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

摘要 人类心脏在诸如心肌梗死（MI）的损伤之后显示出很少的再生能力。 相反，心脏疤痕，降低心脏功能，并导致心力衰竭。没有临床 为心肌梗死患者提供有意义的再生治疗。相比之下，成年斑马鱼可以再生心脏肌肉 没有明显疤痕的情况下死亡这是通过扩散现有的 心肌细胞（CM），由非肌肉细胞提供的环境，如心外膜， 覆盖心脏表面的间皮细胞片。CM增殖的类似再生机制 成年哺乳动物的心脏也有心外膜的贡献，但没有充分激活 进行重要的再生。最近的研究表明，通过应用恢复心外膜因素， 或人干细胞衍生的心外膜细胞与干细胞- 心肌梗死后衍生的CM有益于心脏再生。因此，增强促再生激活的细胞， 心外膜有利于心肌梗死后心脏再生。我们和其他人以前发现， 斑马鱼的心外膜被损伤激活，并通过旁分泌作用帮助肌肉再生， 多能细胞的来源。然而，很少有人知道的细胞和分子机制控制， 心外膜激活导致成功的心脏再生。为了达到这个目的， 在成年斑马鱼中调节心外膜的反应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，我们已经确定了一个 心脏损伤后心外膜内的瞬时成体心外膜祖细胞（aEPC）亚群。 移植测定暗示aEPCs产生血管周围细胞的能力，这对于移植至关重要。 冠状动脉血运重建这些aEPCs的基因切除阻断了心脏再生，这表明 不可或缺的角色。药理学操作和转录组分析产生了候选基因， 是aEPCs激活的基础。此外，使用染色体可及性的无偏全基因组谱分析， ATAC-seq揭示了对这些基因进行转录调控的推定调控元件。我们 假设心外膜中祖细胞状态激活是成功心脏再生的基础。 为了验证这一假设，我们建议：1）确定成年斑马鱼心脏aEPCs的细胞命运和功能 使用遗传命运作图、遗传消融和单细胞移植方法的再生;以及2） 通过基因操作和分析， 几十种转基因品系和突变体。这一建议的结果可能最终为以下方法提供参考： 激活心外膜祖细胞以增强MI后在人类中显示的有限再生。

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.