Insights into Human β-Cardiac Myosin Function from Single Molecule and Single Cell Studies

Abstract

β-Cardiac myosin is a mechanoenzyme that converts the energy from ATP hydrolysis into a mechanical force that drives contractility in muscle. Thirty percent of the point mutations that result in hypertrophic cardiomyopathy are localized to MYH7, the gene encoding human β-cardiac myosin heavy chain (β-MyHC). Force generation by myosins requires a tight and highly conserved allosteric coupling between its different protein domains. Hence, the effects of single point mutations on the force generation and kinetics of β-cardiac myosin molecules cannot be predicted directly from their location within the protein structure. Great insight would be gained from understanding the link between the functional defect in the myosin protein and the clinical phenotypes of patients expressing them. Over the last decade, several single molecule techniques have been developed to understand in detail the chemomechanical cycle of different myosins. In this review, we highlight the single molecule techniques that can be used to assess the effect of point mutations on β-cardiac myosin function. Recent bioengineering advances have enabled the micromanipulation of single cardiomyocyte cells to characterize their force–length dynamics. Here, we briefly review single cell micromanipulation as an approach to determine the effect of β-MyHC mutations on cardiomyocyte function. Finally, we examine the technical challenges specific to studying β-cardiac myosin function both using single molecule and single cell approaches.

Identifying Sarcomere Gene Mutations in Hypertrophic Cardiomyopathy

“…88 Equivalent to disease in human patients, the clinical expression of HCM in mice (ventricular hypertrophy, myocyte disarray and increased myocardial fibrosis) is delayed until early in adulthood. 86 Isolation of mutant myosins from young pre-hypertrophic mice allowed assessment of single molecule mechanics (reviewed by Leinwand, Spudich and colleagues 89 ) of myosins carrying HCM mutations. Surprisingly, mutant myosin heavy chains exhibited enhanced contractile properties, including increased force generation, ATP hydrolysis and action-myosin sliding velocities, indicating that HCM mutations produce a gain in function.…”

How Do Mutations in Contractile Proteins Cause the Primary Familial Cardiomyopathies?

“…Early studies of the R403Q mutation created some confusion since study of the mutation in a skeletal muscle biopsy and expression of the mutation in Baculovirus and in Dictyostelium expression systems indicated a substantially lower rate of crossbridge turnover [22][23][24], whilst a human heart biopsy with this mutation and a transgenic [62] mouse with the mutation introduced into MYH6 showed an increase in crossbridge turnover rate [25][26][27]. Most published studies have addressed the effect of myosin heavy chain mutations on motor functions [28], and only a few studies measured myofibrillar Ca 2+ -sensitivity (Table 2) [29,30]. In the case of MyBP-C, there is no good functional assay available, and most investigations have looked at the effect of MyBP-C mutations on sarcomeric structure [31,32].…”

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

“…The sliding velocity generated by an ensemble of myosin motors is thought to correlate to the shortening velocity measured in muscle (Howard, 2001). In order to examine d uni , f, and t on , the single molecule laser trap motility assay is often used (Simmons and Finer, 1994; Sivaramakrishnan et al, 2009). In this assay a single actin filament is strung between two beads that are each trapped with laser tweezers and when a single myosin molecule is brought close to the actin filament individual displacements (d uni ) are measured.…”