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 HYPERTROPHICCARDIOMYOPATHY MUTATIONS DISPLAY NORMAL ASSEMBLY

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myof ibrillar disorganization. This genetically dominant disease can be cau sed by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether poin t 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 cultu res 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 resul ted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC, Diffuse beta MHC K184R pro tein appeared to colocalize with actin throughout the myocyte, suggest ing a tight interaction of these two proteins, Human beta MHC with S47 2V mutation assembled normally into thick filaments and did not affect sarcomeric structure, Two mutant myosins previously described as caus ing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defin ed I bands, A bands, and H zones. Coexpression and detection of wild-t ype beta MHC and either R249Q or R403Q proteins in the same myocyte sh owed these proteins are equally able to assemble into the sarcomere an d provided no discernible differences in subcellular localization, Thu s, human beta MHC R249Q and R403Q mutant proteins were readily incorpo rated into NRC sarcomeres and did not disrupt myofilament formation, T his study indicates that the phenotype of myofibrillar disarray seen i n HCM patients which harbor either of these two mutations may not be d irectly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary ef fect possibly resulting from the chronic stress of decreased beta MHC function.