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Downregulation of KCNQ5 expression in the rat pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia
Journal of Pediatric Surgery 52 (2017) 702–705
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Downregulation of KCNQ5 expression in the rat pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia Julia Zimmer a, Toshiaki Takahashi a, Alejandro D. Hofmann b, Prem Puri a,c,⁎ a b c
National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Gate 5, Dublin 12, Dublin, Ireland Department of Pediatric Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany School of Medicine and Medical Science and Conway Institute of Biomedical Research, University College Dublin, Belfield, Dublin 4, Dublin, Ireland
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Article history: Received 3 January 2017 Accepted 23 January 2017 Key words: KCNQ5 Voltage-gated potassium channels Congenital diaphragmatic hernia Nitrofen Pulmonary hypertension
a b s t r a c t Purpose: Pulmonary hypertension (PH) is a common complication of congenital diaphragmatic hernia (CDH). Voltage-gated potassium channels KCNQ1, KCNQ4, and KCNQ5 are expressed by rodent pulmonary artery smooth muscle cells, contributing to their vascular tone. We hypothesized that KCNQ1, KCNQ4, and KCNQ5 expression is altered in the pulmonary vasculature of nitrofen-induced CDH rats. Methods: After ethical approval (REC913b), time-pregnant rats received nitrofen or vehicle on gestational day (D)9. D21 fetuses were divided into CDH and control group (n = 22). QRT-PCR and western blotting were performed to determine gene and protein expression of KCNQ1, KCNQ4, and KCNQ5. Confocal microscopy was used to detect these proteins in the pulmonary vasculature. Results: Relative mRNA level of KCNQ5 (p = 0.025) was significantly downregulated in CDH lungs compared to controls. KCNQ1 (p = 0.052) and KCNQ4 (p = 0.574) expression was not altered. Western blotting confirmed the decreased pulmonary KCNQ5 protein expression in CDH lungs. Confocal-microscopy detected a markedly diminished KCNQ5 expression in pulmonary vasculature of CDH fetuses. Conclusions: Downregulated pulmonary expression of KCNQ5 in CDH lungs suggests that this potassium channel may play an important role in the development of PH in this model. KCNQ5 channel activator drugs may be a potential therapeutic target for the treatment of PH in CDH. Level of evidence: 2b (Centre for Evidence-Based Medicine, Oxford) © 2017 Published by Elsevier Inc.
Pulmonary hypertension (PH) is a common cause of morbidity and mortality in patients with congenital diaphragmatic hernia (CDH). PH is characterized by endothelial dysfunction, apoptosis resistance and increased pulmonary artery smooth muscle cell (PASMC) proliferation resulting in adventitial and medial hyperplasia and an increase in pulmonary vascular resistance [1,2]. Although the development of PH is a multifactorial process, potassium channels (KV) have been highlighted as a contributor for PASMC dysfunction [3]. KV are essential in regulating vascular tone, membrane potential, PASMC proliferation and apoptosis and may therefore represent attractive targets in the treatment of PH [3–5]. PH is associated with a loss of KV expression and activity in human and animal PH models [5–9]. The KCNQ family (potassium voltage-gated channel subfamily Q, also known as KV7 channels) consists of 5 members responsible for specific functions when expressed in different tissues [10]. KCNQ channels have been studied primarily in heart, nervous system and auditory pathway [10] but were subsequently identified in several vascular beds
⁎ Corresponding author at: National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Gate 5, Dublin 12, Ireland. Tel.: +353 1 4096420; fax: +353 1 4550201. E-mail address: [email protected] (P. Puri). http://dx.doi.org/10.1016/j.jpedsurg.2017.01.016 0022-3468/© 2017 Published by Elsevier Inc.
and smooth muscles of animals and humans [11–17]. PASMC have been found to express KCNQ 1, 4 and 5, contributing to their resting membrane potential [11]. In models of pulmonary hypertension KCNQ expression has been reported to be decreased [18,19]. We designed this study to investigate the hypothesis that the expression of KCNQ1, KCNQ4 and KCNQ5 expression is altered in the pulmonary vasculature of nitrofen-induced CDH in rats. 1. Materials and methods 1.1. Animals and drugs The Health Products Regulatory Authority approved the following experimental protocol (REC913b) under the Cruelty to Animals Act 1876 (as amended by European Communities Regulations 2002 and 2005). All animal experiments were carried out according to the current guidelines for management and welfare of laboratory animals. Adult pathogen-free, timed-pregnant, Sprague–Dawley rats (Harlan Laboratories, Shardlow, UK) were randomly divided into two experimental groups (“CDH” and “control”). Observation of spermatozoids in the vaginal smear was considered as proof of pregnancy and was determined as gestational day 0 (D0). The CDH group received 100 mg
J. Zimmer et al. / Journal of Pediatric Surgery 52 (2017) 702–705
of nitrofen (2,4-dichloro-p-nitrophenyl ether, WAKO Chemicals, Osaka, Japan) dissolved in 1 ml of olive oil intragastrally on D9. Control group animals were administered only vehicle. On D21, dams were anesthetized with 2% volatile isoflurane (Piramal Healthcare UK, Morpeth, UK), followed by delivery of the fetuses via caesarean section. A standard protocol was used for harvesting the fetal lungs and preparation for further processing [20].
