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Microrna-Mediated Downregulation of K+ Channels in Pulmonary Arterial Hypertension

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Microrna-Mediated Downregulation of K+ Channels in Pulmonary Arterial Hypertension MicroRNA-mediated downregulation of K + channels in pulmonary arterial hypertension Item Type Article Authors Babicheva, Aleksandra; Ayon, Ramon J; Zhao, Tengteng; Ek Vitorin, Jose F; Pohl, Nicole M; Yamamura, Aya; Yamamura, Hisao; Quinton, Brooke A; Ba, Manqing; Wu, Linda; Ravellette, Keeley S; Rahimi, Shamin; Balistrieri, Francesca; Harrington, Angela; Vanderpool, Rebecca R; Thistlethwaite, Patricia A; Makino, Ayako; Yuan, Jason X-J Citation Babicheva, A., Ayon, R. J., Zhao, T., Vitorin, J. F. E., Pohl, N. M., Yamamura, A., ... & Ravellette, K. S. (2019). MicroRNA- mediated downregulation of K+ channels in pulmonary arterial hypertension. American Journal of Physiology-Lung Cellular and Molecular Physiology. DOI 10.1152/ajplung.00010.2019 Publisher AMER PHYSIOLOGICAL SOC Journal AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY Rights Copyright © 2020 the American Physiological Society. Download date 28/09/2021 04:07:06 Item License http://rightsstatements.org/vocab/InC/1.0/ Version Final accepted manuscript Link to Item http://hdl.handle.net/10150/637047 1 1 MicroRNA-mediated Downregulation of K+ Channels in Pulmonary Arterial Hypertension 2 3 Aleksandra Babicheva1,4, Ramon J. Ayon4, Tengteng Zhao1,4, Jose F. Ek Vitorin4, Nicole M. 4 Pohl5, Aya Yamamura6, Hisao Yamamura7, Brooke A. Quinton4, Manqing Ba4, Linda Wu4, 5 Keeley S. Ravellette4, Shamin Rahimi1, Francesca Balistrieri1, Angela Harington1, Rebecca R. 6 Vanderpool4, Patricia A. Thistlethwaite3, Ayako Makino2,4, and Jason X-J. Yuan1,4,5* 7 8 1Section of Physiology, Division of Pulmonary, Critical Care and Sleep Medicine, and 2Division 9 of Endocrinology and Metabolism, Department of Medicine; 3Department of Surgery, University 10 of California, San Diego, La Jolla, CA 92093; 4Departments of Medicine and Physiology, The 11 University of Arizona, Tucson, AZ 85721; 5Department of Medicine, University of Illinois at 12 Chicago, Chicago, IL 60612; 6Kinjo Gakuin University School of Pharmacy, Nagoya, Japan; and 13 7Nagoya City University Graduate School of Pharmaceutical Sciences, Nagoya, Japan 14 15 Running title: miRNA-mediated regulation of K+ channels 16 *Address correspondence to: 17 Jason X.-J. Yuan, M.D., Ph.D. 18 Professor of Medicine 19 Division of Pulmonary, Critical Care and Sleep Medicine 20 Department of Medicine, MC 0856 21 University of California, San Diego 22 9500 Gilman Drive 23 La Jolla, CA 92093-0856 24 Tel: (858)246-5797 25 Fax: (858)534-4812 26 Email: [email protected] 27 28 I 2 29 Abstract 30 Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth 31 muscle cells (PASMC) have been implicated in the development of sustained pulmonary 32 vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial 33 hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in 34 IPAH-PASMC. MicroRNAs (miRNAs) are small noncoding RNAs that are capable of 35 posttranscriptionally regulating gene expression by binding to the 3’-untranslated regions (3’- 36 UTR) of their targeted mRNAs. Here we report that specific miRNAs are responsible for the 37 decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR- 38 29b, miR-138 and miR-222) that were highly expressed in IPAH-PASMC in comparison to 39 normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the 40 decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR- 41 138 or miR-222 in normal PASMC significantly decreased whole-cell K+ currents and + 42 downregulated voltage-gated K channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of + 43 miR-29b in IPAH-PASMC completely recovered K channel function and KV1.5 expression, 44 while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that 45 KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC 2+ + 46 decreased large-conductance Ca -activated K (BKCa) channel currents and downregulated 47 BKCa channel β1 subunit (BKCaβ1 or KCNMB1) expression, while inhibition of miR-29b in 48 IPAH-PASMC increased BKCa channel activity and BKCaβ1 level. These data indicate 49 upregulated miR-29b contributes, at least partially, to the attenuated function and expression of 50 KV and BKCa channels in PASMC from patients with IPAH. 51 Key words: posttranscriptional regulation; potassium channels; microRNA; KCNA5; KCNMB1. 52 I 3 53 Introduction 54 Sustained pulmonary vasoconstriction is an important early cause for elevated pulmonary 55 vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH) (25, 61). 