Mutations in the motor domain modulate myosin activity and myofibril organization

Wang, Qun; Moncman, Carole L.; Winkelmann, Donald A.

doi:10.1242/jcs.00709

Cited by 67 publications

(69 citation statements)

References 62 publications

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“…Histological analyses of heterozygous and homozygous cardiac tissue, although consistent with the diagnosis of DCM and elongated myocytes, demonstrated no myofibrillar disarray or fibrosis in young mice ( Fig. 1 and Table 2) or in 50-week-old mice (data not shown), as observed previously in models of hypertrophic cardiomyopathy (HCM) (9,10) or in other forms of DCM (11).…”

Section: Resultssupporting

confidence: 85%

“…Do the various myosin mutations differentially impact myosin performance to account for such dramatically different clinical phenotypes associated with DCM and HCM? HCM-causing mutations, especially the R403Q mutant, enhance rather than compromise function (10,15,19,28,29). Direct comparison of the mutant myosins described here with data obtained by our laboratories from mutant R403Q myosin isolated from a mouse model using all of the identical assays (29) demonstrates that V actin , F avg , and actomyosin ATPase activity all are enhanced for R403Q myosin compared with controls.…”

Section: Resultsmentioning

confidence: 54%

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

Schmitt

Debold²,

Ahmad³

et al. 2006

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

108

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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...

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 54%

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

Schmitt

Debold²,

Ahmad³

et al. 2006

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

108

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“…S3 B and D), strongly suggesting a direct functional requirement for NMI in the nucleus, rather than an indirect effect caused by a disrupted cytoskeleton. To document the involvement of NMI-based nuclear motors in mediating E 2 -induced interchromosomal interactions, we preformed rescue experiments, using NMI mutants that impair actin binding (e.g., R353C) or the ATPase activity (e.g., S397L) in the nuclear myosin-I ''head'' (34,35). We found that the interchromosomal interactions abolished with anti-NMI IgG could be restored using the expression vector expressing WT, but not mutant NMI defective in actin binding or lacking ATPase activity (Fig.…”

Section: Identification Of Estrogen-induced Interchromosomal Interactmentioning

confidence: 99%

Enhancing nuclear receptor-induced transcription requires nuclear motor and LSD1-dependent gene networking in interchromatin granules

Kwon

Núñez

et al. 2008

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

251

253

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Fig. 2. Rapid induction of interchromosomal interactions by nuclear hormone signaling. (A) 3D-FISH confirmation of E 2 -induced (60 min) TFF1:GREB1 interchromosomal interactions in HMECs with the distribution of loci distances measured (box plot with scatter plot) and quantification of colocalization (bar graph) before and after E 2 treatment. Cells exhibiting mono-or biallelic interactions were combined for comparison with cells showing no colocalization; statistical significance in the bar graph was determined by χ 2 test (**, P < 0.001). (B) 2D FISH confirmation of the interchromosomal interactions in HMEC cells by combining chromosome paint (aqua) and specific DNA probes (green and red). (Upper) Illustrates two examples of mock-treated cells. (Lower) Shows the biallelic interactions/ nuclear reorganization after E 2 treatment for 60 min, exhibiting kissing events between chromosome 21 and chromosome 2. (C) Similar analysis on HMECs, but in this case using 3D FISH to paint chromosome 2 (red) and chromosome 21 (green), showing E 2 -induced chromosome 2-chromosome 21 interaction. Both assays revealed neither chromosome 21-chromosome 21 nor chromosome 2-chromosome 2 interactions in response to E 2. (D) Temporal kinetics of GREB1:TFF1 interactions by 3D FISH in HMECs (**, P < 0.001 by χ 2 ). (E-G) Nuclear microinjection of siRNA against ERα, CBP/p300, or SRC1/pCIP prevented E 2 -induced interchromosomal interactions, counting both mono-and biallelic interactions (**, P < 0.001 by χ 2 ). The injection of siER and siDLC1 were done in the same experiment, sharing the same control group. (H) Nuclear microinjection of siRNA against LSD1, which was shown to be required for estrogen-induced gene expression (22), did not block E 2 -induced interchromosomal interactions. The injection of siLSD1 and SRC1/pCIP were done in a single experiment, sharing the same control group.

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“…The cDNA of the GFP-myosin was cloned between an enhanced CMV promoter and a SV40 polyadenylation signal in the AdEasy shuttle vector pCMVShuttle for construction of the replication-deficient adenovirus AdGFP-MHC (He et al, 1998). The details of construction of GFP::MHC chimeric cDNA and the production and amplification of the recombinant adenovirus AdGFP-MHC using the AdEasy vector system are described elsewhere (Wang et al, 2003).…”

Section: Methodsmentioning

confidence: 99%

“…A replication-defective adenovirus containing a GFP::myosin chimeric gene driven by a CMV promoter has been developed and used to infect post mitotic C2C12 myocytes in culture (Wang et al, 2003). This cell line is an established model for studying the expression of muscle-specific genes and the assembly of muscle proteins into striated myofibrils (McMahon et al, 1994).…”

Section: Myosin Transits Through Intermediates Before Assembling In Mmentioning

confidence: 99%

Chaperone-mediated folding and assembly of myosin in striated muscle

Srikakulam¹,

Winkelmann²

2004

Journal of Cell Science

Self Cite

137

131

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De novo folding and assembly of striated muscle myosin was analyzed by expressing a GFP-tagged embryonic myosin heavy chain (GFP-myosin) in post-mitotic C2C12 myocytes using replication defective adenoviruses. In the early stages of muscle differentiation, the GFP-myosin accumulates in bright globular foci and short filamentous structures that are later replaced by brightly fluorescent myofibrils. Time-lapse microscopy shows that the intermediates are dynamic and are present in elongating and fusing myocytes and in multinucleated myotubes. Immunostaining reveals the co-localization of the molecular chaperones Hsc70 and Hsp90 with the GFP-myosin in the intermediates, but not in the mature myofibrils. Uninfected cells have similar intermediates suggesting a common pathway for myosin maturation. Two conformation-sensitive antibodies that bind the unfolded motor domain and the coiled-coil conformation of the rod demonstrate that in the intermediates, the myosin rod is folded but the motor domain is not folded. Electron microscopy reveals that the intermediates contain loose filament bundles surrounded by a protein rich matrix. Geldanamycin, a specific inhibitor of Hsp90, reversibly blocks myofibril assembly and triggers accumulation of myosin folding intermediates. We conclude that multimeric complexes of nascent myosin filaments associated with Hsc70 and Hsp90 are intermediates in the folding and assembly pathway of muscle myosin.

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Mutations in the motor domain modulate myosin activity and myofibril organization

Cited by 67 publications

References 62 publications

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

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

Enhancing nuclear receptor-induced transcription requires nuclear motor and LSD1-dependent gene networking in interchromatin granules

Chaperone-mediated folding and assembly of myosin in striated muscle

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