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.
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