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（由适用提供）：这项研究的长期目标是定义负责恶性高温综合征的机制。我们先前已经表明，RYR1 MH突变的通用特征是增加静息肌胞质的CA2+浓度（[Ca2+] i），并且它们与静息Na+浓度的增加相关（[Na+] i）。我们还表明，肌浆网（SR）泄漏是控制肌质量Ca2+浓度的一个因素，该因素与肌膜通道，泵和交换器一起工作。此外，由于与细胞外池相比，内部商店中的Ca2+量受到限制，因此在MH触发了这些相同的肌毛孔通道，泵和交换器之后，以及其他可能涉及MH综合征的维持。由于MH肌肉中的[Na+] I的增加，TRPC是最可能的候选者。我们的直接目的是使用我们创建的两个鼠标模型，RyR1-R163C人类MH的鼠标模型和具有像表型这样的MH的CSQ1 NULL小鼠，用于研究如何改变细胞内Ca2+稳态的突变在暴露于挥发性动作的情况下会导致Funminant MH响应。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是由由于突变而导致的RyR1的会议变化引起的，或者是由RyRR1的会议变化或通过CAV1.1中的突变与RyRR1与RyRR1密切相关的突变诱导的（与Ryr1的突变）诱导的（与CASS相似的case case case case case case case case case case的case case case中的相​​关性很大，而case则是MH的现象（case）。这里要研究的一个变革性概念是，导致RYR1泄漏增加的Ca2+释放单元蛋白之间信号传导缺陷是导致所有MH敏感性的常见收敛途径。假设1：MH敏感性是由RYR1突变引起的RyR1构象变化的结果，或者是由于与RYR1密切相关的其他蛋白质突变间接诱导的，从而导致RyR1 SR CA2+泄漏和肌甲菌具有NA+和Ca2+进入。具体目标1。确定MH肌肉纤维中SR Ca2+泄漏速率的填充状态。具体目的2。确定TRPC在引起肌膜Na+和Ca2+输入RyR1-R163C和CSQ1无效肌肉中引起异常的作用，然后确定骨骼肌肉特异性的过表达是否对主要的非导不导致TRPC6通道可以修改RYR1-R163C的MH现象。假设2：除了阻止RYR1 SR Ca2+释放外，Dantrolene还通过调节RYR1 SR CA2+泄漏和肌符号Na+和Ca2+条目来促进MH表型。具体目的3。确定丹特洛烯会减少异常CA2+信号传导的机制。这些特定目标的成功完成将为MH提供更全面的了解。