Mechanisms controlling epicardial-dependent promotion of heart regeneration in zebrafish.

摘要 人类心脏在心肌梗死(MI)等损伤后几乎没有再生能力。 相反，心脏会留下疤痕，降低心脏功能，导致心力衰竭。在临床上没有 有意义的再生疗法可用于心肌梗死患者。相比之下，成年斑马鱼能再生心肌 在严重的心脏损伤后没有明显的疤痕。这是通过扩展现有的 心肌细胞(CMS)，在非肌肉细胞提供的环境的帮助下，如心外膜， 覆盖在心脏表面的间皮细胞薄片。一种类似CM增殖的再生机制 在成年哺乳动物心脏中也有心外膜的贡献，但它还没有被充分激活 以实现显著的再生。最近的研究表明，通过应用恢复心外膜因子 心外膜补片或将人类干细胞来源的心外膜细胞与干细胞共同移植- 心肌梗死后衍生的CMS有利于心脏再生。因此，增强促进再生的激活 心外膜可能有利于心肌梗死后哺乳动物心脏的再生。我们和其他人之前发现 斑马鱼的心外膜被损伤激活，并通过旁分泌效应和作为一种 多能细胞的来源。然而，对细胞和分子机制的控制知之甚少。 心外膜激活，导致成功的心脏再生。为此，了解再生是如何 在成年斑马鱼中，心外膜的反应受到调节，这将导致新的治疗靶点 哺乳动物的再生缺陷。为了解决这个问题，使用单细胞RNA测序，我们识别了一种 心脏损伤后心外膜内的暂时性成人心外膜祖细胞亚群。 移植试验表明aEPC具有产生血管周围细胞的能力，而血管周围细胞对 冠状动脉血运重建。这些aEPC的基因消融阻止了心脏再生，这表明 不可或缺的角色。药理操作和转录组分析产生了候选基因 这是激活aEPC的基础。此外，使用以下方法对染色质可及性进行无偏见的全基因组分析 ATAC-SEQ揭示了对这些基因施加转录调控的假定调控元件。我们 假设心外膜祖细胞状态的激活是心脏再生成功的基础。 为了验证这一假设，我们建议1)确定成年斑马鱼心脏中aEPC的细胞命运和功能 使用遗传命运图谱、基因消融和单细胞移植方法进行再生； 通过基因操作和分析确定AEPC激活的分子机制 数十个转基因品系和突变体。这项提案的结果可能最终会为 激活心外膜祖细胞以增强人类心肌梗死后有限的再生。

Abstract The human heart shows little regenerative capacity following an injury such as myocardial infarction (MI). Instead, the heart scars, decreasing cardiac function, and leading to heart failure. There is no clinically meaningful regenerative therapy available for MI patients. By contrast, adult zebrafish regenerate heart muscle after severe cardiac damage without significant scarring. This is achieved through proliferation of existing cardiomyocytes (CMs), aided by the environment provided by non-muscle cells, such as the epicardium, a mesothelial cell sheet covering the surface of the heart. An analogous regenerative machinery of CM proliferation and epicardium contributions also exists in the adult mammalian heart; however, it is not sufficiently activated for significant regeneration. Recent studies demonstrated that restoring epicardial factors through the application of epicardial patches or co-transplantation of human stem cell-derived epicardial cells together with stem cell- derived CMs after an MI benefit heart regeneration. Thus, enhancing the pro-regenerative activation of the epicardium may benefit mammalian heart regeneration after MI. We and others previously found that the zebrafish epicardium is activated by injury and aids muscle regeneration through paracrine effects and as a source of multipotent cells. However, little is known about the cellular and molecular mechanisms controlling epicardial activation that lead to successful heart regeneration. To this end, understanding how regenerative responses of the epicardium are regulated in adult zebrafish will lead to new therapeutic targets that underlie the regenerative deficiencies in mammals. To address this, using single-cell RNA-sequencing, we have identified a transient adult epicardial progenitor cell (aEPC) subpopulation within the epicardium after heart injury. Transplantation assays implicate a capacity of aEPCs to give rise to perivascular cells, which are critical for coronary revascularization. Genetic ablation of these aEPCs blocks heart regeneration, suggesting an indispensable role. Pharmacological manipulations and transcriptome analyses yielded candidate genes that underlie the activation of aEPCs. Further, unbiased genome-wide profiling of chromatin accessibility using ATAC-seq revealed putative regulatory elements that exert transcriptional regulation of these genes. We hypothesize that activation of a progenitor cell state in the epicardium underlies successful heart regeneration. To test this hypothesis, we propose to 1) define the cell fates and functions of the aEPCs in adult zebrafish heart regeneration using genetic fate mapping, genetic ablation, and single-cell transplantation approaches; and 2) define the molecular mechanisms underlying aEPC activation through genetic manipulations and analyzing dozens of transgenic lines and mutants. The outcome of this proposal may ultimately inform approaches for activating the epicardial progenitors to enhance the limited regeneration displayed in humans after MI.