MicroRNA-378 suppresses myocardial fibrosis through a paracrine mechanism at the early stage of cardiac hypertrophy following mechanical stress.

Abstract

<b>Rationale</b>: Excessive myocardial fibrosis is the main pathological process in the development of cardiac remodeling and heart failure; therefore, it is important to prevent excessive myocardial fibrosis. We determined that microRNA-378 (miR-378) is cardiac-enriched and highly repressed during cardiac remodeling. We therefore proposed that miR-378 has a critical role in regulation of cardiac fibrosis, and examined the effects of miR-378 on cardiac fibrosis after mechanical stress. <b>Methods:</b> Mechanical stress was respectively imposed on mice through a transverse aortic constriction (TAC) procedure and on cardiac fibroblasts by stretching silicon dishes. A chemically modified miR-378 mimic (Agomir) or an inhibitor (Antagomir) was administrated to mice by intravenous injection and to cells by direct addition to the culture medium. MiR-378 knockout mouse was constructed. Cardiac fibroblasts were cultured in the conditioned media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869. Quantitative real-time polymerase chain reaction analysis of gene and miRNA expression, Western blot analysis, immunochemistry and electron microscopy were performed to elucidate the mechanisms. <b>Results</b>: Mechanical stress induced significant increases in fibrotic responses, including myocardial fibrosis, fibroblast hyperplasia, and protein and gene expression of collagen and matrix metalloproteinases (MMPs) both <i>in vivo</i> and <i>in vitro</i>. All these fibrotic responses were attenuated by treatment with a chemically modified miR-378 mimic (Agomir) but were exaggerated by treatment with an inhibitor (Antagomir). MiR-378 knockout mouse models exhibited aggravated cardiac fibrosis after TAC. Media from the cardiomyocytes with either miR-378 depletion or treatment with sphingomyelinase inhibitor GW4869 enhanced the fibrotic responses of stimulated cardiac fibroblasts, confirming that miR-378 inhibits fibrosis in an extracellular vesicles-dependent secretory manner. Mechanistically, the miR-378-induced anti-fibrotic effects manifested partially through the suppression of p38 MAP kinase phosphorylation by targeting MKK6 in cardiac fibroblasts. <b>Conclusions</b>: miR-378 is secreted from cardiomyocytes following mechanical stress and acts as an inhibitor of excessive cardiac fibrosis through a paracrine mechanism.