A critical role of retinoic acid concentration for the induction of a fully human-like atrial action potential phenotype in hiPSC-CM

Figures

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  Figure 1. Concentration dependency of RA on outward currents in hiPSC-CM (A) Original current traces recorded in individual hiPSC-CM differentiated in the absence (0 RA) or presence of different concentration of RA in in the absence (basal) and in the presence of 50 μM 4-AP and 5 mM 4-AP. For 0 RA, the initial part of outward currents is given on an extended scale. Pulse protocol given as inset. (B) Summary of the results for peak currents measured in the absence of 4-AP (basal) (B) and in the presence of 50 μM and 5 mM 4-AP, recorded in hiPSC-CM cultured with different concentrations of RA (concentration given on top). Gray lines indicate individual cells (number of cells given in brackets, dissociated from three EHTs, generated from three different batches). Open circles indicate mean values ± SD. ∗p < 0.05 (repeated measures ANOVA) vs respective basal values. #p < 0.05 (one-way ANOVA of log-transformed data).

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  Figure 2. Concentration dependency of RA on AP in EHT (Top) Superimposed original traces of AP recorded in EHT (ERC001) based on hiPSC-CMs cultured in the absence (0) or presence of RA concentrations. Summary of results: Mean values ± SD for beating rate (BR). (Bottom) AP duration (APD20 and APD90) and repolarization fraction (APD90 – APD50/APD90). ∗p < 0.05 vs 0 RA (one-way ANOVA of log-transformed data). Number of EHTs resulting from one differentiation run is given in brackets. Individual data points represent single AP recording from one individual EHT.

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  Figure 3. Concentration dependency of RA on the AP response to IKur block (Top) Superimposed original traces of AP before (control) and after exposure to 50 μM 4-AP in EHT based on hiPSC-CM (ERC001) cultured in the absence (0 RA) or in the presence of RA (concentration given in brackets). (Bottom) Summary of results for the effects of 4-AP on APD20 (left), on VPlateau (middle), and on APD90; (right). Open circles indicate mean values ± SD. Numbers in brackets indicate number of EHTs resulting from one differentiation run. ∗p < 0.05 vs 0 RA (one-way ANOVA of log-transformed data). Individual data points represent single AP recording from one individual EHT.

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  Figure 4. In aEHT (1 μM RA)m the IKur block-induced shortening of APD90 depends on IKr (Top left) Superimposed original traces of AP in EHT based on hiPSC-CM (ERC001) treated with 1 μM RA before (basal) (B) and after exposure to 50 μM 4-AP in the presence of the IKr blocker E-4031 (1 μM) (right). Summary of results for the effects of 4-AP in the absence and presence of E-4031 on APD90 (top left), on APD20 (bottom left), and on VPlateau (bottom right). Gray lines indicate individual EHT. Numbers in brackets indicate number of EHTs resulting from one differentiation run. Open circles indicate mean values ± SD. ∗p < 0.05 vs basal or E-4031, respectively (paired t-test).

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  Figure 5. Concentration dependency of RA on CCh effects on APD90 in EHT (Top) Superimposed original traces of AP before (basal) (B) and after exposure to 10 μM CCh recorded in EHT based on hiPSC-CM (ERC001) cultured in the presence of 0.01, 0.1, and 1 μM RA. (Bottom) Summary of results for the effects of CCh on beating rate (BR) (left), TOP (middle), and APD90 (right). Gray lines indicate individual EHT. Numbers in brackets indicate number of EHTs resulting from one differentiation run. Open circles indicate mean values ± SD. ∗p < 0.05 vs basal (paired t-test). Number of EHTs is given in brackets. Individual data point represents single AP recording from one individual EHT.

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  Figure 6. Consistent increase of inward rectifier currents upon CCh in hiPSC-CM (1 μM RA) (Top left) Superimposed original current traces evoked by a slow voltage ramp in a hiPSC-CM cultured (ERC001) in the presence of 1 μM RA before (basal) and after exposure to 10 μM CCh. Pulse protocol given as inset. (Bottom left) Time course of the current at −100 mV in response to CCh. (Right) Summary of results for the current at −100 mV. Gray lines indicate individual hiPSC-aCM, open circles indicate respective mean values ± SD. Numbers in brackets indicate number of cells/number of dissociated EHTs (three different batches). ∗p < 0.05 vs basal (paired t-test).

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  Figure 7. Reproducibility of RA-effects on AP shape (Top) Original recordings of AP recorded in EHT based on hiPSC-CM differentiated with 0 and 1 μM RA from three different cell lines (ERC001, ERC018, and ERC021). (Bottom) Individual data points and mean values ± SD for AP duration at APD20, VPlateau, and repolarization fraction (APD90 – APD20/APD90) measured in EHT from three different batches of RA-treated (1 μM RA) vs untreated hiPSC-CM (0 RA) from three different cell lines. Individual data point represents single AP recording from one individual EHT. ∗p < 0.05 for nested ANOVA vs 0 RA of the same cell line. #No significant difference between single batches (ANOVA, every batch against another). n/N indicate number of EHTs/number of different batches.