Effects of cardioactive drugs on human induced pluripotent stem cell derived long QT syndrome cardiomyocytes

Kuusela et al. SpringerPlus (2016) 5:234 DOI 10.1186/s40064-016-1889-y RESEARCH Effects ofcardioactive drugs onhuman induced pluripotent stem cell derived long QT syndrome cardiomyocytes Jukka Kuusela1†, Ville J. Kujala1,2†, Anna Kiviaho1, Marisa Ojala1, Heikki Swan3, Kimmo Kontula3 and Katriina Aalto‑Setälä1,4,5* Abstract Human induced pluripotent stem cells (hiPSC) have enabled a major step forward in pathophysiologic studies of inherited diseases and may also prove to be valuable in in vitro drug testing. Long QT syndrome (LQTS), characterized by prolonged cardiac repolarization and risk of sudden death, may be inherited or result from adverse drug effects. Using a microelectrode array platform, we investigated the effects of six different drugs on the electrophysiological characteristics of human embryonic stem cell‑derived cardiomyocytes as well as hiPSC‑derived cardiomyocytes from control subjects and from patients with type 1 (LQT1) and type 2 (LQT2) of LQTS. At baseline the repolarization time was significantly longer in LQTS cells compared to controls. Isoprenaline increased the beating rate of all cell lines by 10–73 % but did not show any arrhythmic effects in any cell type. Different QT‑interval prolonging drugs caused prolongation of cardiac repolarization by 3–13 % (cisapride), 10–20 % (erythromycin), 8–23 % (sotalol), 16–42 % (qui‑ nidine) and 12–27 % (E‑4031), but we did not find any systematic differences in sensitivity between the control, LQT1 and LQT2 cell lines. Sotalol, quinidine and E‑4031 also caused arrhythmic beats and beating arrests in some cases. In summary, the drug effects on these patient‑specific cardiomyocytes appear to recapitulate clinical observations and provide further evidence that these cells can be applied for in vitro drug testing to probe their vulnerability to arrhythmia. Keywords: Induced pluripotent stem cell, Patient‑specific, Long QT syndrome, Cardiomyocytes, Multielectrode array, Cardioactive drug, Arrhythmia © 2016 Kuusela et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Background Inherited long QT syndrome (LQTS) is a potentially severe arrhythmic disease that affects the electrical repolarization of the myocardium and manifests as an abnormally long QT interval on electrocardiogram (ECG) recordings. LQTS may result in polymorphic ventricular tachycardias known as torsades de pointes (TdP), which can ultimately lead to life-threatening ventricular fibrillation and sudden cardiac death (Schwartz etal. 2013). Inherited forms of LQTS typically result from mutations in cardiac ion channel coding genes. In the case of LQT type 1 (LQT1), the mutations are present in the KCNQ1 gene which encodes the α-subunit of the slow component of the delayed rectifier potassium current (Kv7.1) (Wang etal. 1996), whereas LQT2 is caused by mutations of the KCNH2 gene which encodes the α-subunit of the rapid component of the delayed rectifier potassium current (Kv11.1) (Sanguinetti etal. 1995). Four founder mutations in these two genes have been found to explain the high (0.4%) prevalence of LQTS in the Finnish population (Marjamaa etal. 2009). The most prevalent of these founder mutations is the C-terminal KCNQ1 G589D missense mutation, resulting in a dysfunctional assembly domain and preventing the formation of fully functional potassium channel tetramers (Piippo et al. Open Access *Correspondence: katriina.aalto‑[email protected] †Jukka Kuusela and Ville J. Kujala contributed equally to this work 1 BioMediTech, University of Tampere, Finn‑Medi 5, Biokatu 12, 33014 Tampere, Finland Full list of author information is available at the end of the article Page 2 of 13 Kuusela et al. SpringerPlus (2016) 5:234 2001). Another prevalent but apparently milder founder mutation, the KCNH2 