KV7 channel activation inhibits human and murine myometrium contractility and delays delivery in a mouse model of preterm birth

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We hypothesized that uterine smooth muscle (myometrium) Kv7 channels could be a therapeutic target for preventing preterm labor. Building on our previous work, we confirmed that K V 7 channels, proteins encoded by KCNQ2-5 genes and associated accessory KCNE1-5, are expressed and functional in pregnant human myometrium prior to and after the onset of labor. K V 7.2-5 activators (retigabine and ML213) effectively inhibit pregnant human and mouse myometrium contractions in vitro , and in vivo significantly delayed PTB in a non-infection preterm labor mouse model. This supports our hypothesis that augmenting K V 7 activity represents a viable mechanism to suppress uterine contractility and delay of PTB. Addressing the current drive to repurpose existing drugs for treating PTB, we propose this as a new avenue of clinical exploration. One Sentence Summary : K V 7 channel activators inhibit human uterine contractions and delay delivery in preterm mice, identifying a new target for preterm labor prevention. Biological sciences/Physiology/Reproductive biology Health sciences/Pathogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Preterm birth (< 37 weeks’ gestation) affects approximately 15 million pregnancies worldwide every year and results in unacceptable levels of neonatal death and morbidity 1 . The resultant social, economic, and healthcare burden for surviving babies is considerable 2 . A major cause of preterm birth is the spontaneous onset of preterm labor, typified by inappropriate early onset of uterine contractions and premature cervical shortening. Due to an incomplete understanding of the mechanisms driving preterm labor and the lack of effective prophylactic interventions, many women still present in threatened preterm labor and require intervention 3 – 5 . Most will receive, when not contraindicated due to the presence of overt infection, tocolytic therapy to delay labor and corticosteroids to improve neonatal survival and morbidity 5 – 7 . However, there are few tocolytic therapies available e.g., nifedipine and atosiban (a mixed oxytocin/vasopressin receptor antagonist), but the effectiveness of these agents in terms of delaying birth and impact on neonatal outcomes is limited 5 , 8 . As a result, there is a clinical need to identify new uterine smooth muscle (myometrium) targets to develop more effective tocolytic agents. Plasma membrane potassium (K + ) channels are particularly apposite for targeting, as K + channel expression and function underpin uterine quiescence in pregnancy and contribute to the generation of rhythmic contractions in labor 9 . Gene expression of several K V channel sub-types, and related KCNE ancillary units, have been identified in human and rodent uterine myocytes including K V 7 and K V 11 9,10 . We have demonstrated that myometrial K V 7 channels encoded by KCNQ1-5 genes are expressed robustly throughout mouse gestation. In addition, activators of K V 7.2 to K V 7.5, such as retigabine, inhibited myometrial contractions in mouse and human myometrium 11 , 12 . Considering this functional impact, we hypothesize that K V 7 channels have potential as a novel therapeutic target to delay preterm birth. Consequently, the aim of this study was to establish which K V 7 channels are present in human myometrium from pregnant women after the onset of labor, and to use a mouse model of preterm birth to provide ‘proof of principle’ data that targeting K V 7 in vivo can delay preterm delivery. RESULTS K V 7 channel expression in late pregnant human myometrium For K V 7 channel activators to be considered as a potential treatment of preterm labor, KCNQ and KCNE mRNA and associated proteins must be expressed and active in pregnant human myometrium and remain in tissues after labor onset. Using myometrium from women at the time of elective cesarean section (term not in labor: TNL) (Fig. 1 A, n = 51), mRNA expression of all KCNQ isoforms except KCNQ2 were detected. Similar to our previous mouse studies, KCNQ4 was the most highly expressed mRNA compared to KCNQ1,3,5 (p > KCNQ3 > KCNQ1 > KCNQ5; Kruskal-Wallis tests confirmed significant differences between the individual KCNQ isoforms (Fig. 1 A). Whilst there was a relatively wide spread of KCNQ4 expression values, individual profiles of KCNQ expression were remarkably similar, with KCNQ4 being the predominant isoform in 42 out of 51 tissues (and second highest in the remaining 9 samples). We also assessed KCNQ expression in myometrium from women at term in active labor (TAL) (Fig. 1 B; n = 46). KCNQ profiles in TAL were similar to those found in TNL (KCNQ4 > KCNQ3 > KCNQ1 > KCNQ5), with KCNQ4 remaining the most predominant transcript, providing evidence that at the mRNA level, K V 7 channel components are still detectable, and hence available for pharmacological targeting, in labor. Accessory subunits KCNE1-5 were analyzed in the same TNL and TAL samples (Fig. 1 C, D). All KCNE genes were detected in both TNL and TAL samples, with KCNE4 being the most highly expressed isoform in both groups. The expression profile of KCNE genes was the same for both groups (TNL and TAL, KCNE4 > KCNE3 > KCNE5 = KCNE1 > KCNE2). KCNE2 expression was particularly low compared with other KCNE isoforms. KCNE3 showed a slightly lower expression in TNL [median, quartile range KCNE3: TNL: 737.3 (286.0, 1166) vs TAL: 915.0 (502.5, 2708). In a separate experiment, using mRNA from a small set of myometrial tissues (provided by author D Slater), we compared expression from women who had Cesarean sections from preterm deliveries not in labor (PNL, n = 4) and after spontaneous onset of preterm labor (PAL, n = 5, < 37 weeks of gestation pregnancy), with TNL (n = 6) and TAL (n = 6) controls. We focused on the most highly expressed KCNQ and KCNE genes identified in Fig. 1 . Both KCNQ4 and KCNE4 mRNA (Fig. 2 A and 2 B respectively) were detected in preterm tissues. Expression of KCNQ4 was high in PAL as it was in TNL and TAL samples. KCNQ4 expression in the PNL group was lower than that in TNL women (P < 0.05). A similar profile of mRNA expression was seen for KCNE4 and there were no significant differences between the groups. Overall, these data shown in human myometrium that KCNQ4 tissue expression remains consistent across term and preterm (in active labour) gestations. Kv7.1-5 and KCNE1-5 proteins in human myometrium Following the successful detection of KCNQ1, 3–5 and KCNE1-5 transcripts in human myometrium, we determined whether transcripts were associated with protein expression in human myometrium using Western blot and commercially available antibodies Kv7.1-5/KCNE1-5 proteins 12 – 19 . We were able to detect Kv7.1, Kv7.3, Kv7.4, KCNE2, KCNE3 and KCNE4 proteins. Kv7.2, Kv7.5, KCNE1 and KCNE5 were not detected (Fig. 3 A). Of the two KCNE2 antibodies, only one antibody (APC-054, Alomone; epitope – 88–107 aa, rat origin) detected a faint protein band (Fig. 3 A). APC-054 appears to detect the fully glycosylated protein, whereas the second antibody (sc-25703, Santa Cruz, epitope – 1–70 aa, human origin) detects the semi-mature monoglycosylated protein. Focusing on K V 7.4, we quantified expression in whole tissue lysates from human myometrium in TNL and TAL samples. Figure 3 B demonstrates that protein expression was similar in both groups. Immunohistochemistry provided further confirmation of the presence and localization of K V 7.1, K V 7.3, K V 7.4, KCNE3 and KCNE4 proteins in pregnant human myometrium smooth muscle (Fig. 4 ). All were expressed in smooth muscle cells and widely distributed across cells. Semi-quantification of the staining indicated that K V 7.3 was slightly increased in TAL v TNL samples and K V 7.4 reduced (Fig. 4 B and C). Both proteins were present in laboring samples suggesting functional availability. Pharmacological evidence for functional K7 channels in human myometrium Evidence for functional K V 7.2-5 channels was gained through a series of in vitro protocols which assessed the impact of pharmacological K V 7 activators and inhibitors on TNL human myometrium contractility (Fig. 5 ). Application of the K V 7.2-5 activator retigabine (10 µM and 20 µM) to spontaneously contracting pregnant human myometrium (TNL) resulted in a reduction in mean integral tension (MIT) compared to vehicle time controls (dimethylsulfoxide, DMSO; Fig. 5 A) providing evidence of functional K V 7.2-5 channels in these tissues. ML213, a K V 7.2/K V 7.4 and K V 7.5 activator caused a reduction in MIT at doses 5, 10 and 20 µM, indicating it is an effective uterine relaxant (Fig. 5 B). There was no significant effect of chromanol 293B (0.5–20 µM), an inhibitor of K V 7.1/KCNE1, on spontaneously contracting myometrium (Fig. 5 C) indicating a limited contribution from this channel in human myometrium consistent with our previous mouse myometrium data 11 . In contrast, the pan Kv7 blocker XE991, at 10 and 20 µM had no significant effect on mean integral tension but significantly enhanced contractile frequency (Fig. 5 D-E) and decreased contraction amplitude (Fig. 5 F) compared to vehicle control in TNL pregnant human myometrium. This is consistent with XE991 causing a small depolarization of the resting membrane potential due to inhibition of K V 7 channels. These data indicate that channels formed by K V 7.2-7.5 are the major regulators of contractility in term pregnant human myometrium, and that modulators reported to have greater specificity for K V 7.4 have greatest effect. K V 7.1 has a limited functional contribution to contraction and an explanation for this may be that K V 7.1 is forming channels with KCNE4 (which is the most abundant isoform) in myometrium; this combination is reported to suppress K V 7.1 currents in model systems 20 , 21 . K7 channels in myometrium from a mouse model of preterm birth We have previously demonstrated the presence of functional of K V 7 channels in non-pregnant and pregnant mouse myometrium (day 6–7 and term) and in myometrium from mice injected with lipopolysaccharide (day 15, preterm gestation, but not in labor) 11 . However, there is limited knowledge of expression or function of myometrial K V 7 channels related to preterm labor. To rectify this, Kcnq and Kcne profiles were assessed in myometrium harvested from a preterm mouse model (RU486 treated on day 15 of gestation to induce progesterone withdrawal and preterm labor and birth on ~ day 16) and vehicle (DMSO) treated control mice (Suppl. Figure 1). Myometrium was obtained from mice when they showed signs of labor (PTAL) and from control non-laboring pregnant mice on day 16 (14 hours after DMSO treatment, PTNL) for experiments shown in Figs. 6 and 7 Using myometrium harvested on day 16 of pregnancy, transcripts for all Kcnq genes were detected in both PTNL and PTAL myometrium (Fig. 6 A and B). Kcnq1 , Kcnq4 and Kcnq5 were highly expressed. A separate comparison individual Kcnq genes expression data from PTNL versus PTAL myometrium revealed decreases in Kcnq4 (median [IQR]; PTNL, 139.5 [114.6, 259.7]; PTAL, 83.6 [56.7, 106.2], p = 0.02) and Kcnq5 (PTNL, 630.8 [443.3, 1025.0]; PTAL, 113.2 [104.6, 351.1], p = 0.01) mRNA expression after the onset of preterm labor. Although Kcnq1 showed a t reduction in PTAL, this did not reach significance (p = 0.065). No differences were observed for Kcnq2 or Kcnq3 . All Kcne genes were detected in PTNL (Fig. 6 C) and PTAL (Fig. 6 D) samples with Kcne4 being the most highly abundant in both groups (p = 0.0001 for both). Comparison of Kcne gene expression data from PTNL versus PTAL myometrium showed significant reductions in Kcne2 (median [IQR]; PTNL, 18040 [16183, 226228]; PTAL, 319.3 [231.4, 471.4]; p = 0.002) and Kcne1 expression (PTNL, 27.7 [13.1, 35.8]; PTAL, 5.4 [2.8, 8.6]; p = 0.02). Contribution of K V 7 channels to regulating ex vivo myometrium contractile activity in a mouse model of preterm birth After characterizing the expression of Kcnq and Kcne genes, the functional expression of these channels was assessed using two K V 7 activators, retigabine and ML213. Figure 7 shows baseline spontaneous contractility in vitro in PTNL and PTL mice (n = 6 for each concentration). Application of retigabine to spontaneously contracting myometrium from PTAL mice significantly decreased MIT by more than 60% at all concentrations tested (mean ± SEM; DMSO, 79.2 ± 4.7%; retigabine 5 µM, 32.7 ± 4.5%; 10 µM, 28 ± 7.4%; 20 µM, 32.6 ± 8%; p < 0.05 for all as indicated by Dunn’s multiple comparison post hoc test performed after Kruskal-Wallis test; Fig. 7 B). In PTNL samples, significant decreases in MIT were only observed with 10 and 20 µM (DMSO, 91.2 ± 2.7%; retigabine 10 µM, 41.4 ± 10.6%; 20 µM, 16.9 ± 6.1%; Fig. 7 A). Retigabine caused complete abolition of contractions in 45% and 50% of PTNL and PTAL samples, respectively. Similar to retigabine, ML213 inhibited contractile activity of PTAL myometrium at all concentrations (DMSO: 75.8 ± 4.5%; ML213 5 µM, 11.9 ± 3.3%; 10 µM: 10.4 ± 2.8%; 20 µM: 11.4 ± 5%; p < 0.05 for all; Fig. 7 D). In PTNL myometrium, a significant relaxatory effect was only observed with 10 and 20 µM (DMSO: 94.5 ± 2.1%; ML213 10 µM: 11.9 ± 2.1%; 20 µM: 11.9 ± 3.6%; p < 0.05 for both; Fig. 7 C). The effect of retigabine and ML213 at 20 µM was not different between PTNL and PTAL (p < 0.05). In vivo measurement of intrauterine pressure (IUP) With confirmation that mRNA expression for all K V 7 channel components could be detected in myometrium from PTAL and the pharmacological evidence that the channels were functional in myometrium assessed in vitro , the next step was to determine the effect of K V 7 channel activators on myometrial contractility in vivo, assessed with intrauterine pressure telemetry measurements and timing of birth (delivery of first pup as recorded on CCTV) in RU486 treated mice in vivo . Acute gavage of two doses of retigabine, 90 minutes apart starting at 14 hours post RU486 demonstrated little impact on timing of delivery and hourly averaged IUP measurements in RU486 treated mice compared to administration of retigabine vehicle (0.5% hydroxymethylcellulose) (Suppl Fig. 2.). However, since time of delivery post RU486 showed some variation that potentially could have masked effects of retigabine, data were also analyzed to account for time of delivery (IUP displayed relative to time of delivery of first pup labor; t = 0) and revealed a temporal decrease in IUP following the gavage treatment period. This suggested that short term exposure to retigabine altered uterine contractility and IUP in vivo, but not sufficiently to delay birth. Repeat dosing of with Kv7 activators delays birth in mouse model of preterm birth To address the above observations, a repeat dosing regimen comprising of up to six gavage treatments (retigabine, ML213, or vehicle) was given every 60 minutes starting ~ 13 hours post RU486 injection was employed. Retigabine significantly delayed preterm birth, by increasing the median [IQR] time to delivery of first pup to 21.5 [20.5, 24.0] hours v 15.5 [15.1, 16.5] (p < 0.001) hours for RU486 vehicle treated animals; this equated to an average of 39% (36 to 59%) delay in time to delivery (Fig. 8 ). This is physiologically relevant if compared to the 1 hour it takes a mouse to deliver all pups and the ~ 16 hours it takes from RU486 administration to induce preterm delivery. ML213 also delayed delivery (18.0 [16.5,20.1], p < 0.01) by ~ 3 hours (Fig. 8 ); this was slightly less effective at delaying delivery in vivo compared to the tocolytic effect displayed in vitro . However, this was anticipated as it is reported the drug had poor metabolic stability following incubation with rat or human liver microsomes and was predicted to be rapidly cleared after systemic exposure 22 . Fetal viability and fetal ductus arteriosus function We were unable to assess the impact on neonatal outcomes as in this PTB model, premature neonates are either born without signs of life/cannibalized by the mother due to their prematurity. This occurred even when gestation was prolonged by K V 7 activator treatment. However, in a separate experimental protocol, we assessed the impact of treatment with K V 7 channel activators on ex vivo murine fetal ductus arteriosus (DA) contractility. This is an important consideration, as agents that constrict the ductus in utero would impact on fetal perfusion and oxygen delivery 23 . Suppl. Figure 3 shows that under deoxygenated organ bath conditions that mimic in utero oxygen status, fetal ductus arteriosus segments exposed to ML213 had no significant change in lumen diameter (10 − 8 to 10 − 4 M). DAs exposed to retigabine were similarly unaffected over the pharmacologic range (10 − 8 to 10 − 6 M) although at the highest concentration applied (a concentration unlikely to be used in in vivo ), a significant (p = 0.0002) constriction was observed at the highest concentration (10 − 4 M) compared to baseline diameter. Because of this, there was a significant (p = 0.004) difference in E max -values between these two drugs. Discussion This study provides a clear rationale for pursuing K V 7 channels as a potential target for tocolytic therapy and the prevention of preterm labor. Two structurally different activators of K V 7.2-7.5 delayed early birth in a recognized mouse model of preterm birth. This important finding is supported by comprehensive molecular data and functional data from human pregnant myometrium and a preterm birth mouse model. These novel findings build upon our previous work which described the physiological function of K V 7 channels in the non-pregnant and pregnant mouse 11 , 12 . K7 channels as a plausible target for tocolytic targeting Ion channels have long been viewed as important targets for therapeutic intervention due to their fundamental role in cellular physiology and human disease. Indeed, an estimated 10–20% of small molecular targets are ion channels 24 , 25 . K V channels represent a large gene superfamily that has been studied intensively in smooth muscles, including the myometrium, in terms of function 9 , 26 – 33 and in relation to K V 7 channels this has led to a number of compounds being considered for treatment of epilepsy, hypertension, stroke, lung disease, overactive bladder, and neuropathic pain 27 , 34 – 40 . However, as far as we are aware, apart from our previous work 11 , 12 , 41 and information in RNAseq datasets (e.g., 42 ) here is a paucity of knowledge concerning the functional role of K V 7 channels in the uterus. In general, K channel activity contributes to suppression of uterine contractility in pregnancy through promoting a low resting potential, modifying action potentials (and hence contraction amplitude) through repolarization, and hence limiting calcium entry and contractility 41 . However, as gestation nears term