Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a “pseudo” QRS complex suggesting reduced inward current(s). Voltage clamp analysis of ICa showed no difference in the magnitude of current. Measurements of INa reveal a 60% reduction in INa density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of INa was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of INa due to a mutation in PKP2 coupled with and a gain of function in IK,ATP due to a mutation in ABCC9.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used for safety pharmacology and to investigate genetic diseases affecting cardiac ion channels. We have previously shown that the maximum diastolic potential (MDP) in hiPSC-CMs depends on the rapid delayed rectifier K+ current (IKr). We examined the effect of a dual Ito and IKr agonist (NS3623) on hiPSC-CMs and determined its contribution to action potentials (APs), extracellular field potentials (EFPs) and ionic currents. hiPSC-CM monolayers were used to record EFPs using CardioExcyte 96, and AP recordings were made using high resistance electrodes. Whole cell patch clamp was used to record IKr in single hiPSC-CMs. All measurements were made at 36° C. EFP signals from hiPSC-CMs monolayers showed a pseudo-QRS complex and T-wave similar to that observed in native ventricular tissue. Application of NS3623 (5 μM) resulted in a small shortening of the QT interval. Similarly, AP recordings in the presence of NS3623 resulted in hyperpolarization of the MDP (from −70.4±1.9 to −73.9±1.9 mV) and shortening of APD (from 196.6±28.0 to 176.6±25.7 ms). Voltage clamp analysis of IKr tail currents revealed a 47±9% increase in IKr following application of NS3623. A minor inhibition of Ito was observed following NS3623. AP clamp experiments revealed the IKr transient was largest during repolarization with some contribution during phase 4 depolarization. A robust IKr is present in hiPSC-CMs which can be augmented by the agonist NS3623. Application of the agonist resulted a slowing of the spontaneous rate and shortening of the APD but minimal effect on MDP. Although we have previously shown that the MDP in hiPSC-CMs critically depends on IKr, increasing the magnitude of IKr resulted only in a small hyperpolarization of the MDP.

BACKGROUND:Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for many applications including safety pharmacology. However, a deficiency or complete absence of several K+ currents suggests repolarization reserve is low in hiPSC-CMs. We determined whether a dual Ito and IKr activator can improve repolarization reserve in hiPSC-CMs resulting in a more electrophysiologically mature phenotype.METHODS AND RESULTS:Human iPSC were maintained on growth factor and differentiated into the cardiac phenotype by addition of selective Wnt molecules. Current and voltage clamp recordings in single cells were made using patch electrodes. Extracellular field potentials were made using a microelectrode array on hiPSC monolayers. Action potential recordings from hiPSC-CMs following application of an IKr inhibitor resulted in depolarization of the membrane potential and prolongation of the APD. A flattening of the T-wave was noted on the pseudo-ECG. In contrast, application of the IKr and Ito agonist, NS3623, resulted in hyperpolarization of the membrane, slowing of the spontaneous rate and shortening of the APD. Voltage clamp recording showed a significant increase in IKr; no enhancement of Ito in hiPSC-CMs was noted. AP clamp experiments revealed that IKr plays a role in both phase 3 repolarization and phase 4 depolarization. mRNA analysis revealed that KCNH2 is abundantly expressed in hiPSC-CM, consistent with electrophysiological recordings.CONCLUSIONS:Although NS3623 is a dual Ito and IKr activator in ventricular myocytes, application of this compound to hiPSC-CMs enhanced only IKr and no effect on Ito was noted. Our results suggest IKr enhancement can improve repolarization reserve in this cell type. The disconnect between a dramatic increase in Ito in adult myocytes versus the lack of effect in hiPSC-CMs suggest that the translation of pharmacological effects in hiPSC-CM to adult myocytes should be viewed with caution.

Human induced pluripotent stem cells (hiPSCs) are relevant for cardiac safety testing due to their validated predictivity as described in recent publications derived from the comprehensive in vitro proarrhythmia assay (CiPA) study. We combined automated patch clamp (APC), impedance and extracellular field potential (EFP) measurements to study cardiac ion channels in cell lines and hiPSC-CMs. Cell lines expressing different cardiac ion channels were recorded on two different APC instruments (8 or 384 wells simultaneously) at room and physiological temperature, at 4 different sites. Within the myocyte validation study of the CiPA initiative, hiPSC-CMs from different providers were used on a device combining impedance-based contractility and extracellular field potential (EFP) recordings. Within the ion channels working group of the CiPA initiative, ion channel data on 7 cardiac ion channel currents were measured. The effects of >20 CiPA reference compounds deemed low, intermediate and high risk by the FDA were investigated. In parallel to APC investigations, arrhythmogenicity of this and other compounds were investigated via contractility and field potential recordings in 2D monolayers of iPSC-CM. Our results show that high risk compounds such as dofetilide prolong field potential duration which resulted in the detection of arrhythmic events in both impedance and EFP recordings. Datasets from multiple sites and cell types will be shown and compared. Cross-site/cell comparisons of APC ion channel data and myocyte repolarization data was combined in order to investigate the mode of action of reference compounds. This promotes a good understanding of drug-induced arrhythmia and thus represents a valuable approach in drug development efforts.

