Molecular mechanics of E525K dilated cardiomyopathy mutant human cardiac myosin

Dilated cardiomyopathy (DCM) is associated with point mutations in beta-cardiac myosin and characterized by cardiac hypo-contractility. Myocardial power generation can be tuned by modulation of the number of functionally available myosin motors through myosin's ability to adopt an autoinhibited “super relaxed” state (SRX). Therefore, hypo-contractility of DCM hearts may result from: 1) reduced function of individual myosin heads, and/or 2) decreased myosin availability through increased SRX stabilization. To define the molecular impact of an established DCM myosin mutation, E525K, we expressed both wildtype (WT) and E525K myosin in several GFP-tagged human cardiac myosin constructs. We then determined actin filament velocity versus myosin surface density (GFP fluorescence) in the in vitro motility assay (1 mM ATP, 25 mM KCl, 30oC). The SRX state is thought to be stabilized by intramolecular head-tail interactions, which is where the E525K mutation exists. Accordingly, myosin constructs incapable of these interactions, such as single headed (S1) and short-tailed, double-headed myosin (heavy meromyosin with 2-heptad tail) constructs generated similarly fast velocities (2298±298 nm/s and 2352±234 nm/s, respectively). At the same myosin density, longer-tailed, SRX-capable constructs generated progressively slower velocities: 961±379 nm/s with a 15-heptad tail and 635±195 nm/s with a 25-heptad tail. These SRX-capable myosin constructs required 4-fold more myosin surface density to generate velocity equivalent to non-SRX constructs. Therefore, we propose that the 15- and 25-heptad constructs adopt the SRX state with a 75% probability. Unexpectedly, the E525K had no apparent impact on velocity, suggesting that under the low salt, unloaded in vitro motility assay conditions, the E525K mutation does not alter ensemble motor function. However, power generation is dependent on both force (F) and velocity (V), so future measurements of F:V relationships may uncover the true impact of this DCM myosin mutation.