activin A-induced fibrosis and play an important role in the treatment of fibrosis.The purpose of this study was to determine whether the imbalance of activin A-follistatin systme is involved in the myocardial fiborsis and the subsequent heart failure after MI. For this purpose, we analyzed the relationship between the imbalance of activin A-follistatin system and the left ventricular remodeling in the rat model of heart failure after MI. Non-infarcted ventricular remodeling in heart failure plays an important role, so this study focused on non-infarcted area of left ventricular remodeling.1 Clinical study.1.1 MethodsBetween March and December 2008, we enrolled 87 inpatients and outpatients with congestive heart failure (CHF) and 44 healthy control subjects. The CHF patients were 63 to 85 years of age (mean age, 72.88±7.35 years); 46 were men and 41 were women. CHF was diagnosed according to ESC Guidelines for the Diagnosis and Management of Chronic Heart Failure in the Adult 2005. According to the New York Heart Association (NYHA) functional classification system released in 1994, the CHF patients were classified into NYHA II group (n=30), NYHA III group (n=26), and NYHA IV group (n=31). The healthy control subjects were 63 to 84 years of age (mean age, 73.77±7.07 years); 20 were men and 24 were women. None of the participants had concomitant disease such as lung, liver, and kidney diseases, diabetes, and other endocrine diseases. Written informed consent was obtained from each participant.The venous blood of 3 ml was drawn from each fasting individual in the morning and centrifuged at 1500 r/min for 15 minutes. The serum was stored at -80℃until analyzed. Serum levels of activin A,FS and BNP were analyed by enzyme-linked immunosorbent assay according to the instruction (ADL ELISA Kit, USA).1.2 ResultsThe levels of activin A and BNP were significantly higher in sera of all patients with NYHA II, NYHA III, and NYHA IV than that in healthy control subjects (P<0.05～P<0.001); and it was significantly higher in NYHA IV groups, compared with NYHA II group (P<0.05). Although the levels of follistatin increased slightly in sera of all patients with NYHA III, the differences in all proteins were not significant, compared with that in healthy control subjects (P>0.05). The data show that patients with heart failure significantly increased the levels of Activin A in serum, Activin A levels and degree of heart failure and BNP levels were positively correlated, the correlation coefficient and P values were (R = 0.9350, P <0.001), (R = 0.8918 , P <0.001).2 The role of activin A and follistatin in left ventricular remodeling in a rat model of heart failure2.1 MethodsFemale Wistar rats were subjected to the ligation of the anterior descending branch of the left coronary artery or sham operation during halothane anesthesia (1% halothane in a mixture of one third O2 and two thirds N2O) anesthesia as previously described . Post-operated rats were divided into 6 groups (n=6 each group): SO group, sham-operated rats receiving 0.5% CMC; SO-ramipril group, sham-operated rats treated with ramipril (3 mg/kg/day); SO-telmisartan group, sham-operated rats treated with telmisartan (30 mg/kg/day); MI group, MI rats receiving 0.5% CMC; MI-ramipril group, MI rats treated with ramipril (3mg/kg/day), and MI-telmisartan group, MI rats treated with telmisartan (30 mg/kg/day). All treatment regimens were initiated 6 weeks after surgery and continued for 5 weeks. Ramipril (C7002) and telmisartan (602328) were provided by Sanofi-Aventis Pharma. Beijing Co., Ltd and Boehringer Ingelheim International Trade (Shanghai) Co., respectively.After 11 weeks,the Hemodynamic measurements were observed ,the rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30mg/kg). A catheter was inserted into the right carotid artery and then further advanced into the left ventricular chamber to record left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and rate of contraction and relaxation (±dp/dt). The systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were also recored. After the cardiac function was recorded, 3 ml of the arterial blood was drawn from abdominal aorta and centrifuged at 2500 r/min for 10 minutes. The serum was stored at -70℃. The rats were then killed by decapitation, and the hearts were immediately removed. The heart weight and left ventricular mass were measured, and the ratio of heart weight to body weight (HW/BW) and the ratio of left ventricular mass to body weight (LVM/BW) were calculated.The infarcted and noninfarcted left ventricular tissue was separated. The noninfarcted left ventricular tissue was cut into two parts along the longitudinal axis of the left ventricle. One part were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5-μm sections. morphological changes in the myocardium, sections (5μm) were stained by hematoxylin and eosin (H&E) and Masson's trichrome.The expressions of activin A and follistatin proteins were detected immunohistochemical stain. Another part was stored at -70℃for subsequent determination of the protein expressions of activin A, follistatin, and angiotensin II (Ang II) and the mRNA expressions of activin A, type II A and II B activin receptor (ActR II A and ActR II B), follistatin, and type I and III collagen (col-I and col-III).2.2 results2.2.1 Gross morpholgical evidenceRamipriland telmisartan could decrease the volume of heart of VR rats, limit the infarct area, lower the rate of aneurysm and reduce the expansion of