Mapping the cell specific DNA damage-induced molecular and bioelectrical responses in the 3D cardiac unit

绘制 3D 心脏单元中细胞特异性 DNA 损伤诱导的分子和生物电反应

• 批准号: 10344373
• 负责人: Tzahi Cohen-Karni
• 金额: $ 61.99万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10344373
• 关键词: 3-Dimensional; Action Potentials; Address; Age; Anthracycline; Architecture; Biochemical; Biological; CRISPR/Cas technology; Calcium; Cancer Survivor; Cardiac; Cardiac Myocytes; Cardiac health; Cardiovascular Diseases; Cell Aging; Cell Communication; Cell Culture Techniques; Cells; Coculture Techniques; Crista ampullaris; Cultured Cells; DNA; DNA Damage; DNA Markers; DNA Repair; DNA Repair Endonuclease; DNA mapping; Data; Data Science; Deterioration; Dilated Cardiomyopathy; Disease; Dissection; ERCC1 gene; Electrophysiology (science); Event; Exhibits; Fibroblasts; Frequencies; Functional disorder; Genes; Genotoxic Stress; Heart Diseases; Heart failure; Human; Intervention; Investigation; Ionizing radiation; Life; Link; Lipids; Machine Learning; Maps; Measurement; Mediating; Membrane Potentials; Metabolic; Mitochondria; Molecular; Mus; Myocardial dysfunction; Myocardium; Myofibrils; Nature; Nuclear; Optics; Organoids; Oxidative Stress; Pathogenesis; Pathologic; Pathology; Patients; Pattern; Pharmaceutical Preparations; Pharmacology; Prevention strategy; Primary idiopathic dilated cardiomyopathy; Production; Public Health; Reactive Oxygen Species; Role; Series; Source; Specificity; Surface; System; TP53 gene; Target Populations; Testing; Therapeutic Intervention; Time; Tissues; Treatment Protocols; Work; autocrine; bioelectricity; biological adaptation to stress; cardiac tissue engineering; cardiovascular disorder risk; cell dimension; cell type; design; falls; genotoxicity; heart damage; heart electrical activity; heart function; imaging modality; in vivo; induced pluripotent stem cell; induced pluripotent stem cell technology; inhibitor; innovation; insight; live cell imaging; millisecond; mitochondrial dysfunction; mitochondrial membrane; nanofabrication; novel; paracrine; response; senescence; sensor; spatiotemporal; temporal measurement; tool; treatment strategy; two-dimensional

PROJECT SUMMARY This project will test the hypothesis that DNA damage in cardiomyocytes activates p53 leading to mitochondrial alterations and secretion of paracrine factors that drive heart failure. The premise for this has been established from our preliminary data and from the work of others. First, DNA damage and activated DNA damage response (DDR) have been observed in cardiovascular disease (CVD) in humans. Second, studies also show evidence that multiple cell types in the cardiac unit, including cardiomyocytes (CM) and cardiac fibroblasts (CF) display markers of DNA damage and cellular senescence in several disease pathologies. Third, we have recently identified that nuclear DNA damage drives dilated cardiomyopathy. Specifically, cardiomyocyte-depletion of the DNA repair endonuclease, ERCC1-XPF in mice, upregulates the DNA damage response gene, p53, and leads to irregular mitochondrial cristae, accumulation of lipids and increased oxidative stress. Additionally, there is an increase in several cardiac failure and senescence associated markers. However, the exact molecular underpinnings and cell-specificity of these DNA damage-induced changes is poorly understood. One barrier to addressing this question in vivo has been lack of appropriate tools, where DNA damage can be introduced in only one cell type (e.g., CM) and its effect on CF and cardiac function can be investigated. Additionally, 2D cell culture and co-culture systems fall short, as they cannot reproduce tissue dynamics present in a cardiac unit. Herein, we have developed several tools enable the study of cell-cell communication of 3D multicellular system. Specific Aim 1 will map the molecular, functional, and architectural changes upon loss of ERCC1 in CM. In this aim, we will test the mechanistic role of p53 and reactive oxygen species on a number of cellular and mitochondrial parameters, as well as cardiomyocyte electrophysiology. Specific Aim 2 will test whether stochastic, spontaneous DNA damage in the CM or CF drives cardiac electromechanical dysfunction in a cell- autonomous or cell non-autonomous manner through a paracrine effect on neighboring cells. Here, we will analyze the pathological secretome upon genotoxic stress, as well as test the role of eliminating senescent cells on cardiac health. This work is technically innovative as it uses a number of unique tools including concomitant optical and bioelectrical measurements in 3D cardiac organoids. These contributions will be significant because DNA damage is unavoidable and intimately linked to cardiac health and disease. Our team is uniquely qualified to perform this work, with expertise in DNA damage/ repair, cellular senescence, nanofabrication, human iPSC- derived cardiac tissue engineering, and data science. This analysis, we believe, will increase our fundamental understanding of the connection between DNA damage and heart disease and potentially pave the way for new treatment strategies.

项目概要 该项目将测试心肌细胞 DNA 损伤激活 p53 导致线粒体损伤的假设。 导致心力衰竭的旁分泌因子的改变和分泌。这样做的前提已经成立 根据我们的初步数据和其他人的工作。一、DNA损伤和激活的DNA损伤反应 (DDR) 已在人类心血管疾病 (CVD) 中观察到。其次，研究还表明 心脏单位中的多种细胞类型，包括心肌细胞 (CM) 和心脏成纤维细胞 (CF) 显示 多种疾病病理中 DNA 损伤和细胞衰老的标志物。第三，我们最近 发现核 DNA 损伤会导致扩张型心肌病。具体而言，心肌细胞耗竭 小鼠体内的 DNA 修复核酸内切酶 ERCC1-XPF 上调 DNA 损伤反应基因 p53 和导联 线粒体嵴不规则、脂质积累和氧化应激增加。此外，还有一个 一些心力衰竭和衰老相关标志物的增加。然而，确切的分子 这些 DNA 损伤引起的变化的基础和细胞特异性尚不清楚。一个障碍 在体内解决这个问题一直缺乏适当的工具，可以在体内引入DNA损伤 只能研究一种细胞类型（例如 CM）及其对 CF 和心脏功能的影响。此外，二维细胞 培养和共培养系统存在不足，因为它们无法再现心脏单元中存在的组织动力学。 在此，我们开发了多种工具来研究 3D 多细胞系统的细胞间通信。 具体目标 1 将绘制 CM 中 ERCC1 丢失后的分子、功能和结构变化。在这个 目的，我们将测试 p53 和活性氧对许多细胞和细胞的机械作用。 线粒体参数以及心肌细胞电生理学。具体目标 2 将测试是否 CM 或 CF 中随机、自发的 DNA 损伤会导致细胞中的心脏机电功能障碍。 通过对邻近细胞的旁分泌作用实现自主或细胞非自主方式。在这里，我们将 分析基因毒性应激下的病理分泌组，并测试消除衰老细胞的作用 关于心脏健康。这项工作在技术上具有创新性，因为它使用了许多独特的工具，包括伴随的 3D 心脏类器官中的光学和生物电测量。这些贡献将是巨大的，因为 DNA 损伤是不可避免的，并且与心脏健康和疾病密切相关。我们的团队拥有独特的资质 执行这项工作，具有 DNA 损伤/修复、细胞衰老、纳米加工、人类 iPSC 方面的专业知识 衍生出心脏组织工程和数据科学。我们相信，这一分析将增强我们的基本面 了解 DNA 损伤与心脏病之间的联系，并可能为新的研究铺平道路 治疗策略。