Up-regulated expression of two-pore domain K+ channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

Background Pulmonary arterial hypertension (PAH) is a severe and rare disease in the cardiopulmonary system. Its pathogenesis involves vascular remodeling of the pulmonary artery, which results in progressive increases in pulmonary arterial pressure. Chronically increased pulmonary arterial pressure causes right ventricular hypertrophy and subsequent right heart failure. Pulmonary vascular remodeling is attributed to the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are induced by enhanced Ca2+ signaling following the up-/down-regulation of ion channel expression. Objectives In the present study, the functional expression of two-pore domain potassium KCNK channels was investigated in PASMCs from idiopathic PAH (IPAH) patients and experimental pulmonary hypertensive (PH) animals. Results In IPAH-PASMCs, the expression of KCNK1/TWIK1 and KCNK2/TREK1 channels was up-regulated, whereas that of KCNK3/TASK1 and KCNK6/TWIK2 channels was down-regulated. The similar up-regulated expression of KCNK1 and KCNK2 channels was observed in the pulmonary arterial smooth muscles of monocrotaline-induced PH rats, Sugen 5416/hypoxia-induced PH rats, and hypoxia-induced PH mice. The facilitated proliferation of IPAH-PASMCs was suppressed by the KCNK channel blockers, quinine and tetrapentylammonium. The migration of IPAH-PASMCs was also suppressed by these channel blockers. Furthermore, increases in the proliferation and migration were inhibited by the siRNA knockdown of KCNK1 or KCNK2 channels. The siRNA knockdown also caused membrane depolarization and subsequent decrease in cytosolic [Ca2+]. The phosphorylated level of c-Jun N-terminal kinase (JNK) was elevated in IPAH-PASMCs compared to normal-PASMCs. The increased phosphorylation was significantly reduced by the siRNA knockdown of KCNK1 or KCNK2 channels. Conclusion Collectively, these findings indicate that the up-regulated expression of KCNK1 and KCNK2 channels facilitates the proliferation and migration of PASMCs via enhanced Ca2+ signaling and JNK signaling pathway, which is associated with vascular remodeling in PAH.

Background: Pulmonary arterial hypertension (PAH) is a severe and rare disease in the cardiopulmonary system.Its pathogenesis involves vascular remodeling of the pulmonary artery, which results in progressive increases in pulmonary arterial pressure.Chronically increased pulmonary arterial pressure causes right ventricular hypertrophy and subsequent right heart failure.Pulmonary vascular remodeling is attributed to the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are induced by enhanced Ca 2+ signaling following the up-/down-regulation of ion channel expression.Objectives: In the present study, the functional expression of two-pore domain potassium KCNK channels was investigated in PASMCs from idiopathic PAH (IPAH) patients and experimental pulmonary hypertensive (PH) animals.Results: In IPAH-PASMCs, the expression of KCNK1/TWIK1 and KCNK2/TREK1 channels was up-regulated, whereas that of KCNK3/TASK1 and KCNK6/TWIK2 channels was down-regulated.The similar up-regulated expression of KCNK1 and KCNK2 channels was observed in the pulmonary arterial smooth muscles of monocrotaline-induced PH rats, Sugen 5416/hypoxia-induced PH rats, and hypoxia-induced PH mice.The facilitated proliferation of IPAH-PASMCs was suppressed by the KCNK channel blockers, quinine and tetrapentylammonium.The migration of IPAH-PASMCs was also suppressed by these channel blockers.Furthermore, increases in the proliferation and migration were inhibited by the siRNA knockdown of KCNK1 or KCNK2 channels.The siRNA knockdown also caused membrane depolarization and subsequent decrease in cytosolic [Ca 2+ ].The phosphorylated level of c-Jun N-terminal kinase (JNK) was elevated in IPAH-PASMCs compared to normal-PASMCs.The increased phosphorylation was significantly reduced by the siRNA knockdown of KCNK1 or KCNK2 channels.

