Application Note

Channels: hERG, CaV1.2, NaV1.5
Cells:
human iPSC
Cardiomyocytes
Tools:
CardioExcyte 96

Impedance and EFP recordings of Pluricyte®
Cardiomyocytes on the CardioExcyte 96
The impedance team at Nanion Technologies GmbH, Munich.
Pluricyte® Cardiomyocytes kindly provided by Ncardia.
Summary

Results

Cardiomyocytes
pluripotent

derivedfrom

humaninduced

Pluricyte® Cardiomyocytes were grown on the NSP-96

stem cells (hiPSCs) are gaining interest inplates of the CardioExcyte 96. The cardiomyocytes formed

cardiac safety screening. Given their recapitulationa monolayer and started to beat synchronously, at whichof native behavior, availability, ease of use andpoint they were used for pharmacological experiments,

standardized production, they are likely to provide a

typically 7 - 12 days after plating. Figure 1 shows theviable alternative to acutely isolated cardiomyocytes

effect of nifedipine, a CaV channel blocker, and dofetilide,toassessthepro-arrhythmic

potentials

ofdruga hERG channel blocker, on the impedance and EFP

candidates. Although automated patch clamp cansignals of Pluricyte® Cardiomyocytes.

provideA

excellent information about theeffects ofcompounds on cardiac ion channels and possible
effectsonthecardiac

actionpotential1,

Nifedipine

Dofetilide

B

Nifedipine

Dofetilide

other

outputs such as extracellular field potential (EFP) andimpedance, also provide crucial and complementary
information about complex physiological parameterssuch as beat rate, amplitude and duration. The
CardioExcyte

96

isa

hybridscreeningtool

combining impedance (cell contractility) and EFP
recordings2,3. These measurements are non-invasive,
label-free and have a temporal resolution of 1 ms. The
recordings are made from cells within a network thus
providing a physiologically relevant environment formeasuring

drug-inducedchangesincontractile

parameters. This hybrid technology is an easy-to-use
screening tool which permits the reliable investigation ofshort- and long-term pharmacological effects.
Here we present data recorded on the CardioExcyte 96
using Pluricyte® Cardiomyocytes from Ncardia. The effects of the CaV blocker, nifedipine, hERG blockers, dofetilide and E4031, and NaV blocker, mexiletine, on EFP andimpedance parameters are shown.

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Figure 1: Effect of nifedipine and dofetilide on the impedance and EFP
signals of Pluricyte® Cardiomyocytes. A Impedance traces of six example
wells in control solution (red trace) and in the presence of 10 nM nifedipine(left, black trace) or 10 nM dofetilide (right, black trace). B EFP traces of six
example wells in control solution (red trace) and in the presence of 10 nM
nifedipine (left, black trace) or 10 nM dofetilide (right, black trace).

Application NoteNifedipine is an L-type calcium channel blocker which

Dofetilide is a hERG inhibitor which has been shown tocauses QT shortening in isolated heart4. In impedance

prolong cardiac action potential duration and induce

mode nifedipine caused a concentration-dependent

Torsade de Pointes (TdP)7,8,9. High concentrations (1 μM)

decrease in amplitude and pulsewidth 20 and an

induce early afterdepolarizations (EADs) in isolated rabbit

increase in beat rate as previously reportedandcardiomyocytes9. In impedance mode, dofetilide caused

shown in Figure 2A & C. Figure 2C shows the impedance

an increase in pulsewidth 20 which resulted in a decrease

parameters: amplitude, pulsewidth 20, beat rate andin beat rate (Figure 3A, C). In EFP mode, dofetilide caused

beat rate regularity displayed as a spider chart for bettera dramatic and concentration-dependent increase invisualization of the effect of the compound. Nifedipine

FPD which also resulted in a decrease in beat rate (Figure

had no effect on beat regularity. In EFP mode, nifedipine

3B, D).

3,5,6

caused a decrease in field potential duration (FPD) and an
and shown in Figure 2B & D. Figure 2D shows the EFP

A

regularity displayed as a spider chart for better
visualization of the effect of the compound.

