SUMMARYNatural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.

SUMMARYRecent lineage-tracing studies have produced conflicting results about whether the epicardium is a source of cardiac muscle cells during heart development. Here, we examined the developmental potential of epicardial tissue in zebrafish during both embryonic development and injury-induced heart regeneration. We found that upstream sequences of the transcription factor gene tcf21 activated robust, epicardium-specific expression throughout development and regeneration. Cre recombinase-based, genetic fate-mapping of larval or adult tcf21 + cells revealed contributions to perivascular cells, but not cardiomyocytes, during each form of cardiogenesis. Our findings indicate that natural epicardial fates are limited to non-myocardial cell types in zebrafish.

Unlike adult mammals, adult zebrafish vigorously regenerate lost heart muscle in response to injury. The epicardium, a mesothelial cell layer enveloping the myocardium, is activated to proliferate after cardiac injury and can contribute vascular support cells or provide mitogens to regenerating muscle. Here, we applied proteomics to identify secreted proteins that are associated with heart regeneration. We found that Fibronectin, a main component of the extracellular matrix, is induced and deposited after cardiac damage. In situ hybridization and transgenic reporter analyses indicated that expression of two fibronectin paralogues, fn1 and fn1b, are induced by injury in epicardial cells, while the itgb3 receptor is induced in cardiomyocytes near the injury site. fn1, the more dynamic of these paralogs, is induced chamber-wide within one day of injury before localizing epicardial Fn1 synthesis to the injury site. fn1 loss-of-function mutations disrupted zebrafish heart regeneration, as did induced expression of a dominant-negative Fibronectin cassette, defects that were not attributable to direct inhibition of cardiomyocyte proliferation. These findings reveal a new role for the epicardium in establishing an extracellular environment that supports heart regeneration.

In response to cardiac damage, a mesothelial tissue layer enveloping the heart called the epicardium is activated to proliferate and accumulate at the injury site. Recent studies have implicated the epicardium in multiple aspects of cardiac repair: a source of paracrine signals for cardiomyocyte survival or proliferation; a supply of perivascular cells and possibly other cell types like cardiomyocytes; and, a mediator of inflammation1-9. Yet, the biology and dynamism of the adult epicardium is poorly understood. Here, we created a transgenic line to ablate this cell population in adult zebrafish. We find that genetic depletion of epicardium after myocardial loss inhibits cardiomyocyte proliferation and delays muscle regeneration. The epicardium vigorously regenerates after its ablation, through proliferation and migration of spared epicardial cells as a sheet to cover the exposed ventricular surface in a wave from the chamber base toward its apex. By reconstituting epicardial regeneration ex vivo, we show that extirpation of the bulbous arteriosus (BA), a distinct, smooth muscle-rich tissue structure that distributes outflow from the ventricle, prevents epicardial regeneration. Conversely, experimental repositioning of the BA by tissue recombination initiates epicardial regeneration and can govern its direction. Hedgehog (Hh) ligand is expressed in the BA, and treatment with Hh signaling antagonist arrests epicardial regeneration and blunts the epicardial response to muscle injury. Transplantation of Shh-soaked beads at the ventricular base stimulates epicardial regeneration after BA removal, indicating that Hh signaling can substitute for the BA influence. Thus, the ventricular epicardium has pronounced regenerative capacity, regulated by the neighboring cardiac outflow tract and Hh signaling. These findings extend our understanding of tissue interactions during regeneration and have implications for mobilizing epicardial cells for therapeutic heart repair.

