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

Abstract

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 α-smooth muscle actin (α-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 α-SMA expression levels were significantly repressed by CaCCinh-A01. T16Ainh-A01 showed a marked inhibitory effect on the mRNA levels of ANO1 and α-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.

TMEM16A Inhibition Preserves Blood–Brain Barrier Integrity After Ischemic Stroke

“…As previously described, T16ainh-A01 and Caccinh-A01 have different inhibitory effects on TMEM16A. Caccinh-A01 was used to facilitate the degradation of TMEM16A in many types of tissues and T16ainh-A01 could only inhibit the channel activity of TMEM16A (Bill et al, 2014; Tian et al, 2018). In our present study, Caccinh-A01 obviously attenuated brain infarct size and neurological deficits by reducing the expression of TMEM16A.…”

Epigenetic Repression of Chloride Channel Accessory 2 Transcription in Cardiac Fibroblast: Implication in Cardiac Fibrosis

“…Alternatively, chloride intracellular channel 4 (CLIC4) has been shown to promote TGF-β induced FMyT by inducing a dominant negative SMAD7 splicing isoform ( Shukla et al, 2016 ). Despite the fact that several studies have provided evidence to show that chloride channel inhibitors/blockers can potentially attenuate the activation of cardiac fibroblasts ( El Chemaly et al, 2014 ; Tian et al, 2018 ; Chen P. H. et al, 2021 ), no consensus seems to exist regarding the underlying mechanisms. It is therefore imperative for future investigators to focus on delineating the mode of action for Clca2 in the process of FMyT so that the plethora of data, including the ones presented here, can be exploited in the development of novel therapeutic solutions to treat adverse cardiac remodeling and heart failure.…”

Transmembrane protein 16A/anoctamin 1 inhibitor T16Ainh‐A01 reversed monocrotaline‐induced rat pulmonary arterial hypertension

“…In addition, we choose a TMEM16A inhibitor, which considerably decreased [Cl À ] in the nucleus, exhibited a minimal effect on [Ca 2þ ], and had a negligible effect on TMEM16A at the protein level. 18 PAH and vessel remodeling were still improved by TMEM16A inhibition; this means that quantitative decrease, or functionally inhibition of TMEM16A, can both reverse vessel remodeling and PAH.…”

“…T16Ainh-A01 had a negligible effect on TMEM16A at the protein level. 18 We choose the fourth week post MCT treatment to administer T16Ainh-A01, to make sure that T16Ainh-A01 could functionally and not quantitatively inhibit TMEM16A. We consider the improvement of vascular remodeling by T16Ainh-A01 treatment was not dependent on the TMEM16A protein level.…”