Cannabinoid Receptor Agonist Inhibits Atrial Electrical Remodeling in a Tachypaced Ex Vivo Rat Model

FIGURE 1. Example recordings from the ex vivo experimental setup. (A) Baseline (left) and 1 h (right) recordings demonstrating the atrial and ventricular electrical signals and LVP signal used to evaluate the effects of CB-13 under standard conditions without pacing. (B) Conventional S1S2 programmed stimulation protocol applied using a quadripolar electrode attached to the right atrium. Left, Atrial capture following S1S2 of 60 ms. Right, Failure of atrial capture following S1S2 of 59 ms. (C) Continuous assessment of atrial and ventricular signals during atrial tachypacing study. (D) Enlarged segment of the recording in C demonstrating the clear and precise ability to determine 1:1 atrial capture during the tachypacing protocol used in this study. Note beat-to-beat oscillation in the shape of the atrial signal (alternans) often observed when the fast pacing cycle length was reaching the minimal value that was able to maintain 1:1 atrial capture.

FIGURE 2. Absence of chronotropic, dromotropic or hemodynamic effects of CB13 in the non-paced rat heart. Isolated rat heart preparations were exposed to either 1 μM of CB13 or vehicle alone, and (A) RR interval, (B) atrioventricular (AV) interval, (C) developed pressure (D) maximal rate of rise of LVP (+dP/dt), and (E) maximal rate of decay of LVP (−dP/dt) were assessed. All parameters demonstrated stability over time with no difference between the CB13 and vehicle preparations. (n = 5). Data presented as mean ± SEM.

FIGURE 3. CB13-treatment inhibited tachypacing-induced atrial electrical remodeling. (A) AERP before and after 90 min of atrial tachypacing; (B) Scatter plot demonstrating the effect of atrial tachypacing on the AERP represented as % of baseline value in each experiment. CB13 treatment prevented reduction in AERP following tachypacing. (C) Conduction time analysis demonstrated a trend of lengthening in vehicle treated tachypaced hearts relative to the CB13 treated hearts. (D) Average pacing CL over the 90 min of atrial tachypacing. The CL was adjusted continuously and maintained 5 ms above the minimal CL to maintain 1:1 atrial capture. Note no difference between the CB13 group and vehicle group in this regard. (E) Spontaneous RR interval measured before and after 90 min of atrial tachypacing in both groups. Note a mild tendency of RR interval prolongation which reached significance in the CB13 group only. n = 5–8 for each group. Data are presented as mean ± SEM. *p ≤ 0.05. CL, cycle length; AERP, atrial effective refractory period. Based on the normality test statistical significance was determined using two-way ANOVA with Newman-Keuls test for multiple comparisons (A,E), Mann-Whitney test…

FIGURE 4. Cannabinoid receptor protein levels remain unaltered in atrial tissue. (A) CB1R and (B) CB2R protein levels within atrial tissue exhibit no significant changes between groups. n = 3–8. Data presented as mean ± SEM. Data was tested for normality using Shapiro-Wilk normality test. Statistical significance was determined for non-normal data by Kruskal-Wallis with Dunn’s post-hoc for multiple comparisons (A,B).

FIGURE 5. Tachypacing induced reduction of phosphorylated AMPK, while CB13 treatment abrogated tachypacing effects and increased PGC1α signaling. (A) Tachypacing for 1.5 h significantly reduced pAMPK compared to non-paced atrial tissue, while CB13 perfusion during tachypacing rescued pAMPK. (B) pLKB1 levels did not change between groups. (C) Protein expression levels of PGC1α were unaltered in both non-paced and tachypaced atrium whereas CB13 perfusion during tachypacing significantly upregulated PGC1α. *p ≤ 0.05. n = 3–8. Data presented as mean ± SEM. Data was tested for normality using Shapiro-Wilk normality test. Accordingly, statistical analysis was performed by one-way ANOVA with Holm-Sidak post-hoc for multiple comparisons (A, C) and by Kruskal-Wallis with Dunn’s post-hoc for multiple comparisons (B).

FIGURE 6. Total AMPK and total LKB1 were not altered in atrial tissue. (A) Tachypacing for 1.5 h significantly reduced pAMPK compared to non-paced atrial tissue (B) but did not alter total AMPK. (C) Phospho-LKB1 and (D) total LKB1 protein expression did not change between groups. See Figures 5A,B for representative blots. *p ≤ 0.05. ns; not significant. n = 3–8. Data presented as mean ± SEM. Data was tested for normality using Shapiro-Wilk normality test. Accordingly, statistical significance was determined by one-way ANOVA with Holm-Sidak post-hoc for multiple comparisons (A).

FIGURE 7. Tachypacing-induced reduction of connexin 43 is diminished by CB13. (A) Cx43 is downregulated in tachypaced atrium compared to non-paced atrium. This effect of tachypacing demonstrated a lower tendency in the presence of CB13. *p ≤ 0.05. n = 3–5. Data presented as mean ± SEM. Data was tested for normality using Shapiro-Wilk normality test. Accordingly, statistical analysis was performed by Kruskal-Wallis with Dunn’s post-hoc for multiple comparisons.