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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.

See related article, pages 629–638 In response to stress signals, the mammalian heart responds by an increase in its size.1 This is largely accomplished by an increase in the size of myocytes (hypertrophy) rather than by increasing their numbers (hyperplasia). At the molecular level, pathological stresses induce multiple changes, including genetic reprogramming—the reexpression of a battery of fetal genes and the downregulation of multiple adult genes.2 Together, the changes in gene expression result in substantial phenotypic changes, including changes in size, contractility, metabolic state, and electric conductance. Indeed, the reexpression of fetal genes, including β-myosin heavy chain (β-MyHC), atrial natriuretic factor (ANF), and alpha-skeletal actin, has for many years been looked on as an important molecular indicator of pathological hypertrophy. In spite of this use of β-MyHC reexpression as a marker of pathological hypertrophy, until recently there have been surprisingly few reports on β-MyHC reexpression at the level of individual myocytes within the hypertrophic heart. Nevertheless, previous work using in situ hybridization against β-MyHC mRNA has shown that reexpression of β-MyHC does not take place in all areas of the hypertrophic heart; rather, it is distributed in distinct regions.3 More recently, it has been possible to study individual cells from the heart using genetically altered mice in which a fluorescent protein (yellow fluorescent protein [YFP]) is tagged onto the native β-MyHC gene. This procedure confirmed that β-MyHC reexpression takes place in cells located in distinct regions, including regions adjacent to areas of …