Cardiac muscle contraction results from cyclic interaction of actin with myosin cross-bridges under the influence of thick and thin filament regulatory proteins. Of the thin filament regulatory proteins, troponin T (TnT), tropomyosin, and troponin I undergo normal developmental isoform switching in heart. Additionally, TnT isoform expression is altered in cardiac disease states including mutations associated with hypertrophic cardiomyopathy (HCM) and altered TnT isoform distribution in heart failure. While effects of TnT isoform expression upon steady-state Ca2+-sensitivity of tension are recognized, the role of TnT isoforms in regulating actin-myosin cross-bridge kinetics and in regulating cooperative activation of the thin filament by strongly-bound cross-bridges are largely unknown. Experiments proposed in this application will address the mechanisms by which TnT isoform expression influences kinetics of myocardial force generation. Rates of Ca2+-activated tension development (kCa), relaxation (kr), and tension redevelopment (ktr) of permeabilized multicellular ventricular preparations will be measured to determine the effects TnT isoform expression upon transition of cross-bridges to the strongly-bound force-generating state, and upon rates of cross-bridge detachment. Velocity of loaded shortening of permeabilized myocytes at maximal and submaximal Ca2+ will be measured in order to determine TnT isoform-mediated effects upon force-velocity and power-load curves and upon optimum force for mechanical output. The tension-pCa relationship, kCa, kr, and ktr of permeabilized multicellular ventricular preparations treated with the non-force generating strong-binding myosin derivative, N-ethylmaleimide myosin S1 (NEM-S1) will be measured to determine the influence TnT isoform expression upon cooperative activation of thin filament by bound cross-bridges. Overall, the results of the proposed experiments will provide new information regarding the role of TnT in influencing myocardial contractility and will provide insight as well into thin filament regulatory protein modification to improve function of diseased hearts.
