- Email: [email protected]
Connexin 43 expression in normal versus dysfunctional labor
Connexin 43 expression in normal versus dysfunctional labor Brian T. Pierce, MD,a Byron C. Calhoun, MD,a Kimberly R. Adolphson, BS,b Alan F. Lau, PhD,d and Lisa M. Pierce, DScc Fort Lewis, Wash, and Honolulu, Hawaii OBJECTIVE: We sought to determine whether connexin 43 (Cx43), the major myometrial gap junction protein, is differentially expressed in normal versus dysfunctional labor. STUDY DESIGN: Myometrial biopsies were obtained from 28 patients undergoing cesarean section and grouped into the following categories: (1) no labor, (2) dysfunctional labor, and (3) normal labor. Northern and Western analyses were performed to determine Cx43 messenger RNA (mRNA) and protein expression, respectively. Localization of Cx43 protein was determined by immunohistochemistry. RESULTS: Labor was associated with increased Cx43 mRNA expression (P < .05). This association was not true for protein. There was no difference in mRNA or protein expression between patients with normal labor versus those with dysfunctional labor. The extent of Cx43 immunohistochemical staining was not significantly different among the groups (P > .05). CONCLUSION: Dysfunctional labor is not associated with aberrant Cx43 mRNA or protein expression or with a reduction in immunodetectable Cx43 gap junctions. (Am J Obstet Gynecol 2002;186:504-11.)
Key words: Connexin 43, gap junction, myometrium, labor, dysfunctional labor
Gap junctions are thought to be one of several essential components for effective labor and delivery.1,2 Gap junctions mediate cell-to-cell communication, maintain normal cell function, and play an important role in embryonic development and possibly growth control.3,4 They also provide low-resistance pathways between smooth muscle cells, thereby increasing their electrical coupling to allow increased coordination of contractile activity. Connexins are a family of gap junction proteins that are named for their molecular weights, with connexin 43 (molecular weight, 43 kD) being the major gap junction protein present in myometrium.5-8 Gap junctions are formed when a connexon (hemichannel) from 1 cell pairs with a symmetrically opposed connexon from an adjacent cell, forming a channel that permits the passage of
From the Division of Maternal-Fetal Medicine,a the Department of Pathology,b and the Department of Clinical Investigation,c Madigan Army Medical Center, and the Cancer Research Center of Hawaii, University of Hawaii.d Supported by the Department of the Air Force Clinical Investigations, grant No. SGO 2000-0015H. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Air Force, the Department of the Army, or the Department of Defense. Received for publication June 14, 2001; revised October 1, 2001; accepted October 18, 2001. Reprint requests: Brian T. Pierce, MD, Department of Obstetrics and Gynecology, Darnall Army Community Hospital, 36000 Darnall Loop, Fort Hood, TX 76544-5063. Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/121108 doi:10.1067/mob.2002.121108
504
ions and small molecules (<1000-1700 D) between neighboring cells.9 Each connexon consists of an oligomer of 6 connexin proteins that form the central aqueous pore.9 During most of pregnancy, gap junction plaques (clusters of numerous gap junctions) are undetectable or are present in very low numbers as measured by electron microscopy or immunohistochemistry.7,10-12 This is consistent with the presence of poorly coordinated, lowamplitude contractions (Braxton Hicks) during pregnancy. The onset of labor is associated with a dramatic increase in the number and size of the plaques, an increase in the electrical conductivity of the myometrium, and the development of spontaneous, well-coordinated labor contractions.7,10-12 Correlating to the appearance of gap junction plaques, connexin 43 (Cx43) messenger RNA (mRNA) levels increase in the human myometrium toward term and are highest during delivery.12 Effective labor may require the synthesis and assembly of Cx43 into functional gap junctions at the myometrial cell surface.1,2 The exact mechanisms that regulate the synthesis and assembly of gap junctions in the human myometrium are not known. Hendrix et al13 found in rats that Cx43 was synthesized several days before labor but accumulated within the cytoplasm until parturition, when it was rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These Cx43-positive gap junctions disappeared within 24 hours after delivery. Cx43 phosphorylation may regulate gap junctional protein trafficking, gap junction assembly, channel gating, and gap junction turnover.14 Laird et al14 demonstrated in rat mammary tumor cells that Cx43 is transiently phosphorylated in the endoplas-
