The applicant states that normal cardiac function depends on the force and motion generating capacity of the myosin molecular motor. Alterations to myosin's structure compromises its mechanical function, leading to sudden death in patients with familial hypertrophic cardiomyopathy (FHCM). Thus, the applicant concludes that an understanding of cardiac myosin's molecular structure and how it relates to its hydrolytic and mechanical function is of significant clinical importance. The applicant indicates that two recent technical advances now allow investigators to begin addressing this question. First, by applying the laser optical trap to the in vitro motility assay, myosin's molecular force (pico-Newton range) and displacement (nano-meter range) can be directly measured. The second advance is the crystal structure of the myosin head region determined by Rayment and coworkers. Using the laser trap in conjunction with the knowledge of myosin's molecular structure, the applicant will begin to carry out a functional analysis of the cross-bridge cycle in terms of its structure for V1 and V3 cardiac myosin as well as mutant myosins from FHCM patients. To measure myosin's molecular mechanics, the applicant will attach an actin filament at each end to polystyrene beads and then trap each bead in separate laser traps. The actin filament is maneuvered so that it interacts with a single myosin molecule. The applicant's strategy is to compare the mechanics of V1 and V3 myosin molecules and to relate the observed differences to the known structural differences that exist in key regions of the molecule. The V1 and V3 myocardial isoforms are 95% homologous with differences residing near the base of the catalytic domain that abuts the essential light chain region, the nucleotide binding pocket, the actin binding domain, and the neck, which may function as a lever arm. More precise identification of key amino acids within the myosin molecule that are important to myosin's enzymatic and mechanical performance will be obtained by studying cardiac myosin mutants from FHCM patients. The mutants the applicant initially plans to study are in: A) the nucleotide binding pocket (T1241, Y162C); B) the actin myosin interface area (R403Q); C) the essential light chain binding surface (R719W, G741F); D) the rod segment (L908V).
| Yamashita, H; Tyska, M J; Warshaw, D M et al. (2000) Functional consequences of mutations in the smooth muscle myosin heavy chain at sites implicated in familial hypertrophic cardiomyopathy. J Biol Chem 275:28045-52 |
| Palmiter, K A; Tyska, M J; Haeberle, J R et al. (2000) R403Q and L908V mutant beta-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level. J Muscle Res Cell Motil 21:609-20 |
| Palmiter, K A; Tyska, M J; Dupuis, D E et al. (1999) Kinetic differences at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms. J Physiol 519 Pt 3:669-78 |
