Functional Analysis of Myosin Mutations That Cause Familial Hypertrophic Cardiomyopathy

Roopnarine, Osha; Leinwand, Leslie A.

doi:10.1016/s0006-3495(98)77743-4

Cited by 47 publications

(40 citation statements)

References 49 publications

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“…The majority of studies in the decade after the original discovery prepared myosin from biopsies of patients with FHC (23,24) or from a variety of expression systems (25)(26)(27)(28). With the introduction of the mouse model in 1996 (6), attention shifted to myosin isolated from homozygous (R403Q/R403Q) mouse hearts.…”

Section: Discussionmentioning

confidence: 99%

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

Lowey

Lesko

Rovner

et al. 2008

Journal of Biological Chemistry

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The R403Q mutation in the ␤-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly ␣-MHC as opposed to the ␤-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q ␣-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous ␣-MHC was largely replaced with transgenically encoded ␤-MHC. A His 6 tag was cloned at the N terminus of the ␣-and ␤-MHC to facilitate protein isolation by Ni 2؉ -chelating chromatography. Characterization of the R403Q ␣-MHC by the in vitro motility assay showed a 30 -40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a ␤-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q ␣-S1 is ϳ30% higher than for wild type, whereas the enzymatic activity for R403Q ␤-S1 is reduced by ϳ10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.The arginine to glutamine mutation at amino acid 403 (R403Q) in the ␤-myosin heavy chain (MHC) 2 was the first MHC missense mutation linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle (1). Since that time several hundred mutations in genes encoding sarcomeric proteins have been identified, leading to the designation of FHC as primarily a "disease of the sarcomere" (2, 3). However, the pathways linking the various genetic defects to the characteristic human disease phenotype remain largely unknown. Even the fundamental question of whether these mutations cause a gain or a loss of function in myosin remains unresolved. We have confined our study to the R403Q mutation, because it is one of the most extensively studied FHC mutations and because it has a poor clinical prognosis (2, 4). The majority of the earlier biochemical/biophysical results, derived from expression systems and biopsies, supported the hypothesis that the R403Q mutation leads to a large decrease in motor function, which ultimately results in a compensatory hypertrophic response (for review, see Ref. 5).The first murine model for FHC replaced one allele of the endogenous cardiac mouse ␣-MHC with the mutant ␣-MHC (R403Q) gene by homologous recombination (6); this heterozygous mouse (R403Q/ϩ) resembled the human cardiac phenotype in many ways, except cardiac hypertrophy was notably milder. Subsequent studies on myosin extracted from a Ͻ1-week-o...

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Section: Discussionmentioning

confidence: 99%

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

Lowey

Lesko

Rovner

et al. 2008

Journal of Biological Chemistry

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“…(vii) Targeted disruption of fast MyHC genes in mice causes myopathy with muscle weakness and disorganization of myofilaments (18), and point mutations in MyHC genes cause muscle dysfunction in Drosophila, Caenorhabditis elegans, and Dictyostelium (16). MyHC gene missense mutations may cause dominant negative effects on muscle function by various mechanisms, including interference with filament assembly and sarcomeric structure, functional defects in ATPase activity, impaired myosin-actin interaction, and perturbation of conformational changes during ATP hydrolysis (16,19,20). In familial hypertrophic cardiomyopathy associated with ␤ MyHC mutations it has been considered that myocardial hypertrophy and disarray of myocytes are secondary changes caused by defective sarcomeric function (20).…”

Section: Discussionmentioning

confidence: 99%

Autosomal dominant myopathy: Missense mutation (Glu-706 → Lys) in the myosin heavy chain IIa gene