R176W missense mutation, resides in the N-terminus of the channel and accelerates the deactivation of the IKr tail current, thus leading to reduced potassium efflux (Fodstad etal. 2006). Research on the exact effects of these mutations on the functional properties of cardiomyocytes has been hampered by the lack of suitable techniques that can adequately recapitulate the full spectrum of the disease invitro. Human induced pluripotent stem cell (hiPSC) technology has recently made it possible to produce human cell lines with characteristics similar to those of embryonic stem cells from somatic cells of various origins (Takahashi etal. 2007; Yu etal. 2007). To date, multiple disease-specific hiPSC lines have already been established (Wu and Hochedlinger 2011). Several studies have focused on LQTS using the hiPSC technology (Bellin etal. 2013; Itzhaki etal. 2011; Kiviaho etal. 2015; Lahti etal. 2012; Matsa etal. 2011, 2014; Moretti etal. 2010). These studies suggest that hiPSC technology may be useful for modeling LQTS invitro. Until now, large-scale drug effects have been studied with human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and wildtype hiPSC-CMs (Braam etal. 2010; Caspi etal. 2009; Harris etal. 2013; Mehta etal. 2013; Navarrete etal. 2013; Yokoo etal. 2009). In this study, we investigated the effect of six clinically used drugs in control hESCand wildtype hiPSC-CMs and in hiPSC-derived LQT-CMs. Additional value will be gained by studying drug responses at clinically relevant concentrations (Schulz and Schmoldt 2003), since many of the mutation carriers can have a latent, subclinical form of LQTS that may be exacerbated upon pharmacological challenge (Roden 2004). We created hiPSC lines from the dermal fibroblasts of patients with KCNQ1 G589D and KCNH2 R176W mutations. We investigated how these patient-specific hiPSCs respond to pharmacological challenges in comparison with cardiomyocytes derived from control hESCs and hiPSCs. Methods Patient‑specific pluripotent stem cell induction The LQT1and 2-specific hiPSCs were induced as previously described (Takahashi etal. 2007) from fibroblasts of patients carrying the KCNQ1 (G589D) or the HERG (R176W) mutation (Fodstad et al. 2004) (see Additional file1: Materials and methods). The study has been approved by Pirkanmaa Hospital District ethical committee (R08070). Details of the hiPSC lines are listed in Table1. The subjects volunteered for the study gave their consent for skin biopsy to be taken. Stem cell culture The control pluripotent stem cell lines used in this study were H7 (hESC, WiCell), UTA.00112.hFF [fetal wild-type from ATCC (WT)-hiPSC], and UTA.01006.WT (WThiPSC derived from a healthy adult). For LQT1, 4 patientspecific hiPSC lines were used: UTA.00208.LQT1, UTA.00211.LQT1, UTA.00313.LQT1 and UTA.00303. LQT1. For LQT2 2 patient-specific hiPSC lines were used: UTA.00514.LQT2 and UTA.00525.LQT2. The pluripotent stem cells were cultured on a mouse embryonic fibroblast (MEF, Millipore) feeder cell layer in KSR medium consisting of KnockOut Dulbecco’s Modified Eagle’s Medium (KO-DMEM, Invitrogen) supplemented with 20% KnockOut serum replacement (KO-SR, Invitrogen), 1% non-essential amino acids (NEAA, Lonza), 1% Glutamax (Invitrogen), 50U/mL penicillin/streptomycin (Lonza), and recombinant human basic fibroblast growth factor (R&D Systems). The cells were passaged once a week by treating the pluripotent stem cell colonies with collagenase IV (Gibco) and seeding them onto a fresh MEF feeder layer. Table 1 Details ofthe control andpatient-specific pluripotent stem cell lines used fordrug testing ICM inner cell mass of blastocyst, hFF human foreskin fibroblast, hADF human adult dermal fibroblast, hESC human embryonic stem cell, hiPSC human induced pluripotent stem cell *Siblings ‡,θ,ψ clonal lines from same individual ssymptomatic aasymptomatic Cell line Line