Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology

Drugs targeting atrial-specific ion channels, Kv1.5 or Kir3.1/3.4, are being developed as new therapeutic strategies for atrial fibrillation. However, current preclinical studies carried out in non-cardiac cell lines or animal models may not accurately represent the physiology of a human cardiomyocyte (CM). In the current study, we tested whether human embryonic stem cell (hESC)-derived atrial CMs could predict atrial selectivity of pharmacological compounds. By modulating retinoic acid signaling during hESC differentiation, we generated atrial-like (hESC-atrial) and ventricular-like (hESC-ventricular) CMs. We found the expression of atrial-specific ion channel genes, KCNA5 (encoding Kv1.5) and KCNJ3 (encoding Kir 3.1), in hESC-atrial CMs and further demonstrated that these ion channel genes are regulated by COUP-TF transcription factors. Moreover, in response to multiple ion channel blocker, vernakalant, and Kv1.5 blocker, XEN-D0101, hESC-atrial but not hESC-ventricular CMs showed action potential (AP) prolongation due to a reduction in early repolarization. In hESC-atrial CMs, XEN-R0703, a novel Kir3.1/3.4 blocker restored the AP shortening caused by CCh. Neither CCh nor XEN-R0703 had an effect on hESC-ventricular CMs. In summary, we demonstrate that hESC-atrial CMs are a robust model for pre-clinical testing to assess atrial selectivity of novel antiarrhythmic drugs.


Materials and Methods
Preparation of atRA: atRA (Sigma) was diluted in DMSO to make a solution of 50 mmol/L which was further diluted in distilled water to have a stock solution of 0.0001 mol/L. atRA was used in a final concentration of 1 µmol/L in BPEL medium to direct atrial differentiation.

Human Material
Human fetal hearts of 12-15 weeks of gestation (n=3) were collected in phosphate buffered saline (PBS) and either fixed for immunohistochemistry or lysed for RNA isolation. Total RNA from adult heart was purchased (United States Biological, Massachusetts).

Collection and preparation of samples for microarray analysis:
Differentiated cells from Control and RA-treated conditions were collected at day 31 from two independent differentiations. Cells were dissociated with 10X TypLE TM select (Life technologies) and filtered through falcon tubes containing 35um cell strainer lids (BD Biosciences). Cells were sorted for GFP on FACS ARIA TM III (BD Biosciences). RNA was isolated (NucleoSpin RNA, Macherey-Nagel) from the recovered GFP + and GFPfractions from both hESC-ventricular and hESC-atrial CMs.
For RNA isolation from human fetal tissue, atria and ventricles were separated from one heart at 15 weeks of gestation and lysed with TriZol (Invitrogen). Total RNA was extracted using NucleoSpin kit (Macherey Nagel).

Gene expression analysis:
Gene expression was determined on the HumanHT-12 v4 Expression BeadChip at ServiceXS, Leiden, The Netherlands. Biotin-labeled cRNA samples were amplified with Illumina® TotalPrep TM -96 RNA Amplification Kit. Hybridization and scanning were performed according to standard Illumina protocols. Expression profiles were established from two independent samples each for GFP+ and GFP-populations of hESC-ventricular and hESC-atrial differentiations, isolated at day 31. Data normalization and analysis was performed using GeneSpring v12.6 (Agilent Technologies). Genes differentially expressed in both the replicates and satisfying the fold change cutoff of >2.0 were selected for further analysis. ConsensusPathDB-human (http://cpdb.molgen.mpg.de/) web server was used for gene ontology analysis. Microarray data has been deposited in the GEO database with the accession number GSE61154.

