Gpnmb is required for immune and fibrotic responses during zebrafish heart regeneration

Myocardial infarction occurs when coronary supply of oxygen and nutrients to a part of the heart is interrupted. In contrast to adult humans, adult zebrafish have a unique ability to regenerate their heart after cardiac injury. To identify potential regulators of the regenerative response, we performed transcriptomics profiling of regenerating coronary endothelial cells (cECs) at 7 days post cryoinjury, when revascularization is vigorous, and identified gpnmb to be upregulated. Gpnmb (glycoprotein non-metastatic melanoma protein B) is a transmembrane glycoprotein implicated in inflammation resolution and tissue regeneration. Expression analyses at earlier time points revealed gpnmb specific expression in infiltrating macrophages post cryoinjury in zebrafish ventricles. Using newly generated gpnmb mutants, we analyze its role during heart regeneration and find that animals lacking gpnmb exhibit neutrophil retention and decreased macrophage recruitment as well as reduced myofibroblasts numbers. Moreover, loss of gpnmb impairs coronary endothelial cell regeneration and cardiomyocyte dedifferentiation. Transcriptomic analyses identified enhanced collagen production and extracellular matrix (ECM) related pathway activated in cryoinjured gpnmb mutant hearts. Furthermore, gpnmb mutant hearts exhibit larger fibrotic scars revealing defects in cardiac regeneration. Overall, these data suggest that gpnmb expressing macrophages modulate inflammation and ECM deposition after cardiac cryoinjury and highlight the importance of this subset of immune cells to support a regenerative response. Overall design: To investigate the role of gpnmb during zebrafish heart regeneration, we generated gpnmb mutants and performed comparative RNAseq analysis on WT and MUT

心肌梗死（Myocardial infarction）是指心脏局部区域的冠状动脉血氧与营养供给中断所引发的疾病。与成年人类不同，成年斑马鱼在心脏损伤后具备独特的心脏再生能力。为鉴定心脏再生反应的潜在调控因子，本研究在冷冻损伤后7天（此时血管新生活动旺盛）对再生性冠状动脉内皮细胞（coronary endothelial cells, cECs）开展转录组谱分析，筛选得到表达上调的gpnmb基因。Gpnmb（糖蛋白非转移性黑色素瘤蛋白B，glycoprotein non-metastatic melanoma protein B）是一种跨膜糖蛋白，参与炎症消退与组织再生过程。对更早时间点的表达分析显示，冷冻损伤后斑马鱼心室中的浸润型巨噬细胞特异性表达gpnmb。本研究利用新构建的gpnmb突变体，探究其在心脏再生中的功能，发现缺失gpnmb的个体出现中性粒细胞滞留、巨噬细胞招募减少以及肌成纤维细胞数量降低的表型。此外，gpnmb缺失会损伤冠状动脉内皮细胞再生与心肌细胞去分化过程。转录组分析显示，冷冻损伤的gpnmb突变体心脏中，胶原蛋白合成与细胞外基质（extracellular matrix, ECM）相关通路被过度激活。进一步研究发现，gpnmb突变体心脏的纤维化瘢痕面积更大，表明其心脏再生存在缺陷。综上，本研究数据表明，表达gpnmb的巨噬细胞可在心脏冷冻损伤后调控炎症反应与细胞外基质沉积，凸显了该类免疫细胞亚群对再生反应的支持作用。
实验整体设计：为探究gpnmb在斑马鱼心脏再生中的功能，本研究构建了gpnmb突变体，并对野生型（wild type, WT）与突变型（mutant, MUT）样本开展比较RNAseq分析。