2.1 Maintenance and expansion of hiPSCs

hiPSCs (WiCell, IMR90‐1) were maintained and expanded in mTeSR1 medium and feeder‐free culture using 6‐well plates coated with Matrigel. Using Versene (EDTA), hiPSCs were passaged every 4 days or ~85% confluency at 1:15 ratio. Passaged hiPSCs were cultured with mTeSR1 supplemented with 10 μM Y27632 for the first 24 hours and the mTeSR1 was exchanged daily during cell culture maintenance.

2.2 Directed differentiation of hiPSCs into atrial and ventricular subtypes

hiPSC‐derived ventricular cardiomyocytes were differentiated by employing a modified GiWi protocol¹¹ that we previously published.¹² In brief, hiPSCs were seeded at a density of 87 500 cells/cm². At day 0, differentiation was initiated using 12 μM CHIR99021. At day 3, the cells were incubated with 5 μM IWP‐4. At day 5, the media were refreshed with RPMI‐1640 supplemented with B27 minus insulin. At day 7, the medium was replaced with cardiomyocyte maintenance media (RPMI‐1640 supplemented with B27 with insulin). Thereafter, cardiomyocyte maintenance media were replaced every 4 days. For the atrial differentiation protocol, retinoic acid (RA) addition was first optimized in pilot studies (Figure S2 and S3) and determined to be 0.75 μM RA every 24 hours from days 4‐6.

2.3 Flow cytometry

hiPSC‐aCMs and hiPSC‐vCMs at Day 20‐30 postdifferentiation were dissociated into single cells as described in the Supplemental Information. The harvested cells were fixed in 4.1% PFA solution for 25 minutes and then washed and permeabilized in Saponin/FBS. Cells were subsequently incubated overnight in primary mouse‐cTnT (1:2000) and rabbit‐MLC2V (1:1000) antibodies. Subsequently, the cells were washed and incubated in secondary goat anti mouse Alexa‐488 (1:500) and goat anti rabbit Alexa‐647 (1:2000) antibodies for 1 hour, respectively. Cells were then washed and suspended in PBS for analysis. All analyses were performed using the BDJAZZ Fluorescence Activated Cell Sorter.

2.4 mRNA expression profiling

Gene expression profiling was conducted using multiplexed NanoString and real time quantitative PCR (qPCR). Pooled total RNA was used in both assays. The extracted RNA was reverse transcribed into cDNA which was used in the qPCR assay. Oligonucleotide sequences are described in Table S7. The multiplexed mRNA profiling was conducted using NanoString Technologies (Seattle, Washington) platform with a custom Codeset containing 250 gene probes. Analysis was performed on the Sprint instrument and nSolver analysis software with the Advanced Analysis module.

2.5 Atrial natriuretic peptide measurement

The levels of atrial natriuretic peptide (ANP) of hiPSC‐aCMs and ‐vCMs were measured by a competitive enzyme‐linked immunosorbent assay (ELISA) using a commercially available kit (Invitrogen, California). The assay was conducted according to the manufacturer’s protocol and was measured using a spectrophotometric plate reader.

2.6 Cardiomyocyte enrichment

For cardiac enrichment, hiPSC‐aCMs and ‐vCMs at day 20‐30 postdifferentiation were dissociated into single cells which were then enriched using a MidiMACS PSC‐derived Cardiomyocyte Isolation Kit (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. Enriched hiPSC‐CMs were seeded on Matrigel‐coated 24‐well plates at a seeding density of 600 000 cells per well.

2.7 Patch‐clamp recordings

Single hiPSC‐aCMs and ‐vCMs were plated on gelatin (0.1%) and Geltrex (1:10) at 30 000 cells per well. After 48 hours in culture, glass electrodes were used to achieve the whole‐cell configuration with single hiPSC‐CMs and only cells with gigaohm seals were used for further analysis. The formulation for internal and external recordings solutions are outlined in the Supplemental Information. Current recordings were performed using the Axon Instruments 700B amplifier and digitized at 20 kHz. All recordings were performed at 33‐35°C as maintained. For pacing at 1 Hz, gradually increasing amounts of current were injected with a 1 ms pulse width until reliable action potentials (APs) were triggered. The maximal upstroke velocity was determined by calculating the maximum derivative and the resting membrane potential was measured during a 5 second epoch without spontaneous activity 1 minute after break‐in. Further details on data analysis are found in the Supplemental Information.

