Troponin structure & function in cardiomyopathy

描述(申请人提供)：心肌收缩是由钙离子与肌钙蛋白复合体结合调节的，肌钙蛋白复合体由肌钙蛋白C、肌钙蛋白I和肌钙蛋白T三个亚基组成。本研究旨在研究异常剪接的心肌肌钙蛋白T(CTnT)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(CTnI)多态对这种异常cTnT的补偿作用。我们之前在土耳其和哺乳动物的DCM模型中发现了具有异常剪接N-末端区域的cTnT变体。由于cTnT剪接异常也存在于肌病心脏和衰竭心脏中，这些cTnT变异体可能在扩张型心肌病和心力衰竭的发病机制和病理生理学中发挥作用。我们假设，在正常均匀的成人心肌中，由于存在两个或更多功能上不同的cTnT变体而导致的cTnT异质性会使收缩失去同步性，并降低心肌效率。我们已经开发出在心肌中表达与DCM相关的cTnT变体的转基因小鼠，用于功能鉴定。我们将检测cTnTs异常对肌动球蛋白ATPase活性、单个肌细胞和去皮肌条的收缩性能、离体心功能和在体小鼠心功能的影响。与cTn1结合亲和力增强是DCM相关cTnT的主要异常。我们最近在野生火鸡心脏中发现了一种新的cTn1(Arg111Cys)基因多态性。Arg-m在所有心肌和骨骼肌TnL中以Arg或Lys的形式保守，并位于TnL和TnT之间的螺旋线圈界面上。CTn1中的Arg111Cys替换降低了其与cTnT的结合亲和力，这可能是对DCM相关cTnT异常的补偿。因此，野生火鸡中cTn1-Cysm等位基因的存在可能会阻止DCM的发生，因此具有显著的选择价值。为了研究这一假设，我们将在转基因小鼠中对火鸡cTn1-Cysm多态进行生化和生理学表征。然后，我们将在双转基因小鼠中将该cTn1多态与DCM cTnT共表达，以检测其代偿作用。利用这些集成的生理系统，本研究将有助于我们理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.