Cardiac ryanodine receptor and oxidative stress

项目摘要 心脏ryanodine受体（RyR 2）的异常活性导致Ca 2+从心肌细胞中释放增加和过早释放。 肌浆网（SR），驱动Ca 2+依赖的促细胞生成，可导致猝死， 许多心脏疾病。RyR 2通过活性氧（ROS）的氧化修饰长期以来一直是研究的热点。 建立了增强通道对衰竭心脏中SR（管腔内Ca 2+）内Ca 2+的敏感性。 然而，细胞内ROS的来源，以及RyR 2的特异性氧化还原敏感残基， 控制管腔内Ca 2+敏感性，仍然难以捉摸。我们的初步研究暗示了SR氧化还原酶的作用 在这种控制中，分子伴侣和促进蛋白质折叠的酶也调节 RyR 2的活性。我们已经鉴定了RyR 2的管腔内半胱氨酸，其引起对通道的功能作用， 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，我们发现在啮齿动物心脏病模型中氧化还原酶上调，并观察到 RyR 2活性稳定与该酶的药理学抑制。因此，我们假设， SR氧化还原酶系统的失调通过“管腔内SR”损害RyR 2的管腔Ca 2+调节 氧化还原传感器'，并促进促细胞生成。我们将通过以下方式来检验我们的假设：1）定义分子 控制RyR 2的管腔Ca 2+敏感性的SR氧化还原传感器的组分，以及2）确定 钙离子依赖性肿瘤发生中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 建议使用异源系统、生物化学方法和人诱导多能干细胞 心肌细胞（hiPSC-CM）技术来鉴定RyR 2氧化还原传感器。我们也建议研究疾病- 遗传性和获得性Ca 2 +-钙缺乏的啮齿动物模型中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)功能获得和丧失方法。与心脏EC偶联、蛋白质 俄亥俄州州立大学提供了一个特殊的培训环境， 为实现这些目标，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-CM基因编辑专家合作研究 这些机制在相关的人体模型中。所提出的目标的实现将揭示新的 RyR 2调节的调节机制，具有治疗开发的潜力。因此，这项建议 解决了一个富有成效的和未开发的研究领域，有关的一系列心血管疾病，这将 为心血管生理学的独立研究生涯奠定坚实的基础。

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.