Cardiac Myosin Binding Protein-C: Structure and Function

心肌肌球蛋白结合蛋白-C：结构和功能

基本信息

批准号：
8207226
负责人：
Sakthivel Sadayappan
金额：
$ 33.64万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
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/8207226
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

项目摘要

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.

7. 项目概要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) 在蛋白水解是 I-R 损伤期间心肌收缩发病机制的重要决定因素。简要地， cMyBP-C 是一种粗丝相关蛋白，可稳定肌球蛋白，肌球蛋白是肌动蛋白的重要组成部分。收缩机制，调节心脏的肌节结构和功能。 cMyBP-C 突变基因约占所有心肌病病例的 34%，其中 70% 预计会产生不稳定的心肌病截短的蛋白质。在 I-R 损伤期间，我们证明了 cMyBP-C 的广泛碎片与肌节结构改变和收缩功能障碍。因此，虽然短期目标是阐明 cMyBP-C 在心脏保护临床中的蛋白水解和致病特性在缺血再灌注（I-R）损伤期间，长期目标是确定缺血再灌注（I-R）损伤的机制 cMyBP-C 稳定肌节结构和功能，以在 I-R 损伤期间提供心脏保护作用。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 片段是体外的主要片段。这种蛋白水解导致释放小鼠 I-R 损伤期间 29-kDa 片段进入血流。此外，质谱分析确认 29 kDa 片段的释放与钙蛋白酶靶向位点 (CTS) 相关，该位点是可能调节其裂解的保守磷酸化基序。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抵抗蛋白水解，从而消除 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，从而保留收缩结构和功能。然而，cMyBP-C 降解的临床和致病意义以及其蛋白水解特性尚未确定，因此代表了临床上重要的领域转化研究。因此，目标是确定 29- 血液中的 kDa 片段和收缩功能障碍，证明其对心肌细胞和收缩功能的毒性作用研究抑制 cMyBP-C 中的 CTS 裂解如何保护心脏免受 I-R 损伤。总体而言，拟议的研究旨在确定 cMyBP-C 在支持治疗背景下的稳定性和功能一般而言，在 I-R 损伤期间，特别是心肌收缩力。为了实现我们的目标，具体目标 1 将根据梗塞面积和收缩功能确定血液中 29-kDa 片段的水平在 I-R 损伤期间。具体目标 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 进行位点特异性抑制，如如上所述，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 片段的动力学验证该肽是在诱导 I-R 损伤的野生型非转基因小鼠的血清中可定量，我们可以证实其作为临床上有用的缺血后心肌梗死读数的潜力。我们的实验方法是全面，从分子相互作用分析到肌节功能评估体外和体内的排列和功能。野生型非转基因会诱导 I-R 损伤小鼠以确定 29-kDa 片段的顺序释放及其与梗塞相关的血清水平与对照组相比，大小、钙蛋白酶活性和心肌功能。成年小鼠心肌细胞具有被选为模型系统，通过使用研究 29-kDa 片段的致病特性重组腺病毒和肽。确定 cMyBP-C 和 CTS 之间的关联心脏保护，我们将使用表达 cMyBP-C 的转基因小鼠，其中 CTS 已被消除，并且与表达磷酸模拟物和表达磷酸化的转基因小鼠相比，培育到cMyBP-C无效背景中野生型 cMyBP-C 对照。终点测量包括 29-kDa 片段的量血液与梗塞面积和心脏功能、钙蛋白酶活性、cMyBP-C 磷酸化水平相关，细胞内 Ca2+ 瞬变、Mg2+-ATP 酶活性、肌丝 Ca2+ 敏感性、分子结合研究、肌节的结构和功能。