Basis of Muscle Dysfunction in Malignant Hyperthermia and Central Core Disease

描述(申请人提供)：骨骼肌兰尼定受体(RYR1)调节肌浆网(SR)储存的钙释放，并在人类中枢核心疾病(CCD)和药物遗传综合征恶性高热(MH)中发生突变。虽然RyR1的MH和Ccd突变被认为改变了SR钙释放通道功能和肌肉兴奋-收缩(EC)耦合，但这些影响导致这些疾病的肌肉特征的表型变化的机制尚不清楚。该项目将使用转基因MH和CCD敲入小鼠来详细分析RYR1疾病突变改变体内肌肉功能的基本机制。该项目的长期目标是确定MH/CD突变改变完整肌肉中钙稳态和兴奋-收缩(EC)偶联的细胞/分子机制和原理。我们的总体假设是：MH/CCD区1和2上的MH和Ccd突变通过改变RYR1内以及RYR1与T小管膜上的电压依赖性钙通道之间的重要分子内和分子间相互作用来促进电压和钙门控的SR的释放，而RyR1区3孔区的Ccd选择性突变破坏了钙通过通道的渗透。为了验证这一假设，我们建议：1.建立三个新的MH/CD小鼠系，分析突变对肌肉收缩特性和RYR1单通道特性的影响，对咖啡因和温度的反应；2.定位MH/CD区域1和2在三维结构中的位置，并分析突变对RYR1结构的影响，S.分析RyR1的MH/CD突变对肌管和成年肌纤维的钙稳态和双向DHPR-RyR1偶联的影响以及4评估MH/CD突变对原位释放通道对RyR1配体激活和局部结合钙增加的敏感性的影响。这项申请将两位合作者聚集在一起，他们都高度致力于阐明MH和CCD病理生理学的基本机制，但他们以截然不同但互补的方式处理问题。这一联合将导致一个独特的跨学科项目，该项目将确定MH/CCD病突变改变RyR1结构和调节、亚细胞钙转运/处理机制、肌肉EC偶联和SR钙储存/隔离的机制。这一结果将对相关临床领域的其他钙调节失调疾病具有广泛的指导意义，包括心律失常、心力衰竭、高血压以及包括中风和神经变性在内的广泛的中枢神经系统疾病。

DESCRIPTION (provided by applicant): The skeletal muscle ryanodine receptor (RYR1) regulates Ca2+ release from the sarcoplasmic reticulum (SR) stores and is mutated in human central core disease (CCD) and in the pharmacogenetic syndrome, malignant hyperthermia (MH). Although MH and CCD mutations in RyR1 are thought to alter SR Ca2+ release channel function and muscle excitation-contraction (EC) coupling, the mechanisms by which these effects result in phenotypic changes in muscle characteristic of these disorders are unknown. This project will use transgenic MH and CCD knock-in mice to provide detailed analyses of the fundamental mechanisms by which RYR1 disease mutations alter in vivo muscle function. The long-term goal of this project is to define the cellular/molecular mechanisms and principles by which MH/CCD mutations alter Ca2+ homeostasis and excitation-contraction (EC) coupling in intact muscle. Our overall hypothesis is: MH and CCD mutations in MH/CCD regions 1 and 2 enhance voltage- and Ca2+-gated SR release by altering crucial intra and intermolecular interactions within RYR1 and between RYR1 and the voltage dependent Ca2+ channel in the t-tubule membrane, while CCD-selective mutations in the region 3 pore region of RyR1 disrupt Ca2+ permeation through the channel. To test this hypothesis, we propose to: 1. Create three new MH/CCD mouse lines and analyze the effects of the mutations on muscle contractile properties and RYR1 single channel properties in response to caffeine and temperature, 2. Map the location of MH/CCD regions 1 and 2 in the 3D structure and analyze the effects of the mutations on RYR1 structure, S.Analyze the effects of MH/CCD mutations in RyR1 on Ca2+ homeostasis and bi-directional DHPR-RyR1 coupling in myotubes and adult muscle fibers obtained from MH/CCD knock-in mice, and 4 Evaluate the effects of MH/CCD mutations on in situ release channel sensitivity to activation by RyR1 ligands and local increases in junctional Ca2+. This application brings together two collaborators, both highly committed to elucidating fundamental mechanisms of MH and CCD pathophysiology, but who approach the problems in very different, but complimentary ways. This union will result in a uniquely interdisciplinary project that will determine the mechanisms by which MH/CCD disease mutations alter RyR1 structure and regulation, subcellular Ca2+ transport/handling mechanisms, muscle EC coupling, and SR Ca2+ storage/sequestration. Results will have broad implications for other disorders of Ca2+ dysregulation in related clinical arenas including cardiac arrhythmias, heart failure, hypertension, and wide of central nervous system disorders including stroke and neurodegeneration.