Quantitative PCR (qPCR)
For assessing gene expression by qPCR, cDNA was synthesized from approximately 1µg of RNA obtained from three independent differentiations (iScript cDNA Synthesis Kit, BIO-RAD). Target identification was performed using SYBR Green (Applied Biosystems, Life technologies) and detected by CFX96 Real-Time PCR system (Bio-Rad). Each reaction was performed in triplicates and non-template reaction (replacing cDNA with water) was used as negative control. The cycling parameters were 95°C for 3 minutes followed by 95°C for 10 seconds, 60°C for 10 seconds and 72°C for 30 seconds for 40 cycles. Absolute mRNA levels were normalized to human acidic ribosomal phosphoprotein (hARP) which was used a reference. Primer sequences are enclosed (Table S10)
Nuclei were counterstained with DAPI (Life technologies). Images were captured using Leica TCS SP8 microscope (Leica Microsystems, Germany) and acquisition was performed with LAS AF software (Leica, Germany).

Immunohistochemistry of human heart tissue:
Expression of COUP-TFI or COUP-TFII was analyzed by immunohistochemistry in a human fetal heart (hFHs) at 12 weeks of gestation. Briefly, intact hearts were fixed with 4% PFA. Paraffin-embedded hearts were sectioned at 5 µm. Sections were mounted onto silane-coated slides, deparaffinized in xylene, rehydrated in graded ethanol series and washed in PBS. Serial sections were used for immunostaining of COUP-TFI (clone H8132, Perseus proteomics) or COUP-TFII (clone H7147, Perseus Proteomics) in combination with Troponin I (clone H-170, Santa Cruz). An additional hFH at 14 weeks of gestation was used to confirm findings observed in the 12 week-old hFH.
COUP-TFI and COUP-TFII were stained by indirect immunohistochemistry. After over night incubation with primary antibodies, slides were washed and incubated with biotin coupled Horse anti-mouse secondary antibody (Vector Labs) and 1.5% normal horse serum (Vector Labs) in PBS supplemented with 0.05% Tween-20. COUP-TF binding was visualized by using Streptavidin Alexa-488 (Molecular Probes, Life technologies) antibody.

Transcription factor (TF) binding site analysis:
TF binding sites in the promoter regions of KCNA5 and KCNJ3 were analyzed using MatInspector (Genomatix, Munich, Germany) and weight matrices used for prediction were described previously (1). MatInspector is accessible at http://www.genomatix.de/en/index.html and MatBase matrix library version 9.0 was used.

ChIP-qPCR assays:
ChIP assays were performed with Transcription ChIP kit (Diagenode) according to manufacturers instructions. 2*10 6 cells from day 30 EBs derived from control or retinoic acid treated differentiations were used for each experiment. Intact EBs were washed twice with PBS and fixed for 10 min with 1% PFA followed by quenching with 0.125 M glycine. Samples were sonicated using Bioruptor (Diagenode) for 20 cycles of 30 sec ON/OFF each. Sheared chromatin was analysed on gel to ensure optimal sonication and a portion of total chromatin was set aside as input DNA. Protein A/G beads were used for immunoprecipitation (IP) carried out over night at 4°C. 10 µg of the following antibodies were used -COUP-TFI (Clone H8132, Perseus Proteomics); COUP-TFII (Clone H7147, Perseus Proteomics); Mouse IgG2a Isotype control (MAB003, R&D systems). After washing and elution of the beads, cross-links were reversed for 4 hours at 65°C. DNA was recovered by Phenol-Chloroform extraction.
For qPCR, samples were analyzed with promoter-specific primers and results are displayed as fold enrichment over mock. Results from three independent experiments were averaged and compared against IgG control. Primer sequences are enclosed (Table  S11)

Drugs
The effects of drugs were tested in paired measurements 5 minutes after the onset of bath application. 4-AP was prepared as a 50 mmol/L stock solution in Tyrode's solution, and pH adjusted to 7.4, with the addition of HCl. CCh, a muscarinic receptor agonist, was prepared as a 10 mmol/L stock solution in Tyrode's solution. Nifedipine was prepared as a 10 mmol/L stock solution in DMSO and stored at 4 ºC. Stock solutions for the other compounds were prepared in DMSO: Vernakalant (30 mmol/L), XEN-D0101 (3 mmol/L), XEN-R0703 (2 mmol/L) and stored at-20 0 C. All stock solutions were diluted appropriately before use.

