Mechanisms controlling Ca2+ dyshomeostasis in MH susceptible mice

MH 易感小鼠 Ca2 稳态失衡的控制机制

• 批准号: 9480595
• 负责人: Paul D Allen
• 金额: $ 23.76万
• 依托单位: UNIVERSITY OF LEEDS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-11-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9480595
• 关键词: Adult; Affect; Aftercare; Anesthetics; Calcium; Cations; Chronic; Dantrolene; Defect; Diglycerides; Dominant-Negative Mutation; Electric Stimulation; Event; Exposure to; Family; Fiber; Goals; Halothane; Homeostasis; Human; Individual; Ions; Knockout Mice; Lead; Location; Maintenance; Malignant hyperpyrexia due to anesthesia; Measures; Methods; Microelectrodes; Modeling; Molecular Conformation; Mus; Muscle; Muscle Fibers; Mutation; Myopathy; Neuromuscular Depolarizing Agents; Pathway interactions; Phenotype; Predisposition; Process; Protein Isoforms; Proteins; Pump; Research; Rest; Role; RyR1; Sarcoplasmic Reticulum; Signal Transduction; Skeletal Muscle; Specificity; Syndrome; Testing; analog; azumolene; extracellular; in vivo; knock-down; member; mouse model; overexpression; patch clamp; prevent; public health relevance; response; skeletal; small hairpin RNA

 DESCRIPTION (provided by applicant): The long-term goal of this research is to define the mechanisms responsible for the malignant hyperthermia syndrome. We have previously shown that a universal feature of RyR1 MH mutations is an increased resting myoplasmic free Ca2+ concentration ([Ca2+]i) and more recently that they are associated with an increased resting Na+ concentration ([Na+]i). We have also shown that sarcoplasmic reticulum (SR) leak is a factor in controlling myoplasmic Ca2+ concentration at rest working in concert with sarcolemmal channels, pumps and exchangers. Furthermore, because the amount of Ca2+ in internal stores is limited compared to the extracellular pool, after MH is triggered these same sarcolemmal channels, pumps and exchangers and perhaps others must be involved in the maintenance of the MH syndrome. Because of the observed increase in [Na+]i in MH muscles, TRPCs are the most likely candidates. Our immediate objectives are to use two mouse models that we have created, RyR1-R163C a mouse model of human MH, and CSQ1 null mice, which have an MH like phenotype, to study how mutations that alter intracellular Ca2+ homeostasis cause a fulminant MH response when exposed to volatile anesthetics. These models will allow us to test a unified general hypothesis applicable to any and all MH mutations regardless of the location of the mutation: MH is caused by conformational changes in RyR1 as a result of a mutation, or by conformational changes in RyR1 induced indirectly by a mutation in CaV1.1 or another protein closely associated with RyR1 (as demonstrated by an MH like phenotype in Casq1 null mice). A transformative concept to be investigated here is that a defect in signaling among Ca2+ release unit proteins leading to increased RyR1 leak is a common convergent pathway leading to all MH susceptibility. Hypothesis 1: MH susceptibility is the result of a conformational change in RyR1 caused either by a RyR1 mutation, or induced indirectly by mutations in other proteins closely associated with RyR1 which results in increased RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry. Specific Aim 1. To determine the filling state of the SR and rate of SR Ca2+ leak in MH muscle fibers. Specific Aim 2. To determine the role of TRPCs in causing abnormalities in sarcolemmal Na+ and Ca2+ entry RyR1-R163C and CSQ1 null muscles and then to determine if skeletal muscle specific over-expression of a dominant negative non-conducting TRPC6 channel can modify RyR1-R163C's MH phenotype. Hypothesis 2: In addition to blocking RyR1 SR Ca2+ release, dantrolene abrogates the MH phenotype by modulating RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry. Specific Aim 3. To determine the mechanisms by which dantrolene diminishes aberrant Ca2+ signaling. Successful completion of these specific Aims will provide a more comprehensive understanding of MH.

 描述（由申请人提供）：本研究的长期目标是确定恶性高热综合征的机制。我们以前已经表明RyR 1 MH突变的普遍特征是静息肌浆游离Ca 2+浓度（[Ca 2 +]i）增加，最近发现它们与静息Na+浓度（[Na+]i）增加相关。我们还表明，肌浆网（SR）泄漏是一个因素，在休息时控制肌浆钙离子浓度与肌膜通道，泵和交换。此外，由于与细胞外池相比，内部储存的Ca 2+的量是有限的，因此在MH被触发后，这些相同的肌膜通道、泵和交换器以及可能的其他必须参与MH综合征的维持。由于观察到MH肌肉中[Na+]i增加，TRPC是最可能的候选者。我们的近期目标是使用我们已经创建的两个小鼠模型，RyR 1-R163 C人类MH的小鼠模型，和CSQ 1 null小鼠，其具有MH样表型，以研究当暴露于挥发性麻醉剂时，改变细胞内Ca 2+稳态的突变如何引起暴发性MH反应。这些模型将使我们能够测试适用于任何和所有MH突变的统一的一般假设，无论突变的位置如何：MH是由突变导致的RyR 1的构象变化引起的，或者由CaV1.1或与RyR 1密切相关的另一种蛋白质的突变间接诱导的RyR 1的构象变化引起的（如Casq 1 null小鼠中的MH样表型所示）。这里要研究的一个变革性概念是，导致RyR 1泄漏增加的Ca 2+释放单位蛋白之间信号传导缺陷是导致所有MH易感性的常见会聚途径。假设1：MH易感性是由RyR 1突变引起的RyR 1构象变化的结果，或由与RyR 1密切相关的其他蛋白质中的突变间接诱导的，其导致RyR 1 SR Ca 2+泄漏和肌膜Na+和Ca 2+进入增加。具体目标1.测定MH肌纤维SR的充盈状态和SR Ca ~（2+）漏出率。具体目标2。确定TRPC在导致肌膜Na+和Ca 2+进入RyR 1-R163 C和CSQ 1缺失肌肉中的异常中的作用，然后确定骨骼肌特异性过表达显性负非传导TRPC 6通道是否可以改变RyR 1-R163 C的MH表型。假设二：除了阻断RyR 1 SR Ca 2+释放外，丹曲林还通过调节RyR 1 SR Ca 2+泄漏和肌膜Na+和Ca 2+进入来消除MH表型。具体目标3。确定丹曲林减少异常Ca 2+信号传导的机制。成功完成这些具体目标将提供对MH更全面的了解。