Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function

Background/Aims: Calcium-activated chloride channels (CaCCs) regulate numerous physiological processes including cell proliferation, migration, and extracellular matrix secretion. T16Ainh-A01 and CaCCinh-A01 are selective inhibitors of CaCCs. But it is unknown whether these two compounds have functional effects on cardiac fibroblasts (CFs). Methods: Primary CFs were obtained by enzymatic dissociation of cardiomyocytes from neonatal rat hearts. Intracellular Ca2+ ([Ca2+](i)) and Cl ([Cl-](i)) were measured using the fluorescent calcium indicators (Fluo-4 AM) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide respectively. The expression of anoctamin-1 (ANO1) and alpha-smooth muscle actin (alpha-SMA) was detected by quantitative RT-PCR, immunofluorescence, and western blotting. A hydroxyproline assay was used to examine collagen secretion. Cell proliferation, cell cycle distribution, and cell migration were assessed by Cell Counting Kit-8, flow cytometry, and Transwell assays, respectively. Results: ANO1 was preferentially expressed on the nuclear membrane and partially within intracellular compartments around the nucleus. T16Ainh-A01 and CaCCinh-A01 displayed different inhibitory effects on [Cl-] i in CFs. T16Ainh-A01 considerably decreased [Cl-] i in the nucleus, whereas CaCCinh-A01 reduced [Cl-](i) in intracellular compartments around the nucleus, and both inhibitors exhibited a minimal effect on [Ca2+](i) in CFs. ANO1 and alpha-SMA expression levels were significantly repressed by CaCCinh-A01. T16Ainh-A01 showed a marked inhibitory effect on the mRNA levels of ANO1 and alpha-SMA, but had a negligible effect on ANO1 at the protein level. T16Ainh-A01 and CaCCinh-A01 led to the significant repression of cell proliferation, cell migration, and collagen secretion in CFs. Conclusion: Our findings indicate that T16Ainh-A01 and CaCCinh-A01 have the potential to inhibit the proliferation and collagen secretion of CFs and may serve as novel anti-fibrotic therapeutic drugs in the future. (C) 2018 The Author(s) Published by S. Karger AG, Basel.