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Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca2+ imaging of cells loaded with the Ca2+ indicator Fluo-4 enabled us to examine intracellular Ca2+ handling properties, and we found a significantly higher incidence of irregular Ca2+ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca2+ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na+/Ca2+exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca2+ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (INCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of INCX.


Introduction
In 1971, Andersen et al. [1] reported a patient with intermittent muscular weakness, extrasystoles, and multiple developmental anoma-lies. Then in 1994, Tawil et al. [2] denoted a novel and distinct clinical syndrome showing potassium-sensitive periodic paralysis, cardiac arrhythmia, and dysmorphic features as Andersen syndrome, warranting attention due to the potentially lethal cardiac arrhythmic events.
Thereafter, Andersen syndrome was generally renamed as Andersen-Tawil syndrome (ATS). Early reports also described ATS patients with QT interval prolongation, and consequently ATS was proposed as long QT syndrome type 7 (LQTS7) [3]. However, accumulating subsequent findings showed that QT interval prolongation is not a common feature of ATS, although prominent U wave and QU interval prolongation can be hallmarks of ATS, distinguished it from long QT syndrome [4]. Approximately 40% of ATS patients show cardiac arrhythmias on the baseline electrocardiogram, half of which are diagnosed as nonsustained ventricular tachycardia, and 3% of ATS patients show Torsade de pointes [4]. However, a French cohort showed that a severe clinical presentation with a very high rate of ventricular arrhythmias might be unrelated to the fatal arrhythmic prognosis of ATS patients [5].
Several clinical cues and case reports of ATS have provided hints for future standardized treatments. Exercise is an important trigger of ventricular tachyarrythmia and syncope in some patients with ATS, therefore patients are commonly treated with beta-blocker therapy [6]. Other drugs such as flecainide, calcium blockers, acetazolamide, and amiodarone have been proposed by anecdotal experiences [7][8][9]. An implantable cardiac defibrillator (ICD) is often necessary when patients showed fatal cardiac arrhythmias [10]. However, the rarity of ATS hampers the analytical interrogation of clinical data and thus the development of effective treatments, and it remains unclear who should be treated, as well as when and how [11,12].
ATS is an autosomal dominant genetic or sporadic disorder and has been linked to mutations in KCNJ2, a genotype labeled as ATS type 1 (ATS1) [13]. Patients with such mutations in KCNJ2 accounts for about 70% of ATS cases, with the remaining 30% of cases labeled as type 2 (ATS2), for which the genetic cause remains unknown [14]. KCNJ2 encodes for a voltage-gated, inward-rectifying potassium channel (Kir2.1) that contributes to the cardiac inward rectifier current I K1 . Most KCNJ2 mutations in ATS1 cause loss of function and dominant-negative suppression of the Kir2.1 channel function, leading to less I K1 [15,16]. Most of the electrophysiological studies have used a heterologous expression system, with human mutated genes in nonhuman/non-cardiac cells, such as Xenopus oocyte and HEK293 cells. I K1 is present in human ventricular and atrial cardiomyocytes and might play a major role in repolarizing the action potential and stabilizing the resting potential in humans [17,18]. Simulation studies suggest that a reduction in I K1 during the terminal phase of repolarization and during diastole would induce delayed after-depolarizations (DADs) and spontaneous arrhythmias [3,19]. To elucidate the pathophysiological function of mutated genes in the human heart, patient cardiomyocytes can be compared to human control cardiomyocytes. However, human cardiomyocytes from living patients are not an easily obtained or abundant enough tool for basic research. Reprogramming of human somatic cells into induced pluripotent stem cells (iPSCs) potentially enables us to generate patient-specific, disease-specific iPSCs as in vitro genetic disease models. To this end, several studies have yielded disease models using patient-specific iPSCs [20][21][22][23][24].
This study sought to establish a model for ATS using patientspecific iPSCs, to elucidate the pathogenesis of ATS and search for potential drug candidates for therapy. We thus generated iPSCs from three ATS patients carrying one of the KCNJ2 mutations (R218W, R218Q, R67W, respectively). Multi-electrode array (MEA) analysis revealed strong arrhythmic events in these ATS-iPSC-derived cardiomyocytes, and Ca 2+ imaging unveiled a highly irregular Ca 2+ release. Drug testing subsequently revealed that both of these effects were significantly suppressed in the ATS-iPSC-derived cardiomyocytes by flecainide and a reverse-mode Na + /Ca 2+ exchanger (NCX) inhibitor. Finally, whole-cell voltage clamping of isolated guinea-pig ventricular cardiomyocytes showed that flecainide could directly affect I NCX . Our results indicate that ATS-iPSC-derived cardiomyocytes could recapitulate abnormal electrophysiological phenotypes and thus might serve as a useful model for exploring disease mechanisms and drug screening.

