Determinants of Cardiac Thin Filament Regulation

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DESCRIPTION (provided by applicant): Cardiac muscle contraction is initiated by Ca2+ binding to the thin filament regulatory unit comprised of 3 troponin (Tn) subunits: troponin C (TnC), troponin I (TnI), troponin T (TnT), and 1 tropomyosin (Tm) and 7 actins. The heart normally functions at submaximal activation levels where cooperative activation mechanisms are most pronounced and thus are a significant determinant of contractility. The long term goal of these studies is to provide a precise description of how Ca2+ binding leads to the cooperative interaction of regulatory complexes, how the rate of these processes control the tension time course and how Tn mutation alter this mechanism and leads to cardiomyopathies. The proposed studies will test the hypotheses that the activation state of the cardiac thin filament and [Ca2+] are in rapid equilibrium and regulatory units function, not individually, but as an ensemble. Thus, during activation at submaximal [Ca2+], and during relaxation, the extent of thin filament activation and the tension time course are dictated primarily by positive cross-bridge feedback and their kinetics. We also hypothesize that the action of cTnI mutations that increase Ca2+- sensitivity of tension are mediated by altered thin filament activation and relaxation kinetics and cross-bridge mechanics. To test these hypotheses, we will monitor tension simultaneously with conformational changes of Tn subunits via a rhodamine probe bound to specific sites. Alterations in Tn conformation and mobility will be measured using fluorescence polarization (FP) as changes in probe orientation (probe angle) with respect to the thin filament axis and angular dispersion. Endogenous cTnC or Tn of skinned cardiac trabeculae will be exchanged for labeled cTnC and a novel labeled Tn complex with an inactive TnC Ca2+regulatory site. This complex mimics a Ca2+-free regulatory unit while the attached probe allows monitoring of Tn structural changes arising solely through its interactions with neighboring regulatory units and NOT from direct binding of Ca2+. Activation will be elicited by incubation in solutions containing varying [Ca2+] and via rapid jumps in [Ca2+] by laser photolysis of a novel caged Ca2+. Relaxation will be elicited by photolysis of caged-Ca2+ chelator, diazo-2. The long term goal of this project is to understand the mechanism of thin filament regulation in cardiac muscle and thus form the foundation from which the action of Tn mutations can be definitively assessed and therapeutic modalities developed to treat the resulting cardiomyopathies.