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 miRNAs 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 microRNAs (miRNA) 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 microRNAs 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 upon JAK inhibitor I treatment. Importantly, administration of microRNAs 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 non-cardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. Conclusion The findings from this study provide the first 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.

Stem cells play an important role in restoring cardiac function in the damaged heart. In order to mediate repair, stem cells need to replace injured tissue by differentiating into specialized cardiac cell lineages and/or manipulating the cell and molecular mechanisms governing repair. Despite early reports describing engraftment and successful regeneration of cardiac tissue in animal models of heart failure, these events appear to be infrequent and yield too few new cardiomyocytes to account for the degree of improved cardiac function observed. Instead, mounting evidence suggests that stem cell mediated repair takes place via the release of paracrine factors into the surrounding tissue that subsequently direct a number of restorative processes including myocardial protection, neovascularization, cardiac remodeling, and differentiation. The potential for diverse stem cell populations to moderate many of the same processes as well as key paracrine factors and molecular pathways involved in stem cell-mediated cardiac repair will be discussed in this review.

Rationale A major goal for the treatment of heart tissue damaged by cardiac injury is to develop strategies for restoring healthy heart muscle through the regeneration and repair of damaged myocardium. We recently demonstrated that administration of a specific combination of micro-RNAs (miR combo) into the infarcted myocardium leads to direct in vivo reprogramming of non-cardiac myocytes to cardiac myocytes. However, the biologic and functional consequences of such reprogramming are not yet known. Objective The aim of this study was to determine whether non-cardiac myocytes directly reprogrammed using miRNAs in vivo develop into mature functional cardiac myocytes in situ, and whether reprogramming leads to improvement of cardiac function. Methods and Results We subjected FSP1-Cre mice/tdTomato mice to cardiac injury by permanent ligation of the left anterior descending coronary artery (LAD) and injected lentiviruses encoding miR combo or a control nontargeting miRNA. miR combo significantly increased the number of reprogramming events in vivo. Five-to-six weeks following injury, morphological and physiological properties of tdTomato− and tdTomato+ cardiac myocyte-like cells were analyzed ex vivo. tdTomato+ cells expressed cardiac myocyte markers, sarcomeric organization, excitation-contraction coupling, and action potentials characteristic of mature ventricular cardiac myocytes (tdTomato− cells). Reprogramming was associated with improvement of cardiac function, as analyzed by serial echocardiography. There was a time delayed and progressive improvement in fractional shortening and other measures of ventricular function, indicating that miR combo promotes functional recovery of damaged myocardium. Conclusions The findings from this study further validate the potential utility of miRNA-mediated reprogramming as a therapeutic approach to promote cardiac regeneration following myocardial injury.