Fig 6.

cAMP standards (10 pM-1 μM; half-log increments) were used to create a concentration calibration based on the decreasing time-resolved fluorescent resonance energy transfer (TR-FRET) in hiPSC-CMs (A). Forskolin (1nM-100 μM; half-log increments) caused a concentration-dependent decrease in TR-FRET and thus an increase in cAMP. The dose response curve was drawn using the mean values (B). COA-Cl (100 μM-10M; half-log increments) caused a concentration-dependent decrease in TR-FRET and thus an increase in cAMP. The dose response curve was drawn using the mean values (C). In hiPSC-CMs, COA-Cl (1 mM), ISO (10 nM), and FSK (10 μM) substantially increased the cAMP level compared to the basal cAMP level in untreated controls. Treatment with SEW2871 (20 nM) and PRP (10 nM) did not significantly alter the cAMP levels compared to control. *P values of < 0.05 were considered to represent a statistically significant difference compared to control (n = 5–8) (D). HiPSC-CMs treated with Suramin (100 μM), COA-Cl (1 mM), ISO (10 nM), and FSK (10 μM) showed significantly increased cAMP levels compared to the basal cAMP level in untreated controls. Treatment with SEW2871 (20 nM) and PRP (10 nM) did not significantly alter the cAMP level compared to control. *P values of < 0.05 were considered to represent a statistically significant difference compared to control (n = 5–8) (E, F). *P < 0.05 significant compared to control. These results showed that COA-Cl increased the cAMP levels of hiPSC-CMs similarly to Isoproterenol and Forskolin, but not through A1R or S1P1R activation (n = 5–8).