Mechanisms controlling Ca2+ dyshomeostasis in MH susceptible mice

 描述(申请人提供)：这项研究的长期目标是确定恶性高热综合征的机制。我们以前已经证明，RyR1 MH突变的一个普遍特征是静息肌浆游离钙浓度([Ca+]i)增加，最近发现它们与静息Na+浓度([Na+]i)增加有关。我们还表明，肌浆网(SR)泄漏是控制静息状态下肌浆钙浓度的一个因素，与肌膜通道、泵和交换器协同工作。此外，由于与胞外池相比，体内钙离子的量是有限的，因此在MH被触发后，这些相同的肌膜通道、泵和交换器以及其他可能必须参与MH综合征的维持。由于观察到MH肌肉中[Na+]i的增加，TRPC是最有可能的候选者。我们的直接目标是使用我们创建的两个小鼠模型，RyR1-R163C，人类MH的小鼠模型，以及CSQ1缺失小鼠，它们具有MH样表型，以研究当暴露于挥发性麻醉剂时，改变细胞内钙稳态的突变如何导致暴发性MH反应。这些模型将使我们能够检验一个统一的一般假设，该假设适用于任何和所有MH突变，而不考虑突变的位置：MH是由突变引起的RyR1的构象变化引起的，或者由CaV1.1或与RyR1密切相关的另一种蛋白质的突变间接引起的RyR1的构象变化引起的(Casq1缺失小鼠的MH样表型证明了这一点)。这里要研究的一个变革性的概念是，导致RyR1泄漏增加的钙释放单位蛋白之间的信号缺陷是导致所有MH易感性的常见收敛途径。假设1：MH的易感性是由RyR1突变引起的RyR1构象改变的结果，或者是由与RyR1密切相关的其他蛋白质的突变间接引起的，这导致RyR1 SR钙离子泄漏增加，肌膜Na+和Ca2+内流增加。具体目的1.测定肌浆网的充盈状态和肌浆网钙离子在肌纤维中的漏出率。具体目的2.确定TRPC在肌膜Na+和Ca~(2+)进入RyR1-R163C和CSQ1缺陷肌异常中的作用，进而确定骨骼肌特异性过表达显性负性非传导TRPC6通道是否可以改变RyR1-R163C的MH表型。假设2：丹曲林除了阻断RyR1肌浆网钙离子释放外，还通过调节RyR1肌浆网钙离子泄漏和肌膜Na+和钙离子内流来消除MH表型。具体目的3.确定丹曲林抑制钙信号异常的机制。成功完成这些具体目标将使我们对MH有更全面的了解。

 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.