Sub-Project #4

恶性高热(MH)和中枢性核心病(CCD)是由骨骼肌突变引起的 兰尼定受体(RyR1)。尽管RyR1中的MH和Ccd突变改变了 肌膜二氢吡啶受体(DHPR)与肌浆钙反向释放通道 这些突变对多个亚细胞钙转运过程的综合影响是 人们对此知之甚少。该项目的长期目标是通过以下方式确定细胞/分子机制 RyR1的哪些MH和Ccd突变改变了肌膜、SR和肌膜之间的钙信号相互作用 线粒体(“钙信号三联体”)。具体地说，这个项目将检验这样一种假设：“MH/CD RyR1突变增强ECCE活性，使电压门控和配体门控敏化 肌浆网钙离子释放，并在EC偶联过程中改变线粒体钙离子摄取。 几种常见的RyR1 MH/CD突变对DHPR-RyR1双向偶联的影响 骨骼肌管和完全分化的肌纤维来源于CoreB基因敲除小鼠的MH。 目的#2将测试RyR1的MH/CD突变是否通过促进细胞内钙离子的耗竭而提高稳态静息钙离子的水平 SR、Ca~(2+)和增加肌膜ECCE通道的活性。实验将使用SR靶向、钙离子- 直接报告肌质网钙离子和全细胞膜片钳变化的敏感荧光“骆驼” 监测幼儿保育和教育活动变化的措施。目标3将确定线粒体的程度 RyR1的MH/Ccd突变改变了三联体靶向和局部SR-线粒体钙信号转导。 与Core D合作的实验将使用电子显微镜来评估线粒体的形态， 正常和MH/CD敲入小鼠FOB纤维的定位和三联体靶向。机能实验 将使用共聚焦显微镜、高速钙成像和线粒体靶向比率测量仪来 MH突变对线粒体钙离子的幅度、动力学和电压依赖性的影响 EC耦合过程中的变化。此外，与AIMS 1-3中描述的实验平行的实验将是 在对照组和MHS患者的肌肉样本中产生的人肌管中进行 在RyR1(例如，R163C和G2435R)中含有与用于制造敲入小鼠的相似的突变。为 这些实验，核心C收集的人类肌肉样本来自对照组和已知 基因分型和IVCT结果将由Core B用于繁殖人类成肌细胞，这是产生 项目4中的肌管培养。该项目将结合分子生物学、小鼠遗传学、 电生理学、共聚焦/电子显微镜和高速钙成像通过以下方法评估其机制 哪些MH/Ccd突变改变了钙信号三联体的功能。

Malignant hyperthermia (MH) and central core disease (CCD) arise from mutations in the skeletal muscle ryanodine receptor (RyR1). Although MH and CCD mutations in RyR1 alter mechanical coupling between sarcolemmal dihydropyridine receptors (DHPRs) and opposing Ca2+ release channels of the sarcoplasmic reticulum (SR), the integrated effects of these mutations on multiple subcellular Ca2+ transport processes are poorly understood. The long-term goal of this project is to determine the cellular/molecular mechanisms by which MH and CCD mutations in RyR1 alter Ca2+ signaling interactions between the sarcolemma, SR, and mitochondria (the "Ca2+ signaling triad"). Specifically, this project will test the hypothesis that "MH/CCD mutations in RyR1 enhance excitation coupled Ca2+ entry (ECCE) activity, sensitize voltage- & ligand-gated SR Ca2+ release, and alter mitochondrial Ca2+ uptake during EC coupling." Aim #1 will characterize effects of several common RyR1 MH/CCD mutations on bi-directional DHPR-RyR1 coupling in skeletal myotubes and fully differentiated muscle fibers derived from MH knock-in mice generated by Core B. Aim #2 will test if MH/CCD mutations in RyR1 elevate steady-state resting Ca2+ by promoting a depletion of SR Ca2+ and increasing the activity of sarcolemmal ECCE channels. Experiments will use SR-targeted, Ca2+- sensitive fluorescent "cameleons" to directly report changes in SR Ca2+ and whole-cell patch clamp measurements to monitor changes in ECCE activity. Aim #3 will determine the degree to which mitochondrial triad targeting and local SR-mitochondrial Ca2+ signaling is altered by MH/CCD mutations in RyR1. Experiments in collaboration with Core D will use electron microscopy to assess mitochondrial morphology, localization, and triad targeting in FOB fibers of normal and MH/CCD knock-in mice. Functional experiments will use confocal microscopy, high-speed Ca2+ imaging and mitochondrial-targeted ratiometric pericam to report effects of MH mutations on the magnitude, kinetics, and voltage-dependence of mitochondrial Ca2+ changes during EC coupling. Additionally, parallel experiments to those described in Aims 1-3 will be conducted in human myotubes generated from muscle samples of control individuals and MHS patients harboring analogous mutations in RyR1 (e.g. R163C and G2435R) to those used to make knock-in mice. For these experiments, human muscle samples collected by Core C from control individuals and patients of known genotypes and IVCT results will be used by Core B to propagate human myoblasts required for generating myotube cultures in Project 4. This project will combine the tools of molecular biology, mouse genetics, electrophysiology, confocal/electron microscopy, and high-speed Ca2+ imaging to asses the mechanisms by which MH/CCD mutations alter the function with the Ca2+ signaling triad.