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.

摘要 间盘(ICD)将相邻心肌细胞(CM)的末端物理地、电地和 化学上的。面对数十亿次的强力循环，ICD在保持CM完整性方面的结构作用-- 牵引和放松是很受欢迎的；然而，ICD作为基本的CM信号中枢的功能是 只是现在才出现。致心律失常的心肌病(ACM)提供了了解心脏功能的独特窗口。 ICD，特别是桥粒。ACM是一种潜在的致命性疾病，其特征是高度心律失常。 DEN，收缩心肌丧失，取而代之的是纤维脂肪组织。桥粒基因的突变 (PKP2、DSG2、DSC2、DSP、JUP)发生在大约一半的ACM患者中。尽管知识在不断增长 关于ACM病的发病机制，桥粒突变与心律失常之间的机制联系，我的. 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；我们的主要假设是桥粒是整合的。 GRAL通过结构和信号途径维持正常心肌细胞动态平衡 活动。ACM突变破坏了这些活性，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 应用并发单核技术检测人急性心肌梗死心肌细胞组成及基因调控 RNA-seq和atac-seq，以及具有大规模平行的单分子序列的空间转录组学(sn多序列)。 月形原位杂交(MerFish)；(2)我们将使用嵌合体、成体、心肌细胞特异性灭活 利用数字信号处理器来探索桥粒的细胞自主功能。该模型将使用SNMULT-SEQ和 MerFish，然后使用体内功能增减AP询问关键预测的调节因子- (3)利用ICD组分N-钙粘附素的邻近蛋白质组学，我们鉴定了新的ICD组分 以及通过DSP消融而改变的ICD组件。我们将使用体内获得和失去功能的AP- 研究通过此筛选确定的选定候选人的功能的方法；(4)定义 在DSP突变体hiPSC-CMS中，WNT和GSK3信号转导ACM表型。在生物基因组中使用遗传方法- 结合HIPSC-CMS，我们将剖析GSK3和WNT信号在ACM发病机制中的作用。 影响：这项建议将促进我们对桥粒和ICD在CM中的功能的理解 动态平衡与ACM的分子发病机制。这一知识将加速努力开发 针对性的ACM疗法。