Stem Cell Reviews and Reports

, Volume 13, Issue 5, pp 631–643 | Cite as

A Singular Role of IK1 Promoting the Development of Cardiac Automaticity during Cardiomyocyte Differentiation by IK1 Induced Activation of Pacemaker Current

  • Yu Sun
  • Valeriy Timofeyev
  • Adrienne Dennis
  • Emre Bektik
  • Xiaoping Wan
  • Kenneth R. Laurita
  • Isabelle Deschênes
  • Ronald A. LiEmail author
  • Ji-Dong FuEmail author


The inward rectifier potassium current (IK1) is generally thought to suppress cardiac automaticity by hyperpolarizing membrane potential (MP). We recently observed that IK1 could promote the spontaneously-firing automaticity induced by upregulation of pacemaker funny current (If) in adult ventricular cardiomyocytes (CMs). However, the intriguing ability of IK1 to activate If and thereby promote automaticity has not been explored. In this study, we combined mathematical and experimental assays and found that only IK1 and If, at a proper-ratio of densities, were sufficient to generate rhythmic MP-oscillations even in unexcitable cells (i.e. HEK293T cells and undifferentiated mouse embryonic stem cells [ESCs]). We termed this effect IK1-induced If activation. Consistent with previous findings, our electrophysiological recordings observed that around 50% of mouse (m) and human (h) ESC-differentiated CMs could spontaneously fire action potentials (APs). We found that spontaneously-firing ESC-CMs displayed more hyperpolarized maximum diastolic potential and more outward IK1 current than quiescent-yet-excitable m/hESC-CMs. Rather than classical depolarization pacing, quiescent mESC-CMs were able to fire APs spontaneously with an electrode-injected small outward-current that hyperpolarizes MP. The automaticity to spontaneously fire APs was also promoted in quiescent hESC-CMs by an IK1-specific agonist zacopride. In addition, we found that the number of spontaneously-firing m/hESC-CMs was significantly decreased when If was acutely upregulated by Ad-CGI-HCN infection. Our study reveals a novel role of IK1 promoting the development of cardiac automaticity in m/hESC-CMs through a mechanism of IK1-induced If activation and demonstrates a synergistic interaction between IK1 and If that regulates cardiac automaticity.


IK1 If Rhythmic oscillation Automaticity Embryonic stem cell Cardiomyocyte differentiation 



We are grateful to Dr. Jill Dunham for editorial assistance. This work was supported by the Start-up Fund from The MetroHealth System (to J.D.F.) and grants from the American Heart Association-13SDG14580035 (to J.D.F.), the Research Grant Council (TRS T13-706/11 to R.A.L.) and the National Institutes of Health (NIH)-R01HL096962 (to I.D.), NIH-R21HL123012 (to K.R.L.).

Compliance with Ethical Standards


No disclosure of conflict interest.

Supplementary material

12015_2017_9745_MOESM1_ESM.docx (629 kb)
Figure S1 (DOCX 629 kb)
12015_2017_9745_MOESM2_ESM.docx (835 kb)
Figure S2 (DOCX 834 kb)
12015_2017_9745_MOESM3_ESM.docx (322 kb)
Figure S3 (DOCX 322 kb)
12015_2017_9745_MOESM4_ESM.docx (118 kb)
Supplemental Table 1 (DOCX 117 kb)


