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

项目摘要/摘要 强直性肌营养不良(DM)是成人起病的最常见原因，其次是 总体上是肌肉营养不良的最常见原因。至少50%的人受1型糖尿病影响 (DM1)有心脏损害，主要是传导异常和危及生命的心律失常。 心脏受累是DM1死亡的第二大原因，占疾病相关死亡的25% 死亡。DM1的致病原因被认为是扩展的CUG重复序列的毒性。 包含从扩展的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，特定的分子机制通过 CUGexp RNA在心脏组织中的哪种表达会导致传导异常、心律失常和 心功能不全是未知的。应用于DM1的治疗方法主要集中在骨骼 肌肉和心脏在疾病机制的细节方面提出了一系列不同的问题 和治疗方法。我们建立了四环素(Tet)诱导表达的小鼠模型。 CUGexp RNA在心肌细胞中特异表达。Tet诱导的转基因表达960中断的CUG 在包含外显子11-15的人类DMPK基因组片段中重复。CUG960的表达 RNA导致大多数心脏传导异常和心律失常的倾向，这些 很可能是DM1心脏猝死的罪魁祸首。老鼠也表现出强劲的剪接变化，因为 在DM1心脏组织中观察到。重要的是，分子和电生理异常是可逆的 阻断CUG960 RNA的表达。在本项目中，我们将使用鼠标模型来识别和测试 DM1心脏发病的分子基础假说和应用新的治疗方法。 第一个目标是将体内和体外电生理分析与 对细胞内结构、转录组和信号变化的详细分析，以确定 CUG960核糖核酸诱导的心脏表现。关键结果将在DM1组织样本中进行测试 验证。第二个目标是利用心脏特征的遗传挽救来测试假想的疾病。 机制，并期望多种机制在发病机制中起作用，以确定 不同机制的贡献。第三个目标是将使用停用的Cas9的方法应用于 靶向转录或转录后下调的CUG960 RNA。在这个项目完成后 我们预计将了解CUGexp RNA与 心功能障碍，并优化了一种逆转DM1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.