56 Pulmonary arterial tone and vasoconstriction are controlled by the resting membrane potential 57 (Em) in pulmonary artery smooth muscle cells (PASMC) (30, 49, 73). A change in Em plays a 58 key role in excitation-contraction coupling in PASMC by regulating the cytosolic free Ca2+ 2+ 2+ 59 concentration ([Ca ]cyt). Membrane depolarization leads to an increase in [Ca ]cyt by opening 2+ 2+ 60 voltage-dependent Ca channels (VDCC) in PASMC (31, 32, 69). Increased [Ca ]cyt not only 61 causes PASMC contraction and pulmonary vasoconstriction, but also stimulates PASMC 62 proliferation and migration, which are the major contributors to concentric pulmonary arterial 63 wall remodeling (10, 15). + 64 K channel activity in PASMC contributes significantly to Em regulation (2, 41). 65 Decreased K+ currents by downregulating channel expression (leading to decreased number of 66 K+ channels in the plasma membrane) and/or inhibiting channel activity would depolarize 67 PASMC, open VDCC and increase Ca2+ influx. Downregulated expression of K+ channels and 68 decreased K+ currents in PASMC have been implicated in the development and progression of 69 pulmonary hypertension, however, the underlying mechanisms are still unknown. 70 To date, more than eight different K+ channel families have been identified in the 2+ + 71 pulmonary vasculature; voltage-gated (KV) and Ca -activated (KCa) K channels appear to 72 participate in the regulation of Em in PASMC. Each KV channel comprises four pore-forming α 73 subunits and four regulatory cytoplasmic β subunits that modulate channel activity via 74 inactivating α subunits (47). The diversity of mammalian KV channels is derived from genetic 75 diversity, with over 40 genes encoding KV channel α subunits from 12 subtypes (KV1-12)(18, 42, I 4 2+ + 76 74). There are five families of Ca -activated K channels (KCa1-KCa5) including the large- 77 conductance KCa channels (MaxiK or BKCa) (60). The BKCa channel consists of four α subunits 78 (BKCaα) that create a pore in the membrane associated with four ancillary β (BKCaβ1-4) subunits. 79 BKCaβ2 (KCNMB2), BKCaβ3 (KCNMB3), and BKCaβ4 (KCNMB4) subunits are predominantly 80 expressed in endocrine tissue, testis, and brain tissue respectively, however, BKCaβ1 (KCNMB1) 81 appears to be solely expressed in smooth muscle tissue (5, 12). The function of BKCa channels in 82 PASMC is finely tuned by its regulatory β-subunits via enhancing α subunit sensitivity to 2+ 83 intracellular Ca and voltage (13). The presence of BKCa channels has been established in 84 human and animal PASMC (3, 9, 20, 26). Downregulated KV channel expression (e.g., 85 KV1.5/KCNA5) and decreased whole-cell KV currents in PASMC have been established in PH 86 including IPAH (8, 63, 75) while the role of BKCa channels in PH is not well established (1, 4, 87 19, 45). 88 MicroRNAs (miRNAs) are small non-coding regulatory RNAs that posttranscriptionally 89 regulate gene expression by binding to the 3’-untranslated region (UTR) of their targeted mRNA, 90 thereby preventing translation and/or decreasing stability of target mRNAs. miRNAs have been 91 implicated in the development and progression of pulmonary hypertension by using lung 92 specimen and isolated PASMC from patients with IPAH as well as genetically engineered mice 93 including miR-204, miR-21, miR-130/301 and the miR-17/92 cluster (11, 28, 46, 56, 76). It has + 94 been found that certain miRNAs target multiple K channels including KV7.5/KCNQ5 (34), 95 KV4.2/KCND2 (38), KCa2.3/KCNN3 (36) and KIR2.1/KCNJ2 (40) channels in vascular smooth 96 muscle cells and cardiac myocytes. The aim of this study is to investigate the role of miRNAs in 97 the regulation of KV channels and BKCa channels in PASMC from patients with IPAH. 98 I 5 99 Materials and Methods 100 Cell Culture: The approval for using human cells was granted by the University Institutional 101 Review Board. PASMC from 6 healthy (normal) subjects without pulmonary hypertension and 6 102 IPAH patients were provided by the Pulmonary Hypertension Breakthrough Initiative (PHBI). 103 Normal and IPAH PASMC were cultured in 5% fetal bovine serum (FBS) smooth muscle 104 growth media (LifeLine) and incubated in a humidified 5% CO2 atmosphere at 37°C. Pulmonary 105 arterial endothelial cells (PAEC) isolated from 4 normal subjects and 4 IPAH patients (PHBI) 106 were cultured in 2% FBS growth media (LifeLine) and incubated in a humidified 5% CO2 107 atmosphere at 37°C. Human PASMC and PAEC were isolated at PHBI facility as previously 108 described (27, 67). PASMC authentication was carried out with fluorescence-activated cell 109 sorting (FACS) and immunocytochemistry (ICC) with smooth muscle actin-α (SMA), smooth 110 muscle 22-α (SM22α) and smooth muscle myosin heavy chain (SMMHC). The percentage of 111 positive cells that exhibit SMA, SM22α, and SMMHC signal was over 95%. PAEC 112 authentication was carried out with FACS and ICC using CD31 (FACS), von Willebrand factor 113 (vWF), and vascular endothelial cadherin (VE-cadherin) (ICC). The percentage of positive cells 114 that exhibit CD31, vWF, and VE-cadherin signal was over 93% in cultures. The cells were 115 acquired at passages 2-3. Passages 4-6 were used for the experiments. The demographic 116 information of human PASMC and PAEC is listed in Table 1.
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