Cardiac myosin binding protein-C: Structure and Function

描述（由申请人提供）：心肌肌球蛋白结合蛋白- c （cMyBP-C）是一种跨丝蛋白，在其N'区连接粗丝和细丝，调节心脏收缩力。R01HL-105826更新中包含的研究的总体目标是将cMyBP-C的N'-区域定义为心脏收缩性的关键调节因子。特别是C0-C1f，仅与肌动蛋白相互作用，在缺血/再灌注（I/R）损伤和心力衰竭时由cMyBP-C蛋白水解产生。另一方面，C0-C2与肌动蛋白和肌凝蛋白相互作用，连接细丝和粗丝，调节肌节功能。基于这些发现，我们将1)使用一种新的表达cMyBP-C的转基因小鼠模型（cMyBP-C110kDa），其中C0-C1f区域被切除，以研究该区域在调节心脏收缩性中的必要性和充分性；2)使用腺相关病毒9 （AAV9）介导的(i)重组C0-C2的表达，以确定体内N'端C0-C2在捆绑粗丝和细丝中的充分性；其中calpain-targeted site （CTS）被切除，以确定其在i /R损伤前后改善心功能的治疗潜力。cMyBP-C调控的分子机制及其对肌节结构和功能的影响在很大程度上是未知的。我们的短期目标是阐明cMyBP-C的N′区在心脏功能调节中的具体作用，而我们的长期目标是确定cMyBP-C稳定肌肉结构和功能的机制，从而在I/R损伤期间赋予心脏保护作用。SPECIFIC AIM 1将验证cMyBP-C的C0-C1f结构域的假设，该结构域在I/R损伤期间被切割和释放，是调节心功能所必需的。与对照非转基因小鼠相比，转基因cMyBP-C110kDa小鼠将用于确定cMyBP-C的N'-区域在肌节和全心水平上的关键作用。SPECIFIC AIM 2将验证cMyBP-C的C0-C2结构域足以捆绑粗和细纤维并调节正常心功能的假设。aav9介导的重组C0-C2表达将用于预防或挽救体内缺乏N'区（C0-C1f）或全长cMyBP-C的小鼠模型的收缩功能障碍。SPECIFIC AIM 3将验证重组C0-C2ΔCTS在I/R损伤期间在体内表达具有心脏保护作用的假设。重组C0-C2ΔCTS蛋白免受calpain介导的降解。我们预计aav9介导的重组C0-C2ΔCTS的表达可以显著减少梗死面积和细胞凋亡，并在I/R损伤期间保持收缩功能。总之，这些研究将确定cMyBP-C的N′区（C0-C1f和C0-C2）调节心功能的必要性和充分性，从而为I/R损伤期间发生的心肌损伤和心力衰竭提供治疗。

DESCRIPTION (provided by applicant): Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein which, at its N'-region, connects thick and thin filaments to regulate cardiac contractility. The overall objective of the studies contained in this renewal of R01HL-105826 is to define the N'-region of cMyBP-C as a critical regulator of cardiac contractility. In particular, C0-C1f, which interacts only with actin, is generated by proteolysis of cMyBP-C during ischemia/reperfusion (I/R) injury and heart failure. On the other hand, C0-C2 interacts with both actin and myosin, connecting thin and thick filaments to regulate sarcomere function. On the basis of these findings, we will 1) use a novel transgenic mouse model expressing cMyBP-C (cMyBP-C110kDa) in which the C0-C1f region was ablated to study the necessity and sufficiency of this domain in regulating cardiac contractility and 2) use adeno-associated virus 9 (AAV9)-mediated expression of (i) recombinant C0-C2 to determine the sufficiency of N'-terminal C0-C2 in bundling thick and thin filaments in vivo and (ii) recombinant C0-C2ΔCTS, in which the calpain-targeted site (CTS) has been ablated, to determine its therapeutic potential in improving cardiac function pre- and post-I/R injury. The molecular mechanisms underlying the regulation of cMyBP-C and, in turn, its impact on sarcomere structure and function, are largely unknown. Our short-term goal is to elucidate the specific role(s) of the N'-region of cMyBP-C in the regulation of cardiac function, whereas our long-term goal is to determine the mechanisms by which cMyBP-C stabilizes sarcomeric structure and function, thereby conferring cardioprotection during I/R injury. SPECIFIC AIM 1 will test the hypothesis that the C0-C1f domain of cMyBP-C, which is cleaved and released during I/R injury, is necessary for regulating cardiac function. Transgenic cMyBP-C110kDa mice will be used to determine the critical role of the N'-region of cMyBP-C at the sarcomere and whole-heart levels, compared to the control non-transgenic mice. SPECIFIC AIM 2 will test the hypothesis that the C0-C2 domains of cMyBP-C are sufficient to bundle thick and thin filaments and to regulate normal cardiac function. AAV9-mediated expression of recombinant C0-C2 will be used to prevent or rescue contractile dysfunction in mouse models that lack either the N'-region (C0-C1f) or full-length cMyBP-C in vivo. SPECIFIC AIM 3 will test the hypothesis that expression of recombinant C0-C2ΔCTS in vivo confers cardioprotection during I/R injury. Recombinant C0-C2ΔCTS protein is protected from calpain-mediated degradation. We expect AAV9-mediated expression of recombinant C0-C2ΔCTS to significantly reduce infarct size and apoptosis, as well as preserve contractile function during I/R injury. Together, these studies will determine the necessity and sufficiency of the N'-region (both C0-C1f and C0-C2) of cMyBP-C to regulate cardiac function, and, hence, provide therapy for myocardial injury and heart failure occurring during I/R injury.