type Mutation Disease Affected current Source Purpose H7 (hESC) hESC Wild type None None ICM hESC control UTA.00112.hFF (WTa) hiPSC Wild type None None hFF Control UTA.01006.WT (WTb) hiPSC Wild type None None hADF Adult control UTA.00208.LQT1θ,*,s hiPSC G589D LQT1 IKs hADF Symptomatic, patient‑specific UTA.00211.LQT1θ,*,s hiPSC G589D LQT1 IKs hADF Symptomatic, patient‑specific UTA.00303.LQT1ψ,*,a hiPSC G589D LQT1 IKs hADF Asymptomatic mutation carrier‑specific UTA.00313.LQT1ψ,*,a hiPSC G589D LQT1 IKs hADF Asymptomatic mutation carrier‑specific UTA.00514.LQT2‡,a hiPSC R176W LQT2 IKr hADF Asymptomatic mutation carrier‑specific UTA.00525.LQT2‡,a hiPSC R176W LQT2 IKr hADF Asymptomatic mutation carrier‑specific Page 3 of 13 Kuusela et al. SpringerPlus (2016) 5:234 Cardiomyocyte differentiation Cardiomyocytes were differentiated from hESCs as well as from the control and LQT1 and -2-specific hiPSCs using the mouse visceral endoderm-like (END-2) cell coculture method, as described elsewhere (Mummery etal. 2003). Briefly, mitomycin C (Sigma) -inactivated END-2 cells were trypsinized and plated onto 12-well plates in 0% KO-SR hES medium. The pluripotent stem cells were plated on top of the END-2 cell monolayer the following day. The 0% KO-SR hES medium was refreshed on days 5, 8 and 12. At day 14, the medium was changed to 10% KO-SR hES medium, and it was refreshed three times a week thereafter. Pharmacological testing Drugs Cisapride monohydrate, quinidine and (±)-sotalol hydrochloride were obtained from Sigma-Aldrich and dissolved in dimethyl sulfoxide (Sigma-Aldrich) at 10mM. E-4031 (Alomone Labs) and erythromycin (Abboticin i.v., Amdipharm) were dissolved in sterile H2O at 1 and 50mg/mL, respectively. Isoprenaline (Isuprel i.v., Hospira) was supplied in ready-to-use ampoules. The drug concentrations were chosen based on their therapeutic blood serum concentration range (Schulz and Schmoldt 2003). The following concentrations of cisapride monohydrate, quinidine, (±)-sotalol hydrochloride, and erythromycin were tested: half the concentration of the lower limit for the therapeutic blood serum concentration, the lower limit of the therapeutic blood serum concentration range, the average therapeutic serum concentration, the upper limit of the therapeutic range, and twice the upper limit of the therapeutic concentration (Schulz and Schmoldt 2003). The isoprenaline concentrations were chosen according to previously published values and multiples thereof (Pekkanen-Mattila et al. 2009). The drugs were serially diluted in embryoid body (EB)- medium with 5% fetal bovine serum (FBS, see below). The test concentrations for each drug are listed in Table2. Multielectrode array recordings The hESC-CMs and the control, LQT1-, and LQT2specific hiPSC-CM aggregates were mechanically excised from the cultures and plated onto fetal bovine serum (PAA) and 0.1% gelatin- (Sigma-Aldrich) coated 6-well-MEAs (6wellMEA200/30iR-TI-mr, MultiChannel Systems MCS GmbH). The FPs were recorded with a USB-MEA1060 amplifier, and the temperature was kept at +37°C using a TC02 heating element (both from Multi Channel Systems MCS GmbH). The MEAs were covered with gas-permeable MEA membranes (ALA MEASHEET, ALA Scientific) during the recordings. A wash-in time of 2min was allowed for the drugs before recordings. The beating areas were cultured in EB medium consisting of KO-DMEM (Invitrogen) supplemented with 20% FBS (Invitrogen), 1% NEAA (Lonza), 1% Glutamax (Invitrogen), and 50U/mL penicillin/streptomycin (Lonza). The drug tests were performed in 5% FBS containing EB medium; the medium was changed at least an hour prior to the baseline recordings, and the cells were allowed to stabilize in the incubator (+37°C, 5% CO2). The field potential signals were recorded with MC_Rack v.4.0.0 software (Multi Channel Systems MCS GmbH). Data andstatistical analysis The field