Mechanism of Skeletal Muscle Calcium Dysregulation in Myotonic Dystrophy

摘要： 调节兴奋性和兴奋-收缩偶联(ECC)的骨骼肌关键成分经历 发育过程中异构体表达的主要变化。这种围产期ECC重塑的过程是高度的 在脊椎动物的整个进化过程中都是保守的，主要是由转录后机制引起的，其中 CLC-1、CaV1.1、RyR1和SERCA1的特定外显子的选择性剪接发生。在强直性肌营养不良(DM)中， 由于MBNL剪接因子在细胞核中的隔离，这些剪接开关恢复到它们的胎儿设定点 RNA焦点。我们使用基因编辑技术重建了小鼠个体的DM剪接缺陷，并系统地分析了 通过饲养隔离和组合的小鼠的效果。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∆E29)，与观察到的DM相当 患者，导致严重的肌肉无力和呼吸障碍，并在小鼠到9周龄时是致命的。这 通过食品和药物管理局(FDA)批准的口服长期治疗来挽救效果 钙通道阻滞剂。在这里，我们建议进行研究，以定义机制并探索药物 针对肌强直和Cav1.1通道的治疗可以缓解糖尿病患者的肌肉无力。在目标1中，我们将 探讨强直性Cav1.1∆E29小鼠早期死亡的机制，包括 Cav1.1∆E29通道影响膜兴奋性和钙稳态，下游效应器包括 钙蛋白、转录和线粒体健康。此外，我们将确定强直性Cav1.1∆E29小鼠是否表现出 骨骼肌无力和呼吸功能改变。在目标2中，我们将通过以下方法治疗肌强直Cav1.1∆E29小鼠 通过析因设计口服FDA批准的针对钙通道或肌强直的药物(一， 其他，两者兼而有之)，看看哪种疗法在延长生命和改善肌肉方面最有效 呼吸功能。在目标3中，我们将把治疗转移到CUG重复扩增DM1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 侵入性技术测量肌肉和呼吸功能以确定纵向治疗的益处 学习。这项建议的最终目标是确定DM1治疗干预措施，可以迅速 转到了诊所。

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.