Skip to main content
SpringerLink
Log in

Influence of Electromechanical Activity on Cardiac Differentiation of Mouse Embryonic Stem Cells

  • Published:
Cardiovascular Engineering and Technology Aims and scope Submit manuscript

Abstract

During differentiation, mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) receive electromechanical cues from spontaneous beating. Therefore, promoting electromechanical activity via electrical pacing or suppressing it by drug treatment might affect the cellular functional development. Electrical pacing was applied to confluent monolayers of mESC-CMs during late-stage differentiation (days 16–18). Alternatively, spontaneous contraction was suppressed by (a) blocking ion currents with CsCl (HCN channel), trazodone (T-type Ca2+ channel), or both CsCl and trazodone on days 11–18; or (b) applying blebbistatin (excitation–contraction uncoupler) on days 11–14. Electrophysiological properties and gene expression were examined on day 19 and 18, respectively. Optical mapping revealed no significant difference in conduction velocity (CV) in paced vs. non-paced monolayers, nor were there significant changes in gene expression of connexin-43, Na–Ca exchanger (NCX), or myosin heavy chain (MHC). However, CV variability among differentiation batches and CV heterogeneity within individual monolayers were significantly lower in paced mESC-CMs. Alternatively, while the four drug treatments suppressed contraction with varying degrees (up to complete inhibition), there was no significant difference in CV for any of the treatments compared with controls. Trazodone treatment significantly reduced CV variability as compared to controls, whereas CsCl treatment significantly reduced CV heterogeneity. Distinct changes in gene expression of connexin-43, MHC, HCNl, Cav3.1/3.2 were not observed. Electrical pacing, but not suppression of spontaneous contraction, during late-stage differentiation reduces the intrinsic variability of CV among differentiation batches and across individual monolayers, which can be beneficial in the application of ESCs for myocardial tissue repair.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Anwar, A., G. Taimor, H. Korkususz, R. Schreckenberg, T. Berndt, Y. Abdallah, H. M. Piper, and K. D. Schlüter. PKC-independent signal transduction pathways increase SERCA2 expression in adult rat cardiomyocytes. J. Mol. Cell Cardiol. 39:911–919, 2005.

    Article  Google Scholar 

  2. Au, H. T., I. Cheng, M. F. Chowdhury, and M. Radisic. Interactive effects of surface topography and pulsatile electrical field stimulation on orientation and elongation of fibroblasts and cardiomyocytes. Biomaterials 28:4277–4293, 2007.

    Article  Google Scholar 

  3. Berger, H. J., S. K. Prasad, A. J. Davidoff, D. Pimental, O. Ellingsen, J. D. Marsh, T. W. Smith, and R. A. Kelly. Continual electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture. Am. J. Physiol. 266:H341–H349, 1994.

    Google Scholar 

  4. Caspi, O., A. Lesman, Y. Basevitch, A. Gepstein, G. Arbel, I. H. Habib, L. Gepstein, and S. Levenberg. Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circ. Res. 100:263–272, 2007.

    Article  Google Scholar 

  5. Chen, M. Q., X. Xie, R. Hollis Whittington, G. T. Kovacs, J. C. Wu, and L. Giovangrandi. Cardiac differentiation of embryonic stem cells with point-source electrical stimulation. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2008:1729–1732, 2008.

    Google Scholar 

  6. Fedorov, V. V., I. T. Lozinsky, E. A. Sosunov, E. P. Anyukhovsky, M. R. Rosen, C. W. Balke, and I. R. Efimov. Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts. Heart Rhythm 4:619–626, 2007.

    Article  Google Scholar 

  7. Fu, J. D., H. M. Yu, R. Wang, J. Liang, and H. T. Yang. Developmental regulation of intracellular calcium transients during cardiomyocyte differentiation of mouse embryonic stem cells. Acta Pharmacol. Sin. 27:901–910, 2006.

    Article  Google Scholar 

  8. Genovese, J. A., C. Spadaccio, J. Langer, J. Habe, J. Jackson, and A. N. Patel. Electrostimulation induces cardiomyocyte predifferentiation of fibroblasts. Biochem. Biophys. Res. Commun. 370:450–455, 2008.

    Article  Google Scholar 

  9. Genovese, J. A., C. Spadaccio, E. Chachques, O. Schussler, A. Carpentier, J. C. Chachques, and A. N. Patel. Cardiac pre-differentiation of human mesenchymal stem cells by electrostimulation. Front Biosci. 14:2996–3002, 2009.