potential durations (FPDs) from the recorded files were measured offline using our in-house developed CardioMDA software (Pradhapan etal. 2013) and AxoScope10 (Molecular Devices). The baseline data are presented as mean±standard deviation (SD). Linear mixed-effect models with either Bazett’s corrected field potential duration (cFPD) or beating rate (BR) as a dependent variable were fitted using the function lme in R (Software environment for statistical computing and graphics, version 2.13.0, The R Foundation for Statistical Computing). The factorial variable for the concentration level was used as an independent variable. The lowest concentration level was used as a reference group. Random intercept for different aggregates was used together with independent random errors. Results LQT patient characteristics Skin biopsies were obtained from two siblings, a 28-year old female asymptomatic LQT1 patient (QTc interval, 428ms) and a 41-year old female symptomatic LQT1 patient (QTc interval, 456 ms), both with the KCNQ1 G589D mutation. In addition, skin biopsies were obtained from a 61-year old male asymptomatic LQT2 patient with the KCNH2 R176W mutation (QTc interval, 437ms). The symptomatic LQT1 patient had experienced seizures before beta-blocker medication, while the asymptomatic sibling has never had any cardiac Table 2 Drugs andtheir concentrations used withcardiomyocytes derived from human embryonic and induced pluripotent stem cells Drug Concentrations Isoprenaline nM: 81, 402 Cisapride monohydrate nM: 40, 83, 120, 165, 330 Erythromycin µM: 1.5, 3, 5.5, 8, 16 Sotalol µM: 0.8, 1.6, 5.7, 9.7, 19.4 Quinidine µM: 1.5, 3, 9, 15, 30 E‑4031 nM: 10, 100, 300, 500, 700 Page 4 of 13 Kuusela et al. SpringerPlus (2016) 5:234 symptoms. The asymptomatic LQT2 individual has only experienced occasional palpitations, which although are not specific symptoms of LQTS. Molecular characteristics ofcardiomyocytes Reverse-transcription PCR and immunofluorescence microscopy showed that the control and LQT-hiPSCs expressed the pluripotency markers NANOG, OCT3/4, SOX2, SSEA4, TRA-1-60, and TRA-1-81 (Additional file1: Fig. S1A, Table S1). Furthermore, the hiPSC colony morphology resembled those of hESCs, the hiPSCs differentiated into cardiomyocytes at efficiencies previously reported for hESCs upon END-2 co-culture (data not shown) (Pekkanen-Mattila etal. 2009), showed nuclear localization of the cardiac-specific transcription factor NKX2.5 which is also expressed in hESC-CMs (Asp etal. 2010), and exhibited a distinct striated pattern in cardiac troponin T staining (Additional file1: Fig. S1B). The electrophysiological properties and drug responses were recorded with microelectrode arrays (MEAs). Effects ofcardioactive drugs tothe cFPD ofhiPSC‑CMs We compared the drug responses of the control cardiomyocytes derived from hESC line H7 (hereafter called hESC) and two wild-type hiPSC lines UTA.00112.hFF (hereafter called WTa) and UTA.01006.WT (hereafter called WTb) to the hiPSC-derived LQT-CMs (for the cell line properties, see Table1). The results of the clonal cell lines derived from the same individual were pooled together (UTA.00208.LQT1 and UTA.00211.LQT1 [=LQT1A], UTA.00303.LQT1 and UTA.00313.LQT1 [=LQT1B], UTA.00514.LQT2 and UTA.00525.LQT2 [=LQT2]). LQT1A were derived from a symptomatic patient, LQT1B from an asymptomatic mutation carrier and LQT2 from asymptomatic mutation carrier. Baseline characteristics The baseline beating rates (BR) showed large differences between cell populations, ranging from 59 to 105 beats per minute (Fig. 1a). However, these BRs did not differ significantly among cardiomyocytes differentiated from hESCs, control-, or LQT-hiPSCs. In contrast, the beating rate-corrected field potential duration (cFPD, Bazett´s) showed significant differences between controland LQT-CMs (Fig.1b). At baseline level, the LQT-CMs showed