Mechanism of Skeletal Muscle Calcium Dysregulation in Myotonic Dystrophy

摘要： 调节兴奋性和兴奋-收缩偶联（ECC）的骨骼肌的关键成分经历了 发育过程中同种型表达的主要变化。围产期ECC重塑的这一过程是高度 在整个脊椎动物进化过程中是保守的，主要来自转录后机制， 发生C1 C-1、CaV1.1、RyR 1和SERCA 1的特定外显子的选择性剪接。在强直性肌营养不良（DM）中， 这些剪接开关由于核内MBNL剪接因子的隔离而恢复到它们的胎儿设定点， RNA病灶。我们使用基因编辑在小鼠中重建了单个DM剪接缺陷，并系统地分析了 通过育种，在单独和组合中对小鼠进行效果测试。我们的初步研究表明， ClC-1功能结合CaV1.1外显子29排除（Cav1.1外显子29），与DM中观察到的结果相当 患者，导致严重的肌肉无力和呼吸缺陷，并且在约9周龄的小鼠中是致命的。 经美国食品药品监督管理局（FDA）批准， 钙通道阻滞剂。在这里，我们提出研究，以确定机制，并探讨药物的可能性， 靶向肌强直和Cav1.1通道的治疗可以减轻DM中的肌无力。在目标1中， 研究肌强直Cav1.1小鼠早期死亡的机制，包括研究如何 Cav1.1通道影响膜兴奋性和Ca 2+稳态，下游效应子包括 钙蛋白酶、转录和线粒体健康。此外，我们将确定肌强直性Cav1.1小鼠是否表现出 骨骼肌无力和呼吸功能改变。在目标2中，我们将通过以下方式治疗肌强直性Cav1.1小鼠29： 通过析因设计口服FDA批准的靶向钙通道或肌强直的药物（一种， 其他，两者或两者都没有），看看哪种治疗方法在延长寿命和改善肌肉方面最有效， 呼吸功能在目标3中，我们将治疗转移到CUG重复扩增DM 1小鼠模型中， 表现出严重的肌肉无力、肌病特征和寿命缩短。我们将使用一系列非- 测量肌肉和呼吸功能的侵入性技术，以确定纵向治疗获益 问题研究该提案的最终目标是确定可以快速治疗的DM 1治疗干预措施。 转移到诊所。

ABSTRACT: Key components of skeletal muscle that regulate excitability and excitation-contraction coupling (ECC) undergo major shifts of isoform expression during development. This process of perinatal ECC remodeling is highly conserved throughout vertebrate evolution and results mainly from post-transcriptional mechanisms in which alternative splicing of specific exons for ClC-1, CaV1.1, RyR1 and SERCA1 occurs. In myotonic dystrophy (DM), these splicing switches revert to their fetal set points due to sequestration of MBNL splicing factors in nuclear RNA foci. We used gene editing to recreate individual DM splicing defects in mice and systematically analyzed mice for effects in isolation and in combination through breeding. Our preliminary studies indicate that loss of ClC-1 function combined with CaV1.1 exon 29 exclusion (Cav1.1∆e29), comparable to that observed in DM patients, results in severe muscle weakness and respiratory deficits and is lethal in mice by age ~9 wks. This effect is rescued by long-term treatment by oral feeding with a Food & Drug Administration (FDA) approved calcium channel blocker. Here we propose studies to define mechanisms and explore the possibility that drug treatments that target myotonia and Cav1.1 channels can mitigate muscle weakness in DM. In Aim 1 we will investigate the mechanism for the early demise of myotonic Cav1.1∆e29 mice, including the study of how Cav1.1∆e29 channels impact membrane excitability and Ca2+ homeostasis, and downstream effectors that include calpain, transcription and mitochondrial health. Further, we will determine if myotonic Cav1.1∆e29 mice exhibit skeletal muscle weakness and altered respiratory function. In Aim 2 we will treat myotonic Cav1.1∆e29 mice by oral feeding of FDA approved drugs that target the calcium channel or myotonia by factorial design (one, the other, both or neither) to see which treatment is most effective at extending life and improving muscle and respiratory function. In Aim 3 we will move the treatment into a CUG repeat expansion DM1 mouse model that exhibits severe muscle weakness, myopathic features and shortened lifespan. We will use a series of non- invasive techniques to measure muscle and respiratory function to determine treatment benefit in longitudinal studies. The ultimate goal of this proposal is to identify DM1 therapeutic interventions that can be rapidly transitioned to the clinic.