Reprogramming Non-myocytes to Cardiomyocytes in vivo

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There is intense interest in approaches to generating new cardiomyocytes, including not only through laboratory generation of cardiomyocytes but also by promoting cardiomyocyte formation therapeutically through endogenous cardiogenesis. The endogenous cardiogenesis approaches would not need delivery of cells with issues of engraftment and survival, and thus could have advantages. One of these exciting endogenous cardiogenesis approaches is direct reprogramming of non-cardiac cells to cardiomyocyte. To study reprogramming in vivo, the inducible cre approach in mice is the most widely used method for genetic fate-mapping of cells. However, inducible cre and other genetic lineage mapping approaches may be limited by even very transient leakage of promoters or spontaneous recombinase activity in the absence of the inducer molecule, and these studies can currently only be performed in mice. To gain confidence in the study of endogenous cardiogenesis, approaches that complement genetic fate mapping could provide compelling evidence that our field is headed toward the best regeneration strategy. We have now developed an entirely new approach to marking the identity of cells in vivo using non-radioactive isotopes in a cell-specific metabolic compound. This “Metabolic Fate-Mapping” approach utilizes an isotope-enriched metabolic tracer, specifically creatine, which is taken up by muscle cells, phosphorylated, and utilized in the cytoplasmic phosphocreatine shuttle. Cellular uptake of creatine by muscle cells is rapid, while subsequent turnover of creatine is slow, making it a suitable metabolic label for myocytes. Cells that are creatine-positive can then be identified via Multi-Isotope Imaging Mass Spectrometry (MIMS), a high resolution approach that we have adapted for myocardial biology. We have previously demonstrated usage of labeled thymidine in vivo to demonstrate rare proliferation of cardiomyocytes over months, and we have also demonstrated the stable isotope imaging approach in human volunteers. We will use this new Metabolic Fate-Mapping approach to cell lineage mapping along with an inducible cardiomyocyte cre mouse and an inducible fibroblast cre mouse to study reprogramming of non-myocytes to cardiomyocytes in vivo. Unlike genetic fate-mapping strategies, the stable isotope lineage mapping approach is amenable to any species on any genetic background, and this will enable future large animal experiments of reprogramming. Finally, because this approach can be applied with stable, non-radioactive isotopes such as 13C and 15N, which have been widely used in humans and are regarded by the FDA as safe, studies of human regeneration in diverse tissues could be enabled with Metabolic Fate- Mapping by identification of cellular labels specific to cell type.