Regulation of the Epicardial Injury Response During Heart Regeneration in Zebrafish

 描述（由申请人提供）：心肌梗死（MI）是一种常见的损伤，可导致数亿个心肌细胞（CM）的永久性损失，增加心力衰竭和猝死的易感性。再生医学的主要目标是提高心肌梗死后CM恢复和恢复心力衰竭患者的心脏功能的方法。在其自然环境中，心脏再生通过备用CM的增殖来构建肌肉，通过支持细胞类型如心外膜、内皮细胞、脉管系统和炎性细胞的影响来促进。覆盖心脏的心外膜被损伤激活，并通过旁分泌效应和作为多能细胞来源来帮助肌肉再生，并且最近作为心脏修复策略中的靶点而受到广泛关注。然而，直到最近，人们对任何物种的心外膜稳态--这些细胞是否以及如何再生--几乎一无所知。这种缺陷已经成为理解心外膜和将其用于治疗目标的障碍。在一项新的研究中，我们建立了斑马鱼心外膜的再生能力，并确定了心脏流出道和Hedgehog信号对这一过程的影响。虽然这项工作在成人心外膜生物学方面开辟了新天地，但同时也揭示了新的挑战。首先，我们对 心外膜细胞紧密连接在一个连续的片内的细节响应损伤和/或有丝分裂原的来源。其次，我们对心外膜再生以及心外膜细胞如何促进肌肉再生的分子理解是原始的。在这里，我们将这些解决心外膜生物学和心脏再生的核心问题，使用斑马鱼的尖端筛选和分析工具。1)我们将应用一种新的离体心外膜再生模型和一组新的转基因报告者，这些报告者在真实的时间内可视化心外膜细胞阶段，以定义心脏损伤后成人心外膜细胞的时空增殖动力学。2)我们将利用我们生成的单个心外膜细胞转录组数据来定义新的心外膜标记物，开始我们使用新试剂的工作，以定义心外膜标记物caveolin-1在心外膜损伤反应中的作用。3)我们将确定心外膜损伤反应的小分子调节剂，使用体外筛选策略和新的转基因工具来可视化和操纵心外膜。通过这些方法，我们将检验一个假设，即一个有力的心外膜损伤反应是心脏再生的关键。通过确定动态心外膜损伤反应及其对肌肉再生影响的关键调节因子，我们的工作将为理解和增强MI后人类显示的有限再生反应提供信息。

 DESCRIPTION (provided by applicant): Myocardial infarction (MI) is a common injury that causes permanent loss of hundreds of millions of cardiomyocytes (CMs), increasing susceptibility to heart failure and sudden death. Major goals of regenerative medicine are methodologies to enhance CM recovery after MI and to restore cardiac function to heart failure patients. Heart regeneration in its natural context builds muscle by proliferation of spared CMs, facilitated by influences of supporting cell types like epicardium, endocardium, vasculature, and inflammatory cells. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received much recent attention as a target in cardiac repair strategies. Yet, until recently, virtually nothing was known about epicardial homeostasis - whether and how these cells regenerate - in any species. This deficiency has presented a barrier to understanding the epicardium and developing it for therapeutic goals. In a new study, we established the capacity of the zebrafish epicardium to vigorously regenerate, and we identified influences on this process by the cardiac outflow tract and Hedgehog signaling. While this work broke new ground in adult epicardial biology, it simultaneously revealed new challenges. First, we understand little of the details by which epicardial cells tightly connected within a contiguous sheet respond to injury and/or mitogen sources. Second, our molecular understanding of epicardial regeneration, and how epicardial cells promote muscle regeneration, is primitive. Here, we will these address central issues in epicardial biology and heart regeneration, using cutting edge screening and analysis tools in zebrafish. 1) We will apply a new ex vivo model of epicardial regeneration and a panel of new transgenic reporters that visualize epicardial cell phases in real time, to define spatiotemporal proliferation dynamics of adult epicardial cells after cardiac injury. 2) We will harness single epicardial cell transcriptome data we have generated to define new epicardial markers, initiating our work with new reagents to define the role of the epicardial marker caveolin-1 in epicardial injury responses. 3) We will identify small molecule modulators of the epicardial injury response, using an ex vivo screening strategy and new transgenic tools to visualize and manipulate the epicardium. With these approaches, we will test the hypothesis that a vigorous epicardial injury response is critical for heart regeneration. By identifying key regulators of the dynamic epicardial injury response and its impact on muscle regeneration, our work will inform approaches for comprehending and enhancing the limited regenerative response displayed by humans after MI.