Robeson, K. Z. | Search bioRxiv

bioRxiv2026

The Dilated Cardiomyopathy E525K β-Myosin Mutation Causes Hypocontractility in Cardiomyocytes Without Altering Crossbridge Cycling

The cardiac {beta}-myosin (MYH7) mutation E525K was identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered constructs has shown that this mutation stabilizes the interacting heads motif (IHM) of myosin and increases the ATPase activity of mutant motor S1 heads. However, no measurements have been made in myofilaments or cardiomyocytes to determine its effect on contractile function. Here, we present force and contractile kinetics measurements from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) engineered for heterozygous expression of E525K. Single-cell contraction for E525K hiPSC-CMs decreased by 65%, and isometric twitch force in engineered heart tissues (EHTs) decreased by 39%. In contrast, maximal Ca2+ activated isometric force in isolated myofibrils increased by 45% and sub-maximal Ca2+ activated force was similar to WT myofibrils. Structural analysis revealed reduced myofibril content (13.7% decrease) and decreased organization (increased z-disk dispersion angle) in E525K cells. We confirmed that E525K S1 myosin has higher actin affinity than WT S1 and elevated ATPase activity. However, there was no change in S1 ADP release rate. There was also no change in either the rate of force development or relaxation in myofibrils, cells, or EHTs. These findings suggest that E525K myosin crossbridge cycling is not altered during loaded contractions. To understand how twitch force of myocytes was reduced but maximal isometric force was increased, we used a spatially explicit sarcomere model. The results were explained by changing three rates: reduced recruitment from the OFF/IHM state, and increased rates of actin binding and Pi release. Additional force deficits in cells and EHTs likely result from the myofibrillar disorganization. This study demonstrates the value of multi-scale analysis and coupled, computational modeling to understand the molecular mechanisms of sarcomere mutations in cardiomyocytes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/733270v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1a64b4aorg.highwire.dtl.DTLVardef@98f053org.highwire.dtl.DTLVardef@c9f839org.highwire.dtl.DTLVardef@1c795c6_HPS_FORMAT_FIGEXP M_FIG C_FIG A model for how the E525K mutation impacts contracting myofibrils Here, we show that the E525K mutation impacts contraction in multiple ways: (1) Decreased sarcomere organization in cells and tissues drives decreased force generation. (2) Increased binding affinity of E525K myosin for actin and faster Pi release contributes to increased force generation in isolated myofibrils. (3) Increased IHM stability reduces twitch force. (4) The rate-limiting step of loaded crossbridge cycling, ADP release, is unchanged by the E525K mutation, and the rates of loaded contraction and relaxation are unchanged at all scales of contraction.

biophysics↗

bioRxiv2025

Conformational State of Myosins Disordered Loop 2 Structure Mediates Actomyosin Association During Crossbridge Formation

The binding of myosin to actin to form crossbridges is a critical step for force generation by sarcomeres. A recent cryo-electron microscopy structure has resolved the weakly-bound actomyosin complex (AM.ADP.Pi); however, the structural and dynamic factors that influence actin-myosin association are unclear. The disordered loop 2 of myosin is thought to mediate actomyosin interactions in complex, chemomechanical state-dependent fashion; however, the loop is usually unresolved in structural studies due to its intrinsic disorder. Here, we utilize a combination of molecular dynamics simulations and electrostatic calculations to investigate the dynamics of these actin binding regions of myosin. Our results show that loop 2 experiences disordered dynamics and that specific conformations sampled by loop 2 modulate the strength of the associative electrostatic force between actin and myosin. Variation in the actin-myosin associative force was associated with the presentation and orientation of positively charged residues in loop 2. We provide an in-depth analysis of the conformational state space occupied by loop 2 during nine 500 ns molecular dynamics simulations of pre-powerstroke human {beta}-myosin S1, with three replicates each of wildtype and two different mutant (E525K and V606M) myosin structures. This dataset allowed for exploration of how loop 2 conformational sampling is altered by these two mutations which have been clinically and experimentally associated with cardiomyopathy and altered actin binding affinity. The E525K and V606M mutations altered the conformational ensemble sampled by loop 2 and were associated with associative actin binding strength. These results highlight the importance of the positive charges on loop 2 for actomyosin interactions and demonstrate how disease-causing mutations outside of loop 2 can still affect it.

molecular biology↗