Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant inherited disease characterized by ventricular hypertrophy, myofibriller disarray and a spectrum of clinical symptoms. Recent molecular genetic studies demonstrated a number of missense mutations in the human beta-cardiac myosin heavy chain gene located on chromosome 14. Approximately 30 point mutations have been identified in patients suffering from HCM. Most of them are found in the motor domain, while several are in the S-2 region. Although genetic studies have provided evidence that the mutations in myosin heavy chain gene are responsible for HCM, the molecular basis of this disease is not known. The applicant's hypothesis is that mutations of cardiac beta-myosin heavy chain in the motor domain or the head-rod junction diminish the motor function of myosin. Cardiac hypertrophy would result as compensation for the reduced contractile function resulting from the low individual cross-bridge activity. Their goal is to elucidate at a molecular level how the point mutations in beta-myosin heavy chain change myosin function. To achieve this goal, human cardiac beta-myosin will be functionally expressed in Sf9 cells using a Baculovirus expression system. The function of human cardiac beta-myosin wild type and mutants will be investigated by means of the following assay systems; (1) The shortening velocity will be determined by measuring in vitro actin sliding velocity; (2) The force production of individual myosin molecules will be measured by using a feedback enhanced laser trap system; (3) Kinetics of the actomyosin ATPase reaction will be analyzed by means of various biochemical techniques in order to determine what step of the cross-bridge cycle is altered by the mutation. Point mutation at the S2 region may cause changes in the assembly of myosin molecules thus reducing the efficiency of contractility of cardiac cells. The effect of these mutations on the myosin filament assembly and filament structure will be investigated. 3D structure of chicken fast skeletal myosin S1 determined recently has been utilized to understand the structure-function relationship of myosin. However, to understand how the mutations found in HCM change myosin function, it is crucial to determine 3D structure of human cardiac beta-myosin since the motor activity of beta-myosin is significantly different from fast skeletal myosin. Human cardiac beta-myosin will be crystallized and the 3D structure will be determined by x-ray crystallography. The flexible regions will be modified using recombinant DNA technology in aid of making good quality crystals. The itemized Specific Aims are; (1) Functional expression of human adult cardiac beta-myosin; (2) Mutagenesis of human cardiac beta-myosin heavy chain and expression of mutant myosins; (3) Characterization of motor activity of human cardiac beta-myosin by in vitro motility assay: Identification of the effects of missense mutation on motor function; (4) Kinetic analysis of human cardiac beta-myosin ATPase cycle: Identification of effects of missense mutation on actomyosin ATPase cycle; (5) To determine the effect of mutation on thick filament formation and the dependence of actomyosin contractility on the fraction of mutant myosin in thick filaments; (6) Crystallization and 3D structural analysis of human cardiac beta-myosin.
