Human stem cell-derived cardiomyocytes detect drug-mediated changes in action potentials and ion currents

Abstract

Introduction

It has been proposed that proarrhythmia assessment for safety pharmacology testing includes the use of human pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to detect drug-induced changes in cardiac electrophysiology. This study measured the actions of diverse agents on action potentials (AP) and ion currents recorded from hiPSC-CM.

Methods

During AP experiments, the hiPSC-CM were paced at 1 Hz during a baseline period, and when increasing concentrations of test compound were administered at 4-minute intervals. AP parameters, including duration (APD₆₀ and APD₉₀), resting membrane potential, rate of rise, and amplitude, were measured throughout the entire experiment. Voltage clamp experiments with E-4031 and nifedipine were similarly conducted.

Results

E-4031 produced a dose-dependent prolongation of cardiac action potential and blocked the hERG/IKr current with an IC₅₀ of 17 nM. At 3 nM, dofetilide significantly increased APD₉₀. Astemizole significantly increased APD₆₀ and APD₉₀ at 30 nM. Terfenadine significantly increased APD₉₀ at concentrations greater than 10 nM. Fexofenadine, a metabolite of terfenadine, did not produce any electrophysiologic changes in cardiac action potentials. Flecainide produced a dose-dependent prolongation of the cardiac action potential at 1 and 3 μM. Acute exposure to nifedipine significantly decreased APD₆₀ and APD₉₀ and produced a dose-dependent block of calcium current with an IC₅₀ of 0.039 μM. Verapamil first shortened APD₆₀ and APD₉₀ in a dose-dependent manner, until a compensating increase in APD₉₀, presumably via hERG blockade, was observed at 1 and 3 μM. Following a chronic exposure (20–24 h) to clinically relevant levels of pentamidine, a significant increase in action potential duration was accompanied by early afterdepolarizations (EADs).

Discussion

These experiments show the ability of AP measured from hiPSC-CM to record the interactions of various ion channels via AP recording and avoid the limitations of using several single ion channel assays in a noncardiac tissue.

Introduction

The generation of pluripotent stem cells by reprogramming mature somatic cells from adult human patients has produced an unlimited supply of stem cells which can be differentiated into a variety of cell types, including induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). These cells can be used for a variety of applications, such as the production of patient-specific models of arrhythmia; in vitro models to evaluate the effects of drugs for their causative or preventative actions on human disease such as congestive heart failure; and/or as a preclinical safety pharmacology cells to predict the outcome of clinical trials (Davis et al., 2012, Pugsley et al., 2011, Terrenoire et al., 2013, Uesugi et al., 2013).

Cardiac safety pharmacology must be determined during non-clinical and clinical drug development to identify changes in cardiovascular function and is mandated by regulatory agencies in the ICH S7B and E14 guidelines (2005). At present, there is no in vitro pre-clinical system using human tissue for cardiac safety testing. The unpredictable availability of human donors, including variations in age, sex, disease and drug treatment, make it impossible to use native human cardiac tissue as a routine tissue source for a testing model. The development of an in vitro system based on hiPSC-CM provides tissue for drug safety assessment using cells from the most relevant species in a timely and reproducible manner (Qu, Gao, Fang, & Vargas, 2013).

In regard to preclinical safety pharmacology, the actions of several reference compounds have been reported on action potentials and ion channels recorded from embryonic H7 stem cells as preclinical biomarkers of cardiac safety and efficacy. In general, these experiments accurately predicted the risk of torsades de pointes (TdP) with an overall sensitivity greater than animal tissue assays (Peng, Lacerda, Kirsch, Brown, & Bruening-Wright, 2010). However, a reduced latency for development of channel block and increased potency for IKr block by terfenadine and quinidine was observed in these studies. An additional study using the same embryonic H7 stem cells also reported sensitivity for detecting repolarization delay induced by IKr blockade (Qu et al., 2013). Although, in this study, these cells were less sensitive for detecting Na + channel blockade. These limitations may result from the embryonic source of these stem cells.

iCell® Cardiomyocytes are generated from an hiPSC line developed from adult volunteers and engineered to permit selection for high cardiomyocyte purity by expressing blasticidin resistance under the control of a cardiac myosin heavy chain promoter (Ma et al., 2011). Study of gene expression demonstrates that these cells express relevant cardiac markers such as ion channels, tissue-specific structural markers, and transcription factors (Puppala et al., 2013). A comprehensive investigation of the electrophysiologic properties of these highly pure hiPSC-CM also demonstrated three separate types of action potentials with atrial-like, nodal-like, and ventricular-like action potentials with gating properties of seven major cardiac ion currents (Ma et al., 2011).

To further validate ventricular-like iCell® Cardiomyocytes as a preclinical model of safety pharmacology, we investigated the actions of diverse reference agents at different concentrations on action potentials and ion currents recorded from ventricular-like cells while paced at a constant pacing rate under physiologic temperatures. We would emphasize that these experiments were designed to measure safety pharmacology in hiPSC-CM, not to investigate specific electrophysiologic mechanisms of drug action. These experiments included agents known to block both individual and multiple cardiac ion channels, inhibit ion channel trafficking and produce EADs. We found that hiPSC-CM can serve as a useful tool in early drug screening or cardiovascular safety frontloading.