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

项目总结 该项目将检验心肌细胞DNA损伤激活P53导致线粒体的假说 导致心力衰竭的旁分泌因子的变化和分泌。这样做的前提已经确立。 从我们的初步数据和其他人的工作中。第一，DNA损伤和激活的DNA损伤反应 (DDR)在人类的心血管疾病(CVD)中被观察到。其次，研究还表明，有证据表明 心脏单位的多种细胞类型，包括心肌细胞(CM)和心脏成纤维细胞(CF) 几种疾病病理中DNA损伤和细胞衰老的标记物。第三，我们最近 确定了核DNA损伤会导致扩张型心肌病。具体地说，心肌细胞-耗尽 小鼠的DNA修复内切酶ERCC1-XPF上调DNA损伤反应基因P53和Lead 导致线粒体脊不规则、脂质堆积和氧化应激增加。此外，还有一个 几种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损伤引起的改变的基础和细胞特异性还知之甚少。一个障碍就是 在活体内解决这个问题一直缺乏适当的工具，在哪里可以引入DNA损伤 只能研究一种细胞类型(例如，CM)及其对CF和心功能的影响。此外，2D单元 培养和共培养系统是不够的，因为它们不能复制心脏单位中存在的组织动力学。 在此，我们开发了几种工具来研究三维多细胞系统的细胞-细胞通讯。 特定目标1将绘制在CM中失去ERCC1后的分子、功能和结构变化。在这 目的，我们将测试P53和活性氧物种对一些细胞和 线粒体参数，以及心肌细胞电生理学。《特定目标2》将测试 CM或CF中随机的、自发的DNA损伤导致细胞中的心脏机电功能障碍- 自主或细胞非自主的方式，通过旁分泌作用于相邻细胞。在这里，我们将 分析基因毒性应激下的病理分泌体，以及检测其对消除衰老细胞的作用 心脏健康的问题。这项工作在技术上是创新的，因为它使用了许多独特的工具，包括伴随 三维心脏器官的光学和生物电学测量。这些贡献将是重大的，因为 DNA损伤是不可避免的，与心脏健康和疾病密切相关。我们队是独一无二的合格球队 为了进行这项工作，拥有DNA损伤/修复、细胞衰老、纳米制造、人类IPSC- 衍生心脏组织工程和数据科学。我们相信，这一分析将增加我们的基本面 了解DNA损伤和心脏病之间的联系，并可能为新的 治疗策略。

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.