Introduction
Pulmonary arterial hypertension (PAH), which is known as clinical classification Group 1 of pulmonary hypertension (PH), is a rare and life-threatening disease in the cardiovascular/ respiratory systems.It is characterized by the vascular remodeling of pulmonary arterioles (<500 μm in diameter).These pathological events induce constitutive increases in pulmonary arterial pressure.Chronically increased pulmonary arterial pressure causes right ventricular hypertrophy, and ultimately, right heart failure with high mortality (1).PAH has been categorized by its underlying etiologies: idiopathic (IPAH, 46.2%), associated (45.4%), drug/toxin-induced (5.3%), and heritable (2.7%) PAH (2).The etiological causes of IPAH remain unknown or there is no family history.Heritable PAH causes familial mutations in the genes encoding activin A receptor-like type 1, bone morphogenetic protein receptor type 2, caveolin 1, endoglin, mothers against decapentaplegic homolog 9, and two-pore domain potassium channel subfamily K member 3 (KCNK3/TASK1) (3).Associated PAH occurs with congenital heart disease, connective tissue disease, human immunodeficiency virus infection, schistosomiasis, and portal hypertension (1).
In PAH, the progression of irreversible vascular remodeling are predominantly caused by the facilitated proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) composing the medial layer of the pulmonary artery.The proliferation and migration of PASMCs are elicited by a rise in cytosolic [Ca 2+ ] ([Ca 2+ ] cyt ), which is regulated by Ca 2+ influx through Ca 2+ -permeable ion channels: e.g., voltage-dependent Ca 2+ channels (VDCCs), receptor-operated Ca 2+ (ROC) channels, and store-operated Ca 2+ (SOC) channels.It is also caused by Ca 2+ release from intracellular Ca 2+ stores: e.g., the sarcoplasmic reticulum (4,5).The activity of K + and Cl − channels has also been shown to participate in the modulation of [Ca 2+ ] cyt through the membrane potential in PASMCs (6,7).
The two-pore domain potassium KCNK channel family contains 15 genes (KCNK1 to 18, except for 8, 11, and 14) that are classified into six subfamilies based on sequence similarity and functional resemblance: TWIK, TREK, TASK, TALK, THIK, and TRESK (8)(9)(10).The KCNK subunit has four transmembrane structures containing two pore-forming regions that form a functional ion channel as a homomeric or heteromeric dimer.KCNK channels produce background or leak K + currents, thereby maintaining the resting membrane potential and [Ca 2+ ] cyt in several types of cells.Pharmacologically, KCNK channels are sensitive to lipids, temperature, membrane stretch, pH, and volatile anesthetics.In the pathological profile, KCNK channels are associated with depression, epilepsy, cardiac arrhythmia, nociception, and cancers (8,9).Furthermore, missense mutations in the KCNK3/TASK1 gene are associated with heritable PAH (3).Limited information is, however, available on the involvement of other KCNK channels in PAH.
Mitogen-activated protein kinases (MAPKs) are a group of serine/threonine protein kinases that play a pivotal role in regulating the growth, proliferation, differentiation, migration, and apoptosis of vascular myocytes (4,5).MAPKs include extracellular signal-regulated protein kinase 1/2, p38 MAPK, and c-Jun N-terminal kinase (JNK), which are activated by mitogen, hormones, growth factors, cytokines, and environmental stresses (11).Previous studies reported the activation of JNK in the pulmonary artery of experimental PH animals (12,13) and PAH patients (14) and also in hypoxia-treated PASMCs (15).Among the three isoforms of JNK (JNK1 to 3), JNK2 is predominantly responsible for vascular remodeling in hypoxia-induced PH (16,17).Therefore, JNK signaling has been supposed to participate in the process of vascular remodeling in PAH.
In the present investigation, the expression of KCNK channels in PASMCs from IPAH patients and experimental PH animals (monocrotaline (MCT)-induced PH rats, Sugen 5416/hypoxia (SuHx)-induced PH rats, and hypoxia-induced PH mice) was analyzed using quantitative real-time PCR (qPCR), Western blotting, and immunohistochemical staining.The contribution of KCNK channels to the enhanced proliferation of IPAH-PASMCs was assessed by WST-8 and bromodeoxyuridine (BrdU) incorporation assays.The role of KCNK channels in the migration of IPAH-PASMCs was investigated by Transwell assays, and their involvement in the phosphorylation of JNK in IPAH-PASMCs was also evaluated.In addition, the involvement of KCNK channels in the regulation of the resting membrane potential and [Ca 2+ ] cyt in IPAH-PASMCs was examined by fluorescence DiBAC 4 (3) and fura-2 imaging, respectively.The present investigation clearly showed that the expression of KCNK1/TWIK1 and KCNK2/TREK1 channels was up-regulated in PASMCs from IPAH patients, MCT-PH rats, SuHx-PH rats, and hypoxia-PH mice.The proliferation and migration of IPAH-PASMCs were inhibited by KCNK channel blockers and by the siRNA knockdown of KCNK1 or KCNK2 channels.These siRNA knockdown also caused membrane depolarization and decreased resting [Ca 2+ ] cyt .The phosphorylation level of JNK was reduced by the siRNA knockdown of KCNK1 or KCNK2 channels.Collectively, these findings indicate that the up-regulated expression of KCNK1 and KCNK2 channels is associated with vascular remodeling through enhanced Ca 2+ signaling and JNK signaling pathway in PAH.

