Limits...
Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly.

Becker KD, Gottshall KR, Hickey R, Perriard JC, Chien KR - J. Cell Biol. (1997)

Bottom Line: Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure.Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation.This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, American Heart Association Bugher Foundation Center for Molecular Biology, University of California, San Diego, La Jolla 92093, USA. dbecker@ucsd.edu

ABSTRACT
Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether point mutations in human beta MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human beta MHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human beta MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse beta MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type beta MHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

Show MeSH

Related in: MedlinePlus

Thick filaments containing R403Q have normal organization within the sarcomere. NRC transfected with human R403Q plasmid  were costained for exogenous βMHC (B) with the epitope specific antibody and myomesin (A). The merged image (C) shows that myomesin staining is completely localized to the M-line, indicating normal thick filament and sarcomere structure. Bars: (A) 20 μm; (inset), 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139848&req=5

Figure 6: Thick filaments containing R403Q have normal organization within the sarcomere. NRC transfected with human R403Q plasmid were costained for exogenous βMHC (B) with the epitope specific antibody and myomesin (A). The merged image (C) shows that myomesin staining is completely localized to the M-line, indicating normal thick filament and sarcomere structure. Bars: (A) 20 μm; (inset), 5 μm.

Mentions: There was no detectable difference in the distribution of phenotypes comparing expression of wild-type βMHC with the three βMHC mutations R249Q, R403Q, or S472V in NRC. All three of these mutant proteins assembled into extremely organized myofibrils (Fig. 5) at the same frequency as observed for the wild-type βMHC protein (Table I). R249Q, R403Q, and S472V mutant proteins assembled into A band regions as defined via costaining for endogenous myofilament proteins (Figs. 5 and 6). This result indicated that the three mutants are readily incorporated into thick filaments, and such inclusion did not produce an observable defect in sarcomere organization. Unlike the K184R mutant there was little excess accumulation or abnormal subcellular localization of the R249Q, R403Q, or S472V molecules even in cells with little sarcomeric structure. These three mutant proteins always colocalized with the endogenous MHC in transfected cells.


Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly.

Becker KD, Gottshall KR, Hickey R, Perriard JC, Chien KR - J. Cell Biol. (1997)

Thick filaments containing R403Q have normal organization within the sarcomere. NRC transfected with human R403Q plasmid  were costained for exogenous βMHC (B) with the epitope specific antibody and myomesin (A). The merged image (C) shows that myomesin staining is completely localized to the M-line, indicating normal thick filament and sarcomere structure. Bars: (A) 20 μm; (inset), 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139848&req=5

Figure 6: Thick filaments containing R403Q have normal organization within the sarcomere. NRC transfected with human R403Q plasmid were costained for exogenous βMHC (B) with the epitope specific antibody and myomesin (A). The merged image (C) shows that myomesin staining is completely localized to the M-line, indicating normal thick filament and sarcomere structure. Bars: (A) 20 μm; (inset), 5 μm.
Mentions: There was no detectable difference in the distribution of phenotypes comparing expression of wild-type βMHC with the three βMHC mutations R249Q, R403Q, or S472V in NRC. All three of these mutant proteins assembled into extremely organized myofibrils (Fig. 5) at the same frequency as observed for the wild-type βMHC protein (Table I). R249Q, R403Q, and S472V mutant proteins assembled into A band regions as defined via costaining for endogenous myofilament proteins (Figs. 5 and 6). This result indicated that the three mutants are readily incorporated into thick filaments, and such inclusion did not produce an observable defect in sarcomere organization. Unlike the K184R mutant there was little excess accumulation or abnormal subcellular localization of the R249Q, R403Q, or S472V molecules even in cells with little sarcomeric structure. These three mutant proteins always colocalized with the endogenous MHC in transfected cells.

Bottom Line: Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure.Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation.This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, American Heart Association Bugher Foundation Center for Molecular Biology, University of California, San Diego, La Jolla 92093, USA. dbecker@ucsd.edu

ABSTRACT
Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether point mutations in human beta MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human beta MHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human beta MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse beta MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type beta MHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

Show MeSH
Related in: MedlinePlus