Patient consent
All subjects provided informed consent for blood testing for genetic abnormalities associated with ATS. The Ethics Committee of Keio University approved the isolation and use of patient and control somatic cells for iPSC studies (approval no. 20-92-5), which was performed only after the patients and controls had provided written informed consent. Our study also conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2-3) for use of human tissue or subjects. Patient 1 is 10 years old, male with a KCNJ2 R218W mutation. Patient 2 is 27 years old, male with a KCNJ2 R67W mutation. Patient 3 is 47 years old, male with a KCNJ2 R218Q mutation. Control 1 is 29 years old, male and control 2 is 24 years old, male.

Human iPSCs generation
The iPSCs were established from T lymphocytes with Sendai virus encoding OCT3/4, SOX2, KLF4, and c-MYC [25,26]. Briefly, peripheral blood mononuclear cells (PBMCs) were collected and transferred at 1.5×10 6 cells per well to a fresh anti-CD3 antibody-coated 6-well plate, and incubated for an additional 24 h. Then, solutions containing the Sendai virus vectors individually carrying each of OCT3/4, SOX2, KLF4, and c-MYC were added at 10 MOI. After 24 h of infection, the medium was changed to fresh KBM 502 medium (KOHJIN BIO, Saitama, Japan), and the cells were collected and split at 5×10 4 cells into 10 cm plates pre-seeded with mouse embryonic fibroblasts (MEFs). After an additional 24 h of incubation, the medium was changed to hiPSC medium supplemented with 4 ng/ml basic fibroblast growth factor (bFGF). Approximately 20-30 days after infection, iPSC colonies appeared and were picked up. Sendai virus vectors carrying the same four specific transcriptional factors independently were commercially obtained (Life Technologies; CytoTune-iPS reprogramming kit).

Teratoma formation
To confirm pluripotency in vivo, teratoma formation was assessed in accordance with the Institutional Animal Care and Use Committee of Keio University. Approximately 1-2×10 6 iPSCs were injected into the testis of anesthetized immune-compromised NOD-SCID mice (CREA-Japan, Tokyo, Japan). At 10-12 weeks after the injection, mice were euthanized and the teratomas were excised, fixed overnight in formalin, embedded in paraffin, and analyzed by haematoxylin-eosin staining. The mice were anesthetized using a mixture of ketamine (50 mg/kg), xylazine (10 mg/kg), and chlorpromazine (1.25 mg/kg). The adequacy of anesthesia was monitored by heart rate, muscle relaxation, and the loss of sensory reflex responses, i.e., nonresponsive to tail pinching. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publications number 23-80 revised in 2011) and was approved by university review board in Keio University.

Genome sequencing
DNA sequencing was used to confirm the mutations in patientderived iPSCs. Genomic DNA was isolated using a Gentra Puregene Cell Kit (Qiagen, Venlo, Netherland) and the region encoding the mutation was amplified by polymerase chain reaction (PCR). The PCR product was electrophoresed on a 1% agarose gel and purified using a Wizard SV Gel and PCR Clean-Up System (Promega, WI, USA). The purified PCR product was sequenced with original primers (Supplementary Table 1) [27].

