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Effects of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers on the proliferation and cell cycle progression of embryonic stem cells

  • Signaling and Cell Physiology
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Abstract

Embryonic stem cells (ESCs) can uniquely proliferate indefinitely and differentiate into all cell lineages. ESCs may therefore provide an unlimited supply of cells for cell-based therapies. Previous study reported the presence of hyperpolarization-activated inward currents in undifferentiated mouse (m) ESCs, but the functional role of this hyperpolarization-activated current in mESCs is unknown. In this study, the role of this current in maintaining the proliferative capacity and the cell cycle progression of ESCs was investigated. In D3 mESCs, this hyperpolarization-activated inward current can be blocked by HCN channel blocker ZD7288. Application of the HCN channel blockers, cesium (1–10 mM) or ZD7288 (0.1–30 μM), attenuated cell proliferation in a concentration-dependent manner. Both HCN blockers were found to be non-cytotoxic to mESCs as determined by cell viability test. Interestingly, ZD7288 at 10 and 30 μM was found to decrease the proportion of cells in G0/G1 phase and increase the proportion of cells in S phase. This suggests that this hyperpolarization-activated current can affect the cell cycle progression in mESCs. In summary, the present investigation suggests that ESC proliferation and cell cycle progression can be regulated by this hyperpolarization-activated current.

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Abbreviations

ESCs:

Embryonic stem cells

HCN channel:

Hyperpolarization-activated cyclic nucleotide-gated channel

TBP:

TATA-box binding protein

References

  1. Aponte Y, Lien CC, Reisinger E, Jonas P (2006) Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus. J Physiol 574:229–243

    Article  CAS  PubMed  Google Scholar 

  2. Barrow AJ, Wu SM (2009) Low-conductance HCN1 ion channels augment the frequency response of rod and cone photoreceptors. J Neurosci 29:5841–5853

    Article  CAS  PubMed  Google Scholar 

  3. Bartek J, Lukas J (2001) Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr Opin Cell Biol 13:738–747

    Article  CAS  PubMed  Google Scholar 

  4. Baruscotti M, Bucchi A, Difrancesco D (2005) Physiology and pharmacology of the cardiac pacemaker (“funny”) current. Pharmacol Ther 107:59–79

    Article  CAS  PubMed  Google Scholar 

  5. Behfar A, Perez-Terzic C, Faustino RS, Arrell DK, Hodgson DM, Yamada S, Puceat M, Niederlander N, Alekseev AE, Zingman LV, Terzic A (2007) Cardiopoietic programming of embryonic stem cells for tumor-free heart repair. J Exp Med 204:405–420

    Article  CAS  PubMed  Google Scholar 

  6. Biel M, Wahl-Schott C, Michalakis S, Zong X (2009) Hyperpolarization-activated cation channels: from genes to function. Physiol Rev 89:847–885

    Article  CAS  PubMed  Google Scholar 

  7. Bolivar JJ, Tapia D, Arenas G, Castanon-Arreola M, Torres H, Galarraga E (2008) A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells. Am J Physiol Cell Physiol 294:C893–906

    Article  CAS  PubMed  Google Scholar 

  8. Chen J, Mitcheson JS, Tristani-Firouzi M, Lin M, Sanguinetti MC (2001) The S4-S5 linker couples voltage sensing and activation of pacemaker channels. Proc Natl Acad Sci USA 98:11277–11282

    Article  CAS  PubMed  Google Scholar 

  9. Felix R, Sandoval A, Sanchez D, Gomora JC, De la Vega-Beltran JL, Trevino CL, Darszon A (2003) ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function. Biochem Biophys Res Commun 311:187–192

    Article  CAS  PubMed  Google Scholar 

  10. Fujii-Yamamoto H, Kim JM, Arai K, Masai H (2005) Cell cycle and developmental regulations of replication factors in mouse embryonic stem cells. J Biol Chem 280:12976–12987

    Article  CAS  PubMed  Google Scholar 

  11. Gonzalez-Iglesias AE, Kretschmannova K, Tomic M, Stojilkovic SS (2006) ZD7288 inhibits exocytosis in an HCN-independent manner and downstream of voltage-gated calcium influx in pituitary lactotrophs. Biochem Biophys Res Commun 346:845–850

