Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Aug 21;7(1):8362.
doi: 10.1038/s41598-017-08947-2.

Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation

David C Zebrowski  1 , Charlotte H Jensen  2   3 , Robert Becker  1 , Fulvia Ferrazzi  4 , Christina Baun  5 , Svend Hvidsten  5 , Søren P Sheikh  2   3   6 , Brian D Polizzotti  7   8 , Ditte C Andersen  2   6   9 , Felix B Engel  10   11
Affiliations

Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation

David C Zebrowski et al. Sci Rep. .

Abstract

After birth cardiomyocytes undergo terminal differentiation, characterized by binucleation and centrosome disassembly, rendering the heart unable to regenerate. Yet, it has been suggested that newborn mammals regenerate their hearts after apical resection by cardiomyocyte proliferation. Thus, we tested the hypothesis that apical resection either inhibits, delays, or reverses cardiomyocyte centrosome disassembly and binucleation. Our data show that apical resection rather transiently accelerates centrosome disassembly as well as the rate of binucleation. Consistent with the nearly 2-fold increased rate of binucleation there was a nearly 2-fold increase in the number of cardiomyocytes in mitosis indicating that the majority of injury-induced cardiomyocyte cell cycle activity results in binucleation, not proliferation. Concurrently, cardiomyocytes undergoing cytokinesis from embryonic hearts exhibited midbody formation consistent with successful abscission, whereas those from 3 day-old cardiomyocytes after apical resection exhibited midbody formation consistent with abscission failure. Lastly, injured hearts failed to fully regenerate as evidenced by persistent scarring and reduced wall motion. Collectively, these data suggest that should a regenerative program exist in the newborn mammalian heart, it is quickly curtailed by developmental mechanisms that render cardiomyocytes post-mitotic.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Effect of AR on cardiomyocyte centrosome disassembly. (a) Representative images of centrioles (γ-tubulin) in heart cryosections of P0 rat heart ventricles. Nuclei: DAPI. Cardiac nuclei: Nkx2.5. Arrowheads indicate paired centrioles in non-myocytes. Asterisk indicates centrioles in cardiomyocytes. Paired-centrioles: doublet γ-tubulin signals within 2 μm of one another; split-centrioles: γ-tubulin signals greater than 2 μm of one another; single-centriole: single γ-tubulin signal with no identifiable pair (e.g. other centriole is either overlapping or split to the extent of no longer residing in the section); no-centriole: no identifiable, nuclear-proximal, γ-tubulin signal in the section. Scale bars: 2 μm. (b,c) Quantitative analysis of centriole signals and configurations in non-myocytes (b) and cardiomyocytes (c) from cryosections of P0 (MOCK) and P3 or P6 (SHAM and AR) rat heart ventricles. Results are from three independent animals. ≥200 cardiomyocytes and ≥100 non-myocytes, collectively, from basal and apical regions were analyzed per experimental condition (see also Supplementary Fig. 1). (d) Quantitative analysis of cardiomyocytes proximal (within 1 mm) to base or apex/resection with paired centrioles from cryosections of P3 or P6 (SHAM and AR) rat hearts. Data are ± SD. p-values were calculated using two-tailed Student’s t-test.
Figure 2
Figure 2
Analysis of cardiomyocyte binucleation in AR hearts. (a) Representative images of mononucleated and binucleated P8-isolated rat cardiomyocytes. Nuclei were counterstained with propidium iodide. Yellow scale bars: 10 μm. (b,c) Percent binucleated cardiomyocytes at different time points after AR and SHAM in P1 rat (n = 4 for each time point, (b) and mouse (n = 3 for each time point, (c), see also Supplementary Fig. 2) hearts. p-values were calculated using two-tailed Student’s t-test comparing AR and SHAM. Inflection points reflect mean values. ΔR = fold difference in the rate of binucleation of AR- compared to SHAM-operated hearts. P1 data point for rat is from Li et al. (JMCC, 1996) and for mouse from Soonpaa et al. (AJP, 1996). p-values considered here significant (p < 0.05) are indicated in red.
Figure 3
Figure 3
Analysis of H3P-positive cardiomyocytes in AR rat hearts. (a) Representative immunofluorescence images of a P6 H3P-positive cardiomyocyte and non-myocyte (scale bar: 50 µm). Red: cardiomyocytes (sarcomeric-α-actinin); green: cells in mitosis (H3P); blue: nuclei (DAPI). Scale bar: 50 µm (see also Supplementary Fig. 3). (b) Quantification of cardiomyocytes in mitosis at P3 and P6 based on anti-H3P and anti-sarcomeric-α-actinin staining. Nuclei were counterstained with DAPI. Data are ± SD. n = 4 for MOCK, AR, and SHAM at each time point. (c,d) Quantification of H3P-positive cardiomyocytes at P3 (c) and P6 (d) in apical, mid, and base zones. Data are ± SD. n = 4 for MOCK, AR, and SHAM at each time point. p-values have been calculated using Fisher’s LSD test after one-way Anova.
Figure 4
Figure 4
Characterization of cardiomyocyte cytokinesis in vivo. Representative images of rat heart sections stained for cytokinesis proteins Aurora B (purple) and Anillin (green) and cardiomyocytes (Troponin I, red). Nuclei were visualized with DAPI (blue). (a–c) Representative images of cardiomyocytes undergoing midbody formation in hearts from E15 (a) cytokinesis results in cell division), P3 SHAM (b) cytokinesis results in binucleation) and P3 post-AR (c) rats. Scale bars: 10 µm.
Figure 5
Figure 5
Characterization of AR rat hearts. (a) Representative stereomicroscopic images of SHAM- and AR-operated rat hearts at different postnatal time points. Arrow heads indicate myocardial scar. Scale bars: 1 mm. (b–d) Representative images of Hematoxylin & Eosin staining (b), Masson’s trichrome staining (c), and immunofluorescence analysis of non-myocytes (Collagen I/Desmin/DAPI) (d) of SHAM- and AR-operated hearts at different postnatal time points. P3–P22 (n = 4–6) and P56 (n = 3) for each group. Red scale bars: 1 mm. Black/yellow scale bars: 200 µm. (e) The mean wall motion of each heart segment was compared between SHAM and AR hearts (n = 3). (f) Summary of wall motion depicting heart segments in AR hearts versus those of SHAM demonstrates defects in the apex region. (g) Quantitation of heart axes by PET scanning (n = 3). Right-left (RL). Anterior-posterior (AP). Data are ± SEM. *p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jopling C, et al. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature. 2010;464:606–609. doi: 10.1038/nature08899. - DOI - PMC - PubMed
    1. King RS, Newmark PA. The cell biology of regeneration. J Cell Biology. 2012;196:553–562. doi: 10.1083/jcb.201105099. - DOI - PMC - PubMed
    1. Knopf F, et al. Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev Cell. 2011;20:713–724. doi: 10.1016/j.devcel.2011.04.014. - DOI - PubMed
    1. Li F, Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol. 1996;28:1737–1746. doi: 10.1006/jmcc.1996.0163. - DOI - PubMed
    1. Engel FB, Schebesta M, Keating MT. Anillin localization defect in cardiomyocyte binucleation. J Mol Cell Cardiol. 2006;41:601–612. doi: 10.1016/j.yjmcc.2006.06.012. - DOI - PubMed

Publication types

LinkOut - more resources