Cardiac Myosin Binding Protein-C: Structure and Function

心肌肌球蛋白结合蛋白-C：结构和功能

• 批准号: 8023964
• 负责人: Sakthivel Sadayappan
• 金额: $ 32.55万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8023964
• 关键词: Ablation; Accounting; Actins; Adenoviruses; Adult; Amino Acid Sequence; Antibodies; Area; Binding; Biological Markers; Biological Models; Blood; Blood specimen; Breeding; Ca(2+) Mg(2+)-ATPase; Calpain; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cardiomyopathies; Cardiovascular system; Cleaved cell; Clinical; Complex; Custom; Cyclic AMP-Dependent Protein Kinases; Data; Development; Familial Hypertrophic Cardiomyopathy; Functional disorder; Generations; Genes; Goals; Heart; In Vitro; Infarction; Injury; Ischemia; Kinetics; Link; Mass Spectrum Analysis; Measurement; Mediating; Microfilaments; Modification; Molecular; Morbidity - disease rate; Mus; Muscle; Mutation; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myosin ATPase; Organ; Pathogenesis; Peptides; Phosphorylation; Post-Translational Protein Processing; Property; Protein Dephosphorylation; Proteins; Proteolysis; Recombinants; Reperfusion Injury; Reperfusion Therapy; Research; Resistance; Role; Sarcomeres; Secure; Serum; Severities; Site; Stream; Structure; Supportive care; Testing; Therapeutic; Thick Filament; Thin Filament; Time; Toxic effect; Transgenic Mice; Translational Research; Western Blotting; base; c-Myc Staining Method; citrate carrier; heart function; in vivo; injured; mimetics; mortality; myosin-binding protein C; novel; prevent

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.

描述（由申请人提供）：缺血性损伤引起的心肌梗塞是心血管发病率和死亡率的突出且常见的特征。心脏肌球蛋白结合蛋白-C（CMYBP-C）通过其在蛋白水解过程中的降解是I-R损伤期间心肌发病机理的重要决定因素。简而言之，CMYBP-C是一种厚细丝相关的蛋白质，稳定肌球蛋白是收缩机械的重要组成部分，可调节心脏中的肉类结构和功能。 CMYBP-C基因中的突变占所有心肌病病例的34％，其中70％被预测会产生不稳定的截短蛋白质。在I-R损伤期间，我们证明了CMYBP-C的广泛碎片与肉类结构改变和收缩功能障碍相关。因此，虽然短期目标是在缺血 - 重新灌注（I-R）损伤过程中阐明CMYBP-C的蛋白水解和致病性能，但长期目标是确定CMYBP-C稳定肉瘤结构的机制，以稳定肉体性结构并在I-RENIGERCERECTION期间稳定肉体性治疗。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，而29 kDa片段是体外主要片段。这种蛋白水解导致在小鼠I-R损伤期间将29 kDa片段释放到血流中。此外，质谱分析证实，29 kDa片段的释放与靶向钙蛋白酶的位点（CTS）相关，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的消融可能导致对钙蛋白酶介导的蛋白水解的抗性，从而消除了29 kDa片段的释放。因此，我们建议抑制CTS裂解将确保CMYBP-C的结构完整性，从而保留收缩的结构和功能。但是，尚未确定CMYBP-C降解的临床和致病意义及其蛋白水解的特性，因此代表了转化研究的临床重要领域。因此，目标是确定血液和收缩功能障碍29-KDA片段的释放之间的相关性，证明了其在心肌细胞中的毒性作用，并研究CMYBP-C中CTS裂解的抑制如何保护心脏造成I-R损伤。总体而言，拟议的研究旨在在I-R损伤期间（通常是心脏肌肉收缩力）中定义CMYBP-C的稳定性和功能。为了实现我们的目标，特定的目标1将根据I-R损伤期间的梗塞大小和收缩功能来确定血液中29 kDa片段的水平。特定的目标2将在肌球蛋白功能的背景下确定29 kDa片段的致病性能。特定的目标3将确定上述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。重要的是，一旦29 kDa片段的动力学验证了该肽在具有诱导I-R损伤的野生型非转基因小鼠的血清中是可量化的，我们可以证实其潜在的潜在潜力，是临床上有用的异常心肌心肌梗死的临床读数。我们的实验方法是全面的，从分子相互作用的分析到体外和体内的肌膜排列和功能的功能评估。与对照组相比，野生型非转基因小鼠将诱导I-R损伤，以定义29 kDa片段的顺序释放及其血清水平与梗死大小，钙蛋白酶活性和心肌功能相比，与对照组相比。成年小鼠心肌细胞已被选为模型系统，以使用重组腺病毒和肽来研究29 kDa片段的致病性质。为了确定CMYBP-C中的CTS与心脏保护区中的CTS之间的关联，我们将使用表达CMYBP-C的转基因小鼠，其中CTS已被消融并繁殖到CMYBP-C NULL背景中，与表达磷酸化和野生型CMYBP-C对照的转基因小鼠相比。终点测量值包括血液中29-KDA片段的量与梗死区域和心脏功能相关，钙蛋白酶活性，CMYBP-C磷酸化水平，细胞内Ca2+瞬态，MG2+ -ATPase活性，肌毛丝CA2+敏感性，肌肉纤维敏感性，分子结合研究，分子结合研究，萨科尔脑结构和功能。 公共卫生相关性：长期目标是了解心脏肌球蛋白结合蛋白-C蛋白对心脏功能的功能后果。具体而言，拟议的研究将研究心脏肌球蛋白结合蛋白-C降解与心脏功能障碍之间的关联，从而导致通过位点特异性蛋白质修饰发展潜在的心脏保护治疗方法。