Paul JW, et al., J Hum Endocrinol 2017, 2: 008 DOI: 10.24966/HHE-9640/10008 HSOA Journal of Human Endocrinology Research Article Conclusion Expression of KCNH2 KCNH2 and KCNE2 expression correlate with the expression of key myometrial genes implicated in parturition, further strengthening a role for K 11.1 channels in human pregnancy. Neither gene was (hERG1) and KCNE2 Cor- v correlated with BMI, suggesting that previously reported effects of relates With Expression of Key obesity likely affect protein levels rather than gene expression. Keywords: ESR1; hERG; KCNE2; KCNH2; Key myometrial genes; Myometrial Genes in Term Myometrium; OXTR; PGR; PTGS2 Pregnant Human Myometrium Introduction Jonathan W Paul1,2,3*†, Marina Ilicic1,2,3†, Tamas Zakar1,2,3,4 and A number of studies have demonstrated that ion channels encoded Roger Smith1,2,3,5 by the ether-à-go-go-related gene 1 (ERG1) play a role in regulating 1School of Medicine and Public Health, Faculty of Health and Medicine, the contractile activity of cardiac myocytes (reviewed by Vandenberg University of Newcastle, NSW, Australia et al. [1]). Moreover, there is increasing evidence demonstrating a role 2Priority Research Centre for Reproductive Science, University of Newcas- for ERG1 in regulating smooth muscle cell contractility. ERG1 en- tle, NSW, Australia codes the pore-forming α-subunit of the delayed rectifier voltage-gat- 3 Hunter Medical Research Institute, NSW, Australia ed potassium channel, Kv11.1. Herein we refer to the ERG gene and 4Department of Obstetrics and Gynaecology, John Hunter Hospital, NSW, the cognate mRNA as ‘KCNH2’, and ‘KCNH2 mRNA’, respectively, to Australia the channel protein as ERG and to the functional channel as Kv11.1. 5 Department of Endocrinology, John Hunter Hospital, NSW, Australia In addition to ERG, Kv11.1 channels contain regulatory β-subunits †Co-first authors: These authors contributed to the work equally [2,3], such as the single transmembrane domain protein potassium voltage-gated channel subfamily E members 2, encoded by the gene Abstract KCNE2 (mRNA referred to as ‘KCNE2 mRNA’, and channel protein referred to as KCNE2). Background K 11.1 channels are activated following action potentials and func- Loss of activity of Kv11.1 potassium channels (encoded by v KCNH2) facilitates labor, and is associated with expression of an tion to repolarize the cell membrane by conducting potassium ions inhibitory subunit encoded by KCNE2. (K+) out of the cell, which constitutes the rapid component of the de- Objective layed rectifier current IKr [4,5]. Repolarization terminates the action To determine whether KCNH2 and KCNE2 expression was linked potential and the associated contraction. to expression of key genes involved in myometrial contractility, in- K 11.1 channels have been shown in rat stomach and murine por- cluding the oxytocin receptor (OXTR), estrogen receptor 1 (ESR1), v tal vein [6-8], as well as in opossum oesophagus [9]. In addition, selec- progesterone receptor (PGR) and prostaglandin-endoperoxidase synthase 2 (PTGS2). We further aimed to examine KCNH2 and tive Kv11.1 channel blockers have been shown to increase contractility KCNE2 expression in preterm samples. in rat stomach [6], mouse portal vein [8], opossum oesophagus [9], Study Design mouse and guinea pig gall bladder [10], bovine epididymis [11] and human and equine jejunum [12,13]. These smooth muscles all exhibit Biopsies of term or preterm, non-laboring human myometrium were analysed by qPCR to determine KCNH2 and KCNE2 mRNA spontaneous contractile activity, which is also a property of uterine abundance, as well as abundance of OXTR, ESR1, PGR and smooth muscle [14]. PTGS2 mRNAs. Kv11.1 channels were initially linked to myometrial contractili- Results ty by Aaronson et al., who demonstrated that tetraethylammonium KCNH2 and KCNE2 expression were significantly correlated at (TEA)- and 4-aminopyridine (4-AP)-sensitive voltage-dependent term, but not correlated with BMI. KCNH2 expression significantly + K (Kv) channels played a role in regulating action potential duration correlated with OXTR, ESR1, PGR and PTGS2 expression, while in rat myometrium [15]. Greenwood et al. later examined mouse ERG KCNE2 expression correlated with the expression of ESR1, PGR (mERG) expression and function, and confirmed the presence of both and PTGS2. Preterm samples revealed no difference in KCNH2 and KCNE2 expression compared to term. the ERG1a and ERG1b splice variants, with ERG1a expression being more abundant than that of ERG1b [16]. They found that KCNH2 *Corresponding author: Jonathan W Paul, Hunter Medical Research Insti- mRNA abundance did not change throughout gestation or with the tute, 1 Kookaburra Circuit, New Lambton Heights 2305, NSW, Australia, Tel: onset of