  1. 1.
    Christoffels, V. M., Smits, G. J., Kispert, A., & Moorman, A. F. (2010). Development of the pacemaker tissues of the heart. Circulation Research, 106, 240–254.CrossRefPubMedGoogle Scholar
  2. 2.
    Mangoni, M. E., & Nargeot, J. (2008). Genesis and regulation of the heart automaticity. Physiological Reviews, 88, 919–982.CrossRefPubMedGoogle Scholar
  3. 3.
    Wilders, R. (2007). Computer modelling of the sinoatrial node. Medical & Biological Engineering & Computing, 45, 189–207.CrossRefGoogle Scholar
  4. 4.
    Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F., & Biel, M. (1998). A family of hyperpolarization-activated mammalian cation channels. Nature, 393, 587–591.CrossRefPubMedGoogle Scholar
  5. 5.
    DiFrancesco, D., & Noble, D. (2012). The funny current has a major pacemaking role in the sinus node. Heart Rhythm, 9, 299–301.CrossRefPubMedGoogle Scholar
  6. 6.
    DiFrancesco, D. (2010). The role of the funny current in pacemaker activity. Circulation Research, 106, 434–446.CrossRefPubMedGoogle Scholar
  7. 7.
    Cohen, I. S., & Robinson, R. B. (2006). Pacemaker current and automatic rhythms: Toward a molecular understanding. Handbook of Experimental Pharmacology, 41–71.Google Scholar
  8. 8.
    Qu, J., Barbuti, A., Protas, L., Santoro, B., Cohen, I. S., & Robinson, R. B. (2001). HCN2 overexpression in newborn and adult ventricular myocytes: Distinct effects on gating and excitability. Circulation Research, 89, E8–14.CrossRefPubMedGoogle Scholar
  9. 9.
    Qu, J., Plotnikov, A. N., Danilo Jr., P., et al. (2003). Expression and function of a biological pacemaker in canine heart. Circulation, 107, 1106–1109.CrossRefPubMedGoogle Scholar
  10. 10.
    Tse, H. F., Xue, T., Lau, C. P., et al. (2006). Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN Channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation, 114, 1000–1011.CrossRefPubMedGoogle Scholar
  11. 11.
    Kubo, Y., Baldwin, T. J., Jan, Y. N., & Jan, L. Y. (1993). Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature, 362, 127–133.CrossRefPubMedGoogle Scholar
  12. 12.
    Tourneur, Y., Mitra, R., Morad, M., & Rougier, O. (1987). Activation properties of the inward-rectifying potassium channel on mammalian heart cells. The Journal of Membrane Biology, 97, 127–135.CrossRefPubMedGoogle Scholar
  13. 13.
    Ibarra, J., Morley, G. E., & Delmar, M. (1991). Dynamics of the inward rectifier K+ current during the action potential of guinea pig ventricular myocytes. Biophysical Journal, 60, 1534–1539.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Miake, J., Marban, E., & Nuss, H. B. (2003). Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression. The Journal of Clinical Investigation, 111, 1529–1536.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Shinagawa, Y., Satoh, H., & Noma, A. (2000). The sustained inward current and inward rectifier K+ current in pacemaker cells dissociated from rat sinoatrial node. The Journal of Physiology, 523(Pt 3), 593–605.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Cho, H. S., Takano, M., & Noma, A. (2003). The electrophysiological properties of spontaneously beating pacemaker cells isolated from mouse sinoatrial node. The Journal of Physiology, 550, 169–180.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Guo, J., Mitsuiye, T., & Noma, A. (1997). The sustained inward current in sino-atrial node cells of guinea-pig heart. Pflügers Archiv, 433, 390–396.CrossRefPubMedGoogle Scholar
  18. 18.
    Zaritsky, J. J., Redell, J. B., Tempel, B. L., & Schwarz, T. L. (2001). The consequences of disrupting cardiac inwardly rectifying K(+) current (I(K1)) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes. The Journal of Physiology, 533, 697–710.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Plaster, N. M., Tawil, R., Tristani-Firouzi, M., et al. (2001). Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell, 105, 511–519.CrossRefPubMedGoogle Scholar
  20. 20.
    Liu, A., Tang, M., Xi, J., et al. (2010). Functional characterization of inward rectifier potassium ion channel in murine fetal ventricular cardiomyocytes. Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology, 26, 413–420.CrossRefGoogle Scholar
