Allele-specific Effects: Single Amino Acid Exchanges/cTnT

描述（由申请人提供）：心脏肌节的调节功能存在于细丝中。它是由多个蛋白质亚基组成的复杂大分子结构，这些蛋白质亚基相互作用并调节收缩功能以响应慢性和急性生理应激。Ca 2+与肌钙蛋白C的结合引发肌钙蛋白（cTnC、cTnI和cTnT）和原肌球蛋白-肌动蛋白复合物内蛋白质相互作用的变构变化级联，促进放线肌球蛋白复合物的形成和肌肉收缩的动力冲程。虽然正常心脏保留了改变这些复杂相互作用的能力，例如，通过收缩蛋白亚型变化和翻译后修饰，但许多自然发生的细丝突变的耐受性较差。事实上，心肌肌钙蛋白T（cTnT）突变导致一种特别严重的家族性肥厚性心肌病（FHC），其特征是在没有明显心室肥大的情况下发生高频率的早期心脏性猝死。基因突变和复杂临床表型之间的直接联系仍然未知。肌钙蛋白T是一种高度伸长的蛋白质，与细丝的所有其他组分相互作用，并被描述为调节系统的“胶水”。完整的三元结构是未知的，因此这些复杂的蛋白质-蛋白质相互作用的细节仍然不清楚。本申请的假设是，单个cTnT突变破坏了这些蛋白质-蛋白质相互作用的离散方面，导致细丝功能的特定变化，并导致不同的心血管表型。为了解决这一假设，我们已经开发了三个独立的转基因小鼠模型，仅基于密码子92（Arg 92 GIn，Arg 92 Trp和Arg 92 Leu），一个已知的突变热点在人类cTnT的氨基酸取代。初步表征揭示了心脏质量、收缩力和肥大信号通路的等位基因特异性变化。在这些初步研究中，我们选择了观察到的表型的特定方面，作为每个突变引起的细丝功能差异的生理“指标”。建立特定cTnT突变等位基因与其细胞表型之间的机制联系将为细纤维功能的基础生物学提供新的见解，并进一步了解细纤维相关心肌病中涉及的致病途径。

DESCRIPTION (provided by applicant): The regulatory function of the cardiac sarcomere resides in the thin filament. It is a complex macromolecular structure comprised of multiple protein subunits, which interact and modulate contractile function in response to both chronic and acute physiologic stress. The binding of Ca2+ to Troponin C initiates a cascade of allosteric changes in the interactions of the proteins within the troponin (cTnC, cTnl and cTnT) and tropomyosin-actin complexes, facilitating the formation of the actinomyosin complex and the power stroke of muscle contraction. While normal hearts retain the ability to alter these complex interactions, for example, via contractile protein isoform shifts and post-translational modifications, many naturally-occurring thin filament mutations are poorly tolerated. In fact, mutations in cardiac Troponin T (cTnT) result in a particularly severe form of Familial Hypertrophic Cardiomyopathy (FHC) characterized by a high frequency of early sudden cardiac death in the absence of overt ventricular hypertrophy. The direct link between the gene mutation and the complex clinical phenotype remains unknown. Troponin T is a highly elongated protein that interacts with all other components of the thin filament and has been described as the "glue" of the regulatory system. The full ternary structure is unknown and thus the details of these complex protein-protein interactions remain unclear. The hypothesis for this application is that individual cTnT mutations disrupt discrete aspects of these protein-protein interactions causing specific changes in thin filament function and leading to distinct cardiovascular phenotypes. To address this hypothesis we have developed three independent transgenic mouse models based solely on amino acid substitutions at Codon 92 (Arg92GIn, Arg92Trp and Arg92Leu), a known mutational hotspot in human cTnT. Initial characterization has revealed allele-specific changes in cardiac mass, contractility and hypertrophic signaling pathways. In these initial studies we have selected specific aspects of the observed phenotypes to serve as physiologic "indicators" of the differences in thin filament function caused by each mutation. Establishing the mechanistic link between the specific cTnT mutant allele and it's cellular phenotype will both provide new insights into the fundamental biology of thin filament function and further our understanding of the pathogenic pathways involved in thin filament related cardiomyopathies.