Introduction: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines. Methods: Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed. Results: Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values. Discussion With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

Hepatic toxicity has accounted for 15 of the 47 drugs withdrawn from the market in the last two decades1. More specifically, Drug Induced Liver Injury (DILI) is the major cause of acute liver failure. DILI is classified as intrinsic (or dose-dependent) or as idiosyncratic2. A prominent example of idiosyncrasy is acetaminophen2,3, with a variable time of onset and not directly dependent on dose. We present a non-invasive DILI assay approach based on impedance measurements in monocyte-derived hepatocyte-like (MH) cells from MetaHeps®. MH cells were used on a 96-well screening system that monitors changes in impedance (or cell monolayer resistance). Cells are monitored under physiological conditions for temperature, humidity and CO2. We investigated hepatotoxic effects of paracetamol on MH cells when exposed for 24 and 48 hours. In agreement with other standard toxicity assays, such as the lactate dehydrogenase release assay, low doses of paracetamol caused transient toxicity and 'adaptation' was observed. At higher doses, hepatotoxic effects of paracetamol could be reversed upon washout after 24 hours, but continued exposure caused increased hepatotoxicity. In addition to hepatotoxicity, another validation of the principle is shown for chronic proliferation of cancer cells. In this study, murine mammary carcinoma cells (H8N8 and H8N8 T3.2) were used and changes in impedance, were used as a measure of toxicity. Intrinsic (dose-dependent) effects of the standard clinical regimen cyclophosphamide, doxorubicin and 5-fluouracil could be identified consistent with other methods of live cell analysis systems. Therefore, the utilized 96-well impedance system in combination with murine mammary carcinoma cells provides a novel tool for investigating therapy resistance of cancer cells in vitro. References: 1. Stevens, J.L, 2008. Drug Disc.Tod. 2.Roth, R.A, et al.2010, J Pharmacol Exp Ther. 3.McGill, M.R. et al 2013. Pharm Res. 30(9).

Optical in-vitro platforms will be of particular relevance in the early stages drug discovery processes. We show recordings on impedance and extracellular field potential (EFP)-based devices with induced pluripotent stem cell (iPSC)-derived cardiomyocytes as well as automated patch clamp data. Optogenetic stimulation and the recording of electrophysiological and contractile parameters of ChR2 (channelrhodopsin 2) transfected iPS Cor.4U cardiomyocytes were performed in a new assay approach, which allows a parallel investigation of impedance and EFP signals. This allowed a mechanistic understanding of cardiomyocyte cell physiology, which has been investigated over a physiological frequency range (60-180 ppm). Frequency dependent effects on cell physiology with reference compounds such as Ranolazine and Mexiletine will be presented. Furthermore, automated patch clamp investigations in the voltage-and current-clamp mode on blue-light activated ChR2 (channelrhodopsin 2) transfected cells will be presented and discussed in association with impedance/EFP results.

INTRODUCTION:While extracellular field potential (EFP) recordings using multi-electrode arrays (MEAs) are a well-established technique for monitoring changes in cardiac and neuronal function, impedance is a relatively unexploited technology. The combination of EFP, impedance and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has important implications for safety pharmacology as functional information about contraction and field potentials can be gleaned from human cardiomyocytes in a beating monolayer. The main objectives of this study were to demonstrate, using a range of different compounds, that drug effects on contraction and electrophysiology can be detected using a beating monolayer of hiPSC-CMs on the CardioExcyte 96. METHODS:hiPSC-CMs were grown as a monolayer on NSP-96 plates for the CardioExcyte 96 (Nanion Technologies) and recordings were made in combined EFP and impedance mode at physiological temperature. The effect of the hERG blockers, E4031 and dofetilide, hERG trafficking inhibitor, pentamidine, β-adrenergic receptor agonist, isoproterenol, and calcium channel blocker, nifedipine, was tested on the EFP and impedance signals. RESULTS:Combined impedance and EFP measurements were made from hiPSC-CMs using the CardioExcyte 96 (Nanion Technologies). E4031 and dofetilide, known to cause arrhythmia and Torsades de Pointes (TdP) in humans, decreased beat rate in impedance and EFP modes. Early afterdepolarization (EAD)-like events, an in vitro marker of TdP, could also be detected using this system. Isoproterenol and nifedipine caused an increase in beat rate. A long-term study (over 30h) of pentamidine, a hERG trafficking inhibitor, showed a concentration and time-dependent effect of pentamidine. DISCUSSION:In the light of the new Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative to improve guidelines and standardize assays and protocols, the use of EFP and impedance measurements from hiPSCs may become critical in determining the proarrhythmic risk of potential drug candidates. The combination of EFP offering information about cardiac electrophysiology, and impedance, providing information about contractility from the same area of a synchronously beating monolayer of human cardiomyocytes in a 96-well plate format has important implications for future cardiac safety testing.

