Single-Molecule Mechanics of R403Q Cardiac Myosin Isolated From the Mouse Model of Familial Hypertrophic Cardiomyopathy

Abstract

Abstract-Familial hypertrophic cardiomyopathy (FHC) is an inherited cardiac disease that can result in sudden death in the absence of any overt symptoms. Many of the cases documented to date have been linked with missense mutations in the ␤-myosin heavy chain gene. Here we present data detailing the functional impact of one of the most deadly mutations, R403Q, on myosin motor function. Experiments were performed on whole cardiac myosin purified from a mouse model of FHC to eliminate potential uncertainties associated with protein expression systems. The R403Q mutant myosin demonstrated 2.3-fold higher actin-activated ATPase activity, 2.2-fold greater average force generation, and 1.6-fold faster actin filament sliding in the motility assay. The force-and displacement-generating capacities of both the normal and mutant myosin were also characterized at the single molecule level in the laser trap assay. Both control and mutant generated similar unitary forces (Ϸ1 pN) and displacements (Ϸ7 nm) without any differences in event durations.On the basis of the distribution of mean unitary displacements, this mutation may possibly perturb the mechanical coordination between the 2 heads of cardiac myosin. Any of these observations could, alone or possibly in combination, result in abnormal power output and potentially a stimulus for the hypertrophic response. (Circ Res. 2000;86:737-744.)Key Words: familial hypertrophic cardiomyopathy Ⅲ cardiac myosin Ⅲ R403Q mouse model Ⅲ laser trap Ⅲ molecular motor F amilial hypertrophic cardiomyopathy (FHC) is an inherited cardiac disease that frequently results in the sudden death of young and otherwise healthy individuals. 1,2 Although the clinical manifestation of FHC is widely varied, common features include asymmetric septal hypertrophy, potential outflow tract obstruction, myocyte disarray, interstitial fibrosis, and arrhythmia. [1][2][3] Approximately 70% of all FHC cases documented to date have been linked to single point mutations in various contractile proteins of the cardiac sarcomere. These include ␤-myosin heavy chain (MHC), myosin regulatory light chain, myosin essential light chain, troponin-T, troponin-I, ␣-tropomyosin, and myosin binding protein-C. [3][4][5][6][7][8][9] Although a large portion of these point mutations have been localized to the ␤-MHC gene (nϾ50 specifically in the motor domain or "head" region), 10 few have been characterized in terms of their effect on myosin motor function to the extent of the R403Q substitution. This mutation causes malignant disease, with 50% of the affected individuals dying by 40 years of age. 6,11 Investigators seeking to characterize the effects of R403Q have worked with muscle fibers from afflicted individuals, myosin purified from human patients, and genetically engineered fragments of myosin. Because ␤-MHC is expressed in slow skeletal muscle fibers in addition to adult cardiac tissue, 12 Lankford et al 13 were able to isolate fibers from the soleus muscles of R403Q FHC patients for mechanical characterization. These fibers...

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

“…Compromised coronary flow due to hypertrophy, microvascular dysfunction (42), increased oxidative stress (43), and increased metabolic demands imposed by abnormal biophysical properties of mutant sarcomeres (7,8) are factors that contribute to premature myocyte death and the emergence of focal fibrosis in HCM. Far less is known about mechanisms that expand the extracellular matrix in HCM hearts.…”

“…Activated non-myocyte cells may also be derived from circulating cells (14,(46)(47)(48). Alternatively, the enhanced biomechanical forces resulting from sarcomere protein mutations (7,8) could also provide a local mechanism for activating resident non-myocyte cells. Lineage studies are underway to assess the source of activated non-myocyte cells in HCM hearts.…”

“…Previous studies of HCM mutations in the myosin heavy chain gene showed that these enhance sarcomere biophysical properties (7)(8)(9) and alter cardiac relaxation (10) and calcium signaling in myocytes (11), events that are associated with widespread transcriptional changes (12). In addition to inducing changes within myocytes, the characteristic histopathology found in HCM hearts implies that sarcomere gene mutations also impact non-myocyte cells.…”

Molecular genetics of familial hypertrophic cardiomyopathy (FHC)

“…The symptoms, including premature death, were similar to those observed in human, thus showing that the rabbit could be a desirable model for studying FHC. The mouse model was also used to understand the basis for the highly malignant phenotype of the p.R403Q mutation (Tyska et al 2000). The work of Tyska et al (2000) revealed that the mutant protein actually exhibits a 2.3-fold higher actin-activated ATPase activity, a 2.2-fold greater average force generation, and a 1.6-fold faster actin filament sliding in the motility assay.…”

Hypertrophic cardiomyopathy: from gene defect to clinical disease

“…In transgenic models, levels of mutant sarcomere protein expressed within the heart show some correlation with the severity of myocyte dysfunction [57]. Analyses of the mechanics of Arg403Gln myosin derived from heart muscle indicate enhanced actin-activated ATPase activity, increased generated force, and accelerated actin filament sliding [61]. Taken together with data derived from in vitro systems, these findings collectively indicate HCM myosin mutations produce a gain of function.…”