1.2. RNA isolation from left lungs, cDNA synthesis and quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen, Carlsbad, California, United States) was used for the acid guanidinium-thiocyanate–phenol–chloroform extraction method to isolate total RNA from D21 lungs (n = 22) according to the manufacturer's instructions. Spectrophotometrical quantification of total RNA was performed using a NanoDrop ND-1000 UV–Vis spectrophotometer (Thermo Scientific Fisher, Wilmington, USA). The RNA solution was stored at −20 °C until further use. Reverse transcription of total RNA for cDNA synthesis and qRT-PCR followed a standard protocol as previously described [21] using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics, UK) and LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturers protocol. Following gene-specific primer sequences were used (Eurofins Genomics, Ebersberg, Germany): rat KCNC5 sense primer 5′- ACGGAAGCAGAGTCAGAAGC-3′, rat KCNQ5 anti-sense primer 5′- ACCTACCGATGCTTGTCTGC-3′, rat KCNQ4 sense primer 5″-ACCAGTGTGAGCTTACGGTG-3, rat KCNQ4 antisense primer 5″-GCTTGCAGGCTCTTGATTCG-3′, rat KCNQ1 sense primer 5′-ACCTCATCGTGGTTGTAGCC-3′, rat KCNQ1 anti-sense primer 5′-TCCT GGCGGTGAATGAAGAC-3′, rat GAPDH sense primer 5’ATGACTCTACC CACGGCAAG-3′, rat GAPDH anti-sense primer 5’GATCTCGCTCCTGG AAGATG-3′. Relative changes in gene expression levels of KCNQ5, KCNQ4 and KCNQ1 were normalized against the level of GAPDH gene expression in each sample (DDCT method). Experiments were carried out in duplet for each sample and primer.
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1.4. Immunofluorescence staining and confocal microscopy Fetal left lungs (n = 6 for each group) were fixed with 10% buffered formalin (Santa Cruz Biotechnology), washed overnight in PBS, embedded in OCT Mounting Compound (VWR International, Leuven, Belgium), frozen at − 80 °C, sectioned transversely at a thickness of 10 μm and mounted on SuperFrost Plus slides (VWR International). After washing, sections were permeabilized with 1% Triton X-100 for 20 min at room temperature, washed and blocked with 3% BSA for 30 min to avoid nonspecific absorption of immunoglobulin. Sections were then incubated with primary antibodies against KCNQ5 (rabbit polyclonal, sc-50416, 1:100 dilution in PBST, Santa Cruz Biotechnology, Germany), KCNQ4 (goat polyclonal, sc-20,882, 1:100 dilution in PBST, Santa Cruz Biotechnology, Germany), KCNQ1 (goat polyclonal, sc-10,646, 1:100 dilution in PBST, Santa Cruz Biotechnology, Germany) and alpha smooth muscle actin (α-SMA, mouse monoclonal, M0851, dilution 1:400 in PBST, DAKO Diagnostics Ireland) overnight at 4 °C. After washing, sections were incubated with corresponding secondary antibodies (dilution 1:200 in PBST, anti-rabbit Alexa 647 A-150067, anti-goat Alexa 555 A-150134 and anti-mouse Alexa 488 A-150105, Abcam, UK) for 1 h at room temperature. After washing, sections were counterstained with DAPI antibody (1:1000 in PBST, Roche), washed again and mounted using Sigma Mounting Medium (Sigma-Aldrich, St. Louis, MO, USA).
