Mechanisms controlling epicardial-dependent promotion of heart regeneration in zebrafish.

Abstract
The human heart shows little regenerative capacity following an injury such as myocardial infarction (MI).
Instead, the heart scars, decreasing cardiac function, and leading to heart failure. There is no clinically
meaningful regenerative therapy available for MI patients. By contrast, adult zebrafish regenerate heart muscle
after severe cardiac damage without significant scarring. This is achieved through proliferation of existing
cardiomyocytes (CMs), aided by the environment provided by non-muscle cells, such as the epicardium, a
mesothelial cell sheet covering the surface of the heart. An analogous regenerative machinery of CM proliferation
and epicardium contributions also exists in the adult mammalian heart; however, it is not sufficiently activated
for significant regeneration. Recent studies demonstrated that restoring epicardial factors through the application
of epicardial patches or co-transplantation of human stem cell-derived epicardial cells together with stem cell-
derived CMs after an MI benefit heart regeneration. Thus, enhancing the pro-regenerative activation of the
epicardium may benefit mammalian heart regeneration after MI. We and others previously found that the
zebrafish epicardium is activated by injury and aids muscle regeneration through paracrine effects and as a
source of multipotent cells. However, little is known about the cellular and molecular mechanisms controlling
epicardial activation that lead to successful heart regeneration. To this end, understanding how regenerative
responses of the epicardium are regulated in adult zebrafish will lead to new therapeutic targets that underlie the
regenerative deficiencies in mammals. To address this, using single-cell RNA-sequencing, we have identified a
transient adult epicardial progenitor cell (aEPC) subpopulation within the epicardium after heart injury.
Transplantation assays implicate a capacity of aEPCs to give rise to perivascular cells, which are critical for
coronary revascularization. Genetic ablation of these aEPCs blocks heart regeneration, suggesting an
indispensable role. Pharmacological manipulations and transcriptome analyses yielded candidate genes that
underlie the activation of aEPCs. Further, unbiased genome-wide profiling of chromatin accessibility using
ATAC-seq revealed putative regulatory elements that exert transcriptional regulation of these genes. We
hypothesize that activation of a progenitor cell state in the epicardium underlies successful heart regeneration.
To test this hypothesis, we propose to 1) define the cell fates and functions of the aEPCs in adult zebrafish heart
regeneration using genetic fate mapping, genetic ablation, and single-cell transplantation approaches; and 2)
define the molecular mechanisms underlying aEPC activation through genetic manipulations and analyzing
dozens of transgenic lines and mutants. The outcome of this proposal may ultimately inform approaches for
activating the epicardial progenitors to enhance the limited regeneration displayed in humans after MI.

Grant Number: 5R01HL155607-05
NIH Institute/Center: NIH
Principal Investigator: Jingli Cao