Abstract 19629: Expression and Role of TRPC Channels in Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Abstract

Store-operated Ca 2+ entry (SOCE) is a mechanism for increasing cytosolic Ca 2+ (Ca i 2+ ) load after the SR/ER Ca 2+ depletion in many cell types. Our previous study has implicated SOCE, which is at least partially mediated by transient receptor potential canonical (TRPC) channels, exists in adult mouse ventricular myocytes, and contributes to the generation of spontaneous SR Ca 2+ release and triggered arrhythmias. However, it is unclear if this mechanism also exists in human cardiac myocytes. In the present study, we investigate the expression and role of TRPC channels in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) (purchased from Cellular Dynamics). Abundant distributions of TRPC3 and 6 channels were detected in the plasma membrane of hiPSC-CMs using immunocytochemical staining. Ca i 2+ was imaged in fluo-4-AM loaded cells. A SOCE (F/F 0 = 1.75 ± 0.08, n = 47) was observed in hiPSC-CMs when the external Ca 2+ concentration ([Ca 2+ ] o ) was switched from 0 to 1 mM after SR Ca 2+ was completely depleted by caffeine (20 mM) and thapsigargin (10 µM). The SOCE was inhibited by non-selective TRPC blockers gadolinium (F/F 0 = 1.05 ± 0.03, n = 13) and SKF-96265 (F/F 0 = 1.04 ± 0.03, n = 17), respectively, compared to control (p<0.05). Whereas, the SOCE in hiPSC-CMs was significantly reduced only by pretreatment with anti-TRPC6 antibody (F/F 0 = 1.38 ± 0.06, n = 15, p<0.05), but not with anti-TRPC1 (F/F 0 = 1.93 ± 0.19, n = 8) or anti-TRPC3 antibodies (F/F 0 = 1.49 ± 0.05, n = 14), indicating TRPC6 channels are essential for SOCE in hiPSC-CMs. In addition, the hiPSC-CMs exhibit a property of spontaneous rhythmic beating. After SR Ca 2+ depletion, spontaneous Ca 2+ waves reappeared via refilling from the extracellular Ca 2+ source, suggesting the involvement of SOCE in the generation of spontaneous Ca 2+ waves. This idea was confirmed by the application of TRPC blockers, including gadolinium, SKF-96265 and Pyr3, all of which showed mild to potent inhibitory effects on the spontaneous Ca 2+ waves. In conclusion, we demonstrated the existence of SOCE in hiPSC-CMs. SOCE mediated by TRPC channels, especially the TRPC6, may act as an important mechanism of controlling Ca 2+ handling and arrhythmogenesis in the developing human heart.