and labor (in both rodents and humans), there is a transition to greater myometrial cell excitability due to a gradual increase of the resting in resting membrane potential, most likely due to change in K + channel populations and activity 43 , 44 . For a K + channel to be useful for inhibiting preterm labor, its expression must be maintained in labor. K V 7.11 channels, for example, are expressed in late pregnancy, but their ability to influence repolarization is suppressed at labor onset through a variety of pathways 31 , 41 , 45 , 46 . Our previous pilot work in human myometrium suggested that K V 7.2-5 were expressed in human myometrium in late pregnancy, but here we confirm through molecular profiling that KCNQ and KCNE mRNA are expressed both at the end of pregnancy and after the onset of labor. Of the different isoforms found, KCNQ4 and KCNE4 mRNAs were the most highly expressed transcripts in term labor and were also expressed in myometrium from women in preterm labor. This was reflected in K V 7.4 and KCNE4 protein expression in myometrium from women at term and in labor. This identifies K V 7.4 as a potential K V 7 channel target in human myometrium 23 . Whilst transcripts for KCNQ5 were found and maintained in labour, we did not detect K V .5 protein. This does not fully rule out this as a target but more work on understanding protein expression is needed. KCNQ3 whilst expressed, is a key component of the neuronal M channel, so a less favorable target 47 along with Kv7.1 which is a cardiac associated K V channel. Pharmacological approaches using retigabine and ML213 provide clear evidence that activation of K V 7.2-5 channels can suppress contractions in human myometrium in vitro , an observation similar to that in term pregnant mice 11 . ML213 is reported as a K V 7.4, K V 7.2 and K V 7.5 channel activator, but given that K V 7.2 was expressed at low levels in myometrium, these findings support the notion that K V 7.4 channels is a plausible functional human myometrium target. The K V 7 channel inhibitor, XE991 also altered human uterine contractility in vitro providing additional evidence for functional K V 7. In contrast, Kv7.1, whilst detectable in human myometrium as KCNQ1 transcripts, did not contribute functionally to myometrial contractility (i.e., not affected by chromanol application). K V 7.2-5 channel activation prevented preterm labour Two options for inducing PTB were considered, a lipopolysaccharide induced PTB (inflammation driven) and RU486 (progesterone/glucocorticoid block). The latter was chosen because it may be more reflective of idiopathic PTB, a clinical scenario that would benefit from an effective tocolytic. RU486 induction of PTB is rapid in the mouse due to dependence on progesterone for maintenance of pregnancy, so any delay in labor in this scenario provides a strong proof of principle effect. The effectiveness of Kv7.2-5 activation to prevent preterm labor was demonstrated in a mouse model where K V 7 channels components, particularly kcnq4 and kcnq5 , were highly expressed in myometrium from preterm mice (prior to and after the onset of labour). Both retigabine and ML213 suppressed contractility in tissue ex vivo from preterm mice (not in labor and in labor). This effect was also confirmed in vivo where intrauterine pressure monitoring indicated that retigabine reduced pressure (indication of reduced uterine contractility) in RU486 treated mice in the hours preceding delivery. Whilst a single dose of retigabine was insufficient to delay birth, repeated gavage of retigabine significantly delayed labor and birth of the first pup by six hours compared to controls. ML213 was also assessed, although we were aware prior to starting experimentation that it would quickly be metabolized by the liver when given orally. With this caveat, as a K V 7.4 (and Kv7.4/5, Kv7.5) targeting agent it showed significant promise as it delayed preterm birth by three hours. Focus on the development of more favorable administration routes of an ML213 drug may be preferable. In the context of human pregnancy, this delay of preterm birth using KV7.2-5 activators has real potential to translate to prolongation of delivery for > 7 days or more, a delay that could significantly enhance human fetal maturity. Limitations and future work In terms of limitations, there are several avenues available for further exploration. There is scope for further interrogation of the composition of the K V 7.4 channels (homo or heterotetramers with other KCNQ isoform such as KCNQ5) 48 , 49 , or KCNE accessory subunits using proximity assays, imaging, patch clamp electrophysiology and transgenic mouse models. In the current study, we restricted functional studies to myometrium from women in late pregnancy as samples from women in spontaneous preterm labor are rare, but with time these experiments could be collated. The work in mouse models of PTB were designed to circumnavigate this issue and to provide an in vivo solution. Uterine contraction mechanisms are similar between species, and tissues exhibited relevant KCNQ and KCNE expression profiles. However, before work can be translated into pregnant women this may necessitate additional studies in another animal model, particularly if more specific K V 7.4 activators are to be developed. The side effects on other tissues and smooth muscles 10 , 50 , which also express K V 7.2-5 would also have to be monitored, although in the mouse model, retigabine was well tolerated. There is still a need to assess the impact of K V 7 activators on neonatal outcome. In our study, neonatal wellbeing could not be reviewed due to the gestation-based immaturity of the mice in this model even when gestation was prolonged. However, we considered the impact of retigabine and ML213 ex vivo on the murine fetal ductus arteriosus, and whilst retigabine at the highest concentration caused some constriction, this was at concentrations not likely to be achieved in vivo . In future studies, we plan to assess compounds in term pregnancy or a modified PTB model (induced later in gestation or milder stimulus) where neonates survive to determine neonatal developmental and behavior outcomes. To support translation of these findings into K V 7.4 (or KV7.4/5) based tocolytics, protocols investigating different routes and drug delivery mechanisms may be useful. In summary, this study identifies K V 7.2-5 activators as emerging candidates for tocolytic drug development. These activators can clearly suppress uterine contractility in human tissues and delay preterm birth in mice. There is compelling evidence that K V 7.4 and associated accessory subunit KCNE4 are the targets for uterine specific drug development. Many pharmaceutical companies avoid investing in maternity, but with continued increase in PTB and the greatest burden being within the low-risk pregnant population there is a need that should be addressed 5 . Whilst opportunities to explore drug repurposing have been affected by concerns about the long-term effects of retigabine and flupirtine 50 , 51 , release of compounds from existing drug libraries and/or further development 23 , 34 , 52 could potentially facilitate research and drug development for more short-term use in this major area of health care. This could be underpinned with more precise uterine targeting using emerging and novel drug delivery systems 53 , 54 . Materials and Methods Study design This was an experimental laboratory study using human myometrial tissue, a mouse (C57BL/6J) model of preterm delivery, and an isolated fetal ductus arteriosus preparation (from CD-1 pregnant mice). Human myometrium studies Human myometrium was obtained from 143 women with informed written consent, from women at term (> 37 weeks’ gestation) at the time of elective not in labor (TNL) or emergency in labor (TAL) cesarean section, as approved by St Thomas’ Hospital Ethics Committee (and subsequently regulated NRES Committee London-Westminster (EC00/137). For molecular and isometric tension measurement all were singleton pregnancies with exclusion criteria of adverse medical and obstetric conditions (including asthma requiring steroids, chronic hypertension requiring medication, lupus/systemic lupus erythematosus, diabetes, and pre-eclampsia) and the use of any medication that had potential to alter myometrial contractility (e.g., anti-hypertensives and inhaled bronchodilators). Biopsies were obtained from the upper edge of the lower segment incision and placed into ice-cold phosphate buffered saline. Tissue was rinsed and dissected on ice into small segments which were either snap frozen and stored at -80°C for RNA and protein analysis; formalin fixed, and wax embedded for immunohistochemical analysis; or kept at 4°C until used for isometric tension recording and cell isolation for patch clamp electrophysiology. Participant and pregnancy outcome data was retrieved from electronic and hand-held patient notes. cDNA from a smaller number of human myometrium samples were obtained from D Slater we compared expression from women who had cesarean sections from preterm deliveries not in labor (PNL, n = 4) and after spontaneous onset of preterm labor (PAL, n = 5, < 37 weeks of gestation pregnancy), with TNL (n = 6) and TAL (n = 6) controls (Conjoint Health Research Ethics Board REB15-1110). Mouse model of preterm birth Acclimatized female C57BL/6J mice (Charles River) were mated and a copulation plug visualized one day post copulation (1 dpc) and subsequently weighed regularly. Mice were housed in constant temperature rooms (21 ± 1°C) with fixed 12-hour light: dark intervals and ad libitum access to standard chow and water. The progesterone receptor/glucocorticoid receptor antagonist RU486 (mifepristone; Merck Life Science, UK) was used to artificially induce preterm birth as previously published 55 (Suppl. Figure 1). On 15 dpc, mice were restrained and a subcutaneous injection containing 150 µg RU486 [dissolved in DMSO (Merck) at a concentration of 1 µg/µl] administered in the nape of the neck. Control mice were injected with 150 µl of DMSO. All injections were administered at the same time of day and mice were continuously monitored by video so that the timing of gestation (defined as time to deliver first pup) could be accurately determined. For in vitro contraction studies, myometrium was harvested in RU486 mice when they showed signs of labor (preterm in labor, PTL) and in parallel with tissue from control non-laboring pregnant mice at the same gestation (preterm not in labor, PTNL). Measurement of intrauterine pressure using radio telemetry in mouse model of preterm birth This followed the method developed by Pierce and colleagues 56 , 57 . C57BL/6J mice were anesthetized with 3% isoflurane/oxygen in an induction chamber at day 8–10 dpc. Once anesthesia had been achieved, mice were transferred to a warming pad, placed in a supine position and a nose cone was used to deliver maintenance anesthesia (2% isoflurane/oxygen) for the duration of the surgical procedure. The abdomen was shaved and disinfected with a 0.5% (w/v) chlorhexidine gluconate solution (Medlock Medical Ltd., UK) and the work surface was replaced with a sterile cover. The fore- and hind-limbs were secured to the work surface, and 3 µg of buprenorphine (Vetergesic) (Sogeval UK Limited, York, UK) was injected intra-muscularly (I.M.) before a midline incision approximately 3 cm in length was made. The right and left uterine horns were identified, carefully exteriorized and the pups in each horn counted. One uterine horn was chosen for implantation of the pressure radio-telemetry probe, whilst the other horn was covered in saline-soaked gauze to prevent drying. Dumont tweezers were used to hold a small fold of the uterine wall, and a 1 mm incision was made using 3 mm Vanna scissors. Vessel cannulation forceps were used to feed the catheter of the probe into the incision of the uterus. Blunt-end forceps were used to manipulate the path of the catheter until the opening of the catheter was located between two fetal sacs. The catheter was kept in place by applying a drop of Vetbond (3M, USA) to the site of catheter insertion and the uterus was carefully placed back into the abdominal cavity, followed by the probe body, which was placed in the lower portion of the abdominal cavity. The probe was carefully placed so as to not introduce any acute bending or twisting of the catheter that could interfere with pressure measurements. The abdominal muscle layer and skin were each closed with a simple continuous suture pattern using a braided absorbable suture. Anesthesia was withdrawn and mice placed into a recovery chamber held at 28°C overnight with access to softened standard chow. Additional analgesia (3 µg buprenorphine, S.C.) was given to mice the following morning. Mice were then placed in a cage over a ‘receiver mat,’ which was connected via a matrix to a computer for remote data acquisition. Mice were free to move during the recording period. From day 13–14 dpc, intrauterine pressure (IUP) was recorded continuously until one day post-partum (approximately 19.5 dpc) or until delivery occurred. Continuous IUP data was averaged over one-minute intervals for all treatment groups using the manufacturers supplied software (Dataquest A.R.T, UK). A baseline period prior to any intervention was used to standardise intrauterine pressure. All data are expressed as a fold change in IUP from baseline. For mice delivering at term, a baseline period of 12 hours prior to delivery was used so that data from all groups was comparable. Every 60 minutes, pressure recordings were averaged to obtain a mean hourly pressure over a 17-hour period. For all groups, labor was defined as time 0 as indicated by the complete delivery of first pup. Some mice displayed negative IUP values in certain hours which were due to movement and occasional blockage of the catheter (DSI, personal communication), therefore for any hourly averages which included less than 75% positive values, negative values were excluded from the analysis. In addition, mice that had a negative average IUP baseline were excluded from data analysis. In vivo administration of K V 7 activators in preterm birth mouse model Acute treatment: On 16 dpc, 14 hours after RU486 administration, C57BL/6J mice were administered a single 20 mg/kg retigabine (Merck) or vehicle (10% (v/v) DMSO in 0.5% hydroxymethylcellulose solution) via oral gavage. For retigabine or ML213, 5 mg was first dissolved into 100 µl DMSO to which 900 µl 0.5% hydroxymethylcellulose was added to achieve a final concentration of 5 mg/ml. A second administration was given 90 minutes later. This was based on pharmacokinetic profiles of retigabine administration to female non-pregnant mice (carried out by Quotient Bioresearch, UK, data not shown) which reported that peak plasma concentrations of retigabine (6930 ng/ml) were achieved approximately 60 minutes after oral gavage. Repeated gavage: retigabine or ML213 (20 mg/kg, P.O.) or vehicle were prepared similarly as described for acute treatment above and were administered at 60-minute intervals starting 13 hours after RU486 injection. Doses were repeated for a maximum of 6 treatments, or until delivery of the first pup had occurred. Preterm birth mouse model myometrial tissue collection For murine myometrium, pregnant mice were sacrificed by cervical dislocation and the gravid uterus was carefully removed from the peritoneum. Uterine horns were cut along the axis of placental attachment, and the fetus and placenta were excised. Endometrial tissue was gently removed by brushing with a cotton bud. Tissues were either snap frozen in liquid nitrogen for mRNA extraction or used immediately for isometric tension measurement studies. Preterm birth mouse model isometric tension recording This is a modified protocol based on a method published previously 12 , 58 . In brief, small longitudinal myometrial strips (human: ~7x2x2 mm; mouse: ~ 5x2x2 mm) were mounted and maintained at 37°C in organ baths (oxygenated, 95% O2, 5% CO2) in physiological salt solution (PSS, pH 7.4 in mM: NaCl 119, KCl 4.7, MgSO4 1.17, NaHCO3 25, KH2PO4 1.18, EDTA 0.025, glucose 6 and CaCl2 2.5). Mouse and human myometrial tissues were subjected to stretch to approximately 1.5-fold their slack length (by applying between 29.34 to 38.25 mN respectively). After development of regular spontaneous contractile activity with a stable baseline, a control period of contractile activity was recorded prior to the addition of either KV7 activators [retigabine, ICA-069673 (Santa Cruz) and ML213 (Tocris)] or inhibitors [XE991 and chromanol 293B (Sigma)] (0.5–20 µM for all compounds). Vehicle control (DMSO) was carried out in parallel. Contractility data were recorded and analyzed using LabChart 6 software (ADInstruments, UK) and mean integral tension (MIT) for an experimental period, addition of Kv7 activator or vehicle, MIT (the sum of the integrals for each contraction divided by the duration of the period assessed) was expressed as a percentage of the preceding control (baseline) period MIT. Contraction frequency and amplitude of contractions were also assessed. Human and mouse myometrium mRNA extraction and real-time qRT-PCR Frozen myometrial tissue (~ 100 mg of human or mouse) was homogenized using a Qiagen Tissue Lyser. Total RNA was extracted using the RNeasy mini kit (Qiagen, UK) according to the manufacturer's instructions. Complementary deoxyribonucleic acid (cDNA) was synthesized using an Omniscript RT Kit (Qiagen, UK). Real-time polymerase chain reaction (PCR) was carried out with the use of SYBR Green chemistry (Bioline) on a RotorGene 6000 (Qiagen, UK) using human primers as reported in 59 and for mouse as listed in Supplementary Table 1. A pre-PCR cycle was run for 10 min at 95°C followed by 35 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 50 s followed by a final extension at 72°C for 15 s. Melt curve analysis was performed to confirm the presence of one single product. Cycle threshold (CT) values were used for analysis, and abundance data were obtained using quantified cDNA to generate a standard curve. All unknowns fell within the dynamic range of the standard curve. Standards were quantified using densitometry, and tenfold serial dilution from 1010 to 101 copies was run in parallel with the samples. Abundance data for the genes of interest were expressed as normalized copy number following normalization using GeNORM ( http://medgen.ugent.be/~jvdesomp/genorm ), with stably expressed reference genes (glyceraldehyde 3-phosphate dehydrogenase (GADPH). Data for the genes of interest were normalized (GeNorm) to mRNA copy number of a panel of genes (GAPDH, β-actin and β-2 microglobulin). All PCR products were sequenced to confirm identity. For analysis of human myometrium KCNQ4 and KCNE4 mRNA expression comparing preterm and term samples, because of limited material, only the housekeeper GAPDH was used. Immunohistochemistry studies of pregnant human myometrium Serial sections of paraffin embedded human myometrial tissues were cut (5 µm) and dewaxed from paraffin-embedded tissue blocks (using a Leica RM 2065 microtome, Leica Biosystems, UK) as previously described 59 . Immunohistochemical staining was performed using the Vector Stain Elite ABC kit (Vector Laboratories, UK). The optimal dilution for each antibody was established (Supplementary Tables 2 & 3). A positive control (human midbrain) was used to verify specificity. A negative control was performed for each test section by incubation with goat IgG. All slides were assessed by the same observer, blinded to labor group. Digital images of 5 randomly selected, high-power (×400 magnification) fields were captured on NIS-Elements F2.20 microscope (Nikon United Kingdom Ltd, UK). Quantifications of KCNQx and KCNEx expression was performed as described previously 60 using the Positive Pixel Algorithm of Aperio ImageScope software. A visual check was also performed to ensure accurate discrimination of immunolabeled regions. SDS-PAGE and Western Immunoblotting Protein samples for Western Blot were prepared by homogenisation of human myometrial tissues (TNL samples, n = 4; TAL samples, n = 4) using a Tissue Lyser II (Qiagen, UK) at 25hZ for 3 min in 20 µl lysis buffer/mg of tissue (10 mM of Hepes-KOH [pH 7], 1 mM of dithiothreitol, 1% nonident-P40, and protease-inhibitor cocktail (COMPLETE tablets; Boehringer-Mannheim Biochemicals, UK) and subsequent centrifugation for 1 min at 12470 x g to remove any tissue debris. Mouse brain, heart or aorta tissues were used as a positive control for a detection of variety KV7x/KCNEx proteins and were prepared in the same way as the human myometrium samples. Total protein concentration was calculated using BCA assay (Pierce, UK) and 30 µg of proteins were loaded to each lane of 12% Tris-Glycine precast gels (Generon, UK) and separated using the XCell SureLock™ Mini-Cell system (Invitrogen, UK). Following electrophoresis, proteins were transferred to Immobilion™-P transfer membrane (Millipore) using the XCell SureLock™ Mini-Cell blotting module wet transfer blotting system. 