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© The Rockefeller University Press,
0021-9525/1997//131 $5.00
The Journal of Cell Biology, Volume 137, Number 1,
, 1997 131-140
Article |
Point Mutations in Human β 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
Institute for Cell Biology, Swiss Federal Institute of Technology, 8093 Zurich, Switzerland
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 β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC 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 βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC 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 βMHC 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 βMHC 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 βMHC function.
Abbreviations used in this paper: HCM, hypertrophic cardiomyopathy; MHC, myosin heavy chain; NRC, neonatal rat cardiomyocytes.
The authors are very much indebted to Mahmoud Itani for excellent technical assistance in preparation of neonatal rat cardiomyocytes. The authors thank the Institute for Biomedical Engineering Confocal and Imaging Core for the use of the confocal microscope. We also thank Drs. Sanford Bernstein, Pieter Doevendans, Peter Gruber, Kirk Knowlton, and Howard Rockman for discussions and critical reading of the manuscript.
The Core facility is supported by the Whittaker Foundation Development Award (Grants Section). K.D. Becker is supported by an individual National Research Service Award from the National Institutes of Health. This work was supported by funds to K.R. Chien from the National Institutes of Health/National Heart, Lung, and Blood Institute (1 RO1 HL51549; PO1 HL46345; HL53773; 1 RO1 Hl 55926) and American Heart Association (HL 91-022170).
Please address all correspondence to K. David Becker, Department of Medicine, 0613-C, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093. Tel.: (619) 534-8207; Fax.: (619) 534-8081; E-mail: dbecker{at}ucsd.edu
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