REGULATING MUSCLE CONTRACTION: DETERMINING THE RELATIONSHIP BETWEEN MYOSIN KINETICS, CALCIUM SENSITIVITY AND COOPERATIVE ACTIVATION
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The objective of this dissertation is to characterize the relationship between myosin ATPase kinetics, calcium sensitivity and cooperativity of thin filament activation to explain the role of calcium- and myosin-dependent activation processes observed during muscle contraction. The findings will be extended to a cardiomyocyte system developed to investigate the relationship between myosin kinetics, force generated from the sarcomere, and maladaptive phenotypic changes. Using the in vitro motility assay to simulate unloaded muscle shortening, the effect of inhibition of intermediate states of the myosin ATPase cycle was characterized. We determined that calcium sensitivity and cooperativity were modulated via perturbation of myosin duty ratio. A cardiomyocyte system for mechanical studies was developed to further investigate the role of ATPase kinetics in cardiac remodeling. This system allows for the perturbation of ATPase kinetics, as well as manipulation of various signaling pathways (which can be monitored fluorescently), while monitoring force generated internally and transferred to the extracellular environment, which is critical in the study of hypertrophic phenotypes. Collectively, our findings indicate that targeted therapeutics which modulate myosin ATPase kinetics can normalize calcium sensitivity and cooperativity in compromised cardiac tissue; and these therapeutics can be accurately evaluated and modeled in the in vitro environment.