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Stem Cell Reviews and Reports

, Volume 6, Issue 2, pp 178–185 | Cite as

Hyperpolarization Induces Differentiation in Human Cardiomyocyte Progenitor Cells

  • Patrick van Vliet
  • Teun P. de Boer
  • Marcel A. G. van der Heyden
  • Mazen K. El Tamer
  • Joost P. G. Sluijter
  • Pieter A. Doevendans
  • Marie-José GoumansEmail author
Article

Abstract

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. These cells can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells and are therefore of great value for investigation of the mechanisms that drive progenitor cell function and plasticity, drug testing and, potentially, therapeutical purposes. In this respect, most studies have focused on enhancing differentiation with chemicals or growth factors, or co-culture with other cell types. Although they have revealed important mechanisms, protocols need to be established that exclude the need for such factors when one considers using progenitor cells to repair the human heart. In this study we tested whether we could induce cardiomyogenic differentiation of human cardiomyocyte progenitor cells (CMPCs) by altering their membrane potential. We induced hyperpolarization in CMPCs by either co-culturing them with a Kir2.1-overexpressing cell line or by overnight culture in medium containing low potassium concentrations. Hyperpolarization led to increased intracellular calcium concentrations, activation of calcineurin signaling, increased cardiac-specific gene and protein expression levels and, ultimately, to the formation of spontaneously beating cardiomyocytes. Thus, hyperpolarization is sufficient to induce differentiation of CMPCs, thereby revealing a novel mechanism for cardiomyogenic differentiation of heart-derived progenitor cells.

Keywords

Progenitor cell Cardiomyocyte Differentiation Membrane potential Electrophysiology Biophysical signaling Calcineurin 

Abbreviations

CMPC

cardiomyocyte progenitor cell

ES cell

embryonic stem cell

IK1

inward rectifier current

Kir

potassium inward rectifier

KWGF cell

HEK 293 cell stably expressing murine wild-type Kir2.1-GFP fusion protein

NFAT

nuclear factor of activated T-cells

RCAN1

regulator of calcineurin 1

RMP

resting membrane potential

TGFβ

transforming growth factor beta

Notes

Acknowledgements

We are very grateful to Corina Metz, Tom Korfage, Pieter Glerum and Lukas Nalos for technical assistance and Dr. Marta Roccio for valuable comments. We want to thank Dr. Leon de Windt for helpful discussions and for providing us with the adenovirus. This work was supported by a VIDI grant (016.056.319) from the Netherlands Organization for Scientific Research (NWO), the Van Ruyven foundation, the BSIK program “Dutch Program for Tissue Engineering” and the Netherlands Heart Foundation (2003B073 and 2005T102).

Conflict of Interest

The authors declare no potential conflicts of interest.

Supplementary material

12015_2010_9142_MOESM1_ESM.doc (32 kb)
Supplemental Table 1 Primer sequences and annealing temperatures (DOC 32 kb)
12015_2010_9142_Fig5_ESM.gif (34 kb)
Supplemental figure 1

IK1-mediated differentiation in CMPCs. (A) Inhibition of gap junctional coupling by halothane did not result in a change of the RMP in CMPCs alone (n=6). (B) Co-culture of CMPCs with an increased relative number of KWGF cells (from 0 to 5 or 25%) resulted in a dose-dependent increased expression of troponin T, βMHC and αHCA mRNA 2 weeks later. (GIF 34 kb)

12015_2010_9142_MOESM2_ESM.tif (516 kb)
High resolution image (TIFF 516 kb)
12015_2010_9142_Fig6_ESM.gif (10 kb)
Supplemental figure 2

Effect of low potassium on CMPC resting membrane potential. Representative patch-clamp traces of data in figure 2A showing the RMP of CMPCs exposed to normal (5 mM, Ctrl) or low (1.5 mM, LowK) extracellular potassium concentrations. (GIF 10 kb)

12015_2010_9142_MOESM3_ESM.tif (185 kb)
High resolution image (TIFF 184 kb)
12015_2010_9142_Fig7_ESM.gif (16 kb)
Supplemental figure 3

Dose-dependent hyperpolarization-induced gene expression. RNA was isolated from cardiomyocytes that were derived from CMPCs that underwent one (1x), two (2x) or three (3x) hyperpolarizations in week one. Subsequent quantitative RT-PCR analysis showed a dose-dependent increase in expression of troponin T and βMHC in CMPC-derived cardiomyocytes (*** p<0.001 versus Ctrl). (GIF 16 kb)

12015_2010_9142_MOESM4_ESM.tif (268 kb)
High resolution image (TIFF 267 kb)
12015_2010_9142_Fig8_ESM.gif (10 kb)
Supplemental figure 4

Effect of low potassium on CMPC resting membrane potential. Representative patch-clamp traces of data in figure 2E showing the RMP of CMPC control (Ctrl) and CMPC-derived cardiomyocytes after hyperpolarization-induced differentiation (LowK). (GIF 9 kb)