  21. 21.
    Masuda, H., & Sperelakis, N. (1993). Inwardly rectifying potassium current in rat fetal and neonatal ventricular cardiomyocytes. The American Journal of Physiology, 265, H1107–H1111.PubMedGoogle Scholar
  22. 22.
    Fu, J. D., Jiang, P., Rushing, S., Liu, J., Chiamvimonvat, N., & Li, R. A. (2010). Na+/Ca2+ exchanger is a determinant of excitation-contraction coupling in human embryonic stem cell-derived ventricular cardiomyocytes. Stem Cells and Development, 19, 773–782.CrossRefPubMedGoogle Scholar
  23. 23.
    Lieu, D. K., Fu, J. D., Chiamvimonvat, N., et al. (2013). Mechanism-based facilitated maturation of human pluripotent stem cell-derived cardiomyocytes. Circulation. Arrhythmia and Electrophysiology, 6, 191–201.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Fu, J. D., Rushing, S. N., Lieu, D. K., et al. (2011). Distinct roles of microRNA-1 and -499 in ventricular specification and functional maturation of human embryonic stem cell-derived cardiomyocytes. PloS One, 6, e27417.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kehat, I., Kenyagin-Karsenti, D., Snir, M., et al. (2001). Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. The Journal of Clinical Investigation, 108, 407–414.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Mummery, C., Ward-van Oostwaard, D., Doevendans, P., et al. (2003). Differentiation of human embryonic stem cells to cardiomyocytes: Role of coculture with visceral endoderm-like cells. Circulation, 107, 2733–2740.CrossRefPubMedGoogle Scholar
  27. 27.
    Xue, T., Cho, H. C., Akar, F. G., et al. (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.CrossRefPubMedGoogle Scholar
  28. 28.
    He, J. Q., Ma, Y., Lee, Y., Thomson, J. A., & Kamp, T. J. (2003). Human embryonic stem cells develop into multiple types of cardiac myocytes: Action potential characterization. Circulation Research, 93, 32–39.CrossRefPubMedGoogle Scholar
  29. 29.
    Miake, J., Marban, E., & Nuss, H. B. (2002). Biological pacemaker created by gene transfer. Nature, 419, 132–133.CrossRefPubMedGoogle Scholar
  30. 30.
    Chan, Y. C., Siu, C. W., Lau, Y. M., Lau, C. P., Li, R. A., & Tse, H. F. (2009). Synergistic effects of inward rectifier (I) and pacemaker (I) currents on the induction of bioengineered cardiac automaticity. Journal of Cardiovascular Electrophysiology, 20, 1048–1054.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Saito, Y., Nakamura, K., Yoshida, M., et al. (2015). Enhancement of spontaneous activity by HCN4 overexpression in mouse embryonic stem cell-derived Cardiomyocytes - a possible biological pacemaker. PloS One, 10, e0138193.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Wobus, A. M., Guan, K., Yang, H. T., & Boheler, K. R. (2002). Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods in Molecular Biology, 185, 127–156.PubMedGoogle Scholar
  33. 33.
    Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., & Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nature Biotechnology, 18, 399–404.CrossRefPubMedGoogle Scholar
  34. 34.
    Xue, T., Siu, C. W., Lieu, D. K., Lau, C. P., Tse, H. F., & Li, R. A. (2007). Mechanistic role of I(f) revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels. Circulation, 115, 1839–1850.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wang, K., Xue, T., Tsang, S. Y., et al. (2005). Electrophysiological properties of pluripotent human and mouse embryonic stem cells. Stem Cells, 23, 1526–1534.CrossRefPubMedGoogle Scholar
  36. 36.
    Ma, J., Guo, L., Fiene, S. J., et al. (2011). High purity human-induced pluripotent stem cell-derived cardiomyocytes: Electrophysiological properties of action potentials and ionic currents. American Journal of Physiology. Heart and Circulatory Physiology, 301, H2006–H2017.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Dokos, S., Celler, B., & Lovell, N. (1996). Ion currents underlying sinoatrial node pacemaker activity: A new single cell mathematical model. Journal of Theoretical Biology, 181, 245–272.CrossRefPubMedGoogle Scholar
  38. 38.
