Dissection and Rescue of Mechanical and Transcriptional Defects in Desmoplakin Cardiomyopathy

摘要： 桥粒斑蛋白（DSP）基因的变异是扩张型子宫内膜异位症的一种常见遗传原因。 心肌病DSP变异导致一种致瘤性心肌病， 室性心律失常和进行性心力衰竭，并且没有可用的治疗。DSP对关键的 将收缩机制的力传递到细胞间连接的结构蛋白质。以前的工作有 证明DSP心肌病几乎总是由截短遗传变异引起的， 通过DSP mRNA丰度降低而丧失功能。与DSP心肌病不同，这些截短 变异导致心脏纤维化在疾病过程的早期发展，先于左心室纤维化的发展。 收缩功能障碍基于心脏损伤修复反应导致纤维化的基本原理，我们 假设由于截短突变导致DSP丰度降低使得心肌组织 由于不能正常处理心脏工作负荷，易受损伤和纤维化修复的影响。我们 主要目的是在体外和体内测试这种机制，同时在临床前研究中建立证据。 新的治疗策略的模型，可用于患者，以防止DSP患者的心脏损伤。 我们的具体目标将测试以下具体假设：（目的1）生物力学应力诱导 心肌细胞损伤是DSP遗传变异的结果，其可以通过收缩性心肌细胞损伤来减少。 抑制作为上游预防方法;（目的2）DSP变体的功能丧失后果可以是 通过DSP表达的转录拯救完全废除。严格审查关系 DSP心肌病中生物力学应力和损伤之间的关系，我们将利用两种体外生物工程 利用DSP衍生的诱导多能干细胞（iPSC）的心肌组织平台 患者此外，收缩拮抗剂将在小鼠模型中作为体内预防方法进行测试。 DSP心肌病。尽管看起来很矛盾，但这些实验将测试抑制剂是否 使用再利用药物的收缩调节实际上是对纤维化重塑发展的预防 在DSP心肌病通过减少心肌细胞水平的生物力学应变。同时，我们将使用 这些相同的体外和体内系统来剖析DSP mRNA减少和 生物力学损伤反应受损。CRISPR-Cas9工具能够激活和抑制 内源mRNA表达将靶向DSP启动子。CRISPR-Cas9激活将在 体内与腺相关病毒作为一种新的基因治疗方法，具有很高的潜力，为临床翻译。 总之，这一建议将产生基本的见解DSP损失的机制， 功能遗传变异导致心肌细胞损伤和纤维化，同时直接转化临床观察结果 两种新的治疗方法。

Abstract: Variants in the gene desmoplakin (DSP) are one of the more common genetic causes of dilated cardiomyopathy. DSP variants cause an arrhythmogenic form of cardiomyopathy that can lead to both lethal ventricular arrhythmias and progressive heart failure, and no treatments are available. DSP encodes a critical structural protein that transduces force from the contractile machinery to intercellular junctions. Prior work has demonstrated the DSP cardiomyopathy is almost always caused by truncating genetic variants that cause a loss of function through reduced DSP mRNA abundance. Distinct to DSP cardiomyopathy, these truncating variants cause cardiac fibrosis to develop early in the disease course, preceding development of left ventricular systolic dysfunction. Based on the rationale that fibrosis occurs due to the cardiac injury-repair response, we hypothesize that reduced DSP abundance due to truncating mutations renders heart muscle tissue susceptible to injury and fibrotic repair due to an incapacity to normally handle the cardiac workload. Our primary objective is to test this mechanism in vitro and in vivo while also building evidence in pre-clinical models for novel treatment strategies that can be used in patients to prevent cardiac injury in DSP patients. Our specific aims will test the following specific hypothesis: (Aim 1) biomechanical stress induced cardiomyocyte damage is a consequence of DSP genetic variants that can be reduced through contractile inhibition as an upstream preventive approach; (Aim 2) loss of function consequences of DSP variants can be completely abrogated through transcriptional rescue of DSP expression. To rigorously examine relationships between biomechanical stress and injury in DSP cardiomyopathy, we will utilize two in vitro bioengineered cardiac muscle tissue platforms that leverage induced pluripotent stem cells (iPSCs) derived from DSP patients. Further, contractile antagonists will be tested as an in vivo preventive approach in a mouse model of DSP cardiomyopathy. Although seemingly paradoxical, these experiments will test whether inhibitory contractile modulation using re-purposed drugs is actually preventive to the development of fibrotic remodeling in DSP cardiomyopathy by reducing biomechanical strain at the cardiomyocyte level. In parallel, we will use these same in vitro and in vivo systems to dissect the relationships between DSP mRNA reduction and impaired biomechanical injury response. CRISPR-Cas9 tools that enable activation and repression of endogenous mRNA expression will be targeted to the DSP promoter. CRISPR-Cas9 activation will be tested in vivo with adeno-associated virus as a novel gene therapy approach with high potential for clinical translation. Taken together, this proposal will yield fundamental insights into the mechanisms by which DSP loss of function genetic variants cause cardiomyocyte injury and fibrosis while directly translating clinical observations towards two novel therapeutic approaches.