Expression of ANO1 during cardiac fibroblasts differentiation and its electrophysiological characteristics as calcium-activated chloride channel protein

doi:10.12047/j.cjap.5940.2020.077

Abstract

Abstract: Objective: To investigate the expression and electrophysiological characteristics of calcium-activated chlorine channel anoctamin-1 (ANO1) protein during the differentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFs), and to elucidate the role of ANO1 in myocardial fibrosis. Methods: The primary CFs from neonatal rats were isolated and the cells differentiated into MFs by subculture. The Ca²⁺-activated Cl^- current (I_Cl_(Ca)) in CFs and MFs were measured by whole-cell patch clamp, and the expressions of ANO1, α-smooth muscle actin(α-SMA)and vimentin in CFs and MFs were detected by immunofluorescence assay and Western blot, respectively. Results: The current density in the early adherent CFs was stronger than that in MFs. ANO1 was expressed preferentially within and around the nuclei, and a small amount of ANO1 was expressed on the cell membrane. Moreover, ANO1 expression was weak in the early adherent CFs and displayed stronger expression in the MFs with proliferation tendency. Conclusion: The expression of ANO1 is closely related to the differentiation of MFs and it may be involved in modulation myocardial fibrosis.

Key words: cardiac fibroblasts, calcium-activated chloride channels, ANO1, whole-cell patch clamp, myocardial fibrosis

CLC Number:

R331.3

TIAN Xiang-qin, TAN Chao-yang, LI Na-na, CHANG Yu-qiao, ZHANG Ai-mei, ZHOU Ying, MA Ke-tao, SI Jun-qiang. Expression of ANO1 during cardiac fibroblasts differentiation and its electrophysiological characteristics as calcium-activated chloride channel protein[J]. CJAP, 2020, 36(4): 358-362.

References

[1] Yang YD, Cho H, Koo JY, et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance [J]. Nature, 2008, 455(7217): 1210-1215.
[2] Schroeder BC, Cheng T, Jan YN, et al. Expression cloning of TMEM16A as a calcium-activated chloride channel subunit [J]. Cell, 2008, 134(6): 1019-1029.
[3] Caputo A, Caci E, Ferrera L, et al. TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity [J]. Science, 2008, 322(5901): 590-594.
[4] Ji Q, Guo S, Wang X, et al. Recent advances in TMEM16A: Structure, function, and disease [J]. J Cell Physiol, 2019, 234(6): 7856-7873.
[5] Oh U, Jung J. Cellular functions of TMEM16/anoctamin [J]. Pflugers Arch, 2016, 468(3): 443-453.
[6] 谷小雨, 方婷婷, 高琴, 等. 高糖对线粒体乙醛脱氢酶2在大鼠心肌成纤维细胞中表达的影响 [J]. 中国应用生理学杂志, 2017, 33(3): 267-270.
[7] 冯惠清, 崔炜, 冯惠平, 等. 醛固酮对新生大鼠心肌成纤维细胞分泌ET、NO功能的影响 [J]. 中国应用生理学杂志, 2005, 21(1): 79-81.
[8] Hiraoka K, Miyazaki H, Niisato N, et al. Chloride ion modulates cell proliferation of human androgen-independent prostatic cancer cell [J]. Cell Physiol Biochem, 2010, 25(4-5): 379-388.
[9] El Chemaly A, Norez C, Magaud C, et al. ANO1 contributes to angiotensin-II-activated Ca²⁺-dependent Cl- current in human atrial fibroblasts [J]. J Mol Cell Cardiol, 2014, 68(1): 12-19.
[10] 李辞霞, 常玉巧, 郭志坤. 新生大鼠心脏成纤维细胞体外分裂方式 [J]. 解剖学报, 2016, 47(1): 55-61.
[11] Yamamura H, Nishimura K, Hagihara Y, et al. TMEM16A and TMEM16B channel proteins generate Ca(2+)-activated Cl(-) current and regulate melatonin secretion in rat pineal glands [J]. J Biol Chem, 2018, 293(3): 995-1006.
[12] Bebarova M. Advances in patch clamp technique: towards higher quality and quantity [J]. Gen Physiol Biophys, 2012, 31(2): 131-140.
[13] Sucher NJ, Deitcher DL, Baro DJ, et al. Genes and channels: patch/voltage-clamp analysis and single-cell RT-PCR [J]. Cell Tissue Res, 2000, 302(3): 295-307.
[14] Kamaleddin MA. Molecular, biophysical, and pharmacological properties of calcium-activated chloride channels [J]. J Cell Physiol, 2018, 233(2): 787-798.
[15] Horvath B, Vaczi K, Hegyi B, et al. Sarcolemmal Ca(2+)-entry through L-type Ca(2+) channels controls the profile of Ca(2+)-activated Cl(-) current in canine ventricular myocytes [J]. J Mol Cell Cardiol, 2016, 97(1): 125-139.
[16] Tian XQ, Ma KT, Wang XW, et al. Effects of the calcium-activated chloride channel inhibitors T16Ainh-A01 and CaCCinh-A01 on cardiac fibroblast function [J]. Cell Physiol Biochem, 2018, 49(2): 706-716.
[17] Sundelacruz S, Levin M, Kaplan DL. Role of membrane potential in the regulation of cell proliferation and differentiation [J]. Stem Cell Rev, 2009, 5(3): 231-246.
[18] Ferrera L, Caputo A, Galietta LJV. TMEM16A protein: A new identity for Ca²⁺-dependent Cl-channels [J]. Physiology, 2010, 25(6): 357-363.
[19] Duvvuri U, Shiwarski DJ, Xiao D, et al. TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression [J]. Cancer Res, 2012, 72(13): 3270-3281.
[20] Bohn AA, de Morais HA. A quick reference on chloride [J]. Vet Clin North Am Small Anim Pract, 2017, 47(2): 219-222.
[21] Travers JG, Kamal FA, Robbins J, et al. Cardiac fibrosis: The fibroblast awakens [J]. Circ Res, 2016, 118(6): 1021-1040.