Border-zone cardiomyocytes and macrophages contribute to remodeling of the extracellular matrix to promote cardiomyocyte invasion during zebrafish cardiac regeneration
Despite numerous advances in our understanding of zebrafish cardiac regeneration, an aspect that remains less studied is how newly proliferated cardiomyocytes invade, and eventually replace, the collagen-containing fibrotic tissue following injury.  Here, we provide an in-depth analysis of the process of cardiomyocyte invasion and migration using live-imaging and histological approaches.  We observed a close interaction between protruding cardiomyocytes and macrophages at the wound border zone, and irf8 mutant zebrafish, which largely lack macrophages, exhibited defects in extracellular matrix (ECM) remodeling and cardiomyocyte protrusion into the injured area.  Using a resident macrophage ablation model, we show that defects in ECM remodeling at the border zone and subsequent cardiomyocyte protrusion can be partly attributed to a population of resident macrophages.  Single-cell RNA-sequencing analysis of cells at the wound border revealed a population of cardiomyocytes and macrophages with fibroblast-like gene expression signatures, including the expression of genes encoding ECM structural proteins and ECM-remodeling proteins.  The expression of mmp14b, which encodes a membrane-anchored matrix metalloproteinase, was restricted to cells in the border zone and genetic deletion of mmp14b led to a decrease in 1) collagen degradation at the border zone, 2) macrophage recruitment to the border zone, and 3) subsequent cardiomyocyte invasion.  Furthermore, cardiomyocyte-specific overexpression of mmp14b was sufficient to enhance cardiomyocyte invasion both into the injured area and along the apical surface of the wound.  Altogether, our data shed important insights into the process of cardiomyocyte invasion of the collagen-containing injured tissue during cardiac regeneration.  They further suggest that cardiomyocytes and resident macrophages contribute to ECM remodeling at the border zone to promote cardiomyocyte replenishment of the fibrotic injured tissue. Overall design: The border zone of regenerating wild-type zebrafish hearts at 7 dpci was microdissected and subjected to scRNA-seq