Mechanisms of contractile dysfunction of the G256E HCM-associated mutation

Abstract

Hypertrophic cardiomyopathy (HCM) affects approximately 1 in 500 individuals in the U.S. and is associated with adverse outcomes conditions such as atrial fibrillation, heart failure, and sudden cardiac death. About 60% of HCM cases result from inherited mutations with the MYH7 gene, encoding β-myosin, the molecular motor of the sarcomere, is the second most common mutational hotspot. Despite advancements in genetic sequencing, connecting specific mutations to clinical outcomes remains challenging, with many identified variants having unknown significance. Current therapeutic strategies for HCM are geared towards managing symptoms rather than targeting the underlying molecular causes of the disease. Our goal is to elucidate how HCM mutations in myosin lead to alterations at the protein and contractile organelle level, and how these alterations manifest at the cell and tissue level in order to find direct, druggable targets for more effective therapies.In the first body of work, we collaborated with academic groups from Stanford, UC Santa Barbara, Institut Curie, and others at UW to form a multi-institutional, multidisciplinary group to study how single missense mutations in myosin lead to HCM. We collaborated with the Allen Institute for Cell Science to engineer a CRISPR/Cas9-edited human induced pluripotent stem cell line (hiPSC) with the MYH7 G256E mutation as a HCM disease model. We (the Regnier Lab) focused on the contractile organelle, or myofibril, and molecular scale. In myofibrils isolated from hiPSC-cardiomyocytes (CMs), we saw greater and faster force development accompanied by impairment of the slow, early phase of relaxation. In molecular dynamics (MD) simulations of post-rigor human cardiac myosin (M.ATP), the G256E mutation caused instability in the transducer region, possibly altering signal transduction from the nucleotide pocket and the actin-binding cleft. Altogether, these data suggest that G256E leads to hypercontractility through impairment of relaxation resulting in a greater population of force generating myosin. The second body of work addresses how the G256E mutation alters ADP nucleotide handling. We challenged isolated hiPSC-CM myofibrils with elevated ADP to assess their response to product inhibition. G256E myofibrils demonstrated reduced sensitivity to ADP inhibition of the slow, early phase of relaxation, suggesting that ADP release is already impaired by the G256E mutation. In MD simulations of post-powerstroke myosin (A.M.ADP), we saw changes in ADP coordination in the nucleotide pocket due to the G256E mutation. Furthermore, we performed steered MD simulations to determine that G256E myosin requires on average 1.5x as much work to displace ADP compared to WT due to alterations in the molecular release pathway. This finding was validated through stopped-flow biochemistry with an observed 25% increase in ADP affinity with G256E sS1 compared to WT. In summary, by combining experimental approaches with molecular dynamics simulations, we uncovered the molecular structural alterations resulting from the G256E HCM mutation and related them to functional findings, pinpointing a specific step in the cross-bridge cycle that is impacted by this mutation–ADP release. This work serves as a framework for how identifying the structural basis of disease provides insights into genotype-specific therapeutic targets, paving the way for more precise and effective treatments for HCM.