The contribution of Kv7 channels to pregnant mouse and human myometrial contractility

Abstract

Premature birth accounts for approximately 75% of neonatal mortality and morbidity in the developed world. Despite this, methods for identifying and treating women at risk of preterm labour are limited and many women still present in preterm labour requiring tocolytic therapy to suppress uterine contractility. The aim of this study was to assess the utility of Kv7 channel activators as potential uterine smooth muscle (myometrium) relaxants in tissues from pregnant mice and women. Myometrium was obtained from early and late pregnant mice and from lipopolysaccharide (LPS)-injected mice (day 15 of gestation; model of infection in pregnancy). Human myometrium was obtained at the time of Caesarean section from women at term (38-41 weeks). RT-PCR/qRT-PCR detected KCNQ and KCNE expression in mouse and human myometrium. In mice, there was a global suppression of all KCNQ isoforms, except KCNQ3, in early pregnancy (n= 6, P < 0.001 versus late pregnant); expression subsequently increased in late pregnancy (n= 6). KCNE isoforms were also gestationally regulated (P < 0.05). KCNQ and KCNE isoform expression was slightly down-regulated in myometrium from LPS-treated-mice versus controls (P < 0.05, n= 3-4). XE991 (10 μM, Kv7 inhibitor) significantly increased spontaneous myometrial contractions in vitro in both human and mouse myometrial tissues (P < 0.05) and retigabine/flupirtine (20 μM, Kv7 channel activators) caused profound myometrial relaxation (P < 0.05). In summary, Kv7 activators suppressed myometrial contraction and KCNQ gene expression was sustained throughout gestation, particularly at term. Consequently, activation of the encoded channels represents a novel mechanism for treatment of preterm labour.

Fig 1

Expression of (A) KCNQ and (B) KCNE genes in non-pregnant, early and late pregnant mouse myometrial tissue. Non-pregnant (oestrous, white bars, n= 6; data taken from McCallum et al., for comparison), early pregnant (black bars, n= 6, gestation days 6–7) and late pregnant mouse myometrium (grey bars, n= 6, gestation days 17–18). Data are expressed as log geometric mean of copy number (± S.E.) normalized to housekeeping genes. (*P < 0.001, +P < 0.01, ΔP < 0.05).

Fig 2

Effect of XE991 (10 μM) on spontaneous myometrial contractions in tissues from mice in early (A) and late pregnancy (B). (C) Mean effect of XE991 (10 μM) (±S.E.) in early pregnancy (n= 10 from 9 animals) versus vehicle control (n= 10 from 9 animals). (D) Mean effect of XE991 (10 μM) (±S.E.) in late pregnancy (n= 14 from 8 animals) compared to vehicle controls (n= 14 from 8 animals). (*P < 0.05, **P < 0.01).

Fig 3

The effect of flupirtine (20 μM) on spontaneous myometrial contractions from mice in early (A) and late pregnancy (B). The impact of flupirtine (20 μM, mean ± S.E.) and reversal by XE991 (10 μM) in myometrium from animals in early (C, n= 6) and late pregnancy (D, n= 10 from 9 animals) compared to time and gestation-matched vehicle controls (C, n= 6; D, n= 9 from 8 animals). (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4

The effect of retigabine (20 μM) on spontaneous (A) and oxytocin induced (10⁻⁹ M, C) myometrial contractions in tissue from mice in late pregnancy. (B); mean data (±S.E.) for retigabine (20 μM) and reversal by XE991 (10 μM, n= 10 from 9 animals) compared to vehicle controls (n= 9 from 8 animals). (D); Mean (± S.E.) for retigabine versus oxytocin period (n= 3). (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 5

The expression of KCNQ (A) and KCNE (B) mRNA transcripts in myometrial tissue taken from mice 8 hrs after being injected (IP) with LPS (100 ng). Vehicle control (grey bars, n= 3) and LPS (black bars, n= 4). Data are expressed as log mean of copy number (± S.E.) normalized to geometric mean of two housekeeping genes (*P < 0.05).

Fig 6

The expression (A) and function (B–C) of KCNQ isoforms in myometrial tissue from pregnant women at term (n= 3). KCNQ1 (124 bp), KCNQ2 (123 bp), KCNQ3 (119 bp), KCNQ4 (119 bp), KCNQ5 (120 bp). The effect of (B) XE991 (10 μM) and (C) retigabine (20 μM) on spontaneous myometrial contractions in vitro (not in labour, representative of n= 3).