Allele-Specific Effects of Single Amino Acid Exchange in cTnT

描述（由申请人提供）：本申请中概述的研究项目的长期目标是建立将细丝蛋白结构突变与家族性肥厚性心肌病（FHC）发生联系起来的机制。在最初的授权期间，我们确定了cTnT残基92（R92 Q，R92 W和R92 L）的独立疾病突变导致cTnT结构和蛋白动力学，能量和收缩储备，肾上腺素能反应性和肌细胞Ca 2+稳态的离散，突变特异性改变。此外，许多这些下游细胞过程表现出突变特异性的时间变化，决定了所产生的心肌病的进展。因此，cTnT的单个残基处的独立氨基酸取代足以引起不同的生理表型，并且这些表型是cTnT分子的生物物理性质的特定变化的最终结果。我们现在已经开发了基于分子、计算、细胞和全心脏的工具来直接解决这一假设，它们定义了一种综合方法来建立并最终修改cTnT突变与FHC恶性临床过程之间的机制联系。这些研究旨在进一步了解FHC的发病机制，并为细丝的基本生理学和生物物理学提供新的见解。为了完成这项研究计划，我们将实现以下三个具体目标：目标1：鉴定、评估和功能性排序已知TNT 1突变对关键铰链残基104周围的70-170肽的柔性和结构的影响。目标二：确定降低收缩成本是否挽救了R92 Q、R92 L和R92 W cTnT突变心脏的突变特异性能量-机械表型。目标3：在肌丝水平确定R92 cTnT突变心脏中观察到的β-肾上腺素能反应性改变的潜在机制。这些研究的完成将扩展我们对cTnT突变如何在蛋白质动力学、收缩成本、能量储备和关键肌丝对肾上腺素能刺激的反应水平上与其复杂的恶性表型机械联系的理解。此外，我们相信，这些研究将建立一个新的计算功能的研究范式细丝心肌病，并进一步扩大我们的心肌肌节水平的肌丝激活的理解。 家族性肥厚性心肌病是年轻人心脏性猝死的最常见原因之一，由细丝蛋白心肌肌钙蛋白T突变引起的疾病形式包括特别恶性的子集。该研究项目的目标是开发一种综合方法，利用计算建模，开发严格的基因型-分子表型相关性，并最终在动物模型中进行全心脏研究。最终结果将是更好地了解肌节蛋白中的这些个体突变如何导致严重的心脏疾病，并最终导致针对这种目前无法治疗的疾病的基因型特异性治疗。

DESCRIPTION (provided by applicant): The long-term goal of the research program outlined in this application is to establish the mechanisms that link structural mutations in thin filament proteins to the development of Familial Hypertrophic Cardiomyopathy (FHC). During the original granting period we established that independent disease mutations at Residue 92 of cTnT (R92Q, R92W and R92L) result in discrete, mutation-specific alterations in cTnT structure and protein dynamics, energetics and contractile reserve, -adrenergic responsiveness and myocellular Ca2+ homeostasis. Moreover, many of these downstream cellular processes exhibit mutation-specific temporal changes that determine the progression of the resultant cardiomyopathy. Independent amino acid substitutions at a single residue of cTnT are thus sufficient to cause disparate physiologic phenotypes, and these phenotypes are the eventual result of specific changes in biophysical properties of the cTnT molecule. We have now developed the molecular, computational, cellular and whole-heart based tools to directly address this hypothesis and they define an integrative approach to establishing and eventually modifying the mechanistic link between mutations in cTnT and the malignant clinical course of FHC. These proposed studies are designed to both further our understanding of the pathogenesis of FHC and to provide new insights into the fundamental physiology and biophysics of the thin filament. In order to complete this research program we will implement the following three Specific Aims: Aim 1: To identify, evaluate and functionally }rank} the effects of known TNT1 mutations on the flexibility and structure of the 70-170 peptide around the critical }hinge} residue 104. Aim 2: To determine whether reducing the cost of contraction rescues the mutation-specific energetic-mechanical phenotypes of R92Q, R92L and R92W cTnT mutant hearts. Aim 3: To determine the mechanism(s) underlying the observed alterations in -adrenergic responsiveness in the R92 cTnT mutant hearts at the myofilament level. The completion of the studies will extend our understanding of how mutations in cTnT are mechanistically linked to their complex, malignant phenotype at the level of protein dynamics, cost of contraction, and energy reserve and the crucial myofilament response to -adrenergic stimulation. Moreover, we believe that these studies will both establish a new computational-functional paradigm for the study of thin filament cardiomyopathies and further expand our understanding of myofilament activation at the level of the cardiac sarcomere. Familial Hypertrophic Cardiomyopathy is one of the most common causes of sudden cardiac death in young people and the form of the disease caused by mutations in the thin filament protein cardiac Troponin T comprises a particularly malignant subset. The goal of this research project is to develop a integrated approach that utilizes computational modeling, development of rigorous genotype-molecular phenotype correlations and eventual whole-heart studies in animal models. The end result will be a better understanding of how these individual mutations in sarcomere proteins lead to severe cardiac disease and eventually lead to genotype-specific therapeutics for this currently untreatable disorder.