2.8 Optical mapping

Optical mapping recordings were performed on enriched monolayers of hiPSC‐aCMs and ‐vCMs cultured in a 24‐well plate format at Day 45‐60 postdifferentiation. Imaging experiments were conducted using Ca²⁺ Tyrode’s solution (formulation found in Supplemental Information). The hiPSC‐CMs were loaded with RH‐237, blebbistatin, and Rhod‐2AM sequentially before imaging as described.^12, 13 Both RH‐237 and Rhod‐2AM were excited by 530 nm LEDs. Images were acquired at a frame rate of 100 frames/second by a sCMOS camera (Orca Flash 4.0V2, Hamamatsu Photonics, Japan) equipped with an optical splitter. The cells were paced using programmable stimulation. Data collection, image processing, and initial data analysis were accomplished using custom software. The multiwell optical mapping system was custom engineered in the lab based on a system as described previously.^12, 13 Further details are found in the Supplemental Information.

2.9 Pharmacological analyses

The drugs used in this study are listed Table S8. Drug stocks were further diluted in Ca²⁺ Tyrode’s solution prior to pharmacological testing with the final DMSO concentration in the experimental solution not exceeding 0.03% (v/v). Drug effects were studied in serum‐free conditions (ie, Ca²⁺ Tyrode’s and drug only) at four doses by sequentially increasing the drug concentration in the same well with recordings at 20‐minute intervals.

2.10 Statistical analysis

Further details on data and statistical analysis can be found in the Supplemental Information. Unpaired t tests were conducted to compare two groups (ie, hiPSC‐aCMs vs hiPSC‐vCMs) in the analysis of qPCR, ELISA, patch clamp recordings, and optical mapping (baseline condition and normalized drug effects). Analysis of dose‐dependent effects was performed using one‐way ANOVA and Dunnett’s post hoc test. All data are presented as mean ± SEM unless noted otherwise. Significance level for all statistical analysis was set at p < .05 with the following notation: *p < .05, **p < .01, ***p < .001.

3.1 RA treatment drives cardiac differentiation into atrial phenotype

We first optimized the atrial differentiation protocol by altering the concentration and timing of retinoic acid (RA) based on the molecular signatures of atrial phenotype as measured by qPCR and flow cytometry (Figures S2 and S3). Higher dose of RA reduced cardiac differentiation efficacy defined by the decrease in the cTnT⁺ proportion of the total cell population as measured by flow cytometry (Figure S2A). The finalized protocol to generate hiPSC‐aCMs included RA addition at 0.75 μM every 24 hours on days 4, 5, and 6 (Figure 1A) which was found as a balance between sufficiently driving atrial differentiation as defined by decreased ventricular marker myosin light chain 2 ‐ ventricular paralog (MLC‐2v) while having no impact cardiac differentiation efficacy Figures S2 and S3).

Compared with hiPSC‐vCMs, hiPSC‐aCMs were found to have no significant difference in pan cardiac phenotype. Expression of the pan cardiac transcript NKX 2.5 measured by qPCR was similar between hiPSC‐aCMs and ‐vCMs (Figure 1B), as was cardiac troponin T (cTnT) protein expression measured by flow cytometry (Figures 1C and S1). The protein expression of MLC‐2v was reduced in hiPSC‐aCMs compared with hiPSC‐vCMs (8.0 ± 1.1% vs 57.0 ± 0.5%; p < .05) (Figure 1C). Furthermore, hiPSC‐aCMs displayed higher concentrations (increased by 91%) of atrial natriuretic peptide (ANP) at 65 ± 2 compared with 34 ± 6 ng/mL in hiPSC‐vCMs as measured by ELISA (p < .05).

The qPCR assay revealed that atrial‐specific transcripts such as atrial natriuretic peptide (NPPA), connexin 40 (GJA5), the calcium channel Ca_V1.3 (CACNA1D), and the K⁺ channels K_v1.5 (KCNA5) and K_ir3.1 (KCNJ3) transcripts were all expressed at a significantly higher levels in hiPSC‐aCMs compared with hiPSC‐vCMs (p < .05, Figure 1B). Another ventricular marker, IRX4, also had decreased expression in hiPSC‐aCMs (Figure 1B). Furthermore, consistent with previous studies,^{8–10, 14, 15} hiPSC‐aCMs started beating at day 10 or earlier and exhibited an increased beating frequency relative to hiPSC‐vCMs, which started beating around day 10‐12 postdifferentiation.