Cloned cardiac ion channel automated electrophysiology
Experiments were performed using a QPatch48. For Na v 1.5 a train of 10 voltage clamp pulses (V Hold -100mV, step -20mV / 20 ms, 1Hz) was appled to elicit inward Na + current. The train of 10 pulses was applied every minute for 4 minutes (4 x train) first in vehicle then again in presence of increasing concentrations of drug (1, 3 & 10 µM). Total duration of the cumulative concentration-response experiment was 16 minutes. Peak amplitude of the inward Nav1.5 current elicited by the tenth pulse of the fourth train in each condition was measured. Percentage inhibition of current by drug was calculated relative to current in vehicle.

Isolation of native human atrial myocytes
Studies reported here conform to the principles outlined in the Declaration of Helsinki, were reviewed and approved by the local research ethical approval committee (H03/035). Tissue was obtained from consenting patients (from Papworth Hospital NHS Trust, Cambridge, UK) and human atrial myocytes were mechano-enzymatically dissociated using a previously described protocol ( Briefly, male Beagles (10-15kg) were anaesthetized and ECG leads and bi-polar pacing leads were secured on the heart via a right thoracotomy under sterile conditions. Two bipolar pacing electrodes were attached to the right atrial appendage for pacing and recording of atrial effective refractory period (AERP). ECG electrodes were placed on the left ventricular epicardium near the ventricular apex (positive) and the musculature near the 9 th -10 th rib (negative). Wires were externalized in between the scapula. Animals recovered and 1-week post-surgery dogs were connected to an external pacemaker and the right atrium was paced at 400 bpm. After 2 weeks, conscious-dogs were placed in a sling and cardiac electrophysiology and AF inducibility were evaluated. ECGs were continuously recorded throughout the experiment via telemetry for all dogs during the dosing periods. For determination of atrial effective refractory period (AERP), hearts experienced cycles of 8 paced beats (3 Hz) of atrial origin (s1: at twice electrical diastolic threshold, using two discrete fixed cycle length) followed by an extra atrial stimulus (s2) delivered at varying coupling intervals from the eight beat train of s1. The coupling time that failed to elicit an s2 stimulus was noted as the AERP. AF inducibility was determined using three seconds of burst atrial pacing using 50 Hz pulses with a 2 ms duration at four times diastolic threshold current. The incidence of successful initiation over 25 attempts was recorded and percentage inducibility calculated as number of successful attempts divided by 25. AF inducibility greater than 25% was used as the arbiter to entry into the dosing protocol. Prior to electrophysiology experimentation, the conscious dog was placed in a sling restraint for the entire dosing period. Test article was administered in escalating doses every 60 minutes (15 minute loading period followed by a 45 minute maintenance dosing period). Atrial refractory period measurements were made at the end of loading-dose period. During the dosemaintenance period, AF inducibility testing was performed.

ECG Parameters Analysis
Measurements were assessed by two different investigators and compared to assure agreement. Van de Water's rate-corrected (QTc) interval of the ECG was taken as the mean value from 10-15 cardiac cycles at the end of the drug loading period.
Supplemental Tables: Table S1: Gene lists represented in Venn diagrams of Fig. 2D-E. Please see enclosed excel file.  Please see enclosed excel file. Recombinant ion channel pharmacology of XEN-R0703 was investigated using either conventional whole-cell patch-clamp (CP), automated electrophysiology using the Sophion QPatch platform (QP) or using a Ca 2+ -sensitive dye, fluorescence-based assay (Flex). Native ion channel pharmacology was investigated using freshly dissociated human atrial myocytes and the conventional whole-cell patch-clamp technique.