    Article  CAS  PubMed  Google Scholar 

  12. Jiang YQ, Sun Q, Tu HY, Wan Y (2008) Characteristics of HCN channels and their participation in neuropathic pain. Neurochem Res 33:1979–1989

    Article  CAS  PubMed  Google Scholar 

  13. Jirmanova L, Afanassieff M, Gobert-Gosse S, Markossian S, Savatier P (2002) Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells. Oncogene 21:5515–5528

    Article  CAS  PubMed  Google Scholar 

  14. Kapur N, Mignery GA, Banach K (2007) Cell cycle-dependent calcium oscillations in mouse embryonic stem cells. Am J Physiol Cell Physiol 292:C1510–1518

    Article  CAS  PubMed  Google Scholar 

  15. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 108:407–414

    CAS  PubMed  Google Scholar 

  16. Leor J, Gerecht S, Cohen S, Miller L, Holbova R, Ziskind A, Shachar M, Feinberg MS, Guetta E, Itskovitz-Eldor J (2007) Human embryonic stem cell transplantation to repair the infarcted myocardium. Heart 93:1278–1284

    Article  PubMed  Google Scholar 

  17. Li YL, Tran TP, Muelleman R, Schultz HD (2008) Blunted excitability of aortic baroreceptor neurons in diabetic rats: involvement of hyperpolarization-activated channel. Cardiovasc Res 79:715–721

    Article  CAS  PubMed  Google Scholar 

  18. Liu N, Lu M, Tian X, Han Z (2007) Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells. J Cell Physiol 211:279–286

    Article  CAS  PubMed  Google Scholar 

  19. Maric D, Maric I, Barker JL (1998) Buoyant density gradient fractionation and flow cytometric analysis of embryonic rat cortical neurons and progenitor cells. Methods 16:247–259

    Article  CAS  PubMed  Google Scholar 

  20. Mistrik P, Mader R, Michalakis S, Weidinger M, Pfeifer A, Biel M (2005) The murine HCN3 gene encodes a hyperpolarization-activated cation channel with slow kinetics and unique response to cyclic nucleotides. J Biol Chem 280:27056–27061

    Article  CAS  PubMed  Google Scholar 

  21. Ng SY, Chin CH, Lau YT, Luo J, Wong CK, Bian ZX, Tsang SY (2010) Role of voltage-gated potassium channels in the fate determination of embryonic stem cells. J Cell Physiol 224:165–177

    CAS  PubMed  Google Scholar 

  22. Nussbaum J, Minami E, Laflamme MA, Virag JA, Ware CB, Masino A, Muskheli V, Pabon L, Reinecke H, Murry CE (2007) Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J 21:1345–1357

    Article  CAS  PubMed  Google Scholar 

  23. Petkova-Kirova P, Gagov H, Krien U, Duridanova D, Noack T, Schubert R (2000) 4-Aminopyridine affects rat arterial smooth muscle BK(Ca) currents by changing intracellular pH. Br J Pharmacol 131:1643–1650

    Article  CAS  PubMed  Google Scholar 

  24. Proenza C, Angoli D, Agranovich E, Macri V, Accili EA (2002) Pacemaker channels produce an instantaneous current. J Biol Chem 277:5101–5109

    Article  CAS  PubMed  Google Scholar 

  25. Proenza C, Yellen G (2006) Distinct populations of HCN pacemaker channels produce voltage-dependent and voltage-independent currents. J Gen Physiol 127:183–190

    Article  CAS  PubMed  Google Scholar 

  26. Robinson RB, Siegelbaum SA (2003) Hyperpolarization-activated cation currents: from molecules to physiological function. Annu Rev Physiol 65:453–480

    Article  CAS  PubMed  Google Scholar 

  27. Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, Tibbs GR (1998) Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93:717–729

    Article  CAS  PubMed  Google Scholar 

  28. Stead E, White J, Faast R, Conn S, Goldstone S, Rathjen J, Dhingra U, Rathjen P, Walker D, Dalton S (2002) Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E and E2F activities. Oncogene 21:8320–8333

    Article  CAS  PubMed  Google Scholar 

  29. Stieber J, Stockl G, Herrmann S, Hassfurth B, Hofmann F (2005) Functional expression of the human HCN3 channel. J Biol Chem 280:34635–34643

    Article  CAS  PubMed  Google Scholar 

  30. Swijnenburg RJ, Tanaka M, Vogel H, Baker J, Kofidis T, Gunawan F, Lebl DR, Caffarelli AD, de Bruin JL, Fedoseyeva EV, Robbins RC (2005) Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium. Circulation 112:I166–172