    Article  Google Scholar 

  10. Griffin, M. A., S. Sen, H. L. Sweeney, and D. E. Discher. Adhesion-contractile balance in myocyte differentiation. J. Cell Sci. 117:5855–5863, 2004.

    Article  Google Scholar 

  11. Gryshchenko, O., I. R. Fischer, M. Dittrich, S. Viatchenko-Karpinski, J. Soest, M. M. Bohm-Pinger, P. Igelmund, B. K. Fleischmann, and J. Hescheler. Role of ATP-dependent K(+) channels in the electrical excitability of early embryonic stem cell-derived cardiomyocytes. J. Cell Sci. 112(Pt 17):2903–2912, 1999.

    Google Scholar 

  12. Guo, A., and H. T. Yang. Ca2+ removal mechanisms in mouse embryonic stem cell-derived cardiomyocytes. Am. J. Physiol. Cell Physiol. 297:C732–C741, 2009.

    Article  Google Scholar 

  13. Heng, B. C., H. Haider, E. K. Sim, T. Cao, and S. C. Ng. Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. Cardiovasc. Res. 62:34–42, 2004.

    Article  Google Scholar 

  14. Heron, M., D. L. Hoyert, S. L. Murphy, J. Q. Xu, K. D. Kochanek, and B. Tejada-Vera. Deaths: Final Data for 2006. Hyattsville, MD: U.S. Department of Health and Human Services, National Center for Health Statistics, 2009.

    Google Scholar 

  15. Hescheler, J., B. K. Fleischmann, S. Lentini, V. A. Maltsev, J. Rohwedel, A. M. Wobus, and K. Addicks. Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc. Res. 36:149–162, 1997.

    Article  Google Scholar 

  16. Inoue, N., T. Ohkusa, T. Nao, J. K. Lee, T. Matsumoto, Y. Hisamatsu, T. Satoh, M. Yano, K. Yasui, I. Kodama, and M. Matsuzaki. Rapid electrical stimulation of contraction modulates gap junction protein in neonatal rat cultured cardiomyocytes: involvement of mitogen-activated protein kinases and effects of angiotensin II-receptor antagonist. J. Am. Coll. Cardiol. 44:914–922, 2004.

    Google Scholar 

  17. Itzhaki, I., J. Schiller, R. Beyar, J. Satin, and L. Gepstein. Calcium handling in embryonic stem cell-derived cardiac myocytes: of mice and men. Ann. NY Acad. Sci. 1080:207–215, 2006.

    Article  Google Scholar 

  18. Kapur, N., and K. Banach. Inositol-1, 4, 5-trisphosphate-mediated spontaneous activity in mouse embryonic stem cell-derived cardiomyocytes. J. Physiol. 581:1113–1127, 2007.

    Article  Google Scholar 

  19. Klug, M. G., M. H. Soonpaa, G. Y. Koh, and L. J. Field. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin. Invest. 98:216–224, 1996.

    Article  Google Scholar 

  20. Kolossov, E., Z. Lu, I. Drobinskaya, N. Gassanov, Y. Duan, H. Sauer, O. Manzke, W. Bloch, H. Bohlen, J. Hescheler, and B. K. Fleischmann. Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes. FASEB J. 19:577–579, 2005.

    Google Scholar 

  21. Kolossov, E., T. Bostani, W. Roell, M. Breitbach, F. Pillekamp, J. M. Nygren, P. Sasse, O. Rubenchik, J. W. Fries, D. Wenzel, C. Geisen, Y. Xia, Z. Lu, Y. Duan, R. Kettenhofen, S. Jovinge, W. Bloch, H. Bohlen, A. Welz, J. Hescheler, S. E. Jacobsen, and B. K. Fleischmann. Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium. J. Exp. Med. 203:2315–2327, 2006.

    Article  Google Scholar 

  22. Kraus, R. L., Y. Li, A. Jovanovska, and J. J. Renger. Trazodone inhibits T-type calcium channels. Neuropharmacology 53:308–317, 2007.

    Article  Google Scholar 

  23. Lammers, W. J., M. J. Schalij, C. J. Kirchhof, and M. A. Allessie. Quantification of spatial inhomogeneity in conduction and initiation of reentrant atrial arrhythmias. Am. J. Physiol. 259:H1254–H1263, 1990.

    Google Scholar 

  24. Martens, J. C., and M. Radmacher. Softening of the actin cytoskeleton by inhibition of myosin II. Pflugers Arch. 456:95–100, 2008.

    Article  Google Scholar 

  25. Nichol, J. W., G. C. Engelmayr, Jr., M. Cheng, and L. E. Freed. Co-culture induces alignment in engineered cardiac constructs via MMP-2 expression. Biochem. Biophys. Res. Commun. 373:360–365, 2008.