1.3. Western blot Fresh frozen left lungs were thawed, sonicated and proteins were isolated in lysis buffer containing 25 mM Tris–HCl, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% NP-40, 10% glycerol and 1% protease inhibitor cocktail (Sigma-Aldrich Ireland, Wicklow, Ireland). Protein concentrations were measured using Bradford assays (Sigma-Aldrich, Ireland) and diluted with gel loading buffer Laemmli (Sigma-Aldrich, Ireland) prior to gel loading. Gel electrophoresis for protein separation was performed using precast 10% SDS polyacrylamide gels (NuPAGE NovexBis–Tris gels, Invitrogen) in NuPAGE MES SDS running buffer (Invitrogen). Proteins were transferred to 0.45-lm nitrocellulose membranes (Millipore Corporation, Billerica, USA) by western blotting. Following western blotting, the membranes were blocked in 3% BSA + 0.05% Tween for 60 min or overnight before antibody detection. Primary antibodies against KCNQ5 (rabbit polyclonal, sc-50416, 1:500 dilution in PBST, Santa Cruz Biotechnology, Germany), KCNQ4 (goat polyclonal, sc-20882, 1:500 dilution in PBST, Santa Cruz Biotechnology, Germany) and KCNQ1 (goat polyclonal, sc-10646, 1:500 dilution in PBST, Santa Cruz Biotechnology, Germany) were incubated overnight at 4 °C. After extensive washing, the membranes were incubated with the secondary antibodies in a dilution of 1:5000 (goat anti-rabbit IgG-HRP: #7074S, Cell Signaling Technology, USA and donkey anti-goat IgG HRP SC-2020, Santa Cruz Biotechnology, Germany) followed again by extensive washing. Detection was performed with a PIERCE chemiluminescence kit (Thermo, Fisher Scientific, Dublin, Ireland). GAPDH (Anti-GAPDH antibody, ab9484, Abcam UK) was used to control equal loading and transfer of the samples.
Fig. 1. qRT-PCR detected a significantly decreased relative mRNA expression level of KCNQ5 (p = 0.025) in CDH lungs compared to normal lung tissue. Relative expression levels of KCNQ1 (p = 0.052) and KCNQ4 (p = 0.574) were not found to be altered between both experimental groups.
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J. Zimmer et al. / Journal of Pediatric Surgery 52 (2017) 702–705
2.2. Western blot for KCNQ5, KCNQ4 and KCNQ1 The protein expression of KCNQ5, KCNQ1 and KCNQ4 was analyzed to confirm the qRT-PCR results. Western blot results demonstrated a decreased pulmonary protein expression of KCNQ5 in nitrofenexposed lungs compared to normal lung tissue. Equal loading of electrophoresis gels was confirmed by GAPDH staining of the stripped membranes (Fig. 2). Pulmonary protein expression of KCNQ4 and KCNQ1 was not found to be altered between CDH lungs and controls (data not shown). Fig. 2. Western blot results revealed a decreased expression of KQNQ5 in CDH lungs compared to control lungs. GAPDH was used to control equal loading of electrophoresis gels.
Sections were scanned and evaluated with a ZEISS LSM 700 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) independently by two investigators.
2.3. Immunofluorescence evaluation of KCNQ5, KCNQ4 and KCNQ1 Confocal laser scanning microscopy revealed a markedly diminished KCNQ5 vascular expression co-localized with α-SMA expression in CDH lungs compared to control lung tissue (Fig. 3). No alteration in vascular expression of KCNQ4 and KCNQ1 was detected between both experimental groups (data not shown). 3. Discussion
1.5. Statistical analysis All numerical data are presented as mean ± standard error of the mean. Student's t test was used for evaluation of differences between two normal distributed groups on D21. The confidence interval was set at 95%.
2. Results 2.1. Relative mRNA expression levels of KCNQ5, KCNQ4 and KCNQ1 We observed a significantly decreased relative mRNA expression level of KCNQ5 (p = 0.025) in CDH lungs compared to normal lung tissue using qRT-PCR. Relative expression levels of KCNQ4 (p = 0.574) and KCNQ1 (p = 0.052) were not found to be altered between both experimental groups (Fig. 1).
KV play an essential role in the underlying pathomechanisms for abnormal PASMC development [3,5]. Altered KV expression for selected channels has been previously shown in the lungs of nitrofen-induced CDH model and their down-regulation has been suggested to contribute to vascular remodeling leading to PH in this model [22]. Recently, KCNQ expression by PASMC was discovered in a rat model [11]. The identification of KCNQ gene products in human arteries was not reported until 2010, when Ng et al. demonstrated their presence as well as their contribution to vascular tone in humans [13]. Although the investigated models and vessels vary, the common findings are that KCNQ1, 4 and 5 genes are consistently expressed in smooth muscle cells [23] and have been proven essential for maintaining the smooth muscle tone [17]. Our study provides evidence for the first time, that KCNQs are also altered in the lungs of the nitrofen-induced CDH rat model. We showed not only that KCNQs are detectable in CDH rat lung tissue but also that
Fig. 3. Immunofluorescence evaluation of pulmonary tissue for KCNQ5 and α-SMA. An increased medial and adventitial thickness in pulmonary arteries of all sizes were observed in the CDH lungs compared to control lungs using α-SMA to identify pulmonary arteries. Furthermore, confocal microscopy revealed a markedly decreased pulmonary vascular expression of KCNQ5 co-localized with α-SMA. Scale bar equals 25 μm, original magnification ×63.
