Abstract
Current drug development efforts for the treatment of atrial fibrillation are hampered by the fact that many preclinical models have been unsuccessful in reproducing human cardiac physiology and its response to medications. In this study, we demonstrated an approach using human induced pluripotent stem cell‐derived atrial and ventricular cardiomyocytes (hiPSC‐aCMs and hiPSC‐vCMs, respectively) coupled with a sophisticated optical mapping system for drug screening of atrial‐selective compounds in vitro. We optimized differentiation of hiPSC‐aCMs by modulating the WNT and retinoid signaling pathways. Characterization of the transcriptome and proteome revealed that retinoic acid pushes the differentiation process into the atrial lineage and generated hiPSC‐aCMs. Functional characterization using optical mapping showed that hiPSC‐aCMs have shorter action potential durations and faster Ca2+ handling dynamics compared with hiPSC‐vCMs. Furthermore, pharmacological investigation of hiPSC‐aCMs captured atrial‐selective effects by displaying greater sensitivity to atrial‐selective compounds 4‐aminopyridine, AVE0118, UCL1684, and vernakalant when compared with hiPSC‐vCMs. These results established that a model system incorporating hiPSC‐aCMs combined with optical mapping is well‐suited for preclinical drug screening of novel and targeted atrial selective compounds.
Significance statement
Current in vitro drug screening systems for treatment of atrial fibrillation are confounded by cell type heterogeneity, specificity, and translatability to human physiology. In this study, we developed a drug screening platform using human induced pluripotent stem cell‐derived atrial cardiomyocytes (hiPSC‐aCMs) and a multiwell optical mapping system. The high‐content optical mapping system reports on membrane voltage and Ca2+ transients which serve as critical biomarkers of cardiac function in vitro. The hiPSC‐aCMs generated by this protocol possess atrial‐specific molecular profiles, functional signatures, and pharmacological response. These findings demonstrate that the platform can be readily applied as a relevant preclinical model for drug screening for atrial fibrillation therapies.1 INTRODUCTION
The advent of human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) has revolutionized the field of cardiac research. It has enabled the study of cardiac diseases in a patient‐specific and human‐relevant in vitro model system which provides a unique opportunity for clinical translation.1 Furthermore, the ability to differentiate chamber‐specific cardiomyocytes allows for a more precise study of cardiac disease physiology and pharmacology. The cardiomyocytes of the lower (ventricles) and upper (atria) chambers have distinct characteristics that arise from differential developmental pathways. Previous work in vivo has shown that the expression patterns of retinoic acid and retinaldehyde dehydrogenase 2 (RALDH2) are important determinants of the atrial fate.2-5 These results were later recapitulated in a pivotal study by Lee and Protze et al6 who determined that atrial cardiomyocytes (aCMs) differentiated from human embryonic stem cells (hESCs) originate from a unique mesoderm characterized by robust RALDH2 expression. This study established an atrial differentiation protocol that included the addition of retinoic acid. Retinoic acid has also been utilized to selectively differentiate hESCs and hiPSCs into aCMs in other studies.6-10 The distinct properties of the atrial and ventricular cardiomyocytes are determined by the differential expression of unique sets of ion channels and other proteins that optimize their specific function. Drugs that target atrial ion channels selectively can therefore produce differences in pharmacological function in the two chambers. This atrial‐selective pharmacology is of utmost interest in the study and treatment of atrial‐specific diseases such atrial fibrillation (AF), which is the most common heart rhythm disorder. Investigating atrial‐selective pharmacology can assist and guide novel cardiac drug development as well as improving both safety and efficacy by avoiding potential toxic electrophysiologic effects on the ventricular chambers. The differential pharmacology of stem cell‐derived aCMs was studied previously by Laksman et al7 who showed that flecainide can rescue the AF phenotype in a dish. Other studies have also studied the selective pharmacological effects of agents on hiPSC‐derived aCMs but have largely focused on proof‐of‐concepts using limited number of test compounds and standard measurement systems that are low in throughput.9, 10 With a focus on translation, a preclinical model platform that characterizes pharmacological activity must capture the main cardiac functional signatures that most closely mimic and predict human cardiac physiology and drug responses. As such, we established in this study an in vitro assay platform by combining hiPSC‐derived atrial cardiomyocytes (hiPSC‐aCMs) and high‐content optical mapping, a noninvasive all‐optical system that simultaneously measures membrane potential (Vm) and Ca2+ transients at a high‐resolution in a monolayer tissue format. We first demonstrate a selective hiPSC‐aCM differentiation protocol by modifying the well characterized GiWi protocol11 through the controlled introduction of retinoic acid. The recapitulation of the human atrial phenotype of the hiPSC‐aCMs was validated with assays that measure the expression of gene transcripts and proteins, as well as functional signatures. We then demonstrate the utility of our platform as an atrial‐selective drug screening tool by using existing clinical and experimental drugs. The model established in this study adds to our current understanding of the utility of stem cell‐derived cardiomyocytes in preclinical and translational research focused on screening new pharmacological agents.2 METHODS AND MATERIALS
A detailed methods section is available in the Supplemental Information.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 RESULTS
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).
