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.

总结 夹层盘（ICD）将相邻心肌细胞（CM）的末端物理地、电地和生物学地连接在一起。 化学上ICD在面对数十亿次强有力的控制周期时保持CM完整性的结构性作用， 牵引和放松是很好的赞赏;然而，ICD作为重要的CM信号中枢的功能， 现在才刚刚出现。致心律失常性心肌病（ACM）提供了一个独特的窗口， ICD，特别是桥粒。ACM是一种潜在的致命疾病，其特征是高心律失常， DEN、收缩性心肌的丧失和被纤维脂肪组织替代。桥粒基因突变 (PKP2、DSG 2、DSC 2、DSP、JUP）在大约一半的ACM患者中发生。尽管知识不断增长 关于ACM疾病的发病机制，桥粒突变和心律失常之间的机制联系，我- 心脏功能障碍和纤维脂肪替代仍然知之甚少。 这项提案的总体目标是深入了解桥粒突变的机制， 导致心律失常和心肌功能障碍;我们的总体假设是桥粒是介导的， GRAL通过其结构和信号传导维持正常心肌细胞稳态 活动ACM突变破坏了这些活性，导致结构完整性的丧失和异常的蛋白质表达。 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 用并行单核细胞技术检测人ACM心肌的细胞组成和基因调控 RNA-seq和ATAC-seq，以及空间转录组学（snMulti-seq）与大规模平行单分子荧光检测， （2）我们将使用嵌合体、成体、心肌细胞特异性失活的 利用Dsp探讨桥粒的细胞自主功能。该模型将使用snMulti-seq进行研究， MERFISH，然后使用体内获得和丧失功能的ap-1询问关键的预测调节因子。 方法;（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 以及通过Dsp消融改变的ICD组件。我们将使用体内功能获得和丧失的ap- 研究通过该屏幕识别的所选候选人的功能;（4）定义 DSP突变体hiPSC-CM中WNT和GSK 3信号传导至ACM表型。在生物工程中使用遗传方法- 我们将详细分析GSK 3和WNT信号通路在ACM发病机制中的作用。 影响：该提案将促进我们对CM中桥粒和ICD功能的理解 动态平衡和ACM的分子发病机制。这些知识将加速开发 针对性ACM治疗。