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the KCNQ5 channel is significantly downregulated in this CDH model. As the observed alteration was seen at gene and protein level and KCNQ5 expression correlated with α-SMA expression in confocal microscopy we speculate that this decrease in KCNQ 5 expression participates in the progression of vascular remodeling resulting in the development of PH in this model. However, the significant downregulation of KCNQ5 and unaltered expression of KCNQ1 and KCNQ4 in the rat CDH vasculature in our study is in contrast to studies using other models of hypertension, which reported reduced KCNQ4 expression and unaltered KCNQ1 expression [19,24,25], suggesting variations in KCNQ regulation due to the multifactorial pathomechanisms of hypertension. Modulation of KCNQ activity and expression include interaction with calmodulin and phosphatidylinositol (4,5) bisphosphate (PIP2), the β-adrenoceptor pathway and vasopressin [23,24,26–29]. KV channel opener can act as pulmonary vasodilators and may be a potential therapeutic target in PH treatment [6,30]. A problem of KV openers is that they can cause systemic vasodilation due to general activation of KV in the systemic circulation [6]. Therefore, KCNQ channels are under ongoing investigation for the development of pulmonaryselective vasodilators [6,30]. Flupirtine (analgesic), retigabine (anticonvulsant) and the acrylamide S-1 are known KCNQ activators [11,23,30], leading to relaxation of precontracted vessels and hyperpolarisation of membrane potential [23]. Promisingly, flupirtine markedly attenuated PH and right ventricular hypertrophy in two models of arterial PH [30]. Selective KCNQ blockers such as linopirdine (a cognition enhancing drug) and XE991 (linopirdine analogue) have been identified to cause pulmonary vasoconstriction [11,31]. Our study provides a promising approach to conduct further research on the influence of KCNQ channels on CDH related PH in animal models and humans likewise. KCNQ5 channel activator drugs may be a potential therapeutic target for the treatment of PH in CDH. Funding This work was supported and funded by the National Children's Research Centre Dublin [grant number I/15/1]. Conflicts of interest None. References [1] Kool H, Mous D, Tibboel D, et al. Pulmonary vascular development goes awry in congenital lung abnormalities. Birth Defects Res C Embryo Today 2014;102(4): 343–58. http://dx.doi.org/10.1002/bdrc.21085. [2] Guignabert C, Tu L, Le Hiress M, et al. Pathogenesis of pulmonary arterial hypertension: lessons from cancer. Eur Respir Rev 2013;22(130):543–51. http://dx.doi.org/10.1183/ 09059180.00007513. [3] Boucherat O, Chabot S, Antigny F, et al. Potassium channels in pulmonary arterial hypertension. Eur Respir J 2015;46(4):1167–77. http://dx.doi.org/10.1183/13993003. 00798-2015. [4] Park WS, Firth AL, Han J, et al. Patho-, physiological roles of voltage-dependent K+ channels in pulmonary arterial smooth muscle cells. J Smooth Muscle Res 2010; 46(2):89–105. [5] Burg ED, Remillard CV, Yuan JX. Potassium channels in the regulation of pulmonary artery smooth muscle cell proliferation and apoptosis: pharmacotherapeutic implications. Br J Pharmacol 2008;153(Suppl. 1):S99–S111. http://dx.doi.org/10.1038/sj. bjp.0707635. [6] Gurney AM, Joshi S, Manoury B. KCNQ potassium channels: new targets for pulmonary vasodilator drugs? In: Yuan JX-J, Ward JPT, editors. Membrane receptors, channels and transporters in pulmonary circulation. Totowa, NJ: Humana Press; 2010. p. 405–17.