Volume 186, Number 3 Am J Obstet Gynecol
mic reticulum/Golgi apparatus, where it is available for the assembly of new gap junction channels. The authors also showed that the phosphorylation of Cx43 is necessary for the trafficking of Cx43 to the membrane and that the rapid turnover of gap junction plaques correlates with dephosphorylated forms of Cx43.14 Studies that investigated the transcriptional regulation of the human Cx43 gene identified activator protein 1 and specificity protein 1 consensus sites in the promoter sequence of the Cx43 gene.15-17 Upregulation of Cx43 gene expression appears to involve induction of the transcription factors c-jun and c-fos (which bind to the activator protein 1 site on the Cx43 promoter) and specificity protein 1 (which binds to the specificity protein 1 site) after protein kinase C activation.15-17 In this study, we explored the hypothesis that aberrant Cx43 expression, which may lead to decreased gap junction formation and disrupted gap junctional communication, may be responsible, at least in part, for the lack of high amplitude, well-coordinated contractions occurring in patients with dysfunctional labor. The goal of the present investigation was to determine whether Cx43 is differentially expressed in normal versus dysfunctional labor. Material and methods Subjects. The human subjects protocol for this study was reviewed and approved by the institutional review board of Madigan Army Medical Center. Written informed consent was obtained from all subjects after they were consented for, but before cesarean section was performed. Study patients included all women undergoing cesarean section at >35 weeks’ gestation. Patients with chorioamnionitis were excluded from this study. Clinical information for each patient was recorded and included maternal age, gestational age, birth weight, length of labor, cervical dilation, Montevideo units for the final 10 minutes of available tracing, number of contractions in the final 10 minutes of available tracing, and presence and duration of rupture of membranes. For purposes of analysis, study patients were divided into the following 3 groups on the basis of indication for cesarean section and the characteristics of the first stage of labor: 1. No labor (elective) cesarean section group: cesarean section before the onset of labor 2. Dysfunctional labor group: laboring patients undergoing cesarean section with the labor course classified as failure to progress, protracted active phase, or secondary arrest of dilation 3. Normal labor group: laboring patients undergoing cesarean section for indications unrelated to the first stage of labor characteristics. These included nonreassuring fetal heart rate tracing, desired repeat cesarean section presenting in labor, and contraindications for vaginal delivery presenting in labor.
Pierce et al 505
Failure to progress was defined as the inability to progress past latent phase labor, despite adequate uterine contractions and with or without pitocin augmentation. Secondary arrest of dilation was defined as cessation of cervical dilation for a period of 2 hours, despite adequate uterine contractions and with or without pitocin augmentation. Nonreassuring fetal heart rate tracing was defined as a fetal heart rate tracing demonstrating characteristics necessitating operative intervention as determined by the attending staff obstetrician. Contraindications for vaginal delivery included prior classical cesarean section and suspected fetal macrosomia in a pregnancy complicated by diabetes mellitus. Montevideo units were determined by internal uterine pressure catheter (IUPC) and defined as the summation of the amplitude of each uterine contraction minus the baseline in a 10-minute period. Average contraction strength was defined as Montevideo units divided by the number of contractions in a 10-minute period. The use of IUPCs was determined by the attending physician and was not a criterion for study enrollment, although all (11 of 11) patients with dysfunctional labor and 4 of 7 patients with normal labor had IUPCs. The final 10 minutes of available IUPC tracing was used to calculate Montevideo units and average contraction strength for study purposes. Tissue collection. Myometrial biopsies (approximately 2 cm 1 cm 1 cm) from the lower uterine segment were excised from the superior middle margin of the uterine incision during cesarean section after delivery of the fetus and placenta. Human myometrial sampling at cesarean delivery has been shown not to increase overall maternal morbidity in the presence or absence of labor.18 Tissue samples were washed in physiologic saline solution and divided into thirds for Northern analysis, Western analysis, and