Regulation Of Blockade Of TGF-β1 And Wnt Signal Pathway On The Process Of Myocardial Fibrosis After Myocardial Infarction In Rats

Myocardial infarction (MI), an ischemic cardiomyopathy caused by occlusion of coronary arteries or its branches, often leads to left ventricular remodeling, advances on heart failure (HF) gradually, and is a threat against human health and lives. Myocardial fibrosis (MF), one of the most characteristic structural changes, is the common pathologic change of multiple heart diseases and one of the principal manifestations of left ventricular remodeling, and contributes to both systolic and diastolic dysfunction. Prolonged activation of cardiac fibroblasts (CFs), defined by increased cell proliferation, myofibroblast differentiation and subsequent extracellular matrix (ECM) secretion, leads to myocardial fibrosis.Transforming growth factor-β1 (TGF-β1), the major contributor to tissue fibrosis, is the mutual pathway of myocardial fibrosis caused by a variety of factors. The expression of TGF-β1 is markedly increased in both infarcted and non-infarcted areas of the hearts after MI. In vivo gene transfer of TGF-β1 can induce myocardial fibrosis. To clarify the regulatory mechanism of TGF-β1 signaling inhibition on myocardial fibrosis after MI, we first designed in vitro experiments.CFs were cultured and treated respectively with PBS, TGF-β1, soluble transforming growth factor-β1 receptorⅡ(sTβRⅡ), and TGF-β1+sTβRⅡ. CFs proliferation was measured by MTT assay. Myofibroblast differentiation was examined by a-smooth muscle actin (a-SMA) immunocytochemical staining. The expression of P-Smad2 and Smad2/Smad3, the critical signaling molecules, were determined by immunocyto-chemical staining and Western blot analysis. Results demonstrated that TGF-β1 significantly increased CFs proliferation, myofibroblast differentiation and the expression of P-Smad2 compared with PBS group (P<0.01); CFs proliferation, myofibroblast differentiation and the expression of P-Smad2 in TGF-β1+sTβRⅡgroup were reduced markedly compared with TGF-β1 group (P<0.01). There were no significant differences in the expression of total Smad2/Smad3 among the 4 groups (P>0.05). These results showed that TGF-β1 induced the activation of Smad signaling, CFs proliferation and myofibroblast differentiation; while pretreatment with sTβRⅡinhibited TGF-β1-induced the activation of Smad signaling and its downstream events, I.e. cell proliferation and myofibroblast differentiation, and consequently inhibited CFs function.In addition, Wnt signal pathway is thought to be functionally conserved in vertebrates and plays an important role in organogenesis, and is found to participate in the development of fetal heart. In cardiovascular pathology, re-expression of a fetal gene expression pattern is a generally observed phenomenon. Several studies suggested that Wnt signal pathway might be involved in the repair process after MI. To illustrate the regulatory mechanism of Wnt signaling inhibition on myocardial fibrosis after MI, we designed the following experiments in vitro.CFs were cultured and treated respectively with PBS, Wnt-1, secreted frizzled related protein-1 (sFRP-1), and Wnt-1+sFRP-1. Cell proliferation was measured by MTT assay. Myofibroblast differentiation was examined byα-SMA immunocytochemical staining. The expression of dishevelled-1 (DVL-1) andβ-catenin, two critical signaling molecules, were determined by immunocytochemical staining and Western blot analysis. Results demonstrated that Wnt-1 significantly increased CFs proliferation, myofibroblast differentiation, and the expression of DVL-1 andβ-catenin compared with PBS group (P <0.01); CFs proliferation (P<0.05), myofibroblast differentiation, and the expression of DVL-1 andβ-catenin in Wnt-l+sFRP-1 group were reduced markedly compared with Wnt-1 group (P<0.01). These results indicated that Wnt-1 induced the activation of Wnt signal pathway, CFs proliferation and myofibroblast differentiation; whereas pretreatment with sFRP-1 antagonized the action of Wnt-1, inhibited the activation of Wnt signal pathway and its downstream events, that is CFs proliferation and myofibroblast differentiation, and thus inhibited CFs function.To clarify the mechanism that blockade of TGF-β1 signal pathway and Wnt signal pathway may control the process of myocardial fibrosis after MI, we prepared MI model by ligation of the left anterior descending coronary artery.4 days after the coronary ligation, MI rats that had survived were treated and divided into groups at random: Sham-operated group (equal PBS, n=10), MI group (equal PBS, n=10), MI+sTβRⅡgroup (100ng sTβRⅡ/20μl PBS was injected into the infarct myocardium border and the infarct area, n=10), MI+sFRP-1 group (100ng sFRP-1/20μl PBS was injected into the infarct myocardium border and the infarct area, n=10), and MI+sTβRⅡ+sFRP-1 group (100ng sTβRⅡ/10μl PBS and 100ng sFRP-1/10μl PBS, n=10).2 weeks after treatment, 4 rats per group were killed and made frozen section, and myofibroblast differentiation was measured with a-SMA immunohistochemical staining.4 weeks after treatment, cardiac function was evaluated by hemodynamic measurement, weight parameters and infarct size were detected with a previous experimental method. Results demonstrated that compared with MI group, MI+sTβRⅡ, MI+sFRP-1, and MI+sTβRⅡ+sFRP-1 significantly reduced myofibroblast differentiation (P<0.01), improved left ventricular systolic pressure and the maximum rate of left ventricular systolic pressure rise and fall, but decreased left ventricular end-diastolic pressure (P<0.01), markedly reduced the ratio of heart weight and body weight, and the ratio of left ventricular weight and body weight (P<0.05), and significantly reduced infarct size (P<0.05). However, these changes are more significant in MI+sT(3RⅡ+sFRP-1 group. These results suggested that combination of sTβRⅡand sFRP-1 blocked TGF-β1 and Wnt signal pathway could inhibit myofibroblast differentiation, alleviate myocardial fibrosis and left ventricular remodeling, and improve ischemic cardiac function effectively.For all the above, the present study verified the manipulation of blockade of TGF-β1 and Wnt signal pathway to the process of myocardial fibrosis after MI both in vivo and in vitro, and thus represented a new approach for future therapeutic interventions.