2-Cl-C.OXT-A stimulates contraction through the suppression of phosphodiesterase activity in human induced pluripotent stem cell-derived cardiac organoids

Background 2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates Sphingosine-1-phosphate 1 receptor (S1P1R) and combines with the adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle. Methods and results Cardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half. Conclusions COA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.