The CardioExcyte 96 system provides a non-invasive, label-free, high temporal resolution approach for safety screening on iPSCMs. It is a hybrid screening instrument that combines impedance with MEA-like extracellular field potential (EFP) recordings. Furthermore, it can be either used in an incubator or, by utilizing the dedicated incubation system, directly on a lab bench. The system is capable of electrically pacing the cells, which allows for screening of cells which beat with individual frequencies and, in addition, investigations of frequency dependent compound inhibition.

Drug induced arrhythmia is one of the most common causes of drug development failure. Human induced pluripotent stem cell-derived cardiomyocytes (iPSCMs) show great promise for cardiovascular research and predictive in-vitro cardiac safety screening. We present data recorded on the CardioExcyte 96, a hybrid screening instrument that combines impedance (cell contractility) with MEA-like extracellular field potential (EFP) recordings. A case study showing impaired excitation-contraction coupling will be presented. This indicates the importance of testing compounds in assays complementary to patch clamp electrophysiology to provide a more complete safety profile. Furthermore, in accordance with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) guidelines, standard reference compounds were tested on iPS-derived cardiomyocytes. Changes in the impedance signal indicate effects on cell contractility and shape whereas the field potential parameters provide information about the electrophysiological activity of the beating network of cells. Pharmacological effects of a number of reference compounds including those designated high risk (e.g. Dofetilide), intermediate risk (e.g. Cisapride) or low risk (e.g. Verapamil) were evaluated and summarized in this study. The CardioExcyte 96 system provides a non-invasive, label-free, high temporal resolution approach for safety screening. It can be either used in an incubator or, by utilizing the dedicated incubation system, directly on a lab bench. Furthermore, the system is capable of electrically pacing the cells, which allows for screening of cells which beat with individual frequencies and, in addition, investigations of frequency dependent compound inhibition.

The need for predictive, in vitro cardiac safety screening drives further development of automated, high-throughput-compatible drug evaluation based on cardiac cell preparations. Recently, pluripotent stem cells are evaluated as a new, more predictive model for cardiovascular risk assessment pertaining to in vitro assays. We present a new screening platform, the CardioExcyte 96, a hybrid instrument that combines impedance (cell contractility) with extracellular field potential (EFP) recordings. The electrophysiological measurements are noninvasive, label free and have a temporal resolution of 1 ms. This hybrid technology addresses the lack of easy-to-use high-throughput screening for in vitro assays and permits the reliable investigation of short- and long-term pharmacological effects. Several models of cardiomyocyte preparations were successfully validated for use with the CardioExcyte96. Furthermore, the pharmacological effects of a number of reference compounds were evaluated. Compound effects on cell monolayers of human-induced pluripotent stem cell-derived cardiomyocytes are evaluated using a quasi-simultaneous hybrid recording mode that combines impedance and EFP readouts. A specialized software package for rapid data handling and real-time analysis was developed, which allows for comprehensive investigation of the cellular beat signal. Combining impedance readouts of cell contractility and EFP (microelectrode array-like) recordings, the system opens up new possibilities in the field of in vitro cardiac safety assessment.

A new trend in the industry utilizes induced pluripotent stem cells (iPSCs) for functional assays to identify cardiac pathologies and their genetic underpinnings (such as Long QT Syndrome (LQTS)), in addition to cardiotoxicity profiling of pharmaceutical compounds. The here utilized CardioExcyte® 96 system is a hybrid instrument that combines impedance readout (a correlate of cell contractility) with electric field potential recordings of the compound signal that is generated by cellular action potentials. These electrophysiological measurements are label-free and allow investigations in High Throughput format. In addition, the instrument is capable of electrical stimulation of the cellular monolayer, while the rapid acquisition rate of 1ms allows a more accurate description of signal features and nuances stemming from pharmacological effects. A dedicated software package for rapid data handling and real-time analysis is based on novel algorithms that allow a comprehensive investigation of the cellular beat signal. A broad range of cell lines was successfully validated, such as stem cell-derived cardiomyocytes from Axiogenesis (human: Cor4U®, murine: CorAt®), GE Healthcare (Cytiva), Cellectis (human, 3D-clusters: hES-CMC™) and CDI (iCell®). Furthermore, the pharmacological effects of a number of reference compounds on these cell types were evaluated and their phenotype correctly identified. For example, the effects of compounds like Blebbistatin, which blocks contraction without disrupting the electric field potential can be easily identified since it is an inhibitor of the myosin II which is mainly responsible for the contraction of cardiomyocytes, while it has no effect to the ion channels. Other compound classes like hERG inhibitors were validated as well and example data will be shown. They induce typical arrhythmia since hERG ion channels are affected.