FIGURE 1
Directed differentiation of hiPSC‐derived atrial and ventricular CMs. A, Schematic depicting the atrial differentiation protocol. Doses of 0.75 μM retinoic acid (RA) were added to the cells every 24 hours on days 4, 5, and 6 with media exchanged to RPMI1640 + B27 with insulin at day 7. Cells were harvested for analysis at day 20. B, qPCR analysis of ventricular markers MYL2 and IRX4, cardiac marker NKX2.5, and atrial markers NPPA, GJA5, CACNA1D, KCNA5, and KCNJ3. n = 3, unpaired t test, *p < .05. C, Flow cytometric analysis of cardiac troponin T (cTnT) and myosin light chain 2v (normalized to cTnT expression) in hiPSC‐aCMs and ‐vCMs. n = 4, unpaired t test, ***p < .001. D, Average beating rates of hiPSC‐aCMs and ‐vCMs from the day they begin to beat until day 20. n = 4 independent differentiation batches. E, Atrial Natriuretic peptide (ANP) concentration between hiPSCs, and hiPSC‐aCMs and ‐vCMs determined by competitive ELISA. n = 3 and n = 2 hiPSC lines, unpaired t test *p < .05, **p < .01, ***p < .001. Data are presented as mean ± SEM One n represents one independent differentiation batch
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 Kv1.5 (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 Cav1.2 (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).
FIGURE 2
Gene expression analysis of hiPSC‐aCMs and ‐vCMs using NanoString. Global gene expression pattern of hiPSC‐aCMs and ‐vCMs shown in A, heat map of the expression of the 250 genes across samples of hiPSC‐aCMs and ‐vCMs. The cluster dendrogram shows the unsupervised hierarchical clustering that was conducted using the agglomerative algorithm and the Euclidian distance criterion. B, Differentially expressed genes between hiPSC‐aCMs and ‐vCMs expressed in volcano plot shows 14 upregulated (red) and 27 downregulated (blue) genes in hiPSC‐aCMs. Solid horizontal line represents the Benjamini‐Hochberg false discovery rate (FDR) adjusted p‐value <.05 (−log10 = 1.3). Dashed vertical lines represent the arbitrary log2 fold change cut‐off of −0.5 and 0.5. C, Forty‐two differentially expressed genes identified from the statistical criteria of FDR adjusted p‐value <.05 and log2 fold change of <−0.5 and >0.5. Data are presented as mean ± SEM. n = 5 independent differentiation batches
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).