    Article  Google Scholar 

  26. Pedrotty, D. M., J. Koh, B. H. Davis, D. A. Taylor, P. Wolf, and L. E. Niklason. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. Am. J. Physiol. Heart Circ. Physiol. 288:H1620–H1626, 2005.

    Article  Google Scholar 

  27. Radisic, M., H. Park, H. Shing, T. Consi, F. J. Schoen, R. Langer, L. E. Freed, and G. Vunjak-Novakovic. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds. Proc. Natl Acad. Sci. USA 101:18129–18134, 2004.

    Article  Google Scholar 

  28. Sathaye, A. S. Effects of Long Term Electrical Stimulation on the Electrophysiological Development of Neonatal Rat Ventricular Myocytes [Master’s thesis]. Johns Hopkins University, 2003.

  29. Sauer, H., G. Rahimi, J. Hescheler, and M. Wartenberg. Effects of electrical fields on cardiomyocyte differentiation of embryonic stem cells. J. Cell Biochem. 75:710–723, 1999.

    Article  Google Scholar 

  30. Tung, L., and Y. Zhang. Optical imaging of arrhythmias in tissue culture. J. Electrocardiol. 39:S2–S6, 2006.

    Article  Google Scholar 

  31. Weiss, J. N., Z. Qu, P. S. Chen, S. F. Lin, H. S. Karagueuzian, H. Hayashi, A. Garfinkel, and A. Karma. The dynamics of cardiac fibrillation. Circulation 112:1232–1240, 2005.

    Article  Google Scholar 

  32. Wobus, A. M., K. Guan, H. T. Yang, and K. R. Boheler. Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods Mol. Biol. 185:127–156, 2002.

    Google Scholar 

  33. Xia, Y., J. B. McMillin, A. Lewis, M. Moore, W. G. Zhu, R. S. Williams, and R. E. Kellems. Electrical stimulation of neonatal cardiac myocytes activates the NFAT3 and GATA4 pathways and up-regulates the adenylosuccinate synthetase 1 gene. J. Biol. Chem. 275:1855–1863, 2000.

    Article  Google Scholar 

  34. Yanagi, K., M. Takano, G. Narazaki, H. Uosaki, T. Hoshino, T. Ishii, T. Misaki, and J. K. Yamashita. Hyperpolarization-activated cyclic nucleotide-gated channels and T-type calcium channels confer automaticity of embryonic stem cell-derived cardiomyocytes. Stem Cells 25:2712–2719, 2007.

    Article  Google Scholar 

  35. Zhang, Y. M., L. Shang, C. Hartzell, M. Narlow, L. Cribbs, and S. C. Dudley, Jr. Characterization and regulation of T-type Ca2+ channels in embryonic stem cell-derived cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. 285:H2770–H2779, 2003.

    Google Scholar 

  36. Zimmermann, W. H. Tissue engineering: polymers flex their muscles. Nat. Mater. 7:932–933, 2008.

    Article  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Peter He for providing statistical advice. This work was supported by a Johns Hopkins Provost’s Undergraduate Research Award (W.L.), NIH training grant T32-HL07581 (E.A.L.; A. Shoukas, principal investigator), funds from the Johns Hopkins Institute for Cell Engineering (J.G.), NIH R01 HL066239 (L.T.), and grant 0855330E from the Mid-Atlantic Affiliate of the American Heart Association (L.T.).

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Worawan Limpitikul, Susan A. Thompson & Leslie Tung

  2. Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA

    Nicolas Christoforou

  3. Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    John D. Gearhart

  4. Department of Chemical Engineering, Auburn University, Auburn, AL, 36849, USA

    Elizabeth A. Lipke

Authors
  1. Worawan Limpitikul
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicolas Christoforou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susan A. Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John D. Gearhart
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Leslie Tung
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elizabeth A. Lipke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Leslie Tung.

