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


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.


Progenitor cell Cardiomyocyte Differentiation Membrane potential Electrophysiology Biophysical signaling Calcineurin 



cardiomyocyte progenitor cell

ES cell

embryonic stem cell


inward rectifier current


potassium inward rectifier

KWGF cell

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


nuclear factor of activated T-cells


regulator of calcineurin 1


resting membrane potential


transforming growth factor beta



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)


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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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