Advertisement

Cytotechnology

, Volume 68, Issue 4, pp 763–770 | Cite as

Cardiac cell proliferation assessed by EdU, a novel analysis of cardiac regeneration

  • Bin ZengEmail author
  • Suiyang Tong
  • Xiaofeng Ren
  • Hao XiaEmail author
Original Research

Abstract

Emerging evidence suggests that mammalian hearts maintain the capacity for cardiac regeneration. Rapid and sensitive identification of cardiac cellular proliferation is prerequisite for understanding the underlying mechanisms and strategies of cardiac regeneration. The following immunologically related markers of cardiac cells were analyzed: cardiac transcription factors Nkx2.5 and Gata 4; specific marker of cardiomyocytes TnT; endothelial cell marker CD31; vascular smooth muscle marker smooth muscle myosin IgG; cardiac resident stem cells markers IsL1, Tbx18, and Wt1. Markers were co-localized in cardiac tissues of embryonic, neonatal, adult, and pathological samples by 5-ethynyl-2′-deoxyuridine (EdU) staining. EdU was also used to label isolated neonatal cardiomyocytes in vitro. EdU robustly labeled proliferating cells in vitro and in vivo, co-immunostaining with different cardiac cells markers. EdU can rapidly and sensitively label proliferating cardiac cells in developmental and pathological states. Cardiac cell proliferation assessed by EdU is a novel analytical tool for investigating the mechanism and strategies of cardiac regeneration in response to injury.

Keywords

Cardiac cells Proliferation EdU Regeneration Injury 

Notes

Acknowledgments

This work was supported by the Chinese National Nature Science Foundation (30900609, 81270271).

Conflict of interest

The authors do not have any possible conflicts of interest.

Supplementary material

10616_2014_9827_MOESM1_ESM.ppt (1000 kb)
Supplementary Fig. 1: EdU-labeled cardiac proliferating cells in adult hearts at E10.5, E17.5, and neonatal mice at 7 days (×40). The highest red fluorescence in the atrial and ventricular walls and ventricular septum (VS) in the fetus; staining was noticeably reduced after birth (PPT 1000 kb)

