Automated Patch Clamp System Introducing Simulated IK1 Intostem cell-derived Cardiomycoytes Using Dynamic Clamp
George Okeyo1, András Horváth2, Nadine Becker2, Alan Fabbri3, Christian Grad2, Michael George2, Teun P. de Boer3, Niels
Fertig2
1Nanion Technologies Inc, Livingston, NJ, USA, 2Nanion Technologies, Munich, Germany, 3Department of Medical Physiology, Division of Heart & Lungs, University Medical CenterUtrecht, Utrecht, Netherlands.

Introduction

Combining dynamic clamp with APC

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 typically expressed at low
levels in these cells4, hence their membrane potential is more
depolarized than that of primary cardiomyocytes4,5, limiting theiruse in safety pharmacology. Introducing simulated IK1 into hiPSCCMs may render them a viable alternative to scarcely available
adult human cardiomyocytes.
In this study, we combined dynamic clamp with an automatedpatch clamp (APC) system to demonstrate that IK1 conductancecan be added to hiPSC-CMs on this platform, while at the sametime, applying automatic Rseal compensation (SC)6,7. Our results
show that virtual IK1 can be successfully injected into hiPSC-CMs in
up to 8 cells simultaneously and that Rseal is correctly compensatedavoiding overcompensation. Our approach results in more stable
resting membrane potentials and action potential duration (APD)
values. Action potential (AP) shape is also improved. „L”-Typecalcium channel opener BayK 8644 and channel blocker
nifedipine were also tested. In addition, we compared the shapeof APs at physiological versus ambient temperature.

Figure 1 Block diagram showing connections between Patchliner, patch
clamp amplifier and dynamic clamp system. Reproduced from Ref. 7.

Theory of Seal Resistancecompensation

Figure 2 In current clamp mode (CC), VM can dissipate overmembrane resistance (RM) or seal resistance (Rseal). By injecting VM x
Rseal-1, leak through Rseal can be compensated for.

BC

Figure 3 A: Example of an action potential (AP) recorded from aCardiosight-S® cell (Nexel) when only holding current is injected(black), when 70% seal compensation is injected (dark blue), andwhen 70% seal compensation and IK1 is injected (light blue). B:
Injected seal compensation for the cell shown in A. C: Injected seal
compensation and IK1 for the cell shown in A. B and C are shown on
the same scale (data from Ref. 7).

Conclusions

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

A

Effect of seal compensation and IK1in the shape of hiPSC-CM• IK1 and seal compensation were successfully applied to hiPSC-CMs in
multiple cells simultaneously and effects on RMP and AP duration.
• Combined with the Patchliner Octo, Dynamite8 enabled the recordingof record stable APs from hiPSC-CM at a higher throughput.
• Ca2+ channel activator, BayK 8644, and blocker, nifedipine, prolonged
and shortened AP duration, respectively, as expected and a complete
concentration-response curve was recorded on an individual cell.

DEF• APs at physiological temperature were also measured.
• Future goals:• Test effects of the set of drugs defined by CiPA on action
potentials using dynamic clamp.
• Upgrade technique to higher throughput devices, e.g.
SyncroPatch 384i.

Figure 4 Screenshot taken from the PatchMaster softwarewith 8 APs recorded in parallel from hiPSC-cardiomyocyteson the Patchliner Octo. Seal compensation combined withsimulated IK1 using dynamic clamp resulted in longer APs.

Figure 5 A: APs recorded from Cardiosight–S® hiPSC-CMs in the presence of BayK 8644 and nifedipine. B: Effect of 10 µM BayK 8644 and nifedipine on APD90 inCardiosight-S® hiPSC-CMs (*P<0.05, paired Student’s T-test). C: Time course (left) and short term variability of APD90 recorded from Cardiosight-S®. D: APs recorded fromiCell Cardiomyocyte2 hiPSC-CMs (Fujifilm Cellular Dynamics, Inc.) under control conditions and after addition of 10 µM BayK 8644 and 10 µM nifedipine. E: Doseresponse curve of BayK 8644 on APD90 in iCell Cardiomyocyte2. F: Effect of physiological temperature on the shape of the AP (Data from Ref 7).

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