Martinsson

Oldfors

Darín

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

140

102

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We here report on a human myopathy associated with a mutation in a fast myosin heavy chain (MyHC) gene, and also the genetic defect in a hereditary inclusion body myopathy. The disorder has previously been described in a family with an ''autosomal dominant myopathy, with joint contractures, ophthalmoplegia, and rimmed vacuoles.'' Linkage analysis and radiation hybrid mapping showed that the gene locus (Human Genome Map locus name: IBM3) is situated in a 2-Mb region of chromosome 17p13, where also a cluster of MyHC genes is located. These include the genes encoding embryonic, IIa, IIx͞d, IIb, perinatal, and extraocular MyHCs. Morphological analysis of muscle biopsies from patients from the family indicated to us that the type 2A fibers frequently were abnormal, whereas other fiber types appeared normal. This observation prompted us to investigate the MyHC-IIa gene, since MyHC-IIa is the major isoform in type 2A fibers. The complete genomic sequence for this gene was deduced by using an ''in silico'' strategy. The gene, found to consist of 38 exons, was subjected to a complete mutation scan in patients and controls. We identified a missense mutation, Glu-706 3 Lys, which is located in a highly conserved region of the motor domain, the so-called SH1 helix region. By conformational changes this region communicates activity at the nucleotide-binding site to the neck region, resulting in the lever arm swing. The mutation in this region is likely to result in a dysfunctional myosin, compatible with the disorder in the family.

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“…Other hydrophobic interactions also occur. The critical Arg 405 Gln mutation (identical with the Arg 403 Gln mutation in human cardiac myosin), which occurs in a severe familial hypertrophic cardiomyopathy and results in a decreased actin-activated myosin ATPase (22), is not in the interface: It faces away from actin. However, it may make a salt bridge with Glu 631 in the now structured part of loop 2.…”

Section: Fig 2 (A)mentioning

confidence: 99%

The actin-myosin interface

Lorenz

Holmes

2010

Proc. Natl. Acad. Sci. U.S.A.

108

165

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In order to understand the mechanism of muscle contraction at the atomic level, it is necessary to understand how myosin binds to actin in a reversible way. We have used a novel molecular dynamics technique constrained by an EM map of the actin-myosin complex at 13-Å resolution to obtain an atomic model of the strong-binding (rigor) actin-myosin interface. The constraining force resulting from the EM map during the molecular dynamics simulation was sufficient to convert the myosin head from the initial weak-binding state to the strong-binding (rigor) state. Our actin-myosin model suggests extensive contacts between actin and the myosin head (S1). S1 binds to two actin monomers. The contact surface between actin and S1 has increased dramatically compared with previous models. A number of loops in S1 and actin are involved in establishing the interface. Our model also suggests how the loop carrying the critical Arg 405 Glu mutation in S1 found in a familial cardiomyopathy might be functionally involved.cryoelectron microscopy | myosin II | myosin V T he binding of myosin to actin can be weak or strong. The affinity, which changes over 5 orders of magnitude, is controlled by ATP binding to the myosin head at a position remote from the actin binding site. ATP binding produces "weak binding." In the absence of ATP the binding is "strong." The mechanism of this interaction and its control by ATP is central to an understanding of muscle contraction (1). Thus we need to know the structure of the strong-binding state of myosin to actin in atomic detail. Attempts to crystallize the complex have been unsuccessful. However, incubating myosin heads (subfragment 1, S1) with filamentous actin (f-actin) produces "decorated actin" in which each myosin head binds to the actin filament in the strong-binding or "rigor" configuration. Decorated actin may be used as a model of the actin-myosin "strong-binding" mode. Cryoelectron microscopy and 3D reconstructions of actin decorated with myosin II have produced a density map at 13-Å resolution (2). Combining this with structural data from the head of myosin V, which can be crystallized in the strong-binding myosin conformation (3), gave a near-atomic view of the actin-myosin rigor interaction (4). The actin binding site comprises parts of the upper and lower 50-K domains that are separated by a cleft in the weak-binding form. The cleft closes on strong binding. However, the interface appears incomplete: Some additional local refolding of myosin and actin appears to be necessary. We have now used constrained molecular dynamics-molecular dynamics flexible fitting (MDFF) (5) to fit atomic models into the interface using the EM density map as a constraint. In MDFF the gradient of the density is used as an extra force field. It appears that, in addition to the elements of the upper and lower 50-K domains recognized in the earlier study, part of the flexible loop 2 between the upper and lower 50-K domains in S1 and the actin-DNase I binding loop in actin form essential parts of the interface...

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Functional Analysis of Myosin Mutations That Cause Familial Hypertrophic Cardiomyopathy

Cited by 47 publications

References 49 publications

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

Autosomal dominant myopathy: Missense mutation (Glu-706 → Lys) in the myosin heavy chain IIa gene

The actin-myosin interface

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