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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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K184R protein colocalizes with actin. A transfected  NRC expressing K184R was costained with monoclonal antibody  A20 (anti-actin, A) and the epitope specific antibody (anti-EE,  B). The composite image (C) shows the high degree of colocalization of these two proteins throughout the cell. Bar, 20 μm.
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Figure 4: K184R protein colocalizes with actin. A transfected NRC expressing K184R was costained with monoclonal antibody A20 (anti-actin, A) and the epitope specific antibody (anti-EE, B). The composite image (C) shows the high degree of colocalization of these two proteins throughout the cell. Bar, 20 μm.

Mentions: The K184R mutation in βMHC resulted in a severe alteration in the subcellular localization of this mutant protein in transfected cardiomyocytes (Fig. 3). The distribution of K184R protein was visualized as diffuse and somewhat uniform throughout the cell or occurred as cable-like structures. Alternatively, K184R accumulated as brightly staining globules of various size. NRC with large amounts of K184R protein (i.e., stain very brightly for this molecule) were small and irregularly shaped with many pointed lamellipodia (data not shown). As seen in Fig. 3 B, K184R mutant protein formed cable-like structures similar to those described as the premyofibril (Rhee et al., 1994). In this same cell, endogenous MHC (Fig. 3 A) displayed nearly complete loss of sarcomeric organization. The presence of K184R βMHC in cardiomyocytes caused disorganization of endogenous myomesin (Fig. 3, C and D), indicating that thick filament structure is disturbed. Thin filament order was also disrupted in cells expressing K184R (Fig. 3, E and F). In a series of experiments, >2,000 individual cells successfully transfected with K184R were analyzed by immunostaining. In more than half of these cells sarcomeric organization of thick or thin filament markers was completely absent. The remaining K184R positive cells contained few sarcomeres, except for rare cells in which the entire cell volume was filled with highly organized myofibrils (Table I). In NRC with a few recognizable myofilaments, K184R βMHC sometimes appeared to decorate thin filaments or occurred in A band regions, albeit in a sporadic pattern that never filled the entire sarcomere. Even though organized phalloidin staining was lost in many cardiomyocytes with K184R, G actin was readily detectable (Fig. 4). The scattered distribution of K184R protein within the cell appears to colocalize with G actin, indicating that these molecules may be in close association with one another.


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)

K184R protein colocalizes with actin. A transfected  NRC expressing K184R was costained with monoclonal antibody  A20 (anti-actin, A) and the epitope specific antibody (anti-EE,  B). The composite image (C) shows the high degree of colocalization of these two proteins throughout the cell. Bar, 20 μm.
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Related In: Results  -  Collection

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Figure 4: K184R protein colocalizes with actin. A transfected NRC expressing K184R was costained with monoclonal antibody A20 (anti-actin, A) and the epitope specific antibody (anti-EE, B). The composite image (C) shows the high degree of colocalization of these two proteins throughout the cell. Bar, 20 μm.
Mentions: The K184R mutation in βMHC resulted in a severe alteration in the subcellular localization of this mutant protein in transfected cardiomyocytes (Fig. 3). The distribution of K184R protein was visualized as diffuse and somewhat uniform throughout the cell or occurred as cable-like structures. Alternatively, K184R accumulated as brightly staining globules of various size. NRC with large amounts of K184R protein (i.e., stain very brightly for this molecule) were small and irregularly shaped with many pointed lamellipodia (data not shown). As seen in Fig. 3 B, K184R mutant protein formed cable-like structures similar to those described as the premyofibril (Rhee et al., 1994). In this same cell, endogenous MHC (Fig. 3 A) displayed nearly complete loss of sarcomeric organization. The presence of K184R βMHC in cardiomyocytes caused disorganization of endogenous myomesin (Fig. 3, C and D), indicating that thick filament structure is disturbed. Thin filament order was also disrupted in cells expressing K184R (Fig. 3, E and F). In a series of experiments, >2,000 individual cells successfully transfected with K184R were analyzed by immunostaining. In more than half of these cells sarcomeric organization of thick or thin filament markers was completely absent. The remaining K184R positive cells contained few sarcomeres, except for rare cells in which the entire cell volume was filled with highly organized myofibrils (Table I). In NRC with a few recognizable myofilaments, K184R βMHC sometimes appeared to decorate thin filaments or occurred in A band regions, albeit in a sporadic pattern that never filled the entire sarcomere. Even though organized phalloidin staining was lost in many cardiomyocytes with K184R, G actin was readily detectable (Fig. 4). The scattered distribution of K184R protein within the cell appears to colocalize with G actin, indicating that these molecules may be in close association with one another.

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