ventricular,and could increase the number of residual cardialcytes in infarct area and reduce the cytopathic around the infarct area.2.2.2 Morphological changes in the myocardiumH&E stain showed that in SO, SO-telmisartan, and SO-ramipril groups, the cardiomyocytes were intact, and the arrangement of myocardial fibers was regular. In MI group, the arrngement of myocardial fibers were irregular, and various degrees of cardiomyocyte degeneration and hypertrophy and interstitial fibrosis were found. Compared with MI group, the above changes were less severe in MI-telmisartan and MI-ramipril groups.2.2.3 Cardiac functionHW/BW and LVM/BW were significantly higher in MI group than in SO group (both P<0.001) 11 weeks after the surgery, and they were also significantly higher in MI-telmisartan and MI-ramipril groups than in MI group (P<0.001, P<0.01). There were significant differences in both HW/BW and LVM/BW between MI-telmisartan and MI-ramipril groups (both P<0.05). Compared with SO group, SBP, DBP, LVSP, and±dp/dt significantly decreased and LVEDP significantly increased in MI group (P<0.01, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). In MI-telmisartan and MI-ramipril groups, LVSP, +dp/dt, and -dp/dt were significantly higher and LVEDP significantly lower, as compared with MI group (P<0.05, P<0.01, P<0.05, and P<0.05, respectively),but no significant between-group difference was found in HR, SBP, and DBP.2.2.4 Expressions of activin A, ActR II A, ActR II B, follistatin, col-I, and col-III mRNA in the noninfarcted area of the left ventricleThe expression levels of activin A, ActRIIA, ActRIIB, col-I, and col-III mRNA in the noninfarcted area of the left ventricle and the ratio of col-I to col-III were higher in MI group than in SO group, but the expression level of follistatin mRNA in MI group was significantly lower than that in SO group. Compared with MI group, the expression levels of activin A, ActR II A, ActR II B, col-I, and col-III mRNA and the ratio of col-I to col-III were lower and but the expression level of follistatin mRNA was higher in MI-telmisartan and MI-ramipril groups.2.2.5 Serum level of activin A, Ang II, and BNPThe serum level of activin A, Ang II, and BNP in MI group were significantly higher than those in SO group, and compared with MI group, their levels significantly decreased in MI-telmisartan and MI-ramipril groups. In MI-telmisartan and MI-ramipril groups, the expressions of these 3 proteins were down-regulated. In all group, the serum level of activin A was positively correlated to the levels of Ang II and BNP. The data indicated that expression of activin A protein increased after myocardial infarction, ramipril and telmisartan could improved cardiac function which may be associated with down-regulated expression of activin A protein.2.2.6 Levels of activin A, follistatin, and Ang II in the noninfarcted area of the left ventriucle.The levels of activin A and Ang II in MI group were significantly higher than those in SO group 11 weeks after the surgery, but the level of follistatin was signficantly lower in MI group than in SO group. Compared with MI group, the expressions of activin A and Ang II were down-regulated and the expression of follistatin was up-regulated in MI-telmisartan and MI-ramipril groups.The differences in the expression of these 3 proteins were more signficant between MI-telmisartan and MI groups .2.2.7 Expressions of activin A and follistatin identified by immunohistochemistry.Low expressions of activin A and follistatin were found in the noninfarcted area of the left ventricle in SO, SO-ramipril, and SO-telmisartan groups . In MI group, the expression level of activin A was up-regulated, and the expression of follistatin was down-regualted, while the converse was true in MI-telmisartan and MI-ramipril groups .2.2.8 Myocardial fibrosis after MIMasson's trichrome stain demonstrated the formation of myocardial fibrosis after MI. In SO,SO-ramipril, and SO-telmisartan groups, the arrangement of cardiomyocytes was regular, and no obvious green collagen fiber was found. In the noninfarcted area of rats in MI group, collagen fiber deposition and cardiomyocyte hypertrophy was found, the number of cardiomyocytes decreased, and the arrangement of cardiomyocytes was irregular. The above changes in MI-telmisartan and MI-ramipril groups were less severe than those in MI group.Conclusions1.Serum activin A was in a state of high expression in patients with heart failure,which was positively correlated with the degree of heart failure,furthermore,activin A level could be expected as a clinical serological indicator to make a diagnosis of heart failure, therapy and monitor this disease.2.The imbalance of activin A-follistatin system which is characterized by up-regulated expression of activin A is an important predisposing factor for myocaridal fibrosis and ultimately heart failure after MI. The down-regulated expression of follistatin could promote the development of heart failure after MI. Thus, up-regulating follistatin expression or exogenous follistatin and reversing the imbalance of activin A-follistatin system may be the effective ways to improve heart failure and prevent activin A-induced myocardial fibrosis.3.Activin A and type II activin receptors (ActR II A and ActR II B) were down-regulated, the expression of follistatin increased, and the collagen deposition decreased by using telmisartan and ramipril,which may be a new mechanism of action to telmisartan and ramipril in heart failure.