Connective tissue growth factor—a novel mediator of angiotensin II-stimulated cardiac fibroblast activation in heart failure in rats

Abstract

The pathophysiologic mechanisms of myocardial remodeling in heart failure (HF) remain poorly understood. Using differential mRNA display of myocardial tissue from rats with ischemic HF vs. controls we identified robust myocardial induction of the mRNA encoding connective tissue growth factor (CTGF). The aim of this study was to investigate the sites of synthesis and the mechanisms of induction of CTGF in failing myocardial tissue. The study demonstrates that myocardial expression of CTGF mRNA and protein is substantially elevated in non-ischemic tissue from both the left and the right ventricles of rats with experimentally induced myocardial infarction (MI). The induction of myocardial CTGF mRNA was shown to transcend from early post-infarction HF to chronic HF. In situ hybridization and immunohistochemical analysis of myocardial tissue sections demonstrated expression of CTGF confined to fibroblasts and endothelial cells of non-ischemic myocardial tissue. In subsequent experiments rats subjected to MI were randomized to treatment with the AT₁ angiotensin receptor antagonist losartan (12.5 mg/kg b.i.d. per os) or vehicle. Losartan attenuated ventricular hypertrophy, improved hemodynamics, and prevented the induction of myocardial CTGF mRNA observed in rats post-MI. To provide the cellular basis of Ang II-stimulated CTGF mRNA expression, primary cultures of rat myocardial fibroblasts were stimulated with Ang II (10^–7 M). Real-time reverse transcription–polymerase chain reaction and western blot analysis demonstrate that Ang II induces rapid, AT₁ receptor-mediated elevations of CTGF mRNA and protein in rat cardiac fibroblasts. Furthermore, CTGF was shown to stimulate fibroblast proliferation in vitro. In conclusion, this study demonstrates that CTGF is a myocardial effector of Ang II-induced myocardial remodeling in HF mediated via AT₁ receptors situated on cardiac fibroblasts.

Introduction

Congestive heart failure (CHF) is associated with structural alterations of myocardial tissue collectively termed myocardial remodeling. The most characteristic features of these structural alterations include compensatory hypertrophy of cardiac myocytes and myocardial fibrosis [1]. The latter involves both increased deposition of extracellular matrix (ECM) as well as altered composition of the ECM [2]. Although such remodeling of myocardial tissue may initially be regarded as compensatory responses to increased wall stress and neurohormonal activation, the alterations ultimately decrease left ventricular (LV) function and act as strong predictors of mortality in patients with heart failure (HF). Despite considerable scientific data on the biochemical characteristics of myocardial remodeling, the growth factors initiating these structural alterations of the heart still remain poorly understood. We employed differential mRNA display of myocardial tissue from rats with ischemic HF to identify putative candidate genes in the mechanisms of myocardial remodeling. One of the genes that were strongly induced in the failing hearts was the 38 kDa cysteine-rich immediate early gene product, connective tissue growth factor (CTGF). Several features of CTGF prompted us to investigate the mechanisms of induction of this growth factor in HF. CTGF is a member of the CCN (acronym of Cyr61/CEF-10, CTGF/Fisp-12, and Nov) family of growth factors [3]. These growth factors are secreted, heparin-binding, and ECM-associated proteins involved in multiple cellular events including ECM production, cell adhesion, cell proliferation, or in some cell types apoptosis, as well as more complex biological processes such as tumorigenesis, angiogenesis, and wound healing [4], [5]. CTGF was found to be highly expressed in fibroblasts and to trigger many of the cellular processes underlying fibrosis [6]. Indeed, several reports provide evidence implicating CTGF in the pathogenesis of fibrotic disorders such as advanced atherosclerosis, scleroderma, as well as liver- and kidney-fibrosis [7], [8], [9], [10]. The profibrotic cytokine TGF-β has also been shown to increase the mRNA levels of CTGF in fibroblasts in vitro [11]. Furthermore, TGF-β-stimulated secretion of collagen from fibroblasts could be inhibited by neutralizing anti-CTGF antibodies [12]. Although, considerable evidence points to a role of TGF-β in the pathophysiologic mechanisms of myocardial remodeling in HF, corresponding involvement of CTGF is unknown. In addition, certain G protein-coupled receptors have also recently been shown to induce expression of CTGF [13]. Thus, the major aims of this study were (1) to elucidate the cellular site(s) of synthesis of CTGF in failing myocardial tissue, and (2) to determine the mechanisms of induction of CTGF mRNA in non-ischemic myocardial tissue in HF. Substantial evidence implicates the neurohormonal renin–angiotensin II (Ang II)-AT₁ receptor axis in remodeling of failing myocardial tissue. However, to what extent CTGF is an autocrine/paracrine effector of Ang II was an issue that remained to be resolved. In this study, investigations of myocardial tissue and isolated cardiac fibroblasts from rats with ischemic HF provide strong evidence that CTGF is a mediator of Ang II-stimulated myocardial remodeling.