Epicardial regeneration is guided by cardiac outflow tract and Hh signaling

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. Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to K.D.P. ([email protected]).. Author Contributions. J.W., J.C., and K.D.P. designed experimental strategy, analyzed data, and prepared the manuscript. J.W. generated the transgenic system for epicardial cell ablation and performed in vivo regeneration experiments and analysis. J.C. developed the ex vivo culture assay and performed ex vivo regeneration experiments and analysis. A.D. performed histology and data analysis. All authors commented on the manuscript. Author Information. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. HHS Public Access Author manuscript Nature. Author manuscript; available in PMC 2015 December 11. Published in final edited form as: Nature. 2015 June 11; 522(7555): 226–230. doi:10.1038/nature14325. A uhor M anscript