Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life1,2. Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction3. By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced4,5. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre-recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labelled most cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury, primarily through activation and expansion of cardiomyocyte populations. These findings have implications for promoting regeneration of the injured human heart.
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We thank J. Burris and A. Eastes for zebrafish care, X. Meng and the Developmental Studies Hybridoma Bank for antibodies, M. Gignac for help with electron microscopy, laboratory members for comments on the manuscript, and G. Burns, P. Chambon and G. Felsenfeld for plasmids. This work was supported by postdoctoral fellowships from AHA (K.K. and Y.F.), JDRF (R.M.A.), and JSPS (K.K.); NIH training grants HL007208 at Massachusetts General Hospital (A.A.W.) and HL007101 at Duke University Medical Center (G.F.E.); grants from NHLBI (HL064282 to T.E., HL054737 to D.Y.R.S., and HL081674 to K.D.P.), NIGMS (GM075846 to C.A.M.), and March of Dimes (C.A.M.); and grants from AHA, Pew Charitable Trusts and Whitehead Foundation (K.D.P.).
Author Contributions K.K. and K.D.P. designed experimental strategy, analysed data, and prepared the manuscript. K.K., J.E.H. and Y.F. generated and characterized transgenic lines for lineage-tracing. R.M.A. and D.Y.R.S. provided unpublished reagents for lineage-tracing. K.K., J.E.H. and K.D.P. performed regeneration experiments. J.E.H. performed electron microscopy. A.A.W., G.F.E. and C.A.M. designed physiology experiments and interpreted data. A.A.W. performed optical mapping assays. T.E. helped design strategy and provided key reagents. All authors commented on the manuscript.
The authors declare no competing financial interests.
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Kikuchi, K., Holdway, J., Werdich, A. et al. Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes. Nature 464, 601–605 (2010). https://doi.org/10.1038/nature08804
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