Inhibition of EPAC1 signaling pathway alters atrial electrophysiology and prevents atrial fibrillation

FIGURE 1. In vivo evaluation of AF inducibility, probability and duration by modulation of EPAC activity in WT mice. (A) Number of animals in percent (%) with AF inducibility under EPAC1&2 activation (N = 11), EPAC1 inhibition (N = 14) and EPAC2 inhibition (N = 10). Insets below show representative ECG recordings. (B) AF probability (%) after EPAC1&2 activation and EPAC1 and EPAC2 inhibition. (C) Mean ± SEM of AF duration before and after pharmacologic modulations of EPAC1 and EPAC2 activation and inhibition. Statistical differences were determined by Fisher’s exact tests (A), and by ANOVA with Tukey post hoc test and Kruskal Wallis with Dunn post hoc test [(B,C), #p < 0.05; ##p < 0.01].

FIGURE 2. Impact of EPAC1 and EPAC2 expressions on AF. (A) AF vulnerability. (B) AF probability. (C) AF episode duration. Statistical differences were determined by Fisher’s exact tests (A) and Mann Whitney (B,C); N = 10 WT, 12 EPAC1 KO, 12 EPAC2 KO.

FIGURE 3. Ex vivo measurements of APD of WT and KO mice using optical mapping during 7 Hz pacing. (A) Means ± SEM of APD from LA and RA surfaces on WT (N = 7) and EPAC1 KO mice (N = 6). (B) Means ± SEM of APD from LA and RA surfaces on WT and EPAC2 KO mice (N = 5). Statistical differences were determined by unpaired t-tests (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 4. Impact of various EPAC isoforms on LA and RA APD. (A) Activation of EPAC1&2 by 8-CPT increases APD20, 50, 80. (N = 7) (B) Activation of EPAC2 pathway by inhibition of EPAC1 following by the activation of EPAC2 using AM-001 and 8-CPT increases APD20, 50, 80. (N = 5) (C) Activation of EPAC1 pathway by inhibition of EPAC2 following by the activation of EPAC1 using ESI-05 and 8-CPT tends to increase APD20, 50 and 8-CPT increases APD80. (N = 10). Statistical differences were determined by paired t-tests [(A), *p < 0.05; **p < 0.01], and by ANOVA with Tukey post hoc test and Kruskal Wallis with Dunn post hoc test [(B,C), #p < 0.05].

FIGURE 5. Quantification of conduction velocity (CV) using activation time (AT) maps during 7 Hz pacing. (A) Activation of EPAC1&2 leads to a slowing of CV and an increase of total activation time (TAT). (N = 7) (B) Activation of EPAC2 pathway do not lead to any change in CV and TAT. (N = 5) (C) Activation of EPAC1 pathway induced a slowing of CV and increased TAT. (N = 10) Statistical differences were determined by paired t-tests [(A); *p < 0.05], and by ANOVA with Tukey post hoc test and Kruskal Wallis with Dunn post hoc test [(B,C); #p < 0.05; ##p < 0.01].

FIGURE 6. Effect of pharmacological EPAC activation and selective EPAC1 inhibition in APD on WT and EPAC1 deleted atrial myocytes. (A) Representative traces of AP in response to EPAC activation and EPAC1 selective inhibition. (B) Mean ± SEM of APD20, APD50 and APD90, relative to control (n = 10, N = 5 for WT and n = 12, N = 6 for EPAC1−/−). At each percentage of repolarization, EPAC activation by 8-CPT-AM (10 μmol/L) increases APD on WT myocytes, and this effect is partially corrected by EPAC1 selective inhibition. In EPAC1−/− myocytes, effect of 8-CPT-AM is reduced compared to WT and not corrected by AM-001 treatment. Statistical differences were determined by two-way repeated measure ANOVA with Holm-Sidak post hoc test and Friedman’s ANOVA on ranks with Dunn’s multiple comparisons post -hoc. *p < 0.05 vs. control, **p < 0.01 vs. control, ***p < 0.001 vs. control. $ is for p < 0.05 vs. WT 8-CPTAM condition.

FIGURE 7. Selective inhibition of EPAC1 by AM-001 treatment reduces 8-CPT-AM dependent APD lengthening in WT myocytes. (A) Representative traces of AP in response to EPAC pharmacological activation with 8-CPT-AM, consecutive to pre-treatment with the EPAC1 selective non-competitive inhibitor AM-001 in WT and EPAC1−/− atrial myocytes. (B) Mean ± SEM of APD20, APD50 and APD90, relative to control (n = 9, N = 3 for WT, n = 9, N = 5 for EPAC1−/−). Treatment with AM-001 (20 μmol/L) seems to have no effect on APD at each percentage of repolarization for each genetic background. EPAC activation with 8-CPT-AM (10 μmol/L) consecutive with this pre-treatment lengthens the APD similarly for WT and EPAC1−/−. Statistical differences were determined by two-way repeated measure ANOVA with Holm-Sidak post hoc test. **p < 0.01 vs. control, ***p < 0.001 vs. control. $ is for p < 0.05 vs. AM-001 condition, $$ is for p < 0.01 vs. AM-001 condition and $$$ is for p < 0.001 vs. AM-001 condition.