Cardiac ryanodine receptor and oxidative stress

项目总结 心肌Ryanodine受体(RyR2)活性异常导致钙离子释放增加 肌浆网(SR)，驱动钙依赖性心律失常的发生，可导致猝死。 很多心脏疾病。长期以来，RyR2的RyR2被活性氧物种(ROS)氧化修饰 旨在增强心力衰竭患者心脏肌浆网内钙离子通道的敏感性。 然而，ROS的胞内来源以及RyR2对氧化还原敏感的特定残基 控制血管内钙敏感性，仍然难以捉摸。我们的初步研究涉及SR氧化还原酶的作用 在这个控制系统中，分子伴侣和促进蛋白质折叠的酶也调节 RyR2的活性。我们已经确定了RyR2的腔内半胱氨酸可以在通道上产生功能效应， 以及以氧化还原依赖的方式与通道相关联的氧化还原酶伴侣。 此外，我们还发现，在心脏病的啮齿动物模型中，氧化还原酶的表达上调，并观察到 RyR2的活性通过药物抑制该酶而稳定。因此，我们假设 肌质网氧化还原酶系统的失调通过内质网损伤RyR2的腔内钙调节 “氧化还原传感器”，促进心律失常的发生。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，控制RyR2的管腔钙敏感性，以及2)确定RyR2的作用 钙依赖型心律失常发生中的SR氧化还原动态平衡失调。为了达到这些目标，我们将 采用多层次实验方法，在分子、细胞和整个心脏水平进行研究。我们 建议使用异源系统、生化方法和人类诱导的多能干细胞 心肌细胞(HiPSC-CM)技术识别RyR2氧化还原传感器。我们还建议研究疾病-- 遗传性和获得性Ca~(2+)-啮齿动物模型中SR氧化还原酶系统的相关扰动 依赖性心律失常，利用新的遗传生物传感器以及腺病毒(AV)和腺相关病毒 (AAV)功能的得失方法。与心脏EC偶联、蛋白质等领域的知名专家 在生物化学和HiPSC-CM技术方面，俄亥俄州立大学提供了卓越的培训环境 为了达到这些目标，该奖项的指导阶段。此外，基于我在 分子生物学，我将与CRISPR介导的hiPSC-CMS基因编辑专家合作研究 这些机制在一个相关的人体模型中。拟议目标的实现将揭开小说的面纱 RyR2调节的调控机制，具有潜在的治疗开发潜力。因此，这项提议 解决与一系列心血管疾病相关的富有成效和未被探索的研究领域，这将是 为心血管生理学的独立研究事业奠定坚实的基础。

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.