Role of the cardiac cytoskeleton in mRNA localization and hypertrophy

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PROJECT SUMMARY In the adult heart, remodeling in response to changing hemodynamic demands occurs primarily through hypertrophy, the addition of new sarcomeres to individual cardiomyocytes. Physiological hypertrophy, triggered by pregnancy or exercise, maintains normal organization of cardiac structure and can improve cardiac function. Pathological hypertrophy, however, often precedes heart failure. This can induce drastic changes to the cardiomyocyte cytoskeleton. However, with the exception of cellular mechanics, the role of the cardiomyocyte cytoskeleton in both health and disease has remained understudied. Recent data suggest that both the actinomyosin and microtubule network may play a role in mRNA and ribosomal localization in cardiomyocytes, though the mechanism remains unknown. In non-muscle cell types, actin-based and microtubule-based directed mRNA transport is well characterized. Additionally, myosin and other sarcomeric mRNAs, ribosomes, and protein degradation machinery appear to localize to the sarcomere in the cardiomyocyte, supporting a model of local translation for sarcomere maintenance and/or de novo formation. But how these new sarcomeres are formed remains particularly unclear, and the dependence of hypertrophy on proper mRNA transport and localization is unknown. The goal of my research proposal is to determine the relationship between mRNA localization and cardiac hypertrophy both in health and disease. My preliminary data establishes that the microtubule network is essential for mRNA localization in the rat cardiomyocyte in vivo and in vitro. To determine the specific mechanism of mRNA transport, I will utilize pharmacological reagents to destabilize the actin network and specifically inhibit motor proteins in isolated adult rat cardiomyocytes to test if mRNA localization changes. I will also use the MS2-MCP system to live track single mRNA transcripts to unambiguously define the mode of mRNA transport. I will leverage molecular biological tools to mislocalize specific sarcomeric transcripts and to visualize sites of new sarcomere deposition in growth-responsive neonatal rat cardiomyocytes to test if mRNA sublocalization is required for cardiac hypertrophy. Finally, I will evaluate if hypertrophy disrupts proper mRNA localization, and if so, dissect the relative contribution of pathophysiological microtubule network changes to this mislocalization using both in vitro and in vivo models of hypertrophy. Taken together, the proposed research will definitively establish the mechanism of mRNA transport and localization in the cardiomyocyte and its function in cardiac hypertrophy.