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Participation of KCNQ (Kv7) potassium channels in myogenic control of cerebral arterial diameter. J Physiol 2010;588(Pt 17): 3277–93. http://dx.doi.org/10.1113/jphysiol.2010.192823. [16] Hedegaard ER, Nielsen BD, Kun A, et al. KV 7 channels are involved in hypoxiainduced vasodilatation of porcine coronary arteries. Br J Pharmacol 2014;171(1): 69–82. http://dx.doi.org/10.1111/bph.12424. [17] Greenwood IA, Ohya S. New tricks for old dogs: KCNQ expression and role in smooth muscle. Br J Pharmacol 2009;156(8):1196–203. http://dx.doi.org/10.1111/j.14765381.2009.00131.x. [18] Li SS, Ran YJ, Zhang DD, et al. MicroRNA-190 regulates hypoxic pulmonary vasoconstriction by targeting a voltage-gated K(+) channel in arterial smooth muscle cells. J Cell Biochem 2014;115(6):1196–205. http://dx.doi.org/10.1002/jcb.24771. [19] Sedivy V, Joshi S, Ghaly Y, et al. Role of Kv7 channels in responses of the pulmonary circulation to hypoxia. 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Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Downregulation of KCNQ5 expression in the rat pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia Julia Zimmer a, Toshiaki Takahashi a, Alejandro D. Hofmann b, Prem Puri a,c,⁎ a b c
National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Gate 5, Dublin 12, Dublin, Ireland Department of Pediatric Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany School of Medicine and Medical Science and Conway Institute of Biomedical Research, University College Dublin, Belfield, Dublin 4, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 3 January 2017 Accepted 23 January 2017 Key words: KCNQ5 Voltage-gated potassium channels Congenital diaphragmatic hernia Nitrofen Pulmonary hypertension
a b s t r a c t Purpose: Pulmonary hypertension (PH) is a common complication of congenital diaphragmatic hernia (CDH). Voltage-gated potassium channels KCNQ1, KCNQ4, and KCNQ5 are expressed by rodent pulmonary artery smooth muscle cells, contributing to their vascular tone. We hypothesized that KCNQ1, KCNQ4, and KCNQ5 expression is altered in the pulmonary vasculature of nitrofen-induced CDH rats. Methods: After ethical approval (REC913b), time-pregnant rats received nitrofen or vehicle on gestational day (D)9. D21 fetuses were divided into CDH and control group (n = 22). QRT-PCR and western blotting were performed to determine gene and protein expression of KCNQ1, KCNQ4, and KCNQ5. Confocal microscopy was used to detect these proteins in the pulmonary vasculature. Results: Relative mRNA level of KCNQ5 (p = 0.025) was significantly downregulated in CDH lungs compared to controls. KCNQ1 (p = 0.052) and KCNQ4 (p = 0.574) expression was not altered. Western blotting confirmed the decreased pulmonary KCNQ5 protein expression in CDH lungs. Confocal-microscopy detected a markedly diminished KCNQ5 expression in pulmonary vasculature of CDH fetuses. Conclusions: Downregulated pulmonary expression of KCNQ5 in CDH lungs suggests that this potassium channel may play an important role in the development of PH in this model. KCNQ5 channel activator drugs may be a potential therapeutic target for the treatment of PH in CDH. Level of evidence: 2b (Centre for Evidence-Based Medicine, Oxford) © 2017 Published by Elsevier Inc.
Pulmonary hypertension (PH) is a common cause of morbidity and mortality in patients with congenital diaphragmatic hernia (CDH). PH is characterized by endothelial dysfunction, apoptosis resistance and increased pulmonary artery smooth muscle cell (PASMC) proliferation resulting in adventitial and medial hyperplasia and an increase in pulmonary vascular resistance [1,2]. Although the development of PH is a multifactorial process, potassium channels (KV) have been highlighted as a contributor for PASMC dysfunction [3]. KV are essential in regulating vascular tone, membrane potential, PASMC proliferation and apoptosis and may therefore represent attractive targets in the treatment of PH [3–5]. PH is associated with a loss of KV expression and activity in human and animal PH models [5–9]. The KCNQ family (potassium voltage-gated channel subfamily Q, also known as KV7 channels) consists of 5 members responsible for specific functions when expressed in different tissues [10]. KCNQ channels have been studied primarily in heart, nervous system and auditory pathway [10] but were subsequently identified in several vascular beds
⁎ Corresponding author at: National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Gate 5, Dublin 12, Ireland. Tel.: +353 1 4096420; fax: +353 1 4550201. E-mail address: [email protected] (P. Puri). http://dx.doi.org/10.1016/j.jpedsurg.2017.01.016 0022-3468/© 2017 Published by Elsevier Inc.