In vitro cardiomyocyte differentiation
Differentiation of iPSCs to cardiomyocytes was induced via embryoid bodies (EBs) [28,29]. iPSCs colonized on MEF feeders were detached by 1 mg/ml type IV collagenase (Invitrogen), and suspended on ultra-low attachment plates (Corning, NY, USA) with differentiation medium, consisting of 80% Minimum Essential Medium Alpha Medium (Gibco, CA, USA), 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol, 50 U/ml penicillin and 50 mg/ml streptomycin (Invitrogen), and 20% fetal bovine serum (Gibco). During suspension culture, the medium was changed every 2 days during the first week, after then every 4-5 days. For immunofluorescence and Ca 2+ imaging, single cardiomyocytes were obtained by dissociating spontaneously beating EBs using 0.25% trypsin (Invitrogen) and 1 mg/ml type IV collagenase (Invitrogen). Cardiomyocytes were plated on fibronectin (Sigma-Aldrich)-coated dishes. All assays were performed using beating EBs and cardiomyocytes, which were cultured 60-80 days after differentiation. All experiments we have done include the mixed data from 2 clones of each 2 control individual and 2 clones of each patient-derived iPSC.

Drug testing
Isoproterenol hydrochloride (Tokyo Chemical Industry, Tokyo, Japan), Flecainide acetate salt (Sigma-Aldrich), Pilsicainide hydrochloride (Sigma-Aldrich), KB-R7943 (Sigma-Aldrich), SEA0400 (MedChem Express, NJ, USA) and JTV519 (Sigma-Aldrich) were used for the drug testing assays. In drug testing experiments, we added prewarmed medium containing each drug, mildly by pipetting and mixed well gently. The temperature of medium was maintained at 37°C during experiment.

Multi-electrode array recordings
A multi-electrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany) was used to characterize the electrophysiological properties of human iPSC-derived cardiomyocytes. The spontaneously beating EBs were plated on fibronectin-coated MEA plates and incubated at 37°C. The extracellular electrograms were recorded in DMEM/HEPES (D5796; Sigma-Aldrich) at 37°C, and the obtained data were subsequently analyzed with MC_Rack (Multichannel Systems). The extracellular electrograms were used to determine field potential duration (FPD) and detect arrhythmic events. FPD was defined as the time interval between the initial deflection of the field potential and its return to baseline. FPD measurements were normalized (cFPD) to the rate using Bazett's correction formula: cFPD=FPD/(RR interval) 1/2 .

Ca 2+ imaging
Cardiomyocytes were placed on 35 mm glass-bottom dishes and loaded with 5 μM Fluo-4 AM (Invitrogen) in Tyrode's solution for 30 min at 37°C. Ca 2+ imaging was performed with a confocal microscope (LSM 510 Duo, Carl Zeiss, Jena, Germany) using a ×40 objective (NA=0.75). Line scans were acquired at a sampling rate of 2 ms/line (total of 10,000 times for a 20 s recoding). Cardiomyocytes spontaneously beating and electrically stimulated at 1 Hz were analyzed.

Action potential measurement in iPSC-derived cardiomyocytes
Cardiomyocytes were isolated from the spontaneously beating EBs by enzymatic dissociation, and action potentials were recorded in own beating condition by a conventional whole-cell configuration of patchclamp techniques at 37°C, using an Axopatch 200B patch clamp amplifier and a Digidata 1550 digitizer (Axon Instruments, Foster City, CA, USA). Action potentials were evoked by 2 ms supra-threshold current pulses at 1 Hz in the current-clamp mode. Pipettes were filled with the following solution (in mM): KCl 20, L-Glutamic acid monopotassium salt 120, NaCl 10, HEPES 10 (pH 7.2 with KOH), while Tyrode's solution contained (in mM): NaCl 140, KCL5.4, CaCl 2 1.8, MgCl 2 0.33, NaH 2 PO 4 5.5, glucose 5.5, HEPES 5.0 (pH7.4 with NaOH). Ventricular-type action potentials are distinguished by the presence of a marked plateau phase and an APD 90 /APD 50 ratio up to 1.3 [30].