12015_2010_9142_MOESM5_ESM.tif (217 kb)
High resolution image (TIFF 217 kb)
Supplemental Movie

Spontaneous beating clusters of CMPC-derived cardiomyocytes 5 weeks after induction of differentiation by hyperpolarization. (MPG 9004 kb)

References

  1. 1.
    Harvey, R. P. (2002). Patterning the vertebrate heart. Nature Reviews Genetics, 3, 544–556.CrossRefPubMedGoogle Scholar
  2. 2.
    Bergmann, O., Bhardwaj, R. D., Bernard, S., et al. (2009). Evidence for cardiomyocyte renewal in humans. Science, 324, 98–102.CrossRefPubMedGoogle Scholar
  3. 3.
    Messina, E., De Angelis, L., Frati, G., et al. (2004). Isolation and expansion of adult cardiac stem cells from human and murine heart. Circulation Research, 95, 911–921.CrossRefPubMedGoogle Scholar
  4. 4.
    Smith, R. R., Barile, L., Cho, H. C., et al. (2007). Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens. Circulation, 115, 896–908.CrossRefPubMedGoogle Scholar
  5. 5.
    Goumans, M. J., de Boer, T. P., Smits, A. M., et al. (2007). TGF-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro. Stem Cell Research, 1, 138–149.CrossRefPubMedGoogle Scholar
  6. 6.
    Bearzi, C., Rota, M., Hosoda, T., et al. (2007). Human cardiac stem cells. Proceedings of the National Academy of Sciences of the United States of America, 104, 14068–14073.CrossRefPubMedGoogle Scholar
  7. 7.
    Bu, L., Jiang, X., Martin-Puig, S., et al. (2009). Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature, 460, 113–117.CrossRefPubMedGoogle Scholar
  8. 8.
    van Vliet, P., Sluijter, J. P., Doevendans, P. A., & Goumans, M. J. (2007). Isolation and expansion of resident cardiac progenitor cells. Expert Review of Cardiovascular Therapy, 5, 33–43.CrossRefPubMedGoogle Scholar
  9. 9.
    Roccio, M., Goumans, M. J., Sluijter, J. P., & Doevendans, P. A. (2008). Stem cell sources for cardiac regeneration. Panminerva Medica, 50, 19–30.PubMedGoogle Scholar
  10. 10.
    Heng, B. C., Haider, H. K., Sim, E. K., Cao, T., & Ng, S. C. (2004). Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. Cardiovascular Research, 62, 34–42.CrossRefPubMedGoogle Scholar
  11. 11.
    van Vliet, P., Roccio, M., Smits, A. M., et al. (2008). Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy. Netherlands Heart Journal, 16, 163–169.PubMedGoogle Scholar
  12. 12.
    Adams, D. S. (2008). A new tool for tissue engineers: ions as regulators of morphogenesis during development and regeneration. Tissue Engineering Part A, 14, 1461–1468.CrossRefPubMedGoogle Scholar
  13. 13.
    Sundelacruz, S., Levin, M., & Kaplan, D. L. (2009). Role of membrane potential in the regulation of cell proliferation and differentiation. Stem Cell Reviews and Reports, 5, 231–246.CrossRefPubMedGoogle Scholar
  14. 14.
    Smits, A. M., van Vliet, P., Metz, C. H., et al. (2009). Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nature Protocols, 4, 232–243.CrossRefPubMedGoogle Scholar
  15. 15.
    de Boer, T. P., van Veen, T. A., Houtman, M. J., et al. (2006). Inhibition of cardiomyocyte automaticity by electrotonic application of inward rectifier current from Kir2.1 expressing cells. Medical & Biological Engineering & Computing, 44, 537–542.CrossRefGoogle Scholar
  16. 16.
    Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., Huet, S., & Gauthier, J. M. (1998). Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO Journal, 17, 3091–3100.CrossRefPubMedGoogle Scholar
  17. 17.
    de Boer, T. P., van Veen, T. A., Jonsson, M. K., et al. (2010). Human cardiomyocyte progenitor cell-derived cardiomyocytes display a maturated electrical phenotype. Journal of Molecular and Cellular Cardiology, 48, 254–260.CrossRefPubMedGoogle Scholar
  18. 18.
    Konig, S., Beguet, A., Bader, C. R., & Bernheim, L. (2006). The calcineurin pathway links hyperpolarization (Kir2.1)-induced Ca2+ signals to human myoblast differentiation and fusion. Development, 133, 3107–3114.CrossRefPubMedGoogle Scholar
  19. 19.
    Lagostena, L., Avitabile, D., De Falco, E., et al. (2005). Electrophysiological properties of mouse bone marrow c-kit+ cells co-cultured onto neonatal cardiac myocytes. Cardiovascular Research, 66, 482–492.CrossRefPubMedGoogle Scholar
  20. 20.
    Yasuda, T., Bartlett, P. F., & Adams, D. J. (2008). K(ir) and K(v) channels regulate electrical properties and proliferation of adult neural precursor cells. Molecular and Cellular Neuroscience, 37, 284–297.CrossRefPubMedGoogle Scholar