and smooth muscles of animals and humans [11–17]. PASMC have been found to express KCNQ 1, 4 and 5, contributing to their resting membrane potential [11]. In models of pulmonary hypertension KCNQ expression has been reported to be decreased [18,19]. We designed this study to investigate the hypothesis that the expression of KCNQ1, KCNQ4 and KCNQ5 expression is altered in the pulmonary vasculature of nitrofen-induced CDH in rats. 1. Materials and methods 1.1. Animals and drugs The Health Products Regulatory Authority approved the following experimental protocol (REC913b) under the Cruelty to Animals Act 1876 (as amended by European Communities Regulations 2002 and 2005). All animal experiments were carried out according to the current guidelines for management and welfare of laboratory animals. Adult pathogen-free, timed-pregnant, Sprague–Dawley rats (Harlan Laboratories, Shardlow, UK) were randomly divided into two experimental groups (“CDH” and “control”). Observation of spermatozoids in the vaginal smear was considered as proof of pregnancy and was determined as gestational day 0 (D0). The CDH group received 100 mg
J. Zimmer et al. / Journal of Pediatric Surgery 52 (2017) 702–705
of nitrofen (2,4-dichloro-p-nitrophenyl ether, WAKO Chemicals, Osaka, Japan) dissolved in 1 ml of olive oil intragastrally on D9. Control group animals were administered only vehicle. On D21, dams were anesthetized with 2% volatile isoflurane (Piramal Healthcare UK, Morpeth, UK), followed by delivery of the fetuses via caesarean section. A standard protocol was used for harvesting the fetal lungs and preparation for further processing [20].
1.2. RNA isolation from left lungs, cDNA synthesis and quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol reagent (Invitrogen, Carlsbad, California, United States) was used for the acid guanidinium-thiocyanate–phenol–chloroform extraction method to isolate total RNA from D21 lungs (n = 22) according to the manufacturer's instructions. Spectrophotometrical quantification of total RNA was performed using a NanoDrop ND-1000 UV–Vis spectrophotometer (Thermo Scientific Fisher, Wilmington, USA). The RNA solution was stored at −20 °C until further use. Reverse transcription of total RNA for cDNA synthesis and qRT-PCR followed a standard protocol as previously described [21] using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics, UK) and LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturers protocol. Following gene-specific primer sequences were used (Eurofins Genomics, Ebersberg, Germany): rat KCNC5 sense primer 5′- ACGGAAGCAGAGTCAGAAGC-3′, rat KCNQ5 anti-sense primer 5′- ACCTACCGATGCTTGTCTGC-3′, rat KCNQ4 sense primer 5″-ACCAGTGTGAGCTTACGGTG-3, rat KCNQ4 antisense primer 5″-GCTTGCAGGCTCTTGATTCG-3′, rat KCNQ1 sense primer 5′-ACCTCATCGTGGTTGTAGCC-3′, rat KCNQ1 anti-sense primer 5′-TCCT GGCGGTGAATGAAGAC-3′, rat GAPDH sense primer 5’ATGACTCTACC CACGGCAAG-3′, rat GAPDH anti-sense primer 5’GATCTCGCTCCTGG AAGATG-3′. Relative changes in gene expression levels of KCNQ5, KCNQ4 and KCNQ1 were normalized against the level of GAPDH gene expression in each sample (DDCT method). Experiments were carried out in duplet for each sample and primer.
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1.4. Immunofluorescence staining and confocal microscopy Fetal left lungs (n = 6 for each group) were fixed with 10% buffered formalin (Santa Cruz Biotechnology), washed overnight in PBS, embedded in OCT Mounting Compound (VWR International, Leuven, Belgium), frozen at − 80 °C, sectioned transversely at a thickness of 10 μm and mounted on SuperFrost Plus slides (VWR International). After washing, sections were permeabilized with 1% Triton X-100 for 20 min at room temperature, washed and blocked with 3% BSA for 30 min to avoid nonspecific absorption of immunoglobulin. Sections were then incubated with primary antibodies against KCNQ5 (rabbit polyclonal, sc-50416, 1:100 dilution in PBST, Santa Cruz Biotechnology, Germany), KCNQ4 (goat polyclonal, sc-20,882, 1:100 dilution in PBST, Santa Cruz Biotechnology, Germany), KCNQ1 (goat polyclonal, sc-10,646, 1:100 dilution in PBST, Santa Cruz Biotechnology, Germany) and alpha smooth muscle actin (α-SMA, mouse monoclonal, M0851, dilution 1:400 in PBST, DAKO Diagnostics Ireland) overnight at 4 °C. After washing, sections were incubated with corresponding secondary antibodies (dilution 1:200 in PBST, anti-rabbit Alexa 647 A-150067, anti-goat Alexa 555 A-150134 and anti-mouse Alexa 488 A-150105, Abcam, UK) for 1 h at room temperature. After washing, sections were counterstained with DAPI antibody (1:1000 in PBST, Roche), washed again and mounted using Sigma Mounting Medium (Sigma-Aldrich, St. Louis, MO, USA).
