Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Cardiomyocytes marked by gata4 :EGFP are activated by injury and proliferate at the injury site.
Figure 2: Major contribution of gata4 + cardiomyocytes to heart regeneration.
Figure 3: Electrical coupling of regenerated cardiomyocytes.
Figure 4: Restoration of gata4 :EGFP expression and a new ventricular wall after scarring.


  1. 1

    Bergmann, O. et al. Evidence for cardiomyocyte renewal in humans. Science 324, 98–102 (2009)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Quaini, F. et al. Chimerism of the transplanted heart. N. Engl. J. Med. 346, 5–15 (2002)

    Article  PubMed  Google Scholar 

  3. 3

    Rubart, M. & Field, L. J. Cardiac regeneration: repopulating the heart. Annu. Rev. Physiol. 68, 29–49 (2006)

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Poss, K. D., Wilson, L. G. & Keating, M. T. Heart regeneration in zebrafish. Science 298, 2188–2190 (2002)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Raya, A. et al. Activation of notch signaling pathway precedes heart regeneration in zebrafish. Proc. Natl Acad. Sci. USA 100, 11889–11895 (2003)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Cai, C. L. et al. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877–889 (2003)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Dor, Y., Brown, J., Martinez, O. I. & Melton, D. A. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429, 41–46 (2004)

    ADS  CAS  Article  PubMed  Google Scholar 

  8. 8

    Hsieh, P. C. et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nature Med. 13, 970–974 (2007)

    CAS  Article  Google Scholar 

  9. 9

    Laugwitz, K. L. et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433, 647–653 (2005)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10

    Meilhac, S. M. et al. A retrospective clonal analysis of the myocardium reveals two phases of clonal growth in the developing mouse heart. Development 130, 3877–3889 (2003)

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Zhou, B. et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454, 109–113 (2008)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Holtzinger, A. & Evans, T. Gata4 regulates the formation of multiple organs. Development 132, 4005–4014 (2005)

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Crispino, J. D. et al. Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes Dev. 15, 839–844 (2001)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Molkentin, J. D., Lin, Q., Duncan, S. A. & Olson, E. N. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 11, 1061–1072 (1997)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Kuo, C. T. et al. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 11, 1048–1060 (1997)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Zeisberg, E. M. et al. Morphogenesis of the right ventricle requires myocardial expression of Gata4 . J. Clin. Invest. 115, 1522–1531 (2005)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Pu, W. T., Ishiwata, T., Juraszek, A. L., Ma, Q. & Izumo, S. GATA4 is a dosage-sensitive regulator of cardiac morphogenesis. Dev. Biol. 275, 235–244 (2004)

    CAS  Article  Google Scholar 

  18. 18

    Heicklen-Klein, A. & Evans, T. T-box binding sites are required for activity of a cardiac GATA-4 enhancer. Dev. Biol. 267, 490–504 (2004)

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Lepilina, A. et al. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127, 607–619 (2006)

    CAS  Article  Google Scholar 

  20. 20

    Laflamme, M. A., Zbinden, S., Epstein, S. E. & Murry, C. E. Cell-based therapy for myocardial ischemia and infarction: pathophysiological mechanisms. Annu. Rev. Pathol. 2, 307–339 (2007)

    CAS  Article  Google Scholar 

  21. 21

    Passier, R., van Laake, L. W. & Mummery, C. L. Stem-cell-based therapy and lessons from the heart. Nature 453, 322–329 (2008)

    ADS  CAS  Article  Google Scholar 

  22. 22

    de Pater, E. et al. Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart. Development 136, 1633–1641 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Bersell, K., Arab, S., Haring, B. & Kuhn, B. Neuregulin1/erbb4 signaling induces cardiomyocyte proliferation and repair of heart injury. Cell 138, 257–270 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Kühn, B. et al. Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair. Nature Med. 13, 962–969 (2007)

    Article  PubMed  Google Scholar 

  25. 25

    Engel, F. B., Hsieh, P. C., Lee, R. T. & Keating, M. T. Fgf1/p38 map kinase inhibitor therapy induces cardiomyocyte mitosis, reduces scarring, and rescues function after myocardial infarction. Proc. Natl Acad. Sci. USA 103, 15546–15551 (2006)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Engel, F. B. et al. p38 map kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Genes Dev. 19, 1175–1187 (2005)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Wills, A. A., Holdway, J. E., Major, R. J. & Poss, K. D. Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish. Development 135, 183–192 (2008)

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Matsuda, T. & Cepko, C. L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl Acad. Sci. USA 104, 1027–1032 (2007)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Thermes, V. et al. I-scei meganuclease mediates highly efficient transgenesis in fish. Mech. Dev. 118, 91–98 (2002)

    CAS  Article  Google Scholar 

  30. 30

    Indra, A. K. et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-Er(T2) recombinases. Nucleic Acids Res. 27, 4324–4327 (1999)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Rottbauer, W. et al. Reptin and pontin antagonistically regulate heart growth in zebrafish embryos. Cell 111, 661–672 (2002)

    CAS  Article  PubMed  Google Scholar 

  32. 32

    Waxman, J. S., Keegan, B. R., Roberts, R. W., Poss, K. D. & Yelon, D. Hoxb5b acts downstream of retinoic acid signaling in the forelimb field to restrict heart field potential in zebrafish. Dev. Cell 15, 923–934 (2008)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Sidorov, V. Y., Holcomb, M. R., Woods, M. C., Gray, R. A. & Wikswo, J. P. Effects of unipolar stimulation on voltage and calcium distributions in the isolated rabbit heart. Basic Res. Cardiol. 103, 537–551 (2008)

    Article  PubMed  PubMed Central  Google Scholar 

  34. 34

    Brette, F. et al. Characterization of isolated ventricular myocytes from adult zebrafish (Danio Rerio). Biochem. Biophys. Res. Commun. 374, 143–146 (2008)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35

    Fast, V. G. & Kleber, A. G. Cardiac tissue geometry as a determinant of unidirectional conduction block: assessment of microscopic excitation spread by optical mapping in patterned cell cultures and in a computer model. Cardiovasc. Res. 29, 697–707 (1995)

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Bayly, P. V. et al. Estimation of conduction velocity vector fields from epicardial mapping data. IEEE Trans. Biomed. Eng. 45, 563–571 (1998)

    CAS  Article  PubMed  Google Scholar 

Download references


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.

Author information



Corresponding author

Correspondence to Kenneth D. Poss.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-10 with legends. (PDF 17172 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kikuchi, K., Holdway, J., Werdich, A. et al. Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes. Nature 464, 601–605 (2010).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing