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

Abstract

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.

Keywords: COUP‐TF; arrhythmias; atrial cardiomyocytes; atrial fibrillation; ion channels.

Figure 1. Treatment of differentiating hESCs with RA promotes atrial specification

1. A Schematic of the cardiac differentiation protocol. Beating embryoid bodies (EBs) were observed at day 10. Differentiation efficiency in each experiment was assessed by flow cytometry (FC) for GFP at day 15. Further characterization of EBs derived from control (CT) and RA-treated (RA) cultures was carried out by transcriptional or functional analysis between days 27 and 31.

2. B GFP⁺ EBs derived from CT and RA cultures at day 10; scale bar: 100 μm.

3. C Representative FC plots depicting percentage of GFP⁺ cells obtained at day 15, from CT and RA cultures in a typical experiment.

4. D Heat map demonstrating enrichment of cardiac genes in GFP⁺ fractions (CT+, RA+) compared to GFP⁻ fractions (CT−, RA−) at day 31.

5. E, F Heat map of a select list of genes (E) upregulated and (F) downregulated in RA+ compared to CT+ at day 31. Fold change > 2.

Figure 2. Transcriptional analysis of CT+ and RA+ CMs

1. A–C qPCR of selected transcripts at day 31 to validate (A) enrichment of cardiac markers in GFP⁺ fractions against GFP⁻ fractions, (B) upregulation of atrial and (C) downregulation of ventricular genes in RA+ compared to CT+ (n = 3).

2. D, E Venn diagram to illustrate overlap of gene lists upregulated (UP) in CT+ CMs (D) and RA+ CMs (E) with genes expressed in atria and ventricles of 15-week-old fetal heart. Red square indicates higher overlap of CT+ with gene list of fetal ventricles or higher overlap of RA+ with gene list of fetal atria.

3. F Pie chart illustrates major classes of gene ontology terms enriched in gene lists upregulated in CT+ and RA+ CMs.

Data information: Data are presented as mean ± SEM. In (A–C), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), for TNNT2, P = 0.0001 for CT− against CT+ and P = 0.0002 for RA− against RA+; for NKX2.5, P = 0.00005 for CT− against CT+ and P = 0.00007 for RA− against RA+. In (B), P = 0.0006 for NPPA and P = 0.0002 for PITX2. In (C), P = 0.02 for HEY2 and P = 0.007 for IRX4.

Figure 3. AP characterization of CMs generated from control and RA-treated differentiations

1. A AP illustrating the analyzed parameters.

2. B Representative APs of day 31 CMs from control (CT) and RA-treated (RA) groups at 1 Hz.

3. C–E RMP, APA_max and APA_plat (C), dV/dt_max (D) and APD₂₀, APD₅₀ and APD₉₀ of CT and RA CMs (E).

4. F Plot showing all measured APA_plat values of CT and RA CMs.

5. G Representative APs of CT and RA CMs at 0.5–4 Hz.

6. H Average APA_plat at 0.5–4 Hz. Please note that the AP differences in morphology are present at all measured frequencies.

Data information: Data are presented as mean ± SEM. *P < 0.05 by unpaired t-test or Mann–Whitney rank-sum test for (C–E). In (C), P = 0.238 for RMP; P < 0.001 for APA_max and APA_plat. In (D), P = 0.598 for dV/dt_max. In (E), P ≤ 0.001 for APD₂₀; P = 0.009 for APD_50; P = 0.04 for APD₉₀. Two-way repeated measures ANOVA followed by pairwise comparison using the Student–Newman–Keuls test for (H). *P = 0.002, 0.006, 0.003, 0.004 and 0.003, respectively, for comparison of APA_plat between CT and RA groups at frequencies of 0.5, 1.0, 2.0, 3.0 and 4.0 Hz. AP = action potential; APA_max = maximum AP amplitude; APA_plat = AP plateau amplitude; APD₂₀, APD₅₀ and APD₉₀ = AP duration at 20, 50, and 90% repolarization, respectively; CMs = cardiomyocytes; dV/dt_max = maximum upstroke velocity; RMP = resting membrane potential.

Figure 4. Retinoic acid induces COUP-TFI and COUP-TFII during atrial differentiation

1. A, B Line plot illustrating relative mRNA levels of (A) COUP-TFI and (B) COUP-TFII in VM and AM differentiations from day 5 through day 9 (left) and in GFP⁺ CMs at day 31 (right); n = 3.

2. C, D COUP-TFI (C) and COUP-TFII (D) immunofluorescence at day 31 in AM (top) and VM (bottom). Scale bars: 40 μm.

Data information: Data are presented as mean ± SEM. In (A, B), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), left panel, P = 0.03, 0.02, 0.03, 0.005 and 0.0004 for comparison of COUP-TFI expression between AM and VM at days 5, 6, 7, 8 and 9 of differentiation. In (A), right panel, P = 0.0002 for comparison of COUP-TFI expression at day 31 between AM and VM. In (B), left panel, P = 0.02, 0.03, 0.006, 0.004 and 0.003 for comparison of COUP-TFII expression between AM and VM at days 5, 6, 7, 8 and 9 of differentiation. In (B), right panel, P = 0.0001 for comparison of COUP-TFII expression at day 31 between AM and VM. CT = control differentiation; hESC-atrial (AM); hESC-ventricular (VM).

