Calcium release channel dysfunction: molecular mechanisms

钙释放通道功能障碍：分子机制

基本信息

批准号：
9204856
负责人：
Aleksey V Zima
金额：
$ 37.75万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-15 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9204856
关键词：
Affect Arrhythmia Binding Sites Blinking Calcium Calmodulin Cardiac Cardiac Myocytes Clinical Complex Confocal Microscopy Cysteine Defect Dissociation Ensure Fatal Outcome Functional disorder Future Generations Goals Heart Diseases Heart failure Homeostasis Image In Situ In Vitro Infarction Life Lipid Bilayers Measurement Measures Mediating Modification Molecular Molecular Target Muscle Cells Mutagenesis Mutation Myocardial Contraction Myocardial Infarction Optics Outcome Study Oxidation-Reduction Oxidative Stress Pathology Pilot Projects Play Post-Translational Protein Processing Process Public Health Regulation Reporting Research Proposals Resistance Resolution Role Ryanodine Receptor Calcium Release Channel Sarcoplasmic Reticulum Site Site-Directed Mutagenesis System Techniques Testing Therapeutic Therapeutic Intervention Time Treatment Efficacy UV induced Ventricular Work crosslink design disulfide bond free radical oxygen heart function improved mutant novel oxidation prevent public health relevance receptor binding receptor function receptor structure function response targeted treatment

项目摘要

DESCRIPTION (provided by applicant): Calcium (Ca) release through the ryanodine receptor (RyR) is essential for regular heart contraction. Defects in RyR regulation cause imbalance in Ca homeostasis and contractile dysfunction in a variety of cardiac diseases. Since the most common cardiac pathologies (e.g. infarction, heart failure) are associated with oxidative stress, the main goal of this proposal is to define the molecular mechanisms of RyR dysfunction during oxidative stress. The RyR contains a large number of cysteine residues that can couple the cytosolic redox potential and Ca homeostasis. However, the functionally important redox-sensing sites on the RyR have not yet been identified. As a result, the molecular mechanisms of RyR dysfunction during oxidative stress remain largely unknown. This delays our progress in designing effective therapeutic interventions that can improve Ca homeostasis during cardiac diseases. Thus, more direct work identifying functionally important redox-sensing cysteines on RyR is essential to advance the field. We have recently discovered that oxidative stress activates the RyR by forming disulfide bonds between two neighboring subunits: intersubunit crosslinking. In this proposal we will test the hypothesis that intersubunit crosslinking is the mot functionally important redox modification of RyR responsible for the imbalance in Ca homeostasis during oxidative stress. This hypothesis will be tested using cutting-edge experimental techniques, such as RyR mutagenesis, single RyR channel recordings, and high resolution Ca imaging. In aim 1 we will identify specific cysteine residues on RyR that are involved in the crosslinking. Then, we will determine if mutation of these cysteines can maintain normal RyR function and Ca homeostasis during oxidative stress. In aim 2 we will define the molecular mechanisms of RyR dysfunction induced by the crosslinking. Calmodulin (CaM) bound to the RyR plays an important role in negative control of RyR activity. Our pilot studies suggest that the crosslinking causes dissociation of CaM from the RyR. Here, we will define if mutation of crosslinking cysteines can normalize Ca homeostasis during oxidative stress by preventing the CaM-RyR uncoupling. We will also explore whether stabilizing the CaM-RyR binding can protect the RyR function against oxidative stress in cardiomyocytes. By accomplishing these studies, we expect to define novel targets for future therapies that can improve Ca homeostasis during cardiac diseases associated with oxidative stress.

描述（由适用提供）：通过ryanodine接收器（RYR）释放钙（CA）对于常规心脏收缩至关重要。 RYR调节的缺陷导致CA稳态和收缩功能障碍的各种心脏疾病的失衡。由于最常见的心脏病理（例如梗塞，心力衰竭）与氧化应激有关，因此该提案的主要目标是定义氧化应激期间RYR功能障碍的分子机制。 RYR包含大量的半胱氨酸保留，可以将胞质氧化还原电位和CA稳态造成。但是，尚未确定在RYR上功能上重要的氧化还原敏感位点。结果，氧化应激期间RYR功能障碍的分子机制在很大程度上未知。这延迟了我们在设计有效的治疗干预措施方面的进展，这些干预措施可以改善心脏病期间的CA稳态。这是在RYR上识别功能上重要的氧化还原敏感性半胱氨酸的更直接的工作对于推进该领域至关重要。我们最近发现，氧化应激通过在两个相邻亚基之间形成二硫键来激活RYR：subusubunit交联。在此提案中，我们将检验以下假设：subumunit交联是MOT在氧化应激期间CA稳态失衡的RYR功能上重要的氧化还原修饰。该假设将使用尖端的实验技术进行检验，例如RYR诱变，单一RYR通道记录和高分辨率CA成像。在AIM 1中，我们将确定与交联有关的RYR上的特定半胱氨酸保留。然后，我们将确定这些半胱氨酸的突变是否可以在氧化应激期间维持正常的RYR功能和Ca稳态。在AIM 2中，我们将定义由交联引起的RYR功能障碍的分子机制。与RYR结合的钙调蛋白（CAM）在RYR活性的负面控制中起着重要作用。我们的试点研究表明，交联导致CAM与RYR的解离。在这里，我们将通过防止CAM-RYR解耦在氧化应激期间的交联突变是否可以在氧化应激过程中归一化。我们还将探索稳定CAM-RYR结合是否可以保护RYR功能免受心肌细胞中氧化应激的影响。通过完成这些研究，我们期望为未来的疗法定义新的靶标，这些疗法可以改善与氧化应激相关的心脏疾病期间CA稳态。