Dissection and Rescue of Mechanical and Transcriptional Defects in Desmoplakin Cardiomyopathy

桥粒斑蛋白心肌病机械和转录缺陷的剖析和挽救

• 批准号: 10181155
• 负责人: ADAM S HELMS
• 金额: $ 47.83万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10181155
• 关键词: Arrhythmia; Biomechanics; Biomedical Engineering; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Characteristics; Clinical; Complex; Data; Defect; Dependovirus; Development; Dilated Cardiomyopathy; Disease; Dissection; Dose; Engineering; Exhibits; Failure; Fibrosis; Functional disorder; Genes; Genetic; Genetic Transcription; Geometry; Heart; Heart Injuries; Heart failure; Human; Impairment; In Vitro; Inflammatory Response; Injury; Intercalated disc; Intercellular Junctions; Lead; Left; Link; Mechanical Stress; Mechanics; Membrane; Messenger RNA; Methods; Morbidity - disease rate; Mus; Muscle; Mutation; Myocardial dysfunction; Myocardial tissue; Myocardium; Pathogenesis; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Plant Roots; Play; Pre-Clinical Model; Predisposition; Preventive; Proteins; Repression; Role; Stress; Stretching; Structural Protein; Structure; Subcategory; System; Testing; Tissue Model; Tissues; Transcription Coactivator; Transcriptional Activation; Translating; Variant; Ventricular; Ventricular Arrhythmia; Virus; Work; Workload; arrhythmogenic cardiomyopathy; base; clinical translation; coronary fibrosis; cytokine; desmoplakin; experimental study; gene replacement therapy; gene therapy; genetic variant; high risk; in vivo; in vivo evaluation; induced pluripotent stem cell; injury and repair; insight; loss of function; mRNA Expression; mortality; mouse model; novel; novel therapeutic intervention; personalized medicine; prevent; promoter; repaired; resilience; response; response to injury; restoration; stem cell model; stoichiometry; tool; treatment strategy

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.

摘要： 桥粒蛋白(Dsp)基因变异是扩张症较为常见的遗传原因之一。 心肌病。DSP变异体导致一种致心律失常的心肌病，可导致致命的 室性心律失常和进行性心力衰竭，并且没有可用的治疗方法。DSP编码了一个关键的 将力从收缩机械传递到细胞间连接的结构蛋白。之前的工作已经完成 证明了DSP心肌病几乎总是由截断引起 通过减少DSP mRNA丰度而导致的功能丧失。与DSP心肌病不同，这些截断 变异导致心脏纤维化在病程的早期发展，先于左心室的发展 收缩功能障碍。基于心脏损伤修复反应导致纤维化发生的理论基础，我们 截断突变导致DSP丰度降低的假说呈现心肌组织 由于无法正常处理心脏工作负荷，容易受到损伤和纤维化修复。我们的 主要目标是在体外和体内测试这一机制，同时在临床前建立证据 新的治疗策略模型，可用于患者预防数字信号处理器患者的心脏损伤。 我们的具体目标将检验以下具体假设：(目标1)生物力学应力诱导 心肌细胞损伤是DSP基因变异的结果，这种变异可以通过收缩来减少 作为上游预防方法的抑制；(目标2)DSP变体的功能损失后果可能是 通过转录挽救DSP的表达而完全被取消。严谨审视人际关系 在生物力学应力和损伤之间的DSP心肌病，我们将利用两个体外生物工程 利用来自数字信号处理器的诱导多能干细胞(IPSCs)的心肌组织平台 病人。此外，收缩拮抗剂将作为体内预防方法在小鼠模型中进行测试。 数字信号处理器心肌病。尽管看似自相矛盾，但这些实验将测试抑制性 使用重新调整用途的药物进行收缩调节实际上可以预防纤维化重塑的发展 通过在心肌细胞水平上降低生物力学应变来治疗DSP心肌病。同时，我们将使用 这些相同的体外和体内系统，以剖析数字信号处理器的mRNA减少和 生物力学损伤反应受损。CRISPR-Cas9工具，支持激活和抑制 内源mRNA的表达将针对DSP启动子。CRISPR-CAS9激活将在 腺相关病毒活体作为一种新的基因治疗方法，具有很高的临床翻译潜力。 综上所述，这项提议将对DSP损失的机制产生基本的见解 功能遗传变异导致心肌细胞损伤和纤维化，直接转化为临床观察 走向两种新的治疗方法。