3.2 Gene expression analysis of hiPSC‐aCMs

We performed an extensive gene expression analysis of hiPSC‐aCMs and ‐vCMs using NanoString technology in which each mRNA copy was digitally counted for accurate and sensitive detection of gene expression.¹⁶ Five independent differentiation batches of each cardiac subtype were included in the analysis. The unsupervised hierarchical clustering analysis showed clear grouping of hiPSC‐aCM samples that were segregated relative to hiPSC‐vCMs (Figure 2A). The gene expression profile of the hiPSC‐vCM samples were more variable with 2 samples closer in distance to the hiPSC‐aCMs while three samples displayed clear segregation (Figure 2A). The overall difference in global gene expression and lineage between hiPSC‐aCMs and ‐vCMs was also captured in the principal component analysis (PCA, Figure S4A). Out of the 250 transcripts analyzed, 200 genes were detected above background noise defined by a threshold of 50 raw digital counts as determined by the negative controls of the assay. In the hiPSC‐aCMs, 14 and 27 genes were significantly upregulated and downregulated, respectively (Figure 2C). As expected, hiPSC‐aCMs displayed significantly higher expression profiles of atrial‐specific markers including atrial‐specific K⁺ channel K_v1.5 (KCNA5) and transcription factors (NR2F2 and TBX18) (Figure 2C). Meanwhile, hiPSC‐vCMs displayed higher expression of ventricular‐specific genes such as those encoding for contractile proteins MYL2, MYH7, and the L‐type Ca²⁺ channel isoform Ca_v1.2 (CACNA1C) (Figure 2C). The genes encoding for the proteins in the sarcoplasmic reticulum complex such as TRDN, CASQ2, and RYR2 were expressed in significantly lower amounts in the hiPSC‐aCMs samples (Figure 2C). Meanwhile, pan‐cardiac markers NKX2‐5 and TNNT2 were expressed at similar levels in both hiPSC‐aCMs and ‐vCMs, further corroborating the efficiency of the differentiation protocol (Figure S4B).

3.3 Functional phenotyping of hiPSC‐derived atrial cardiomyocytes

We compared the electrophysiological characteristics of the differentiated hiPSC‐aCMs and ‐vCMs using whole‐cell patch clamp. Confirming our observations in tissue culture, the spontaneous beating rates were higher in the single hiPSC‐aCMs than in ‐vCMs (Figure 3A,C). Whole cell current clamp recordings demonstrated the ventricular‐like AP morphology of hiPSC‐vCMs with a clear and prolonged plateau phase while the AP of the hiPSC‐aCMs displayed atrial‐like morphology with a shorter action potential duration (APD) and a lack of prolonged plateau phase at both spontaneous beating rates (Figure 3B,D, left panel) and paced at 1 Hz (Figure 3B,D, right panel). No statistical differences were observed in the resting membrane potential and the maximum upstroke velocity of hiPSC‐aCMs and ‐vCMs. The APD at 50% (APD₅₀) and 90% (APD₉₀) of the peak voltage were significantly shorter in hiPSC‐aCMs than ‐vCMs at both spontaneous beating rates (APD₅₀: 157 ± 16 ms vs 349 ± 35 ms, p‐value <.005; APD₉₀: 249 ± 34 ms vs 484 ± 30 ms, p‐value <.005) and paced at 1 Hz (APD₅₀: 157 ± 16 ms vs 264 ± 44 ms, p‐value <.05; APD₉₀: 242 ± 22 ms vs 341 ± 48 ms, p‐value <.05).

We further assessed the functional properties of hiPSC‐aCMs and ‐vCMs using optical mapping with simultaneous measurement of APs and calcium transients (CaT). Like the patch clamp recordings, optical membrane voltage measurements revealed similar atrial‐like and ventricular‐like AP morphology in the hiPSC‐aCMs and ‐vCMs, respectively (Figure 4A). AP and CaT durations were quantified at early, mid, and late repolarization (APD₂₀, APD₅₀, and APD₈₀) and Ca²⁺ decay (CaTD₂₀, CaTD₅₀, and CaTD₈₀), respectively. These stages reflect different phases of ionic currents across the plasma membrane and the extrusion of Ca²⁺ handling mechanics.

