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...

Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), a disease characterized by left-ventricular hypertrophy, angina, and dyspnea that can result in sudden death. We report here that a murine model of FHC bearing a cardiac myosin heavy-chain gene missense mutation (alphaMHC(403/+)), when treated with calcineurin inhibitors or a K(+)-channel agonist, developed accentuated hypertrophy, worsened histopathology, and was at risk for early death. Despite distinct pharmacologic targets, each agent augmented diastolic Ca(2+) concentrations in wild-type cardiac myocytes; alphaMHC(403/+) myocytes failed to respond. Pretreatment with a Ca(2+)-channel antagonist abrogated diastolic Ca(2+) changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated alphaMHC(403/+) mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca(2+) responses that initiate a hypertrophic response. These data define an important Ca(2+)-dependent step in the pathway by which mutant sarcomere proteins trigger myocyte growth and remodel the heart, provide definitive evidence that environment influences progression of FHC, and suggest a rational therapeutic approach to this prevalent human disease.

Dominant mutations in sarcomere protein genes cause hypertrophic cardiomyopathy, an inherited human disorder with increased ventricular wall thickness, myocyte hypertrophy, and disarray. To understand the early consequences of mutant sarcomere proteins, we have studied mice (designated αMHC 403/+ ) bearing an Arg403Gln missense mutation in the α cardiac myosin heavy chain. We demonstrate that Ca 2+ is reduced in the sarcoplasmic reticulum of αMHC 403/+ mice, and levels of the sarcoplasmic reticulum Ca 2+ -binding protein calsequestrin are diminished in advance of changes in cardiac histology or morphology. Further evidence for dysregulation of sarcoplasmic reticulum Ca 2+ in these animals is seen in their decreased expression of the ryanodine receptor Ca 2+ -release channel and its associated membrane proteins and in an increase in ryanodine receptor phosphorylation. Early administration of the L-type Ca 2+ channel inhibitor diltiazem restores normal levels of these sarcoplasmic reticular proteins and prevents the development of pathology in αMHC 403/+ mice. We conclude that disruption of sarcoplasmic reticulum Ca 2+ homeostasis is an important early event in the pathogenesis of this disorder and suggest that the use of Ca 2+ channel blockers in advance of established clinical disease could prevent hypertrophic cardiomyopathy caused by sarcomere protein gene mutations. cytoplasm, sarcoplasmic reticulum (SR), and sarcomereaccount for excitation-contraction coupling. Depolarization triggers entry of small amounts of Ca 2+ through the L-type Ca 2+ channels located on the cell membrane, which in turn prompts SR Ca 2+ release by cardiac ryanodine receptors (RyR's), a process termed calcium-induced Ca 2+ release. The resulting rapid rise in cytosolic levels fosters Ca 2+ -troponin-C interactions and triggers sarcomere contraction. Activation of the ATP-dependent calcium pump (SERCA) recycles cytosolic Ca 2+ into the SR to restore sarcomere relaxation. To understand the mechanism by which calcium dysregulation occurs in αMHC 403/+ myocytes, we studied Ca 2+ -binding proteins in specific myocyte compartments and monitored the hypertrophic response to the Arg403Gln missense mutation. We report that changes in Ca 2+ -binding protein levels occur in advance of disease, and we demonstrate that restoration of these protein levels by the L-type Ca 2+ channel inhibitor diltiazem prevents clinical expression of hypertrophic cardiomyopathy in αMHC 403/+ mice. MethodsMice. αMHC 403/+ mice were generated as described (13) and were bred and maintained on the 129/SvEv genetic background. Selected mice were treated with diltiazem added to their drinking water (450 mg/l) corresponding to 1.8 mg of diltiazem per day. Short-term studies involved treatment of mice with either enalapril, atenolol, or fludrocortisone added to their drinking water to achieve a dose of 25 mg/kg/d. All mice were maintained according to protocols approved by the Institutional Animal Care and Use Committee of Harvard Medical School.RNA and protein analyses. Nor...

Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.