Modulators of cardiomyocyte structure to promote functional recovery during cardiac regeneration and repair

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PROJECT SUMMARY The governing premise of this application is that cardiac injury response falls along a continuum, depending on species, developmental stage, as well as therapeutic/experimental manipulation. Lower organisms such as zebrafish and neonatal mice are capable of complete heart regeneration following partial amputation or ischemic- induced cardiomyocyte (CM) death. Remuscularization is facilitated by proliferation of pre-existing cardiomyocytes (CMs). On the other hand, in response to cardiac injury or stress in adult mammals, CMs fail to proliferate and instead undergo adverse hypertrophic remodeling that can result in cardiac dysfunction. There is great interest in identifying molecules and signaling cues that guide CMs to undergo a proliferative versus hypertrophic response so that we can modulate these factors to promote adult mammalian cardiac regeneration and repair in response to injury. In this proposal we investigate the hypothesis that junctional and cytoarchitectural proteins are key effectors that regulate how CMs respond to injury. Modulation of these proteins coordinate the proliferative state of CMs, but also facilitate proper re-integration of CMs with neighboring cells during cardiac regeneration. In preliminary studies, we initiated a screen for cardiac development and regeneration in zebrafish mutants that disrupt factors known to regulate cell junctional and cytoskeletal proteins. The selected proteins have known interactions with Hippo-Yap signaling, a pathway of significant interest for promoting cardiac regeneration. From this screen, we identified Llgl1 as critical for zebrafish heart morphogenesis and development, and Shroom3 as a factor that is critical for regulation of CM proliferation during zebrafish heart regeneration. Subsequently, we found that CM specific deletion of either Llgl1 or Shroom3 impairs intercalated disc integrity which was associated with aberrant CM cell cycle activity in uninjured hearts. Here, we propose to delineate the cellular and physiological role of Llgl1 and Shroom3 in mammalian CMs during development and the post cardiac injury response in neonatal and adult mice, and to further investigate the mechanism by which these proteins interact with Hippo-Yap pathway components. Aims 1 and 2 employ transgenic mice with CM specific deletion of Llgl1 or Shroom3, respectively, to investigate the role of these two proteins in cardiac development and the post injury response with emphasis on cell morphology and establishment of junctional complexes and intercalated discs. We employ the zebrafish model to investigate the mechanisms underlying Llgl1 and Shroom3 functions, and whether these factors are co-regulated by the Hippo kinase, Lats1/2. Collectively, our proposed studies will elucidate the role of key effectors of the cardiac injury response, capable of modulating CM cytoarchitecture and cell-cell junctions to promote functional regeneration and restore a healthy myocardium.