Cardiac Myosin Binding Protein-C: Structure and Function

7.项目总结 由缺血性损伤引起的心肌梗死是心血管疾病的一个突出而常见的特征 发病率和死亡率。心肌肌球蛋白结合蛋白-C(cMyBP-C)是由其在 蛋白降解是I-R损伤过程中心肌收缩发生的重要决定因素。简单地说， CMyBP-C是一种厚丝相关蛋白，可稳定肌球蛋白，肌球蛋白是肌球蛋白的重要组成部分。 收缩机械，调节心脏中肌节的结构和功能。CMyBP-C基因的突变 基因约占所有心肌病病例的34%，其中70%被预测为产生不稳定 截短的蛋白质。在I-R损伤过程中，我们证明了cMyBP-C的广泛碎裂与 肌节结构改变和收缩功能障碍。因此，虽然短期目标是 在心脏保护的临床背景下阐明cMyBP-C的蛋白分解和致病特性 在缺血-再灌注(I-R)损伤过程中，长期目标是确定 CMyBP-C稳定肌节的结构和功能，从而在I-R损伤时提供心脏保护。 更具体地说，我们的初步研究表明，钙蛋白酶将cMyBP-C降解成几个片段，并 29 kDa片段是体外表达的主要片段。这种蛋白质分解会导致细胞释放 小鼠I-R损伤过程中29 kDa片段进入血流。此外，质谱分析 确认29 kDa片段的释放与Calain靶点(CTS)相关，即 一个保守的磷酸化基序，可能调节其切割。从治疗的角度来看， 这些发现表明，消融CTS可能会导致对钙蛋白酶介导的抵抗。 蛋白分解，因此取消了29 kDa片段的释放。因此，我们认为抑制CTS 切割将确保cMyBP-C的结构完整性，从而保留收缩结构和 功能。然而，cMyBP-C降解的临床和发病意义以及 其蛋白分解的性质尚未确定，因此代表了临床上重要的领域 翻译研究。因此，目标是确定29人获释之间的相关性- 血液中的KDA片段和收缩功能障碍，表明其对心肌细胞和 研究cMyBP-C中CTS裂解的抑制如何保护心脏免受I-R损伤。总体而言， 拟议的研究旨在确定cMyBP-C在支持性治疗背景下的稳定性和功能 在I-R损伤期间，一般情况下，和心肌收缩能力，特别是。为了实现我们的目标，明确的目标 1将根据梗塞面积和收缩功能来确定血液中29 kDa片段的水平 在I-R损伤期间。特异靶2将确定29 kDa片段的致病特性 肌球蛋白功能的背景。具体目标3将确定是否对CTS进行特异性抑制，如 上面定义的，可以在I-R损伤过程中保持cMyBP-C的稳定性和功能，从而赋予 心脏保护。重要的是，一旦29 kDa片段的动力学验证了该肽是 在诱导I-R损伤的野生型非转基因小鼠血清中可定量表达，我们可以证实其 有可能成为临床有用的缺血后心肌梗死的读数。我们的实验方法是 全面，从分子相互作用的分析到肌瘤的功能评估 在体外和体内的排列和功能。野生型非转基因将诱导I-R损伤 小鼠确定29 kDa片段的序贯释放及其血清水平与脑梗塞的关系 与对照组相比，大小、钙蛋白酶活性和心肌功能。成年小鼠心肌细胞有 被选为模型系统，用于研究29 kDa片段的致病特性 重组腺病毒和多肽。确定cMyBP-C中的CTS与 为了保护心脏，我们将使用表达cMyBP-C的转基因小鼠，其中CTS已被消融并 培育到cMyBP-C缺失背景中，与表达模拟磷酸化和 野生型cMyBP-C对照。终点测量包括29-kDa片段在 血与心肌梗死面积、心功能、钙蛋白酶活性、cMyBP-C磷酸化水平、 细胞内钙瞬变，镁-三磷酸腺苷酶活性，肌丝钙敏感性，分子结合研究， 肌节的结构和功能。

7. PROJECT SUMMARY Myocardial infarction resulting from ischemic injury is a prominent and common feature of cardiovascular morbidity and mortality. Cardiac myosin binding protein-C (cMyBP-C) is, by its degradation during proteolysis, an important determinant of myocardial contractile pathogenesis during I-R injury. Briefly, cMyBP-C is a thick filament-associated protein that stabilizes myosin, an important component of the contractile machinery, to regulate sarcomeric structure and function in the heart. Mutations in the cMyBP-C gene account for ~34% of all cardiomyopathy cases, 70% of which are predicted to produce unstable truncated proteins. During I-R injury, we demonstrated that extensive fragmentation of cMyBP-C correlates with altered sarcomeric structure and contractile dysfunction. Therefore, while the short-term goal is to elucidate the proteolytic and pathogenic properties of cMyBP-C in the clinical context of cardioprotection during ischemia-reperfusion (I-R) injury, the long-term goal is to determine the mechanisms by which cMyBP-C stabilizes sarcomeric structure and function in order to confer cardioprotection during I-R injury. More specifically, our preliminary studies show that calpains degrade cMyBP-C into several fragments and that the 29-kDa fragment is the predominant fragment in vitro. Such proteolysis leads to the release of the 29-kDa fragment into the blood stream during I-R injury in mice. Moreover, mass spectrometry analyses confirm that the release of the 29-kDa fragment is associated with the calpain-targeted site (CTS), which is a conserved