The role of the RHOA/ROCK pathway in the regulation of myometrial stages throughout pregnancy

Abstract

Background: Controlling uterine contractile activity is essential to regulate the duration of pregnancy. During most of the pregnancy, the uterus does not contract (i.e., myometrial quiescence). The myometrium recovers its contractile phenotype at around 36 weeks (i.e., myometrial activation) through several mechanisms. The RHOA/ROCK pathway plays a vital role in facilitating muscular contractions by calcium sensitization in humans. Yet, the role of this pathway during different myometrial stages, including quiescence, has not been elucidated.

Objective: we aimed to study the role of the RHOA/ROCK pathway in the regulation of the different myometrial stages throughout pregnancy. Specifically, we hypothesized that the inhibition of the components of the RHOA/ROCK pathway play an important role in maintaining uterine quiescence.

Study design: Myometrial samples were obtained from pregnant individuals who underwent cesarean section. Pregnant individuals who delivered preterm without labor (myometrial quiescence), preterm with labor (nonphysiological myometrial stimulation), term not in labor (activation), and term in labor (physiological myometrial stimulation) were included. The mRNA and protein expression of RHOA, ROCK I, ROCK II, RND1-3, and ROCK activity through pMYTP1 were evaluated.

Results: We found that the human myometrium constitutively expressed RHOA/ROCK pathway components throughout pregnancy. No changes in the components of the RHOA/ROCK pathway were found during quiescence. Moreover, the RHOA protein and ROCK activity increased in the myometrium during labor, supporting the hypothesis that this pathway participates in maintaining the contractile activity of the myometrium. This study provides insight into the role of the RHOA/ROCK pathway in controlling myometrial contractile activity during pregnancy.

Keywords: labor; myometrial activation; myometrial quiescence; myometrium; uterine contraction.

Figure 1

Expression of myometrial RHOA throughout pregnancy (A) Study design. Myometrial samples were taken from 4 groups of pregnant individuals: term not in labor (T-NL), term in labor (T-L), preterm not in labor (PT-NL), and preterm in labor (PT-L). (B) Top, agarose gel electrophoresis of the RT-PCR; bottom, relative abundance (compared to PT-NL mRNA) of mRNA encoding RHOA. (C) Western blot analysis of RHOA. Top, bands, and representative immunoblots revealing the expression of RHOA protein; bottom, quantification of Western Blot signals compared to PT-NL. Data are expressed as the median + standard error. N=8 in each group. PT-NL, preterm not in labor; PT-L, preterm in labor; TN-L, term not in labor; T-L, term in labor. *P<.05, **P<.01.

Figure 2

Expression of myometrial ROCK I and ROCK II throughout pregnancy (A) Top, agarose gel electrophoresis of the RT-PCR; bottom, relative abundance (compared to PT-NL mRNA) of mRNA encoding ROCK I. (B) Top, agarose gel electrophoresis of the RT-PCR; bottom, relative abundance (compared to PT-NL mRNA) of mRNA encoding ROCK II. Data are expressed as the median + standard error. N=6 in each group. PT-NL, preterm not in labor; PT-L, preterm in labor; TN-L, term not in labor; T-L, term in labor.

Figure 3

Expression of myometrial RHOA throughout pregnancy (A) Top, agarose gel electrophoresis of the RT-PCR; bottom, relative abundance (compared to PT-NL mRNA) of mRNA encoding RND1. (B) Western blot analysis of RND1. Top, bands and representative immunoblots revealing the expression of RND1 protein; bottom, quantification of Western Blot signals compared to PT-NL. (C) Top, agarose gel electrophoresis of the RT-PCR; bottom, relative abundance (compared to PT-NL mRNA) of mRNA encoding RND2. (D) Western blot analysis of RND2. Top, bands, and representative immunoblots revealing the expression of RND2 protein; bottom, quantification of Western Blot signals compared to PT-NL. (E) Top, agarose gel electrophoresis of the RT-PCR; bottom, relative abundance (compared to PT-NL mRNA) of mRNA encoding RND3. (F) Western blot analysis of RND3. Top, bands, and representative immunoblots revealing the expression of RND3 protein; bottom, quantification of Western Blot signals compared to PT-NL. Data are expressed as the median + standard error. N=7 in each group. PT-NL, preterm not in labor; PT-L, preterm in labor; TN-L, term not in labor; T-L, term in labor. *P<.05.

Figure 4

ROCK protein's activity is increased in labor Top, bands, and representative immunoblots revealing the expression of p-MYPT1 and MYPT1 proteins; bottom, quantification of the p-MYPT1/MYPT1 ratio of Western Blot signals compared to PT-NL. Data are expressed as the median + standard error. N=7 in each group. PT-NL, preterm not in labor; PT-L, preterm in labor; TN-L, term not in labor; T-L, term in labor. *P<.05.