Cardiac ryanodine receptor and oxidative stress

心脏兰尼碱受体与氧化应激

基本信息

批准号：
10632861
负责人：
Shanna Hamilton
金额：
$ 5.4万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-09 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10632861
关键词：
Achievement Address Area Arrhythmia Automobile Driving Award Biochemical Biosensor Cardiac Cardiac Myocytes Cardiovascular Diseases Cardiovascular Physiology Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Coupled Coupling Cysteine Disease Environment Enzyme Inhibition Enzymes Foundations Genes Genetic Goals Health Heart Heart Diseases Homeostasis Human Impairment Inherited Lead Link Mediating Mentors Modification Molecular Molecular Biology Molecular Chaperones Ohio Oxidation-Reduction Oxidative Stress Oxidoreductase Pharmacology Phase Protein Biochemistry Reactive Oxygen Species Regulation Research Rodent Model Role Ryanodine Receptor Calcium Release Channel Sarcoplasmic Reticulum Source Stress Sudden Death System Technology Testing Therapeutic Training United States Universities Up-Regulation Viral career human model induced pluripotent stem cell insight loss of function novel novel therapeutic intervention protein folding sensor

项目摘要

PROJECT SUMMARY Abnormal activity of the cardiac ryanodine receptor (RyR2) leads to increased and untimely release of Ca2+ from the sarcoplasmic reticulum (SR), driving Ca2+-dependent arrhythmogenesis that can lead to sudden death in many cardiac disorders. Oxidative modification of RyR2 by reactive oxygen species (ROS) has long been established to enhance the sensitivity of the channels to Ca2+ within the SR (intraluminal Ca2+) in the failing heart. However, both the intracellular source of ROS, as well as the specific redox-sensitive residues of RyR2 which control intraluminal Ca2+ sensitivity, remain elusive. Our initial studies implicate the role of the SR oxidoreductase system in this control, whereby molecular chaperones and enzymes that facilitate protein folding also modulate activity of RyR2. We have identified intraluminal cysteines of RyR2 that elicit functional effects on the channel, as well as an oxidoreductase chaperone that associates with the channel in a redox-dependent manner. Moreover, we found upregulation of oxidoreductase enzyme in rodent models of cardiac disease, and observed RyR2 activity stabilization with pharmacological inhibition of this enzyme. We therefore hypothesize that dysregulation of the SR oxidoreductase system impairs luminal Ca2+ regulation of RyR2 via an ‘intraluminal SR redox sensor’ and promotes arrhythmogenesis. We will test our hypothesis by 1) defining the molecular components of the SR redox sensor that control luminal Ca2+ sensitivity of RyR2, and 2) determining the role of dysregulated SR redox homeostasis in Ca2+-dependent arrhythmogenesis. To address these aims, we will employ a multilevel experimental approach, investigating at the molecular, cellular, and whole heart level. We propose to use heterologous systems, biochemical approaches and human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) technology to identify the RyR2 redox sensor. We also propose to study disease- associated perturbations of the SR oxidoreductase system in rodent models of inherited and acquired Ca2+- dependent arrhythmia, utilizing novel genetic biosensors, as well as adenoviral (AV) and adeno-associated viral (AAV) gain- and loss- of function approaches. With renowned experts in cardiac EC coupling, protein biochemistry and hiPSC-CM technology, The Ohio State University offers an exceptional training environment for the mentored phase of the award to reach these goals. Furthermore, building on my strong background in molecular biology, I will collaborate with an expert in CRISPR-mediated gene editing of hiPSC-CMs to study these mechanisms in a relevant human model. The achievement of the proposed aims will uncover novel regulatory mechanisms of RyR2 regulation, with potential to be therapeutically exploited. This proposal therefore addresses a fruitful and unexplored research area, relevant to a spectrum of cardiovascular diseases, which will lay strong foundations for an independent research career in cardiovascular physiology.

项目摘要心脏ryanodine受体（RYR2）的异常活性导致Ca2+的增加和不合时宜地释放肌浆网（SR），驱动Ca2+依赖性心律失常发生，可能导致猝死许多心脏疾病。长期以建立的目的是增强在失败的心脏中SR（内腔内Ca2+）内通道对Ca2+的敏感性。但是，ROS的细胞内来源以及特定的氧化还原敏感的RyR2的保留率控制腔内Ca2+灵敏度，仍然难以捉摸。我们的最初研究暗示了SR氧化还原酶的作用在此控制中，系统中有促进蛋白质折叠的分子伴侣和酶也调节 RYR2的活性。我们已经确定了RYR2的腔内半胱氨酸，从而在通道上产生功能作用，以及以氧化还原依赖性方式与通道相关联的氧化链链酶。此外，我们发现心脏病啮齿动物模型中氧化氧化还原酶的上调，并观察到 RyR2活性稳定，该酶的药物抑制作用。因此，我们假设 SR氧化氧化还原酶系统的失调会损害RyR2的腔内Ca2+通过“腔内SR”的调节氧化还原传感器并促进心律失常。我们将通过1）定义分子来检验我们的假设 SR氧化还原传感器的组件，该传感器控制RYR2的腔Ca2+灵敏度，以及2）确定的作用 Ca2+依赖性心律失常发生中的SR氧化还原稳态失调。为了解决这些目标，我们将员工在分子，细胞和整个心脏水平上进行多级实验方法。我们使用异源系统，生化方法和人类诱导多能干细胞的提议心肌细胞（HIPSC-CM）技术以识别RYR2氧化还原传感器。我们还建议研究疾病 - SR氧化还原酶系统的相关扰动在遗传和获得的Ca2+ - 的啮齿动物模型中使用新型遗传生物传感器以及腺病毒（AV）和腺相关病毒（AAV）功能方法的增益和损失。与心脏EC耦合，蛋白质的著名专家生物化学和HIPSC-CM技术，俄亥俄州立大学提供了卓越的培训环境为了实现这些目标的修订阶段。此外，在我强大的背景下建立分子生物学，我将与HIPSC-CMS的CRISPR介导的基因编辑专家合作研究这些机制在相关的人类模型中。提议的目标的成就将揭示小说 RYR2调节的调节机制，有可能被热开发。因此，该建议解决与一系列心血管疾病有关的富有成果和意外的研究领域为心血管生理学的独立研究生涯奠定了坚实的基础。