immunohistochemistry. For Northern and Western analysis, tissue samples were wrapped in foil, snap-frozen in liquid nitrogen or in an isopentane cryobath, and stored at –70°C until further processing. For immunohistochemistry experiments, tissue samples were embedded in ornithine carbamyl transferase, snap-frozen in isopentane, and stored at –70°C before sectioning and staining. Rat heart was prepared in a similar fashion as the human myometrial tissue and was used as a positive control in each experiment performed. Northern analysis. Total cellular RNA was isolated from the myometrium and rat heart using the method of Chomczynski and Sacchi.19 Frozen tissues were placed into 3 mL of Solution D19 in a 50-mL tube on dry ice and were minced with a scalpel. Samples were then homogenized at room temperature with a Polytron hand-held tissue homogenizer. (model pT1200C Brinkman Instruments, Inc, Westbury, NY) and allowed to sit at room temperature for 15 minutes. One-tenth volume of 2 mol/L (2 molar = 2 mol/L) sodium acetate (pH 4.0), one-fifth volume of chloroform-isoamyl alcohol mixture (48:1), and
506 Pierce et al
an equal volume of diethyl pyrocarbonate–treated watersaturated phenol was added to each homogenate with thorough mixing by inversion after the addition of each reagent. The final suspension was shaken vigorously, transferred to a 15-mL tube, and cooled on ice for 20 minutes. Samples were centrifuged at 4°C for 20 minutes at 9,500 rpm in a Beckman GS-15R tabletop centrifuge. (Beckman Coulter, Inc, Fullerton, Calif) The aqueous layer was removed and placed into 1.5-mL microcentrifuge tubes in 600-µL aliquots. An equal volume of isopropanol was added, and samples were either frozen overnight at –70°C or on dry ice for 15 minutes. Samples were centrifuged at 15,300 rpm for 20 minutes, and the resulting RNA pellets were dissolved in 600 µL of Solution D, precipitated again with an equal volume of isopropanol either overnight at –70°C or on dry ice for 15 minutes, centrifuged for 20 minutes, and washed in 80% ice cold ethanol. RNA pellets were centrifuged for 10 minutes, resuspended in 75 µL of diethyl pyrocarbonate–treated water, and quantitated using a ultraviolet spectrophotometer. Total RNA (30 µg/lane) was denatured, resolved in a 1.2% agarose-formaldehyde gel, transferred to a GeneScreen (NEN, Boston, Mass) membrane in 10X saline sodium citrate, and ultraviolet light cross-linked to the membrane. Probes consisted of complementary DNAs (cDNAs) for Cx436 and 18S ribosomal RNA (Ambion, Inc., Austin, Tex) as an internal loading standard. Probes were labeled by random priming (Amersham, Arlington Heights, Ill) with [α-32P] deoxycytidine triphosphate (3,000 Ci/mmol, Amersham, Arlington Heights, Ill) and the Klenow fragment of Escherichia coli DNA polymerase. Unincorporated counts were removed using STE push columns (Stratagene, La Jolla, Calif). Prehybridization and hybridization were performed at 55°C using the SuperTM Hybridization Buffer System (DNA Technologies, Inc., Rockville, MD) following the manufacturer’s instructions. Blots probed with Cx43 were washed 3 5 minutes in 2 saline sodium citrate at room temperature before images were obtained with a BioRad GS-505 Molecular Imager System (Bio-Rad Laboratories, Hercules, Calif). Blots that were stripped and reprobed with the 18S cDNA were washed in the buffers supplied in the SuperTM Hybridization Buffer System kit (DNA Technologies, Inc., Rockville, Md) following the manufacturer’s instructions. Densitometry was used to measure the relative quantity of Cx43 mRNA present in the samples (calculated as Cx43:18S ratio). Western analysis. Myometrial samples and rat heart were homogenized on ice in RIPA lysis buffer (10 mmol/L Tris, pH 7.2/150 mmol/L NaCl/1% sodium deoxycholate/1% Triton X-100/0.1% sodium dodecyl sulfate/1 mmol/L polymethylsulfonyl fluoride/10 mmol/L NaF) containing protease inhibitors. One protease inhibitor tablet (Roche, Palo Alto, Calif) was added
March 2002 Am J Obstet Gynecol
per 10 mL of lysis buffer, and 100 µL of protease inhibitor mix (Sigma, St. Louis, Mo) was added per 40 mL of lysis buffer. Lysis buffer was added to tissue samples at a ratio of 1 g tissue:20 mL lysis buffer (approximate weight of each tissue sample for protein extraction was 200 mg). Homogenates were incubated on ice for 15 to 20 minutes and centrifuged at 14,000 rpm for 10 minutes, and the supernatants containing extracted protein were collected. An aliquot of the supernatant was removed for protein determination using the Lowry protein assay using bovine serum albumin (BSA) as a standard. Aliquots of the protein extracts were frozen at –70°C and saved for Western analysis. Thirty µg of protein from each sample was diluted in 2 loading