Introducing simulated IK1 into human iPSC-cardiomyocytes using

dynamic clamp on an automated patch clamp setup

Introduction Combining dynamic clamp with APC Applying seal compensation to

hiPSC-cardiomyocytes

Effect of applying seal compensation and simulated IK1 to hiPSC-cardiomyocytes on AP shape and pharmacology Conclusions

Gang Lu1, András Horváth2, Nadine Becker2, Sonja Stoelzle-Feix2, Alan Fabbri3, Christian Grad2, Michael George2, Niels Fertig2,

Teun P. de Boer3

1Nanion Technologies Inc, Livingston, NJ, USA, 2Nanion Technologies, Munich, Germany, 3Department of Medical Physiology, Division of Heart & Lungs, University Medical Center

Utrecht, Utrecht, Netherlands.

Dynamic clamp is a powerful tool to inject real-time simulated

currents into patch clamped cells1. This has been shown using

conventional patch clamp whereby the inward rectifier current IK1

was introduced into human induced pluripotent stem cell-derived

cardiomyocytes (hiPSC-CMs)2,3. IK1 is expressed at low levels in

these cells4, hence their membrane potential is more depolarized

than that of primary cardiomyocytes4,5, limiting their use in safety

pharmacology. Introducing simulated IK1 into hiPSC-CMs may

render them a viable alternative to scarcely available adult

human cardiomyocytes.

In this study, we combined dynamic clamp with an automated

patch clamp (APC) system to demonstrate that IK1 conductance

can be added to hiPSC-CMs on this platform, while at the same

time, applying automatic Rseal compensation (SC). Our results show

that virtual IK1 can be successfully injected into hiPSC-CMs in up to

8 cells simultaneously and that Rseal is correctly compensated

avoiding overcompensation. Our approach results in more stable

resting membrane potentials and improved action potential (AP)

shape. „L”-Type calcium channel opener BayK and channel

blocker nifedipine were also tested. Figure 1 Block diagram showing connections between Patchliner, patch

clamp amplifier and dynamic clamp system.

Figure 2 hiPSC Cellartis Cardiomyocytes recorded on the Patchliner.

A Na+ currents and average IV plot (B, n = 7). C Ca2+ currents and

average IV plot (D, n = 18). E Raw traces of Ca2+ current (black:

control, blue: nifedipine). F Nifedipine concentration response curve.

Data from Ref. 6.

Figure 3 Impact of seal compensation on the shape of the cardiac

AP recorded from hiPSC cardiomyocytes. Introducing 100% seal

compensation allowed to remove the majority of the holding

current. Combining seal compensation with various amounts of

virtual IK1 enhanced the 4th phase of the repolarization of the

cardiac AP.

Figure 4 Screenshot taken from the PatchMaster software

with 6 APs recorded in parallel from hiPSC-cardiomyocytes

on the Patchliner Octo. Seal compensation combined with

simulated IK1 using dynamic clamp resulted in longer APs.

Figure 5 A: APs recorded from Cor.4U hiPSC-CMs applying seal compensation and simulated IK1. B: Effect of seal compensation combined with simulated IK1 on APD90

compared to the use of holding current (*P<0.05, unpaired Student’s T-test) in Cor.4U hiPSC-CMs. C: Effect of 10 µM Nifedipine on APD90 in Cor.4U hiPSC-CMs (*P<0.05,

paired Student’s T-test). D: APs recorded from CDI2 hiPSC-CMs under control conditions and after addition of 10 µM BayK 8644 and 10 µM Nifedipine. E: Effect of BayK

8644 and Nifedipine on the APD90 of CDI2 hiPSC-CMs (*P<0.05, paired Student’s T-test). F: Dose-response curve of BayK 8644 on APD90 in CDI2 hiPSC-CMs.

• Dynamic clamp is a powerful tool to inject real-time simulated currents

into cells and here we combine the technique with an automated

patch clamp device, the Patchliner Octo.

• IK1 and seal compensation were successfully applied to hiPSC-CMs in

multiple cells simultaneously and effects on RMP and AP duration were

comparable to those obtained using manual patch clamp2,3.

• Ca2+ channel activator, BayK 8644, and blocker, nifedipine, prolonged

and shortened AP duration, respectively, as expected.

• Future goals:

• Test effects of the set of drugs defined by CiPA on action

potentials under dynamic clamp.

• Upgrade technique to higher throughput devices, e.g.

SyncroPatch 384/768PE.

References

1. Wilders R. J. Physiol. 2006;576:349–359

2. Bett GC, et al. Heart Rhythm. 2013;10:1903–1910.

3. Meijer van Putten RM, et al. Front. Physiol. 2015;6:7. doi: 10.3389/fphys.2015.00007

4. Jonsson MKB, et al. J. Mol. Cell Cardiol. 2012;52:998–1008

5. Rajamohan D, et al. Stem Cells Dev. 2016;25:439–452

6. Goversen B., et al. 2018. Front. Physiol. 6:1094

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Vhalf = -45 mV

n = 18

Vhalf = -5.8 mV

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Voltage clamp characteristics of

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