Cardiac myosin binding protein-C: Structure and Function

说明(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‘区连接粗细纤维以调节心肌收缩能力。此次更新的R01HL-105826中包含的研究的总体目标是将cMyBP-C的N‘区定义为心脏收缩能力的关键调节因子。特别是，C0-C1f只与肌动蛋白相互作用，在缺血/再灌注(I/R)损伤和心力衰竭过程中cMyBP-C的蛋白分解产生。另一方面，C0-C2与肌动蛋白和肌球蛋白相互作用，连接细丝和粗丝，调节肌节功能。在这些发现的基础上，我们将1)使用一种新型的表达cMyBP-C的转基因小鼠模型(cMyBP-C110 kDa)，其中C0-C1f区域被去除，以研究该结构域在调节心肌收缩能力中的必要性和充分性；2)使用腺相关病毒9(AAV9)介导的重组C0-C2的表达来确定N‘端C0-C2在体内捆绑粗细丝的充分性和(Ii)已去除钙痛靶点的重组C0-C2ΔCTS，探讨其在改善I/R损伤前后心功能方面的作用。CMyBP-C调控的分子机制以及它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 N‘区在心脏功能调节中的具体作用(S)，而我们的长期目标是确定cMyBP-C稳定肌节结构和功能的机制，从而在I/R损伤中提供心脏保护。特异性AIM 1将验证cMyBP-C的C0-C1f结构域是调节心脏功能所必需的假设，该结构域在I/R损伤过程中被裂解并释放。转基因cMyBP-C110 kDa小鼠将被用来确定cMyBP-C的N‘区在肌节和全心水平上的关键作用，与对照的非转基因小鼠相比。特异性AIM 2将验证cMyBP-C的C0-C2结构域足以捆绑粗细丝并调节正常心脏功能的假设。AAV9介导的重组C0-C2的表达将用于预防或挽救体内缺乏N‘区(C0-C1f)或全长cMyBP-C的小鼠的收缩功能障碍。特异性AIM3将验证重组C0-C2ΔCTS在体内表达对I/R损伤具有心脏保护作用的假说。重组C0-C2ΔCTS蛋白可被保护而不受钙蛋白的降解。我们期望AAV9介导的重组C0-C2ΔCTs的表达能够显著减少I/R损伤时的心肌梗死面积和细胞凋亡，同时保护心肌的收缩功能。总之，这些研究将确定cMyBP-C的N‘-区(C0-C1f和C0-C2)调节心脏功能的必要性和充分性，从而为I/R损伤时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.