The contractile system of vertebrate skeletal and cardiac muscle is activated by the binding of calcium to troponin C. Considerable information is available on the relationship between free Ca2+ concentration and mechanical response in skinned fiber preparations, as well as on the biochemical properties of the regulatory proteins (troponin C, troponin I, troponin T, tropomyosin) which mediate Ca2+ activation. Less is known about the relationship between bound Ca2+ and mechanical response in the intact myofilament system. Recent investigations have shown that not only is contraction activated by binding of Ca2+ to troponin C but the properties of troponin C are influenced by the mechanical state of the muscle. This is best illustrated by recent studies in this laboratory and elsewhere showing that the steep relationship between contractile force and sarcomere length in cardiac muscle (Frank-Starling relation) is largely determined by a length dependence of Ca2+-troponin C affinity. The goal of this research will be to define the cooperative interactions between contractile and regulatory proteins which determine the Ca2+ binding properties of troponin C in the intact myofilament system and determine where these interactions differ in skeletal and cardiac muscle. A combination of mechanical measurements, isotopic Ca2+ binding, and fluorescence spectroscopy will be used to address the following questions: 1) What are the contributions of cross-bridge interactions and length-dependent Ca2+ binding to the shapes of the ascending limb of the force-length curves in skeletal and cardiac muscle? 2) Are apparent length dependent changes in Ca2+ sensitivity or Ca2+ binding based on changes in length per se, cross-bridge attachments, or interfilament spacing? 3) What are the quantitative differences between skeletal and cardiac troponin C with respect to cooperative effects of cross-bridge interaction and shortening? 4) In interactions between adjacent troponin units play a role in cross-bridge mediated changes in the Ca2+-troponin C interaction, and 5) What structural changes take place in cardiac troponin C in association with Ca2+ binding and cross-bridge interactions?
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