Application Note

Channels:
Cells:

hERG, CaV1.2
human iPS
Cardiomyocytes
CardioExcyte 96

Tools:

Impedance and EFP recordings of Cor.4U®
cells using Nanion’s CardioExcyte 96
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cells kindly provided by Ncardia.

Summary

Results
inducedBlebbistatin affects the contraction of cardiomyocytes

stem cells (hiPSCs) are gaining interest in(impedance signal), but does not interfere with the EFP3,4.

Cardiomyocytes
pluripotent

derivedfrom

humanrelative

Having no effect on ion channels, blebbistatin is anandinhibitor of myosin II which is the regulatory subunit ofstandardized production, they are likely to provide athe myosin filament and primarily responsible for theviable alternative to acutely isolated cardiomyocytes

contraction of cardiomyocytes.

cardiacsafetyabundance,

toassessscreening.

availability,

theGivenease

pro-arrhythmic

theirofuse

potentials

of

drug

before compound application
after compound application

excellent information about theeffects ofcompounds on cardiac ion channels and possible
effectsontechniques,crucialandthecardiacsuchasactionpotential1,impedance,complementary

other

also

provideinformation

about

complex physiological parameters such as beat rate,

600
EFP [µV]

provideImpedance [Ohm]candidates. Although automated patch clamp can-2-4-6

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-induced changes in beat parameters.
This hybrid technology addresses the lack of easy-touse high-throughput screening for in-vitro assays, andpermits the reliable investigation of short- and long-termpharmacological effects.
Herewepresentdata

recordedontheCardioExcyte 96 using Cor.4U hiPSCs from Ncardia.
The effects of the compounds blebbistatin, E4031 andnifedipine on the impedance and EFP signals are shown.

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0.4
0.8
Time [s]1.20.0Blebbistatin

1.2

0.40.8Time [s]1.2

Blebbistatin
1.2
Normalised EFP

recordings2. These measurements are non-invasive,

0-400.0

Normalised Impedancecontractility) and extracellular field potential (EFP)

20-20-8

amplitude and duration. The CardioExcyte 96 is a newhybrid screening tool combining impedance (cell400.80.80.4Control3 µM Blebbistatin
6 µM Blebbistatin

0.40.00.0050100
Time [min]1500100200300
Time [min]400

Figure 1: Blebbistatin blocks the impedance but not EFP signal. Impedance
signal (left) and EFP signal (right) during application of blebbistatin (6 µM), amyosin II inhibitor. Blebbistatin affects the contractility of the cells but does
not affect ion channels. Therefore, the impedance signal is blocked whilst
the EFP signal remains unchanged. Bottom panels show the timecourseof the experiment after addition of blebbistatin (3 µM and 6 µM) for theimpedance (left) and EFP (right) signals.

Application NoteFigure

1showstheimpedance

andEFPsignalsAC

B

D

recorded from Cor.4U Cardiomyocytes under control
conditions and after application of blebbistatin. As
expected, the contraction of the cells was blocked as
revealed by the impedance signal, whilst the EFP signal
remained unchanged.

Figure 3: The effect of the hERG channel blocker, E4031, on the impedanceand EFP of Cor.4U cells. Shown are raw traces in impedance (A) and EFP (B)mode in control conditions or in the presence of E4031 at the concentration
indicated. C Effect of increasing concentrations of E4031 on the impedance
parameters amplitude, pulsewidth 30, BRRI and beat rate displayed ona spider chart. D Effect of increasing concentrations of E4031 on the EFPparameters amplitude, FPDmax, BRRI and beat rate displayed on a spiderchart.
Figure 2: 96-well view of an exemplary experimental layout. Different
concentrations of compounds were tested on Cor.4U Cardiomyocytes. The
compound-induced effects on the beat rate, amplitude or beat irregularity(arrhythmia), amongst other parameters, can be viewed online during theexperiment.

Asapotent

blockerofhERG,

E4031

is

considered a high risk compound by the FDA for
Figure 2 shows a screenshot of the CardioExcyte 96
software (CardioExcyte Control) during an experiment.
In this case, six different compounds acting on ion
channels were tested at different concentrations andthe impedance signal is shown.

causingarrhythmia.

E4031

hasbeenshowntoblock hERG in patch clamp experiments at low
concentrations6.

Furthermore,

E4031

causesearly afterdepolarizations (EADs) in stem cell-derived
cardiomyocytes7.

