Cardiac Myosin Binding Protein-C: Structure and Function

描述（申请人提供）：缺血性损伤引起的心肌梗死是心血管疾病发病率和死亡率的一个突出而共同的特征。心肌肌球蛋白结合蛋白c （cMyBP-C）在蛋白水解过程中降解，是I-R损伤时心肌收缩发病机制的重要决定因素。简而言之，cMyBP-C是一种厚纤维相关蛋白，可稳定肌球蛋白，调节心脏中的肌肉结构和功能，肌球蛋白是收缩机制的重要组成部分。cMyBP-C基因突变约占所有心肌病病例的34%，其中70%预计会产生不稳定的截断蛋白。在I-R损伤期间，我们证明了cMyBP-C的广泛断裂与肌肉结构改变和收缩功能障碍有关。因此，虽然短期目标是阐明cMyBP-C在缺血再灌注（I-R）损伤期间心脏保护的临床背景下的蛋白水解和致病特性，但长期目标是确定cMyBP-C在I-R损伤期间稳定肌肉结构和功能以赋予心脏保护的机制。更具体地说，我们的初步研究表明，calpain将cMyBP-C降解为几个片段，并且29kda片段是体外的主要片段。在小鼠I-R损伤期间，这种蛋白水解导致29kda片段释放到血流中。此外，质谱分析证实，29kda片段的释放与calpain-targeted site （CTS）有关，这是一个保守的磷酸化基序，可能调节其裂解。从治疗的角度来看，这些发现表明CTS的消融可能导致calpain介导的蛋白水解产生抗性，从而取消了29 kda片段的释放。因此，我们认为抑制CTS裂解可以确保cMyBP-C的结构完整性，从而保持其收缩结构和功能。然而，cMyBP-C降解的临床和致病意义及其蛋白水解特性尚未确定，因此是临床重要的转化研究领域。因此，目标是确定29- kDa片段在血液中的释放与收缩功能障碍之间的相关性，证明其对心肌细胞的毒性作用，并研究cMyBP-C中CTS切割的抑制如何保护心脏免受I-R损伤。总的来说，本研究旨在确定I-R损伤期间支持治疗中cMyBP-C的稳定性和功能，特别是心肌收缩性。为了实现我们的目标，Specific Aim 1将根据梗死面积和I-R损伤期间的收缩功能确定血液中29-kDa片段的水平。特异性Aim 2将在肌球蛋白功能的背景下确定29kda片段的致病特性。特异性目的3将确定CTS的位点特异性抑制是否可以在I-R损伤期间保持cMyBP-C的稳定性和功能，从而赋予心脏保护作用。重要的是，一旦29 kda片段的动力学验证了该肽在诱导I-R损伤的野生型非转基因小鼠血清中是可量化的，我们就可以确认其作为缺血性心肌梗死后临床有用的读数的潜力。我们的实验方法是全面的，从分子相互作用的分析到肌肉组织排列和功能的功能评估，在体外和体内都有。将在野生型非转基因小鼠中诱导I-R损伤，以确定与对照组相比，29kda片段的顺序释放及其与梗死面积、钙蛋白酶活性和心肌功能相关的血清水平。以成年小鼠心肌细胞为模型系统，利用重组腺病毒和多肽研究了29kda片段的致病特性。为了确定cMyBP-C中CTS与心脏保护之间的关系，我们将使用表达cMyBP-C的转基因小鼠，其中CTS已被切除并饲养到cMyBP-C零背景中，与表达模拟磷酸化和野生型cMyBP-C的转基因小鼠进行比较。终点测量包括血液中与梗死面积和心功能相关的29-kDa片段的量、钙蛋白酶活性、cMyBP-C磷酸化水平、细胞内Ca2+瞬态、Mg2+- atp酶活性、肌丝Ca2+敏感性、分子结合研究、肌节结构和功能。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The long-term objective is to understand the functional consequences of cardiac myosin binding protein-C protein on heart function. Specifically, the proposed studies will examine the association between cardiac myosin binding protein-C degradation and cardiac dysfunction, leading to the development of potential cardioprotective therapeutic approaches by site-specific protein modification.