1.3. Western blot Fresh frozen left lungs were thawed, sonicated and proteins were isolated in lysis buffer containing 25 mM Tris–HCl, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% NP-40, 10% glycerol and 1% protease inhibitor cocktail (Sigma-Aldrich Ireland, Wicklow, Ireland). Protein concentrations were measured using Bradford assays (Sigma-Aldrich, Ireland) and diluted with gel loading buffer Laemmli (Sigma-Aldrich, Ireland) prior to gel loading. Gel electrophoresis for protein separation was performed using precast 10% SDS polyacrylamide gels (NuPAGE NovexBis–Tris gels, Invitrogen) in NuPAGE MES SDS running buffer (Invitrogen). Proteins were transferred to 0.45-lm nitrocellulose membranes (Millipore Corporation, Billerica, USA) by western blotting. Following western blotting, the membranes were blocked in 3% BSA + 0.05% Tween for 60 min or overnight before antibody detection. Primary antibodies against KCNQ5 (rabbit polyclonal, sc-50416, 1:500 dilution in PBST, Santa Cruz Biotechnology, Germany), KCNQ4 (goat polyclonal, sc-20882, 1:500 dilution in PBST, Santa Cruz Biotechnology, Germany) and KCNQ1 (goat polyclonal, sc-10646, 1:500 dilution in PBST, Santa Cruz Biotechnology, Germany) were incubated overnight at 4 °C. After extensive washing, the membranes were incubated with the secondary antibodies in a dilution of 1:5000 (goat anti-rabbit IgG-HRP: #7074S, Cell Signaling Technology, USA and donkey anti-goat IgG HRP SC-2020, Santa Cruz Biotechnology, Germany) followed again by extensive washing. Detection was performed with a PIERCE chemiluminescence kit (Thermo, Fisher Scientific, Dublin, Ireland). GAPDH (Anti-GAPDH antibody, ab9484, Abcam UK) was used to control equal loading and transfer of the samples.
Fig. 1. qRT-PCR detected a significantly decreased relative mRNA expression level of KCNQ5 (p = 0.025) in CDH lungs compared to normal lung tissue. Relative expression levels of KCNQ1 (p = 0.052) and KCNQ4 (p = 0.574) were not found to be altered between both experimental groups.
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2.2. Western blot for KCNQ5, KCNQ4 and KCNQ1 The protein expression of KCNQ5, KCNQ1 and KCNQ4 was analyzed to confirm the qRT-PCR results. Western blot results demonstrated a decreased pulmonary protein expression of KCNQ5 in nitrofenexposed lungs compared to normal lung tissue. Equal loading of electrophoresis gels was confirmed by GAPDH staining of the stripped membranes (Fig. 2). Pulmonary protein expression of KCNQ4 and KCNQ1 was not found to be altered between CDH lungs and controls (data not shown). Fig. 2. Western blot results revealed a decreased expression of KQNQ5 in CDH lungs compared to control lungs. GAPDH was used to control equal loading of electrophoresis gels.
Sections were scanned and evaluated with a ZEISS LSM 700 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) independently by two investigators.
2.3. Immunofluorescence evaluation of KCNQ5, KCNQ4 and KCNQ1 Confocal laser scanning microscopy revealed a markedly diminished KCNQ5 vascular expression co-localized with α-SMA expression in CDH lungs compared to control lung tissue (Fig. 3). No alteration in vascular expression of KCNQ4 and KCNQ1 was detected between both experimental groups (data not shown). 3. Discussion
1.5. Statistical analysis All numerical data are presented as mean ± standard error of the mean. Student's t test was used for evaluation of differences between two normal distributed groups on D21. The confidence interval was set at 95%.
2. Results 2.1. Relative mRNA expression levels of KCNQ5, KCNQ4 and KCNQ1 We observed a significantly decreased relative mRNA expression level of KCNQ5 (p = 0.025) in CDH lungs compared to normal lung tissue using qRT-PCR. Relative expression levels of KCNQ4 (p = 0.574) and KCNQ1 (p = 0.052) were not found to be altered between both experimental groups (Fig. 1).
