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.
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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.).
Associate Editor Ajit P. Yoganathan oversaw the review of this article.
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Real-time movie showing spontaneously contracting confluent monolayer of mESC-CMs. Movie was taken at 10×. (MPG 3632 kb)
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)
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)
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)
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)
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)
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)
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)
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)
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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) doi:10.1007/s13239-010-0020-8
- Electrical stimulation
- Cell culture
- Optical mapping
- Cardiac regeneration