Pathogenic mechanisms and therapeutics for the cardiac manifestations of myotonic dystrophy type 1

项目概要/摘要 强直性肌营养不良（DM）是成人发病的肌营养不良的最常见原因， 最常见的原因是肌肉萎缩症。至少50%的人患有1型DM (DM1)心脏受累，主要是传导异常和危及生命的心律失常。 心脏受累是DM患者死亡的第二大原因1，占疾病相关死亡的25%。 死亡DM 1的致病原因已被确定为扩增的CUG重复序列的毒性， 含有从扩增的DMPK等位基因表达的（CUGexp）RNA。CUGexp RNA破坏RNA 加工、信号通路和microRNA调节。然而，特定的分子机制， 其中CUGexp RNA在心脏组织中的表达引起传导异常、心律失常和 心室功能障碍未知。应用于DM 1的治疗方法集中在骨骼肌 肌肉和心脏在疾病机制的细节方面提出了一系列单独的问题， 和治疗方法。我们建立了四环素（泰特）诱导表达的小鼠模型， 心肌细胞中特异性CUGexp RNA。tet诱导型转基因表达960中断的CUG 在含有外显子11-15的人DMPK基因组区段的背景下的重复。CUG 960的表达 RNA产生大多数心脏传导异常和心律失常的倾向， 可能是导致DM 1心脏性猝死的原因。小鼠还显示出强烈的剪接变化， 在DM 1心脏组织中观察到。重要的是分子和电生理异常是可逆的 在关闭CUG 960 RNA的表达后。在这个项目中，我们将使用小鼠模型来识别和测试 DM 1心脏发病机制的分子基础的假设，并应用新的治疗方法。 第一个目的的目标是使用体内和离体电生理学分析结合 详细分析细胞内结构、转录组和信号变化，以确定 CUG 960 RNA诱导的心脏表现。将在DM 1组织样本中检测关键结果， 验证。第二个目标是利用心脏特征的基因拯救来测试假设的疾病 机制，并期望多种机制有助于发病机制，以确定 不同机制的贡献。第三个目标是应用使用失活的Cas9的方法， 靶向CUG 960 RNA进行转录或转录后下调。本项目建成后 我们期望了解CUGexp RNA与 心功能不全，并优化了逆转DM 1心脏特征的治疗方法。

Project Summary / Abstract Myotonic dystrophy (DM) is the most common cause of adult onset muscular dystrophy and the second most common cause of muscular dystrophy overall. At least 50% of individuals affected by DM type 1 (DM1) have cardiac involvement, primarily conduction abnormalities and life threatening arrhythmias. Cardiac involvement is the second leading cause of mortality in DM1 responsible for 25% of disease-related deaths. The pathogenic cause of DM1 is well established to be toxicity of the expanded CUG repeat- containing (CUGexp) RNA expressed from the expanded DMPK allele. The CUGexp RNA disrupts RNA processing, signaling pathways and microRNA regulation. However the specific molecular mechanisms by which expression of CUGexp RNA in heart tissue causes conduction abnormalities, arrhythmias and ventricular dysfunction are unknown. Therapeutic approaches applied to DM1 have focused on skeletal muscle and heart presents a separate set of issues with regard to both the details of disease mechanisms and therapeutic approaches. We developed a mouse model for tetracycline (tet)-inducible expression of CUGexp RNA specifically in cardiomyocytes. The tet-inducible transgene expresses 960 interrupted CUG repeats in the context of a human DMPK genomic segment containing exons 11-15. Expression of CUG960 RNA produces most of the cardiac conduction abnormalities and propensity for the arrhythmias that are likely to be responsible for cardiac sudden death in DM1. The mice also show robust splicing changes as observed in DM1 heart tissue. Importantly molecular and electrophysiological abnormalities are reversible upon shutting off expression of CUG960 RNA. In this project we will use the mouse model to identify and test hypotheses for the molecular basis for heart pathogenesis in DM1 and apply novel therapeutic approaches. The goal of the first aim is to use in vivo and ex vivo electrophysiological analyses in combination with detailed analyses of intracellular structure, transcriptome and signaling changes to identify the basis for the cardiac manifestations induced by CUG960 RNA. The key results will be tested in DM1 tissue samples for validation. The second aim is to use genetic rescue of the cardiac features to test hypothesized disease mechanisms, and with the expectation that multiple mechanisms contribute to pathogenesis, to determine the contributions of different mechanisms. The third aim is to apply approaches using deactivated Cas9 to target CUG960 RNA for transcriptional or post-transcriptional downregulation. Upon completion of this project we anticipate having an understanding of the molecular and physiological pathways linking CUGexp RNA to cardiac dysfunction and to have optimized a therapeutic approach to reverse DM1 cardiac features.