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

The {beta}-cardiac myosin (MYH7) mutation E525K was first identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered myosin constructs has shown that this mutation causes hypocontractility by stabilizing the interacting heads motif (IHM) of myosin despite the mutant E525K motor head exhibiting increased ATPase activity. However, no measurements have been made in myofilaments or cardiomyocytes to determine how this mutation affects contractile function. Here, we present force and contractile kinetics measurements from induced pluripotent stem cell (iPSC)-derived cardiomyocytes engineered for heterozygous expression of E525K. Contraction of E525K single cells decreased by 65%, and isometric twitch force in engineered heart tissues (EHTs) decreased by 39%. In contrast, maximal isometric force in isolated myofibrils increased by 45%. Structural analysis revealed reduced myofibril content (13.7% decrease) and 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, no change was observed in the rate of ADP release. Importantly, there was no change in the rate of force development or relaxation in myofibrils, cells, or EHTs. These findings suggest that myosin crossbridge cycling is not altered under load by E525K. Decreased force generation in EHTs and shortening in cardiomyocytes arise from reduced sarcomere number and myofibrillar disorganization. Additional force deficits likely result from stabilization of the IHM, as recently reported by others. This study demonstrates the value of multi-scale analysis for determining the functional profile of cardiomyocytes containing disease-related sarcomere protein mutations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/733270v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@5f85f5org.highwire.dtl.DTLVardef@153b3b6org.highwire.dtl.DTLVardef@3b8f21org.highwire.dtl.DTLVardef@31d323_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: A Model for how the E525K mutation impacts contracting myofibrils Here we have shown that the E525K mutation impacts contraction in three ways: (1) Decreased sarcomere organization in cells and tissues drives decreased force generation. (2) Increased binding affinity of E525K myosin for actin contributes to increased force generation in isolated myofibrils. (3) Increased IHM stability. (4) The rate limiting step of loaded crossbridge cycling, ADP release, is unchanged by the E525K mutation and the rate of loaded contraction and relaxation is unchanged at all scales of contraction measured here. C_FIG