Patch clamp experiment in the guinea-pig ventricular cardiomyocytes
Patch-clamp experiment was performed under the regulation of the Animal Research Committee of the Hamamatsu University School of Medicine. Single ventricular cells were isolated from guinea-pig heart by enzymatic dissociation. I NCX was recorded by loading Na + and Ca 2+ in both the intracellular and extracellular solutions, as previously described [31]. The external solution was maintained at 36°C ± 0.5°C. I NCX was recorded with a patch-clamp amplifier (TM-1000; Act ME, Tokyo, Japan), filtered at a 2.5-kHz bandwidth, and the series resistance was compensated. Data were stored using pCLAMP8 software (Axon Instruments). The current-voltage (I-V) relationship was obtained by ramp pulses from the holding potential of −60-0 mV, to −140 mV, and then back to −60 mV at a constant rate of 640 mV s −1 . The descending limb (from 0 to −140 mV) was plotted in the I-V relationship without capacitance compensation. The pipette resistance was 2-3 MΩ when filled with the pipette solution. The control external solution contained ouabain, nifedipine, Cs + , and ryanodine to block the Na + /K + pump current, I Ca , K + currents, and Ca 2+ release channels of the sarcoplasmic reticulum, respectively. The animal procedures were performed conform the NIH guidelines (Guide for the care and use of laboratory animals). Adult guinea pig was anesthetized by 100 mg/kg sodium thiopental (Sigma-Aldrich, St Louis, MO, USA) and euthanized by cervical dislocation.

Statistical analyses
Every data shown as "ATS" was obtained by using equal proportion of the data from six ATS-iPSC lines. Values are presented as the mean ± SEM. The significance of differences between two means was evaluated by unpaired and paired t-tests. Chi-square test and Fisher's exact probability test was used for comparisons between groups. Comparisons between more than two groups were determined by ANOVA followed by Tukey-kramer test. P < 0.05 was considered significant. *P < 0.05. **P < 0.01. †P < 0.001.

Generation of Andersen-Tawil syndrome-iPSCs
We selected three unrelated patients with ATS. Each patient has a mutation at KCNJ2, R218W, R67W, R218Q, respectively, which were previously characterized. All cases showed prominent U waves on their electrocardiogram (Fig. 1). Case 1 (R218W) shows frequent premature ventricular contractions (PVCs) by electrocardiogram, while Case 2 (R67W) and Case 3 (R218Q) have a history of periodic paralysis (Supplementary Table 2). As controls, we used two populations of iPSCs derived from healthy volunteers, including a previously characterized control iPSC line [20,25,32]. After normal karyotypes of generated iPSCs were examined, each iPSC was further investigated. The generated ATS-iPSCs and control-iPSCs showed appropriate stem cell marker expression (Fig. 2a, Supplementary Fig. 1) as well as multipotency, based on teratoma formation involving tissues derived from all three germ layers (Fig. 2b, Supplementary Fig. 2). Subsequently, cardiomyocytes were differentiated from the iPSCs by EB formation, and immunostaining revealed that these cells expressed cardiomyocyte-specific markers, α-actinin, atrial natriuretic peptide (ANP), cardiac troponin T (cTnT), and GATA4, and showed normal cardiomyocyte cellular structure (Fig. 2c, Supplementary Fig. 3).

Electrophysiological characterization of ATS-iPSC-derived cardiomyocytes under baseline conditions
To investigate the electrophysiological properties of the derived cells, we used an MEA system to measure the surface electrogenic activities of cell clusters. The MEA analyses revealed that control-iPSC-and ATS-iPSC-derived EBs showed similar rhythmic electrical activity (Fig. 3a), with no significant difference in spontaneous beating rate (Fig. 3b). The field potential duration (FPD) in MEA analysis is analogous to the QT interval in a surface electrocardiogram [20], and there was no significant difference in the cFPD (normalized for beating frequency) between control-iPSCand ATS-iPSC-derived EBs (Fig. 3c). These data are consistent with previous evidence of no significant difference in QT interval between control and ATS patients [4]. Next we investigated the action potentials electrically captured at 1 Hz in control-and ATS-iPSC-derived cardiomyocytes. We observed typical ventricular-type action potentials (Figs. 3d, 3e, 3f, 3g), while phase 3 repolarization was mildly decelerated in the ATS-iPSC-derived cardiomyocytes, although there was no significant difference in action potential duration (APD) 50 and APD 90 compared to control-derived cells (Fig. 3h, i). Additionally, there were no significant differences in maximum diastolic potential (MDP) (Fig. 3j).