For these experiments, both hiPSC‐aCMs and ‐vCMs were paced at 1 Hz. All measured levels of the APD were significantly shorter in hiPSC‐aCMs compared with hiPSC‐vCMs (APD₂₀: 84 ± 8 ms vs 127 ± 6 ms, p < .05; APD₅₀: 131 ± 12 ms vs 191 ± 8 ms, p < .01; APD₈₀: 179 ± 16 ms vs 251 ± 12 ms, p < .05; Figure 4D). The overall CaTD of hiPSC‐aCMs was significantly shorter than that of hiPSC‐vCMs (CaTD₂₀: 180 ± 12 ms vs 266 ± 12 ms, p < .001; CaTD₅₀: 282 ± 18 ms vs 397 ± 16 ms, p < .001; CaTD₈₀: 474 ± 27 ms vs 615 ± 18 ms, p < .001; Figure 4E). Compared with hiPSC‐vCMs, hiPSC‐aCMs displayed significantly faster CaT time‐to‐peak (hiPSC‐aCMs: 116 ± 7 ms vs hiPSC‐vCMs: 246 ± 10 ms, p < .05) and faster decay kinetics (τ; hiPSC‐aCMs: 350 ± 39 ms vs hiPSC‐vCMs: 671 ± 118 ms, p < .05) indicating that Ca²⁺ handling mechanics are accelerated in hiPSC‐aCMs (Figure 4F,G).

The direct comparison between whole‐cell patch clamp and optical mapping read‐outs paced at 1 Hz is shown in Figure S7. We observed no differences in the read‐outs of hiPSC‐aCMs at APD₂₀ (optical: 84 ± 8 ms, patch: 98 ± 12 ms) and APD₅₀ (optical: 131 ± 12 ms, patch: 169 ± 19 ms). However, APD₈₀ of hiPSC‐aCMs measured by patch clamp was longer than the optical APD₈₀ (253 ± 22 ms vs 179 ± 16 ms, p < .05). Similarly, both APD₂₀ (216 ± 22 ms vs 127 ± 6 ms) and APD₈₀ (393 ± 62 vs 251 ± 12 ms) of hiPSC‐vCMs measured by patch clamp were longer than the comparable optical measurements. APD₅₀ of hiPSC‐vCMs did not show a statistical difference between the two assays (optical: 191 ± 8 ms, patch: 308 ± 60 ms).

Rate‐dependent properties are critical in cardiac function. A variable rate protocol (Figure S6) in which the hiPSC‐CMs were electrically paced with increasing frequency at every cycle was used to investigate the electrical restitution dynamics. The electrical restitution curve reflects the ability of the cardiac system to accommodate a higher pacing rate by progressive shortening of APD₈₀ and is described as APD₈₀ in relation to the diastolic interval (DI). Compared with hiPSC‐vCMs, the electrical restitution curve of the hiPSC‐aCMs displayed a flatter portion and did not show APD₈₀ shortening at longer diastolic intervals (Figure 4F). The extensive shortening in APD₈₀ started at shorter diastolic intervals for hiPSC‐aCMs (<275 ms) compared with hiPSC‐vCMs (<500 ms). The maximum slope of the restitution curve was higher in hiPSC‐vCMs compared with hiPSC‐aCMs (1.26 ± 0.08 vs 0.91 ± 0.04, p < .05; Figure 4G) indicating faster kinetics of APD in response to higher pacing rate.

3.4 In vitro screening for atrial‐selective pharmacology

We first established the utility of optical mapping to detect a pan‐cardiac pharmacological response by using dofetilide, a strong blocker of the rapid delayed rectifier K⁺ current (I_Kr),¹⁷ an ionic current expected to be present in both hiPSC‐aCMs and ‐vCMs.¹⁸ Dofetilide elicited a dose‐dependent response in both hiPSC‐aCMs and ‐vCMs. Compared with predrug baseline, dofetilide at 100 nM prolonged APD₈₀ of both hiPSC‐aCMs from 182 ± 16 ms to 355 ± 24 ms (95 + 7% prolongation) and of hiPSC‐vCMs from 238 ± 20 ms to 319 ± 45 ms (34 ± 14% prolongation, p < .05; Table S1 and Figure 5C). The drug prolonged early‐repolarization (APD₂₀) of hiPSC‐vCMs at 10 and 30 nM while having no effect on APD₂₀ of hiPSC‐aCMs at all tested doses (Table S1). Additionally, CaTD₅₀ and CaTD₈₀ of both hiPSC‐aCMs and ‐vCMs were significantly prolonged in response to dofetilide (Table S1). However, hiPSC‐aCMs appeared to be more sensitive to dofetilide as the APD₈₀ was significantly prolonged at the lowest tested dose of 3 nM (from to 182 ± 26 ms to 241 ± 26 ms, p < .05; Table S1) and displayed a larger dose‐response (Figure S8).