KV play an essential role in the underlying pathomechanisms for abnormal PASMC development [3,5]. Altered KV expression for selected channels has been previously shown in the lungs of nitrofen-induced CDH model and their down-regulation has been suggested to contribute to vascular remodeling leading to PH in this model [22]. Recently, KCNQ expression by PASMC was discovered in a rat model [11]. The identification of KCNQ gene products in human arteries was not reported until 2010, when Ng et al. demonstrated their presence as well as their contribution to vascular tone in humans [13]. Although the investigated models and vessels vary, the common findings are that KCNQ1, 4 and 5 genes are consistently expressed in smooth muscle cells [23] and have been proven essential for maintaining the smooth muscle tone [17]. Our study provides evidence for the first time, that KCNQs are also altered in the lungs of the nitrofen-induced CDH rat model. We showed not only that KCNQs are detectable in CDH rat lung tissue but also that
Fig. 3. Immunofluorescence evaluation of pulmonary tissue for KCNQ5 and α-SMA. An increased medial and adventitial thickness in pulmonary arteries of all sizes were observed in the CDH lungs compared to control lungs using α-SMA to identify pulmonary arteries. Furthermore, confocal microscopy revealed a markedly decreased pulmonary vascular expression of KCNQ5 co-localized with α-SMA. Scale bar equals 25 μm, original magnification ×63.
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the KCNQ5 channel is significantly downregulated in this CDH model. As the observed alteration was seen at gene and protein level and KCNQ5 expression correlated with α-SMA expression in confocal microscopy we speculate that this decrease in KCNQ 5 expression participates in the progression of vascular remodeling resulting in the development of PH in this model. However, the significant downregulation of KCNQ5 and unaltered expression of KCNQ1 and KCNQ4 in the rat CDH vasculature in our study is in contrast to studies using other models of hypertension, which reported reduced KCNQ4 expression and unaltered KCNQ1 expression [19,24,25], suggesting variations in KCNQ regulation due to the multifactorial pathomechanisms of hypertension. Modulation of KCNQ activity and expression include interaction with calmodulin and phosphatidylinositol (4,5) bisphosphate (PIP2), the β-adrenoceptor pathway and vasopressin [23,24,26–29]. KV channel opener can act as pulmonary vasodilators and may be a potential therapeutic target in PH treatment [6,30]. A problem of KV openers is that they can cause systemic vasodilation due to general activation of KV in the systemic circulation [6]. Therefore, KCNQ channels are under ongoing investigation for the development of pulmonaryselective vasodilators [6,30]. Flupirtine (analgesic), retigabine (anticonvulsant) and the acrylamide S-1 are known KCNQ activators [11,23,30], leading to relaxation of precontracted vessels and hyperpolarisation of membrane potential [23]. Promisingly, flupirtine markedly attenuated PH and right ventricular hypertrophy in two models of arterial PH [30]. Selective KCNQ blockers such as linopirdine (a cognition enhancing drug) and XE991 (linopirdine analogue) have been identified to cause pulmonary vasoconstriction [11,31]. Our study provides a promising approach to conduct further research on the influence of KCNQ channels on CDH related PH in animal models and humans likewise. KCNQ5 channel activator drugs may be a potential therapeutic target for the treatment of PH in CDH. Funding This work was supported and funded by the National Children's Research Centre Dublin [grant number I/15/1]. Conflicts of interest None. References [1] Kool H, Mous D, Tibboel D, et al. Pulmonary vascular development goes awry in congenital lung abnormalities. Birth Defects Res C Embryo Today 2014;102(4): 343–58. http://dx.doi.org/10.1002/bdrc.21085. [2] Guignabert C, Tu L, Le Hiress M, et al. Pathogenesis of pulmonary arterial hypertension: lessons from cancer. Eur Respir Rev 2013;22(130):543–51. http://dx.doi.org/10.1183/ 09059180.00007513. [3] Boucherat O, Chabot S, Antigny F, et al. Potassium channels in pulmonary arterial hypertension. Eur Respir J 2015;46(4):1167–77. http://dx.doi.org/10.1183/13993003. 00798-2015. [4] Park WS, Firth AL, Han J, et al. Patho-, physiological roles of voltage-dependent K+ channels in pulmonary arterial smooth muscle cells. J Smooth Muscle Res 2010; 46(2):89–105. [5] Burg ED, Remillard CV, Yuan JX. Potassium channels in the regulation of pulmonary artery smooth muscle cell proliferation and apoptosis: pharmacotherapeutic implications. Br J Pharmacol 2008;153(Suppl. 1):S99–S111. http://dx.doi.org/10.1038/sj. bjp.0707635. [6] Gurney AM, Joshi S, Manoury B. KCNQ potassium channels: new targets for pulmonary vasodilator drugs? In: Yuan JX-J, Ward JPT, editors. Membrane receptors, channels and transporters in pulmonary circulation. Totowa, NJ: Humana Press; 2010. p. 405–17.
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