    PubMed  Google Scholar 

  31. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

    Article  CAS  PubMed  Google Scholar 

  32. Tsang SY, Moore JC, Huizen RV, Chan CW, Li RA (2007) Ectopic expression of systemic RNA interference defective protein in embryonic stem cells. Biochem Biophys Res Commun 357:480–486

    Article  CAS  PubMed  Google Scholar 

  33. Varghese A, Tenbroek EM, Coles J Jr, Sigg DC (2006) Endogenous channels in HEK cells and potential roles in HCN ionic current measurements. Prog Biophys Mol Biol 90:26–37

    Article  CAS  PubMed  Google Scholar 

  34. Vemana S, Pandey S, Larsson HP (2008) Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels. Am J Physiol Cell Physiol 295:C557–565

    Article  CAS  PubMed  Google Scholar 

  35. Wang K, Xue T, Tsang SY, Van Huizen R, Wong CW, Lai KW, Ye Z, Cheng L, Au KW, Zhang J, Li GR, Lau CP, Tse HF, Li RA (2005) Electrophysiological properties of pluripotent human and mouse embryonic stem cells. Stem Cells 23:1526–1534

    Article  CAS  PubMed  Google Scholar 

  36. White J, Dalton S (2005) Cell cycle control of embryonic stem cells. Stem Cell Rev 1:131–138

    Article  CAS  PubMed  Google Scholar 

  37. Wong C, Stearns T (2005) Mammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure. BMC Cell Biol 6:6

    Article  PubMed  Google Scholar 

  38. Xue T, Cho HC, Akar FG, Tsang SY, Jones SP, Marban E, Tomaselli GF, Li RA (2005) Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 111:11–20

    Article  PubMed  Google Scholar 

  39. Yanagida E, Shoji S, Hirayama Y, Yoshikawa F, Otsu K, Uematsu H, Hiraoka M, Furuichi T, Kawano S (2004) Functional expression of Ca2+ signaling pathways in mouse embryonic stem cells. Cell Calcium 36:135–146

    Article  CAS  PubMed  Google Scholar 

  40. Zhang YM, Shang L, Hartzell C, Narlow M, Cribbs L, Dudley SC Jr (2003) Characterization and regulation of T-type Ca2+ channels in embryonic stem cell-derived cardiomyocytes. Am J Physiol Heart Circ Physiol 285:H2770–2779

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by the Direct Grant for Research from the Chinese University of Hong Kong (CUHK) and the Lee Hysan Foundation Research Grant. Y.T.L. and C.K.W. were supported by postgraduate studentships from the CUHK. We would like to thank Ms Cecilia Sze-Lee Leung and Dr. Winnie Poon for their excellent technical support.

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Authors and Affiliations

  1. Biochemistry Program, School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China

    Yuen-Ting Lau, Chun-Kit Wong, Pui-Fong Tsang & Suk-Ying Tsang

  2. Cell and Molecular Biology Program, School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China

    Suk-Ying Tsang

  3. State Key Laboratory of Agrobiotechnology, Chinese University of Hong Kong, Hong Kong, China

    Suk-Ying Tsang

  4. Stem Cell and Regeneration Program, School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China

    Suk-Ying Tsang

  5. Key Laboratory for Regenerative Medicine, Ministry of Education, Chinese University of Hong Kong, Hong Kong, China

    Suk-Ying Tsang

  6. Molecular Biotechnology Program, School of Life Sciences, Chinese University of Hong Kong, Hong Kong, China

    Yuen-Ting Lau

  7. School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China

    Jialie Luo & Zhao-Xiang Bian

  8. Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China

    Lok-Hang Leung

  9. School of Life Sciences, Chinese University of Hong Kong, Shatin, NT, Hong Kong, China

    Suk-Ying Tsang

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  1. Yuen-Ting Lau
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Correspondence to Suk-Ying Tsang.

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Lau, YT., Wong, CK., Luo, J. et al. Effects of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers on the proliferation and cell cycle progression of embryonic stem cells. Pflugers Arch - Eur J Physiol 461, 191–202 (2011). https://doi.org/10.1007/s00424-010-0899-9

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  • DOI: https://doi.org/10.1007/s00424-010-0899-9

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