Skip to main content

Thank you for visiting nature.com. 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.

Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish

Abstract

The zebrafish heart has the capacity to regenerate after ventricular resection. Although this regeneration model has proved useful for the elucidation of certain regeneration mechanisms, it is based on the removal of heart tissue rather than on tissue damage. We recently characterized the cellular response and regenerative capacity of the zebrafish heart after cryoinjury (CI), an alternative procedure that more closely models the pathophysiological process undergone by the human heart after myocardial infarction (MI). After anesthesia, localized CI with a liquid nitrogen–cooled copper probe induced damage in 25% of the ventricle, in a procedure requiring <5 min. Here we present a detailed description of the technique, which provides a valuable system for the study of the mechanisms of heart regeneration and scar removal after MI in a versatile vertebrate model.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Detailed description of cardiac cryoinjury in an adult zebrafish.
Figure 2: Constructing a cryoprobe.
Figure 3: Dissection of the adult zebrafish heart.
Figure 4: Effect of cryoinjury on the zebrafish heart ventricle.

References

  1. Jennings, R.B., Murry, C.E., Steenbergen, C. Jr. & Reimer, K.A. Development of cell injury in sustained acute ischemia. Circulation 82, II2–II12 (1990).

    CAS  PubMed  Google Scholar 

  2. Laflamme, M.A. & Murry, C.E. Heart regeneration. Nature 473, 326–335 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sedmera, D. & Thompson, R.P. Myocyte proliferation in the developing heart. Dev. Dyn. 240, 1322–1334 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Raya, A. et al. Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc. Natl. Acad. Sci. USA 100 (suppl. 1), 11889–11895 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schnabel, K., Wu, C.C., Kurth, T. & Weidinger, G. Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS ONE 6, e18503 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gonzalez-Rosa, J.M., Martin, V., Peralta, M., Torres, M. & Mercader, N. Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development 138, 1663–1674 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Chablais, F., Veit, J., Rainer, G. & Jazwinska, A. The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev. Biol. 11, 21 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Yang, Y. et al. Characterization of cryoinjury-induced infarction with manganese-and gadolinium-enhanced MRI and optical spectroscopy in pig hearts. Magn. Reson Imaging 28, 753–766 (2011).

    Article  Google Scholar 

  11. van den Bos, E.J., Mees, B.M., de Waard, M.C., de Crom, R. & Duncker, D.J. A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation. Am. J. Physiol. Heart Circ. Physiol. 289, H1291–H1300 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Schwagten, B., Van Belle, Y. & Jordaens, L. Cryoablation: how to improve results in atrioventricular nodal reentrant tachycardia ablation? Europace 12, 1522–1525 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Jopling, C. et al. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464, 606–609 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhong, H. & Lin, S. Chemical screening with zebrafish embryos. Methods Mol. Biol. 716, 193–205 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Wang, J. et al. The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion. Development 138, 3421–3430 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yang, F. et al. Myocardial infarction and cardiac remodelling in mice. Exp. Physiol. 87, 547–555 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Lawson, N.D. & Weinstein, B.M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Bartolomé and M.Á. Ricote for constructing the cryoprobe, E. Díaz and others at the CNIC animal facility for zebrafish husbandry, and S. Bartlett (CNIC) for text editing. The Tg(cmlc2:GFP) line was provided by A. Raya (Institute for Bioengineering of Catalonia (IBEC), Spain) and the Tg(fli1a:GFP)y1 line was from the Zebrafish International Resource Center, which is supported by grant no. P40 RR012546 from the US National Institutes of Health National Center for Research Resources (NIH-NCRR). Funding was from the Fundación CNIC Carlos III, the Fundación ProCNIC, the Comunidad de Madrid (FIBROTEAM S2011/BMD-2321) and the Spanish Ministry of Economy and Competitiveness (FPU fellowship to J.M.G.-R., RYC-2006-001694 and BFU-2008-0012BMC to N.M.).

Author information

Authors and Affiliations

Authors

Contributions

J.M.G.-R. performed the experiments. J.M.G.-R. and N.M. designed the experiments and prepared the manuscript.

Corresponding author

Correspondence to Nadia Mercader.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Cryoinjury using a platinum cryoprobe. (a and b) Whole mount views of bright field (a) and fluorescence (b) images of freshly cryoinjured dissected Tg(fli1a:GFP) zebrafish hearts. Anterior is to the top, dorsal to the left. The injured area is visible as a pale area in a and as the region lacking coronary vasculature in b. (c) Graph showing the percentage of the ventricle occupied by the injured area at 1 hour postinjury in cryoinjured hearts using a 0.3 mm copper filament (red dots) or a 0.5 mm platinum filament (blue squares). Mean and standard deviation for copper filament was 28% ± 8% (mean ± S.D.) and for platinum 35% ± 11%. at, atrium; ba, bulbus arteriosus; IA, injury area; v, ventricle. Scale bars, 100 µm. (TIFF 19289 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

González-Rosa, J., Mercader, N. Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish. Nat Protoc 7, 782–788 (2012). https://doi.org/10.1038/nprot.2012.025

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.025

This article is cited by

Comments

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.

Search

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