Figure 5. COUP-TFs regulate atrial-specific ion channel genes KCNA5 and KCNJ3

1. A, B mRNA expression of COUP-TFI and COUP-TFII following shRNA-mediated knockdown of (A) COUP-TFI or (B) COUP-TFII in hESC-atrial cardiomyocytes (AM) at day 30.

2. C, D mRNA expression of ion channel genes KCNA5, KCNJ3 and KCNJ5 after knockdown of (C) COUP-TFI or (D) COUP-TFII in AM at day 30.

3. E, F Schematic of NR2F binding sites in (E) KCNA5 and (F) KCNJ3 promoters.

4. G, H ChIP-qPCR analysis at day 30 shows enriched binding of COUP-TFI and COUP-TFII to the promoter region of (G) KCNA5 and (H) KCNJ3, compared to IgG in AM.

Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), P = 0.00004. In (B), P = 0.0001. In (C), P = 0.0001. In (D), P = 0.000004, 0.006 and 0.001, respectively. In (G), P = 0.0004 for COUP-TFI and P = 0.0002 for COUP-TFII. In (H), P = 0.0003 for COUP-TFI and P = 0.0001 for COUP-TFII.

Figure 6. Characterization of I_Kur and I_K,ACh in hESC-ventricular and hESC-atrial CMs

1. A Expression of KCNA5 (left) and KCNJ3 (right) in GFP⁺ pools of VM and AM CMs at day 31, as well as in ventricles and atria of human heart.

2. B, C Typical examples (left) and current–voltage relationships (right) of (B) I_Kur and (C) I_K,ACh in VM and AM CMs.

3. D, E Representative APs of VM and AM at 1 Hz in response to (D) I_Kur block by 4-AP and (E) I_K,ACh activation by CCh. AP parameters are shown in Supplementary Table S5.

Data information: Data are presented as mean ± SEM. In (A), *P < 0.05 by unpaired t-test. In (B, C), *P < 0.05 by two-way repeated measures ANOVA followed by pairwise comparison using the Student–Newman–Keuls test for (B) and Mann–Whitney rank-sum test for (C). In (B), P = 0.778, 0.350, 0.03, 0.02, 0.002, 0.001, < 0.001 and < 0.001, respectively, for comparison between VM and AM within membrane potentials of −20, −10, 0, 10, 20, 30, 40 and 50 mV. In (C), P = 0.01, 0.01, 0.01, 0.01, 0.03, 0.397, 0.397, 0.397, 0.671, 0.207, 0.207, 0.09, 0.01, 0.039 and 0.015, respectively, for comparison between VM and AM within membrane potentials of −120, −110, −100, −90, −80, −70, −60, −50, −40, −30, −20, −10, 0, 10 and 20 mV. CMs = cardiomyocytes; hESC-atrial (AM) and hESC-ventricular (VM) CMs; I_K,ACh = acetylcholine-activated potassium current; I_Kur = potassium ultra-rapid delayed rectifier current. 4-AP = 4-aminopyridine; CCh = carbachol.

Figure 7. Effects of vernakalant on APs of hESC-ventricular and hESC-atrial CMs

1. A Representative APs at 1 Hz of VM and AM CMs in response to vernakalant. Inset shows dV/dt_max.

2. B, C Average APA_plat (B) and dV/dt_max (C)in the absence and presence of vernakalant at 1–4 Hz. AP parameters are shown in Supplementary Table S6. Abbreviations as in Figs3 and 6.

Data information: Data are presented as mean ± SEM. *P < 0.05 by Mann–Whitney rank-sum test for (B). P = 0.01, 0.008, 0.008 and 0.007, respectively, for comparison of APA_plat between VM and AM groups at frequencies of 1.0, 2.0, 3.0 and 4.0 Hz. Two-way repeated measures ANOVA followed by pairwise comparison using the Student–Newman–Keuls test for (C). P = 0.686 between VM and AM groups and hence not statistically significant. For VM, P = 0.06 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.001 for 2 versus 3 Hz; P = 0.002 for 2 versus 4 Hz; and P = 0.857 for 3 versus 4 Hz. For AM, P = 0.03 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.02 for 2 versus 3 Hz; P = 0.02 for 2 versus 4 Hz; and P = 0.621 for 3 versus 4 Hz.

Figure 8. Effects of XEN-D0101 and XEN-R0703 on APs of hESC-ventricular and hESC-atrial CMs

1. A Representative APs of VM and AM CMs in the absence, presence and following washout of 3 μmol/l XEN-D0101. AP parameters are shown in Supplementary Table S7.

2. B Representative APs (1 Hz) of VM and AM in the CCh, to activate I_K,ACh and subsequent addition of XEN-R0703. AP parameters are shown in Supplementary Table S9.

3. C, D Experiments performed in RAP conscious dogs in the presence of vehicle or following 1, 3 and 10 mg/kg XEN-R0703 show (C) mean right AERP values (left), mean Van de Water's QTc (right) and (D) AF inducibility plotted as a function of dose.

Data information: For RAP dog experiments, n = 5; statistical significance tested with paired t-test. Data are presented as mean ± SEM. AERP = atrial effective refractory period; AF = atrial fibrillation; RAP = rapid atrial pacing; N.S. = not significant. Other abbreviations as in Figs3, 6 and 7.