Stefan A Mann1, Juliane Heide1, Thomas Knott2, Razvan Airini3, Florin Bogdan Epureanu3, Alexandru-Florian Deftu3, Antonia-Teona Deftu3, Beatrice Mihaela Radu3, Bogdan Amuzescu4. 1. Cytocentrics Bioscience GmbH, Nattermannallee 1, 50829 Cologne, Germany. 2. CytoBioScience Inc., 3463 Magic Drive, San Antonio, TX 78229, USA. 3. Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, 050095 Bucharest, Romania. 4. Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, 050095 Bucharest, Romania. Electronic address: bogdan@biologie.kappa.ro.
Abstract
INTRODUCTION: 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. METHODS: 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. RESULTS: 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). DISCUSSION: 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.
INTRODUCTION: 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. METHODS: 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. RESULTS: 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). DISCUSSION: 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.