AP-1 Contributes to Chromatin Accessibility to Promote Sarcomere Disassembly and Cardiomyocyte Protrusion During Zebrafish Heart Regeneration.

RATIONALE: The adult human heart is an organ with low regenerative potential. Heart failure following acute myocardial infarction is a leading cause of death due to the inability of cardiomyocytes to proliferate and replenish lost cardiac muscle. While the zebrafish has emerged as a powerful model to study endogenous cardiac regeneration, the molecular mechanisms by which cardiomyocytes respond to damage by disassembling sarcomeres, proliferating, and repopulating the injured area remain unclear. Furthermore, we are far from understanding the regulation of the chromatin landscape and epigenetic barriers that must be overcome for cardiac regeneration to occur. OBJECTIVE: To identify transcription factor regulators of the chromatin landscape, which promote cardiomyocyte regeneration in zebrafish, and investigate their function. METHODS AND RESULTS: Using the Assay for Transposase-Accessible Chromatin coupled to high-throughput sequencing (ATAC-Seq), we first find that the regenerating cardiomyocyte chromatin accessibility landscape undergoes extensive changes following cryoinjury, and that activator protein-1 (AP-1) binding sites are the most highly enriched motifs in regions that gain accessibility during cardiac regeneration. Furthermore, using bioinformatic and gene expression analyses, we find that the AP-1 response in regenerating adult zebrafish cardiomyocytes is largely different from the response in adult mammalian cardiomyocytes. Using a cardiomyocyte-specific dominant negative approach, we show that blocking AP-1 function leads to defects in cardiomyocyte proliferation as well as decreased chromatin accessibility at the fbxl22 and ilk loci, which regulate sarcomere disassembly and cardiomyocyte protrusion into the injured area, respectively. We further show that overexpression of the AP-1 family members Junb and Fosl1 can promote changes in mammalian cardiomyocyte behavior in vitro. CONCLUSIONS: AP-1 transcription factors play an essential role in the cardiomyocyte response to injury by regulating chromatin accessibility changes, thereby allowing the activation of gene expression programs that promote cardiomyocyte dedifferentiation, proliferation, and protrusion into the injured area.

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 regenerating 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 using live-imaging and histological approaches. We observed close interactions between protruding cardiomyocytes and macrophages at the wound border zone, and macrophage-deficient irf8 mutant zebrafish 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, including cardiomyocytes, macrophages, fibroblasts, and endocardial/endothelial cells. Genetic deletion of mmp14b led to a decrease in 1) macrophage recruitment to the border zone, 2) collagen degradation at the border zone, and 3) subsequent cardiomyocyte invasion. Furthermore, cardiomyocyte-specific overexpression of mmp14b was sufficient to enhance cardiomyocyte invasion into the injured tissue 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.