SUMMARYAdult mammalian cardiomyocytes have little capacity to proliferate in response to injury, a deficiency that underlies the poor regenerative ability of human hearts after myocardial infarction. By contrast, zebrafish regenerate heart muscle after trauma by inducing proliferation of spared cardiomyocytes, providing a model for identifying manipulations that block or enhance these events. Although direct genetic or chemical screens of heart regeneration in adult zebrafish present several challenges, zebrafish embryos are ideal for high-throughput screening. Here, to visualize cardiomyocyte proliferation events in live zebrafish embryos, we generated transgenic zebrafish lines that employ fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. We then performed a chemical screen and identified several small molecules that increase or reduce cardiomyocyte proliferation during heart development. These compounds act via Hedgehog, Insulin-like growth factor or Transforming growth factor  signaling pathways. Direct examination of heart regeneration after mechanical or genetic ablation injuries indicated that these pathways are activated in regenerating cardiomyocytes and that they can be pharmacologically manipulated to inhibit or enhance cardiomyocyte proliferation during adult heart regeneration. Our findings describe a new screening system that identifies molecules and pathways with the potential to modify heart regeneration. DEVELOPMENT Drug treatmentsSAG (EMD Chemicals) was either purchased in solution or dissolved in water to a 10 mM stock solution. NBI-31772 (Calbiochem) was dissolved in DMSO for a 30 mM stock solution. SU5402 (Santa Cruz Biotechnology), -secretase inhibitor (Calbiochem), SB-431542 (Selleckchem), RA (Sigma-Aldrich) and DEAB (Sigma-Aldrich) were dissolved in DMSO to a final concentration of 10 mM. CyA (Toronto Research Chemicals), NVP-AEW541 (Cayman Chemical), and GSK-3 Inhibitor IX (BIO, Calbiochem) were dissolved in ethanol to stock solutions of 10 mM. For embryo studies, cmlc2:FUCCI embryos were collected within a 30-minute window from male-female pairings. From 1 day post-fertilization (dpf) onwards, embryos were treated with n-phenylthiourea (PTU; SigmaAldrich) to block development of pigmentation. Drug treatment was performed at 3-4 dpf in 3 ml of buffered embryo water in 6-well plates. For adult studies, injured adult fish were treated in 20 ml of buffered fish water per animal in finger bowls. For embryos, all drugs were used at 5 M except for NBI-31772, RA and BIO, which were used at 2.5 M. In adults, SAG was used at 2.5 µM, NBI-31772 was used at 10 M, CyA was used at 10 M and NVP-AEW541 was used at 2 M. HistologyInitial screening for FUCCI signals was performed using a Leica M205 FA dissecting microscope. z-stacks were acquired from 50 M-thick sections of paraformaldehyde (PFA)-fixed embryos using a Zeiss LSM 700 confocal microscope. Three-dimensional reconstructions were made using Imaris Version 7.3 software. Quantification of mCherry + and/or Venus + n...

SUMMARY Mechanisms that control cell cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By live imaging of cell cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.

No abstract

Embryonic heart formation requires the production of an appropriate number of cardiomyocytes; likewise, cardiac regeneration following injury relies upon the recovery of lost cardiomyocytes. The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in promoting cardiomyocyte formation. It is unclear, however, whether Hand2 plays an instructive or permissive role during this process. Here, we find that overexpression of hand2 in the early zebrafish embryo is able to enhance cardiomyocyte production, resulting in an enlarged heart with a striking increase in the size of the outflow tract. Our evidence indicates that these increases are dependent on the interactions of Hand2 in multimeric complexes and are independent of direct DNA binding by Hand2. Proliferation assays reveal that hand2 can impact cardiomyocyte production by promoting division of late-differentiating cardiac progenitors within the second heart field. Additionally, our data suggest that hand2 can influence cardiomyocyte production by altering the patterning of the anterior lateral plate mesoderm, potentially favoring formation of the first heart field at the expense of hematopoietic and vascular lineages. The potency of hand2 during embryonic cardiogenesis suggested that hand2 could also impact cardiac regeneration in adult zebrafish; indeed, we find that overexpression of hand2 can augment the regenerative proliferation of cardiomyocytes in response to injury. Together, our studies demonstrate that hand2 can drive cardiomyocyte production in multiple contexts and through multiple mechanisms. These results contribute to our understanding of the potential origins of congenital heart disease and inform future strategies in regenerative medicine.