(E-185) A molecular scale investigation of the mechanisms of contractile dysfunction for the hypertrophic cardiomyopathy MYH7 G256E mutation

Introduction:: Studying the molecular mechanisms of hypertrophic cardiomyopathy (HCM) in humans has several challenges, including availability of myectomy samples for specific mutations and history of patient HCM management. To determine the effect of mutation-specific effects on β-myosin heavy chain (MHC) function during contractile behavior, we use CRISPR-Cas9 gene-editing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system.


Materials and Methods:: Tension generation and kinetics of activation and relaxation of isolated hiPSC-CM myofibrils were measured via our custom-built apparatus with fast solution switching which can measure mechanics at the ms timescale. Molecular dynamics were performed on the post-rigor M.ATP structure (PDB: 4DB1) using AMBER20. WT and G256E simulations were both run in triplicate for 500 ns.


Results, Conclusions, and Discussions:: Measurements from individual isolated contractile organelles (myofibrils) from hiPSC-CMs demonstrate that the specific force and rate of force generation for mutant MYH7 G256E myofibrils is greater than isogenic control myofibrils. During relaxation, the initial, slow phase kinetics were decreased, indicating slower cross-bridge detachment rate. Using stopped-flow kinetic analysis, measures of myofibril ATP binding using mant-ATP, were slower for the G256E mutation, Preliminary experiments suggest ADP release may also be slowed by the G256E mutation. Together these data suggest a mechanism for the hypercontractile phenotype with impaired early relaxation with the G256E mutation, e.g. delayed myosin detachment in the crossbridge cycle during calcium activated contractions. To understand the molecular-structural basis of these effects we have used molecular dynamics computational simulations of wild type and mutant β-MHC. In simulations of the post-rigor (M.ATP) and rigor state structures, we observed reduced stability in the transducer region, due to weakened hydrogen bonds between neighboring β-sheet strands as well as significant changes in local contacts. Combining our in silico and experimental data, our results suggest that the G256E mutation affects the myosin transducer region such that it alters communication between the nucleotide binding pocket and the actin binding surface during the acto-myosin chemo-mechanical cycle. This leads to hypercontractility and slowed relaxation.


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