Kinetics of Cardiac Myofilament Activation

描述（由申请人提供）：心力衰竭可能是心脏收缩或舒张受损或两者兼而有之的结果，这是由Ca2+诱导的细丝激活和失活调节的。因此，了解细丝调控机制对预防和治疗心力衰竭具有重要意义。细丝调控的分子基础涉及Ca2+诱导的结构改变以及与肌钙蛋白和肌动蛋白界面蛋白-蛋白相互作用相关的结构动力学和动力学。本提案的目的是定义Ca2+诱导的细丝激活/失活的详细动力学机制，以及它如何受到肌钙蛋白磷酸化和cTnI中发现的心肌病突变的影响。为了实现这一目标，本提案将解决以下问题：1)cTnI的不同功能区域在调节过桥动力学中的动力学作用是什么？2) cTnI c结构域的结构动力学如何在细丝调控中发挥作用？3)剥皮肌纤维和肌原纤维中细丝的结构变化和结构动力学是如何修饰的？该建议的基本假设是，Ca2+诱导的结构转变涉及肌钙蛋白和肌动蛋白之间界面的肌钙蛋白I的不同区域，在调节肌动蛋白和肌球蛋白之间相互作用的动力学中发挥不同的作用，结构转变可以受到强交叉桥、心肌病突变、肌钙蛋白磷酸化以及完整肌肉细胞中发现的细胞晶格环境的影响。该项目利用我们在确定蛋白质结构动力学和动力学方面的FRET和荧光各向异性专业知识，通过将FRET方法应用于肌肉纤维和肌原纤维测量，开发了一种新的方法。这将使我们能够获得迫切需要的与Ca2+诱导的细丝调控肌肉功能相关的动力学和动力学信息。本研究的结果将促进我们对心脏细丝在健康和疾病中对心肌收缩和舒张的调节作用的理解。

DESCRIPTION (provided by applicant): Heart failure can be a result of impaired cardiac contraction or relaxation or both, which are regulated by Ca2+induced thin filament activation and deactivation. Therefore, understanding the mechanism of thin filament regulation is of great importance in prevention and treatment of heart failure. The molecular basis of the thin filament regulation involves Ca2+induced alterations in the structure and structural kinetics and dynamics associated with the protein-protein interactions at the interface between troponin and actin. The objective of this proposal is to define the detailed kinetic mechanism that underlies Ca2+induced thin filament activation/deactivation and how it is affected by troponin phosphorylation and cardiomyopathy mutations found in cTnI. To achieve the goal, this proposal will address the following questions: 1) What is the kinetic role of different functional regions of cTnI in regulating crossbridge kinetics? 2) How do the structural dynamics of the cTnI C-domain play roles in thin filament regulation? and 3) How are the structural changes and structural kinetics of the thin filament modified in the skinned muscle fibers and myofibrils? The underlying hypothesis of this proposal is that the Ca2+induced structural transitions involving the different regions of troponin I at the interface between troponin and actin play different roles in regulating kinetics of the interaction between actin and mysoin, and the structural transitions can be affected by strong crossbridges, cardiomyopathy mutations, and phosphorylation of troponin proteins, as well as cellular lattice environment found in intact muscle cells. This project capitalizes on our FRET and fluorescence anisotropy expertise in determining protein structural kinetics and dynamics to exploit a new approach by implementing FRET methodologies into muscle fibers and myofibril measurements. This will enable us to acquire the urgently needed kinetic and dynamic information associated with the Ca2+induced thin filament regulation on muscle function. The results from this study will have a positive impact by advancing our understanding of the regulatory role of cardiac thin filament in cardiac muscle contraction and relaxation in health and disease.