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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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Coexpression of both mutant and wild-type βMHC  molecules detects no differences in subcellular localization of the  two exogenous proteins. NRC were cotransfected with both wildtype and mutant expression plasmids, each tagged with a different epitope. Sequential staining (see Materials and Methods) allows detection of each protein within the same cell. A and B show  a cardiomyocyte transfected with Hnwt only. This cell was immunostained with anti-HA, goat anti–mouse–FITC (F(ab)), and anti- EE and then donkey anti–mouse–LRSC. A shows the anti-HA  specific staining (FITC channel), and B shows the LRSC channel,  indicating that the anti-EE antibody does not crossreact, and  minimal signal bleed through occurs under these conditions. Using anti-EE to detect Tnwt, the same lack of crossreactivity is observed when costaining with anti-HA (data not shown). Expression and detection of both Hnwt (C) and Tnwt (D) in the same  cell shows identical subcellular localization for both proteins. The  TnR403Q mutant (F) also shows identical distribution to Hnwt  βMHC (E) when coexpressed in the same cell. Similar results are  obtained when coexpressing either R249Q or S472V with wildtype βMHC. The type of epitope does not affect the outcome of  this experiment. Bars: (A, C, and E) 20 μm; (E and inset) 5 μm.
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Figure 7: Coexpression of both mutant and wild-type βMHC molecules detects no differences in subcellular localization of the two exogenous proteins. NRC were cotransfected with both wildtype and mutant expression plasmids, each tagged with a different epitope. Sequential staining (see Materials and Methods) allows detection of each protein within the same cell. A and B show a cardiomyocyte transfected with Hnwt only. This cell was immunostained with anti-HA, goat anti–mouse–FITC (F(ab)), and anti- EE and then donkey anti–mouse–LRSC. A shows the anti-HA specific staining (FITC channel), and B shows the LRSC channel, indicating that the anti-EE antibody does not crossreact, and minimal signal bleed through occurs under these conditions. Using anti-EE to detect Tnwt, the same lack of crossreactivity is observed when costaining with anti-HA (data not shown). Expression and detection of both Hnwt (C) and Tnwt (D) in the same cell shows identical subcellular localization for both proteins. The TnR403Q mutant (F) also shows identical distribution to Hnwt βMHC (E) when coexpressed in the same cell. Similar results are obtained when coexpressing either R249Q or S472V with wildtype βMHC. The type of epitope does not affect the outcome of this experiment. Bars: (A, C, and E) 20 μm; (E and inset) 5 μm.

Mentions: Expressing both mutant and wild-type βMHC within the same transfected cell provided a sensitive measure of detecting minor differences in subcellular localization of the two proteins. Utilization of two different epitopes inserted at the same location within the βMHC sequence facilitated immunostaining of both mutant and wild-type βMHC proteins within the same cell. However, the two epitopes that were useful in the βMHC context were both recognized by mouse monoclonal antibodies. Therefore a sequential staining procedure was used to detect signal specific to each primary antibody (see Materials and Methods). Fig. 7, A and B shows that little or no bleed through signal was detected in NRC expressing only one wild-type βMHC construct that was stained with both epitope specific antibodies. In cotransfected cells, both wild-type constructs were observed, and as would be expected, the subcellular localization was identical for the two wild-type proteins present within the same cell (Fig. 7, C and D). Both proteins were coincorporated into A bands in a pattern that was complementary to thin filament staining.


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)

Coexpression of both mutant and wild-type βMHC  molecules detects no differences in subcellular localization of the  two exogenous proteins. NRC were cotransfected with both wildtype and mutant expression plasmids, each tagged with a different epitope. Sequential staining (see Materials and Methods) allows detection of each protein within the same cell. A and B show  a cardiomyocyte transfected with Hnwt only. This cell was immunostained with anti-HA, goat anti–mouse–FITC (F(ab)), and anti- EE and then donkey anti–mouse–LRSC. A shows the anti-HA  specific staining (FITC channel), and B shows the LRSC channel,  indicating that the anti-EE antibody does not crossreact, and  minimal signal bleed through occurs under these conditions. Using anti-EE to detect Tnwt, the same lack of crossreactivity is observed when costaining with anti-HA (data not shown). Expression and detection of both Hnwt (C) and Tnwt (D) in the same  cell shows identical subcellular localization for both proteins. The  TnR403Q mutant (F) also shows identical distribution to Hnwt  βMHC (E) when coexpressed in the same cell. Similar results are  obtained when coexpressing either R249Q or S472V with wildtype βMHC. The type of epitope does not affect the outcome of  this experiment. Bars: (A, C, and E) 20 μm; (E and inset) 5 μm.
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Figure 7: Coexpression of both mutant and wild-type βMHC molecules detects no differences in subcellular localization of the two exogenous proteins. NRC were cotransfected with both wildtype and mutant expression plasmids, each tagged with a different epitope. Sequential staining (see Materials and Methods) allows detection of each protein within the same cell. A and B show a cardiomyocyte transfected with Hnwt only. This cell was immunostained with anti-HA, goat anti–mouse–FITC (F(ab)), and anti- EE and then donkey anti–mouse–LRSC. A shows the anti-HA specific staining (FITC channel), and B shows the LRSC channel, indicating that the anti-EE antibody does not crossreact, and minimal signal bleed through occurs under these conditions. Using anti-EE to detect Tnwt, the same lack of crossreactivity is observed when costaining with anti-HA (data not shown). Expression and detection of both Hnwt (C) and Tnwt (D) in the same cell shows identical subcellular localization for both proteins. The TnR403Q mutant (F) also shows identical distribution to Hnwt βMHC (E) when coexpressed in the same cell. Similar results are obtained when coexpressing either R249Q or S472V with wildtype βMHC. The type of epitope does not affect the outcome of this experiment. Bars: (A, C, and E) 20 μm; (E and inset) 5 μm.
Mentions: Expressing both mutant and wild-type βMHC within the same transfected cell provided a sensitive measure of detecting minor differences in subcellular localization of the two proteins. Utilization of two different epitopes inserted at the same location within the βMHC sequence facilitated immunostaining of both mutant and wild-type βMHC proteins within the same cell. However, the two epitopes that were useful in the βMHC context were both recognized by mouse monoclonal antibodies. Therefore a sequential staining procedure was used to detect signal specific to each primary antibody (see Materials and Methods). Fig. 7, A and B shows that little or no bleed through signal was detected in NRC expressing only one wild-type βMHC construct that was stained with both epitope specific antibodies. In cotransfected cells, both wild-type constructs were observed, and as would be expected, the subcellular localization was identical for the two wild-type proteins present within the same cell (Fig. 7, C and D). Both proteins were coincorporated into A bands in a pattern that was complementary to thin filament staining.

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