Flecainide has a therapeutic potential on ATS-iPSC-derived cardiomyocytes
Accumulating clinical information suggests that exercise and catecholamine stimulation can be triggers of ventricular tachyarrythmia in patients with ATS [6]. Thus we next examined the effect of catecholamine on iPSC-derived cardiomyocytes by MEA analyses. In control-iPSC-derived cardiomyocytes, isoproterenol administration increased the beating rate, but not the incidence of arrhythmic events (Fig. 4a). In ATS-iPSC-derived cardiomyocytes, isoproterenol administration increased the beating rate and induced arrhythmic events (Fig. 4b,  Supplementary Fig. 4b). We defined the arrhythmic event in this study as the surface electrogenic activity which shows at least one of following characters, different shape, different polarity, different timing from basic rhythm. The incidence of beating EBs with arrhythmic events was significantly increased in ATS-iPSC-derived cardiomyocytes (Fig. 4c,  Supplementary Fig. 4a). Some reports have implicated the therapeutic potential of flecainide for controlling arrhythmic events in ATS patients, although the therapeutic mechanisms underlying such an effect remain elusive. In MEA analyses, flecainide reduced the in-cidence of the EBs with arrhythmic events (Figs. 4b, 4d, Supplementary  Fig. 4b-d).

Abnormal calcium transient in ATS-iPSC-derived cardiomyocytes
To further investigate the mechanisms underlying the arrhythmogenicity of ATS, we analyzed the Ca 2+ -handling properties of iPSCderived single cardiomyocytes, using fluorescent Ca 2+ imaging. In spontaneously self-beating cardiomyocytes, the ATS-iPSC-derived cardiomyocytes often exhibited irregular Ca 2+ release compared to control-iPSC-derived cardiomyocytes (Fig. 5a, Supplementary Fig. 5a), with the rate of cardiomyocytes with irregular Ca 2+ release significantly increased in ATS-iPSC-derived cardiomyocytes (Fig. 5b). To measure the precise properties of Ca 2+ dynamics, the beating of iPSC-derived cardiomyocytes was electrically captured at 1 Hz (Fig. 5c, Supplementary Fig. 5b). The intensity (ΔF/F0) was increased in ATS-iPSC-derived cardiomyocytes compared to controls (Fig. 5d). However, there was no significant difference in time to peak, time to 90% decay of the Ca 2+ transient (CaT 90 ), and τ of Ca 2+ transient decline (τ) between control-and ATS-iPSC-derived cardiomyocytes (Figs. 5e, 5f, 5g). To know whether irregular Ca 2+ release reflects on the increased diastolic cytosolic Ca 2+ concentration, we tested pharmacological sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibitor, cyclopiazonic acid (CPA), to increase the diastolic cytosolic Ca 2+ concentration. CPA significantly increased the proportion of cardiomyocytes with irregular Ca 2+ release in the control-iPSC-derived population ( Supplementary Fig. 5c). This suggests that irregular Ca 2+ release could be caused by diastolic Ca 2+ overload. Next we quantified sarcoplasmic reticulum (SR) Ca 2+ content, because this could affect cytosolic Ca 2+ concentration [33]. SR Ca 2+ content can be measured by the height of the Ca 2+ transient induced by rapid caffeine application, which showed no significant difference in SR Ca 2+ content between control-and ATS-iPSC-derived cardiomyocytes ( Supplementary Fig. 5d, h). The calcium dynamics in cardiomyocyte are regulated by various organelles and proteins, such as ryanodine receptor, and SERCA2a [34]. Then we examined SR Ca 2+ content after administration of flecainide, but found no significant difference between control-and ATS-iPSC-derived cardiomyocytes ( Supplementary  Fig. 5e). To further investigate the mechanisms of flecainide effect on the arrhythmogenicity of ATS, we measured the effect of flecainide on intracellular Ca 2+ dynamics in ATS-iPSC-derived single cardiomyocytes. At baseline, we often observed irregular Ca 2+ release in ATS-iPSC-derived single cardiomyocytes, and this irregularity was significantly reduced by flecainide in ATS-iPSC-derived single cardiomyocytes (Figs. 5i-k, Supplementary Fig. 5f-i).

