Background

During pregnancy, myometrium smooth muscle cells (SMC) relax to accommodate growth of a developing foetus and during labour, develop regular contractions allowing movement of the foetus towards the cervix (Malik et al., 2021). Irregular myometrium contractions during pre-term labour, leads to complications (foetal heart abnormalities, foetal hypoxia) that present unacceptable risks to infant and maternal mortality (Wray & Prendergast, 2019). Clinically, during pre-term labour or uterine hyperstimulation, myometrium contractions are reduced by drugs such as nifedipine, which block voltage-dependent L-type Ca²⁺ channels, preventing Ca²⁺ entry and contraction. However, nifedipine has numerous effects throughout the body (decreased blood pressure, heart palpitations, etc.) that pose additional risks to mother and foetus. In SMC, the sarcoplasmic reticulum (SR) releases Ca²⁺ to enhance contraction. In many tissues, SR refilling occurs when STIM proteins on the SR membrane activate Orai channels in the plasma membrane, allowing store-operated-Ca²⁺-entry (SOCE) and SR refilling. While previous studies suggest myometrium SMC express STIM / Orai (Chin-Smith et al., 2014) and are capable of SOCE (Noble et al., 2014), effects of specific modulators of SOCE on myometrium have not been tested.

 

Results

Using isometric tension recordings of myometrium from non-pregnant wild-type mice (10-16 weeks old), we investigated contributions of SOCE to contractile activity in longitudinal strips isolated from cervical and ovarian regions across different stages of estrous. When myometrium strips (N) equilibrated in an organ bath after application of 10 nM oxytocin and 5 mN of initial stretch, spontaneous contractions developed in cervical and ovarian tissue at all stages of estrous (n=37, N=39), with an average frequency of 2.2 ± 0.7 min^-1 and amplitude of 4.5 ± 2.5 mN. Inhibition of L-type Ca²⁺ channels with nifedipine (1 micromolar) abolished contractions, while addition of Orai channel inhibitors (GSK 7975A, BTP2, Synta66; 10 micromolar) reduced contraction frequency by ~ 40% (n=37, N=39) with minimum effect on amplitude. Greater sensitivity to Orai channel inhibitors was noted in cervical tissues (~ 49% decrease in frequency in cervical tissue (n=37, N=39) vs ~ 31% reduction of frequency in ovarian tissue (n=37, N=39)). Additionally, non-estrous tissue showed greater sensitivity to Orai inhibition compared to estrous tissue from both regions (for example ~  48% reduction in frequency in non-estrous tissue (n=38, N=40) vs 32% reduction in frequency in estrous tissues n=36, N=38). When SOCE was evoked in myometrium by reintroduction of extracellular Ca²⁺ following SR depletion with Ca²⁺ free solutions and blocking the SERCA pump with thapsigargin (10 micromolar), a prolonged transient contraction occurred. Under these conditions, the Orai channel enhancer IA65 (1 – 3 micromolar) evoked phasic contractions superimposed on a tonic contraction. Inhibition of Orai channels with GSK 7975A reduced both tonic and phasic components, while nifedipine only abolished phasic contractions, with no effect on tone. Furthermore, IA65 (1 and 3 micromolar) increased spontaneous contractile duration in both cervical and ovarian regions (n=6, N=18).

 

Conclusions

Collectively, these findings support a regulatory role for SOCE in spontaneous myometrial contractility and that degrees of sensitivity to SOCE modulation depend on uterine region and stage of estrous being targeted.