Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function

Abstract

Dilated cardiomyopathy (DCM) leads to heart failure, a leading cause of death in industrialized nations. Approximately 30% of DCM cases are genetic in origin, with some resulting from point mutations in cardiac myosin, the molecular motor of the heart. The effects of these mutations on myosin's molecular mechanics have not been determined. We have engineered two murine models characterizing the physiological, cellular, and molecular effects of DCM-causing missense mutations (S532P and F764L) in the ␣-cardiac myosin heavy chain and compared them with WT mice. Mutant mice developed morphological and functional characteristics of DCM consistent with the human phenotypes. Contractile function of isolated myocytes was depressed and preceded left ventricular dilation and reduced fractional shortening. In an in vitro motility assay, both mutant cardiac myosins exhibited a reduced ability to translocate actin (V actin) but had similar force-generating capacities. Actin-activated ATPase activities were also reduced. Single-molecule laser trap experiments revealed that the lower V actin in the S532P mutant was due to a reduced ability of the motor to generate a step displacement and an alteration of the kinetics of its chemomechanical cycle. These results suggest that the depressed molecular function in cardiac myosin may initiate the events that cause the heart to remodel and become pathologically dilated.animal models ͉ biophysics ͉ genetics ͉ single molecule ͉ laser trap D ilated cardiomyopathy (DCM) is an early step in the pathway leading to heart failure, the leading cause of human morbidity and mortality (1, 2). Although etiological factors such as ischemia and diabetes can result in DCM, at least 30% of cases are genetic in origin, with mutations found in sarcomeric proteins associated with the cytoskeletal and contractile systems (3). Clinically, DCM is characterized by myocardial hypertrophy, thin-walled ventricles, and myocyte hypoplasia (2, 4). Dilated hearts are hypocontractile, with systolic dysfunction leading to a reduced ejection fraction: a hallmark of a failing heart (2, 3). Only recently have autosomal dominant missense mutations to the ␤-cardiac myosin heavy chain (MHC) been identified that result in DCM (5, 6). Because the primary defect resides within the heart's molecular motor, we hypothesized that altered cardiac contractile function in DCM hearts might reflect changes in the mutant myosin's ability to generate force and motion as it cyclically interacts with actin during its hydrolysis of ATP.To investigate the impact of two distinct DCM-causing MHC mutations (S532P and F764L) on cardiac myosin's molecular performance, we have genetically engineered these missense mutations separately into the murine ␣-cardiac MHC gene, the human ␤-MHC homolog, to generate two separate DCM knockin mouse models. In addition to cardiac dilation and dysfunction, defects in isolated myocyte contractility were observed that are consistent with DCM. Interestingly, defects at the whole-heart and cellular levels were cor...

Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles

“…Disrupted contractile performance of the diseased myocardium may result from alterations in the mutant myosin's ability to generate force and motion (Schmitt et al, 2006;Debold et al, 2007). Mouse models engineered with DCM-causing ␤-cardiac MHC mutations reproduced morphological and functional characteristics consistent with the human phenotype (Schmitt et al, 2006).…”

“…Disrupted contractile performance of the diseased myocardium may result from alterations in the mutant myosin's ability to generate force and motion (Schmitt et al, 2006;Debold et al, 2007). Mouse models engineered with DCM-causing ␤-cardiac MHC mutations reproduced morphological and functional characteristics consistent with the human phenotype (Schmitt et al, 2006). At the molecular level, the mutations depressed ATPase activities, in vitro actin filament sliding velocities, and/or maximal force generating capacity of myosin motors (Schmitt et al, 2006;Debold et al, 2007).…”

“…Mouse models engineered with DCM-causing ␤-cardiac MHC mutations reproduced morphological and functional characteristics consistent with the human phenotype (Schmitt et al, 2006). At the molecular level, the mutations depressed ATPase activities, in vitro actin filament sliding velocities, and/or maximal force generating capacity of myosin motors (Schmitt et al, 2006;Debold et al, 2007). Depressing one or more of these molecular indices of myosin function was considered sufficient to trigger the cascade of events that lead to DCM (Debold et al, 2007).…”

“…Expression of D45 myosin with diminished biomechanical properties produced a Drosophila cardiac phenotype displaying structural and functional characteristics remarkably similar to those found in humans with, and in vertebrate models of, dilated cardiomyopathy (DCM) (Fatkin and Graham, 2002;Ahmad et al, 2005;Chang and Potter, 2005;Schmitt et al, 2006;Debold et al, 2007). DCM is a myocardial disorder characterized by left and/or right ventricular dilation and distended chambers (Fatkin and Graham, 2002;Ahmad et al, 2005;Chang and Potter, 2005).…”

Cardiac fibrosis in mice with hypertrophic cardiomyopathy is mediated by non-myocyte proliferation and requires Tgf-β

“…α-MHC 403/+ mice have been extensively characterized (17,18). α-MHC 719/+ mice were generated by homologous recombination as previously described (17,62,63). …”