Cannabinoid receptor agonist attenuates angiotensin II–induced enlargement and mitochondrial dysfunction in rat atrial cardiomyocytes

FIGURE 1. CB13 suppressed AngII-induced enhancement of NRAM cell surface area in an AMPK-dependent manner. (A) The ability of AngII to induce NRAM enlargement was abolished by CB13 treatment. (B) Treatment of NRAM with compound C, an AMPK inhibitor, prevented the ability of CB13 to suppress NRAM enlargement. Serum-deprived NRAM were pre-treated with CB13 (1 μM, 1 h) in the presence or absence of compound C (1 μM, 1 h), followed by treatment with AngII (10 μM). All treatments remained in media for 24 h. Results are presented with representative fluorescent images. Data are presented as mean ± SEM. n = 3. **p < 0.01.

FIGURE 2. CB13 prevented AngII-induced mitochondrial membrane depolarization in an AMPK-dependent manner. The ratio of red aggregates to green monomers (red:green) reflects membrane depolarization. (A) CB13 treatment prevents AngII-induced mitochondrial membrane depolarization. AngII induces membrane depolarization, as indicated by a decrease in red:green, which is attenuated by CB13. (B) Compound C, an AMPK inhibitor, prevented the ability of CB13 to alter mitochondrial membrane depolarization. Rescue of red:green by CB13 was prevented by compound C treatment. Serum-deprived NRAM were pre-treated with CB13 (1 μM, 1 h) in the presence or absence of compound C (1 μM, 1 h), followed by treatment with AngII (10 μM). All treatments remained in media for 24 h. Results are presented with representative fluorescent images. Data are presented as mean ± SEM. n = 3. *p ≤ 0.05 and ***p < 0.001.

FIGURE 3. Mitochondrial permeability transition pore (mPTP) was not altered by AngII treatment. NRAM treated with AngII did not undergo changes in mPTP opening. Calcein fluorescence was measured as the contrast between the cytosol and mitochondrial puncta. Serum-deprived NRAM were pre-treated with CB13 (1 μM, 1 h) in the presence or absence of compound C (1 μM, 1 h), followed by treatment with AngII (10 μM). All treatments remained in media for 24 h. Results are presented with representative fluorescent images. Data are presented as mean ± SEM. n = 3.

FIGURE 4. CB13 rescued AngII-induced reduction of phosphorylated AMPK. (A) CB13 treatment resulted in phosphorylation of AMPK (normalized by total protein) in NRAM at Thr172 (an indicator of AMPK activation status) compared to control, and rescued AngIIinduced downregulation of p-AMPK. (B) Total AMPK and (C) pAMPK (when normalized by total AMPK) were unaltered by AngII and CB13 treatment. The proportion of pAMPK in the context of total AMPK doesn’t change (panel C), in part because there is seemingly a trend, albeit statistically insignificant, toward reduced total AMPK. (D) p-LKB1, (E) total LKB1, and (F) p-LKB1/total LKB1 were unchanged by AngII and CB13. Data are presented as mean ± SEM. n = 3–4. *p ≤ 0.05 and **p < 0.01.

FIGURE 5. CB13 increased the gap junction, Cx43, compared to AngII controls. (A) CB13 increased Cx43 levels compared to NRAM treated with AngII. CB13 also tended to increase Cx43 levels in the presence of AngII, but the latter effect did not reach significance. (B) NRAM treated with compound C + CB13 did not have different Cx43 levels when compared to control NRAM. Data are presented as mean ± SEM. n = 3–4. *p ≤ 0.05.

FIGURE 6. Cannabinoid receptor 2 expression was downregulated in NRAM by AngII. (A) CB2R was decreased by AngII-treatment. (B) CB1R was unaltered by AngII and CB13. Data are presented as mean ± SEM. n = 3. *p ≤ 0.05.