Application Note

Channels: hERG, NaV1.5, CaV1.2,Cells:
human iPS-derived
cardiomyocytes
Tools: Patchliner, SyncroPatch 96,
SyncroPatch 384PE,
CardioExcyte 96

Combining automated patch clamp,
impedance and EFP of hiPSC-CMs
The electrophysiology team at Nanion Technologies GmbH, Munich.
Cellartis® Cardiomyocytes kindly provided by Takara Bio.
SummaryResults

Cardiomyocytes derived from human induced pluripo-

Voltage-gated Na+ (NaV) and Ca2+ (CaV) currents

behavior, availability, ease of use and standardized

recordedonthePatchliner

Cardiomyocytes (Figure 1).
ABproduction, they are likely to provide a viable alterna--80

tive to acutely isolated cardiomyocytes to assess the-40

1 nA

arrythmic risk by evaluating and implementing currently
available high throughput methods1,2 and evaluating
potential

useofhiPSC-CMs

asatesting has focussed on interaction with the hERG

C-0.8

Voltage (mV)potentially fatal torsades de pointes (TdP). Although this-40

approach has been largely successful in preventing newcause TdP, it is also possible that potentially valuable therapeutics have failed due to this early screening. The CiPA
initiative has proposed an expansion of patch clamp
assessment beyond hERG to include, e.g. NaV1.5 andCaV1.2. In addition, techniques such as multi-electrodearray (MEA) and impedance are being thoroughly evaluated as complementary techniques to patch clamp.
Here we present data recorded using the automatedpatch clamp platforms, the Patchliner, SyncroPatch 96
and SyncroPatch 384PE on Cellartis® Cardiomyocytes(Takara Bio Europe Cat nr. Y10075). Recordings of NaV1.5

and CaV1.2 are shown. I­mpedance and EFP recordings

were also performed using the ­CardioExcyte 96, and theeffects of verapamil and s­ otalol are shown.
Download more Application Notes from www.nanion.de

-1.0

D

channel and QT prolongation which can lead todrugs reaching the market with unexpected potential to40-0.6
5 ms

model

system for cardiac safety testing. Until now, drug safety-0.2-0.4

Comprehensive In-vitro Proarrhythmia Assay (CiPA) was
introduced to provide a more complete assessment of pro-Cellartis®

Voltage (mV)pro-arrhythmic potentials of drug candidates. In 2013 thetheusing

I/Imaxsafety screening. Given their recapitulation of native

were50 pA

50 ms-20-0.2
-0.4
-0.6

2040

I/Imaxtent stem cells (hiPSC-CMs) are gaining interest in cardiac

-0.8
-1.0

Figure 1: Typical recordings from Cellartis Cardiomyocytes recorded onthe Patchliner. A NaV currents in response to increasing voltage steps. B
Corresponding current-voltage plot for an average of 7 cells. A Boltzmann
fit revealed a Vhalf of activation of -46 mV. C CaV currents in response toincreasing voltage steps. D Corresponding current-voltage plot for an
average of 18 cells. A Boltzmann fit revealed a Vhalf of activation of -5.8mV.

Application Note
Cellartis Cardiomyocytes could be captured on theCapture rate (%)% cells with NaV(> -50 pA)% cells with CaV(> -50 pA)

58 (28/48)

71 (20/28)

68 (19/28)

Patchliner NPC-16 chips with a good success rate (seeTable 1). Both NaV and CaV currents could be recorded
in approximately 70% of cells captured (Table 1; Fig-ure 1). Parameters such as seal resistance (RSeal) andcurrent size for Na+ and Ca2+ are given in Table 2. Acurrent-voltage plot for NaV for an average of 7 cells isshown in Figure 1B. A Boltzmann equation was fitted tothe normalized data revealing a Vhalf of activationof -46 mV, in good agreement with the literature valuefor the cardiac sodium channel, NaV1.53,4. A current-voltage plot for CaV for an average of 18 cells is shown in​
Figure 1D. A Boltzmann equation was fitted to thenormalized data revealing a Vhalf of activation of -5.8 mV,in good agreement with the literature value for thecardiac calcium channel, CaV1.25,6.
AControl10 nM Nifedipine
100 nM Nifedipine
1 µM Nifedipine
10 µM Nifedipine

50 pAB

20 ms

1.0

Normalized block

0.8

Table 1: Success rates for cell capture and cells expressing NaV and CaV
currents from 1 experimental day. 6 experiments were performed using
a total of 3 chips, therefore 48 potential sites on the chip were available
and 28 cells were captured (with seal resistance > 150 MΩ) resulting in asuccess rate of 58% for capture. Of the cells captured, 71% showed NaV
current >-50 pA and 68% showed CaV current > -50 pA. Average RSeal,Cm, Rs, NaV and CaV current values are given in Table 2.