InbothimpedanceandEFP

modes, E4031 had little effect on beat rate, but had

Figure 3 shows the effect of the specific hERG blocker,
E4031, on the impedance (A) and EFP (C) signals. The
online analysis parameters for impedance: beat rate,pulsewidth 30 (PW30) and beat rate regularity index (BRRI)
are plotted on the spider chart (B). At higher concentrations, E4031 reduces amplitude, increases pulsewidth

profound effects on amplitude, PW30 and BRRI inimpedancemodeandonamplitude,FPD-max and BRRI in EFP mode. EADs could also be
detected in both impedance and EFP modes (datanot shown), an important indicator for a compound’s
potential to cause Torsades de Pointes (TdP) in humans.

30 and lowers the BRRI. The online analysis parameters
for EFP: amplitude, FPDmax (the time between thenegative deflection to the following positive peak), BRRI
and beat rate are plotted on a spider chart (D). For an
explanation of analysis parameters see Refs 2 & 5.

Nanion Technologies GmbHphone +
 49 89 2190 95-0

Ganghoferstr. 70afax

80339 Munich, Germany

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

Application NoteAC

decrease

inFPD

ina

concentration-dependent

manner, consistent with a shortening of the action
potential
Ca2+

dueto

theinhibitionofvoltage-gatedchannels. This result is in agreement with theliterature8. In impedance mode, nifedipine led to aconcentration-dependent decrease in the amplitude

DBand decrease in pulse width leading to an increase inbeat rate in agreement with reports from other groups9.
In conclusion, the CardioExcyte 96 can be used toreliably perform EFP and impedance measurements
on hiPSCs from Ncardia (Cor.4U). Compounds such as
blebbistatin which affect contractility of the cells with-Figure 6: Concentration-dependent effect of nifedipine on the impedanceand EFP of Cor.4U Cardiomyocytes. Shown are raw traces in impedance (A)and EFP (B) mode in control conditions or in the presence of 300 nM nifedipine.
C Effect of increasing concentrations of nifedipine on the impedance
parameters amplitude, pulsewidth 30, BRRI and beat rate displayed on aspider chart. D Effect of increasing concentrations of nifedipine on the EFPparameters amplitude, FPDmax, BRRI and beat rate displayed on a spiderchart.

out affecting ion channel activity show an effect on
the impedance signal whilst the EFP recording remains
unchanged,

asexpected.

Compounds

which

are known to block the hERG channel showed
profoundeffects

on

both

impedance

andEFPsignals. The L-type calcium channel blocker, nifedipine,reduced impedance and EFP amplitude and caused aFigure 6 shows the effect of nifedipine, an L-type

decrease in the FPD consistent with a shortening of thecalcium channel blocker, on the impedance andcardiac action potential. The CardioExcyte 96 provides

EFP signals of Cor.4U cardiomyocytes. Additionally,a complementary device to automated patch clamp

online analysis parameters amplitude, pulse widthfor high throughput cardiac safety and cardiotoxicity

30%, beat rate and BRRI for impedance, and am-studies.

plitude,FPDmax, BRRI and beatratefor EFP areshown displayed as spider charts. Nifedipine led to a

References

Impedance and EFP measurements

1. Stoelzle, S., et al., 2011. Front. Pharmacol. doi: 10.3389/fphar.2011.00076.

Impedance and EFP measurements were conducted

2. Doerr, L., et al., 2014. J. Lab. Autom. pii: 2211068214562832.

according to Nanion’s standard procedures for the

3. Sprössler, C., et al., 1999. Physical Review E. 60: 2171-2176.

CardioExcyte 96. Cells were seeded on the Sensor Plate

4. Kovács, M., et al. 2004. J. Biol. Chem. 279(34): 35557-63.

at least 1.5 weeks before pharmacology experiments

5. Obergrussberger, A., et al. 2017. In: Stem Cells in Toxicology. DOI:

began to allow the formation of a dense monolayer and10.1007/978-1-4939-6661-5_10

a suitable network of cell-cell interconnections to ensure

6. Kirsch, G.E., et al., 2004. J. Pharmacol.Toxicol. Methods. 50(2): 93–101

a steady propagation of the excitation and synchronized

7. Peng, S., et al., 2010. J. Pharmacol. Toxicol. Methods. 61(3): 277–286

beating. About 2 hours before drug application the8. Navarrete, E. G., et al. 2013. Circulation. 128(11 Suppl 1): S3-13.

medium was completely removed from the wells and 200

9. Guo, L., et al., 2011. Toxicol. Sci. 123(1): 281-289.

μl fresh medium was added (Cor.4U® serum-free BMCCmedium).

MethodsAllsignals(exceptblebbistatin)

werenormalized to a group of control measurements (n=5-11)
on the same plate using DMSO (0.01%) as vehicle.

Cells
Cor.4U cells were kindly provided by Ncardia.

Nanion Technologies GmbHphone + 49 89 2190 95-0

Ganghoferstr. 70afax80339 Munich, Germany

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