Additional information

Associate Editor Ajit P. Yoganathan oversaw the review of this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Real-time movie showing spontaneously contracting confluent monolayer of mESC-CMs. Movie was taken at 10×. (MPG 3632 kb)

Online Resource 2

Optical mapping movie showing wave propagation of non-paced mESC-CMs. Spectrum of colors represents normalized voltage from blue (lowest) to red (highest). Scale bar (2 mm) represents physical length on the coverslip. Wave propagation of nonpaced mESC-CMs is less smooth compared to that of paced mESC-CMs, showing higher local heterogeneity of mESC-CMs which corresponds to higher HI. (MPG 1961 kb)

Online Resource 3

Optical mapping movie showing wave propagation of paced mESC-CMs. Spectrum of colors represents normalized voltage from blue (lowest) to red (highest). Scale bar (2 mm) represents physical length on the coverslip. Wave propagation of paced mESC-CMs is smoother than that of paced mESC-CMs, showing less local heterogeneity of mESC-CMs which corresponds to lower HI. (MPG 2303 kb)

Online Resource 4

Reverse transcription PCR (RT-PCR) of control and paced mESC-CMs. Figures show products of GAPDH, connexin-43 (Cx43), Na-Ca2+ exchanger (NCX), α-myosin heavy chain (α-MHC), and β-myosin heavy chain (β -MHC). “n” represents nonpaced or control and “p” represents paced mESC-CMs. All gene products shown in this figure are from the same differentiation. There is no apparent change in the level of expression in any of the four genes in paced mESC-CMs, compared to the controls. Similar results were observed in other two differentiations. (TIFF 94 kb)

Online Resource 5

Real-time (quantitative) RT-PCR of connexin-43 expression in control and paced mESC-CMs. The amount of connexin-43 expression in paced mESC-CMs increased by 2 to 2.5-fold in 2 differentiations and was relatively the same in 1 differentiation. (TIFF 179 kb)

Online Resource 6

Immunohistochemistry for connexin-43 and cardiac troponin I in control and paced mESC-CMs on day 19 following optical mapping. Left panel is control and right panel is paced mESC-CMs. The distribution of both connexin-43 and cardiac troponin I was similar for control and paced mESC-CMs. The morphology of the mESC-CMs was also similar for control and paced mESC-CMs. Immunofluorescent images were taken at 20×. (TIFF 2258 kb)

Online Resource 7

Reverse transcription PCR (RT-PCR) of control and drug-treated mESC-CMs. Figures show products of GAPDH, α-myosin heavy chain (α-MHC), β-myosin heavy chain (β-MHC), HCN channel type 1 (HCN1), T-type Ca2+ channels subtype 3.1 and 3.2 (Cav3.1, Cav3.2), and connexin-43 (Cx43). “Ct,” “B,” “Cs,” and “T” represent control, blebbistatin-treated, CsCl-treated, and trazodone-treated mESC-CMs, respectively. Gene products shown in the first two rows of this figure are from the same differentiation and the third row is from a different differentiation from the first two rows. Except for connexin-43 (third row), there is no apparent change in the level of expression in any of the other aforementioned genes in drug-treated mESC-CMs, compared to the controls. Similar results to the first two rows were observed in other two differentiations. Although there is no apparent change in level of connexin-43 expression in blebbistatin-treated and trazodone-treated mESC-CMs compared to the control, the level of connexin-43 expression in CsCl-treated mESC-CMs is inconsistent among differentiations- the level increased in two differentiations (as shown in this figure) but decreased in a third differentiation. (TIFF 5991 kb)

Online Resource 8

Phase contrast micrographs of control and blebbistatin-treated mESC-CMs on day 14. In the presence of blebbistatin, mESC-CMs retracted from each other, exhibiting a smaller cell footprint, than in control. However, the morphology of mESC-CMs in the presence of CsCl or trazodone did not change compared with the control. Micrographs were taken at 10×. (TIFF 4500 kb)

Online Resource 9

Local and global cell alignment of mESC-CMs. (A) and (B) are phase-contrast micrographs (taken at 10×) of control and blebbistatin-treated mESC-CMs, respectively. Dotted-line boxes enclose the areas with local alignment of cells with their direction depicted by two-headed arrows. Notice that blebbistatin-treated mESC-CMs show local cell alignment while control mESC-CMs do not. However, local cell alignment of blebbistatin-treated mESC-CMs varies from region to region; hence, the absence of global cell alignment. (C) Fluorescence images (taken at 20×) of blebbistatin-treated mESC-CMs positively stained with α-sarcomeric actinin, a cardiac structural protein. All four images were taken at different locations on the same monolayer. During imaging, the monolayer was maintained in the same orientation relative to the camera. Although local cell alignment was observed, these photomicrographs show regional differences in cell orientation. (TIFF 10449 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Limpitikul, W., Christoforou, N., Thompson, S.A. et al. Influence of Electromechanical Activity on Cardiac Differentiation of Mouse Embryonic Stem Cells. Cardiovasc Eng Tech 1, 179–193 (2010). https://doi.org/10.1007/s13239-010-0020-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13239-010-0020-8

Keywords

Search

Navigation