buffer (20% glycerol/20% sodium dodecyl sulfate (SDS) /0.1% bromophenol blue/0.1 mol/L Tris-HCl, pH 6.8/10% β-mercaptoethanol). Samples were heated at 90°C for 2 minutes, electrophoresed in a 10% polyacrylamide gel with a 6% stacking gel, and transferred for 2 hours at 200 mA (4°C) onto a polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, Mass). The membrane was blocked overnight in 5% nonfat milk/phosphate-buffered saline solution/0.1% Tween20/1% goat serum at 4°C before a 1-hour incubation with a rabbit polyclonal antiserum (diluted 1:1,000) directed against the last 15 amino acids of the Cx43 carboxyl terminal tail (residues 368-382). The membrane was incubated with a peroxidase-conjugated goat anti-rabbit immunoglobin G (IgG) (1:25,000 dilution; Pierce, Rockford, Ill) as the secondary antibody, washed, and then incubated with a mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (G3PD; Chemicon, Temecula, Calif) antibody used as an internal loading standard (1:1,000 dilution). The final incubation step involved a peroxidase-conjugated goat anti-mouse IgG secondary antibody (Chemicon, Temecula, Calif) at 1:25,000 dilution. Immunodetection of the Cx43 and G3PD protein was performed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, Ill) following the manufacturer’s instructions. Densitometry was used to measure the relative quantity of Cx43 protein present in the samples (calculated as Cx43:G3PD ratio). Immunohistochemistry. Frozen myometrial tissues and rat heart were cut into 5 µm sections using a cryostat at –20°C. Sections were collected onto Biocare Kling-On HIER slides (Biocare Medical, Walnut Creek, Calif) and allowed to dry for 15 to 20 minutes at room temperature. Tissue sections were fixed in 100% acetone (4°C) for 10 minutes and dried completely before storage at –70°C. Slides were post-fixed in 100% acetone (4°C) for 10 minutes and transferred to 70% acetone (4°C) and 50% acetone (4°C) for 30 seconds each. Slides were rinsed briefly in tap water and then in wash buffer (tris buffered saline solution with surfactant; Biocare Medical, Walnut Creek, Calif). Immunostaining occurred with the use of the Dako
Pierce et al 507
Volume 186, Number 3 Am J Obstet Gynecol
Table I. Patient demographics and labor characteristics (mean value ± SD) No labor n Age (y) Gestational age (wk) Birth weight (g) Dilation (cm) Nulliparity Length of labor (h)
10 29.0 ± 7.3 38.8 ± 1.4 3446.1 ± 69.3 0-1 10% N/A
Dysfunctional labor 11 25.5 ± 6.9 39.4 ± 1.5 3689.9 ± 799.4 6-7 91% 28.3 ± 10.2
Normal labor 7 30.0 ± 2.3 37.7 ± 1.3 3302.9 ± 656.4 4-5 0% 10.6 ± 6.8
P value
NS* NS* NS* <.0001* <.0001+ .002‡
NS, Not significant. *Analysis of variance. +χ2 analysis. ‡Student t test.
EnVision+ System (Dako Corporation, Carpinteria, Calif). Briefly, endogenous peroxidase activity was quenched by incubating the sections with a 3% hydrogen peroxide solution for 5 minutes. Sections were rinsed in wash buffer and were incubated for 60 minutes at room temperature with a rabbit polyclonal anti-Cx43 antibody applied at a dilution of 1:200 (Zymed Laboratories, Inc., San Francisco, Calif). Sections were rinsed in wash buffer and incubated with a biotin-free, horseradish peroxidase-labeled polymer conjugated to goat anti-rabbit secondary antibodies (Dako Corporation) for 30 minutes. Rinsed sections were then incubated for 5 minutes with a 3,3’-diaminobenzidine chromogen-substrate to form a colored reaction product. Slides were counterstained with Mayer’s hematoxylin (Dako Corporation), dehydrated, and permanently mounted. A positive control of rat heart was used in each experiment performed. Negative controls were achieved by applying normal rabbit serum (normal rabbit immunoglobulin fraction, Dako Corporation) diluted 1:8,000 instead of the anti-Cx43 primary antibody. Each tissue examined possessed its own negative control slide. For the immunohistochemical analysis, the whole area of the specimens was read at objective magnifications from 10 to 40. Two to 4 tissue sections per specimen were scored for intensity of Cx43 staining independently by 2 investigators (Brian T. Pierce, MD, and Lisa M. Pierce, DSc) blinded to the clinical group from which the tissue came. Staining intensity was scored as absent or faint (+), mild (++), moderate (+++), and dense (++++). Statistical analysis. Data were analyzed with the use of the computer statistical program Statview for Windows (version 4.57; SAS, Cary, NC). The Mann-Whitney U test was used to assess differences in mRNA and protein expression among the different groups of patients and to assess differences in Cx43 mRNA levels, Cx43 protein levels, frequency of uterine contractions, Montevideo units, average contraction strength, length of labor, and cervical dilation compared with degree of Cx43 immunohistochemical staining (absent/mild versus moderate/dense). Analysis of variance, the Student t
