The major questions being addressed deal with determining the state of the single Ca2+ regulatory site on cardiac TnC in skinned muscle fibers and how it is modulated by various physiological and pathological states of the heart. This project will focus on the static and dynamic calcium binding properties of troponin in skinned fibers using techniques recently developed in this laboratory. Much is know about the role of Ca2+ binding to troponin in the regulation of cardiac muscle contraction, based primarily upon solution studies of the various proteins involved an these results have been extrapolated to intact muscle. However, recent evidence suggests that the Ca2+ binding properties of the regulatory sites of troponin are altered when troponin is incorporated into thin filaments and, of equal importance, other studies suggest that myosin crossbridge interaction with the actin filament may also affect Ca2+ binding. In addition, on recent preliminary report suggests a role for cardiac TnC in the length dependent autoregulation of the hart. With our new techniques, it is possible to study these question directly. Cardiac TnC (CTnC) can be selectively extracted from skinned fibers and replaced with exogenous fluorescently labeled CTnC. The Ca2+ dependence of the fluorescence change of the incorporated CTnC can be correlated directly with Ca2+ or Sr2+ binding to the single Ca2+-specific regulatory site on CTnC. Through the use of another new technique we can directly measure the Sr2+ affinity of this site using Fura-2 in skinned fibers and thus validate unequivocally the CTnC fluorescence results. Using these techniques we can then follow Ca2+binding, force development and ATPase activity (fluorescence linked enzyme assay) using microspectrofluorometry. This approach has already made it possible to learn much about the regulation of skeletal muscle contraction. In this application we will address questions which are unique to cardiac muscle where the Ca2+ binding properties of CTnC are thought to be modulated by various alterations in the heart (e.g., beta- adrenergic stimulation, ischemia, etc.). Specifically, we will study the effect of troponin I phosphorylation, myosin crossbridge state (e.g., rigor, ADP, Pi, ADP-Pi), sarcomere length and pH on the Ca2+ affinity of the single regulatory site in cardiac muscle. We will also study the mechanism of the unique Sr2+ sensitivity of cardiac muscle. Using the new technique of photolysis of the caged compounds Ca2+ (Nitr- 5 and DM- nitrophen) and Ca2+ -chelator, we will be able to study the various kinetic steps involved in the regulation of force development and relaxation. For example, the rate of Ca2+ dissociation from CTnC and the rate of force development and relaxation can be measured simultaneously upon liberation of caged Ca2+ -chelator and yield important new information about the relationship of bound Ca2+ to relaxation and its modulation by phosphorylation, etc. All of the proposed experiments, which were not previously possible, should lead to a clearer view of the molecular events involved in cardiac muscle regulation and their time course.
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