Project description:Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-pSTAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-pSTAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

Project description:BACKGROUND: Cardiac reprogramming is a technique to directly convert non-myocytes into myocardial cells using genes and/or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. METHODS: Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single AAV. We also created a novel AAV capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating post-translationally modified capsid residues. Since a constitutive promoter was needed to drive high expression of these cell-fate altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3’ UTR of the expression cassette that limits expression to non-myocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid / cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. RESULTS: We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model two weeks after MI, showing that cardiac reprogramming, when delivered in a single, clinically relevant AAV vector, can support functional improvement in the post-remodeled heart. This benefit was not observed with GFP or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. CONCLUSION: Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.

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Direct in vivo cardiac reprogramming improves cardiac function and ventricular remodeling in chronic MI.

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