Functional consequences of FHC mutations in cardiac MyBPC

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The long-range goal of this proposal is to define the mechanisms by which mutations in cardiac myosin binding protein C (MyBPC) cause hypertrophic cardiomyopathy (HCM), a disease that affects up to 1 in 200 individuals, and is the leading cause of sudden death in young adults. Nearly 60% of HCM cases are due to familial inheritance (FHC) of an autosomal dominant disorder caused by mutations in sarcomeric proteins. Mutations in MyBPC are among the most common causes of FHC accounting for half of all known cases. Because MyBPC is a critical modulator of actomyosin interactions, the initial functional deficit caused by mutations in MyBPC is expected to manifest as a defect in the regulation of cardiac muscle contraction at the myofilament level. Whereas 60% of MyBPC truncation mutations are expected to cause haploinsufficiency, the remaining 40% of MyBPC mutations are missense mutations, which are expected to produce full-length MyBPC. A large number of these missense mutations are located in the central domains of MyBPC (i.e., C3-C7), which have no specific known function, and thus it is unclear how FHC mutations located in this region of MyBPC cause disease. Our limited understanding of these critical mechanisms severely limits options for therapeutic intervention for FHC patients. Our preliminary data provide novel evidence that addresses our gap in knowledge and have identified two important regulatory regions within the C4 and C5 domains of MyBPC that modulate cardiac muscle contractile function. Based on these novel observations we have devised an experimental plan that is designed to elucidate molecular mechanisms by which these key regions contribute to regulation of contractile function and how FHC mutations alter this regulation. We have devised a multidisciplinary approach that spans from computational modeling of atomic interactions to whole animal physiology which will accomplished in three principal aims designed to: 1) Establish the functional effects of central domain MyBPC FHC mutations on the magnitude and rate of force in cardiac fibers isolated from mouse hearts expressing HCM causing mutations, and utilize molecular dynamic simulations to elucidate the molecular mechanisms of altered function. 2) Define how MyBPC mutations alter actin and myosin binding properties and rotational dynamics using TPA and FRET based sensors, and 3) Determine the in vivo functional consequences of MyBPC FHC mutations in MyBPC by assessing ventricular contractile and hemodynamic function, and test the efficacy of a MyBPC-specific AAV9 gene-transfer rescue that normalizes contractile function. Parallel studies will utilize FHC patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CM) to determine how these mutations cause disease in humans. It is expected that results from these integrative studies will provide novel insights of the underlying mechanisms by which mutations in MyBPC cause disease and will aid in the development of novel therapeutic strategies for treatment MyBPC related HCM.