REGULATION OF CARDIAC CROSS-BRIDGES BY TROPONIN T

心肌收缩是在粗丝和细丝调节蛋白的作用下，肌动蛋白与肌凝蛋白交叉桥循环相互作用的结果。在细丝调节蛋白中，肌钙蛋白T （TnT）、原肌球蛋白和肌钙蛋白I在心脏中经历正常的发育亚型转换。此外，在心脏病状态下，包括与肥厚性心肌病（HCM）相关的突变和心力衰竭时TnT异构体分布的改变，TnT异构体表达也会改变。虽然TnT异构体表达对稳态Ca2+张力敏感性的影响是公认的，但TnT异构体在调节肌动蛋白-肌球蛋白过桥动力学和调节强结合过桥对细丝的协同活化中的作用在很大程度上是未知的。本应用程序中提出的实验将解决TnT异构体表达影响心肌力产生动力学的机制。将测量渗透性多细胞心室制剂的Ca2+激活张力发展（kCa）、弛豫（kr）和张力再开发（ktr）的速率，以确定TnT异构体表达在跨桥过渡到强结合力产生状态时的影响，以及跨桥脱离的速率。将测量最大和次最大Ca2+时通透性肌细胞的加载缩短速度，以确定TnT同种异型介导的力-速度和功率-负载曲线以及机械输出的最佳力的影响。将测量渗透性多细胞心室制剂经无力产生的强结合肌球蛋白衍生物n-乙酰马来酰亚胺肌球蛋白S1 （NEM-S1）处理后的张力- pca关系、kCa、kr和ktr，以确定TnT异构体表达对结合交叉桥协同激活细丝的影响。总的来说，实验结果将为TnT在影响心肌收缩力方面的作用提供新的信息，并将为细丝调节蛋白修饰改善患病心脏功能提供新的见解。

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.