phosphorylation motif that possibly regulates its cleavage. From a therapeutic perspective, these findings indicate that the ablation of the CTS could result in resistance to calpain-mediated proteolysis, thus abrogating release of the 29-kDa fragment. Therefore, we propose that inhibition of CTS cleavage would secure the structural integrity of cMyBP-C, thus preserving contractile structure and function. However, the clinical and pathogenic significance of cMyBP-C degradation, as well as the properties of its proteolysis, have not been determined and therefore represent a clinically important area of translational research. The goal, therefore, is to determine the correlation between the release of the 29- kDa fragment in the blood and contractile dysfunction, demonstrate its toxic effects in cardiomyocytes and examine how the inhibition of CTS cleavage in cMyBP-C protects the heart from I-R injury. Overall, the proposed research aims to define the stability and function of cMyBP-C in the context of supportive therapy during I-R injury, in general, and heart muscle contractility, specifically. To achieve our goals, Specific Aim 1 will determine the levels of 29-kDa fragment in the blood, according to infarct size and contractile function during I-R injury. Specific Aim 2 will determine the pathogenic properties of the 29-kDa fragment in the context of myosin function. Specific Aim 3 will determine whether site-specific inhibition of the CTS, as defined above, can preserve cMyBP-C stability and function during I-R injury and thus confer cardioprotection. Importantly, once the kinetics of the 29-kDa fragment have validated that this peptide is quantifiable in the serum of wild-type non-transgenic mice with induced I-R injury, we can confirm its potential as a clinically useful readout of post-ischemic myocardial infarction. Our experimental approach is comprehensive, ranging from the analysis of molecular interactions to functional assessments of sarcomeric arrangement and function, both in vitro and in vivo. I-R injury will be induced in wild-type non-transgenic mice to define the sequential release of the 29-kDa fragment and its blood serum levels in relation to infarct size, calpain activities, and myocardial function, compared with controls. Adult mouse cardiomyocytes have been chosen as the model system to investigate the pathogenic properties of the 29-kDa fragment by using recombinant adenoviruses and peptides. To determine the association between the CTS in cMyBP-C and cardioprotection, we will use transgenic mice expressing cMyBP-C in which the CTS has been ablated and bred into the cMyBP-C null background, compared with transgenic mice expressing phospho-mimetic and wild-type cMyBP-C controls. Endpoint measurements include the amount of the 29-kDa fragment in the blood correlated with infarct area and cardiac function, calpain activity, cMyBP-C phosphorylation levels, intracellular Ca2+ transients, Mg2+-ATPase activity, myofilament Ca2+ sensitivity, molecular binding studies, sarcomere structure and function.