significantly longer cFPD (586–600 ms) compared to control-CMs (340–400ms) (p<0.001). Isoprenaline effects To test whether the differentiated cardiomyocytes had proper β-adrenergic responses, we subjected them to an isoprenaline challenge. This treatment had chronotropic effect on cardiomyocytes and increased the BR at the concentration of 81 and 402nM (Fig.2). Isoprenaline did not induce any arrhythmic beats in any of the CMs, including those derived from LQT1and LQT2-hiPSC lines. Effects ofnon‑cardiac drugs cisapride anderythromycin Cisapride increased the repolarization time in all CMs studied, although this effect did not reach statistical significance in the LQT2-CMs, apparently due to large experimental variation (Fig.3a). Erythromycin likewise induced a significant average increase of cFPD in all cell lines studied, in a concentration range of 1.5–16µM (Fig.3b). No arrhythmic beats or beating arrests were observed with cisapride or with erythromycin. Effects ofthe anti‑arrhythmic drugs sotalol andquinidine Upon sotalol (0.8–19.4µM) challenge, the maximal cFPD prolongation ranged from of 8% (WTb-CMs) to 23% (LQT1A-CMs) (Fig.4a). Quinidine also caused a concentration-dependent (1.5–30µM) average cFPD prolongation in all the cardiomyocytes (Fig.4b). Of all clinically used drugs tested (cisapride, erythromycin, sotalol, quinidine), quinidine showed the largest cFPD-increasing effect (31–42%) seen in LQT1-CMs. The effect ofthe hERG channel blocker E‑4031 E-4031 also caused concentration-dependent cFPD prolongation in all cardiomyocyte types (Fig.4c). It is of note that in hESC-CMs all but one of the cardiac aggregates became arrhythmic or stopped beating at concentrations of 500–700 nM. Thus, error bars indicating standard deviation could not be calculated for these experimental points. The cFPD prolongation in the different cell lines reached statistical significance at E-4031 concentration of 100nM indicating that the sensitivities of the different cell lines to E-4031 were very similar (Fig.4c). In summary, Fig.5 illustrates the representative traces of cisapride, erythromycin, sotalol, quinidine and E-4031 in controland LQT-CMs. The proarrhythmic effects ofsotalol, quinidine andE‑4031 Sotalol, quinidine and the hERG blocker E-4031 showed proarrhythmic effects on certain individual cell lines (Table3). With sotalol, only spontaneous afterdepolarization (AD)-like waveform abnormalities (but not beating arrests) were observed (Table3). Sotalol caused AD-like waveforms ranging from concentrations 5.7 µM (average therapeutic serum concentration) to 19.4µM (twice the upper therapeutical serum concentration). None of the LQT1B-CMs showed any arrhythmic behavior upon sotalol challenge. Quinidine showed also beating arrests in addition to AD-like waveform abnormalities (Table3). The AD-like Page 5 of 13 Kuusela et al. SpringerPlus (2016) 5:234 waveforms were observed at the therapeutical serum concentration range (3–15µM) in hESC-, LQT1Aand LQT2-CMs but not in WTor LQT1B-CMs (Table3). On the other hand, the beating arrests occurred ranging from 9µM (average therapeutic serum concentration) to 30µM (twice the upper therapeutical serum concentration) (Table3). The compound E-4031 also caused AD-like waveform abnormalities and beating arrests (Table 3). E-4031 caused AD-like waveform abnormalities in cardiomyocytes differentiated from all of the lines except WTb-CMs. Furthermore, beating arrests were seen in all cardiomyocyte types except LQT1A-CMs. Among the LQTs, the highest frequency for AD-like waveform abnormalities was found in LQT1A-CMs (50% of cardiomyocyte aggregates). On the other hand, the highest frequency for beating arrests were seen in LQT2-CMs (31% of cardiomyocyte aggregates). Discussion The present study further demonstrates the usefulness of the hiPSC-derived cardiomyocytes to serve as a model Fig. 1 Baseline