MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes

TLDR

Repopulating the injured heart with new functional cardiomyocytes remains a major challenge, and an ideal therapy would directly convert damaged tissue into functional myocardium in situ. The study aimed to develop and evaluate microRNAs that can directly reprogram cardiac fibroblasts into cardiomyocytes in vitro and in vivo. Using a combinatorial approach, the authors identified a miRNA cocktail (miR‑1, miR‑133, miR‑208, miR‑499) that induces direct reprogramming of fibroblasts to cardiomyocyte‑like cells, with a ten‑fold efficiency increase when combined with JAK inhibitor I. The miRNA cocktail produced cells expressing mature cardiomyocyte markers, organized sarcomeres, and spontaneous calcium transients; intramyocardial delivery in mice led to fibroblast‑to‑cardiomyocyte conversion confirmed by genetic tracing, proving the feasibility of in vivo reprogramming and suggesting therapeutic potential.

Abstract

Rationale: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. Objective: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. Methods and Results: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusions: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.