Abstract
Mutations in myosin alter its motor functions in diverse ways by affecting different structural and chemo-mechanical events. Multidisciplinary strategies can be used to understand how varying alterations in motor function converge to common phenotypes like hypercontractility and hypertrophic cardiomyopathy (HCM). We combined molecular dynamics (MD) simulations with protein biochemical and myofibril mechanical analyses to study the HCM-causing myosin variant G256E. MD simulations demonstrated that G256E induces structural changes that increase the work required to displace ADP.Mg2+ from actomyosin. Stopped-flow biochemical analysis demonstrated increased ADP affinity and decreased ADP release rate, and single myofibril mechanics analysis demonstrated increased force generation and reduced ADP sensitivity of the early, slow phase of relaxation. Together, these results demonstrate that slower ADP release from myosin during contraction is a significant contributor to pathological contractile nature of the G256E mutation. This study highlights the importance of detailed chemo-mechanical analysis of mutations associated with hereditary cardiac diseases.
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Acknowledgements
This work used the Extreme Science and Engineering Discovery Environment (XSEDE) resource COMET through allocation TG-MCB200100 to M.R. XSEDE was supported by the National Science Foundation grant no. ACI-1548562. Partial funding for M.C.C. was provided by awards T32HL007828 and K99HL173646 from the National Heart, Lung, and Blood Institute. Partial support for K.Y.K. was provided by NIBIB T32EB032787. Partial support for K.Y.K. was provided by NIH-NHLBI R00HL159224 awarded to J.D.P. This research was supported by the University of Washington Center for Translational Muscle Research (CTMR) via the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health award no. P30AR074990 to M.R. This work was initiated and supported by the NIH/NIGMS grant 1RM1 GM131981-03 to J.A.S., K.M.R. and M.R. and supported by NHLBI R01HL128368 to M.R. This work was made possible by the Allen Institute for Cell Science team, which generated the stem cell lines. The parental WT unedited hiPSC line, WTC, was provided by the Bruce R. Conklin Laboratory at the Gladstone Institutes and UCSF. The Allen Institute for Cell Science wishes to thank the Allen Institute for Cell Science founder, Paul G. Allen, for his vision, encouragement, and support. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NHLBI or the NIH.
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M.R. is a consultant for Kardigan Bio and Bristol Myers Squibb; serves on the scientific advisory board for FilamenTech; and has equity in StemCardia, Inc and KineaBio, Inc; none of the current work is in conflict with these associations. J.A.S. is cofounder and on the Scientific Advisory Board of Cytokinetics, Inc., a company developing small molecule therapeutics for treatment of hypertrophic cardiomyopathy, but this work was done independently. J.A.S. is cofounder and Executive Chair, and K.M.R. is cofounder and Research and Clinical Advisor, of Kainomyx, Inc., a company developing small molecule therapeutics targeting cytoskeletal proteins for multiple diseases, but this work was done independently. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
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Kao, K.Y., Childers, M.C., Pathak, D. et al. The hypertrophic cardiomyopathy myosin variant G256E prolongs cardiac muscle relaxation via altered nucleotide handling. Commun Chem (2026). https://doi.org/10.1038/s42004-026-02048-w
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DOI: https://doi.org/10.1038/s42004-026-02048-w
