REGULATION OF CARDIAC CROSS-BRIDGES BY TROPONIN T

心肌收缩是由肌动蛋白和肌球蛋白在粗丝和细丝调节蛋白的影响下形成的周期性相互作用引起的。 在细肌丝调节蛋白中，肌钙蛋白T（TnT）、原肌球蛋白和肌钙蛋白I在心脏中经历正常发育同种型转换。此外，TnT同工型表达在心脏疾病状态中改变，包括与肥厚性心肌病（HCM）相关的突变和心力衰竭中改变的TnT同工型分布。 虽然TnT亚型表达对稳态Ca 2+敏感性的张力的影响是公认的，但TnT亚型在调节肌动蛋白-肌球蛋白跨桥动力学和通过强结合跨桥调节细丝的协同激活中的作用在很大程度上是未知的。 本申请中提出的实验将解决TnT亚型表达影响心肌力产生动力学的机制。 将测量透化多细胞心室制备物的Ca 2+激活张力发展（kCa）、松弛（kr）和张力重建（ktr）的速率，以确定跨桥向强结合力产生状态转变时以及跨桥脱离速率时TnT亚型表达的影响。 将测量最大和次最大Ca 2+下透化肌细胞的负荷缩短速度，以确定TnT亚型介导的对力-速度和功率-负荷曲线以及对机械输出最佳力的影响。 将测量用非产生力的强结合肌球蛋白衍生物N-乙基马来酰亚胺肌球蛋白S1（NEM-S1）处理的透化多细胞心室制备物的张力-pCa关系、kCa、kr和ktr，以确定TnT同种型表达对结合横桥协同激活细丝的影响。 总的来说，拟议的实验的结果将提供新的信息TnT在影响心肌收缩力的作用，并将提供洞察以及细丝调节蛋白修饰，以改善患病心脏的功能。

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.