The CaV current recorded on the Patchliner was blocked
by increasing concentrations of nifedipine (Figure 2). The

concentration response curve for an average of 5 cells
is shown in Figure 2B. The concentration response curve
was fit with a Hill equation revealing an IC50 = 252 ± 186nM (n = 5) in good agreement with calcium current blockin guinea pig ventricular myocytes7.
RSeal (MΩ)

Cm (pF)

Rs (MΩ)

INa at -30
mV (nA)ICa at 10 mV(pA)

976 ± 144(28)

37 ± 6 (28)6.0 ± 0.9 (28)-5.4 ± 1.5 (7)-157 ± 24 (18)

Table 2: Average cell parameters for Cellartis Cardiomyocytes. Shownare values for seal resistance (RSeal), cell capacitance (Cm) and series
resistance (Rs) for Cellartis Cardiomyocytes captured with seal resistance> 150 MΩ. NaV current at -30 mV and CaV current at 10 mV is also shown.
Number of cells shown in brackets. Note that the average current is takenfrom the IV curves and not all cells which had a detectable NaV current
were used for the IV analysis.

Cellartis Cardiomyocytes were also used on the highthroughput automated patch clamp devices, the0.6

SyncroPatch 96 and SyncroPatch 384PE. Figure 3 shows
the NaV and CaV currents from Cellartis Cardiomyocytes

recorded in a pharmaceutical company screening

0.4

laboratory.
Nifedipine (n = 5)
IC50 = 252 ± 186 nM

0.2
0.0
100 pM 1 nM

10 nM 100 nM 1 µM[Nifedipine]

10 µM

Figure 2: Effect of nifedipine on CaV1.2 recorded from Cellartis
Cardiomyocytes on the Patchliner. A Raw current traces of CaV1.2 in control
conditions (black) and inhibition by increasing concentrations of nifedipine(blue). B The concentration response curve (normalized to maximum block)for nifedipine for an average of 5 cells. The average concentration response
curve was fitted with a standard Hill-equation which revealed an IC50 = 252± 186 nM (n = 5).

Figure 3: NaV and CaV currents from Cellartis Cardiomyocytes recorded on
the SyncroPatch 96 and SyncroPatch 384PE. A Raw current traces of NaV1.5 in
response to increasing voltage steps recorded on the SyncroPatch 96. B Raw
current traces of CaV1.2 in response to increasing voltage steps recorded on
the SyncroPatch 384PE.

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application NoteFigures 4 and 5 show screenshots of the SyncroPatch

to patch clamp electrophysiology. Cellartis Cardiomyo-384PE software during a recording using three different

cytes were grown on the NSP-96 plates for approximately

cell types in one experiment, CHO cells expressing NaV1.5

14 days to ensure a synchronously beating monolayeror CaV1.2 or Cellartis Cardiomyocytes. NaV1.5 expressedof cells before experiments were begun. Figure 6 showsin CHO cells and in Cellartis Cardiomyocytes was blocked

the impedance signal (left) and the EFP signal (right) inby tetracaine (Figure 4), CaV1.2 expressed in CHO

control conditions and in increasing concentrations ofcells and in Cellartis Cardiomyocytes was blocked by

the calcium channel blocker, verapamil. Interestingly,

nifedipine (Figure 5). This confirms that Cellartis Cardio-

verapamil has been shown to also block hERG at low

myocytes are suitable for use on high throughput auto-

concentrations8. Verapamil has differential effects on

mated patch clamp devices and cardiac ion channel

the cardiac action potential, prolonging the cardiac

currents, NaV1.5 and CaV1.2, can be reliably measured.

action potential at low concentrations and shorteningpresumably arise due to the action of verapamil on

it at higher concentrations9. These differential effects

Impedance (Ω)

multiple cardiac ion channels.
AEFP (µV)

1.2
0.8
0.4

100-100.0

B20

Impedance (Ω)

1.2

EFP (µV)

Figure 4: Cell lines and Cellartis Cardiomyocytes recorded on the SyncroPatch
384PE in one experiment. Shown are raw current traces recorded from CHO
cells expressing NaV1.5 (left), CaV1.2 (middle) and Cellartis Cardiomyocytes
in response to a single voltage step. NaV1.5 was blocked by tetracaine atdifferent concentrations. Sixteen examples of raw traces and online analysisof the Cellartis Cardiomyocytes is shown on the right.

20100.80.4-100.020
EFP (µV)

1.2

100.8
0.4

-100Impedance (Ω)

D
1.5

Figure 5: Cell lines and Cellartis Cardiomyocytes recorded on the SyncroPatch
384PE in one experiment. Shown are raw current traces recorded from CHO
cells expressing NaV1.5 (left), CaV1.2 (middle) and Cellartis Cardiomyocytes
in response to a single voltage step. CaV1.2 was blocked by nifedipine atdifferent concentrations. Sixteen examples of raw traces and online analysisof the Cellartis Cardiomyocytes is shown on the right.

