Mechanisms of myocardial regeneration mediated by Nkx2.5 in zebrafish

项目总结 由于在受损心肌中启动再生反应的能力有限，心功能障碍仍然存在。 这是导致发病率和死亡率的主要原因。冠状动脉疾病导致心肌梗死，从而导致 纤维性瘢痕、不良重塑和心力衰竭。与成年小鼠和人类相比，新生小鼠 成年斑马鱼最终保留了经历心肌细胞去分化和增殖的能力 刺激损伤后的心脏再生。破译共享的基本转录网络 发育和再生将促进我们唤醒内源性修复机制的能力。在斑马鱼身上， 心脏发生所必需的胚胎心脏转录因子，如NKX2.5，在下列情况下被激活 损伤以刺激心肌细胞的产生。我们的初步研究表明，纤维蛋白5，一种细胞外基质 (ECM)NKX2.5下游的糖蛋白，在受伤的心脏中上调。此外，我们还展示了 纤毛蛋白丧失功能的鱼类表现出再生能力受损，并在迁徙和组织中发挥关键作用 弹性。在本提案中，我们将测试NKX2.5激活是产生ECM所必需的假设 环境由主要的促再生蛋白组成，如纤维蛋白5，可被利用来增强细胞- 以病人为基础的治疗。在目标1中，我们将阐明NKX2.5引导效应器的多种机制 用基于质谱学的蛋白质组学方法调节细胞外基质成分的沉积和重塑 战略。在目标2中，我们假设纤维蛋白5调节转化生长因子-β信号以促进心外膜侵袭和 滴定组织硬度。我们将剖析典型的和非典型的转化生长因子-β通路，以确定关键 利用心脏切片的时间推移成像，我们将探索 当心外膜细胞和心肌细胞深入伤口时，它们会实时相互交织在一起。 我们将受益于原子力显微镜来测量再生心肌的弹性模数来揭示 纤维蛋白5在保持可延展的微环境以促进迁移方面的要求。在《目标3》中，我们将 调查Fiblin 5是再生ECM的重要组成部分的前提，并具有增强 通过评估hiPSC-CMS在体外的迁移率和体内宿主-移植物的整合来解决瘢痕。在一起，我们 预计我们提议的工作将推出一种新的范式，强调胚胎ECM的重要性 Nkx2-5下游纤维蛋白5等成分在促进心外膜和心肌细胞迁移和 组织弹性，以增强患者的治疗策略。

PROJECT SUMMARY Due to the limited ability to mount a regenerative response in injured myocardium, cardiac dysfunction remains a key cause of morbidity and mortality. Coronary artery disease leads to myocardial infarction which results in fibrotic scarring, adverse remodeling, and heart failure. In contrast to adult mice and humans, neonatal mice and adult zebrafish retain the ability to undergo cardiomyocyte dedifferentiation and proliferation, ultimately stimulating cardiac regeneration following injury. Deciphering the fundamental transcriptional networks shared in development and regeneration will fuel our ability to awaken endogenous reparative mechanisms. In zebrafish, embryonic cardiac transcriptional factors necessary for cardiogenesis, such as Nkx2.5, are galvanized following injury to stimulate cardiomyocyte production. Our preliminary studies reveal that Fibulin 5, an extracellular matrix (ECM) glycoprotein functioning downstream of Nkx2.5, is upregulated in the injured heart. Moreover, we show that fibulin loss-of-function fish exhibit impaired regenerative capacity and crucial roles in migration and tissue elasticity. In this proposal, we will test the hypothesis that activation of Nkx2.5 is necessary to produce an ECM milieu comprise of cardinal pro-regenerative proteins such as Fibulin 5 that can be harnessed to augment cell- based therapies for patients. In Aim 1, we will elucidate the myriad mechanisms by which Nkx2.5 directs effectors to mediate deposition and remodeling of ECM elements employing mass spectrometry-based proteomic strategies. In Aim 2, we postulate that Fibulin 5 modulates TGF-β signaling to promote epicardial invasion and titrate tissue stiffness. We will dissect both the canonical and non-canonical TGF-β pathways to identify the key signal transducers downstream of Fibulin 5. Using time lapse imaging of cardiac slices, we will probe the intertwined movements of epicardial and myocardial cells in real time as they penetrate deep into the wound. We will benefit from atomic force microscopy to measure elastic modulus in regenerating myocardium to uncover the requirement of Fibulin 5 in preserving a malleable microenvironment to promote migration. In Aim 3, we will investigate the premise that Fibulin 5 is a vital component of the regenerative ECM with potential to enhance scar resolution by evaluating hiPSC-CMs mobility in vitro and host-graft integration in vivo. Together, we anticipate that our proposed work will launch a novel paradigm highlighting the importance of embryonic ECM components such as Fibulin 5 downstream of Nkx2-5 in promoting epicardial and cardiomyocyte migration and tissue elasticity to enhance therapeutic strategies for patients.