50mM Tris, 150mM NaCl, 0.2% (v/v) Tween-20, pH 7.4) containing 5% fat free milk powder for 1 hour at room temperature. After the transfer, membranes were blocked in TBS-T buffer (50mM Tris, 150mM NaCl, 0.2% (v/v) Tween-20, pH 7.4) supplemented with 5% milk (Sigma-Aldrich) for 1 hour at room temperature, followed by 3 hours incubation with primary antibody at RT. All KV7x and KCNEx antibodies used in this study are listed in Supplementary Table 4. Monoclonal mouse anti-smooth muscle alpha actin (Sigma, A2547) was used as a smooth muscle protein marker and a loading control. After the first antibody incubation, membranes were washed three times in TBS-T and HRP-conjugated anti-rabbit, anti-goat or anti-mouse IgGs secondary antibody was added (1:10000 dilution, Santa Cruz). The secondary antibody was incubated for 45 minutes at room temperature, followed by three washes in TBS-T buffer. To detect the antibody, chemiluminescent reagent (Invitrogen) was added, and the blots were exposed to X-ray film. Mouse ductus arteriosus (DA) vessel studies Animal experiments were conducted in accordance with the National Institutes of Health Animal Care Standards and were approved by the Institutional Animal Care and Use Committee at Vanderbilt University Medical Center. Adult female CD-1 mice were bred by timed mating between 0700–1000 hrs with the presence of a vaginal plug indicating the first day of pregnancy (d1). Pregnant females were anesthetized on the morning of d19 (term) via intraperitoneal injection of 0.4mL of 1.25% avertin (2,2,2-tribromoethanol in tert-amyl alcohol, Sigma-Aldrich, St. Louis, MO), followed by isoflurane inhalation (Baxter) for fetal anesthesia, and cervical dislocation of the dam. The anesthetized fetuses were removed by cesarean section. Their thoracic cavities were opened via partial dissection and puncture of the diaphragm, and the pups were then submerged in chilled, deoxygenated (95% N2, 5% CO2) modified Krebs (in mM; 109 NaCl, 4.7 KCl, 2.5 CaCl22H2O, 0.9 MgSO4, 1.0 KH2PO4, 11.1 glucose, 34 NaHCO3 (pH 7.3)) (2). Any pups with signs of respiration were excluded from vessel myography. DA segments representing at least five different litters were freshly isolated from the d19 fetuses for use in myography studies. Each DA was excised, mounted in custom myography chambers (University of Vermont), and allowed to equilibrate for 40 min at 5 mmHg and 37°C in deoxygenated modified Krebs buffer. The vasoreactivity of each vessel was ascertained using cannulated, pressurized vessel myography as previously described 61 – 64 . Inverted light microscopes with video capture (IonOptix) were used to continuously record lumen diameter. After the equilibration period, distending pressure was raised by 5 mmHg in 10 min increments to 20 mmHg, followed by two 10-minute exposures to deoxygenated 50 mM KCl with a 20 min wash step of deoxygenated Krebs in between to validate viability and reactivity. After a second 20 min wash step, the chambers were changed from a flow-through system to a 20 mL recirculating volume, and vessels were re-equilibrated for 20 min. At the end of this period, the intraluminal diameter was recorded and used as the baseline value to compare drug-related changes in DA tone. Vessels were exposed to increasing concentrations (10 − 8 to 10 − 4 M) of either retigabine (Axon Medchem; n = 10) or ML213 (Tocris Bioscience; n = 11) in 20 min increments, and changes in lumen diameter after each concentration increase were documented and compared. Statistics Formal power calculations determined the samples for KCNQ and KCNE mRNA expression profiles and N numbers for main experiments. For comparison of KCNQ/KCNE genes within pregnant human myometrium tissue, an n of 21 samples per experimental group was calculated to provide 90% power to detect a ratio of five between genes at the group-wise 5% significance level (alpha = 0.001; S.D. = 0.699) correcting for multiple testing using the Sidak-Bonferroni correction. This was based on data generated from previous experiments 12 . To assess changes in KCNQ/KCNE genes in tissues taken from women at term prior to labor (TNL) versus women in labor (TAL), an n of 42 (21 per sample group) was calculated to provide 90% power to detect a ratio of five (corresponding to a standardised ratio of 3.5 from previous work) between groups (TNL Vs. TAL) at the group-wise 5% significance level (alpha = 0.0051; SD = 0.544) correcting for multiple testing as above. To determine the effect of K V 7 modulators on contractile activity of pregnant human myometrium, pilot data has found a consistent standard deviation within treatment groups of 16.6%. With at least n = 4 observations in the control and treatment group, there is 90% power to detect a 33% difference in contractility between the controls and the active treatment. For animal studies, considering the 3R’s, experimental protocols used between n = 6–9 animals. For the DA vessel experiments, changes in lumen diameter were analyzed as percent change from the baseline and plotted as mean ± SEM. A comparison of fit determined whether three parameter non-linear log fit lines were significantly different between ML213 and retigabine. Two-way analysis of variance followed by a post hoc Dunnett’s test was used to determine significant differences in the % change from baseline for each concentration of drug. Two-way analysis of variance followed by a post hoc Sidak’s test was used to determine significant differences between the Emax values of ML213 and retigabine. All tests were performed using SPSS for Windows version 27 and GraphPad PRISM version 9. The Kolmogorov-Smirnov test was used to evaluate normality of data distribution and summary data are presented as means ± SD or median [interquartile range (IQR)] as appropriate for data distribution. Between-group comparisons were made using Kruskal-Wallis and post hoc Dunn’s test or Mann-Whitney. The null hypothesis was rejected where P < 0.05. Declarations Competing interests: Authors declare that they have no competing interest Data and Materials availability All experimental data associated with this study are available in the main text or the supplementary materials. Limited residual human tissue and associated data available (subject to ethical approval and a specifically negotiated MTA with KCL and Guys and St Thomas’ Foundation Trust). Requests to be directed to the corresponding author. Funding: This study was directly funded by an MRC project grant (G1100243), an MRC DPT PhD studentship to YTM and Tommy’s charity and underpinned by preliminary data funded by Action Medical Research and Rosetrees Trust grant (SP4298). HDM was funded during this period by a British Heart Foundation Intermediate Basic Science Fellowship (FS/15/32/31604) and a British Heart Foundation Project Grant (PG/11/22/28800). Author contributions (CRediT) : Conceptualization: RMT, IAG, HDM, MRJ, PDT, SKE, DMS, JLH, JR; Methodology: RMT, PDT, YTM, SKE, DMS, ECCS, HDM, JR; Investigation: YTM, RMT, PDT, MKZ, ECCS, RP, HDM, JR, JLH, DCS; Visualization: YTM, RMT, ECCS, HDM, MKZ, JR, JLH; Funding Acquisition: RMT, IAG, PIA, MRJ, PDT, HDM; Project Administration: RMT; Supervision: RMT, HDM, PDT, JR; Writing original draft: RMT, YTM, HDM; Writing – reviewing and editing: YTM, HDM, ECCS, MKZ, JR, JLH, DCS, RP, PIA, DMS, SKE, IAG, MRJ, PDT, RMT. Acknowledgments: We are particularly grateful to the women who consented to participating in the study and Prof. Andrew Shennan and the NIHR RDN funded team of research midwives and research assistants who supported recruitment, consent, and tissue collection. We also thank the support of staff at the KCL Biological Services facility and Prof Raheela Khan for support with immunohistochemistry. References Chawanpaiboon, S. , et al. Global, regional, and national estimates of levels of preterm birth in 2014: a systematic review and modelling analysis. Lancet Glob Health 7 , e37-e46 (2019). Frey, H.A. & Klebanoff, M.A. The epidemiology, etiology, and costs of preterm birth. Semin Fetal Neonatal Med 21 , 68-73 (2016). Rubens, C.E. , et al. Prevention of preterm birth: harnessing science to address the global epidemic. Sci Transl Med 6 , 262sr265 (2014). Martin, J.N., Jr., D'Alton, M., Jacobsson, B. & Norman, J.E. In pursuit of progress toward effective preterm birth reduction. Obstet Gynecol 129 , 715-719 (2017). McDougall, A.R.A. , et al. New medicines for spontaneous preterm birth prevention and preterm labour management: landscape analysis of the medicine development pipeline. BMC Pregnancy Childbirth 23 , 525 (2023). NICE. Overview: Preterm labour and birth, guidance. (NICE, 2015). Practice bulletin No. 171 summary: management of peterm labor. Obstet Gynecol 128 , 931-933 (2016). Haas, D.M., Caldwell, D.M., Kirkpatrick, P., McIntosh, J.J. & Welton, N.J. Tocolytic therapy for preterm delivery: systematic review and network meta-analysis. BMJ 345 , e6226 (2012). Wray, S. & Arrowsmith, S. Uterine excitability and ion channels and their changes with gestation and hormonal environment. Annu Rev Physiol 83 , 331-357 (2021). Barrese, V., Stott, J.B. & Greenwood, I.A. KCNQ-encoded potassium channels as therapeutic targets. Annu Rev Pharmacol Toxicol 58 , 625-648 (2018). McCallum, L.A., Pierce, S.L., England, S.K., Greenwood, I.A. & Tribe, R.M. The contribution of Kv7 channels to pregnant mouse and human myometrial contractility. J Cell Mol Med 15 , 577-586 (2011). McCallum, L.A., Greenwood, I.A. & Tribe, R.M. Expression and function of K(v)7 channels in murine myometrium throughout oestrous cycle. Pflugers Arch 457 , 1111-1120 (2009). Peretz, A. , et al. Meclofenamic acid and diclofenac, novel templates of KCNQ2/Q3 potassium channel openers, depress cortical neuron activity and exhibit anticonvulsant properties. Mol Pharmacol 67 , 1053-1066 (2005). Roepke, T.K. , et al. KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium. FASEB J 25 , 4264-4273 (2011). Anderson, U.A. , et al. Functional expression of KCNQ (Kv7) channels in guinea pig bladder smooth muscle and their contribution to spontaneous activity. Br J Pharmacol 169 , 1290-1304 (2013). Liu, W.J. , et al. Manipulation of KCNE2 expression modulates action potential duration and Ito and IK in rat and mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 309 , H1288-1302 (2015). Sachse, C.C. , et al. BACE1 and presenilin/gamma-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels. FASEB J 27 , 2458-2467 (2013). Zhang, M. , et al. KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner. Am J Physiol Heart Circ Physiol 302 , H910-922 (2012). Clancy, S.M., Chen, B., Bertaso, F., Mamet, J. & Jegla, T. KCNE1 and KCNE3 beta-subunits regulate membrane surface expression of Kv12.2 K(+) channels in vitro and form a tripartite complex in vivo. PLoS One 4 , e6330 (2009). Grunnet, M. , et al. KCNE4 is an inhibitory subunit to the KCNQ1 channel. J Physiol 542 , 119-130 (2002). Lundquist, A.L. , et al. Expression of multiple KCNE genes in human heart may enable variable modulation of I(Ks). J Mol Cell Cardiol 38 , 277-287 (2005). Yu, H. , et al. A small molecule activator of KCNQ2 and KCNQ4 channels. in Probe Reports from the NIH Molecular Libraries Program (Bethesda (MD), 2010). Villegas-Esguevillas, M. , et al. The novel K(V)7 channel activator URO-K10 exerts enhanced pulmonary vascular effects independent of the KCNE4 regulatory subunit. Biomed Pharmacother 164 , 114952 (2023). Grand View Research. Ion channels in drug discovery, 2023 report. (Martket Trends Reports, 2023). Rogers, M. Ion channel drug discovery and modern medicine. 9-11 (Metrion Biosciences, 2019). Pereira da Silva, E.A., Martin-Aragon Baudel, M., Navedo, M.F. & Nieves-Cintron, M. Ion channel molecular complexes in vascular smooth muscle. Front Physiol 13 , 999369 (2022). Malysz, J. & Petkov, G.V. Detrusor Smooth Muscle K(V)7 Channels: Emerging New Regulators of Urinary Bladder Function. Front Physiol 11 , 1004 (2020). Smith, R.C., McClure, M.C., Smith, M.A., Abel, P.W. & Bradley, M.E. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility. Reprod Biol Endocrinol 5 , 41 (2007). Tribe, R.M. & Taggart, M.J. Cellular ionic mechanisms controlling uterine smooth muscle contraction: effects of gestational state. in New Frontiers in Smooth Muscle Biology and Physiology , Vol. 1 (ed. Savinneau, J.P.) 523-549 (Research Signpost, India, 2007). Bao, J. , et al. BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation micedagger. Biol Reprod 110 , 798-807 (2024). Parkington, H.C. , et al. Diminished hERG K+ channel activity facilitates strong human labour contractions but is dysregulated in obese women. Nat Commun 5 , 4108 (2014). Cowles, C.L. , et al. Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization. Biol Reprod 93 , 122 (2015). Jepps, T.A., Carr, G., Lundegaard, P.R., Olesen, S.P. & Greenwood, I.A. Fundamental role for the KCNE4 ancillary subunit in Kv7.4 regulation of arterial tone. J Physiol 593 , 5325-5340 (2015). Perucca, E. & Taglialatela, M. Targeting Kv7 potassium channels for epilepsy. CNS Drugs (2025). Rejdak, K., Luszczki, J.J., Blaszczyk, B., Chwedorowicz, R. & Czuczwar, S.J. Clinical utility of adjunctive retigabine in partial onset seizures in adults. Ther Clin Risk Manag 8 , 7-14 (2012). Zhang, H. , et al. Neuroprotective Effects, Mechanisms of Action and Therapeutic Potential of the Kv7/KCNQ Channel Opener QO-83 in Ischemic Stroke. Transl Stroke Res (2025). Zhang, B. , et al. Discovery of E0199: A novel compound targeting both peripheral Na(V) and K(V)7 channels to alleviate neuropathic pain. J Pharm Anal 15 , 101132 (2025). Alles, S.R.A. & Smith, P.A. Peripheral Voltage-Gated Cation Channels in Neuropathic Pain and Their Potential as Therapeutic Targets. Front Pain Res (Lausanne) 2 , 750583 (2021). Mondejar-Parreno, G., Perez-Vizcaino, F. & Cogolludo, A. Kv7 Channels in Lung Diseases. Front Physiol 11 , 634 (2020). Jepps, T.A., Olesen, S.P. & Greenwood, I.A. One man's side effect is another man's therapeutic opportunity: targeting Kv7 channels in smooth muscle disorders. Br J Pharmacol 168 , 19-27 (2013). Greenwood, I.A. & Tribe, R.M. Kv7 and Kv11 channels in myometrial regulation. Exp Physiol 99 , 503-509 (2014). Chan, Y.W., van den Berg, H.A., Moore, J.D., Quenby, S. & Blanks, A.M. Assessment of myometrial transcriptome changes associated with spontaneous human labour by high-throughput RNA-seq. Exp Physiol 99 , 510-524 (2014). Casteels, R. & Kuriyama, H. Membrane potential and ionic content in pregnant and non-pregnant rat myometrium. J Physiol 177 , 263-287 (1965). Parkington, H.C., Tonta, M.A., Brennecke, S.P. & Coleman, H.A. Contractile activity, membrane potential, and cytoplasmic calcium in human uterine smooth muscle in the third trimester of pregnancy and during labor. Am J Obstet Gynecol 181 , 1445-1451 (1999). Greenwood, I.A., Yeung, S.Y., Tribe, R.M. & Ohya, S. Loss of functional K+ channels encoded by ether-a-go-go-related genes in mouse myometrium prior to labour onset. J Physiol 587 , 2313-2326 (2009). Tong, W.C., Tribe, R.M., Smith, R. & Taggart, M.J. Computational modeling reveals key contributions of KCNQ and hERG currents to the malleability of uterine action potentials underpinning labor. PLoS One 9 , e114034 (2014). Jepps, T.A., Barrese, V. & Miceli, F. Editorial: Kv7 channels: structure, physiology, and pharmacology. Front Physiol 12 , 679317 (2021). Chadha, P.S. , et al. Contribution of kv7.4/kv7.5 heteromers to intrinsic and calcitonin gene-related peptide-induced cerebral reactivity. Arterioscler Thromb Vasc Biol 34 , 887-893 (2014). Mani, B.K. , et al. Kv7.5 potassium channel subunits are the primary targets for PKA-dependent enhancement of vascular smooth muscle Kv7 currents. Mol Pharmacol 89 , 323-334 (2016). Abou-Khalil, B.W. Update on antiepileptic drugs 2019. Continuum (Minneap Minn) 25 , 508-536 (2019). Wang, L., Qiao, G.H., Hu, H.N., Gao, Z.B. & Nan, F.J. Discovery of novel retigabine derivatives as potent KCNQ4 and KCNQ5 channel agonists with improved specificity. ACS Med Chem Lett 10 , 27-33 (2019). Seefeld, M.A. , et al. Novel K(V)7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction. Bioorg Med Chem Lett 28 , 3793-3797 (2018). Paul, J.W. , et al. Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor. Am J Obstet Gynecol 216 , 283 e281-283 e214 (2017). Paul, J.W. & Smith, R. Preventing preterm birth: New approaches to labour therapeutics using Nanoparticles. Best Pract Res Clin Obstet Gynaecol 52 , 48-59 (2018). Dudley, D.J., Branch, D.W., Edwin, S.S. & Mitchell, M.D. Induction of preterm birth in mice by RU486. Biol Reprod 55 , 992-995 (1996). Rada, C.C., Pierce, S.L., Grotegut, C.A. & England, S.K. Intrauterine telemetry to measure mouse contractile pressure in vivo. J Vis Exp , e52541 (2015). Pierce, S.L., Kutschke, W., Cabeza, R. & England, S.K. In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models. Physiol Genomics 42 , 310-316 (2010). Tribe, R.M., Moriarty, P. & Poston, L. Calcium homeostatic pathways change with gestation in human myometrium. Biol Reprod 63 , 748-755 (2000). Mistry, H.D. , et al. Novel expression and regulation of voltage-dependent potassium channels in placentas from women with preeclampsia. Hypertension 58 , 497-504 (2011). Williams, P.J., Mistry, H.D., Innes, B.A., Bulmer, J.N. & Broughton Pipkin, F. Expression of AT1R, AT2R and AT4R and their roles in extravillous trophoblast invasion in the human. Placenta 31 , 448-455 (2010). Reese, J. , et al. Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall. Prostaglandins Other Lipid Mediat 88 , 89-96 (2009). Crockett, S.L. , et al. Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness. Pediatr Res 87 , 991-997 (2020). Yarboro, M.T. , et al. A novel role for PGE(2)-EP(4) in the developmental programming of the mouse ductus arteriosus: consequences for vessel maturation and function. Am J Physiol Heart Circ Physiol 325 , H687-H701 (2023). Zou, M. , et al. Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility. JCI Insight 8 (2023). Additional Declarations There is NO Competing Interest. Supplementary Files NatMedKv7SupplementaryMaterialsFinalMarch2025.docx KV7 channel activation inhibits human and murine myometrium contractility and delays delivery in a mouse model of preterm birth Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6837828","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":479163473,"identity":"952ff162-f6ba-4a70-aeef-423a4872e89c","order_by":0,"name":"Rachel Tribe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie2RsQrCMBCGTwJ1Oc16pcVniAjWQfoyAacOjk6uLj5ABx+mpeBU944VoVOHjAUdvKK4tdXNId8SCPn4/8sBWCz/iQOw5UMggEnAf10mQ4oCapVRnAD+oPBjgd8owSGrSqNgHxwm6W2dhwjjrBSYdyt+vgnmMaf42VQvokIj4EYJLLoVgsjxkBUSuPQik3CxiBuaHkXWjvd4KcF91SqyHlCIU+CdIqBghdqUvmJULd2jIjcWU+0ec40OVSo99YxPUlfU7NaS5CU1zTmcSamvZX3uVj6/8IHXNLBIi8VisQzyBNnDQK+cGiTSAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3675-9978","institution":"King's College London","correspondingAuthor":true,"prefix":"","firstName":"Rachel","middleName":"","lastName":"Tribe","suffix":""},{"id":479163474,"identity":"53d08dc2-2d4e-4485-9422-d557327fa877","order_by":1,"name":"Yosef Mansour","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Yosef","middleName":"","lastName":"Mansour","suffix":""},{"id":479163475,"identity":"ba82a445-6f5d-4e34-b8a1-0f4c52e0a746","order_by":2,"name":"Hiten Mistry","email":"","orcid":"https://orcid.org/0000-0003-2564-7348","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Hiten","middleName":"","lastName":"Mistry","suffix":""},{"id":479163476,"identity":"a094b0f4-aa01-4c7b-94a0-822d28bd8960","order_by":3,"name":"Evonne Chin-Smith","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Evonne","middleName":"","lastName":"Chin-Smith","suffix":""},{"id":479163477,"identity":"14e701c3-8df7-43e1-b7d8-4ce90f4cc483","order_by":4,"name":"Mariola Zaleska","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Mariola","middleName":"","lastName":"Zaleska","suffix":""},{"id":479163478,"identity":"5ce787c8-002a-40d6-b02f-d558d2d93319","order_by":5,"name":"Deanna Sekulich","email":"","orcid":"https://orcid.org/0009-0001-7920-772X","institution":"Vanderbilt University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Deanna","middleName":"","lastName":"Sekulich","suffix":""},{"id":479163479,"identity":"dee00e75-f46d-437e-99fd-b89c0e4a0f30","order_by":6,"name":"Jennifer Herington","email":"","orcid":"","institution":"Vanderbilt University Medical Centre","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Herington","suffix":""},{"id":479163480,"identity":"2b2b31e3-a613-4b00-ab67-54e2a837e0dd","order_by":7,"name":"Jeff Reese","email":"","orcid":"","institution":"Vanderbilt University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Jeff","middleName":"","lastName":"Reese","suffix":""},{"id":479163481,"identity":"7414f46a-f835-4719-a3c5-dbb07659f1ec","order_by":8,"name":"Rima Patel","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Rima","middleName":"","lastName":"Patel","suffix":""},{"id":479163482,"identity":"cb062e57-c171-4cf7-b8fc-80f8b15f2ed5","order_by":9,"name":"Philip Aaronson","email":"","orcid":"","institution":"King's College London","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Aaronson","suffix":""},{"id":479163483,"identity":"fb7d860d-eaf8-4817-a963-9a6b1c54a0ae","order_by":10,"name":"Donna Slater","email":"","orcid":"","institution":"University of Calgary","correspondingAuthor":false,"prefix":"","firstName":"Donna","middleName":"","lastName":"Slater","suffix":""},{"id":479163484,"identity":"d593f0e0-1e7d-4ef2-9418-a3c51af215d3","order_by":11,"name":"Sarah England","email":"","orcid":"","institution":"WashU Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"England","suffix":""},{"id":479163485,"identity":"04ac4420-0c67-406e-a6a4-61736a9287ae","order_by":12,"name":"Iain Greenwood","email":"","orcid":"","institution":"St George’s University of London","correspondingAuthor":false,"prefix":"","firstName":"Iain","middleName":"","lastName":"Greenwood","suffix":""},{"id":479163486,"identity":"9e621322-054d-4d1d-9c0d-503055e16fa4","order_by":13,"name":"Mark Johnson","email":"","orcid":"","institution":"Department of Metabolism, Digestion \u0026 Reproduction, Faculty of Medicine, Imperial College London","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Johnson","suffix":""},{"id":479163487,"identity":"b7ae6f11-e575-4ac2-a4ea-a8d4dd69f27b","order_by":14,"name":"Paul Taylor","email":"","orcid":"https://orcid.org/0000-0002-4740-4307","institution":"King's College London, School of Life Course Sciences","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Taylor","suffix":""}],"badges":[],"createdAt":"2025-06-06 14:26:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6837828/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6837828/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94829124,"identity":"891956a0-9c29-4963-b334-3a4da54ba901","added_by":"auto","created_at":"2025-10-31 07:04:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormalized mRNA expression profile of KCNQ and KCNE genes in pregnant human myometrium taken at term prior to (TNL n=51, A and C) and during active labor (TAL n=46, B and D). \u003c/strong\u003eNormalized (GeNorm) mRNA copy number expressed as median and quartile ranges. Statistical significance was determined using Kruskal Wallis and Dunn’s multi-comparison post-test. ** P\u0026lt;0.01; *** P\u0026lt; 0.001, # P\u0026lt; 0.001 KCNE2 compared to all other KCNE genes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/7163809f5bd05d63501e148a.png"},{"id":94984986,"identity":"2e3a6730-cf5a-4dce-a1c6-b18d04b3e46d","added_by":"auto","created_at":"2025-11-03 06:57:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormalized mRNA expression profile of KCNQ4 (A) and KCNE4 (B) genes in pregnant human myometrium taken at preterm gestations (\u0026lt;37 weeks) not in labor (PNL) and in active labour (PAL) and at term gestations prior to term (not in labor, TNL) and after during active labor (TAL). \u003c/strong\u003eNormalized (to GAPDH) mRNA copy number expressed as median and quartile ranges. Statistical significance was determined using Kruskal Wallis and Dunn’s multi-comparison post-test. *P\u0026lt;0.05 PNL \u003cem\u003eversus \u003c/em\u003eTNL. PNL, N= 4; PAL, N=5; TNL n=6; TAL n=6.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/10ef32cd1385b7d0877a75db.png"},{"id":94829129,"identity":"c53be46c-1459-4365-9d74-6ec851735c38","added_by":"auto","created_at":"2025-10-31 07:04:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":258336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A). Representative Western blot of protein expression in pregnant human myometrium taken at the time of cesarean section from women at term not in labor (TNL, n=4) and in labor (TAL, n=4) and control (mouse brain, heart, or aorta).\u003c/strong\u003e Proteins include those encoded by KCNQ1, 3, 4 (K\u003csub\u003eV\u003c/sub\u003e7.1, Kv7.3 and KV7.4 respectively), KCNE 1-4 and smooth muscle alpha actin (SMAA, housekeeper). (\u003cstrong\u003eB)\u003c/strong\u003e. Kv7.4 expression normalized to SMAA expressed as median and interquartile range, no significant difference between K\u003csub\u003eV\u003c/sub\u003e7.4 expression in TNL v TAL samples.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/304a01f03f369669afb881ed.png"},{"id":94829125,"identity":"dd8eace1-37b5-466f-a51b-1025c28d922c","added_by":"auto","created_at":"2025-10-31 07:04:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":750624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e7 and KCNE isoform immunostaining in pregnant human myometrium taken at the time of cesarean section from women at term not in labor (TNL) and in labor (TAL).\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e). Representative (of n=14) immunostaining in TNL myometrium a. K\u003csub\u003eV\u003c/sub\u003e7.1, b K\u003csub\u003eV\u003c/sub\u003e7.3, c K\u003csub\u003eV\u003c/sub\u003e7.4, d. negative control for K\u003csub\u003eV\u003c/sub\u003e7.4, no primary antibody; e. kcne1, f kcne3, g, kcne4, h negative control for K\u003csub\u003eV\u003c/sub\u003e7.1 7.3 and all KCNE isoforms, (no primary antibody). (\u003cstrong\u003eB\u003c/strong\u003e). Mean positivity and (\u003cstrong\u003eC\u003c/strong\u003e). representative immunostaining for K\u003csub\u003eV\u003c/sub\u003e7.3 (\u003cstrong\u003eCa\u003c/strong\u003e,\u003cstrong\u003e Cb\u003c/strong\u003e) and K\u003csub\u003eV\u003c/sub\u003e7.4 (\u003cstrong\u003eCe\u003c/strong\u003e, \u003cstrong\u003eCf\u003c/strong\u003e) in pregnant human myometrium TNL (n=14) versus TAL (n=18). Box plots are presented as median [IQR], * p \u0026lt;0.05 and ** p \u0026lt;0.01. (\u003cstrong\u003eCc, Cg\u003c/strong\u003e) positive controls from human rectum and brain; (\u003cstrong\u003eCd\u003c/strong\u003e, \u003cstrong\u003eCh\u003c/strong\u003e) negative controls for K\u003csub\u003eV\u003c/sub\u003e7.3 and K\u003csub\u003eV\u003c/sub\u003e7.4, respectively. Positive staining, shown in brown, was mainly localised to intracellular areas within smooth muscle cells, with some staining around vessels. Black bars are scale bars representing 100 µM. All photomicrographs are taken at x 400 magnification.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/422862daba4e6a6dc5722091.png"},{"id":94829127,"identity":"ac17b245-30bb-447b-9028-9d4875c72d8a","added_by":"auto","created_at":"2025-10-31 07:04:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of K\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e7.2-5 activators (retigabine and ML213) and the pan K\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e7 inhibitor XE991 and chromanol (Kv7.1 inhibitor) on spontaneous contractility of myometrium from women at term prior to labor onset (TNL)\u003c/strong\u003e. Single doses of activator (0.5 – 20 µM) were added to spontaneously contracting myometrium tissue in an organ bath (\u003cstrong\u003eA\u003c/strong\u003e) retigabine; (\u003cstrong\u003eB\u003c/strong\u003e) ML213 and mean integral tension (MIT) recorded over a 60-minute period for each dose compared to vehicle control (10 µl DMSO). Impact of single concentrations (0.5 – 20 µM) of Kv7.1 inhibitor chromanol (C) on MIT and pan K\u003csub\u003eV\u003c/sub\u003e7 activator XE991 (D-F) on MIT, contractile amplitude and frequency versus vehicle control. Mean ± SEM data are expressed as a percentage MIT of the preceding baseline prior to the addition of K\u003csub\u003eV\u003c/sub\u003e7 modulator. *p\u0026lt;0.05, **p\u0026lt;0.01. \u0026nbsp;\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/dd6c02a8187164ab974dbf53.png"},{"id":94829132,"identity":"abbcc619-4d47-489a-8ff4-e14bdc162183","added_by":"auto","created_at":"2025-10-31 07:04:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":96922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormalized mRNA expression profile of KCNQ and KCNE genes in preterm [not in labor (PTNL, n=4-6) and in labor (PTAL, n=5-6), day 16] pregnant mouse myometrium.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e). \u003cem\u003eKcnq1-5\u003c/em\u003emRNA normalized (GeNorm) copy number expression profile in myometrium from PTNL; (\u003cstrong\u003eB\u003c/strong\u003e). \u003cem\u003eKcnq1-5\u003c/em\u003e mRNA normalized copy number expression profile in myometrium from PTAL; (\u003cstrong\u003eC\u003c/strong\u003e). \u003cem\u003eKcne1-5\u003c/em\u003e mRNA normalized copy number expression profile in myometrium from PTNL mice; (\u003cstrong\u003eD\u003c/strong\u003e). \u003cem\u003eKcne1-5\u003c/em\u003emRNA normalized copy number expression profile in myometrium from PTAL mice. Data expressed as median [IQR]. Statistical significance was determined using Kruskal Wallis and Dunn’s multi-comparison post-test. * P\u0026lt;0.05; ** P\u0026lt;0.01; *** P\u0026lt;0.001; **** P\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/153f086a3e162ce65de38a78.png"},{"id":94985020,"identity":"0dfea646-75a2-47f0-ad5d-a27cfb055254","added_by":"auto","created_at":"2025-11-03 06:57:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":64564,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of K\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e7 activators (retigabine and ML213) on spontaneous contractility of myometrium from preterm non laboring mice (PTNL, DMSO controls) and preterm laboring mice (PTAL, RU486 treated) on day 16 post conception. \u003c/strong\u003eSingle concentrations of either retigabine and ML213 (5, 10 and 20 µM) were added to spontaneously contracting myometrium tissues (n=6 for each concentration). Mean integral tension (MIT) recorded over a 20-minute period for each concentration compared to vehicle control (DMSO; 10 µl) in myometrium from PTNL (\u003cstrong\u003eA, C\u003c/strong\u003e) and PTAL (\u003cstrong\u003eB, D\u003c/strong\u003e) mice. Data are expressed as a percentage MIT of the preceding baseline prior to the addition of K\u003csub\u003eV\u003c/sub\u003e7 activator and are presented as mean ± SEM (* p\u0026lt;0.05; ** p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/a69b2e306649c76e591e80a1.png"},{"id":94984615,"identity":"d7f0298a-15ba-4e80-b074-e43a84d4256d","added_by":"auto","created_at":"2025-11-03 06:54:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":23944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of repeat gavage of 20 mg/kg retigabine or ML213 (compared to vehicle) on the onset of preterm labor in RU486 treated mice.\u003c/strong\u003e Mice were treated with six-hourly doses (or until delivery occurred) of retigabine (n=6), ML213 (n=6) (20 mg/kg, P.O.) or vehicle (n=9) from 13 hours post RU486 (150 µg) administration. Mice were continually monitored by CCTV for signs of labor. Labor, defined as delivery of first pup, was determined and the hours to delivery post RU486 were calculated. Data are expressed as median [interquartile range]. Data were analyzed using the Kruskal-Wallis test followed by post-hoc Mann-Whitney U tests. (*p\u0026lt;0.05, **p\u0026lt;0.01)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/09f7b0aba992bc9865d084f2.png"},{"id":94990312,"identity":"8f86a355-b7d9-49ce-a1b8-0c395ef26f78","added_by":"auto","created_at":"2025-11-03 07:16:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3215238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/ec5b7cce-aab5-481f-8405-6b5c7882bbbd.pdf"},{"id":94984773,"identity":"6cfb2c20-6d84-4200-abe3-8ba36d0edd20","added_by":"auto","created_at":"2025-11-03 06:56:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":348925,"visible":true,"origin":"","legend":"KV7 channel activation inhibits human and murine myometrium contractility and delays delivery in a mouse model of preterm birth","description":"","filename":"NatMedKv7SupplementaryMaterialsFinalMarch2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6837828/v1/6edb5e2dad66eb8fbe96c29e.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"KV7 channel activation inhibits human and murine myometrium contractility and delays delivery in a mouse model of preterm birth","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePreterm birth (\u0026lt;\u0026thinsp;37 weeks\u0026rsquo; gestation) affects approximately 15\u0026nbsp;million pregnancies worldwide every year and results in unacceptable levels of neonatal death and morbidity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The resultant social, economic, and healthcare burden for surviving babies is considerable \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA major cause of preterm birth is the spontaneous onset of preterm labor, typified by inappropriate early onset of uterine contractions and premature cervical shortening. Due to an incomplete understanding of the mechanisms driving preterm labor and the lack of effective prophylactic interventions, many women still present in threatened preterm labor and require intervention \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Most will receive, when not contraindicated due to the presence of overt infection, tocolytic therapy to delay labor and corticosteroids to improve neonatal survival and morbidity \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, there are few tocolytic therapies available e.g., nifedipine and atosiban (a mixed oxytocin/vasopressin receptor antagonist), but the effectiveness of these agents in terms of delaying birth and impact on neonatal outcomes is limited \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. As a result, there is a clinical need to identify new uterine smooth muscle (myometrium) targets to develop more effective tocolytic agents.\u003c/p\u003e \u003cp\u003ePlasma membrane potassium (K\u003csup\u003e+\u003c/sup\u003e) channels are particularly apposite for targeting, as K\u003csup\u003e+\u003c/sup\u003e channel expression and function underpin uterine quiescence in pregnancy and contribute to the generation of rhythmic contractions in labor \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Gene expression of several K\u003csub\u003eV\u003c/sub\u003e channel sub-types, and related KCNE ancillary units, have been identified in human and rodent uterine myocytes including K\u003csub\u003eV\u003c/sub\u003e7 and K\u003csub\u003eV\u003c/sub\u003e11 \u003csup\u003e9,10\u003c/sup\u003e. We have demonstrated that myometrial K\u003csub\u003eV\u003c/sub\u003e7 channels encoded by KCNQ1-5 genes are expressed robustly throughout mouse gestation. In addition, activators of K\u003csub\u003eV\u003c/sub\u003e7.2 to K\u003csub\u003eV\u003c/sub\u003e7.5, such as retigabine, inhibited myometrial contractions in mouse and human myometrium \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Considering this functional impact, we hypothesize that K\u003csub\u003eV\u003c/sub\u003e7 channels have potential as a novel therapeutic target to delay preterm birth.