Bridging the gap between mutation & cellular effects: Defining the mechanisms of hypertrophic cardiomyopathy

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Hypertrophic cardiomyopathy (HCM) affects more than 1 in 500 Americans with an extensive burden of morbidity in the form of arrhythmia, heart failure, and sudden death. More than 25 years since the discovery of the genetic underpinnings of HCM, we continue to have limited understanding of the primary effect of genetic mutation on protein function and it is unclear how the genetic mutation leads to hypertrophic signaling in cardiomyocytes. This lack of understanding limits the development of effective pharmacotherapy for HCM. The objective of this proposal is to further advance the knowledge of how mutations affect sarcomere function using biochemical and biophysical approach using purified protein and skinned fiber, as well as human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) as tools for disease modeling to assess the triggers leading to hypertrophy. Given the findings from prior biochemical assessment of myosin heavy chain mutation that cause HCM, it is hypothesized that HCM mutations result in gain of function in sarcomere by increase in number of myosin heads available for cross-bridge formation (Na) through protein interaction and activation state of myosin. It is further hypothesized that increase in Na result in energy imbalance in cells due to increased ATP usage, leading to altered Ca2+ dynamics and mitochondrial dysfunction. In this proposal, 3 mutations in regulatory light chain (RLC) of myosin that are linked to HCM are chosen to test the above hypothesis further: E22K, R58Q and D166V. In addition, D94A mutation that is linked to dilated cardiomyopathy (DCM) is also chosen to assess the effect of mutation that causes the opposite cardiac phenotype for comparison. Aim 1 measures the impact of HCM mutations on myosin's folded state, by assessing protein-protein interaction between the RLC and other sarcomere components (including myosin binding protein C) using novel binding affinity assay. Aim 2 quantifies the effect of HCM mutations on RLC using skinned myofiber from rabbit and purified recombinant human protein, with respect to myosin activation state by measuring the kinetics of myosin using fluorescent ATP. Finally, Aim 3 defines the cellular effect of RLC mutations using hiPSC-CM, by measuring cell mechanics, Ca2+ dynamics and mitochondrial function. I will particularly focus on obtaining properly matured hiPSC-CM by rigorous structural assessment. The current proposal is designed to gain further understanding of molecular pathogenesis of HCM from protein level to myofiber level, focusing on myosin's structural change leading to altered activation state. It will also link these biochemical findings to biomechanical property at the cellular level, and transcriptional profiling will be performed to identify new gene targets involved in hypertrophic signaling pathway. The proposal will also allow me to learn further skills in myofiber and hiPSC-CM as new platforms for performing functional analysis of cardiomyopathy model systems. Moving forward, this proposal will be the basis of my independent R01 grant using these innovative approaches.