    Liu, Q. H., Li, X. L., Xu, Y. W., Lin, Y. Y., Cao, J. M., & Wu, B. W. (2012). A novel discovery of IK1 channel agonist: Zacopride selectively enhances IK1 current and suppresses triggered arrhythmias in the rat. Journal of Cardiovascular Pharmacology, 59, 37–48.CrossRefPubMedGoogle Scholar
  39. 39.
    Johnson, M. A., Weick, J. P., Pearce, R. A., & Zhang, S. C. (2007). Functional neural development from human embryonic stem cells: Accelerated synaptic activity via astrocyte coculture. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 27, 3069–3077.CrossRefGoogle Scholar
  40. 40.
    Kamino, K., Hirota, A., & Fujii, S. (1981). Localization of pacemaking activity in early embryonic heart monitored using voltage-sensitive dye. Nature, 290, 595–597.CrossRefPubMedGoogle Scholar
  41. 41.
    Van Mierop, L. H. (1967). Location of pacemaker in chick embryo heart at the time of initiation of heartbeat. The American Journal of Physiology, 212, 407–415.PubMedGoogle Scholar
  42. 42.
    Konig, S., Hinard, V., Arnaudeau, S., et al. (2004). Membrane hyperpolarization triggers myogenin and myocyte enhancer factor-2 expression during human myoblast differentiation. The Journal of Biological Chemistry, 279, 28187–28196.CrossRefPubMedGoogle Scholar
  43. 43.
    Hoshino, S., Omatsu-Kanbe, M., Nakagawa, M., & Matsuura, H. (2012). Postnatal developmental decline in IK1 in mouse ventricular myocytes isolated by the Langendorff perfusion method: Comparison with the chunk method. Pflügers Archiv, 463, 649–668.CrossRefPubMedGoogle Scholar
  44. 44.
    Lieu, D. K., Chan, Y. C., Lau, C. P., Tse, H. F., Siu, C. W., & Li, R. A. (2008). Overexpression of HCN-encoded pacemaker current silences bioartificial pacemakers. Heart Rhythm, 5, 1310–1317.CrossRefPubMedGoogle Scholar
  45. 45.
    Moore, J. C., Fu, J., Chan, Y. C., et al. (2008). Distinct cardiogenic preferences of two human embryonic stem cell (hESC) lines are imprinted in their proteomes in the pluripotent state. Biochemical and Biophysical Research Communications, 372, 553–558.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Mummery, C. L., Zhang, J., Ng, E. S., Elliott, D. A., Elefanty, A. G., & Kamp, T. J. (2012). Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: A methods overview. Circulation Research, 111, 344–358.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Kim, K., Doi, A., Wen, B., et al. (2010). Epigenetic memory in induced pluripotent stem cells. Nature, 467, 285–290.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Sanchez-Freire, V., Lee, A. S., Hu, S., et al. (2014). Effect of human donor cell source on differentiation and function of cardiac induced pluripotent stem cells. Journal of the American College of Cardiology, 64, 436–448.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Lakatta, E. G., Maltsev, V. A., & Vinogradova, T. M. (2010). A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart's pacemaker. Circulation Research, 106, 659–673.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Yaniv, Y., Lakatta, E. G., & Maltsev, V. A. (2015). From two competing oscillators to one coupled-clock pacemaker cell system. Frontiers in Physiology, 6, 28.PubMedPubMedCentralGoogle Scholar
  51. 51.
    Vaidyanathan, R., Markandeya, Y. S., Kamp, T. J., Makielski, J. C., January, C. T., & Eckhardt, L. L. (2016). IK1-enhanced human-induced pluripotent stem cell-derived cardiomyocytes: An improved cardiomyocyte model to investigate inherited arrhythmia syndromes. American Journal of Physiology. Heart and Circulatory Physiology, 310, H1611–H1621.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Medicine, Heart and Vascular Research CenterMetroHealth Campus, Case Western Reserve UniversityClevelandUSA
  2. 2.Department of Internal MedicineUniversity of CaliforniaDavisUSA
  3. 3.Ph.D. Program in Human Biology, School of Integrative and Global MajorsUniversity of TsukubaTsukubaJapan
  4. 4.Dr. Li Dak-Sum Center for Regenerative MedicineUniversity of Hong Kong, The Hong Kong Jockey Club Building for Interdisciplinary ResearchPokfulamHong Kong
  5. 5.Ming-Wai Lau Center for Regenerative MedicineKarolinska InstitutetSolnaSweden

Personalised recommendations