Recording of multiple ion current components and action potentials in human induced pluripotent stem cell-derived...

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative proposes a three-step approach to evaluate proarrhythmogenic liability of drug candidates: effects on individual ion channels in heterologous expression systems, integrating these data into in-silico models of the electrical activity of human cardiomyocytes, and comparison with experiments on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Here we introduce patch-clamp electrophysiology techniques on hiPSC-CM to combine two of the CiPA steps in one assay.

We performed automated patch-clamp experiments on hiPSC-CM (Cor.4U^®, Ncardia) using the CytoPatch™2 platform in ruptured whole-cell and β-escin-perforated-patch configurations. A combination of three voltage-clamp protocols allowed recording of five distinct ion current components (voltage-gated Na⁺ current, L-type Ca²⁺ current, transient outward K⁺ current, delayed rectifier K⁺ current, and "funny" hyperpolarization-activated current) from the same cell. We proved their molecular identity by either Na⁺ replacement with choline or by applying specific blockers: nifedipine, cisapride, chromanol 293B, phrixotoxin-1, ZD7288. We developed a C++ script for automated analysis of voltage-clamp recordings and computation of ion current/conductance surface density for these five cardiac ion currents.

The distributions from n = 54 hiPSC-CM in "ruptured" patch-clamp vs. n = 35 hiPSC-CM in β-escin-perforated patch-clamp were similar for membrane capacitance, access resistance, and ion current/conductance surface densities. The β-escin-perforated configuration resulted in improved stability of action potential (AP) shape and duration over a 10-min interval, with APD90 decay rate 0.7 ± 1.6%/min (mean ± SD, n = 4) vs. 4.6 ± 1.1%/min. (n = 3) for "ruptured" approach (p = 0.0286, one-tailed Mann-Whitney test).

The improved stability obtained here will allow development of CiPA-compliant automated patch-clamp assays on hiPSC-CM. Future applications include the study of multi ion-channel blocking properties of drugs using dynamic-clamp protocols, adding a valuable new tool to the arsenal of safety-pharmacology.