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。项目摘要缺血性损伤导致的心肌梗塞是心血管的突出特征发病率和死亡率。心脏肌球蛋白结合蛋白-C（CMYBP-C）是通过其在其期间降解的蛋白水解是I-R损伤期间心肌发病机理的重要决定因素。简要地， CMYBP-C是一种厚细丝相关的蛋白质，稳定肌球蛋白，肌球蛋白是一个重要组成收缩机械，以调节心脏中的肉瘤结构和功能。 CMYBP-C中的突变基因占所有心肌病病例的34％，其中70％被预测会产生不稳定截短的蛋白质。在I-R受伤期间，我们证明了CMYBP-C的广泛碎片与随着肌膜结构和收缩功能障碍的改变。因此，而短期目标是在心脏保护的临床背景下阐明CMYBP-C的蛋白水解和致病性能在缺血 - 再灌注（I-R）损伤期间，长期目标是确定该机制 CMYBP-C可以稳定肌膜结构和功能，以便在I-R损伤期间赋予心脏保护。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 29 kDa片段是体外的主要碎片。这种蛋白水解导致释放在小鼠I-R受伤期间，在血流中29 kDa碎片。此外，质谱分析确认29 kDa片段的释放与靶向钙蛋白酶的位点（CTS）相关联，一个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Calpain介导的抗性蛋白水解，从而废除了29 kDa片段的释放。因此，我们建议抑制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。但是，CMYBP-C降解的临床和致病意义以及其蛋白水解的特性尚未确定，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-释放之间的相关性血液和收缩功能障碍中的KDA碎片表明其在心肌细胞和检查CMYBP-C中CTS切割的抑制如何保护心脏免受I-R损伤的影响。总体而言，拟议的研究旨在在支持疗法的背景下定义CMYBP-C的稳定性和功能通常，在I-R受伤期间，特别是心肌收缩力。为了实现我们的目标，特定的目标 1将根据梗塞大小和收缩功能确定血液中29 kDa片段的水平在I-R受伤期间。具体目标2将确定29 kDa片段在肌球蛋白功能的上下文。具体目标3将确定特定于位点特异性抑制CTS的抑制作用上面定义的，可以在I-R受伤期间保持CMYBP-C的稳定性和功能心脏保护。重要的是，一旦29 kDa片段的动力学证实了该肽是可量化的野生型非转基因小鼠具有诱导的I-R损伤，我们可以确认其潜力是临床上有用的缺血后心肌梗塞的读数。我们的实验方法是综合性，从分子相互作用的分析到肉瘤的功能评估布置和功能，体外和体内。 I-R损伤将在野生型非转基因中诱导小鼠定义29 kDa片段的顺序释放及其血清水平与梗塞有关与对照组相比，尺寸，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，以研究29 kDa片段的致病性能重组腺病毒和肽。确定CMYBP-C和CTS之间的关联心脏保护作用，我们将使用表达CMYBP-C的转基因小鼠，其中CTS已被消融并且与表达磷酸化和表达磷酸化和的转基因小鼠相比野生型CMYBP-C对照。端点测量包括29 kDa片段的数量血液与梗塞区域和心脏功能，钙蛋白酶活性，CMYBP-C磷酸化水平相关，细胞内Ca2+瞬变，Mg2+ -ATPase活性，肌丝Ca2+灵敏度，分子结合研究，分子结合研究肌节结构和功能。