Next, we demonstrated the functional differences in the ion channel profiles of hiPSC‐aCMs and ‐vCMs. We aimed to show that the ultrarapid outward current (I_Kur) produced by the channel K_v1.5 (KCNA5) was functional and specific to hiPSC‐aCMs, while the inward Ca²⁺ current (I_Ca,L) produced the voltage‐dependent L‐type Ca²⁺ channel Ca_V1.2 (CACNA1C) was functional and specific to hiPSC‐vCMs. We used two relatively selective compounds, 4‐aminopyridine (4AP) and nifedipine, to dissect the presence of functional I_Kur and I_CaL, respectively. While nifedipine is also known to block Cav1.3, it is expected to have a preferential effect at lower concentrations on Ca_V1.2 based on the literature which indicates ~13‐fold higher block on Ca_V1.2 than Ca_V1.3.¹⁹

At the highest tested dose (300 nM), nifedipine significantly decreased APD₅₀ of hiPSC‐vCMs from 170 ± 14 ms to 121 ± 16 ms (28 ± 4% shortening) and decreased CaTD₅₀ from 357 ± 10 ms to 333 ± 23 ms (30 ± 3% shortening) (Figure 5D; Table S2). We observed a trend in APD₅₀ shortening of hiPSC‐aCMs in response to increasing the nifedipine dose, but the drug elicited a significantly stronger dose‐dependent shortening in both APD and CaTD of hiPSC‐vCMs compared with hiPSC‐aCMs (Figures S8 and S9). Observing the percent change from predrug control, nifedipine induced differential response in overall APD and CaTD between hiPSC‐aCMs and ‐vCMs at 10, 100, and 300 nM (Figure 5D).

In hiPSC‐aCMs, 4AP prolonged APD and CaTD in a dose‐dependent manner with a statistically significant change starting at 30 μM (Figure 6A,C; Table S3). 4AP significantly prolonged early‐repolarization (APD₂₀) of hiPSC‐aCMs by 46 ± 2% and 66 ± 2% at 50 and 100 μM, respectively (APD₂₀ at baseline: 82 ± 8, at 50 μM: 120 ± 9 ms, at 100 μM: 131 ± 9 ms, p < .05) (Figure 6C and Table S3). In contrast, 4AP prolonged APD₂₀ of hiPSC‐vCMs by 23 ± 4% (APD₂₀ at baseline: 138 ± 8 ms, at 100 μM: 170 ± 9 ms) at the highest tested dose of 100 μM (Figure 6C and Table S3). hiPSC‐aCMs showed greater change in APD to relative to predrug control at all concentrations of 4AP compared with hiPSC‐vCMs (Table S3), This is corroborated by the steeper trend of the dose response relationship in hiPSC‐aCMs (Figure S8). Additionally, the overall CaTD of hiPSC‐aCMs were prolonged after exposure to 4AP at 10 μM while the drug had a significant effect on CaTD of hiPSC‐vCMs at 30 μM (CaTD₅₀ elongation from baseline: 68 ± 2% vs 12 ± 2%, p < .05) (Table S3).

We then demonstrated the effectiveness of our drug screening platform in assessing the effects of experimental compounds designed to have targeted effects on atrial‐specific ion channels using AVE0118 and UCL1684.

