Desmosomes in cardiomyocyte homeostasis and disease

桥粒在心肌细胞稳态和疾病中的作用

• 批准号: 10606894
• 负责人: William Tswenching Pu
• 金额: $ 81.65万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606894
• 关键词: ATAC-seq; Ablation; Acceleration; Adhesives; Adipose tissue; Adult; Arrhythmia; Biomedical Engineering; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Nucleus; Cells; Chemicals; Complex; DISC components; Data; Desmosomes; Development; Disease; Dissection; Elements; Experimental Models; Fluorescent in Situ Hybridization; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Goals; Heart Abnormalities; Heart failure; Homeostasis; Human; In Situ Hybridization; Intercalated disc; Knowledge; Lead; Life; Link; Metabolism; Methods; Modeling; Molecular; Mus; Muscle function; Mutation; Myocardial Contraction; Myocardial dysfunction; Myocardium; N-Cadherin; Pathogenesis; Pathway interactions; Patients; Periodicity; Phenotype; Plasma; Proteins; Proteomics; Regulatory Element; Relaxation; Resolution; Role; Sarcomeres; Signal Transduction; Testing; Ventricular Arrhythmia; WNT Signaling Pathway; arrhythmogenic cardiomyopathy; candidate identification; candidate selection; defined contribution; desmoplakin; follow-up; gain of function; genetic approach; heart rhythm; improved; in vivo; induced pluripotent stem cell; inhibitor; innovation; insight; loss of function; mosaic; multiple omics; mutant; novel; preservation; protein function; single molecule; single nucleus RNA-sequencing; transcriptome; transcriptomics

SUMMARY Intercalated disks (ICDs) connect the termini of adjacent cardiomyocytes (CMs) physically, electrically, and chemically. The structural role of ICDs to preserve CM integrity in the face of billions of cycles of forceful con- traction and relaxation is well appreciated; however, the function of ICDs as essential CM signaling hubs is only now emerging. Arrhythmogenic cardiomyopathy (ACM) provides a unique window into the function of ICDs and specifically desmosomes. ACM is a potentially lethal disorder characterized by high arrhythmia bur- den, loss of contractile myocardium, and replacement by fibro-fatty tissue. Mutations of desmosome genes (PKP2, DSG2, DSC2, DSP, JUP) occur in approximately half of ACM patients. Despite growing knowledge about ACM disease pathogenesis, the mechanistic links between desmosome mutations and arrhythmias, my- ocardial dysfunction, and fibrofatty replacement remain poorly understood. The overall goal of this proposal is to gain insights into the mechanisms by which desmosome mutations cause arrhythmia and myocardial dysfunction; Our overarching hypothesis is that desmosomes are inte- gral for maintaining normal cardiomyocyte homeostasis through both their structural and signaling activities. ACM mutations disrupt these activities to cause both loss of structural integrity and aberrant signaling. We will test these hypotheses through four parallel but complementary Specific Aims: (1) We will examine cell composition and gene regulation of human ACM myocardium, using concurrent single nucleus RNA-seq and ATAC-seq, and spatial transcriptomics (snMulti-seq) with massively parallel single molecule fluo- rescent in situ hybridization (MERFISH); (2) We will use mosaic, adult, cardiomyocyte specific inactivation of Dsp to probe the cell autonomous functions of desmosomes. This model will be studied using snMulti-seq and MERFISH, followed by interrogation of key predicted regulators using in vivo gain- and loss-of-function ap- proaches; (3) Using proximity proteomics of ICD component N-cadherin, we identified novel ICD components and ICD components that are altered by Dsp ablation. We will use in vivo gain- and loss-of-function ap- proaches to study the function of selected candidates identified by this screen; (4) Define the contributions of WNT and GSK3 signaling to ACM phenotypes in DSP mutant hiPSC-CMs. Using genetic approaches in bioen- gineered hiPSC-CMs, we will dissect the involvement of GSK3 and WNT signaling to ACM pathogenesis. Impact: This proposal will advance our understanding of the function of desmosomes and ICDs in CM homeostasis and the molecular pathogenesis of ACM. This knowledge will accelerate efforts to develop targeted ACM therapies.

概括 闰盘（ICD）以物理、电学和物理方式连接相邻心肌细胞（CM）的末端。 化学上。 ICD 在面对数十亿次强力连接时保持 CM 完整性的结构性作用 牵引力和放松效果深受赞赏；然而，ICD 作为重要的 CM 信号中枢的功能是 现在才刚刚出现。致心律失常性心肌病 (ACM) 为了解心律失常性心肌病 (ACM) 的功能提供了一个独特的窗口 ICD，特别是桥粒。 ACM 是一种潜在致命性疾病，其特征是高度心律失常 den，收缩性心肌丧失，并被纤维脂肪组织替代。桥粒基因突变 （PKP2、DSG2、DSC2、DSP、JUP）发生在大约一半的 ACM 患者中。尽管知识不断增长 关于 ACM 疾病发病机制、桥粒突变与心律失常之间的机制联系、我的- 心功能障碍和纤维脂肪替代仍然知之甚少。 该提案的总体目标是深入了解桥粒突变的机制 引起心律失常和心肌功能障碍；我们的总体假设是桥粒是相互连接的 gral 通过其结构和信号传导维持正常的心肌细胞稳态 活动。 ACM 突变破坏这些活性，导致结构完整性丧失和异常 发信号。我们将通过四个平行但互补的具体目标来检验这些假设：（1）我们将 使用并发单核检查人 ACM 心肌的细胞组成和基因调控 RNA-seq 和 ATAC-seq，以及具有大规模并行单分子荧光的空间转录组学 (snMulti-seq) 最近的原位杂交（MERFISH）； (2) 我们将使用马赛克、成体、心肌细胞特异性灭活 Dsp 探测桥粒的细胞自主功能。该模型将使用 snMulti-seq 进行研究 MERFISH，然后使用体内功能获得和丧失的应用程序询问关键的预测调节因子 引诱； (3) 利用 ICD 成分 N-钙粘蛋白的邻近蛋白质组学，我们鉴定了新的 ICD 成分 和通过 Dsp 消融改变的 ICD 组件。我们将使用体内功能获得和丧失的应用程序 研究该筛选所确定的选定候选者的功能的方法； (4) 定义贡献 WNT 和 GSK3 信号传导至 DSP 突变体 hiPSC-CM 中的 ACM 表型。在生物工程中使用遗传方法 生成 hiPSC-CM 后，我们将剖析 GSK3 和 WNT 信号传导与 ACM 发病机制的关系。 影响：该提案将增进我们对桥粒和 ICD 在 CM 中功能的理解 稳态和 ACM 的分子发病机制。这些知识将加速开发工作 靶向 ACM 疗法。