qPCR
Total RNA was extracted using RNAiso Plus (Takara Bio, Kusatsu, Japan) and reverse transcribed to cDNA using the ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) (24).A qPCR analysis was carried out using SYBR Premix Ex Taq (Takara Bio) by the LightCycler 96 qPCR system (Roche Diagnostics, Basel, Switzerland).Specific primers were designed as shown in Table 1.

Western blotting
Protein fraction was extracted using RIPA buffer (for human PASMCs) and T-PER Tissue Protein Extraction Reagent (for rat/

Immunohistochemical staining
The lungs of MCT-PH rats were fixed with paraformaldehyde (4%; MilliporeSigma) in PBS.Paraffin-embedded sections from lung lobes were prepared by the Biopathology Institute (Oita, Japan).They were deparaffinized and heat-induced epitope retrieval was carried out with Tris-HCl (10 mM, pH 9.0) containing EDTA (1 mM) at 115°C for 10 min.As the first step, sections were treated with a KCNK1 or KCNK2 antibody (1:100) using an ImmPRESS HRP reagent kit (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h.After washing twice in PBS, they were covered with a secondary antibody contained in the ImmPRESS HRP reagent kit at room temperature for 30 min and rinsed twice with PBS.They were then treated with Fluorescein (1:200) in 1×plus amplification diluent (Akoya Biosciences, Marlborough, MA, USA) at room temperature for 10 min.After heating in a microwave for 1 min and washing with PBS, the same sections were treated with an α-smooth muscle actin (α-SMA) antibody (1:1000; #19245, Cell Signaling Technology), the ImmPRESS HRP reagent kit, and Cyanine 3 (1:400) using the same protocol as the first step.They were also stained with 4',6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories, Kumamoto, Japan).Immunohistochemical images were obtained using the Aperio CS2 image capture device (Leica Biosystems, Wetzlar, Germany).

Cell migration assay
Human PASMCs (5 × 10 4 cells/well) were seeded on a 24-well Transwell insert with a membrane pore size of 8 μm (#3422, Corning) (23).Thereafter, they were treated with culture medium containing FBS (1%) in the upper chamber and that containing FBS (10%) and the vehicle (DMSO) or drug in the lower chamber for 24 h.Transwell inserts were fixed in paraformaldehyde (4%) and stained with crystal violet (1%; Fujifilm Wako Pure Chemical).The number of migratory cells was counted from digital images of Transwell inserts using the SMZ1270 stereomicroscope system equipped with a DS-Vi1 color microscope camera and NIS-Elements imaging software (Nikon, Tokyo, Japan).