\u003c/p\u003e \u003cp\u003eConsequently, the aim of this study was to establish which K\u003csub\u003eV\u003c/sub\u003e7 channels are present in human myometrium from pregnant women after the onset of labor, and to use a mouse model of preterm birth to provide \u0026lsquo;proof of principle\u0026rsquo; data that targeting K\u003csub\u003eV\u003c/sub\u003e7 in vivo can delay preterm delivery.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eK\u003csub\u003eV\u003c/sub\u003e7 channel expression in late pregnant human myometrium\u003c/h2\u003e \u003cp\u003eFor K\u003csub\u003eV\u003c/sub\u003e7 channel activators to be considered as a potential treatment of preterm labor, KCNQ and KCNE mRNA and associated proteins must be expressed and active in pregnant human myometrium and remain in tissues after labor onset. Using myometrium from women at the time of elective cesarean section (term not in labor: TNL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, n\u0026thinsp;=\u0026thinsp;51), mRNA expression of all KCNQ isoforms except KCNQ2 were detected. Similar to our previous mouse studies, KCNQ4 was the most highly expressed mRNA compared to KCNQ1,3,5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The ranked order was KCNQ4\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;KCNQ3\u0026thinsp;\u0026gt;\u0026thinsp;KCNQ1\u0026thinsp;\u0026gt;\u0026thinsp;KCNQ5; Kruskal-Wallis tests confirmed significant differences between the individual KCNQ isoforms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Whilst there was a relatively wide spread of KCNQ4 expression values, individual profiles of KCNQ expression were remarkably similar, with KCNQ4 being the predominant isoform in 42 out of 51 tissues (and second highest in the remaining 9 samples).\u003c/p\u003e \u003cp\u003eWe also assessed KCNQ expression in myometrium from women at term in active labor (TAL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; n\u0026thinsp;=\u0026thinsp;46). KCNQ profiles in TAL were similar to those found in TNL (KCNQ4\u0026thinsp;\u0026gt;\u0026thinsp;KCNQ3\u0026thinsp;\u0026gt;\u0026thinsp;KCNQ1\u0026thinsp;\u0026gt;\u0026thinsp;KCNQ5), with KCNQ4 remaining the most predominant transcript, providing evidence that at the mRNA level, K\u003csub\u003eV\u003c/sub\u003e7 channel components are still detectable, and hence available for pharmacological targeting, in labor.\u003c/p\u003e \u003cp\u003eAccessory subunits KCNE1-5 were analyzed in the same TNL and TAL samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). All KCNE genes were detected in both TNL and TAL samples, with KCNE4 being the most highly expressed isoform in both groups. The expression profile of KCNE genes was the same for both groups (TNL and TAL, KCNE4\u0026thinsp;\u0026gt;\u0026thinsp;KCNE3\u0026thinsp;\u0026gt;\u0026thinsp;KCNE5\u0026thinsp;=\u0026thinsp;KCNE1\u0026thinsp;\u0026gt;\u0026thinsp;KCNE2). KCNE2 expression was particularly low compared with other KCNE isoforms. KCNE3 showed a slightly lower expression in TNL [median, quartile range KCNE3: TNL: 737.3 (286.0, 1166) \u003cem\u003evs\u003c/em\u003e TAL: 915.0 (502.5, 2708).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn a separate experiment, using mRNA from a small set of myometrial tissues (provided by author D Slater), we compared expression from women who had Cesarean sections from preterm deliveries not in labor (PNL, n\u0026thinsp;=\u0026thinsp;4) and after spontaneous onset of preterm labor (PAL, n\u0026thinsp;=\u0026thinsp;5, \u0026lt;\u0026thinsp;37 weeks of gestation pregnancy), with TNL (n\u0026thinsp;=\u0026thinsp;6) and TAL (n\u0026thinsp;=\u0026thinsp;6) controls. We focused on the most highly expressed KCNQ and KCNE genes identified in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Both KCNQ4 and KCNE4 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB respectively) were detected in preterm tissues. Expression of KCNQ4 was high in PAL as it was in TNL and TAL samples. KCNQ4 expression in the PNL group was lower than that in TNL women (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A similar profile of mRNA expression was seen for KCNE4 and there were no significant differences between the groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, these data shown in human myometrium that KCNQ4 tissue expression remains consistent across term and preterm (in active labour) gestations.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKv7.1-5 and KCNE1-5 proteins in human myometrium\u003c/h3\u003e\n\u003cp\u003eFollowing the successful detection of KCNQ1, 3\u0026ndash;5 and KCNE1-5 transcripts in human myometrium, we determined whether transcripts were associated with protein expression in human myometrium using Western blot and commercially available antibodies Kv7.1-5/KCNE1-5 proteins \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We were able to detect Kv7.1, Kv7.3, Kv7.4, KCNE2, KCNE3 and KCNE4 proteins. Kv7.2, Kv7.5, KCNE1 and KCNE5 were not detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Of the two KCNE2 antibodies, only one antibody (APC-054, Alomone; epitope \u0026ndash; 88\u0026ndash;107 aa, rat origin) detected a faint protein band (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). APC-054 appears to detect the fully glycosylated protein, whereas the second antibody (sc-25703, Santa Cruz, epitope \u0026ndash; 1\u0026ndash;70 aa, human origin) detects the semi-mature monoglycosylated protein. Focusing on K\u003csub\u003eV\u003c/sub\u003e7.4, we quantified expression in whole tissue lysates from human myometrium in TNL and TAL samples. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB demonstrates that protein expression was similar in both groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemistry provided further confirmation of the presence and localization of K\u003csub\u003eV\u003c/sub\u003e7.1, K\u003csub\u003eV\u003c/sub\u003e7.3, K\u003csub\u003eV\u003c/sub\u003e7.4, KCNE3 and KCNE4 proteins in pregnant human myometrium smooth muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). All were expressed in smooth muscle cells and widely distributed across cells. Semi-quantification of the staining indicated that K\u003csub\u003eV\u003c/sub\u003e7.3 was slightly increased in TAL v TNL samples and K\u003csub\u003eV\u003c/sub\u003e7.4 reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C). Both proteins were present in laboring samples suggesting functional availability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePharmacological evidence for functional K7 channels in human myometrium\u003c/h3\u003e\n\u003cp\u003eEvidence for functional K\u003csub\u003eV\u003c/sub\u003e7.2-5 channels was gained through a series of \u003cem\u003ein vitro\u003c/em\u003e protocols which assessed the impact of pharmacological K\u003csub\u003eV\u003c/sub\u003e7 activators and inhibitors on TNL human myometrium contractility (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApplication of the K\u003csub\u003eV\u003c/sub\u003e7.2-5 activator retigabine (10 \u0026micro;M and 20 \u0026micro;M) to spontaneously contracting pregnant human myometrium (TNL) resulted in a reduction in mean integral tension (MIT) compared to vehicle time controls (dimethylsulfoxide, DMSO; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) providing evidence of functional K\u003csub\u003eV\u003c/sub\u003e7.2-5 channels in these tissues. ML213, a K\u003csub\u003eV\u003c/sub\u003e7.2/K\u003csub\u003eV\u003c/sub\u003e7.4 and K\u003csub\u003eV\u003c/sub\u003e7.5 activator caused a reduction in MIT at doses 5, 10 and 20 \u0026micro;M, indicating it is an effective uterine relaxant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThere was no significant effect of chromanol 293B (0.5\u0026ndash;20 \u0026micro;M), an inhibitor of K\u003csub\u003eV\u003c/sub\u003e7.1/KCNE1, on spontaneously contracting myometrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) indicating a limited contribution from this channel in human myometrium consistent with our previous mouse myometrium data \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, the pan Kv7 blocker XE991, at 10 and 20 \u0026micro;M had no significant effect on mean integral tension but significantly enhanced contractile frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E) and decreased contraction amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) compared to vehicle control in TNL pregnant human myometrium. This is consistent with XE991 causing a small depolarization of the resting membrane potential due to inhibition of K\u003csub\u003eV\u003c/sub\u003e7 channels.\u003c/p\u003e \u003cp\u003eThese data indicate that channels formed by K\u003csub\u003eV\u003c/sub\u003e7.2-7.5 are the major regulators of contractility in term pregnant human myometrium, and that modulators reported to have greater specificity for K\u003csub\u003eV\u003c/sub\u003e7.4 have greatest effect. K\u003csub\u003eV\u003c/sub\u003e7.1 has a limited functional contribution to contraction and an explanation for this may be that K\u003csub\u003eV\u003c/sub\u003e7.1 is forming channels with KCNE4 (which is the most abundant isoform) in myometrium; this combination is reported to suppress K\u003csub\u003eV\u003c/sub\u003e7.1 currents in model systems \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eK7 channels in myometrium from a mouse model of preterm birth\u003c/h3\u003e\n\u003cp\u003eWe have previously demonstrated the presence of functional of K\u003csub\u003eV\u003c/sub\u003e7 channels in non-pregnant and pregnant mouse myometrium (day 6\u0026ndash;7 and term) and in myometrium from mice injected with lipopolysaccharide (day 15, preterm gestation, but not in labor) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, there is limited knowledge of expression or function of myometrial K\u003csub\u003eV\u003c/sub\u003e7 channels related to preterm labor. To rectify this, \u003cem\u003eKcnq\u003c/em\u003e and \u003cem\u003eKcne\u003c/em\u003e profiles were assessed in myometrium harvested from a preterm mouse model (RU486 treated on day 15 of gestation to induce progesterone withdrawal and preterm labor and birth on ~\u0026thinsp;day 16) and vehicle (DMSO) treated control mice (Suppl. Figure\u0026nbsp;1). Myometrium was obtained from mice when they showed signs of labor (PTAL) and from control non-laboring pregnant mice on day 16 (14 hours after DMSO treatment, PTNL) for experiments shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003c/p\u003e \u003cp\u003eUsing myometrium harvested on day 16 of pregnancy, transcripts for all \u003cem\u003eKcnq\u003c/em\u003e genes were detected in both PTNL and PTAL myometrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). \u003cem\u003eKcnq1\u003c/em\u003e, \u003cem\u003eKcnq4\u003c/em\u003e and \u003cem\u003eKcnq5\u003c/em\u003e were highly expressed.\u003c/p\u003e \u003cp\u003eA separate comparison individual \u003cem\u003eKcnq\u003c/em\u003e genes expression data from PTNL versus PTAL myometrium revealed decreases in \u003cem\u003eKcnq4\u003c/em\u003e (median [IQR]; PTNL, 139.5 [114.6, 259.7]; PTAL, 83.6 [56.7, 106.2], p\u0026thinsp;=\u0026thinsp;0.02) and \u003cem\u003eKcnq5\u003c/em\u003e (PTNL, 630.8 [443.3, 1025.0]; PTAL, 113.2 [104.6, 351.1], p\u0026thinsp;=\u0026thinsp;0.01) mRNA expression after the onset of preterm labor. Although \u003cem\u003eKcnq1\u003c/em\u003e showed a t reduction in PTAL, this did not reach significance (p\u0026thinsp;=\u0026thinsp;0.065). No differences were observed for \u003cem\u003eKcnq2\u003c/em\u003e or \u003cem\u003eKcnq3\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAll \u003cem\u003eKcne\u003c/em\u003e genes were detected in PTNL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and PTAL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) samples with \u003cem\u003eKcne4\u003c/em\u003e being the most highly abundant in both groups (p\u0026thinsp;=\u0026thinsp;0.0001 for both).\u003c/p\u003e \u003cp\u003eComparison of \u003cem\u003eKcne\u003c/em\u003e gene expression data from PTNL versus PTAL myometrium showed significant reductions in \u003cem\u003eKcne2\u003c/em\u003e (median [IQR]; PTNL, 18040 [16183, 226228]; PTAL, 319.3 [231.4, 471.4]; p\u0026thinsp;=\u0026thinsp;0.002) and \u003cem\u003eKcne1\u003c/em\u003e expression (PTNL, 27.7 [13.1, 35.8]; PTAL, 5.4 [2.8, 8.6]; p\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eContribution of K\u003c/b\u003e \u003csub\u003e \u003cb\u003eV\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e7 channels to regulating\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003emyometrium contractile activity in a mouse model of preterm birth\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter characterizing the expression of \u003cem\u003eKcnq\u003c/em\u003e and \u003cem\u003eKcne\u003c/em\u003e genes, the functional expression of these channels was assessed using two K\u003csub\u003eV\u003c/sub\u003e7 activators, retigabine and ML213. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows baseline spontaneous contractility \u003cem\u003ein vitro\u003c/em\u003e in PTNL and PTL mice (n\u0026thinsp;=\u0026thinsp;6 for each concentration). Application of retigabine to spontaneously contracting myometrium from PTAL mice significantly decreased MIT by more than 60% at all concentrations tested (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; DMSO, 79.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7%; retigabine 5 \u0026micro;M, 32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5%; 10 \u0026micro;M, 28\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4%; 20 \u0026micro;M, 32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all as indicated by Dunn\u0026rsquo;s multiple comparison post hoc test performed after Kruskal-Wallis test; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In PTNL samples, significant decreases in MIT were only observed with 10 and 20 \u0026micro;M (DMSO, 91.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7%; retigabine 10 \u0026micro;M, 41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.6%; 20 \u0026micro;M, 16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1%; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Retigabine caused complete abolition of contractions in 45% and 50% of PTNL and PTAL samples, respectively.\u003c/p\u003e \u003cp\u003eSimilar to retigabine, ML213 inhibited contractile activity of PTAL myometrium at all concentrations (DMSO: 75.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5%; ML213 5 \u0026micro;M, 11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%; 10 \u0026micro;M: 10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8%; 20 \u0026micro;M: 11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). In PTNL myometrium, a significant relaxatory effect was only observed with 10 and 20 \u0026micro;M (DMSO: 94.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%; ML213 10 \u0026micro;M: 11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1%; 20 \u0026micro;M: 11.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for both; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The effect of retigabine and ML213 at 20 \u0026micro;M was not different between PTNL and PTAL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003emeasurement of intrauterine pressure (IUP)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWith confirmation that mRNA expression for all K\u003csub\u003eV\u003c/sub\u003e7 channel components could be detected in myometrium from PTAL and the pharmacological evidence that the channels were functional in myometrium assessed \u003cem\u003ein vitro\u003c/em\u003e, the next step was to determine the effect of K\u003csub\u003eV\u003c/sub\u003e7 channel activators on myometrial contractility in vivo, assessed with intrauterine pressure telemetry measurements and timing of birth (delivery of first pup as recorded on CCTV) in RU486 treated mice \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAcute gavage of two doses of retigabine, 90 minutes apart starting at 14 hours post RU486 demonstrated little impact on timing of delivery and hourly averaged IUP measurements in RU486 treated mice compared to administration of retigabine vehicle (0.5% hydroxymethylcellulose) (Suppl Fig.\u0026nbsp;2.). However, since time of delivery post RU486 showed some variation that potentially could have masked effects of retigabine, data were also analyzed to account for time of delivery (IUP displayed relative to time of delivery of first pup labor; t\u0026thinsp;=\u0026thinsp;0) and revealed a temporal decrease in IUP following the gavage treatment period. This suggested that short term exposure to retigabine altered uterine contractility and IUP in vivo, but not sufficiently to delay birth.\u003c/p\u003e\n\u003ch3\u003eRepeat dosing of with Kv7 activators delays birth in mouse model of preterm birth\u003c/h3\u003e\n\u003cp\u003eTo address the above observations, a repeat dosing regimen comprising of up to six gavage treatments (retigabine, ML213, or vehicle) was given every 60 minutes starting\u0026thinsp;~\u0026thinsp;13 hours post RU486 injection was employed.\u003c/p\u003e \u003cp\u003eRetigabine significantly delayed preterm birth, by increasing the median [IQR] time to delivery of first pup to 21.5 [20.5, 24.0] hours v 15.5 [15.1, 16.5] (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) hours for RU486 vehicle treated animals; this equated to an average of 39% (36 to 59%) delay in time to delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This is physiologically relevant if compared to the 1 hour it takes a mouse to deliver all pups and the ~\u0026thinsp;16 hours it takes from RU486 administration to induce preterm delivery.\u003c/p\u003e \u003cp\u003eML213 also delayed delivery (18.0 [16.5,20.1], p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) by ~\u0026thinsp;3 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e); this was slightly less effective at delaying delivery \u003cem\u003ein vivo\u003c/em\u003e compared to the tocolytic effect displayed \u003cem\u003ein vitro\u003c/em\u003e. However, this was anticipated as it is reported the drug had poor metabolic stability following incubation with rat or human liver microsomes and was predicted to be rapidly cleared after systemic exposure \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFetal viability and fetal ductus arteriosus function\u003c/h2\u003e \u003cp\u003eWe were unable to assess the impact on neonatal outcomes as in this PTB model, premature neonates are either born without signs of life/cannibalized by the mother due to their prematurity. This occurred even when gestation was prolonged by K\u003csub\u003eV\u003c/sub\u003e7 activator treatment.