Adaptive reduction of human myometrium contractile activity in response to prolonged uterine stretch during term and twin pregnancy. Role of TREK-1 channel

Abstract

Quiescence of myometrium contractile activity allows uterine expansion to accommodate the growing fetus and prevents preterm labor particularly during excessive uterine stretch in multiple pregnancy. However, the mechanisms regulating uterine response to stretch are unclear. We tested the hypothesis that prolonged uterine stretch is associated with decreased myometrium contractile activity via activation of TWIK-related K⁺ channel (TREK-1). Pregnant women at different gestational age (preterm and term) and uterine stretch (singleton and twin pregnancy) were studied, and uterine strips were isolated for measurement of contractile activity and TREK-1 channel expression/activity. Both oxytocin- and KCl-induced contraction were reduced in term vs preterm pregnancy and in twin vs singleton pregnancy. Oxytocin contraction was reduced in uterine segments exposed to 8 g stretch compared to control tissues under 2 g basal tension. TREK-1 mRNA expression and protein levels were augmented in Singleton-Term vs Singleton-Preterm, and in uterine strips exposed to 8 g stretch. The TREK-1 activator arachidonic acid reduced oxytocin contraction in preterm and term, singleton and twin pregnant uterus. The TREK-1 blocker l-methionine enhanced oxytocin contraction in Singleton-Term and twin pregnant uterus, and reversed the decreases in contraction in uterine strips exposed to prolonged stretch. Carboprost-induced uterine contraction was also reduced by arachidonic acid and enhanced by l-methionine. Thus, myometrium contraction decreases with gestational age and uterine expansion in twin pregnancy. The results suggest that prolonged stretch enhances the expression/activity of TREK-1 channel, leading to decreased myometrium contractile activity and maintained healthy term pregnancy particularly in multiple pregnancy.

Keywords: Contraction; Potassium channel; Pregnancy; Stretch; Uterus.

Fig. 1

Effect of gestational age and pregnancy type on oxytocin-induced uterine contraction. Uterine strips from Singleton-Preterm, Singleton-Term, Twin-Preterm and Twin-Term pregnant subjects were incubated in normal Krebs solution. The uterine strips were stimulated with oxytocin (10⁻¹² to 10⁻⁷ M) and the maintained contraction (in grams) and total contractile response (in AUC) were measured (A). Cumulative oxytocin concentration-contraction curves from uterine strips of different groups were constructed. To correct for differences in the size of uterine segments, maintained contraction was normalized and presented in g/g tissue weight (B), or as % of maximum contraction (C). Because of the variability in the frequency and amplitude of the phasic contractile response, the total contraction area under the curve (AUC) was calculated and presented as AUC/g tissue weight (D) or as % of maximum contractile response (E). Data are presented as means±SEM, n = 7 to 9 subjects. * Significantly different (P<0.05) from Singleton-Preterm. # Significantly different (P<0.05) from Singleton-Term. † Significantly different (P<0.05) from Twin-Preterm

Fig. 2

Effect of gestational age and pregnancy type on KCl-induced uterine contraction. Uterine strips from Singleton-Preterm, Singleton-Term, Twin-Preterm and Twin-Term pregnant subjects were incubated in normal Krebs solution. The uterine strips were stimulated with 96 mM KCl and the contractile response was recorded and presented in g/g tissue weight. The small contractions preceding the KCl response in uterine strips of Singleton-Term and Twin-Preterm subjects represent spontaneous phasic contractions. Data are presented as means±SEM, n = 7 to 9 subjects. * Significantly different (P<0.05) from Singleton-Preterm. # Significantly different (P<0.05) from Singleton-Term. † Significantly different (P<0.05) from Twin-Preterm.

Fig. 3

Effect of prolonged stretch on uterine contraction. Uterine strips from Singleton-Preterm pregnant women were incubated under control 2 g basal tension or high 8 g stretch for 16 h, then stimulated with increasing concentrations of oxytocin (10⁻¹² to 10⁻⁷ M) (A). Cumulative concentration-contraction curves were constructed and the maintained contraction was normalized and presented in g/g tissue weight (B), or as % of maximum contraction (C). To analyze the changes in the total contractile response, the area under the curve (AUC) was calculated and presented as AUC/g tissue weight (D) or as % of maximum contractile response (E). Data are presented as means±SEM, n = 7. * Significantly different (P<0.05) from Singleton-Preterm tissues under control 2 g basal tension.