Measurement of the membrane potential
Human PASMCs were incubated with the voltage-sensitive fluorescent dye, DiBAC 4 (3) (100 nM; Dojindo Laboratories), at room temperature for 30 min.DiBAC 4 (3) at the same concentration was added to the extracellular solution during measurements.Fluorescent signals were measured using the A1R confocal fluorescence imaging system equipped with an ECLIPSE Ti inverted microscope, a Plan Apo VC objective lens (20×/0.75),NIS-Elements imaging software (Nikon), and a solid-state 488-nm laser (Coherent, Santa Clara, CA, USA).PASMCs were illuminated at a 488-nm wavelength, and the fluorescent emissions (>520 nm) were obtained every 5 s.Membrane potential is presented as F/F 140K , where F is the fluorescence intensity and F 140K is the maximum fluorescence intensity in the 140-mM K + HEPES-buffered solution (theoretically 0 mV).Standard HEPES-buffered solution was used as an extracellular solution (in mM): 137 NaCl, 5.9 KCl, 2.2 CaCl 2 , 1.2 MgCl 2 , 14 glucose, 10 HEPES, and pH 7.4 with NaOH.In the 140-mM K + HEPES-buffered solution, the concentrations of NaCl and KCl in standard HEPES-buffered solution were changed to 2.9 and 140 mM, respectively.Human PASMCs were loaded with fura-2 acetoxymethyl ester (fura-2/AM, 10 μM; Thermo Fisher Scientific) at room temperature for 30 min.[Ca 2+ ] cyt measurements were performed using the fluorescence imaging system equipped with an ECLIPSE Ti2 inverted microscope, a S FL objective lens (20×/0.75),NIS-Elements imaging software (Nikon), a pE-340 fura LED illuminator (CoolLED, Hampshire, UK), and a C9100-12 EM-CCD digital camera (Hamamatsu Photonics, Hamamatsu, Japan).PASMCs were illuminated at 340-/380-nm wavelengths, and the fluorescent emissions (510/80 nm) were obtained every 5 s.The fura-2 signal is presented as the fluorescence ratio (F 340 /F 380 ).Standard HEPESbuffered solution was used as an extracellular solution.

Drugs
Pharmacological reagents were obtained from Fujifilm Wako Pure Chemical, except for EDTA, HEPES (Dojindo Laboratories), and quinine (MilliporeSigma).Quinine and tetrapentylammonium (TPA) were dissolved in DMSO at concentrations of 150 and 100 mM, respectively, as a stock solution.

Statistical analysis
Pooled data are shown as the means ± S.E.The significance of differences between two groups was examined using the nonparametric Mann-Whitney U test (n < 10) or Student's t-test (n ≥ 10) using BellCurve software (Social Survey Research Information, Tokyo, Japan).The significance of differences among groups was assessed by Scheffé's or Steel's test after nonparametric Kruskal-Wallis test (n < 10) or Scheffé's test after an analysis of variance (ANOVA) (n ≥ 10) using the same software.

Changes in KCNK1 and KCNK2 channel expression in experimental PH animals
Since the expression of KCNK1 and KCNK2 channels was upregulated in IPAH-PASMCs, their expression changes in PASMs from three types of experimental PH animals (MCT-PH rats, SuHx-PH rats, and hypoxia-PH mice) were also examined.It was confirmed that endothelium was not attached to the dissected PASMs because endothelium-dependent relaxation was not induced by acetylcholine (25).The expression of KCNK1 and KCNK2 channel proteins was examined in PASMs from control and MCT-PH rats by Western blotting.The protein expression of KCNK1 (1.72 ± 0.10-fold, n = 8, p < 0.001 vs. control, 1.00 ± 0.09, n = 8) and KCNK2 (1.35 ± 0.06-fold, n = 8, p = 0.002 vs. control, 1.00 ± 0.06, n = 8) channels was increased in MCT-PASMs (Figures 2A,B).In addition, the expression of KCNK1 and KCNK2 channel proteins was analyzed using the lung sections of control and MCT-PH rats by immunohistochemical staining.Immunohistochemical images revealed that KCNK1 and KCNK2 channels were localized in the medial (smooth muscle) layer of the pulmonary artery and their expression was higher in MCT-PH rats than in the control rats (Figures 2C,D).