\u003c/p\u003e \u003cp\u003eHowever, in a separate experimental protocol, we assessed the impact of treatment with K\u003csub\u003eV\u003c/sub\u003e7 channel activators on \u003cem\u003eex vivo\u003c/em\u003e murine fetal ductus arteriosus (DA) contractility. This is an important consideration, as agents that constrict the ductus \u003cem\u003ein utero\u003c/em\u003e would impact on fetal perfusion and oxygen delivery \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Suppl. Figure\u0026nbsp;3 shows that under deoxygenated organ bath conditions that mimic \u003cem\u003ein utero\u003c/em\u003e oxygen status, fetal ductus arteriosus segments exposed to ML213 had no significant change in lumen diameter (10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M). DAs exposed to retigabine were similarly unaffected over the pharmacologic range (10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M) although at the highest concentration applied (a concentration unlikely to be used in \u003cem\u003ein vivo\u003c/em\u003e), a significant (p\u0026thinsp;=\u0026thinsp;0.0002) constriction was observed at the highest concentration (10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003eM) compared to baseline diameter. Because of this, there was a significant (p\u0026thinsp;=\u0026thinsp;0.004) difference in E\u003csub\u003emax\u003c/sub\u003e-values between these two drugs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides a clear rationale for pursuing K\u003csub\u003eV\u003c/sub\u003e7 channels as a potential target for tocolytic therapy and the prevention of preterm labor. Two structurally different activators of K\u003csub\u003eV\u003c/sub\u003e7.2-7.5 delayed early birth in a recognized mouse model of preterm birth. This important finding is supported by comprehensive molecular data and functional data from human pregnant myometrium and a preterm birth mouse model. These novel findings build upon our previous work which described the physiological function of K\u003csub\u003eV\u003c/sub\u003e7 channels in the non-pregnant and pregnant mouse \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eK7 channels as a plausible target for tocolytic targeting\u003c/h3\u003e\n\u003cp\u003eIon channels have long been viewed as important targets for therapeutic intervention due to their fundamental role in cellular physiology and human disease. Indeed, an estimated 10\u0026ndash;20% of small molecular targets are ion channels \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. K\u003csub\u003eV\u003c/sub\u003e channels represent a large gene superfamily that has been studied intensively in smooth muscles, including the myometrium, in terms of function \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31 CR32\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and in relation to K\u003csub\u003eV\u003c/sub\u003e7 channels this has led to a number of compounds being considered for treatment of epilepsy, hypertension, stroke, lung disease, overactive bladder, and neuropathic pain \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38 CR39\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, as far as we are aware, apart from our previous work \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e and information in RNAseq datasets (e.g., \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e) here is a paucity of knowledge concerning the functional role of K\u003csub\u003eV\u003c/sub\u003e7 channels in the uterus. In general, K channel activity contributes to suppression of uterine contractility in pregnancy through promoting a low resting potential, modifying action potentials (and hence contraction amplitude) through repolarization, and hence limiting calcium entry and contractility \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, as gestation nears term and labor (in both rodents and humans), there is a transition to greater myometrial cell excitability due to a gradual increase of the resting in resting membrane potential, most likely due to change in K\u003csup\u003e+\u003c/sup\u003e channel populations and activity \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. For a K\u003csup\u003e+\u003c/sup\u003e channel to be useful for inhibiting preterm labor, its expression must be maintained in labor. K\u003csub\u003eV\u003c/sub\u003e7.11 channels, for example, are expressed in late pregnancy, but their ability to influence repolarization is suppressed at labor onset through a variety of pathways \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Our previous pilot work in human myometrium suggested that K\u003csub\u003eV\u003c/sub\u003e7.2-5 were expressed in human myometrium in late pregnancy, but here we confirm through molecular profiling that KCNQ and KCNE mRNA are expressed both at the end of pregnancy and after the onset of labor. Of the different isoforms found, KCNQ4 and KCNE4 mRNAs were the most highly expressed transcripts in term labor and were also expressed in myometrium from women in preterm labor. This was reflected in K\u003csub\u003eV\u003c/sub\u003e7.4 and KCNE4 protein expression in myometrium from women at term and in labor. This identifies K\u003csub\u003eV\u003c/sub\u003e7.4 as a potential K\u003csub\u003eV\u003c/sub\u003e7 channel target in human myometrium \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Whilst transcripts for KCNQ5 were found and maintained in labour, we did not detect K\u003csub\u003eV\u003c/sub\u003e.5 protein. This does not fully rule out this as a target but more work on understanding protein expression is needed. KCNQ3 whilst expressed, is a key component of the neuronal M channel, so a less favorable target \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e along with Kv7.1 which is a cardiac associated K\u003csub\u003eV\u003c/sub\u003e channel.\u003c/p\u003e \u003cp\u003ePharmacological approaches using retigabine and ML213 provide clear evidence that activation of K\u003csub\u003eV\u003c/sub\u003e7.2-5 channels can suppress contractions in human myometrium \u003cem\u003ein vitro\u003c/em\u003e, an observation similar to that in term pregnant mice \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. ML213 is reported as a K\u003csub\u003eV\u003c/sub\u003e7.4, K\u003csub\u003eV\u003c/sub\u003e7.2 and K\u003csub\u003eV\u003c/sub\u003e7.5 channel activator, but given that K\u003csub\u003eV\u003c/sub\u003e7.2 was expressed at low levels in myometrium, these findings support the notion that K\u003csub\u003eV\u003c/sub\u003e7.4 channels is a plausible functional human myometrium target.\u003c/p\u003e \u003cp\u003eThe K\u003csub\u003eV\u003c/sub\u003e7 channel inhibitor, XE991 also altered human uterine contractility \u003cem\u003ein vitro\u003c/em\u003e providing additional evidence for functional K\u003csub\u003eV\u003c/sub\u003e7. In contrast, Kv7.1, whilst detectable in human myometrium as KCNQ1 transcripts, did not contribute functionally to myometrial contractility (i.e., not affected by chromanol application).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eK\u003csub\u003eV\u003c/sub\u003e7.2-5 channel activation prevented preterm labour\u003c/h2\u003e \u003cp\u003eTwo options for inducing PTB were considered, a lipopolysaccharide induced PTB (inflammation driven) and RU486 (progesterone/glucocorticoid block). The latter was chosen because it may be more reflective of idiopathic PTB, a clinical scenario that would benefit from an effective tocolytic. RU486 induction of PTB is rapid in the mouse due to dependence on progesterone for maintenance of pregnancy, so any delay in labor in this scenario provides a strong proof of principle effect.\u003c/p\u003e \u003cp\u003eThe effectiveness of Kv7.2-5 activation to prevent preterm labor was demonstrated in a mouse model where K\u003csub\u003eV\u003c/sub\u003e7 channels components, particularly \u003cem\u003ekcnq4 and kcnq5\u003c/em\u003e, were highly expressed in myometrium from preterm mice (prior to and after the onset of labour). Both retigabine and ML213 suppressed contractility in tissue \u003cem\u003eex vivo\u003c/em\u003e from preterm mice (not in labor and in labor).\u003c/p\u003e \u003cp\u003eThis effect was also confirmed \u003cem\u003ein vivo\u003c/em\u003e where intrauterine pressure monitoring indicated that retigabine reduced pressure (indication of reduced uterine contractility) in RU486 treated mice in the hours preceding delivery. Whilst a single dose of retigabine was insufficient to delay birth, repeated gavage of retigabine significantly delayed labor and birth of the first pup by six hours compared to controls.\u003c/p\u003e \u003cp\u003eML213 was also assessed, although we were aware prior to starting experimentation that it would quickly be metabolized by the liver when given orally. With this caveat, as a K\u003csub\u003eV\u003c/sub\u003e7.4 (and Kv7.4/5, Kv7.5) targeting agent it showed significant promise as it delayed preterm birth by three hours. Focus on the development of more favorable administration routes of an ML213 drug may be preferable.\u003c/p\u003e \u003cp\u003eIn the context of human pregnancy, this delay of preterm birth using KV7.2-5 activators has real potential to translate to prolongation of delivery for \u0026gt;\u0026thinsp;7 days or more, a delay that could significantly enhance human fetal maturity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and future work\u003c/h2\u003e \u003cp\u003eIn terms of limitations, there are several avenues available for further exploration. There is scope for further interrogation of the composition of the K\u003csub\u003eV\u003c/sub\u003e7.4 channels (homo or heterotetramers with other KCNQ isoform such as KCNQ5) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, or KCNE accessory subunits using proximity assays, imaging, patch clamp electrophysiology and transgenic mouse models.\u003c/p\u003e \u003cp\u003eIn the current study, we restricted functional studies to myometrium from women in late pregnancy as samples from women in spontaneous preterm labor are rare, but with time these experiments could be collated. The work in mouse models of PTB were designed to circumnavigate this issue and to provide an \u003cem\u003ein vivo\u003c/em\u003e solution. Uterine contraction mechanisms are similar between species, and tissues exhibited relevant KCNQ and KCNE expression profiles. However, before work can be translated into pregnant women this may necessitate additional studies in another animal model, particularly if more specific K\u003csub\u003eV\u003c/sub\u003e7.4 activators are to be developed. The side effects on other tissues and smooth muscles \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, which also express K\u003csub\u003eV\u003c/sub\u003e7.2-5 would also have to be monitored, although in the mouse model, retigabine was well tolerated.\u003c/p\u003e \u003cp\u003eThere is still a need to assess the impact of K\u003csub\u003eV\u003c/sub\u003e7 activators on neonatal outcome. In our study, neonatal wellbeing could not be reviewed due to the gestation-based immaturity of the mice in this model even when gestation was prolonged. However, we considered the impact of retigabine and ML213 \u003cem\u003eex vivo\u003c/em\u003e on the murine fetal ductus arteriosus, and whilst retigabine at the highest concentration caused some constriction, this was at concentrations not likely to be achieved \u003cem\u003ein vivo\u003c/em\u003e. In future studies, we plan to assess compounds in term pregnancy or a modified PTB model (induced later in gestation or milder stimulus) where neonates survive to determine neonatal developmental and behavior outcomes. To support translation of these findings into K\u003csub\u003eV\u003c/sub\u003e7.4 (or KV7.4/5) based tocolytics, protocols investigating different routes and drug delivery mechanisms may be useful.\u003c/p\u003e \u003cp\u003eIn summary, this study identifies K\u003csub\u003eV\u003c/sub\u003e7.2-5 activators as emerging candidates for tocolytic drug development. These activators can clearly suppress uterine contractility in human tissues and delay preterm birth in mice. There is compelling evidence that K\u003csub\u003eV\u003c/sub\u003e7.4 and associated accessory subunit KCNE4 are the targets for uterine specific drug development. Many pharmaceutical companies avoid investing in maternity, but with continued increase in PTB and the greatest burden being within the low-risk pregnant population there is a need that should be addressed \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhilst opportunities to explore drug repurposing have been affected by concerns about the long-term effects of retigabine and flupirtine \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, release of compounds from existing drug libraries and/or further development \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e could potentially facilitate research and drug development for more short-term use in this major area of health care. This could be underpinned with more precise uterine targeting using emerging and novel drug delivery systems \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis was an experimental laboratory study using human myometrial tissue, a mouse (C57BL/6J) model of preterm delivery, and an isolated fetal ductus arteriosus preparation (from CD-1 pregnant mice).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHuman myometrium studies\u003c/h2\u003e \u003cp\u003eHuman myometrium was obtained from 143 women with informed written consent, from women at term (\u0026gt;\u0026thinsp;37 weeks\u0026rsquo; gestation) at the time of elective not in labor (TNL) or emergency in labor (TAL) cesarean section, as approved by St Thomas\u0026rsquo; Hospital Ethics Committee (and subsequently regulated NRES Committee London-Westminster (EC00/137). For molecular and isometric tension measurement all were singleton pregnancies with exclusion criteria of adverse medical and obstetric conditions (including asthma requiring steroids, chronic hypertension requiring medication, lupus/systemic lupus erythematosus, diabetes, and pre-eclampsia) and the use of any medication that had potential to alter myometrial contractility (e.g., anti-hypertensives and inhaled bronchodilators). Biopsies were obtained from the upper edge of the lower segment incision and placed into ice-cold phosphate buffered saline. Tissue was rinsed and dissected on ice into small segments which were either snap frozen and stored at -80\u0026deg;C for RNA and protein analysis; formalin fixed, and wax embedded for immunohistochemical analysis; or kept at 4\u0026deg;C until used for isometric tension recording and cell isolation for patch clamp electrophysiology. Participant and pregnancy outcome data was retrieved from electronic and hand-held patient notes.\u003c/p\u003e \u003cp\u003ecDNA from a smaller number of human myometrium samples were obtained from D Slater we compared expression from women who had cesarean sections from preterm deliveries not in labor (PNL, n\u0026thinsp;=\u0026thinsp;4) and after spontaneous onset of preterm labor (PAL, n\u0026thinsp;=\u0026thinsp;5, \u0026lt;\u0026thinsp;37 weeks of gestation pregnancy), with TNL (n\u0026thinsp;=\u0026thinsp;6) and TAL (n\u0026thinsp;=\u0026thinsp;6) controls (Conjoint Health Research Ethics Board REB15-1110).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMouse model of preterm birth\u003c/h2\u003e \u003cp\u003eAcclimatized female C57BL/6J mice (Charles River) were mated and a copulation plug visualized one day post copulation (1 dpc) and subsequently weighed regularly. Mice were housed in constant temperature rooms (21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) with fixed 12-hour light: dark intervals and ad libitum access to standard chow and water. The progesterone receptor/glucocorticoid receptor antagonist RU486 (mifepristone; Merck Life Science, UK) was used to artificially induce preterm birth as previously published \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e (Suppl. Figure\u0026nbsp;1). On 15 dpc, mice were restrained and a subcutaneous injection containing 150 \u0026micro;g RU486 [dissolved in DMSO (Merck) at a concentration of 1 \u0026micro;g/\u0026micro;l] administered in the nape of the neck. Control mice were injected with 150 \u0026micro;l of DMSO. All injections were administered at the same time of day and mice were continuously monitored by video so that the timing of gestation (defined as time to deliver first pup) could be accurately determined.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e contraction studies, myometrium was harvested in RU486 mice when they showed signs of labor (preterm in labor, PTL) and in parallel with tissue from control non-laboring pregnant mice at the same gestation (preterm not in labor, PTNL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of intrauterine pressure using radio telemetry in mouse model of preterm birth\u003c/h2\u003e \u003cp\u003eThis followed the method developed by Pierce and colleagues \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. C57BL/6J mice were anesthetized with 3% isoflurane/oxygen in an induction chamber at day 8\u0026ndash;10 dpc. Once anesthesia had been achieved, mice were transferred to a warming pad, placed in a supine position and a nose cone was used to deliver maintenance anesthesia (2% isoflurane/oxygen) for the duration of the surgical procedure. The abdomen was shaved and disinfected with a 0.5% (w/v) chlorhexidine gluconate solution (Medlock Medical Ltd., UK) and the work surface was replaced with a sterile cover. The fore- and hind-limbs were secured to the work surface, and 3 \u0026micro;g of buprenorphine (Vetergesic) (Sogeval UK Limited, York, UK) was injected intra-muscularly (I.M.) before a midline incision approximately 3 cm in length was made. The right and left uterine horns were identified, carefully exteriorized and the pups in each horn counted. One uterine horn was chosen for implantation of the pressure radio-telemetry probe, whilst the other horn was covered in saline-soaked gauze to prevent drying. Dumont tweezers were used to hold a small fold of the uterine wall, and a 1 mm incision was made using 3 mm Vanna scissors. Vessel cannulation forceps were used to feed the catheter of the probe into the incision of the uterus. Blunt-end forceps were used to manipulate the path of the catheter until the opening of the catheter was located between two fetal sacs. The catheter was kept in place by applying a drop of Vetbond (3M, USA) to the site of catheter insertion and the uterus was carefully placed back into the abdominal cavity, followed by the probe body, which was placed in the lower portion of the abdominal cavity. The probe was carefully placed so as to not introduce any acute bending or twisting of the catheter that could interfere with pressure measurements. The abdominal muscle layer and skin were each closed with a simple continuous suture pattern using a braided absorbable suture. Anesthesia was withdrawn and mice placed into a recovery chamber held at 28\u0026deg;C overnight with access to softened standard chow. Additional analgesia (3 \u0026micro;g buprenorphine, S.C.) was given to mice the following morning. Mice were then placed in a cage over a \u0026lsquo;receiver mat,\u0026rsquo; which was connected via a matrix to a computer for remote data acquisition. Mice were free to move during the recording period. From day 13\u0026ndash;14 dpc, intrauterine pressure (IUP) was recorded continuously until one day post-partum (approximately 19.5 dpc) or until delivery occurred.