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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.

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Epitope tagged wild-type human βMHC protein assembles into normal myofibrils in transiently transfected NRC. Cells were  transfected with plasmids containing the wild-type human βMHC cDNA tagged with either the EE (A–C) or HA (D–F). The cells were  stained for the presence of actin filaments using rhodamine–phalloidin (A and C) and were costained with the anti-EE (B) or anti-HA (E)  specific antibodies. Sarcomeres containing human βMHC have well defined A bands (a), I bands (i), and M lines (m). The composite  images (C and F) show that the exogenous βMHC (green) fills the A band, except the H zone as expected, in a pattern that is complementary and partially overlapping with F-actin (red, C and F). Bar, 20 μm (inset enlarged 3×).
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Figure 2: Epitope tagged wild-type human βMHC protein assembles into normal myofibrils in transiently transfected NRC. Cells were transfected with plasmids containing the wild-type human βMHC cDNA tagged with either the EE (A–C) or HA (D–F). The cells were stained for the presence of actin filaments using rhodamine–phalloidin (A and C) and were costained with the anti-EE (B) or anti-HA (E) specific antibodies. Sarcomeres containing human βMHC have well defined A bands (a), I bands (i), and M lines (m). The composite images (C and F) show that the exogenous βMHC (green) fills the A band, except the H zone as expected, in a pattern that is complementary and partially overlapping with F-actin (red, C and F). Bar, 20 μm (inset enlarged 3×).

Mentions: An epitope tag was inserted in the NH2-terminal region of human βMHC to facilitate detection of exogenous protein in transfected NRC. Exogenous βMHC proteins containing either haemaglutinin (HA-1 = YPYDVPDYA; Wilson et al., 1984) or glu–glu (EE = EEYMPME; Grussenmeyer et al., 1985) epitopes were specifically recognized by their respective monoclonal antibodies in transiently transfected NRC (Fig. 2). Each epitope was positioned between amino acids 3 and 4 of the βMHC molecule, a domain with no ascribed function. As noted in previous studies, the crystal structure of the myosin S1 head reveals that the NH2 terminus is free in solution, existing as a random coil on the surface molecule (Rayment et al., 1993a,b), making this region a good target for the insertion of short sequences. The exogenous βMHC protein was incorporated into myofibrils in a pattern indistinguishable from the endogenous MLC2v (data not shown), a molecule known to bind MHC.


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)

Epitope tagged wild-type human βMHC protein assembles into normal myofibrils in transiently transfected NRC. Cells were  transfected with plasmids containing the wild-type human βMHC cDNA tagged with either the EE (A–C) or HA (D–F). The cells were  stained for the presence of actin filaments using rhodamine–phalloidin (A and C) and were costained with the anti-EE (B) or anti-HA (E)  specific antibodies. Sarcomeres containing human βMHC have well defined A bands (a), I bands (i), and M lines (m). The composite  images (C and F) show that the exogenous βMHC (green) fills the A band, except the H zone as expected, in a pattern that is complementary and partially overlapping with F-actin (red, C and F). Bar, 20 μm (inset enlarged 3×).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Epitope tagged wild-type human βMHC protein assembles into normal myofibrils in transiently transfected NRC. Cells were transfected with plasmids containing the wild-type human βMHC cDNA tagged with either the EE (A–C) or HA (D–F). The cells were stained for the presence of actin filaments using rhodamine–phalloidin (A and C) and were costained with the anti-EE (B) or anti-HA (E) specific antibodies. Sarcomeres containing human βMHC have well defined A bands (a), I bands (i), and M lines (m). The composite images (C and F) show that the exogenous βMHC (green) fills the A band, except the H zone as expected, in a pattern that is complementary and partially overlapping with F-actin (red, C and F). Bar, 20 μm (inset enlarged 3×).
Mentions: An epitope tag was inserted in the NH2-terminal region of human βMHC to facilitate detection of exogenous protein in transfected NRC. Exogenous βMHC proteins containing either haemaglutinin (HA-1 = YPYDVPDYA; Wilson et al., 1984) or glu–glu (EE = EEYMPME; Grussenmeyer et al., 1985) epitopes were specifically recognized by their respective monoclonal antibodies in transiently transfected NRC (Fig. 2). Each epitope was positioned between amino acids 3 and 4 of the βMHC molecule, a domain with no ascribed function. As noted in previous studies, the crystal structure of the myosin S1 head reveals that the NH2 terminus is free in solution, existing as a random coil on the surface molecule (Rayment et al., 1993a,b), making this region a good target for the insertion of short sequences. The exogenous βMHC protein was incorporated into myofibrils in a pattern indistinguishable from the endogenous MLC2v (data not shown), a molecule known to bind MHC.

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