Abstract 12946: L-Arginine Attenuates Myofibroblast Phenoconversion and Fibrosis in the Human Heart

Abstract

Introduction. L-Arginine (L-Arg) is a semi-essential amino acid involved in a number of biological processes. L-Arg has been recently shown to have beneficial effects on functional capacity in heart failure. However, the molecular mechanism underlying these actions are mostly unknown and a potential direct effect of L-Arg on cardiac fibroblasts has never been investigated hitherto. Transforming growth factor (TGF)-β is one of the main profibrotic cytokines, known to activate fibroblasts into myofibroblasts stimulating the production of extracellular matrix (ECM). In fibrosis condition, excessive ECM alters myocardial structure, eventually impairing cardiac function. Hypothesis. We hypothesized that L-Arg could mitigate TGF-β-dependent phenoconversion of human cardiac fibroblasts (HCFs). Methods. We isolated primary cardiac fibroblasts from human hearts (protocol approved by the Institutional Review Board for protection of human subjects) and tested a model of fibrosis obtained by treatment with TGF-β. Isolated HCFs were characterized by immunostaining. Early passage HCFs were treated with TGF-β or TGF-β + L-Arg at different doses and time points. The mitochondrial generation of reactive oxygen species (ROS) was assessed by MitoSox at the confocal microscopy. Results. TGF-β induced a significant upregulation of established markers of myofibroblast activation and fibrosis (including Fibronectin, Col1A1, Col1A2, and Periostin ), which was significantly attenuated by L-Arg, as confirmed by RT-qPCR, immunostaining, and immunoblot. Moreover, we observed that TGF-β treatment significantly increased the mitochondrial generation of ROS, which was again prevented by co-treatment with L-Arg. In addition, L-Arg significantly reduced SMAD-2 protein expression. Conclusions. Our findings demonstrate, for the first time to our knowledge, the anti-fibrotic role of L-Arg in HCFs, mediated via a TGF-β/SMAD-2 signaling pathway.