The effects of MH mutations on muscle calcium homeostasis

项目1的长期目标是确定由RyR1和Cav1.1突变引起的恶性高热综合征的机制，并利用人类其他基因连锁的新发现，使用新的和已证实的人类疾病小鼠模型和人类肌管来研究RyR1和Cav1.1突变如何改变细胞内钙稳态。假设一：MH“敲入”小鼠模型人类MH易感性。目的1.表型杂合型RyR1R2435H和Cav1.1R174W小鼠。A1.1它们是否会在对挥发性麻醉剂或热应激做出反应时引发MH综合征？易感性受年龄或性别的影响吗？A1.2测定体内[Ca2+]i和[Na+]i A1.3测定对KCI、4CmC和氟烷A1.4的敏感性。Western印迹、免疫组织化学和EM病理检查(核心D)。假设II：导致人类MH的突变增加被动RyR1“泄漏”，并改变肌膜d钙进入的动力学。A2.1.分析杂合型和纯合型MH肌肉在静息和暴露于触发剂后EC偶联和肌膜Na+和Ca~(2+)内流的异常。确定TRPC 1、3和6的作用(S)以及DAG和PKC对它们的控制。A2.2。探索氮杂环烯(丹曲烯)如何减少异常的钙信号。A2.3。验证由Core B提供MH突变的人类获得的肌管中的小鼠MH模型中的异常。假设III：作为MHS突变的后遗症，Ca~(2+)稳态的有害变化可以通过减少肌膜Ca~(2+)进入、减少RyR1泄漏、增加SR Ca~(2+)负荷或清除ROS产生的脂质过氧化的遗传/药物操作来减少/预防。答3.1：我们将在3-6个月大的雄性Het RyR1-T48261 MHS小鼠中研究上述范例，这些小鼠与过度表达SERCA1(增强的SR钙充盈)、dnTPRCG(减少的SOCE)或A3.2的小鼠杂交，并给予4-OH-BDE49(减少RyR1的泄漏)或水杨胺(yKA清除剂)。假设IV：发现新的突变将为MH的发病机制提供新的见解。目的4新的突变将在WT或空肌管中表达，我们将在项目2和3中确定它们是如何干扰[Ca+]i、[Na+]i、RcaE和SR钙负荷补充实验的。

The long-term goal of Project 1 is to define the mechanisms responsible for the malignant hyperthermia syndrome caused by mutations in RyR1 and Cav1 .1 as well as leveraging new discovery of other gene linkage in humans using new and proven mouse models of human disease and human myotubes to study how mutations of RyR1 and Cav1.1alter intracellular Ca2+ homeostasis. Hypothesis I: MH "knock-in" mice model Human MH susceptibility. Aim 1. To phenotype heterozygous and if viable homozygous RyR1 R2435H and Cav1 .1 R174W mice.' A1.1 Do they trigger the MH syndrome in response to volatile anesthetics or heat stress? Is susceptibility affected by age or gender? A1.2 Determine [ Ca2+]I and [Na+]i in vivo A1.3 Determine sensitivity to KCI, 4CmC and halothane A1.4 Western blot, immunohistochemistry and EM for pathology (Core D).Hypothesis II: Mutations responsible for human MH increase passive RyR1 "leak" and alter the dynamics of sarcolemmal d Ca2+ entry. A2.1. Analyze heterozygous and homozygous MH muscles for abnormalities in EC coupling and sarcolemmal Na+ and Ca2+ entry both at rest and after exposure to triggering agents. Determine the role(s) of TRPCs 1,3, and 6 and their control by DAG and PKC. A2.2. Explore how azumolene (dantrolene) diminishes aberrant Ca2+ signaling. A2.3. Validate abnormalities seen in murine MH models in myotubes obtained from humans with MH mutations supplied by Core B. Hypothesis III: Deleterious changes in Ca2+ homeostasis that are sequelae of MHS mutations can be reduced/prevented by genetic/pharmacological manipulations that decrease sarcolemmal Ca2+ entry, reduce RyR1 leak, increase SR Ca2+ load or scavenge lipid peroxides resulting from ROS production. A3.1: We will study the above paradigms in 3-6 month old male Het RyR1-T4826l MHS mice that have been crossed with mice over-expressing SERCA1 (enhanced SR Ca2+ filling), dnTPRCG (reduced SOCE), or A3.2 have been administered 4-OH-BDE49 (reduced RyR1 leak) or salicylamine (yKA scavenger). Hypothesis IV: Discovery - new mutations will provide new insights into the pathogenesis of MH. Aim 4 New mutations will be expressed in WT or null myotubes as they are discovered and we will determine how they disturb [ Ca2+]i, [Na+]i, RcaE and SR Ca2+ load complementing experiments in Projects 2 and 3.