A reverse-mode Na + /Ca 2+ exchanger inhibitor suppresses abnormal calcium transients in ATS-iPSC-derived cardiomyocytes
Flecainide is a cardiac-specific fast-inward Na + current blocker, but has pleiotropic functions. To examine whether the antiarrhythmic mechanism of flecainide would be affected by a Na + current blocker, we tried other a second cardiac fast-inward Na + current blocker, pilisicainide [35]. Unlike with flecainide, pilsicainide administration did not show a therapeutic effect on isoproterenol-induced arrhythmic events in ATS-iPSC-derived cardiomyocytes at various concentrations of pilisicainide (Fig. 6a, b). These data suggested that flecainide might not exert its antiarrhythmic effects in ATS by the Na + current blocking effect. Calcium dynamics are also regulated by a Na + /Ca 2+ exchanger (NCX) in cardiomyocytes [34]. Thus, we tested the effects of pharmacological NCX inhibition by KB-R7943, which at high levels inhibits both the forward-and reverse-mode (Ca 2+ in, Na + out) NCX, but lower KB-R7943 inhibits only reverse-mode NCX [36]. Reverse-mode NCX in turns augments calcium release from the sarcoplasmic reticulum through ryanodine receptors [37], and in our experiments, 500 nM KB-R7943 also suppressed the irregular Ca 2+ release (Fig. 6c). We also used more specific compound to NCX reverse mode, SEA0400 which reduced the incidence of cells with irregular Ca 2+ release ( Supplementary Fig. 6a, Supplementary Table 3). These results are similar to those obtained by flecainide administration. To know the involvement of RyR2, we examined the effect of JTV519 on the incidence of cells with irregular Ca 2+ release but we did not find any difference in the incidence of cells with irregular Ca 2+ release by JTV519 addition (Supplementary Fig. 6b).

Flecainide has a direct effect on NCX current
To elucidate the direct effect of flecainide on I NCX , the whole-cell voltage-clamp experiment was conducted in isolated guinea-pig ventricular cardiomyocytes. I NCX was induced by 1 mM Ca 2+ and 140 mM Na + in the external solution and 20 mM Na + and 226 nM free Ca 2+ in the pipette solution. Under these ionic conditions, the reversal potential of I NCX with a 3Na:1Ca stoichiometry was calculated to be −68 mV. After establishing the whole-cell clamp mode, the external solution was changed to the control external solution. When the  external solution was switched to one containing 30 μM flecainide, I NCX was immediately augmented (Fig. 7a), although 5 mM of Ni, a potent and selective inhibitor of I NCX under the present conditions, was applied to completely inhibit I NCX . The I-V curves of flecainidesensitive (a−c) and Ni-sensitive (b−c) components were obtained by subtraction (Fig. 7b). Both I-V curves are reversed at about −65 mV, indicating that the flecainide-sensitive current is I NCX . Flecainide therefore augmented the bi-directional I NCX in a concentration-dependent manner (Figs. 7c-d).

Discussion
Human genetic studies have successfully revealed the causal genes and mechanisms of inheritable arrhythmic disease [38]. Subsequent in vitro experiments using a heterologous expression system revealed the electrophysiological properties of wild-type and mutated ion channel, while genetically modified mice have played an important role in elucidating the in vivo function of specific genes [39], including in arrhythmic diseases [40]. However, mouse models of human arrhythmic diseases have several limitations. Compared to humans, the mouse has a small heart, a high basal heart rate, a short action potential of ventricular cardiomyocytes, and differential roles for some ion currents [41], and thus, many inheritable arrhythmic diseases cannot be modeled in mouse.
KCNJ2 mutations in ATS1 mostly decrease I K1 in vitro, thus affecting action potential repolarization and resting potential stabilization [17,18]. In ex vivo canine heart tissue, I K1 blockade by CsCl induced DADs and polymorphic ventricular tachycardia (VT) [42], while in ex vivo guinea pig heart, I K1 blockade by BaCl 2 induced high susceptibility to arrhythmic events in hypokalemic conditions due to cytosolic calcium accumulation [43]. In ex vivo rabbit heart, I K1 blockade by CsCl induced bidirectional ventricular tachycardia due to beat-to-beat alteration at the diastolic calcium content and triggered activity [44]. Although such drug-induced disease models are simple and useful, many drugs have pleiotropic effects and in elucidating specific gene functions in vivo, it is preferable to generate genetically modified disease models resembling human disease. Many KCNJ2 mutations in patients with ATS1 have dominantnegative effects on I K1 [45]. To decipher the role of Kir2.1 in cardiomyocytes in vivo, several genetically modified models were generated, with the first KCNJ2 knockout mice showing early lethality at the perinatal period due to a complete cleft of the secondary palate, and that KCNJ2 is required for I K1 -and K + -induced dilations in cerebral arteries [46]. In addition, cardiac-specific overexpression of a dominant-negative KCNJ2 in mice reduces I K1 reduction by 95%, leading to a significant prolongation of action potentials [47]. KCNJ2knockout ventricular cardiomyocytes lack detectable I K1 and show significantly broader action potentials and more frequent spontaneous action potentials than wild-type myocytes [48]. However, KCNJ2 knockout neonates show neither ectopic beats nor re-entry arrhyth- mias, suggesting that the increased automaticity and prolonged action potential were not sufficient causes of arrhythmic events in these mice. These results suggest that genetically modified mice could not fully recapitulate the phenotype of patients with ATS1.