AVE0118 is an experimental drug that blocks I_Kur, the G‐protein‐activated K⁺ current (I_KAch)_, and the transient outward K⁺ current (I_to) at a similar dose range.²⁰ Both I_Kur and I_KAch are atrial‐specific ionic currents. AVE0118 prolonged mid‐ and late‐ repolarization (APD₅₀ and APD₈₀) of both hiPSC‐aCMs and ‐vCMs at the two highest tested doses (3 and 10 μM; Table S4). Similarly, AVE0118 had significant effects on CaTD₅₀ and CaTD₈₀ of hiPSC‐aCMs and ‐vCMs at all tested doses (Table S4). However, the APD₅₀ and APD₈₀ of hiPSC‐aCMs were significantly prolonged at a lower dose of 1 μM (control: 200 ± 14 ms, 1 μM: 244 ± 16 ms; Table S4). Furthermore, the atrial‐selective effects of the drug were demonstrated by a larger proportional prolongation in APD₅₀ and APD₈₀ of hiPSC‐aCMs compared with hiPSC‐vCMs at 1, 3, and 10 μM (APD; Figure 6D). Furthermore, AVE0118 induced a larger proportional prolongation in CaTD of hiPSC‐aCMs compared with hiPSC‐vCMs at all tested doses (Figure 6D). Early repolarization (APD₂₀) of hiPSC‐aCMs also displayed a large dose‐dependent response (Figure S8) with a proportionally larger prolongation at 10 μM (63 ± 2% vs 43 ± 5%, p < .05; Figure 6D).

UCL1684 is purported to be a potent direct pore blocker of the small conductance Ca²⁺ activated K⁺ channel (SK channel)²¹ and was expected to induce a dose‐dependent atrial‐selective response. In hiPSC‐aCMs, UCL1684 treatment resulted in a significantly prolonged APD₈₀ at 3 and 10 μM (from predrug control: 136 ± 11 ms to 3 μM: 188 ± 25 ms or 38 ± 5% prolongation, and to 10 μM: 206 ± 32 ms or 49 ± 11% prolongation, p < .05; Figure 7C and Table S5). UCL1684 prolonged CaTD₈₀ of hiPSC‐aCMs at all tested doses (baseline: 300 ± 15 ms, at 0.3 μM: 372 ± 23 ms, at 1 μM: 387 ± 33 ms, at 3 μM: 413 ± 24 ms, at 10 μM: 416 ± 39 ms, p < .05; Table S5). In contrast, UCL1684 exposure showed no statistically significant effect on overall APD and CaTD of hiPSC‐vCMs. The sensitivity of hiPSC‐aCMs to UCL1684 was also reflected in the dose‐response relationship showing a prolongation APD₈₀, in contrast to the minimal prolongation in APD₈₀ of hiPSC‐vCMs (Figure S8).

Finally, we tested the effects of vernakalant which is a multi‐ion channel blocker that blocks the fast and late inward Na⁺ current (I_Na, I_NaL, respectively), the I_Kur, and the I_KAch.²² The drug is used clinically for intravenous cardioversion of patients in AF²³ and was expected to induce an atrial‐specific effect due to its I_Kur and I_KAch blocking properties.

Vernakalant elicited a positive dose‐dependent response in both APD and CaTD of hiPSC‐aCMs with minimal measurable effects on hiPSC‐vCMs (Table S6; Figures S8 and S9). Vernakalant demonstrated atrial‐selectivity with statistically significant differences between APD and CaTD of hiPSC‐aCMs and ‐vCMs at doses of 3, 10, and 30 μM (Figure 7D). Compared with APD at baseline, vernakalant at 10 μM significantly prolonged APD₂₀, APD₅₀, and APD₈₀ of hiPSC‐aCMs by 84 ± 6%, 70 ± 5%, and 77 ± 4%, respectively (Figure 7D). Additionally, vernakalant at 10 μM prolonged CaTD₂₀, CaTD₅₀, CaTD₈₀ of hiPSC‐aCMs by 58 ± 4%, 50 ± 3%, 35 ± 5%, respectively (Figure 7D). At clinically relevant concentrations (30 μM), vernakalant greatly affected early repolarization of hiPSC‐aCMs (APD₂₀ prolonged by 124 ± 8%; Figure 7D). At 30 μM, vernakalant prolonged APD₈₀ of hiPSC‐vCM by 20 ± 7% (APD₈₀: 238 ± 22 ms at baseline vs 289 ± 30 ms at 30 μM, p < .05; Figure 7D and Table S6). Except for APD₈₀ prolongation at 30 μM, vernakalant had no statistically significant effect on overall APD and CaTD of hiPSC‐vCMs at the lower doses (Table S6).