Single-molecule analysis sheds light on cardiac myosin dysfunction due to hypertrophic cardiomyopathy mutation A57D in ventricular myosin light chain-1 (MLC1v)

Abstract

Ventricular myosin light chain-1 (MLC1v) is a key structural and function-modulating component of the {beta}-cardiac myosin ({beta}M-II) motor complex. Single-point mutations in MLC1v are linked to severe forms of hypertrophic cardiomyopathy (HCM) and sudden cardiac death (SCD) at a young age. However, the molecular mechanisms underlying the motor dysfunction responsible for HCM phenotype development are not fully understood. Here, we investigated native {beta}M-II motors isolated from septal myectomy sample of an HCM patient, harboring a rare homozygous mutation in MLC1v (A57D). Using a pure population of mutant motors (MUT), and sensitive single-molecule functional analysis approach, we directly assessed the primary functional alterations in {beta}M-II bearing A57D MLC1v mutation. In optical trap single-molecules measurements, the mutant motors displayed increased actomyosin (AM) interaction duration in strongly bound state (ton), corresponding to 3-fold reduced AM detachment rate than wild type myosin (WT). The MUT myosin also generated a shorter powerstroke size ({delta}). Ensemble average analysis of AM interaction events demonstrated that both the first powerstroke ({delta}1) associated with Pi release and the second powerstroke ({delta}2) linked to ADP release were reduced in MUT myosin. Moreover, the increased actomyosin cross-bridge stiffness in the AM.ADP state was observed for MUT compared to WT motors. Consistent with slower AM detachment rate and shorter stroke size, reconstituted human mutant {beta}M-II displayed slower actin filament gliding speed. Alterations in sarcomere-level mechanics included increased Ca2+ sensitivity of force generation and prolonged relaxation time, as predicted by FiberSim modelling. Molecular dynamics simulations indicated that the substitution of alanine by aspartate altered MLC1v interactions with myosin heavy chain (MyHC) and light chain 2 (MLC2v), affecting the curvature and flexibility of the lever arm. Overall, these studies establish the molecular mechanism underlying the primary myosin dysfunction due to A57D MLC1v mutation and further highlight the crucial role of MLC1v-mediated regulation of myosin function. Understanding the precise changes in the mutant myosins biomechanical properties is directly relevant to comprehending the initial triggers for pathological cardiac remodeling in HCM patients and designing tailored therapeutic interventions.