test, and χ2 analysis were used to assess demographic differences in labor characteristics among the groups. Simple regression analysis was used to assess for correlation between mRNA expression or protein expression and cervical dilation, length of labor, Montevideo units, frequency of uterine contractions, and average strength of uterine contractions. The relationship between Cx43 staining intensity and labor status was analyzed with the χ2 test. Statistical significance was determined by P < .05. Results Patient demographic information and labor characteristics are listed in Table I. Patients in the various categories differed with respect to cervical dilation, parity, and length of labor (P < .05; Table I). Cx43 mRNA levels. Twenty-eight myometrial specimens were available for Cx43 mRNA determination by Northern analysis. These included 10 patients who were not in labor, 11 patients with dysfunctional labor, and 7 patients with normal labor. The Cx43 cDNA probe hybridized to a transcript of approximately 3.3 kb in size in both the rat heart and human myometrium, similar to that reported in previous studies (Fig 1, A).11,12 Cx43 mRNA expression per labor classification is presented in Fig 1, B. Expression of Cx43 mRNA increased during labor (mean ± SE: mRNA [Cx43:18S ratio] = 0.123 ± 0.031 [normal and dysfunctional labor combined] versus 0.041 ± 0.006 [no labor], P < .05). mRNA analysis by subgroup. There was no difference in mean Cx43 mRNA expression between patients with dysfunctional labor versus patients with normal labor (P > .05). Cx43 mRNA expression was elevated in patients with dysfunctional labor compared with patients not in labor (mean ± SE: mRNA [Cx43:18S ratio] = 0.131 ± 0.047 [dysfunctional labor] versus 0.041 ± 0.006 [no labor], P = .038). Cx43 mRNA expression also tended to be elevated in patients with normal labor compared with patients who were not in labor (mean ± SE: mRNA [Cx43:18S ratio] = 0.109 ± 0.036 [normal labor] versus 0.041 ± 0.006 [no labor], P =.051).
508 Pierce et al
A
March 2002 Am J Obstet Gynecol
B Fig 1. A, Representative Northern blot demonstrating Cx43 mRNA expression in nonlaboring and laboring patients. Lane R = rat heart positive control; lanes 7, 8 = no labor; lane 3 = normal labor; lanes 1, 2, 4-6 = dysfunctional labor. B, Cx43 mRNA expression per clinical group. Values presented as mean Cx43:18S ratio + SE.
Cx43 protein levels. Twenty-six myometrial specimens were available for Cx43 protein determination by Western analysis. These included 10 patients who were not in labor, 10 patients with dysfunctional labor, and 6 patients with normal labor. The anti-Cx43 antibody detected a band of approximately 43 kD in size in both the rat heart and human myometrium (Fig 2, A). The anti-G3PD antibody, used as an internal loading standard, detected a polypeptide of approximately 36 kD in size in both the rat heart and human myometrium (Fig 2, A). Cx43 protein expression per labor classification is presented in Fig 2, B. There was no difference in Cx43 protein expression between patients in labor versus patients not in labor (mean ± SE: protein [Cx43:G3PD ratio] = 0.880 ± 0.181 [normal and dysfunctional labor combined] versus 1.123 ± 0.182 [no labor], P > .05). Protein analysis by subgroup. No significant differences in mean Cx43 protein levels existed between patients with dysfunctional labor versus patients with normal labor, patients with dysfunctional labor versus patients not in labor, or for patients with normal labor versus patients not in labor (P > .05 for all). No associations were observed between Cx43 mRNA or protein expression and length of labor, cervical dilation, Montevideo units, frequency of uterine contractions, or average contraction strength. The lack of association occurred for all patients analyzed together and when broken down by subgroup. Cx43 mRNA expression did not correlate with Cx43 protein expression overall or when analyzed with respect to the clinical grouping. Immunohistochemistry. Twenty-eight myometrial specimens were available for localization of Cx43 protein by immunohistochemistry. These included 10 patients who were not in labor, 11 patients with dysfunctional labor, and 7 patients with normal labor. As expected, the antiCx43 antibody specifically stained the intercalated disk regions between rat myocardial cells (Fig 3, A). In human
Table II. Patient labor characteristics and degree of Cx43 immunohistochemical staining
Absent/mild Moderate/dense
No labor
Normal labor
Dysfunctional labor
4 6
5 2
5 6