labor, however, mRNAs encoding auxiliary subunits, namely +6140420348; Fax: +61 4042 0045; E-mail: [email protected] KCNE2, were significantly up-regulated approaching term [16]. Citation: Paul JW, Ilicic M, Zakar T, Smith R (2017) Expression of KCNH2 More recently, our group reported that human ERG1 (hERG1) (hERG1) and KCNE2 correlates with expression of key myometrial genes in and KCNE2 were present in pregnant human myometrium during term pregnant human myometrium. J Hum Endocrinol 2: 008. late gestation and labor [17]. We found that Kv11.1 activity supressed Received: May 03, 2016; Accepted: June 05, 2017; Published: June 19, 2017 the amplitude and duration of contractions prior to labor, thereby Citation: Paul JW, Ilicic M, Zakar T, Smith R (2017) Expression of KCNH2 (hERG1) and KCNE2 correlates with expression of key myometrial genes in term pregnant human myometrium. J Hum Endocrinol 2: 008. • Page 2 of 7 • supporting a role for Kv11.1 activity in helping to maintain uterine Myometrial tissue acquisition quiescence [17]. Previous reports have indicated that KCNE2 co-ex- These studies were approved by the Hunter and New England Area pression with hERG1 was not necessarily inhibitory of Kv11.1 activity [18], however, we found that the onset of labor was associated with the Human Research Ethics Committee and the University of Newcastle increased expression of KCNE2 and the decreased responsiveness of Human Ethics Committee (02/06/12/3.13). All participants gave in- formed written consent. Human myometrial samples (5 × 5 × 10 mm) myocytes to the Kv11.1 inhibitor, dofetilide [17]. Together these find- ings support an inhibitory role for KCNE2 in the context of regulating were obtained from the lower uterine segment during elective CS of hERG1 in the myometrium. The evidence also suggests that vK 11.1 singleton pregnancies. Preterm samples ranged from 31 - 34 weeks activity contributes to the electrophysiological mechanisms that reg- gestation while term samples were 38.2 - 39.6 weeks gestation. Patient ulate uterine contractions and that inhibition of the α-subunit, ERG1, BMI range was 18.3 - 38.0, and all patients were not-in-labor. The in- by the β-subunit, KCNE2, may facilitate labor [17]. Interestingly, the dications for elective not-in-labor term CS were previous CS, placenta mechanism for reducing K 11.1 activity to facilitate labor appears to v praevia, fetal distress or breach presentation. The indications for elec- be dysregulated in obese women. Maternal obesity is associated with tive not-in-labor preterm CS were placenta increta, pre-eclampsia and increased rates of labor induction, dysfunctional labor requiring Cae- sarean section (CS) delivery, longer pregnancies, as well as postpar- low levels of amniotic fluid. Following delivery of the placenta, 5 units tum haemorrhage [19-21]. We reported that high body mass index of syntocinon were administrated directly into an intravenous line as (BMI) was associated with increased levels of hERG1 and reduced lev- part of standard care for the prevention of postpartum hemorrhage. els of KCNE2 in the myometrium [17]. Furthermore, high BMI was Samples were therefore exposed to oxytocin for a brief period of time associated with heightened Kv11.1 activity in vitro, suggesting that the (3 min). All myometrial samples were placed on ice in a serum-free delayed and protracted labor often observed in obese women is linked media containing DMEM with high glucose, 2 mM L-glutamine, 1 to elevated Kv11.1 activity in the myometrium [17]. mM sodium pyruvate, 40 µg/mL gentamicin and 10 mM HEPES for Genes encoding the progesterone receptor (PGR) [22-24], estro- the transfer to the laboratory. Myometrial tissues were then cleared gen receptor 1 (ESR1) [23,25], oxytocin receptor (OXTR) [24,26,27] of serosa, fibrous or damaged tissue and visible blood vessels before and prostaglandin-endoperoxidase synthase 2 (PTGS2) [24,28] have being dissected into smaller pieces and washed in serum-free media to been identified as key genes involved human parturition. There is now remove excess blood. Approximately 100 mg of tissue was snap frozen extensive published literature linking the myometrial regulation of in liquid nitrogen for subsequent analysis. these genes, among others, to the maintenance of uterine quiescence and the transition to a contractile phenotype at labor [29-31]. Uncov- RNA extraction, Reverse transcription and Real-time quan- ering an association between expression of these key genes and genes titative PCR encoding the Kv11.1 channel would ascertain the involvement of this channel in the increased contractility of the myometrium at term and RNA was extracted from 100 mg of tissue using TRizol Reagent as part of the myometrial transformation leading to labor.