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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.

项目概要心脏兰尼碱受体 (RyR2) 的异常活性导致 Ca2+ 增加和不及时释放肌浆网 (SR)，驱动 Ca2+ 依赖性心律失常，可导致猝死许多心脏疾病。活性氧（ROS）对 RyR2 的氧化修饰长期以来一直被认为是旨在增强衰竭心脏 SR（腔内 Ca2+）内通道对 Ca2+ 的敏感性。然而，ROS 的细胞内来源以及 RyR2 的特定氧化还原敏感残基控制腔内 Ca2+ 敏感性，仍然难以捉摸。我们的初步研究表明 SR 氧化还原酶的作用该控制系统中，促进蛋白质折叠的分子伴侣和酶也能调节 RyR2 的活性。我们已经鉴定出 RyR2 的管腔内半胱氨酸对通道产生功能性影响，以及以氧化还原依赖性方式与通道结合的氧化还原酶伴侣。此外，我们发现心脏病啮齿动物模型中氧化还原酶的上调，并观察到通过药理学抑制该酶来稳定 RyR2 活性。因此我们假设 SR 氧化还原酶系统的失调通过“腔内 SR”损害 RyR2 的腔内 Ca2+ 调节氧化还原传感器”并促进心律失常发生。我们将通过 1）定义分子来检验我们的假设控制 RyR2 的腔内 Ca2+ 灵敏度的 SR 氧化还原传感器的组件，以及 2) 确定 Ca2+ 依赖性心律失常发生中 SR 氧化还原稳态失调。为了实现这些目标，我们将采用多层次的实验方法，在分子、细胞和整个心脏水平上进行研究。我们提议使用异源系统、生化方法和人类诱导多能干细胞心肌细胞 (hiPSC-CM) 技术可识别 RyR2 氧化还原传感器。我们还建议研究疾病—— 遗传性和获得性 Ca2+- 啮齿动物模型中 SR 氧化还原酶系统的相关扰动利用新型遗传生物传感器以及腺病毒 (AV) 和腺相关病毒来治疗依赖性心律失常 (AAV) 功能获得和丧失方法。与心脏 EC 偶联、蛋白质生物化学和 hiPSC-CM 技术，俄亥俄州立大学提供卓越的培训环境在该奖项的指导阶段实现这些目标。此外，基于我强大的背景分子生物学，我将与一位从事 CRISPR 介导的 hiPSC-CM 基因编辑的专家合作进行研究这些机制在相关的人类模型中。实现所提出的目标将发现新的 RyR2 调节的调节机制，具有治疗利用的潜力。因此，本提案涉及与一系列心血管疾病相关的富有成果且尚未探索的研究领域，这将为心血管生理学的独立研究生涯奠定坚实的基础。