REGULATION OF CARDIAC CROSS-BRIDGES BY TROPONIN T

心肌收缩是肌动蛋白与肌球蛋白在粗丝和细丝调节蛋白作用下的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在心脏经历正常的发育异构体转换。此外，在心脏疾病状态下，TNT亚型的表达会发生改变，包括与肥厚型心肌病(HCM)相关的突变和心力衰竭中TNT亚型分布的改变。虽然TnT亚型表达对稳态张力钙敏感性的影响已经被认识到，但TnT亚型在调节肌动蛋白-肌球蛋白跨桥动力学和调节强结合交叉桥对细丝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。测定渗透性多细胞心室标本的钙激活张力发展(KCA)、松弛(KR)和张力再发展(KTR)的速率，以确定跨桥向强结合力产生状态转变时TNT异构体的表达以及对跨桥分离速率的影响。为了确定TNT异构体对力-速度和功率-负荷曲线的影响，以及对机械输出的最佳力的影响，将测量通透性心肌细胞在最大和次最大钙时的负荷缩短速度。本研究将通过测定不产生作用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)处理的渗透性多细胞心肌标本的张力-PCA关系、Kca、KR和Ktr，以确定TNT亚型表达对结合交叉桥协同激活细丝的影响。总体而言，拟议的实验结果将提供关于TNT在影响心肌收缩能力方面的作用的新信息，并将为修改细丝调节蛋白以改善患病心脏的功能提供洞察力。

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.