The effects of MH mutations on muscle calcium homeostasis

Project 1的长期目标是明确RyR1和Cav1突变导致恶性高热综合征的机制。1以及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和人类肌管小鼠模型来研究RyR1和cav1.1突变如何改变细胞内Ca2+稳态。假设一：MH敲入小鼠模型人类MH易感性。目的1。对杂合子RyR1、R2435H和Cav1进行表型分析。1只R174W小鼠。”它们是否会因使用挥发性麻醉剂或热应激而引发MH综合征？易感性是否受年龄或性别的影响？A1.2测定体内[Ca2+]I和[Na+] I A1.3测定对KCI、4CmC和氟烷的敏感性A1.4 Western blot、免疫组织化学和EM病理学（Core D）。假设二：导致人类MH的突变增加了被动RyR1“泄漏”，并改变了肌上皮内Ca2+进入的动力学。A2.1。分析杂合子和纯合子MH肌肉在静止和暴露于触发剂后EC偶联和肌层Na+和Ca2+进入的异常。确定trpc1、3、6的作用及其在DAG和PKC中的控制。A2.2。探索azumolene （dantrolene）如何减少异常Ca2+信号。A2.3。验证从Core b提供的MH突变的人获得的小鼠肌管MH模型中观察到的异常。假设III: MHS突变的后遗症Ca2+稳态的有害变化可以通过遗传/药理学操作减少/预防，减少肌层Ca2+进入，减少RyR1泄漏，增加SR Ca2+负荷或清除由ROS产生的脂质过氧化物。A3.1：我们将在3-6个月大的雄性Het RyR1- t4826l MHS小鼠中研究上述范式，这些小鼠与过表达SERCA1（增强SR Ca2+填充），dnTPRCG（减少SOCE）或A3.2的小鼠杂交，并给予4-羟基- bde49（减少RyR1泄漏）或水杨胺（yKA清除剂）。假设IV：发现-新突变将为MH的发病机制提供新的见解。目标4新突变将在WT或null肌管中被发现时表达，我们将确定它们如何干扰[Ca2+]i， [Na+]i， RcaE和SR Ca2+负载互补实验项目2和3。

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.