FIGURE 3
hiPSC‐aCMs and ‐vCMs have distinct electrophysiological characteristics. Single differentiated hiPSC‐aCMs and ‐vCMs were plated on gelatin and Geltrex after 30 days in culture. A, Whole cell current clamp recordings from a spontaneously beating hiPSC‐vCM. B, Recorded action potential (APs) demonstrates typical prolonged plateau phase in both spontaneous (left) and/or paced at 1 Hz (right). C, Current clamp recording from a spontaneously beating hiPSC‐aCM. D, Single AP from hiPSC‐aCM demonstrates shortened action potential duration (APD) and lack of prolonged plateau phase, spontaneous (left), paced at 1 Hz (right). E, The first differential of voltage recordings from hiPSC‐aCMs and ‐vCMs were used to calculate the maximal upstroke velocities. F, One minute after achieving the whole‐cell configuration, the average resting membrane potential was measured. G, Spontaneously beating and 1 Hz paced APs were assessed for duration at 50% of peak (APD50), and H, 90% of peak (APD90). Statistics were performed by unpaired t test. *p < .05, ***p < .005. Data are presented as mean ± SEM. Two differentiation batches were included in this analysis

FIGURE 4
Functional phenotyping of hiPSC‐derived atrial and ventricular CMs using optical mapping. Representative average traces of A, action potential and B, Ca2+ transients of hiPSC‐aCMs and ‐vCMs electrically paced at 1 Hz. C, Electrical restitution curve measured at APD80 relative to the diastolic interval (DI). D, Quantification of early‐ (APD20), mid‐ (APD50), and late‐ (APD80) repolarization, unpaired t test, *p < .05, **p < .01. E, Quantification of early‐ (CaTD20), mid‐ (CaTD50), and late‐ (CaTD80) Ca2+ transient decay, unpaired t test, ***p < .001. F, Time to peak (TTP) of the Ca2+ transient, unpaired t test, ***p < .001. G, Time constant (τ) of Ca2+ decay, unpaired t test *p < .05. H, Maximum slope of the electrical restitution as shown in panel C, unpaired t test, *p < .05. Electrical restitution curves were measured under a variable rate pacing protocol (60‐200 bpm) as described in the Supplemental Information. n = 4 (four independent differentiation batches) and cardiac enriched hiPSC‐aCMs and ‐vCMs were analyzed in these set of experiments. Data are presented as mean ± SEM
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 (IKr),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).
FIGURE 5
The effects of dofetilide and nifedipine on action potential and Ca2+ transient of hiPSC‐aCMs and ‐vCMs. Representative traces of action potential and Ca2+ transients illustrating the effects of A, dofetilide and B, nifedipine on hiPSC‐aCMs and ‐vCMs. Higher drug doses are presented by a progressively darker shade. The effects of C, 4‐aminopyridine and D, nifedipine on normalized (percent change from predrug baseline) action potential duration (APD) and Ca2+ transient duration (CaTD); both parameters being measured at 20%, 50%, and 80%. Dashed line is the normalized predrug control presented as 0% change. n = 6 from six independent differentiation batches. hiPSC‐derived atrial cardiomyocytes (aCMs) are shown in red while hiPSC‐derived ventricular cardiomyocytes (vCMs) are presented in blue. Data are presented as mean ± SEM. Drug effects were compared between hiPSC‐aCMs and ‐vCMs at each dose using unpaired t test, *p < .05, **p < .001, ***p < .001. NS stands for not significant

FIGURE 6
The effects of 4‐aminopyridine (4AP) and AVE0118 on action potential and Ca2+ transient of hiPSC‐aCMs and ‐vCMs. Representative traces of action potential and Ca2+ transients illustrating the effects of A 4‐aminopyridine (4AP) and B, AVE0118 on hiPSC‐aCMs and ‐vCMs. Higher drug dose is presented by a progressively darker shade. The effects of C dofetilide and D, vernakalant on normalized (percent change from predrug baseline) action potential duration (APD), and B, Ca2+ transient duration (CaTD); both parameters being measured at 20%, 50%, and 80%. Dashed line is the normalized predrug control presented as 0% change. n = 6 from six independent differentiation batches. hiPSC‐derived atrial cardiomyocytes (aCMs) are shown in red while hiPSC‐derived ventricular cardiomyocytes (vCMs) are presented in blue. Data are presented as mean ± SEM. Drug effects were compared between hiPSC‐aCMs and ‐vCMs at each dose using unpaired t test, *p < .05, **p < .001, ***p < .001. NS stands for not significant

FIGURE 7
The effects of UCL1684 and vernakalant on action potential and Ca2+ transient of hiPSC‐aCMs and ‐vCMs. Representative Vm and Ca2+ transients illustrating the effects of A, UCL1684 and B, vernakalant on hiPSC‐aCMs and ‐vCMs. Higher drug doses are presented by a progressively darker shade. The effects of C, AVE0118 and D, UCL1684 on normalized (percent change from predrug baseline) action potential duration (APD) and Ca2+ transient duration (CaTD); both parameters being measured at 20%, 50%, and 80%. Dashed line is the normalized predrug control presented as 0% change. n = 6 from six independent differentiation batches. hiPSC‐derived atrial cardiomyocytes (aCMs) are shown in red while hiPSC‐derived ventricular cardiomyocytes (vCMs) are presented in blue. Data are presented as mean ± SEM. Drug effects were compared between hiPSC‐aCMs and ‐vCMs using unpaired t test at each dose, *p < .05, **p < .001, ***p < .001. NS stands for not significant