Basis of Muscle Dysfunction in Malignant Hyperthermia & Central Core Disease

 描述(申请人提供)：I型Ryanodine受体(RyR1)突变与多种人类肌肉疾病有关，包括恶性高热(MH)、MH伴核心、中枢性核心疾病(CCD)、多微小核心疾病等。RyR1相关肌病是最常见的非营养不良肌肉疾病之一，与严重的临床残疾有关，通常包括轮椅依赖、严重脊柱侧弯、呼吸衰竭，并可导致儿童过早死亡。目前有 这些毁灭性的肌病没有治疗方法。RyR1的MH突变经常与热应激、和/或运动诱导的中暑和/或横纹肌溶解有关。肌萎缩侧索硬化症是一种先天性肌病，与骨骼肌纤维中代谢不活跃的中心核心有关，但核心的存在具有很高的变异性(尽管名称如此)，疾病的严重程度与核心无关。我们已经发现，一些ccd突变增加了肌浆网的钙渗漏，而另一些突变则减少了钙离子通过RyR1的渗透。这些发现提出了一个问题，即RyR1上相反的功能效应如何导致相关疾病。为了回答这个核心的悬而未决的问题，并帮助开发新的干预措施，我们创建了MH的小鼠模型，核心是(Y524S，YS)和电荷耦合器件(I4895T，IT)。YS杂合子小鼠对麻醉剂和热诱导的猝死很敏感，也表现出以线粒体损伤和无定形核心形成为特征的年龄相关性肌病。杂合子IT小鼠对热不敏感，但表现出运动能力降低，肌肉纤维横截面积减少，以及随年龄增长而加重的肌病。在这一新的应用中，我们建议定义功能上相反的RyR1突变产生这些不同表型的细胞和分子机制，然后利用这些发现开发新的、基于机制的治疗MH和CCD的干预措施。我们的工作假设是，YS突变通过增加RyR1钙离子泄漏、激活线粒体通透性转换孔(MPTP)和氧化应激来推动疾病，而IT突变增加了SR钙含量，从而导致ER应激、P53表达、线粒体损伤和细胞凋亡。为了验证这一假说，我们建议：1)确定胞浆、线粒体和肌浆网钙信号的改变以及MPTP激活在YS和IT肌病中的作用。2)定义RyR1翻译后修饰的作用。3)明确P53在YS小鼠IT肌病和热敏性增强中的作用。4)评估S107、NAC、4PBA和匹氟菊酯-µ作为MH和CD干预的潜力。目前还没有治疗RyR1相关肌病的疗法。这一多PI应用程序旨在通过仔细描绘疾病过程背后的特定细胞途径来满足这一需求，然后利用这些信息开发和测试几种具有不同作用机制的新型治疗干预措施。

 DESCRIPTION (provided by applicant): Mutations in the type I ryanodine receptor (RyR1) are associated with a variety of human muscle diseases including malignant hyperthermia (MH), MH with cores, central core disease (CCD), multi-minicore disease, and others. RyR1-related myopathies are among the most common group of non-dystrophic muscle diseases and are associated with significant clinical disabilities, often including wheelchair dependence, severe scoliosis, respiratory failure, and can result in premature death in childhood. Currently there are no therapies for these devastating myopathies. MH mutations in RyR1 are frequently associated with heat stress, and/or exercise-induced heat stroke and/or rhabdomyolysis. CCD is a congenital myopathy associated with metabolically inactive central cores in skeletal muscle fibers, but the presence of cores is highly variable (despite the name) and the severity of the disease does not correlate with cores. We have shown that some CCD mutations increase Ca2+ leak from the sarcoplasmic reticulum, while others decrease Ca2+ permeation through RyR1. These finding raise the question of how opposing functional effects on RyR1 can result in related diseases. To answer this central, unresolved question and aid in the development of new interventions, we created mouse models of MH with cores (Y524S, YS) and CCD (I4895T, IT). Heterozygous YS mice are susceptible to anesthetic and heat-induced sudden death and also exhibit an age-dependent myopathy characterized by mitochondrial damage and the formation of amorphous cores. Heterozygous IT mice are not heat sensitive, but display decreased exercise capacity, deceased muscle fiber cross-sectional area, and a myopathy that increases with age. In this renewal application, we propose to define the cellular and molecular mechanisms by which functionally opposing RyR1 mutations produce these distinct phenotypes and then use these findings to develop new, mechanism-based therapeutic interventions for MH and CCD. Our working hypothesis is that the YS mutation drives the disease via increased RyR1 Ca2+ leak, activation of the mitochondrial permeability transition pore (mPTP), and oxidative stress, while the IT mutation enhances SR Ca2+ content, which leads to increased ER stress, p53 expression, mitochondrial damage and apoptosis. To test this hypothesis, we propose to: 1) Define the role of altered cytosolic, mitochondrial, and SR lumenal Ca2+ signaling and mPTP activation in the YS and IT myopathies. 2) Define the roles of RyR1 post- translational modifications. 3) Define the role of p53 in the IT myopathy and the enhanced heat sensitivity of YS mice. 4) Assess the potential of S107, NAC, 4PBA, and pifithrin-µ as interventions for MH and CCD. There are currently no therapies to treat RyR1-related myopathies. This multi-PI application is designed to address this need by carefully delineating specific cellular pathways that underlie disease processes and then to use this information to develop and test several novel therapeutic interventions with distinct mechanisms of action.