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

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.

Cryopreservation of human pluripotent stem cell-derived cardiomyocytes is not detrimental to their molecular and functional properties

“…3B). While differences in RMP might affect APDs by altering availability of sodium, calcium and potassium currents (Verkerk et al, 2017), we found no correlation between APD90 and RMP (data not shown), suggesting that the longer APs in the frozen LUMC20 hiPSC-CMs may be due to the greater proportion of MLC2v + cardiomyocytes. Nevertheless, these results indicated that cryopreservation is not detrimental to the electrophysiological properties of the hiPSC-CMs in vitro.…”

“…Data acquisition, voltage control, and analysis was performed using custom made software and the potentials were corrected for the calculated liquid junction potential (Barry and Lynch, 1991). The patch pipettes, estimations of cell membrane capacitance, and filtering and digitizing settings were as described previously (Verkerk et al, 2017).…”

Automated Dynamic Clamp for Simulation of IK1 in Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes in Real Time Using Patchliner Dynamite8

“…Dynamic clamp is a powerful tool to inject real-time simulated currents into patchclamped cells. This has been shown in conventional patch clamp experiments, injecting simulated inward rectifier current I K1 into hiPSC-CMs (Meijer van Putten et al, 2015;Verkerk et al, 2017). I K1 is expressed at low levels in these cells; hence, their RMP is more depolarized than that of primary cardiomyocytes, limiting their use in safety pharmacology.…”

Isogenic sets of hiPSC-CMs harboringKCNH2mutations capture location-related phenotypic differences

“…Voltage-clamp recordings of I Kr were made using pipette and bath solutions as previously described 17 . Action potentials (APs) of the hiPSC-CMs were recorded by perforated patchclamp and the dynamic clamp technique with injection of an inward rectifier potassium current (I K1 ) used to achieve a close-to-physiological resting membrane potential (RMP) 21 .…”