Discussion
The present investigation demonstrated that the expression of KCNK1/TWIK1 and KCNK2/TREK1 channels was up-regulated in PASMCs from IPAH patients and experimental PH animals and their up-regulation facilitated the proliferation and migration of IPAH-PASMCs via enhanced Ca 2+ signaling and JNK signaling pathway, resulting in vascular remodeling in PAH.
In vascular myocytes, including PASMCs, [Ca 2+ ] cyt increment is required for cellular contraction, proliferation, migration, apoptosis, and the cell cycle.[Ca 2+ ] cyt is modulated by the resting membrane potential, which is mainly affected by K + channel conductance.Therefore, K + channels are recognized as an important molecule for the modulation of cytosolic Ca 2+ mobilization (4,5).KCNK channels are responsible for background or leak K + currents, which maintain the resting membrane potential (8,35).Among members of the KCNK channel family, the expression of the KCNK2, 3, 5, and 6 channels was detected in PASMCs (6,7,36).Specifically, loss-offunction mutations in KCNK3 channels have been implicated in heritable PAH (3,37,38).Furthermore, the expression of KCNK3 channels was down-regulated in PASMCs treated with hypoxia (39), from IPAH patients and MCT-PH rats (40), and KCNK3-knockdown rats exhibit PH (41).On the other hand, KCNK6-knockout mice developed PH (42), whereas the expression of KCNK6 channels was unchanged in IPAH patients (7).In the present study, KCNK3/TASK1 (29.8 and 30.2% homology with KCNK1 and KCNK2, respectively) and KCNK6/ TWIK2 (46.9 and 33.1%) channel expression at the protein level was slightly down-regulated by 18% and 21%, respectively, in PASMCs from IPAH patients.The most interesting finding of this investigation is that KCNK1/TWIK1 and KCNK2/TREK1 (33.9% homology to each other) channel expression at the protein level was up-regulated to 151% and 129%, respectively, in IPAH-PASMCs.Similar up-regulation was observed in PASMs from MCT-PH rats, SuHx-PH rats, and hypoxia-PH mice.These findings suppose that the mechanism responsible for this upregulation is common between IPAH patients and experimental PH animals.In the right ventricle from MCT-PH rats, the mRNA expression of KCNK2 and KCNK3 channels was upregulated and down-regulated, respectively (Supplementary Table S2).The expression of KCNK1 and KCNK6 channels was unchanged between control and MCT-PH rats.The mRNA expression of KCNK3 channels has been reported to be downregulated in the right ventricle from PAH patients and experimental PH rats (43)(44)(45).To the best of our knowledge, this is the first study to comprehensively demonstrate changes in KCNK channel expression in PASMCs from IPAH patients.These up-regulated and down-regulated expression may be a compensatory mechanism for each other.Therefore, further experiments are required regarding the functional relationship between these expression changes.
An increase in [Ca 2+ ] cyt at physiological ranges triggers the proliferation and migration of PASMCs, however, [Ca 2+ ] cyt overload also facilitate the proliferation and migration of PASMCs and subsequent pulmonary vascular remodeling, resulting in the development and progression of PAH (4,5).In the present investigation, the KCNK channel blockers, quinine and TPA, blocked the proliferation and migration of IPAH-PASMCs.Previous studies reported that quinine blocked the KCNK1 and KCNK2 (also KCNK5, 6, 9, 16, and 18) channels (10,28,29), while TPA blocked the KCNK1 and KCNK2 (also KCNK4, 9, 10, 17, and 18) channels (29-32).These blockers were slightly less selective, but still inhibited the activities of KCNK1 and KCNK2 channels.Therefore, the effects of these blockers on the proliferation and migration of IPAH-PASMCs appear to be mediated by the suppression of up-regulated KCNK1 and KCNK2 channels.The results of siRNA knockdown experiments strongly suggest the involvement of KCNK1 and KCNK2 channel activities in the enhanced proliferation and migration of IPAH-PASMCs following membrane hyperpolarization and [Ca 2+ ] cyt increase.The increased activity of K + channels induces