References

  1. Anversa P, Kajstura J (1998) Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res 13:1–14CrossRefGoogle Scholar
  2. Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R, Nadal-Ginard B, Silvestri F, Leri A, Beltrami CA, Anversa P (2001) Evidence that human cardiac myocytes divided after myocardial infarction. N Eng J Med 344:1750–1757CrossRefGoogle Scholar
  3. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabé-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S, Frisén J (2009) Evidence for cardiomyocyte renewal in humans. Science 324:98–102CrossRefGoogle Scholar
  4. Buck SB, Bradford J, Gee KR, Agnew BJ, Clarke ST, Salic A (2008) Detection of S-phase cell cycle progression using 5-ethynyl-2′-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2′-deoxyuridine antibodies. Biotechniques 44:927–929CrossRefGoogle Scholar
  5. Cappella P, Gasparri F, Pulici M, Moll J (2008) A novel method based on click chemistry, which overcomes limitations of cell cycle analysis byclassical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry A 73:626–636CrossRefGoogle Scholar
  6. Chehrehasa F, Meedeniya AC, Dwyer P, Abrahamsen G, Mackay-Sim A (2009) EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. J Neurosci Methods 15:122–130CrossRefGoogle Scholar
  7. Chen K, Bai H, Arzigian M, Gao YX, Bao J, Wu WS, Shen WF, Wu L, Wang ZZ (2010) Endothelial cells regulate cardiomyocyte development from embryonic stem cells. J Cell Biochem 111:29–39CrossRefGoogle Scholar
  8. Dodou E, Verzi MP, Anderson JP, Xu SM, Black BL (2004) Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development. Development 131:3931–3942CrossRefGoogle Scholar
  9. Formigli L, Francini F, Nistri S, Margheri M, Luciani G, Naro F, Silvertown JD, Orlandini SZ, Meacci E, Bani D (2009) Skeletal myoblasts overexpressing relaxin improve differentiation and communication of primary murine cardiomyocyte cell cultures. J Mol Cell Cardiol 47:335–345CrossRefGoogle Scholar
  10. Guner-Ataman B, Paffett-Lugassy N, Adams MS, Nevis KR, Jahangiri L, Obregon P, Kikuchi K, Poss KD, Burns CE, Burns CG (2013) Zebrafish second heart field development relies on progenitor specification in anterior lateral plate mesoderm and nkx2.5 function. Development 140:1353–1363CrossRefGoogle Scholar
  11. Harvey RP (1996) NK-2 homeobox genes and heart development. Dev Biol 178:203–216CrossRefGoogle Scholar
  12. Hsieh PC, Segers VF, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT (2007) Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 13:970–974CrossRefGoogle Scholar
  13. Hsu TL, Hanson SR, Kishikawa K, Wang SK, Sawa M, Wong CH (2007) Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc Natl Acad Sci USA 20:2614–2619CrossRefGoogle Scholar
  14. Jopling C, Sleep E, Raya M, Martí M, Raya A, Izpisúa Belmonte JC (2010) Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 25:606–609CrossRefGoogle Scholar
  15. Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, Egnaczyk GF, Evans T, Macrae CA, Stainier DY, Poss KD (2010) Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature 25:601–605CrossRefGoogle Scholar
  16. Lagrue E, Abe H, Lavanya M, Touhami J, Bodard S, Chalon S, Battini JL, Sitbon M, Castelnau P (2010) Regional characterization of energy metabolism in the brain of normal and MPTP-intoxicated mice using new markers of glucose and phosphate transport. J Biomed Sci 17:91CrossRefGoogle Scholar
  17. Lepilina A, Coon AN, Kikuchi K, Holdway JE, Roberts RW, Burns CG, Poss KD (2006) A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127:607–619CrossRefGoogle Scholar
  18. Linask KK, Lash JW (1993) Early heart development: dynamics of endocardial cell sorting suggests a common origin with cardiomyocytes. Dev Dyn 196:62–69CrossRefGoogle Scholar
  19. Narmoneva DA, Vukmirovic R, Davis ME, Kamm RD, Lee RT (2004) Endothelial cells promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration. Circulation 110:962–968CrossRefGoogle Scholar
  20. Nassiri SM, Khaki Z, Soleimani M, Ahmadi SH, Jahanzad I, Rabbani S, Sahebjam M, Ardalan FA, Fathollahi MS (2007) The similar effect of transplantation of marrow-derived mesenchymal stem cells with or without prior differentiation induction in experimental myocardial infarction. J Biomed Sci 14:745–755CrossRefGoogle Scholar
  21. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN, Sadek HA (2011) Transient regenerative potential of the neonatal mouse heart. Science 25:1078–1080CrossRefGoogle Scholar
  22. Qu D, Wang G, Wang Z, Zhou L, Chi W, Cong S, Ren X, Liang P, Zhang B (2011) 5-Ethynyl-2′-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells. Anal Biochem 417:112–121CrossRefGoogle Scholar
  23. Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA 19:2415–2420CrossRefGoogle Scholar
  24. Saravanakumar M, Devaraj H (2012) Notch signalling in cardiovasculogenesis: insight into their role in early cardiovasculardevelopment. Mol Biol Rep 40:3537–3547CrossRefGoogle Scholar
  25. Shu T, Zeng B, Ren X, Li Y (2010) HO-1 modified mesenchymal stem cells modulate MMPs/TIMPs system and adverse remodeling in infarcted myocardium. Tissue Cell 42:217–222CrossRefGoogle Scholar
  26. Studzinski GP, Harrison LE (1999) Differentiation-related changes in the cell cycle traverse. Int Rev Cytol 189:1–58CrossRefGoogle Scholar
  27. Wada R, Muraoka N, Inagawa K, Yamakawa H, Miyamoto K, Sadahiro T, Umei T, Kaneda R, Suzuki T, Kamiya K, Tohyama S, Yuasa S, Kokaji K, Aeba R, Yozu R, Yamagishi H, Kitamura T, Fukuda K, Ieda M (2013) Induction of human cardiomyocyte-like cells from fibroblasts by defined factors. Proc Natl Acad Sci USA 110:12667–12672CrossRefGoogle Scholar
  28. Yang J, Xia J, He Y, Zhao J, Zhang G (2013) MSCs transplantation with application of G-CSF reduces apoptosis or increases VEGF in rabbit model of myocardial infarction. Cytotechnology. doi: 10.1007/s10616-013-9655-2 Google Scholar
  29. Zeng B, Chen H, Zhu C, Ren X, Lin G, Cao F (2008) Effects of combined mesenchymal stem cells and heme oxygenase-1 therapy on cardiac performance. Eur J Cardiothorac Surg 34:850–856CrossRefGoogle Scholar
  30. Zeng B, Lin G, Ren X, Zhang Y, Chong H (2010a) Over-expression of HO-1 on mesenchymal stem cells promotes angiogenesis and improves myocardial function in infarcted myocardium. J Biomed Sci 17:80CrossRefGoogle Scholar
  31. Zeng C, Pan F, Jones LA, Lim MM, Griffin EA, Sheline YI, Mintun MA, Holtzman DM, Mach RH (2010b) Evaluation of 5-ethynyl-2′-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system. Brain Res 1319:21–32CrossRefGoogle Scholar
  32. Zeng B, Ren XF, Cao F, Zhou XY, Zhang J (2011) Developmental patterns and characteristics of epicardial cell markers Tbx18 and Wt1 in murine embryonic heart. J Biomed Sci 18:67CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Department of CardiologyRenmin Hospital of Wuhan UniversityWuhanPeople’s Republic of China
  2. 2.Department of Developmental and Regenerative BiologyMount sinai school of medicineNew YorkUSA
  3. 3.College of Veterinary MedicineNortheast Agricultural UniversityHarbinPeople’s Republic of China

Personalised recommendations