Studies of mammalian skeletal and cardiac muscles have shown that the contractile protein myosin undergoes transitions in subunit structure during development from embryo to adult. We will investigate the physiological roles of the isoforms of myosin in rat and rabbit myocardium and in rabbit skeletal muscles. This will be done using single muscle fibers and cardiac myocytes from which the surface membranes have been removed, allowing direct control of activation with Ca2+ . Maximum tension, the rate of tension development, stiffness, and shortening velocity will be measured to determine mechanical properties of myosin cross-bridges and overall kinetics of the cross-bridge interaction cycle. Specific cross-bridge state transitions will be studied by characterizing isometric tension transients in response to rapid photorelease of Pi, ADP or the Ca 2+ chelator BAPTA from chemically caged compounds. The myosin heavy and light chain compositions of these same single cells will then be determined using ultrasensitive gel electrophoretic techniques. (1) These measurements will be made on cardiac muscle and on fast and slow skeletal muscles at fetal, neonatal and adult stages of development, as well as on rabbit fast-twitch skeletal muscles subjected to altered patterns of stimulation in vivo. (2) Further experiments will directly test the hypothesis that the types and proportions of myosin light chains modulate contraction. This will be done by performing mechanical measurements on single cardiac myocytes and skeletal muscle fibers both before and after exchanging various isoforms of the alkali light chains for the endogenous light chains in these cells. (3) Molecular mechanisms of light chain effects on mechanical properties will be investigated by substituting truncated or mutated alkali light chain-1 for endogenous light chains. (4) Mechanical properties of skinned myocytes from senescent myocardium will be characterized to determine whether myosin heavy chains expressed in aged rats account for altered cardiac function in senescence. Measurements of contractile properties in single muscle cells as functions of natural and imposed variations in protein composition should allow straightforward conclusions regarding physiological roles of diverse myosin isoforms. Additional experiments will provide insight regarding cross-bridge state transitions that are sensitive to altered expression of myosin and molecular mechanisms of effects due to altered expression.
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