0.5
0.4
0.3
0.2
0.1
0.0
0.0

Ccontrol B
153 nM
10 nM
1030 nM
5100 nM0-5-10-15
1.5
2.0
0.0

0.25
0.20
0.15
0.10
0.05
0.0

Nifedipine

BC

0.5

1.0
1.5
Time (s)

2.0

D30
20
100
-10-20-30
0.0

Dofetilide

0.5

1.0
1.5
Time (s)2.0

EFP (µV)

Impedance (Ω)

A

Impedance (Ω)

parameters: amplitude, FPD, beat rate and beat rate

control
3 nM
10 nM
30 nM

EFP (µV)

increase in beat rate in agreement with the literature2,3,5,60.5

1.0Time (s)

D0.5

1.0
1.5
Time (s)2.0

Figure 3: Effect of dofetilide on the impedance and EFP signals of Pluricyte®
Cardiomyocytes. A Impedance traces of an example well in increasing
concentrations of dofetilide. B EFP traces of an example well in increasing
concentrations of dofetilide. C Effect of dofetilide on impedance
parameters: amplitude, Pulsewidth 20, beat rate and beat rate regularity
shown as a spider chart. D Effect of dofetilide on EFP parameters: amplitude,FPD, beat rate and beat rate regularity shown as a spider chart.
Figure 2: Effect of nifedipine on the impedance and EFP signals of Pluricyte®
Cardiomyocytes. A Impedance traces of an example well in increasing
concentrations of nifedipine. B EFP traces of an example well in increasing
concentrations of nifedipine. C Effect of nifedipine on impedance
parameters: amplitude, Pulsewidth 20, beat rate and beat rate regularity
shown as a spider chart. D Effect of nifedipine on EFP parameters: amplitude,FPD, beat rate and beat rate regularity shown as a spider chart.

Nanion Technologies GmbHphone + 49 89 2190 95-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note

Impedance (Ω)

concentrations10. Furthermore, E4031 causes EADs in stem
cell-derived cardiomyocytes11. The effects of E4031 in
impedance and EFP modes are complex. In EFP mode,
there is an increase in FPD and a moderate increase inbeat rate, whereas little effect is seen on beat parameters

implemented new CiPA-approved protocols wherecontrol
3 nM
10 nM30 nM

1.2
1.0
0.8
0.6
0.4
0.2
0.00.0in impedance mode (Figure 4). This may be due to theirregular beating patterns induced by E4031. We have

BAblock hERG in patch clamp experiments at lowCMexiletine

20EFP (µV)

Similarly, E4031 is a specific hERG inhibitor shown to0-20-40-60

0.5

1.0Time (s)

1.5

0.0

2.0

0.5

1.0Time (s)

1.5

2.0D

we chose endpoints for analysis taken 30 min after
compound addition. The effects of individual compounds
develop over time and the discrepancies between thespecific hERG blockers, dofetilide and E4031, as depictedin the spider charts may be thus explained. To further
investigate this, analysis at multiple timepoints may be
implemented and coupled with additional patch clamp
experiments where hERG inhibition could be analyzed
directly in the voltage-clamp mode. This highlights theimportance of implementing complementary assays for
cardiac safety screens.
Bcontrol
3 nM
10 nM
30 nM

0.6
0.5
0.4
0.3
0.20.1

Mexiletine is a non-selective NaV channel blocker, simi-

100

lar to lidocaine, used as an anti-arrhythmic compound12.

-10

It is used to treat arrhythmias within the heart and may-20
0.0CE4031

20
EFP (µV)

Impedance (Ω)

A

0.5

1.0
1.5
Time (s)2.0

DFigure 5: Effect of mexiletine on the impedance and EFP signals of Pluricyte®
Cardiomyocytes. A Impedance traces of an example well in increasing
concentrations of mexiletine. B EFP traces of an example well in increasing
concentrations of mexiletine. C Effect of mexiletine on impedance
parameters: amplitude, Pulsewidth 20, beat rate and beat rate regularity
shown as a spider chart. D Effect of mexiletine on EFP parameters: amplitude,FPD, beat rate and beat rate regularity shown as a spider chart.

0.0

0.5

1.0 1.5
Time (s)2.0also be useful as an analgesic. Mexiletine caused amoderate decrease in amplitude in impedance mode
at higher concentrations (30 nM) and concentrationdependent decrease in beat rate (Figure 5A, C). In EFP
mode, mexiletine caused a dramatic, concentration-dependent decrease in spike amplitude, presumably due
to block of the NaV channels, and a decrease in beatrate (Figure 5B, D) as previously reported6.

