STRUCTURE OF VERTEBRATE THICK ELEMENT

University Of Puerto Rico Med Sciences, San Juan PR

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Abstract

The rhythmic beating of the normal heart is vital for life and depends upon the interactions between two sets of microscopic filaments: the myosin-containing thick filaments and the actin-containing thin filaments. The molecular motor for the production of force in this system resides in the myosin of the thick filament. Recent evidence has implicated both the myosin and accessory proteins of the cardiac thick filament in several of hypertrophic cardiomyopathy, and extensive evidence exists that cardiac muscle may differ in a number of its physiological properties from skeletal muscle. Despite its significance, little detailed information is available on the arrangement of myosin and accessory proteins in the cardiac thick filament in several types of hypertrophic cardiomyopathy, and extensive evidence that cardiac muscle may differ in a number of physiological properties from skeletal muscle. Despite its significance, little detailed information is available on the arrangement of myosin and accessory proteins in the cardiac thick filament. Nor are its interactions with the adjacent thin filaments under relaxing conditions clearly understood. We have developed a method for the isolation of relaxed rabbit cardiac muscle thick filaments that preserves the near-helical relaxed arrangement of the myosin heads on the surface of the thick filaments, thus allowing the structure of the filaments to be analyzed by a combination of electron microscopy and Fourier transform-based computer image analysis. In electron micrographs, the filaments appear very period with an axial repeat every third crossbridge level. Fourier transforms of the filaments are very strong and detailed, and show layer lines out at least the 11th or 12th layer lines, thus confirming the near- helical periodicity of the filaments. The isolated filaments in addition to their periodic structure are of interest because of their striking tendency to interact with adjacent thin filaments under relaxing conditions. Because of the striking periodicity of these filaments, we prop9ose to use preparations of these filaments as a model system to determine both the structure of the cardiac thick filament, and the effect of variations in parameters such as myosin isotype and phosphorylation of the filament structure The specific aims of the project are to: 1) Determine the effect of differences in myosin heavy chain type, and phosphorylation of myosin binding protein-C and myosin regulatory light chain on the crossbridge arrangement of the filaments; 2) use electron microscopic methods (negative-staining, platinum-shadowing, stereo-tilts, rapid- freezing freeze-substitution) and computer image analysis to study the interactions between the thick and thin filaments which occur under relaxing conditions, 3) Determining the structure of the filament backbone; and 4) calculate a three-dimensional reconstruction of the structure of the rabbit cardiac muscle thick filaments to provide information on the crossbridge arrangement, accessory proteins, and backbone structure. The long term goal of these studies is to elucidate the structure of the relaxed cardiac muscle thick filament and the relation of its structure to interactions with the thin filament and the normal functioning of cardiac muscle.