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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的：桥粒斑蛋白 (DSP) 基因的变异是扩张性扩张最常见的遗传原因之一。心肌病。 DSP 变异会导致心律失常形式的心肌病，从而导致致命的死亡室性心律失常和进行性心力衰竭，目前尚无治疗方法。 DSP 编码关键将力从收缩机制转导至细胞间连接的结构蛋白。之前的工作有证明 DSP 心肌病几乎总是由截短基因变异引起，这些变异会导致通过减少 DSP mRNA 丰度而丧失功能。与 DSP 心肌病不同，这些截短变异导致心脏纤维化在病程早期发生，先于左心室的发育收缩功能障碍。基于心脏损伤修复反应导致纤维化发生的基本原理，我们假设由于截短突变导致 DSP 丰度减少，导致心肌组织由于无法正常处理心脏负荷，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种机制，同时也在临床前建立证据可用于预防 DSP 患者心脏损伤的新型治疗策略模型。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，可以通过收缩来减少抑制作为上游预防方法；（目标 2）DSP 变体的功能丧失后果可能是通过 DSP 表达的转录拯救完全消除。严格审视关系为了研究 DSP 心肌病的生物力学应激和损伤之间的关系，我们将利用两种体外生物工程技术利用 DSP 衍生的诱导多能干细胞 (iPSC) 的心肌组织平台患者。此外，收缩拮抗剂将作为体内预防方法在小鼠模型中进行测试 DSP心肌病。尽管看似自相矛盾，但这些实验将测试抑制性是否使用重新利用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减少心肌细胞水平的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来剖析 DSP mRNA 减少和生物力学损伤反应受损。 CRISPR-Cas9 工具可激活和抑制内源 mRNA 表达将靶向 DSP 启动子。 CRISPR-Cas9 激活将在体内腺相关病毒作为一种新型基因治疗方法，具有很高的临床转化潜力。总而言之，该提案将对 DSP 丢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，同时直接转化临床观察结果两种新的治疗方法。