\u003c/p\u003e \u003cp\u003eContinuous IUP data was averaged over one-minute intervals for all treatment groups using the manufacturers supplied software (Dataquest A.R.T, UK). A baseline period prior to any intervention was used to standardise intrauterine pressure. All data are expressed as a fold change in IUP from baseline. For mice delivering at term, a baseline period of 12 hours prior to delivery was used so that data from all groups was comparable. Every 60 minutes, pressure recordings were averaged to obtain a mean hourly pressure over a 17-hour period. For all groups, labor was defined as time 0 as indicated by the complete delivery of first pup. Some mice displayed negative IUP values in certain hours which were due to movement and occasional blockage of the catheter (DSI, personal communication), therefore for any hourly averages which included less than 75% positive values, negative values were excluded from the analysis. In addition, mice that had a negative average IUP baseline were excluded from data analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eadministration of K\u003c/b\u003e\u003csub\u003e\u003cb\u003eV\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e7 activators in preterm birth mouse model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAcute treatment: On 16 dpc, 14 hours after RU486 administration, C57BL/6J mice were administered a single 20 mg/kg retigabine (Merck) or vehicle (10% (v/v) DMSO in 0.5% hydroxymethylcellulose solution) via oral gavage. For retigabine or ML213, 5 mg was first dissolved into 100 \u0026micro;l DMSO to which 900 \u0026micro;l 0.5% hydroxymethylcellulose was added to achieve a final concentration of 5 mg/ml. A second administration was given 90 minutes later. This was based on pharmacokinetic profiles of retigabine administration to female non-pregnant mice (carried out by Quotient Bioresearch, UK, data not shown) which reported that peak plasma concentrations of retigabine (6930 ng/ml) were achieved approximately 60 minutes after oral gavage. Repeated gavage: retigabine or ML213 (20 mg/kg, P.O.) or vehicle were prepared similarly as described for acute treatment above and were administered at 60-minute intervals starting 13 hours after RU486 injection. Doses were repeated for a maximum of 6 treatments, or until delivery of the first pup had occurred.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePreterm birth mouse model myometrial tissue collection\u003c/h2\u003e \u003cp\u003eFor murine myometrium, pregnant mice were sacrificed by cervical dislocation and the gravid uterus was carefully removed from the peritoneum. Uterine horns were cut along the axis of placental attachment, and the fetus and placenta were excised. Endometrial tissue was gently removed by brushing with a cotton bud. Tissues were either snap frozen in liquid nitrogen for mRNA extraction or used immediately for isometric tension measurement studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePreterm birth mouse model isometric tension recording\u003c/h2\u003e \u003cp\u003eThis is a modified protocol based on a method published previously \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In brief, small longitudinal myometrial strips (human: ~7x2x2 mm; mouse: ~ 5x2x2 mm) were mounted and maintained at 37\u0026deg;C in organ baths (oxygenated, 95% O2, 5% CO2) in physiological salt solution (PSS, pH 7.4 in mM: NaCl 119, KCl 4.7, MgSO4 1.17, NaHCO3 25, KH2PO4 1.18, EDTA 0.025, glucose 6 and CaCl2 2.5). Mouse and human myometrial tissues were subjected to stretch to approximately 1.5-fold their slack length (by applying between 29.34 to 38.25 mN respectively). After development of regular spontaneous contractile activity with a stable baseline, a control period of contractile activity was recorded prior to the addition of either KV7 activators [retigabine, ICA-069673 (Santa Cruz) and ML213 (Tocris)] or inhibitors [XE991 and chromanol 293B (Sigma)] (0.5\u0026ndash;20 \u0026micro;M for all compounds). Vehicle control (DMSO) was carried out in parallel.\u003c/p\u003e \u003cp\u003eContractility data were recorded and analyzed using LabChart 6 software (ADInstruments, UK) and mean integral tension (MIT) for an experimental period, addition of Kv7 activator or vehicle, MIT (the sum of the integrals for each contraction divided by the duration of the period assessed) was expressed as a percentage of the preceding control (baseline) period MIT. Contraction frequency and amplitude of contractions were also assessed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHuman and mouse myometrium mRNA extraction and real-time qRT-PCR\u003c/h2\u003e \u003cp\u003eFrozen myometrial tissue (~\u0026thinsp;100 mg of human or mouse) was homogenized using a Qiagen Tissue Lyser. Total RNA was extracted using the RNeasy mini kit (Qiagen, UK) according to the manufacturer's instructions. Complementary deoxyribonucleic acid (cDNA) was synthesized using an Omniscript RT Kit (Qiagen, UK). Real-time polymerase chain reaction (PCR) was carried out with the use of SYBR Green chemistry (Bioline) on a RotorGene 6000 (Qiagen, UK) using human primers as reported in \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and for mouse as listed in Supplementary Table\u0026nbsp;1. A pre-PCR cycle was run for 10 min at 95\u0026deg;C followed by 35 cycles of 95\u0026deg;C for 15 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for 50 s followed by a final extension at 72\u0026deg;C for 15 s. Melt curve analysis was performed to confirm the presence of one single product. Cycle threshold (CT) values were used for analysis, and abundance data were obtained using quantified cDNA to generate a standard curve. All unknowns fell within the dynamic range of the standard curve. Standards were quantified using densitometry, and tenfold serial dilution from 1010 to 101 copies was run in parallel with the samples. Abundance data for the genes of interest were expressed as normalized copy number following normalization using GeNORM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://medgen.ugent.be/~jvdesomp/genorm\u003c/span\u003e\u003cspan address=\"http://medgen.ugent.be/~jvdesomp/genorm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with stably expressed reference genes (glyceraldehyde 3-phosphate dehydrogenase (GADPH). Data for the genes of interest were normalized (GeNorm) to mRNA copy number of a panel of genes (GAPDH, β-actin and β-2 microglobulin). All PCR products were sequenced to confirm identity. For analysis of human myometrium KCNQ4 and KCNE4 mRNA expression comparing preterm and term samples, because of limited material, only the housekeeper GAPDH was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry studies of pregnant human myometrium\u003c/h2\u003e \u003cp\u003eSerial sections of paraffin embedded human myometrial tissues were cut (5 \u0026micro;m) and dewaxed from paraffin-embedded tissue blocks (using a Leica RM 2065 microtome, Leica Biosystems, UK) as previously described \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Immunohistochemical staining was performed using the Vector Stain Elite ABC kit (Vector Laboratories, UK). The optimal dilution for each antibody was established (Supplementary Tables\u0026nbsp;2 \u0026amp; 3). A positive control (human midbrain) was used to verify specificity. A negative control was performed for each test section by incubation with goat IgG. All slides were assessed by the same observer, blinded to labor group. Digital images of 5 randomly selected, high-power (\u0026times;400 magnification) fields were captured on NIS-Elements F2.20 microscope (Nikon United Kingdom Ltd, UK). Quantifications of KCNQx and KCNEx expression was performed as described previously \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e using the Positive Pixel Algorithm of Aperio ImageScope software. A visual check was also performed to ensure accurate discrimination of immunolabeled regions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE and Western Immunoblotting\u003c/h2\u003e \u003cp\u003eProtein samples for Western Blot were prepared by homogenisation of human myometrial tissues (TNL samples, n\u0026thinsp;=\u0026thinsp;4; TAL samples, n\u0026thinsp;=\u0026thinsp;4) using a Tissue Lyser II (Qiagen, UK) at 25hZ for 3 min in 20 \u0026micro;l lysis buffer/mg of tissue (10 mM of Hepes-KOH [pH 7], 1 mM of dithiothreitol, 1% nonident-P40, and protease-inhibitor cocktail (COMPLETE tablets; Boehringer-Mannheim Biochemicals, UK) and subsequent centrifugation for 1 min at 12470 x g to remove any tissue debris. Mouse brain, heart or aorta tissues were used as a positive control for a detection of variety KV7x/KCNEx proteins and were prepared in the same way as the human myometrium samples. Total protein concentration was calculated using BCA assay (Pierce, UK) and 30 \u0026micro;g of proteins were loaded to each lane of 12% Tris-Glycine precast gels (Generon, UK) and separated using the XCell SureLock\u0026trade; Mini-Cell system (Invitrogen, UK). Following electrophoresis, proteins were transferred to Immobilion\u0026trade;-P transfer membrane (Millipore) using the XCell SureLock\u0026trade; Mini-Cell blotting module wet transfer blotting system. 50mM Tris, 150mM NaCl, 0.2% (v/v) Tween-20, pH 7.4) containing 5% fat free milk powder for 1 hour at room temperature. After the transfer, membranes were blocked in TBS-T buffer (50mM Tris, 150mM NaCl, 0.2% (v/v) Tween-20, pH 7.4) supplemented with 5% milk (Sigma-Aldrich) for 1 hour at room temperature, followed by 3 hours incubation with primary antibody at RT. All KV7x and KCNEx antibodies used in this study are listed in Supplementary Table\u0026nbsp;4. Monoclonal mouse anti-smooth muscle alpha actin (Sigma, A2547) was used as a smooth muscle protein marker and a loading control. After the first antibody incubation, membranes were washed three times in TBS-T and HRP-conjugated anti-rabbit, anti-goat or anti-mouse IgGs secondary antibody was added (1:10000 dilution, Santa Cruz). The secondary antibody was incubated for 45 minutes at room temperature, followed by three washes in TBS-T buffer. To detect the antibody, chemiluminescent reagent (Invitrogen) was added, and the blots were exposed to X-ray film.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMouse ductus arteriosus (DA) vessel studies\u003c/h2\u003e \u003cp\u003e Animal experiments were conducted in accordance with the National Institutes of Health Animal Care Standards and were approved by the Institutional Animal Care and Use Committee at Vanderbilt University Medical Center. Adult female CD-1 mice were bred by timed mating between 0700\u0026ndash;1000 hrs with the presence of a vaginal plug indicating the first day of pregnancy (d1). Pregnant females were anesthetized on the morning of d19 (term) via intraperitoneal injection of 0.4mL of 1.25% avertin (2,2,2-tribromoethanol in tert-amyl alcohol, Sigma-Aldrich, St. Louis, MO), followed by isoflurane inhalation (Baxter) for fetal anesthesia, and cervical dislocation of the dam. The anesthetized fetuses were removed by cesarean section. Their thoracic cavities were opened via partial dissection and puncture of the diaphragm, and the pups were then submerged in chilled, deoxygenated (95% N2, 5% CO2) modified Krebs (in mM; 109 NaCl, 4.7 KCl, 2.5 CaCl22H2O, 0.9 MgSO4, 1.0 KH2PO4, 11.1 glucose, 34 NaHCO3 (pH 7.3)) (2). Any pups with signs of respiration were excluded from vessel myography.\u003c/p\u003e \u003cp\u003eDA segments representing at least five different litters were freshly isolated from the d19 fetuses for use in myography studies. Each DA was excised, mounted in custom myography chambers (University of Vermont), and allowed to equilibrate for 40 min at 5 mmHg and 37\u0026deg;C in deoxygenated modified Krebs buffer. The vasoreactivity of each vessel was ascertained using cannulated, pressurized vessel myography as previously described \u003csup\u003e\u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Inverted light microscopes with video capture (IonOptix) were used to continuously record lumen diameter. After the equilibration period, distending pressure was raised by 5 mmHg in 10 min increments to 20 mmHg, followed by two 10-minute exposures to deoxygenated 50 mM KCl with a 20 min wash step of deoxygenated Krebs in between to validate viability and reactivity. After a second 20 min wash step, the chambers were changed from a flow-through system to a 20 mL recirculating volume, and vessels were re-equilibrated for 20 min. At the end of this period, the intraluminal diameter was recorded and used as the baseline value to compare drug-related changes in DA tone. Vessels were exposed to increasing concentrations (10\u0026thinsp;\u0026minus;\u0026thinsp;8 to 10\u0026thinsp;\u0026minus;\u0026thinsp;4 M) of either retigabine (Axon Medchem; n\u0026thinsp;=\u0026thinsp;10) or ML213 (Tocris Bioscience; n\u0026thinsp;=\u0026thinsp;11) in 20 min increments, and changes in lumen diameter after each concentration increase were documented and compared.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eFormal power calculations determined the samples for KCNQ and KCNE mRNA expression profiles and N numbers for main experiments. For comparison of KCNQ/KCNE genes within pregnant human myometrium tissue, an n of 21 samples per experimental group was calculated to provide 90% power to detect a ratio of five between genes at the group-wise 5% significance level (alpha\u0026thinsp;=\u0026thinsp;0.001; S.D. = 0.699) correcting for multiple testing using the Sidak-Bonferroni correction. This was based on data generated from previous experiments \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. To assess changes in KCNQ/KCNE genes in tissues taken from women at term prior to labor (TNL) versus women in labor (TAL), an n of 42 (21 per sample group) was calculated to provide 90% power to detect a ratio of five (corresponding to a standardised ratio of 3.5 from previous work) between groups (TNL Vs. TAL) at the group-wise 5% significance level (alpha\u0026thinsp;=\u0026thinsp;0.0051; SD\u0026thinsp;=\u0026thinsp;0.544) correcting for multiple testing as above. To determine the effect of K\u003csub\u003eV\u003c/sub\u003e7 modulators on contractile activity of pregnant human myometrium, pilot data has found a consistent standard deviation within treatment groups of 16.6%. With at least n\u0026thinsp;=\u0026thinsp;4 observations in the control and treatment group, there is 90% power to detect a 33% difference in contractility between the controls and the active treatment. For animal studies, considering the 3R\u0026rsquo;s, experimental protocols used between n\u0026thinsp;=\u0026thinsp;6\u0026ndash;9 animals. For the DA vessel experiments, changes in lumen diameter were analyzed as percent change from the baseline and plotted as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. A comparison of fit determined whether three parameter non-linear log fit lines were significantly different between ML213 and retigabine. Two-way analysis of variance followed by a post hoc Dunnett\u0026rsquo;s test was used to determine significant differences in the % change from baseline for each concentration of drug. Two-way analysis of variance followed by a post hoc Sidak\u0026rsquo;s test was used to determine significant differences between the Emax values of ML213 and retigabine.\u003c/p\u003e \u003cp\u003eAll tests were performed using SPSS for Windows version 27 and GraphPad PRISM version 9. The Kolmogorov-Smirnov test was used to evaluate normality of data distribution and summary data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median [interquartile range (IQR)] as appropriate for data distribution. Between-group comparisons were made using Kruskal-Wallis and post hoc Dunn\u0026rsquo;s test or Mann-Whitney. The null hypothesis was rejected where P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eAuthors declare that they have no competing interest\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData and Materials availability\u003c/strong\u003e \u003cp\u003eAll experimental data associated with this study are available in the main text or the supplementary materials. Limited residual human tissue and associated data available (subject to ethical approval and a specifically negotiated MTA with KCL and Guys and St Thomas\u0026rsquo; Foundation Trust). Requests to be directed to the corresponding author.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was directly funded by an MRC project grant (G1100243), an MRC DPT PhD studentship to YTM and Tommy\u0026rsquo;s charity and underpinned by preliminary data funded by Action Medical Research and Rosetrees Trust grant (SP4298). HDM was funded during this period by a British Heart Foundation Intermediate Basic Science Fellowship (FS/15/32/31604) and a British Heart Foundation Project Grant (PG/11/22/28800).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003e \u003cb\u003e(CRediT)\u003c/b\u003e: Conceptualization: RMT, IAG, HDM, MRJ, PDT, SKE, DMS, JLH, JR; Methodology: RMT, PDT, YTM, SKE, DMS, ECCS, HDM, JR; Investigation: YTM, RMT, PDT, MKZ, ECCS, RP, HDM, JR, JLH, DCS; Visualization: YTM, RMT, ECCS, HDM, MKZ, JR, JLH; Funding Acquisition: RMT, IAG, PIA, MRJ, PDT, HDM; Project Administration: RMT; Supervision: RMT, HDM, PDT, JR; Writing original draft: RMT, YTM, HDM; Writing \u0026ndash; reviewing and editing: YTM, HDM, ECCS, MKZ, JR, JLH, DCS, RP, PIA, DMS, SKE, IAG, MRJ, PDT, RMT.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eWe are particularly grateful to the women who consented to participating in the study and Prof. Andrew Shennan and the NIHR RDN funded team of research midwives and research assistants who supported recruitment, consent, and tissue collection. We also thank the support of staff at the KCL Biological Services facility and Prof Raheela Khan for support with immunohistochemistry.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChawanpaiboon, S.\u003cem\u003e, et al.\u003c/em\u003e Global, regional, and national estimates of levels of preterm birth in 2014: a systematic review and modelling analysis. \u003cem\u003eLancet Glob Health\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e37-e46 (2019).