Y. Kuroda et al.
Biochemistry and Biophysics Reports 9 (2017) [245][246][247][248][249][250][251][252][253][254][255][256] testing revealed that irregular Ca 2+ release and arrhythmic events were significantly suppressed by flecainide, but not by a pure sodium channel blocker, pilsicainide. Flecainide blocks the cardiac fast-inward Na + current [49], but has pleiotropic functions such as inhibiting the rapid component of the delayed rectifier K + current [50], the late Na + current [51], and cardiac ryanodine receptor-mediated Ca 2+ release [33]. Indeed, a drug-induced ATS model in guinea pig heart showed that higher NCX expression and weaker SERCA2a expression correlated with arrhythmic events, and that NCX inhibition by 5 µM KB-R7943 decreased arrhythmic events [52]. This concentration of KB-R7943 fully inhibits both forward-mode and reverse-mode NCX, while lower concentrations inhibit reverse-mode, but not forward-mode, NCX [53]. Reverse-mode NCX augments calcium release from the sarcoplasmic reticulum through ryanodine receptors [37], while inhibiting forward-mode NCX augments cytosolic Ca 2+ overload [54]. If the antiarrhythmic effect of flecainide and KB-R7943 works by blocking forward-mode NCX, irregular Ca 2+ release would not be decreased in ATS-iPSC-derived cardiomyocytes. In contrast, treatment of our ATS-iPSC-derived cardiomyocytes with flecainide and 500 nM KB-R7943 sufficiently suppressed both the increased arrhythmic events and irregular Ca 2+ release, while flecainide also augmented the bidirectional I NCX . These results support the notion that direct I NCX modulation would successfully suppress an arrhythmogenic substrate in ATS-iPSC-derived cardiomyocytes. Previous report showed that flecainide reduces I Na , which results in increased Ca 2+ efflux via NCX across the sarcolemma [55]. We could not observed anti-arrhythmic effect by other sodium channel blocker in ATS-iPSC-derived cardiomyocytes. In our experimental condition, both flecainide and a reversemode NCX inhibitor reduced the arrhythmic events, and flecainide augmented the bi-directional I NCX . NCX is the main pathway for Ca 2+ efflux from cardiomyocytes [56,57]. Ca 2+ removal through the forward mode produces an inward current, while the reverse mode bringing Ca 2+ into the cardiomyocytes produces an outward current. In fact, the mode of NCX dynamically changes throughout the cardiomyocyte cycle, although under normal conditions, net NCX activity in cardiomyocytes uses the forward mode. Now it seems that NCX might have a dual role in arrhythmogenesis. Firstly, reverse-mode NCX is the principal pathway that loads the cardiomyocytes with Ca 2+ and causes Ca 2+ overload. Secondly, the forward mode carries the inward current of a DAD. Thus, balancing the NCX mode in cardiomyocytes would be critical for electrophysiolgical homeostasis. In the ATS-iPSC-derived cardiomyocytes, we observed an increased rate of irregular Ca 2+ release, prompting the speculation that inhibiting reverse-mode NCX would reduce Ca 2+ overload, following an antiarrhythmic effect. Low-dose KB-R7943 also successfully reduced arrhythmic events in our study, thus we hypothesized that flecainide would have a role in reverse-mode NCX inhibition. Of interest, flecainide induced the augmentation of I NCX in both modes, and in Fig. 6. Therapeutic electrophysiological pathway of ATS-iPSC-derived cardiomyocytes, a. Representative MEA recordings showing the arrhythmic events after isoproterenol and pilsicainide administration in ATS-iPSC-derived cardiomyocytes. b. The incidences of EBs with arrhythmic events after isoproterenol and pilsicainide administration in MEA analysis (n=11). c. The incidences of cardiomyocytes with irregular Ca 2+ release after KB-R7943 administration (R218W, n =30, R67W, n=31, R218Q, n=34, 40.0%, 32.3%, 26.5% decreases in incidence, respectively. *P < 0.05, **P < 0.01 vs. baseline by Fisher's exact probability test).
