J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/04/131/10 $2.00
Volume 137, Number 1, April 7, 1997 131-140

Point Mutations in Human  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

K. David Becker,^* Kim R. Gottshall,^* Reed Hickey,^* Jean-Claude Perriard, and Kenneth R. Chien^*

^* Department of Medicine, Center for Molecular Genetics, and American Heart Association Bugher Foundation Center for Molecular Biology, University of California, San Diego, La Jolla, California 92093; and  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  cardiac myosin heavy chain (MHC). To determine whether point mutations in human MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human 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 MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human 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 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 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 MHC function.

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