Troponin structure & function in cardiomyopathy

描述（由申请人提供）：心肌收缩是由Ca2+结合肌钙蛋白复合物调节的，肌钙蛋白复合物由3个亚基组成：肌钙蛋白C、肌钙蛋白I和肌钙蛋白T。本提案探讨了心肌肌钙蛋白T （cTnT）异常剪接在扩张型心肌病（DCM）发病机制中的作用，以及心肌肌钙蛋白I （cTnl）多态性对这种异常cTnT的补偿。我们之前在土耳其和DCM的哺乳动物模型中发现了具有异常拼接n端区域的cTnT变体的存在。由于异常的cTnT剪接也发生在肌病和衰竭的人类心脏中，这些cTnT变异可能在DCM和心力衰竭的发病机制和病理生理中发挥作用。我们假设，正常均匀的成人心肌中存在2种或更多功能不同的cTnT变异，导致cTnT异质性，使收缩不同步，降低心肌效率。我们开发了转基因小鼠，在其心肌中表达dcm相关的cTnT变体，用于功能表征。我们将研究异常的ctnt对肌动球蛋白atp酶活性、单个肌细胞和皮肤肌条的收缩力、离体工作心脏的功能和小鼠体内心功能的影响。增加与cTnl的结合亲和力是dcm相关cTnTs的主要异常。我们最近在野生火鸡心脏中发现了一种新的cTnl多态性（Arg111Cys）。在所有心脏和骨骼肌Tnl中，Arg-m以Arg或Lys的形式保存，位于Tnl和TnT之间的盘绕界面上。Arg111Cys在cTnl中的替换降低了其对cTnT的结合亲和力，这可能补偿dcm相关的cTnT异常。因此，cTnl-Cysm等位基因在野生火鸡中的存在可能会阻止DCM的发生，因此具有重要的选择价值。为了验证这一假设，我们将在转基因小鼠中对火鸡cTnl-Cysm多态性进行生化和生理表征。然后，我们将在双转基因小鼠中与DCM cTnT共表达这种cTnl多态性，以检查其代偿作用。利用这些综合的生理系统，本研究将有助于我们了解肌钙蛋白亚基之间的结构-功能关系，并为cTnT心肌病的预防和治疗奠定基础。

DESCRIPTION (provided by applicant): Cardiac muscle contraction is regulated by Ca2+ binding to the troponin complex, which consists of 3 subunits: troponin C, troponin I and troponin T. This proposal investigates the role of abnormally spliced cardiac troponin T (cTnT) in the pathogenesis of dilated cardiomyopathy (DCM) and the compensation for this abnormal cTnT by a cardiac troponin I (cTnl) polymorphism. We previously discovered the presence of cTnT variants with abnormally spliced N-terminal regions in turkey and mammalian models of DCM. Since abnormal cTnT splicing also occurs in myopathic and failing human hearts, these cTnT variants may play a role in the pathogenesis and pathophysiology of DCM and heart failure. We hypothesize that the cTnT heterogeneity resulting from the presence of 2 or more functionally distinct cTnT variants in the normally uniform adult cardiac muscle desynchronizes contraction and decreases myocardial efficiency. We have developed transgenic mice expressing the DCM-related cTnT variants in their cardiac muscle for functional characterization. We will examine the effects of the abnormal cTnTs on the activity of actomyosin ATPase, the contractility of single myocytes and skinned muscle strips, the function of isolated working hearts, and the in vivo cardiac function of the mice. Increased binding affinity to cTnl is a primary abnormality of the DCM-related cTnTs. We recently found a novel polymorphism of cTnl (Arg111Cys) in wild turkey hearts. Arg-m is conserved as Arg or Lys in all cardiac and skeletal muscle Tnls, and sits in a coiled-coil interface between Tnl and TnT. The Arg111Cys substitution in cTnl lowers its binding affinity for cTnT, which is potentially compensatory for DCM-related cTnT abnormalities. Therefore, the presence of the cTnl-Cysm allele in wild turkeys may prevent the onset of DCM and thus have a significant selection value. To investigate this hypothesis, we will biochemically and physiologically characterize the turkey cTnl-Cysm polymorphism in transgenic mice. We will then coexpress this cTnl polymorphism with the DCM cTnT in double-transgenic mice to examine its compensatory effects. Using these integrated physiological systems, this study will significantly contribute to our understanding of the structure-function relationships among the troponin subunits and lay a foundation for the prevention and treatment of cTnT cardiomyopathies.