cycling cardiomyocytes, the forward-mode and reverse-mode are balanced. An increased I NCX would increase the net forward-mode I NCX , resembling the effect of reverse-mode NCX inhibition.
There are some limitations in this study. The iPSC-derived cardiomyocytes used herein showed an immature phenotype. Unfortunately, despite on-going attempts to obtain mature iPSC-derived cardiomyocytes resembling adult rod-shape cardiomyocytes, current techniques do not accomplish full maturation. We used three unrelated patients with ATS and two unrelated healthy volunteers as controls, all with totally different genetic backgrounds, which can potentially affect disease phenotype. It is therefore preferable to use isogenic control iPSCs generated by gene correction. In this study, we focused on the common phenotype of three patients with ATS that was not observed in two controls. I K1 is very small in iPSC-derived cardiomyocytes, and it is difficult to show the clear difference between patient-and control-derived cells by a patch clamp technique, especially with respect to the loss of function phenotype. But interestingly, we uncovered the electrophysiological findings of ATS-iPSC-derived cardiomyocytes by MEA analysis and Ca 2+ imaging. KB-R7943 is not a specific inhibitor for NCX, and there are more selective inhibitors available, such as SEA-0400 and SN-6. Thus a higher dose of KB-R7943 could inhibit fast Na + , L-type Ca 2+ , inward-rectifier and delayed K + currents in the micromolar range [58], although we used lower concentration of KB-R7943 to minimize such effects. We used flecainide at relatively high concentration for guinea pig experiments. This experiment showed a possible new mechanism of flecainide for NCX augmentation and it remain elusive whether NCX effect solely shows the therapeutic potential on ATS. The underlying precise mechanism for the irregular Ca 2+ releases and arrhythmogenic beating of ATS-iPSC-derived cardiomyocytes remains unclear. In supplementary Fig. 5c, we induced cytosolic Ca 2+ overload by SERCA inhibition using CPA and the incidence of irregular Ca 2+ release is increased, which suggests that irregular Ca 2+ releases in ATS-iPSC-derived cardiomyocytes might be induced by Ca 2+ overload. We speculated that I K1 reduction would result in destabilization of resting membrane potential and depolarized resting membrane potential affects the kinetics of L-type Ca 2+ channel to augment Ca 2+ influx [59], which results in Ca 2+ overload. Ca 2+ efflux through NCX reverse mode would generate I TI , which result in the appearance of DAD.
The rarity of ATS makes it difficult to analyze disease pathogenesis in humans to develop novel therapeutics. In this study we established a disease model of ATS using patient-specific iPSCs. Such iPSCs could be stored and maintained in a cell bank facility, with future efforts recruiting many more patients to facilitate future drug screening. Arrhythmic events in MEA analysis and irregular Ca 2+ releases in Ca 2+ imaging might provide pathogenic hallmarks of ATS-iPSC-derived cardiomyocytes that could in turn be used in high-throughput, automated approaches to drug screening. Flecainide and a reverse-mode NCX inhibitor successfully decreased the incidence of arrhythmic events in ATS-iPSC-derived cardiomyocytes. Finally, we uncovered an unexpected effect of flecainide, I NCX augmentation, which could offer new therapeutic targets for ATS and other such conditions.
Competing financial interests K.F. is a Founding Scientist and a paid SAB of Heartseed Co. Ltd.