membrane hyperpolarization.Since IPAH-PASMCs exhibit a proliferative or synthetic phenotype, Ca 2+ influx is largely mediated by voltageindependent Ca 2+ channels (e.g., ROC and SOC channels), but not by VDCCs (33).Therefore, membrane hyperpolarization due to the up-regulation of KCNK1/KCNK2 channel expression facilitates Ca 2+ influx through ROC and SOC channels in IPAH-PASMCs, similar to that in non-excitable cells, such as epithelial, endothelial, immune, and cancer cells (34).Enhanced Ca 2+ signaling contributes to the facilitated proliferation and migration of IPAH-PASMCs, leading to pulmonary vascular remodeling and the progression of PAH.
Since the activation of JNK signaling, belonging to the MAPK family (11), was identified as one of the mechanisms underlying vascular remodeling in experimental PH animals (12,13,16,17) and PAH patients ( 14), we focused on the JNK signaling pathway in the present study.In addition, KCNK2 channels are necessary for JNK activation in response to pressure overload in cardiomyocytes and fibroblasts, which leads to cardiac remodeling (53).The phosphorylation of JNK was enhanced in IPAH-PASMCs compared to in normal-PASMCs (165%), which is consistent with previous findings (14).The facilitated phosphorylation was markedly suppressed by the knockdown of KCNK1 or KCNK2 channels, suggesting that the activity and/or expression of KCNK1/ KCNK2 channels contribute to the phosphorylation of JNK signaling pathway in IPAH-PASMCs.The up-regulated expression of KCNK1/KCNK2 channels has been suggested to shift the resting membrane potential in a hyperpolarizing direction, enhance Ca 2+ influx, and facilitate Ca 2+ -dependent signaling, including JNK (5).In addition to vascular remodeling, JNK signaling is suggested to contribute to the process of inflammation (11), which is one of the pathological hallmarks of PAH (1).Some KCNK channels (e.g., KCNK2, KCNK3, and KCNK4) have been reported to be associated with inflammatory mechanisms (8,9,35,38,45).Therefore, the increased KCNK1/KCNK2 channels may be also involved in inflammatory processes through JNK signaling pathway in PAH.Further experiments are necessary for elucidating the underlying mechanisms of JNK phosphorylation following the activation of KCNK1 and KCNK2 channels.
Due to the recent development of specific PAH drugs, the fiveyear survival rate of PAH after its diagnosis has increased to 60%-70% in the USA (54), UK, Ireland (55), Spain (56), and France (57).For the treatment of PAH, endothelin receptor antagonists, prostacyclin analogues, a prostaglandin I 2 receptor agonist, phosphodiesterase type 5 inhibitors, and a soluble guanylate cyclase stimulator have been approved (1).Nevertheless, PAH remains incurable and still has a poor prognosis.In the medical management of PAH, monotherapy with an approved drug is used to treat low-risk PAH patients.Since the clinical response is occasionally insufficient, combination therapy using two or three approved drugs with different mechanisms of action is initiated.Combination therapy is also used for intermediate-or high-risk PAH patients (1).Therefore, novel targets for specific PAH drugs are required in therapeutic strategies for PAH (58).KCNK2 channels are expected to become an interactive target for the treatment of depression, cerebral ischemia, general anesthesia, analgesics, ventricular tachycardia, and cancer (35).A recent study reported that treprostinil (prostacyclin analogue), which is used for PAH patients, inhibited KCNK2 channels (59).The effects of treprostinil in PAH patients may be partially mediated by its inhibition of KCNK2 channels.On the other hand, KCNK1 channels may be a molecular target for the treatment of cardiac arrhythmia and cancer (9).The present investigation demonstrated the up-regulated expression of KCNK1/TWIK1 and KCNK2/TREK1 channels in PASMCs from IPAH patients and experimental PH animals, which may be involved in vascular remodeling in PAH.This information provides insights into the underlying mechanisms of PAH and will lead to the development of novel PAH drugs.