cardiac parameters. a The beating rate (BR) of the cardiomyocyte clusters differentiated from each human induced pluripotent stem cell (hiPSC) line. b The rate‑corrected field potential duration (cFPD) for the same cardiomyocyte clusters. Significance level is indicated by ***p < 0.001 Page 6 of 13 Kuusela et al. SpringerPlus (2016) 5:234 for long QT syndrome, showing that the QT intervalprolonging effects of clinically used drugs are replicated in this in vitro system, using concentrations that are within or close to their therapeutic range (Schulz and Schmoldt 2003). We showed that LQT-CMs corrected field potential duration is significantly longer than in control-CMs at baseline conditions, which suggests that long QT syndrome is recapitulated in these cells. This study also reproduces in cell clusters the earlier observation with single cells by patch clamp with prolonged APDs in LQT-CMs compared to controls (Kiviaho etal. 2015; Lahti etal. 2012). In the current study LQTand control CMs were further exposed to various cardioactive drugs to investigate their effects. The cardiomyocytes beating rates were increased by isoprenaline, which is a β-adrenergic agonist that affects calcium (ICa,L) channel activation via β1 receptors. This is in line with the previous studies showing positive chronotropic effect of isoprenaline on both hESCand hiPSC-CMs (Moretti etal. 2010; Pekkanen-Mattila etal. 2009; Yokoo etal. 2009). β-adrenergic stimulation with isoprenaline did not have any arrhythmic effects on the LQT1 lines although LQT1 patients often experience symptoms at increased heart rates. Cisapride is a serotonin 5-HT4 receptor agonist that has been reported to block the hERG potassium channel (Mohammad etal. 1997). It is known to induce TdP in patients (Darpö 2001). Caspi et al. (2009) reported significant (>50 nM) dose-dependent prolongation of cFPD in hESC-CMs (Braam etal. 2010; Caspi etal. 2009). We obtained similar results as Caspi etal. (2009) as the hESC-CMs used in our study showed dose-dependent and significant cFPD prolongation starting at 83 nM. In contrast, Braam etal. (2010) reported no significant cisapride-induced cFPD prolongation in hESC-CMs. However, they judged the therapeutic concentration to be 2.6–4.9nM (Redfern etal. 2003), whereas we chose the therapeutic range as 83–165nM (Schulz and Schmoldt 2003), which may explain the fact that they were not able to detect significant cisapride-induced cFPD prolongation. Mehta etal. (2013) have also investigated the effect of cisapride to the cFPD of 150–170days old EB-based hiPSC-derived CMs. Likewise, they also found concentration-dependent cFPD prolongation (Mehta et al. 2013). Mehta etal. (2013) did not observe any arrhythmias with cisapride, which is in line with our study here. Erythromycin is a macrolide antibiotic that prolongs the QT interval in patients (Shaffer etal. 2002). The therapeutic plasma concentration of erythromycin has been reported to be 3–8µM (Schulz and Schmoldt 2003). In our study, erythromycin prolonged significantly cFPD in hESC-CMs already at the first test concentration (1.5 µM). Our data may suggest a slightly higher sensitivity of LQT2-CMs compared to LQT1-CMs, but high experimental variation precludes firm conclusions. Erythromycin did not induce any arrhythmic effects in the cardiomyocytes. Sotalol is a β-adrenergic receptor antagonist and a Vaughan–Williams class III anti-arrhythmic agent that potently blocks the potassium ion channels in Fig. 2 Isoprenaline response of the control and long QT syndrome (LQT) type 1 and 2‑specific cardiomyocyte clusters (hESC, WTa, WTb, LQT1A, LQT1B and LQT2). Data is presented mean ± SD. Mean beating rates (BR) were normalized to baseline BR. Asterisks mark the statistical significance for mean BR increase compared to baseline values. *p < 0.05, **p < 0.01, ***p < 0.001 Page 7 of 13 Kuusela et