Here we present a carbon nanotube based device to noninvasively and quickly detect mobile single cells with the potential to maintain a high degree of spatial resolution. The device utilizes standard complementary metal oxide semiconductor (CMOS) technologies for fabrication, allowing it to be easily scalable (down to a few nanometers). Nanotubes are deposited using electrophoresis after fabrication in order to maintain CMOS compatibility. The devices are spaced by 6 μm which is the same size or smaller than a single cell. To demonstrate its capability to detect cells, we performed impedance spectroscopy on mobile human embryonic kidney (HEK) cells, neurons cells from mice, and yeast cells (S. pombe). Measurements were performed with and without cells and with and without nanotubes. Nanotubes were found to be crucial to successfully detect the presence of cells. The devices are also able to distinguish between cells with different characteristics.

The murine cardiac action potential waveform can be anthropomorphized into that of a human-like waveform in real time, through a novel dynamic-clamp method known as the cell-type transforming clamp (CTC). In the CTC, a computationally calculated virtual conductance is inserted into the cell in real time, to compensate for the differences between murine and human sarcolemmal currents. By so doing, the CTC anthropomorphizes the membrane potential without clamping it, thereby enabling the investigation of drug- or mutation-induced arrhythmogenic phenotypes in the appropriate human action potential context (but in the experimentally powerful mouse animal model). We are using a real-time implementation of a genetic algorithm that optimizes the morphology of a theoretical model, in order to match the murine action potential recorded from a real cell. We present a comparison of human and guinea pig action potentials anthropomorphized in real time, from neonatal mouse cardiomyocytes.

While the mouse presents an invaluable experimental model organism in biology, its usefulness in cardiac arrhythmia research is limited in some aspects due to major electrophysiological differences between murine and human action potentials (APs). As previously described, these species-specific traits can be partly overcome by application of a cell-type transforming clamp (CTC) to anthropomorphize the murine cardiac AP. CTC is a hybrid experimental-computational dynamic clamp technique, in which a computationally calculated time-dependent current is inserted into a cell in real-time, to compensate for the differences between sarcolemmal currents of that cell (e.g., murine) and the desired species (e.g., human). For effective CTC performance, mismatch between the measured cell and a mathematical model used to mimic the measured AP must be minimal. We have developed a genetic algorithm (GA) approach that rapidly tunes a mathematical model to reproduce the AP of the murine cardiac myocyte under study. Compared to a prior implementation that used a template-based model selection approach, we show that GA optimization to a cell-specific model results in a much better recapitulation of the desired AP morphology with CTC. This improvement was more pronounced when anthropomorphizing neonatal mouse cardiomyocytes to human-like APs than to guinea pig APs. CTC may be useful for a wide range of applications, from screening effects of pharmaceutical compounds on ion channel activity, to exploring variations in the mouse or human genome. Rapid GA optimization of a cell-specific mathematical model improves CTC performance and may therefore expand the applicability and usage of the CTC technique.

Polarization impedance appears at the interface between electrodes and ionic solutions and is a major source of errors in dielectric spectroscopy measurements. This work presents a simple, robust and automated methodology for measuring and analysing the polarization impedance of non-dispersive electrolytes, with a focus on the very low frequency domain from 1 Hz and up. The accuracy of the method is demonstrated by comparing the corrected dielectric permittivity and conductivity of various electrolytes either with their nominal values or with measurements taken with other high precision measuring devices. The dependence of the polarization impedance on several parameters, such as ionic concentration, applied voltage and separation distance between the electrodes, is also presented. For colloidal suspensions, it is argued that a modified protocol of the substitution method is needed due to several shortcomings occurring only in the very low frequency domain. Such a protocol is presented and tested on suspensions of live E. coli cells. As opposed to most of the existing methodologies for polarization removal, the proposed protocol makes no assumptions on the behaviour of the polarization impedance. This could potentially lead to the quantitative resolution of the α-dispersion of live cells in suspension.