Figure 4: Effect of E4031 on the impedance and EFP signals of Pluricyte®
Cardiomyocytes. A Impedance traces of an example well in increasing
concentrations of E4031. B EFP traces of an example well in increasing
concentrations of E4031. C Effect of E4031 on impedance parameters:
amplitude, Pulsewidth 20, beat rate and beat rate regularity shown as aspider chart. D Effect of E4031 on EFP parameters: amplitude, FPD, beatrate and beat rate regularity shown as a spider chart.

Nanion Technologies GmbHphone + 49 89 2190 95-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960

Application Note
In conclusion, the CardioExcyte 96 can be used tocomplementarytechniquessuchasautomatedreliably perform EFP and impedance measurements

patch clamp to obtain a more complete profile foron hiPSCs from Ncardia (Pluricyte® Cardiomyocytes).

cardiac safety and cardiotoxicity studies.

A variety of compounds were used, for example theCaV

blocker,nifedipine,decreased

amplitudeinWeareconfident

that

theCardioExcyte

96

inimpedance mode and increased beat rate in bothcombination with hiPSCs will have a great impact on theimpedance and EFP modes. The hERG blocker, dofetilide,field of pharmacology, toxicology, and cardiac safety

dramatically increased FPD in EFP mode and decreased

screening in the light of the CiPA (Comprehensive Inbeat rate in both impedance and EFP modes. The

Vitro Pro-Arrhythmia Assay) guidelines. This initiative aims

effects of different compounds on the impedance andto define a new, integrated pre-clinical in vitro/in silico

EFP signals can be complex, even though they may

paradigm in which the potential pro-arrhythmic risk of aexert their actions on the same ion channels, eg. dofetilide

new drug will be assessed using hiPSCs.

and E4031 inhibiting hERG. This highlights the importanceof combining experiments on the CardioExcyte 96 with

References

Methods

1. Stoelzle, S., et al., 2011. Front. Pharmacol. doi: 10.3389/

Cardiomyocytes

fphar.2011.00076.

Pluricyte® Cardiomyocytes were kindly provided by

2.

Doerr,L.,etal.,

2014.

J.

Lab.

Autom.

pii:

Ncardia.

2211068214562832.
3. Obergrussberger, A., et al., 2016. J. Pharmacol.

Impedance and EFP measurements

Toxicol. Methods. pii: S1056-8719(16)30033-8.

Impedance and EFP measurements were conducted

4. Guo, L., et al., 2011. Cell. Phys. Biochem. 27: 453-462

according to Nanion’s standard procedures for the5. Guo, L., et al., 2011. Toxicol. Sci. 123(1): 281-289.

CardioExcyte 96. Cardiomyocytes were seeded at 30,000

6. Clements, M., & Thomas, N., 2014. Toxicol. Sci. 140(2):viable cells per well on fibronectin-coated Sensor Plates.

445-461.

Pluricyte Cardiomyocyte Medium was exchanged every

7. Pham, T.V., et al., 2001. Circulation. 103(17): 2207-2212

other day until the conclusion of the assay. The cells were

8. Lu, H.R., et al., 2008. Br. J. Pharmacol. 154(7): 1427-

typically used 7 - 12 days after plating. This enabled the1438

formation of a dense monolayer and a suitable network

9. Nalos, L., et al., 2012. .Br. J. Pharmacol.165: 467-478

of

10. Kirsch, G.E., et al., 2004. J. Pharmacol.Toxicol.

propagation of the excitation and synchronized beating.

Methods. 50(2): 93–101

At least 4 hours before drug application the medium was

11. Peng, S., et al., 2010. J. Pharmacol. Toxicol. Methods.

completely removed from the wells and 100 μl fresh61(3): 277–286

medium was added. For the experiments, a 2x

12. Canavero, S., & Bonicalzi, V., 2011. Central Pain

concentration of compound was prepared, 50 μl of

Syndrome: Pathophysiology, Diagnosis and Manage-solution was removed from the well and 50 μl of the 2xment. Cambridge University Press. pp. 286

compound was added to the wells. Measurements werecell-cellinterconnections

toensureasteadytaken after 30 minutes incubation. All signals werenormalized to a group of control measurements (n=5-11)
on the same plate using DMSO (0.01%) as vehicle.

Nanion Technologies GmbHphone + 49 89 2190 95-0

Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960