The goals of the proposed experiments are to relate the physiology of living muscle fibers more closely to the molecular events of the actomyosin interaction. The relationship between molecular mechanisms and many important aspects of the complex physiological responses of living fibers remain unresolved. Using permeable skeletal muscle fibers, we will measure fiber mechanics and ATPase activities under conditions that more closely simulate physiological conditions. The conditions will include more physiological temperatures, lattice spacing and molecular crowding. In relaxed and partially activated muscle fibers myosin heads in non force producing states can be bound in an ordered array to the core of the thick filament or can be highly disordered, where they presumably can more readily bind to actin. We will explore how the balance between these 2 arrangements may affect fiber function. Myosin heads in these non-force producing states can exert a drag on fiber shortening under some conditions, but not others. The mechanisms that account for this and whether the ordered array is involved will be investigated. Phosphorylation of myosin light chains disrupts the ordered array and its role in potentiating fiber tension will be explored in detail. We will also probe the structural changes that occur in the myosin light chain upon phosphorylation using mutations and functional and structural assays. These studies will be aided by computer simulations to pick likely sites for binding the phosphorylated serine. To make better correlations between fiber mechanics and the configuration of the myosin heads, we will develop spectroscopic probes to monitor the fraction of heads in the ordered array. The extremely low metabolic rate of living, resting muscle is not compatible with the myosin ATPase activity observed in vitro. We will explore several mechanisms that could produce the inhibition of myosin that is required to explain the in vivo data, including the involvement of the ordered array of the thick filament. The metabolic rate of resting muscles plays a role in whole body metabolism, and understanding how it is achieved and how it may be modulated could have important applications to human health. The ability to maintain a very low metabolic rate could also facilitate preservation of skeletal and cardiac muscles for regrafting or transportation. Thus the molecular mechanisms investigated here play important roles in modulating both skeletal and cardiac muscles; and elucidating them will lead to more rational design of therapeutic interventions.
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