Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp.

Arie Verkerk,Christiaan Veerman,Ronald Wilders,Connie Bezzina,Isabella Mengarelli,Jan Zegers

doi:10.3390/ijms18091873

Arie Verkerk, Christiaan Veerman + Show 4 more

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https://doi.org/10.3390/ijms18091873

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Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs.

Highlights

The generation of human induced pluripotent stem cell-derived cardiomyocytes holds great promise for studying inherited cardiac arrhythmias and for the development of drug therapies to treat such arrhythmias, as reviewed elsewhere, e.g., by Davis et al [1] and Hoekstra et al [2], and, more recently, by Kane and Terracciano [3] and Casini et al [4]
We demonstrated that action potential (AP) upstroke velocities, AP amplitudes and AP plateau amplitudes of both ventricular-like and atrial-like Human induced pluripotent stem cells (hiPSCs)-CMs increased upon the presence of an in silico IK1
The action potentials that we recorded from ventricular-like hiPSC-CMs in the present study demonstrate a relatively short duration in comparison with those of freshly isolated adult human cardiomyocytes

Summary

Introduction

The generation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) holds great promise for studying inherited cardiac arrhythmias and for the development of drug therapies to treat such arrhythmias, as reviewed elsewhere, e.g., by Davis et al [1] and Hoekstra et al [2], and, more recently, by Kane and Terracciano [3] and Casini et al [4]. A variety of invasive and non-invasive methods are used for the electrophysiological analysis of hiPSC-CMs, including patch-clamp methodology, sharp electrode measurements, multi-electrode arrays (MEAs), and voltage-sensitive fluorescence, each with specific strengths and limitations [4]. Reliable action potential (AP) measurements of hiPSC-CMs seem hampered by their intrinsic lack of inward rectifier potassium current (IK1) [5]. IK1 is the outward membrane current that modulates the final phase of the AP repolarisation and is essential for the generation of a stable resting membrane potential (RMP) close to the potassium equilibrium potential (EK) of approximately −85 mV. The altered AP configuration is associated with partial inactivation or slowed recovery from inactivation of the fast sodium current (INa) [7,8], transient outward potassium current (Ito1) [6,9] and L-type calcium current (ICa,L) [10]

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