Fig. 4

Changes in TREK-1 mRNA expression and protein levels with gestational age and pregnancy type. Uterine tissue homogenate from Singleton-Preterm, Singleton-Term, Twin-Preterm and Twin-Term pregnant women were prepared for q-PCR analysis and TREK-1 mRNA expression was measured relative to the Singleton-Preterm group (A). Tissue homogenate was also prepared for measurement of TREK-1 protein levels using Western blots and antibody to TREK-1 (1:1000). The intensity of the immunoreactive band corresponding to TREK-1 was analyzed using optical densiometry, and normalized to the housekeeping protein GAPDH (B). Data are presented as means±SEM, n=6-8. * Significantly different (P<0.05) from Singleton-Preterm. # Significantly different (P<0.05) from Singleton-Term. † Significantly different (P<0.05) from Twin-Preterm.

Fig. 5

Effect of prolonged uterine stretch on TREK-1 expression. Uterine strips from Singleton-Preterm pregnancies were placed under control 2 g basal tension or prolonged 8 g stretch for 16 h. The tissues were homogenized and prepared for measurement of TREK-1 mRNA using qPCR (A). Other tissues were homogenized and prepared for Western blots using TREK-1 antibody (1:1000). The intensity of the immunoreactive band corresponding to TREK-1 was analyzed using optical densiometry, and normalized to the housekeeping protein GAPDH (B). Data are presented as means±SEM, n=6–8. * P<0.05, 8 g stretch vs. control 2 g basal tension.

Fig. 6

Effect of modulators of TREK-1 channel on oxytocin-induced uterine contraction. Uterine strips from Singleton-Preterm, Singleton-Term, Twin-Preterm and Twin-term pregnant women were pre-contracted with oxytocin (10⁻⁷ M). Once oxytocin contraction reached steady-state, the tissues were treated with TREK-1 activator arachidonic acid (10⁻⁵ M), or TREK-1 blocker L-methionine (1 mM), or the vehicle, and the effects on oxytocin contraction were recorded at 0, 5, 10 and 15 min (A). Cumulative data from uterine strips of Singleton-Preterm (B), Singleton-Term (C), Twin-Preterm (D) and Twin-term pregnant women (E) were collected and the magnitude of uterine contraction was presented as % of control AUC (100%) in the absence of TREK-1 modulators. Data represent means±SEM, n=6–7. * Oxytocin contraction in arachidonic acid treated tissues is significantly reduced (p<0.05) compared to control non-treated tissues. # Oxytocin contraction in L-methionine treated tissues is significantly enhanced (p<0.05) compared to control non-treated tissues.

Fig. 7

Effect of modulators of TREK-1 on carboprost tromethamine-induced contraction. Uterine strips of Singleton-Term pregnant women were pre-contracted with carboprost tromethamine (3×10⁻⁴ M). Once carboprost tromethamine contraction reached steady-state the tissues were treated with arachidonic acid (10⁻⁵ M), L-methionine (1 mM), or the vehicle, and the changes in uterine contraction were measured after 10 min, and presented as % of control AUC (100%) in the absence of TREK-1 modulators. Data are presented as means±SEM, n=6. * Carboprost contraction in arachidonic acid treated tissues is significantly reduced (p<0.05) compared to control non-treated tissues. # Carboprost contraction in L-methionine treated tissues is significantly enhanced (p<0.05) compared to control non-treated tissues.

Fig. 8

Effect of TREK-1 blockade on contraction in uterine strips exposed to prolonged stretch. Uterine strips from Singleton-Preterm pregnant women were incubated under 2 g basal tension or high 8 g stretch for 16 h .The uterine strips were stimulated with oxytocin (10⁻⁷ M). Once oxytocin contraction reached steady-state the tissues were treated with TREK-1 blocker L-methionine (1 mM) or the vehicle H₂O, and the effects on oxytocin contraction were recorded at 0, 5, 10 and 15 min (A). Cumulative data from uterine strips were collected and the % change of AUC relative to control AUC (100%) in the absence of the TREK-1 blocker L-methionine was measured. Data represent means±weSEM, n=6–7. * Oxytocin contraction in tissues under 8 g stretch is significantly reduced (p<0.05) compared to control tissues under 2 g basal tension. # Oxytocin contraction in tissues under 8 g stretch and treated with L-methionine is significantly enhanced (p<0.05) compared to non-treated tissues under 8 g stretch. † Oxytocin contraction in tissues under 8 g stretch and treated with L-methionine is significantly enhanced (p<0.05) compared to control non-treated tissues under 2 g basal tension.