FIGURE 2 Up
FIGURE 2 Up-regulated expression of KCNK1 and KCNK2 channels in PASMs from MCT-PH rats.The protein expression of KCNK1 and KCNK2 channels in PASMs from MCT-PH rats was examined by Western blotting and immunohistochemical staining.(A,B) Protein expression of KCNK1 (A) and KCNK2 (B) channels in PASMCs from control and MCT-PH rats (n = 8).Protein expression was normalized to that of β-actin and the control group.(C,D) Representative immunohistochemical images of the lung sections of control and MCT-PH rats stained with a KCNK1 (C; green), KCNK2 (D; green), or α-SMA (red) antibody.Cell nuclei were stained with DAPI (blue).Similar results were obtained from six independent experiments.Note that the expression of KCNK1/ TWIK1 and KCNK2/TREK1 was up-regulated in PASMs from MCT-PH rats.Data are presented as means ± S.E.**p < 0.01, ***p < 0.001 vs. the control (Mann-Whitney U test).

FIGURE 3
FIGURE 3 Expression of KCNK1 and KCNK2 channels in PASMs from SuHx-PH rats and hypoxia-PH mice.The protein expression of KCNK1 and KCNK2 channels in PASMs from SuHx-PH rats and hypoxia-PH mice was examined by Western blotting.(A,B) Protein expression of KCNK1 (A) and KCNK2 (B) channels in PASMs from control and SuHx-PH rats (n = 6).Protein expression was normalized to that of β-actin and the control group.(C,D) Protein expression of KCNK1 (C) and KCNK2 (D) channels in PASMs from normoxia and hypoxia-PH mice (n = 6).Protein expression was normalized to that of β-actin and the normoxia group.Data are presented as means ± S.E.*p < 0.05, **p < 0.01 vs. the control or normoxia group (Mann-Whitney U test).

FIGURE 6 siRNA
FIGURE 6 siRNA knockdown of KCNK1 and KCNK2 channels in IPAH-PASMCs.The effects of KCNK1 and KCNK2 siRNAs on the expression of KCNK1, KCNK2, KCNK3, and KCNK6 channels in IPAH-PASMCs were examined by qPCR and Western blotting.(A,B) Knockdown efficiency at the mRNA level of siRNA targeting KCNK1 (A) or KCNK2 (B) in IPAH-PASMCs (n = 6).mRNA expression was normalized to that of β-actin and control siRNA.(C,D) Knockdown efficiency at the protein level of siRNA targeting KCNK1 (C) or KCNK2 (D) in IPAH-PASMCs (n = 6).Protein expression was normalized to that of β-actin and control siRNA.Data are presented as means ± S.E.**p < 0.01 vs. control siRNA (Mann-Whitney U test).

FIGURE 5
FIGURE 5 Effects of KCNK channel blockers on the migration of IPAH-PASMCs.The effects of the KCNK channel blockers, quinine and TPA, on the migration of IPAH-PASMCs were examined using the Transwell assay.(A) Representative images of migrated PASMCs stained with crystal violet after the treatment with vehicle, 300 μM quinine, or 100 μM TPA for 24 h.(B) Effects of quinine and TPA for 24 h on the migration of IPAH-PASMCs (n = 4).Data are presented as means ± S.E.*p < 0.05 vs. the vehicle (Steel's test following Kruskal-Wallis test).

FIGURE 7
FIGURE 7 Contribution of KCNK1 and KCNK2 channels to the proliferation and migration of IPAH-PASMCs.The involvement of KCNK1 and KCNK2 channels in the proliferation and migration of IPAH-PASMCs was examined by siRNA knockdown methods.(A,B) Inhibitory effects of the transfection with KCNK1 (A) or KCNK2 (B) siRNA for 48 h on the growth of IPAH-PASMCs using the Cell Counting Kit-8 assay (n = 4).Absorbance was normalized by control siRNA.(C,D) Inhibitory effects of the transfection with KCNK1 (C) or KCNK2 (D) siRNA for 48 h on the excessive proliferation of IPAH-PASMCs using the BrdU incorporation assay (n = 4).Absorbance was normalized by control siRNA.(E) Anti-migratory effects of the transfection with KCNK1 or KCNK2 siRNA on the migration of IPAH-PASMCs for 24 h using the Transwell assay (n = 4).Data are presented as means ± S.E.*p < 0.05, **p < 0.001 vs. control siRNA (Student's t-test (A-D) or Steel's test following Kruskal-Wallis test (E)).

FIGURE 9
FIGURE 9 Effects of KCNK1 and KCNK2 channel knockdown on the phosphorylation of JNK in IPAH-PASMCs.The expression and phosphorylation levels of JNK in normal-and IPAH-PASMCs and the effects of the siRNA knockdown of KCNK1 and KCNK2 channels were examined by Western blotting.(A) The phosphorylation levels of JNK in normal-and IPAH-PASMCs (n = 6).Protein expression was normalized to that of β-actin and normal-PASMCs.(B) The effects of the siRNA knockdown of KCNK1 channels on the phosphorylation levels of JNK in IPAH-PASMCs (n = 6).Protein expression was normalized to that of βactin and control siRNA.(C) The effects of the siRNA knockdown of KCNK2 channels on the phosphorylation levels of JNK in IPAH-PASMCs (n = 6).Protein expression was normalized to that of βactin and control siRNA.Data are presented as means ± S.E.**p < 0.01, **p < 0.01 vs. normal-PASMCs or control siRNA (Mann-Whitney U test).