  21. 21.
    Chilton, L., Ohya, S., Freed, D., et al. (2005). K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts. American Journal of Physiology - Heart and Circulatory Physiology, 288, H2931–H2939.CrossRefPubMedGoogle Scholar
  22. 22.
    Erdogan, A., Schaefer, C. A., Schaefer, M., et al. (2005). Margatoxin inhibits VEGF-induced hyperpolarization, proliferation and nitric oxide production of human endothelial cells. Journal of Vascular Research, 42, 368–376.CrossRefPubMedGoogle Scholar
  23. 23.
    Bernheim, L., & Bader, C. R. (2002). Human myoblast differentiation: Ca(2+) channels are activated by K(+) channels. News in Physiological Sciences, 17, 22–26.PubMedGoogle Scholar
  24. 24.
    Sundelacruz, S., Levin, M., & Kaplan, D. L. (2008). Membrane potential controls adipogenic and osteogenic differentiation of mesenchymal stem cells. PLoS One, 3, e3737.CrossRefPubMedGoogle Scholar
  25. 25.
    Berridge, M. J., Lipp, P., & Bootman, M. D. (2000). The versatility and universality of calcium signalling. Nature Reviews Molecular Cell Biology, 1, 11–21.CrossRefPubMedGoogle Scholar
  26. 26.
    Schulz, R. A., & Yutzey, K. E. (2004). Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development. Developmental Biology, 266, 1–16.CrossRefPubMedGoogle Scholar
  27. 27.
    Sato, M., Suzuki, K., Yamazaki, H., & Nakanishi, S. (2005). A pivotal role of calcineurin signaling in development and maturation of postnatal cerebellar granule cells. Proceedings of the National Academy of Sciences of the United States of America, 102, 5874–5879.CrossRefPubMedGoogle Scholar
  28. 28.
    Chen, Y., & Cao, X. (2009). NFAT directly regulates Nkx2-5 transcription during cardiac cell differentiation. Biology of the Cell, 101, 335–349.CrossRefPubMedGoogle Scholar
  29. 29.
    Bijlenga, P., Liu, J. H., Espinos, E., et al. (2000). T-type alpha 1H Ca2+ channels are involved in Ca2+ signaling during terminal differentiation (fusion) of human myoblasts. Proceedings of the National Academy of Sciences of the United States of America, 97, 7627–7632.CrossRefPubMedGoogle Scholar
  30. 30.
    Berridge, M. J., Bootman, M. D., & Roderick, H. L. (2003). Calcium signalling: dynamics, homeostasis and remodelling. Nature Reviews Molecular Cell Biology, 4, 517–529.CrossRefPubMedGoogle Scholar
  31. 31.
    Ferreira-Martins, J., Rondon-Clavo, C., Tugal, D., et al. (2009). Spontaneous calcium oscillations regulate human cardiac progenitor cell growth. Circulation Research, 105, 764–774.CrossRefPubMedGoogle Scholar
  32. 32.
    Kawano, S., Shoji, S., Ichinose, S., Yamagata, K., Tagami, M., & Hiraoka, M. (2002). Characterization of Ca(2+) signaling pathways in human mesenchymal stem cells. Cell Calcium, 32, 165–174.CrossRefPubMedGoogle Scholar
  33. 33.
    Sun, S., Liu, Y., Lipsky, S., & Cho, M. (2007). Physical manipulation of calcium oscillations facilitates osteodifferentiation of human mesenchymal stem cells. FASEB Journal, 21, 1472–1480.CrossRefPubMedGoogle Scholar
  34. 34.
    Klagsbrun, M., & D’Amore, P. A. (1991). Regulators of angiogenesis. Annual Review of Physiology, 53, 217–239.CrossRefPubMedGoogle Scholar
  35. 35.
    Scharbrodt, W., Kuhlmann, C. R., Wu, Y., et al. (2004). Basic fibroblast growth factor-induced endothelial proliferation and NO synthesis involves inward rectifier K+ current. Arteriosclerosis, Thrombosis, and Vascular Biology, 24, 1229–1233.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Patrick van Vliet
    • 1
    • 2
    • 3
  • Teun P. de Boer
    • 4
  • Marcel A. G. van der Heyden
    • 4
  • Mazen K. El Tamer
    • 4
  • Joost P. G. Sluijter
    • 1
    • 2
  • Pieter A. Doevendans
    • 1
    • 2
  • Marie-José Goumans
    • 1
    • 5
    Email author
  1. 1.Department of Cardiology, Division Heart & LungsUniversity Medical Center UtrechtUtrechtthe Netherlands
  2. 2.Interuniversity Cardiology Institute Netherlands (ICIN)Utrechtthe Netherlands
  3. 3.Department of Anatomy & EmbryologyLeiden University Medical CenterLeidenthe Netherlands
  4. 4.Department of Medical Physiology, Division Heart & LungsUniversity Medical Center UtrechtUtrechtthe Netherlands
  5. 5.Department of Molecular Cell BiologyLeiden University Medical CenterLeidenthe Netherlands

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