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

Abstract

The cardiac {beta}-myosin (MYH7) variant E525K was identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered myosin constructs has shown that this variant stabilizes the interacting heads motif (IHM) of myosin and increases the ATPase activity of variant 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) gene-edited for heterozygous expression of the E525K variant. Single-cell contraction for E525K hiPSC-CMs was reduced by 65%, and isometric twitch force in engineered heart tissues (EHTs) was reduced by 39%. In contrast, maximal Ca2+ activated isometric force in isolated myofibrils was increased by 45%, and sub-maximal Ca2+ activated force was similar to that of 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 the twitch force of myocytes decreased while the maximal isometric force increased, we used a spatially explicit, stochastic-kinetic sarcomere model. The results were explained by changes in three rates: reduced recruitment from the OFF/IHM state and increased rates of actin binding and Pi release. Finite element modeling was used to demonstrate that additional contractile deficits in cells and EHTs likely result from myofibrillar disorganization. This study demonstrates the value of multi-scale analysis and coupled computational modeling for understanding the molecular mechanisms underlying sarcomere mutations in cardiomyocytes.