Injury-activated endocardium plays structural and signalling roles in zebrafish heart regeneration

The zebrafish heart remarkably regenerates after a severe ventricular damage followed by inflammation, fibrotic tissue deposition and removal concomitant with cardiac muscle replacement. We have investigated the role of the endocardium in this regeneration process. 3D-whole mount imaging in injured hearts revealed that GFP-labelled endocardial cells in ET33mi-60A transgenic fish become rapidly activated and highly proliferative at 3 days post cryoinjury (dpci). Endocardial cells extensively expand within the injury site and organize to form a coherent structure at 9 dpci that persists throughout the regeneration process. Upon injury, endocardial cells strongly up-regulate the Notch pathway ligand delta like4 (dll4) and the Notch receptors notch1b, notch2 and notch3. Expression profiling showed that Notch signalling inhibition affects endocardial gene expression and genes related to extracellular matrix remodelling and inflammation. Gain- and loss-of-function experiments revealed that Notch is required for the organization of the endocardium, attenuation of the inflammatory response and cardiomyocyte proliferation. These results demonstrate a novel structural and signalling role for the endocardium during heart regeneration. RNA was extracted from apical tip of heart ventricles 72h after cryoinjured adult zebrafish heart treated with DMSO (Controls) or RO gamma secretase inhibitor at 24 and 48h post injury.

斑马鱼心脏在遭受严重心室损伤后，可伴随炎症反应、纤维化组织沉积与清除，并同步完成心肌替换，展现出极强的再生能力。本研究探究了心内膜（endocardium）在该再生过程中的作用。对损伤心脏开展全标本三维成像结果显示，ET33mi-60A转基因斑马鱼中，绿色荧光蛋白（GFP）标记的心内膜细胞在冷冻损伤后3天（3 dpci）即被快速激活，且增殖活性显著升高。至冷冻损伤后9天（9 dpci），心内膜细胞在损伤区域大量扩增，并组装形成连贯的组织结构，该结构在整个再生进程中持续存在。损伤发生后，心内膜细胞会显著上调表达Notch信号通路（Notch pathway）配体类delta4（dll4）以及Notch受体notch1b、notch2与notch3。转录组表达谱分析表明，抑制Notch信号会影响心内膜基因的表达，同时改变与细胞外基质重塑及炎症反应相关的基因表达模式。功能获得与功能缺失实验证实，Notch信号对于心内膜的组织组装、炎症反应的缓解以及心肌细胞增殖均不可或缺。本研究结果揭示了心内膜在心脏再生过程中全新的结构与信号调控作用。本研究所用RNA提取自冷冻损伤后72小时的成年斑马鱼心室心尖组织，实验分为两组：对照组以二甲基亚砜（DMSO）处理，实验组则在损伤后24小时与48小时施加ROγ分泌酶抑制剂处理。