\u003c/li\u003e\n\u003cli\u003eFrey, H.A. \u0026amp; Klebanoff, M.A. The epidemiology, etiology, and costs of preterm birth. \u003cem\u003eSemin Fetal Neonatal Med\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 68-73 (2016).\u003c/li\u003e\n\u003cli\u003eRubens, C.E.\u003cem\u003e, et al.\u003c/em\u003e Prevention of preterm birth: harnessing science to address the global epidemic. \u003cem\u003eSci Transl Med\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 262sr265 (2014).\u003c/li\u003e\n\u003cli\u003eMartin, J.N., Jr., D\u0026apos;Alton, M., Jacobsson, B. \u0026amp; Norman, J.E. In pursuit of progress toward effective preterm birth reduction. \u003cem\u003eObstet Gynecol\u003c/em\u003e \u003cstrong\u003e129\u003c/strong\u003e, 715-719 (2017).\u003c/li\u003e\n\u003cli\u003eMcDougall, A.R.A.\u003cem\u003e, et al.\u003c/em\u003e New medicines for spontaneous preterm birth prevention and preterm labour management: landscape analysis of the medicine development pipeline. \u003cem\u003eBMC Pregnancy Childbirth\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 525 (2023).\u003c/li\u003e\n\u003cli\u003eNICE. Overview: Preterm labour and birth, guidance. (NICE, 2015).\u003c/li\u003e\n\u003cli\u003ePractice bulletin No. 171 summary: management of peterm labor. \u003cem\u003eObstet Gynecol\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 931-933 (2016).\u003c/li\u003e\n\u003cli\u003eHaas, D.M., Caldwell, D.M., Kirkpatrick, P., McIntosh, J.J. \u0026amp; Welton, N.J. Tocolytic therapy for preterm delivery: systematic review and network meta-analysis. \u003cem\u003eBMJ\u003c/em\u003e \u003cstrong\u003e345\u003c/strong\u003e, e6226 (2012).\u003c/li\u003e\n\u003cli\u003eWray, S. \u0026amp; Arrowsmith, S. Uterine excitability and ion channels and their changes with gestation and hormonal environment. \u003cem\u003eAnnu Rev Physiol\u003c/em\u003e \u003cstrong\u003e83\u003c/strong\u003e, 331-357 (2021).\u003c/li\u003e\n\u003cli\u003eBarrese, V., Stott, J.B. \u0026amp; Greenwood, I.A. KCNQ-encoded potassium channels as therapeutic targets. \u003cem\u003eAnnu Rev Pharmacol Toxicol\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 625-648 (2018).\u003c/li\u003e\n\u003cli\u003eMcCallum, L.A., Pierce, S.L., England, S.K., Greenwood, I.A. \u0026amp; Tribe, R.M. The contribution of Kv7 channels to pregnant mouse and human myometrial contractility. \u003cem\u003eJ Cell Mol Med\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 577-586 (2011).\u003c/li\u003e\n\u003cli\u003eMcCallum, L.A., Greenwood, I.A. \u0026amp; Tribe, R.M. Expression and function of K(v)7 channels in murine myometrium throughout oestrous cycle. \u003cem\u003ePflugers Arch\u003c/em\u003e \u003cstrong\u003e457\u003c/strong\u003e, 1111-1120 (2009).\u003c/li\u003e\n\u003cli\u003ePeretz, A.\u003cem\u003e, et al.\u003c/em\u003e Meclofenamic acid and diclofenac, novel templates of KCNQ2/Q3 potassium channel openers, depress cortical neuron activity and exhibit anticonvulsant properties. \u003cem\u003eMol Pharmacol\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 1053-1066 (2005).\u003c/li\u003e\n\u003cli\u003eRoepke, T.K.\u003cem\u003e, et al.\u003c/em\u003e KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium. \u003cem\u003eFASEB J\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 4264-4273 (2011).\u003c/li\u003e\n\u003cli\u003eAnderson, U.A.\u003cem\u003e, et al.\u003c/em\u003e Functional expression of KCNQ (Kv7) channels in guinea pig bladder smooth muscle and their contribution to spontaneous activity. \u003cem\u003eBr J Pharmacol\u003c/em\u003e \u003cstrong\u003e169\u003c/strong\u003e, 1290-1304 (2013).\u003c/li\u003e\n\u003cli\u003eLiu, W.J.\u003cem\u003e, et al.\u003c/em\u003e Manipulation of KCNE2 expression modulates action potential duration and Ito and IK in rat and mouse ventricular myocytes. \u003cem\u003eAm J Physiol Heart Circ Physiol\u003c/em\u003e \u003cstrong\u003e309\u003c/strong\u003e, H1288-1302 (2015).\u003c/li\u003e\n\u003cli\u003eSachse, C.C.\u003cem\u003e, et al.\u003c/em\u003e BACE1 and presenilin/gamma-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels. \u003cem\u003eFASEB J\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 2458-2467 (2013).\u003c/li\u003e\n\u003cli\u003eZhang, M.\u003cem\u003e, et al.\u003c/em\u003e KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner. \u003cem\u003eAm J Physiol Heart Circ Physiol\u003c/em\u003e \u003cstrong\u003e302\u003c/strong\u003e, H910-922 (2012).\u003c/li\u003e\n\u003cli\u003eClancy, S.M., Chen, B., Bertaso, F., Mamet, J. \u0026amp; Jegla, T. KCNE1 and KCNE3 beta-subunits regulate membrane surface expression of Kv12.2 K(+) channels in vitro and form a tripartite complex in vivo. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e6330 (2009).\u003c/li\u003e\n\u003cli\u003eGrunnet, M.\u003cem\u003e, et al.\u003c/em\u003e KCNE4 is an inhibitory subunit to the KCNQ1 channel. \u003cem\u003eJ Physiol\u003c/em\u003e \u003cstrong\u003e542\u003c/strong\u003e, 119-130 (2002).\u003c/li\u003e\n\u003cli\u003eLundquist, A.L.\u003cem\u003e, et al.\u003c/em\u003e Expression of multiple KCNE genes in human heart may enable variable modulation of I(Ks). \u003cem\u003eJ Mol Cell Cardiol\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 277-287 (2005).\u003c/li\u003e\n\u003cli\u003eYu, H.\u003cem\u003e, et al.\u003c/em\u003e A small molecule activator of KCNQ2 and KCNQ4 channels. in \u003cem\u003eProbe Reports from the NIH Molecular Libraries Program\u003c/em\u003e (Bethesda (MD), 2010).\u003c/li\u003e\n\u003cli\u003eVillegas-Esguevillas, M.\u003cem\u003e, et al.\u003c/em\u003e The novel K(V)7 channel activator URO-K10 exerts enhanced pulmonary vascular effects independent of the KCNE4 regulatory subunit. \u003cem\u003eBiomed Pharmacother\u003c/em\u003e \u003cstrong\u003e164\u003c/strong\u003e, 114952 (2023).\u003c/li\u003e\n\u003cli\u003eGrand View Research. Ion channels in drug discovery, 2023 report. (Martket Trends Reports, 2023).\u003c/li\u003e\n\u003cli\u003eRogers, M. Ion channel drug discovery and modern medicine. 9-11 (Metrion Biosciences, 2019).\u003c/li\u003e\n\u003cli\u003ePereira da Silva, E.A., Martin-Aragon Baudel, M., Navedo, M.F. \u0026amp; Nieves-Cintron, M. Ion channel molecular complexes in vascular smooth muscle. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 999369 (2022).\u003c/li\u003e\n\u003cli\u003eMalysz, J. \u0026amp; Petkov, G.V. Detrusor Smooth Muscle K(V)7 Channels: Emerging New Regulators of Urinary Bladder Function. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1004 (2020).\u003c/li\u003e\n\u003cli\u003eSmith, R.C., McClure, M.C., Smith, M.A., Abel, P.W. \u0026amp; Bradley, M.E. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility. \u003cem\u003eReprod Biol Endocrinol\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 41 (2007).\u003c/li\u003e\n\u003cli\u003eTribe, R.M. \u0026amp; Taggart, M.J. Cellular ionic mechanisms controlling uterine smooth muscle contraction: effects of gestational state. in \u003cem\u003eNew Frontiers in Smooth Muscle Biology and Physiology\u003c/em\u003e, Vol. 1 (ed. Savinneau, J.P.) 523-549 (Research Signpost, India, 2007).\u003c/li\u003e\n\u003cli\u003eBao, J.\u003cem\u003e, et al.\u003c/em\u003e BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation micedagger. \u003cem\u003eBiol Reprod\u003c/em\u003e \u003cstrong\u003e110\u003c/strong\u003e, 798-807 (2024).\u003c/li\u003e\n\u003cli\u003eParkington, H.C.\u003cem\u003e, et al.\u003c/em\u003e Diminished hERG K+ channel activity facilitates strong human labour contractions but is dysregulated in obese women. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 4108 (2014).\u003c/li\u003e\n\u003cli\u003eCowles, C.L.\u003cem\u003e, et al.\u003c/em\u003e Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization. \u003cem\u003eBiol Reprod\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 122 (2015).\u003c/li\u003e\n\u003cli\u003eJepps, T.A., Carr, G., Lundegaard, P.R., Olesen, S.P. \u0026amp; Greenwood, I.A. Fundamental role for the KCNE4 ancillary subunit in Kv7.4 regulation of arterial tone. \u003cem\u003eJ Physiol\u003c/em\u003e \u003cstrong\u003e593\u003c/strong\u003e, 5325-5340 (2015).\u003c/li\u003e\n\u003cli\u003ePerucca, E. \u0026amp; Taglialatela, M. Targeting Kv7 potassium channels for epilepsy. \u003cem\u003eCNS Drugs\u003c/em\u003e (2025).\u003c/li\u003e\n\u003cli\u003eRejdak, K., Luszczki, J.J., Blaszczyk, B., Chwedorowicz, R. \u0026amp; Czuczwar, S.J. Clinical utility of adjunctive retigabine in partial onset seizures in adults. \u003cem\u003eTher Clin Risk Manag\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 7-14 (2012).\u003c/li\u003e\n\u003cli\u003eZhang, H.\u003cem\u003e, et al.\u003c/em\u003e Neuroprotective Effects, Mechanisms of Action and Therapeutic Potential of the Kv7/KCNQ Channel Opener QO-83 in Ischemic Stroke. \u003cem\u003eTransl Stroke Res\u003c/em\u003e (2025).\u003c/li\u003e\n\u003cli\u003eZhang, B.\u003cem\u003e, et al.\u003c/em\u003e Discovery of E0199: A novel compound targeting both peripheral Na(V) and K(V)7 channels to alleviate neuropathic pain. \u003cem\u003eJ Pharm Anal\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 101132 (2025).\u003c/li\u003e\n\u003cli\u003eAlles, S.R.A. \u0026amp; Smith, P.A. Peripheral Voltage-Gated Cation Channels in Neuropathic Pain and Their Potential as Therapeutic Targets. \u003cem\u003eFront Pain Res (Lausanne)\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 750583 (2021).\u003c/li\u003e\n\u003cli\u003eMondejar-Parreno, G., Perez-Vizcaino, F. \u0026amp; Cogolludo, A. Kv7 Channels in Lung Diseases. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 634 (2020).\u003c/li\u003e\n\u003cli\u003eJepps, T.A., Olesen, S.P. \u0026amp; Greenwood, I.A. One man\u0026apos;s side effect is another man\u0026apos;s therapeutic opportunity: targeting Kv7 channels in smooth muscle disorders. \u003cem\u003eBr J Pharmacol\u003c/em\u003e \u003cstrong\u003e168\u003c/strong\u003e, 19-27 (2013).\u003c/li\u003e\n\u003cli\u003eGreenwood, I.A. \u0026amp; Tribe, R.M. Kv7 and Kv11 channels in myometrial regulation. \u003cem\u003eExp Physiol\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 503-509 (2014).\u003c/li\u003e\n\u003cli\u003eChan, Y.W., van den Berg, H.A., Moore, J.D., Quenby, S. \u0026amp; Blanks, A.M. Assessment of myometrial transcriptome changes associated with spontaneous human labour by high-throughput RNA-seq. \u003cem\u003eExp Physiol\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 510-524 (2014).\u003c/li\u003e\n\u003cli\u003eCasteels, R. \u0026amp; Kuriyama, H. Membrane potential and ionic content in pregnant and non-pregnant rat myometrium. \u003cem\u003eJ Physiol\u003c/em\u003e \u003cstrong\u003e177\u003c/strong\u003e, 263-287 (1965).\u003c/li\u003e\n\u003cli\u003eParkington, H.C., Tonta, M.A., Brennecke, S.P. \u0026amp; Coleman, H.A. Contractile activity, membrane potential, and cytoplasmic calcium in human uterine smooth muscle in the third trimester of pregnancy and during labor. \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e \u003cstrong\u003e181\u003c/strong\u003e, 1445-1451 (1999).\u003c/li\u003e\n\u003cli\u003eGreenwood, I.A., Yeung, S.Y., Tribe, R.M. \u0026amp; Ohya, S. Loss of functional K+ channels encoded by ether-a-go-go-related genes in mouse myometrium prior to labour onset. \u003cem\u003eJ Physiol\u003c/em\u003e \u003cstrong\u003e587\u003c/strong\u003e, 2313-2326 (2009).\u003c/li\u003e\n\u003cli\u003eTong, W.C., Tribe, R.M., Smith, R. \u0026amp; Taggart, M.J. Computational modeling reveals key contributions of KCNQ and hERG currents to the malleability of uterine action potentials underpinning labor. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e114034 (2014).\u003c/li\u003e\n\u003cli\u003eJepps, T.A., Barrese, V. \u0026amp; Miceli, F. Editorial: Kv7 channels: structure, physiology, and pharmacology. \u003cem\u003eFront Physiol\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 679317 (2021).\u003c/li\u003e\n\u003cli\u003eChadha, P.S.\u003cem\u003e, et al.\u003c/em\u003e Contribution of kv7.4/kv7.5 heteromers to intrinsic and calcitonin gene-related peptide-induced cerebral reactivity. \u003cem\u003eArterioscler Thromb Vasc Biol\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 887-893 (2014).\u003c/li\u003e\n\u003cli\u003eMani, B.K.\u003cem\u003e, et al.\u003c/em\u003e Kv7.5 potassium channel subunits are the primary targets for PKA-dependent enhancement of vascular smooth muscle Kv7 currents. \u003cem\u003eMol Pharmacol\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e, 323-334 (2016).\u003c/li\u003e\n\u003cli\u003eAbou-Khalil, B.W. Update on antiepileptic drugs 2019. \u003cem\u003eContinuum (Minneap Minn)\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 508-536 (2019).\u003c/li\u003e\n\u003cli\u003eWang, L., Qiao, G.H., Hu, H.N., Gao, Z.B. \u0026amp; Nan, F.J. Discovery of novel retigabine derivatives as potent KCNQ4 and KCNQ5 channel agonists with improved specificity. \u003cem\u003eACS Med Chem Lett\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 27-33 (2019).\u003c/li\u003e\n\u003cli\u003eSeefeld, M.A.\u003cem\u003e, et al.\u003c/em\u003e Novel K(V)7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction. \u003cem\u003eBioorg Med Chem Lett\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 3793-3797 (2018).\u003c/li\u003e\n\u003cli\u003ePaul, J.W.\u003cem\u003e, et al.\u003c/em\u003e Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor. \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e \u003cstrong\u003e216\u003c/strong\u003e, 283 e281-283 e214 (2017).\u003c/li\u003e\n\u003cli\u003ePaul, J.W. \u0026amp; Smith, R. Preventing preterm birth: New approaches to labour therapeutics using Nanoparticles. \u003cem\u003eBest Pract Res Clin Obstet Gynaecol\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 48-59 (2018).\u003c/li\u003e\n\u003cli\u003eDudley, D.J., Branch, D.W., Edwin, S.S. \u0026amp; Mitchell, M.D. Induction of preterm birth in mice by RU486. \u003cem\u003eBiol Reprod\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 992-995 (1996).\u003c/li\u003e\n\u003cli\u003eRada, C.C., Pierce, S.L., Grotegut, C.A. \u0026amp; England, S.K. Intrauterine telemetry to measure mouse contractile pressure in vivo. \u003cem\u003eJ Vis Exp\u003c/em\u003e, e52541 (2015).\u003c/li\u003e\n\u003cli\u003ePierce, S.L., Kutschke, W., Cabeza, R. \u0026amp; England, S.K. In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models. \u003cem\u003ePhysiol Genomics\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 310-316 (2010).\u003c/li\u003e\n\u003cli\u003eTribe, R.M., Moriarty, P. \u0026amp; Poston, L. Calcium homeostatic pathways change with gestation in human myometrium. \u003cem\u003eBiol Reprod\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, 748-755 (2000).\u003c/li\u003e\n\u003cli\u003eMistry, H.D.\u003cem\u003e, et al.\u003c/em\u003e Novel expression and regulation of voltage-dependent potassium channels in placentas from women with preeclampsia. \u003cem\u003eHypertension\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 497-504 (2011).\u003c/li\u003e\n\u003cli\u003eWilliams, P.J., Mistry, H.D., Innes, B.A., Bulmer, J.N. \u0026amp; Broughton Pipkin, F. Expression of AT1R, AT2R and AT4R and their roles in extravillous trophoblast invasion in the human. \u003cem\u003ePlacenta\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 448-455 (2010).\u003c/li\u003e\n\u003cli\u003eReese, J.\u003cem\u003e, et al.\u003c/em\u003e Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall. \u003cem\u003eProstaglandins Other Lipid Mediat\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, 89-96 (2009).\u003c/li\u003e\n\u003cli\u003eCrockett, S.L.\u003cem\u003e, et al.\u003c/em\u003e Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness. \u003cem\u003ePediatr Res\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 991-997 (2020).\u003c/li\u003e\n\u003cli\u003eYarboro, M.T.\u003cem\u003e, et al.\u003c/em\u003e A novel role for PGE(2)-EP(4) in the developmental programming of the mouse ductus arteriosus: consequences for vessel maturation and function. \u003cem\u003eAm J Physiol Heart Circ Physiol\u003c/em\u003e \u003cstrong\u003e325\u003c/strong\u003e, H687-H701 (2023).\u003c/li\u003e\n\u003cli\u003eZou, M.\u003cem\u003e, et al.\u003c/em\u003e Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility. \u003cem\u003eJCI Insight\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e(2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6837828/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6837828/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite spontaneous preterm birth (PTB, delivery\u0026thinsp;\u0026lt;\u0026thinsp;37 weeks\u0026rsquo; gestation) being a major contributor to perinatal morbidity and mortality worldwide, there is a paucity of treatments for treating preterm labor. We hypothesized that uterine smooth muscle (myometrium) Kv7 channels could be a therapeutic target for preventing preterm labor. Building on our previous work, we confirmed that K\u003csub\u003eV\u003c/sub\u003e7 channels, proteins encoded by KCNQ2-5 genes and associated accessory KCNE1-5, are expressed and functional in pregnant human myometrium prior to and after the onset of labor. K\u003csub\u003eV\u003c/sub\u003e7.2-5 activators (retigabine and ML213) effectively inhibit pregnant human and mouse myometrium contractions \u003cem\u003ein vitro\u003c/em\u003e, and \u003cem\u003ein vivo\u003c/em\u003e significantly delayed PTB in a non-infection preterm labor mouse model. This supports our hypothesis that augmenting K\u003csub\u003eV\u003c/sub\u003e7 activity represents a viable mechanism to suppress uterine contractility and delay of PTB. Addressing the current drive to repurpose existing drugs for treating PTB, we propose this as a new avenue of clinical exploration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOne Sentence Summary\u003c/b\u003e: K\u003csub\u003eV\u003c/sub\u003e7 channel activators inhibit human uterine contractions and delay delivery in preterm mice, identifying a new target for preterm labor prevention.\u003c/p\u003e","manuscriptTitle":"KV7 channel activation inhibits human and murine myometrium contractility and delays delivery in a mouse model of preterm birth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 07:04:16","doi":"10.21203/rs.3.rs-6837828/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0103bf07-ff03-42da-8657-439dd242606f","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50879490,"name":"Biological sciences/Physiology/Reproductive biology"},{"id":50879491,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-10-31T07:04:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 07:04:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6837828","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6837828","identity":"rs-6837828","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}