The CardioExcyte 96 is a hybrid device for recording
impedance (contractility) and extracellular field potential(EFP) from a beating monolayer of cells. The technique isnon-invasive and the data acquired is complementary

01.00.52468Time (s)10control
3 nM
10 nM
30 nM02468Time (s)1050-5-10-15

EFP (µV)

Impedance (Ω)

C0.0

00.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Time (s)0.0 0.2 0.4 0.6 0.8 1.0
Time (s)Figure 6: Effects of verapamil on impedance (left) and EFP (right) signals ofCellartis Cardiomyocytes on the CardioExcyte 96. Impedance (left) andEFP signals (right) in control conditions (A), 10 nM verapamil (B) and 100 nM
verapamil (C) are shown. The mean beats for impedance (left) and EFP(right) are shown in panel (D).

Nanion Technologies GmbHphone +
 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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

Application Note
Using the CardioExcyte 96, we found that at low

has been shown to cause beating irregularities using

concentrations, verapamil caused an increase in beat

impedance measurements of mouse stem cell-derived

rate in both impedance and EFP modes and at higher

cardiomyocytes11. Using the Cellartis Cardiomyocytes,

concentrations, completely disrupted beating of thewe found that sotalol also caused irregular beating

monolayer. This is consistent with effects on multiple ion

in both impedance and EFP modes, indicated by thechannels.

secondary beats that could be detected (Figure 7).
In conclusion, Nanion Technologies offers automatedpatch clamp systems and an impedance/EFP platform
which are compatible with recording from the hiPSCcardiomyocytes,Cellartis

Cardiomyocytes

Takara Bio. Voltage-gated Na and Ca+2+

fromcurrents could

be recorded from these cells on the Patchliner, SyncroPatch 96 and SyncroPatch 384PE. The NaV current wasblocked by tetracaine and the CaV current was blockedby nifedipine. On the CardioExcyte 96, verapamil, aL-type Ca2+ channel and hERG blocker, completely
disrupted beating at higher concentrations. The hERG
blocker, Sotalol caused irregular beating in impedanceand EFP modes as expected11. The combination of Cellartis
Figure 7: Effects of sotalol on impedance (left) and EFP (right) signals ofCellartis Cardiomyocytes on the CardioExcyte 96. Impedance (left) andEFP signals (right) in control conditions (A) and 1 μM sotalol (B) are shown.
Irregular beating can be observed in the presence of sotalol.

Cardiomyocytes, the automated patch clamp systems
Patchliner and SyncroPatch 384PE, and the CardioExcyte 96 provides an excellent assay for drug safety
testing in the light of the CiPA initiative.

Sotalol is a benzenesulfonamide Class III antiarrhythmic agent known to block the hERG channel10. Sotalol

References

Cell culture
Cardiomyocytes

wereculturedandharvested

1. Sager, P.T., et al., 2014. Am. Heart J. 167(3): 292-300

according to Nanion’s standard cell culture protocols for

2. Gintant, G., et al., 2016. Nat. Rev. Drug Discov. 15(7):457-71

electrophysiology or plated directly on the NSP-96 plates

3. Li, R.A., et al., 2002. Mol. Pharmacol. 61:136–141

for impedance/EFP measurements.

4. Catterall, W.A., et al., 2005. Pharmacol. Rev. 57:397–409
5. Bénitah, J. P., et al., 1992. Pflugers Archiv. 421(2–3):176–187

Electrophysiology

6. Li, G-R., et al., 1999. Am. J. Physiol. Heart. Circ. Physiol. 276:H98-H106

Whole cell patch clamp recordings were conducted

7. Shen, J-B., et al., 2000. JPET. 294(2):562-570

according to Nanion’s standard procedures for the

8. Zhang, S., et al., 1999. Circ. Res. 84:989-988

Patchliner, SyncroPatch 96 and SyncroPatch 384PE.

9. Zhan, S., et al., 1997. J. J. Cardiovasc. Pharmacol. 30: 489-496
10. Sanguinetti, M.C. & Jurkiewicz, N.K., 1990. JGP. 96:195-215

Impedance and EFP measurements

11. Nguemo, F., et al., 2012. Cell. Physiol. Biochem. 29:819-832

Impedance and EFP measurements were performed
according to Nanion’s standard procedures for theMethods

CardioExcyte 96.

Cells
Cellartis® Cardiomyocytes were kindly provided by
Takara Bio (Takara Bio Europe, Cat nr. Y10075).

Nanion Technologies GmbHphone + 49 89 218997972

Gabrielenstr. 9fax

80636 Munich, Germany

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