al. SpringerPlus (2016) 5:234 cardiomyocytes (Edvardsson etal. 1980). In hESC-CMs, Braam et al. (2010) reported cFPD prolongations of 15–20% at sotalol concentrations of 1.8–14µM (Braam etal. 2010). In a subsequent study, they also observed 20% cFPD prolongation in hESC-CMs at sotalol concentration of 10 µM (Braam et al. 2013). Our study resulted in similar data. At concentrations of 6–10µM, we observed a cFPD prolongation of 12–18 % in the hESC-CMs, increasing to 21% at 19µM. We observed that sotalol increased cFPD in all the CMs at the therapeutical concentration range (1.6–9.7µM). In line with our data, Mehta etal. (2013) reported similar observations from sotalol-induced cFPD prolongation in hiPSCderived CMs (Mehta etal. 2013). Furthermore, according to our data sotalol significantly increased cFPD at earlier concentrations in LQT-CMs than in WTa or WTb. This may suggest that LQT-CMs may be more vulnerable to the cFPD-prolonging effect of sotalol than hiPSC-derived control cells. Quinidine is a class Ia antiarrhythmic drug that primarily blocks sodium ion channels at high concentrations and hERG channel at low concentrations and is associated with TdP (Bauman et al. 1984). Quinidine prolonged the cFPDs in all of our cell lines ranging from 16 to 42%. Braam etal. (2010) reported a prolongation of 20–50% in hESC-CMs with quinidine concentrations of up to 3.2µM and increases in the cFPD at higher concentrations, and Caspi et al. (2009) reported a cFPD Fig. 3 Non‑cardiac drug responses of the control and long QT syndrome (LQT) type 1 and 2‑specific cardiomyocyte clusters (hESC, WTa, WTb, LQT1A, LQT1B and LQT2). The drugs used were a cisapride and b erythromycin. Changes in the rate‑corrected field potential duration (cFPD) were calculated as relative changes from the baseline for each cardiomyocyte cluster. Data is presented mean ± SD. Mean ΔcFPDs were normal‑ ized to baseline cFPD. Asterisks mark the statistical significance for mean ΔcFPD prolongation compared to baseline values. *p < 0.05, **p < 0.01, ***p < 0.001 Page 8 of 13 Kuusela et al. SpringerPlus (2016) 5:234 Fig. 4 Class I and class III anti‑arrhythmic drug responses of the control and long QT syndrome (LQT) type 1 and 2‑specific cardiomyocyte clusters (hESC, WTa, WTb, LQT1A, LQT1B and LQT2). The drugs used were a sotalol, b quinidine and c E‑4031. During E‑4031 challenge all but one of the cardiac aggregates in hESC‑CMs became arrhythmic or stopped beating at concentrations of 500–700 nM. Thus, error bars indicating standard deviation could not be calculated for these experimental points. Changes in the rate‑corrected field potential duration (cFPD) were calculated as relative changes from the baseline for each cardiomyocyte cluster. Data is presented mean ± SD. Mean ΔcFPDs were normalized to baseline cFPD. Asterisks mark the statistical significance for mean ΔcFPD prolongation compared to baseline values. *p < 0.05, **p < 0.01, ***p < 0.001 Page 9 of 13 Kuusela et al. SpringerPlus (2016) 5:234 prolongation of slightly <30% in hESC-CMs with 8µM quinidine (Braam etal. 2010; Caspi etal. 2009). Mehta etal. (2013) reported a 20 and 55% cFPD prolongation in 150–170days old hiPSC-CMs at 1 and 10µM concentrations, respectively. Here, we noticed an 18% increase in the cFPD of the hESC-CMs at a concentration of 9µM. Quinidine induced beating arrests which emerged at the average or upper therapeutic serum concentration. Quinidine is known to inhibit sodium ion channels at high concentrations (Snyders and Hondeghem 1990), which may be the reason for higher frequency of beating arrest than AD-like waveform abnormalities. The LQT2-CMs, showed significantly prolonged cFPD already at the half of the lower limit of therapeutic concentration (1.5µM), Fig. 5 Representative traces of cisapride, erythromycin, sotalol, quinidine and E‑4031 in control‑ and long QT‑cardiomyocytes (LQT‑CMs)