**Case Report on: Very Early Afterdepolarizations in HiPSC-Cardiomyocytes—An Artifact by Big Conductance Calcium Activated Potassium Current (Ibk,Ca)**

**András Horváth 1,2,3,**†**,**‡**, Torsten Christ 1,2,\*,**†**, Jussi T. Koivumäki 4,**

**Maksymilian Prondzynski 1,2,§, Antonia T. L. Zech 1,2, Michael Spohn 5, Umber Saleem 1,2, Ingra Mannhardt 1,2, Bärbel Ulmer 1,2, Evaldas Girdauskas 2,6, Christian Meyer 3,7, Arne Hansen 1,2, Thomas Eschenhagen 1,2 and Marc D. Lemoine 1,2,7,\***


Received: 28 October 2019; Accepted: 15 January 2020; Published: 20 January 2020

**Abstract:** Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent an unlimited source of human CMs that could be a standard tool in drug research. However, there is concern whether hiPSC-CMs express all cardiac ion channels at physiological level and whether they might express non-cardiac ion channels. In a control hiPSC line, we found large, "noisy" outward K<sup>+</sup> currents, when we measured outward potassium currents in isolated hiPSC-CMs. Currents were sensitive to iberiotoxin, the selective blocker of big conductance Ca2<sup>+</sup>-activated K<sup>+</sup> current (IBK,Ca). Seven of 16 individual differentiation batches showed a strong initial repolarization in the action potentials (AP) recorded from engineered heart tissue (EHT) followed by very early afterdepolarizations, sometimes even with consecutive oscillations. Iberiotoxin stopped oscillations and normalized AP shape, but had no effect in other EHTs without oscillations or in human left ventricular tissue (LV). Expression levels of the alpha-subunit (KCa1.1) of the BKCa correlated with the presence of oscillations in hiPSC-CMs and was not detectable in LV. Taken together, individual batches of hiPSC-CMs can express sarcolemmal ion channels that are otherwise not found in the human heart, resulting in oscillating afterdepolarizations in the AP. HiPSC-CMs should be screened for expression of non-cardiac ion channels before being applied to drug research.

**Keywords:** human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs); iPS cells; stem cells; big conductance calcium activated potassium channel (BK); Maxi-K; slo1; KCa1.1; iberiotoxin; long QT syndrome

#### **1. Introduction**

Human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CMs) have gained interest as a human model to study heart physiology and pathophysiology [1–4], cardiovascular pharmacology [5] and cardiac repair [6]. In this context, it is important that hiPSC-CMs share properties of human adult CMs. Many reports claimed that characteristics of hiPSC-CMs might differ from adult CMs, a finding frequently interpreted as immaturity. On the one hand, immaturity could be caused by the lack of cardiac ion channels or by differences in expression levels such as the inward rectifier potassium current (IK1) [7–10]. On the other hand, hiPSC-CMs can show ion currents, which are absent in adult human CMs such as the T-type calcium current [11,12]. Based on these findings, there is a need for a detailed electrophysiological characterization of hiPSC-CMs before using them as a model for human CMs. Here we report the coincidently found expression of a non-cardiac ion channel in hiPSC-CMs as a peculiarity of a single control cell line: the big conductance calcium activated potassium current (BKCa; alternatively used names: Maxi-K, slo1, KCa1.1).

BKCa is a voltage- and calcium-gated potassium channel (KCa1.1) generating huge conductivity for potassium. BKCa is widely expressed in the human body, mainly in neural cells, blood vessels, kidney, but not in in cardiomyocytes. Recently, artificial expression of BKCa was shown to be able to shorten action potentials (APs) in murine CMs [13]. Consequently, expression of the non-cardiac BKCa current in human CMs was proposed as a genetic therapy for the long QT syndrome. However, the contribution of individual ion channels to repolarization and resulting AP shape differs from murine to human CMs. Putative impact of BKCa to human cardiac electrophysiology remains unclear.

The goal of this study was to draw attention to the fact that hiPSC-CMs can express non-cardiac ion channels. In addition, we had the chance to elucidate how the expression of BKCa may influence the membrane potential of human CMs. Based on our findings, we propose a regular assessment for expression of non-cardiac ion channels as a part of quality control when using hiPSC-CMs.

### **2. Methods**
