The interaction of anabolic mTOR signaling and catabolic autophagic activity during zebrafish heart regeneration

与哺乳动物相比，斑马鱼可以通过心肌细胞的增殖和短暂疤痕组织的完全吸收来再生心脏。损伤后早期发生的巨噬细胞浸润和伤口血运重建对随后的心肌细胞再生至关重要。然而，对这些早期损伤反应的分子调控以及它们如何与心肌细胞损伤反应相关联知之甚少。我们发现，雷帕霉素（mTOR）信号的机制靶点在心脏损伤后1天内在斑马鱼伤口边界心肌细胞中被激活，浸润性白细胞和内皮细胞也被激活。干扰mTOR信号表明该途径是心肌细胞增殖和形态学心脏再生所必需的。因此，我们的初步结果将mTOR信号激活定义为斑马鱼心肌细胞对心脏损伤最早的已知反应之一，并表明mTOR信号在其他细胞类型的早期损伤反应中也具有重要作用。利用遗传学、药理学和生化工具，我们将阐明mTOR信号在中性粒细胞和巨噬细胞浸润、伤口血运重建和心肌细胞去分化中的作用。虽然mTOR通路激活合成代谢，但在许多系统中它抑制具有分解代谢功能的自噬。令人惊讶的是，我们发现斑马鱼损伤心脏的伤口边缘心肌细胞中自噬被激活，自噬通量的抑制导致心肌细胞增殖缺陷和形态再生失败。这表明，合成代谢mTOR信号和分解代谢自噬活性需要紧密平衡才能成功地实现心脏再生。我们将研究心肌细胞去分化是否需要自噬，以及mTOR信号和自噬如何在伤口边缘心肌细胞中相互作用。总之，本提案中的实验将阐明斑马鱼心脏再生中早期损伤反应的分子调控。

In contrast to mammals, zebrafish can regenerate the heart via proliferation of cardiomyocytes and complete resorption of transient scar tissue. Macrophage infiltration and revascularization of the wound, which happen early after injury, are essential for subsequent cardiomyocyte regeneration. However, little is known about the molecular regulation of these early injury responses and how they are linked with injury responses in cardiomyocytes. We have discovered that mechanistic target of rapamycin (mTOR) signaling is activated in zebrafish wound border cardiomyocytes within 1 day after heart injury, as well as in infiltrating leukocytes and in endothelial cells. Interference with mTOR signaling shows that the pathway is required for cardiomyocyte proliferation and morphological heart regeneration. Our preliminary results thus define mTOR signaling activation as one of the earliest known responses of zebrafish cardiomyocytes to heart injury and they suggest important functions for mTOR signaling in early injury responses of other cell types as well. Using genetic, pharmacological and biochemical tools we will elucidate the role of mTOR signaling in neutrophil and macrophage infiltration, wound revascularization and cardiomyocyte dedifferentiation.While the mTOR pathway activates anabolism, it inhibits autophagy, which has catabolic functions, in many systems. Surprisingly, we found that autophagy is activated in wound border cardiomyocytes in injured zebrafish hearts, and that inhibition of autophagic flux results in cardiomyocyte proliferation defects and failure of morphological regeneration. This suggests that anabolic mTOR signaling and catabolic autophagic activity need to be tightly balanced for successful heart regeneration. We will study whether autophagy is required for cardiomyocyte dedifferentiation and how mTOR signaling and autophagy interact in wound border cardiomyocytes.Together, the experiments in this proposal will shed light on the molecular regulation of early injury responses in zebrafish heart regeneration.