myometrium, this antibody also stained punctate regions between cells representative of gap junction plaques (Fig 3, B). Very little background staining occurred with the use of the control antibody (normal rabbit serum) in all rat heart and human myometrial tissue specimens, demonstrating the specificity of the anti-Cx43 antibody (not shown). The extent of immunohistochemical staining per group is presented in Table II. There was no difference among the various clinical groups with regard to degree of staining (χ2 = 1.77, P > .05). There also was no difference in mean Cx43 mRNA levels or mean Cx43 protein levels between tissues graded as absent/mild compared with those graded as moderate/dense (P > .05). Similarly, intensity of Cx43 immunostaining was not associated with frequency of uterine contractions, Montevideo units, average contraction strength, length of labor, or cervical dilation (P > .05). Comment The exact mechanisms of labor, both normal and abnormal, remain unknown. The regulation of uterine activity during pregnancy can be divided into 4 distinct physiologic phases (quiescence, activation, stimulation, and involution) that are influenced by several stimulatory and inhibitory factors.20 Cx43 is one of several contractionassociated proteins involved in myometrial activation. Patch-clamp experiments demonstrated that Cx43 is the major gap junction protein in the human myometrium.8
Pierce et al 509
Volume 186, Number 3 Am J Obstet Gynecol
B
A
Fig 2. A, Representative Western blot demonstrating Cx43 protein expression in nonlaboring and laboring patients. Lane R = rat heart positive control; lanes 1, 2, 7-9, 12 = no labor; lanes 6, 11 = normal labor; lanes 3-5, 10, 13 = dysfunctional labor. B, Cx43 protein expression per clinical group. Values presented as mean Cx43:G3PD ratio + SE.
Our finding that Cx43 mRNA expression increases with the onset of labor is consistent with a prior study investigating Cx43 mRNA levels in the human myometrium12 and suggests a role for Cx43 in labor initiation. The results from this study suggest that ineffective labor does not appear to be caused by aberrant expression of Cx43 mRNA or protein or by a reduction in immunodetectable Cx43 gap junctions. We found no significant difference between mRNA levels, protein levels, or Cx43 immunostaining in the myometrium of women in normal labor compared with those with dysfunctional labor. Similar results were obtained in another study21 that used quantitative immunoconfocal analysis to investigate Cx43 expression in term patients who were not in labor, those with normal labor, or those with oxytocinresistant dystocia; the authors found no difference in immunodetectable Cx43 gap junctions among the 3 patient groups. Our data are further supported by the results of Garfield and Hayashi10 who found that the failure to progress in labor was not caused by an absence of gap junctions as determined by electron microscopy, which remains the most accurate method to visualize gap junctions between myometrial cells. It should be noted that the technique of immunohistochemistry used in this study could not rule out the possibility that the Cx43 protein associated with the myometrial cell membranes (Fig 3, B) may actually be internal to the cell and may not be associated with plaques of organized gap junctions. The results from our study and these 2 others10,21 suggest that the failure of the uterine muscle to successfully expel the fetus and placenta during delivery may be related to the functional capacity of the gap junctions, not just their presence. Consistent with this hypothesis is the fact that our study revealed no increased frequency of uterine contractions, Montevideo units, or average contraction strength with increased intensity of Cx43 immunostaining. Alternatively, it is quite possible that
factors unrelated to myometrial contractions, such as cephalopelvic disproportion and fetal malposition, are involved in dysfunctional labor. This may explain the lack of significant differences in Cx43 expression observed among the patient groups in this study. Although no significant difference was observed in the level of immunodetectable Cx43 gap junctions in the myometrium of women with dysfunctional labor compared with women with normal labor, we could not determine by the techniques used in this study whether a difference existed in the functional capacity of the gap junctions observed in these patients. Traditional methods to examine gap junctional communication to determine the existence of functional channels include electrophysiologic techniques, microinjection of fluorescent molecules, and scrape loading/dye transfer techniques.8,22 Gap junctional communication is regulated by the phosphorylation of Cx43 protein as well as by pH.14,22,23 Cx43 phosphorylation may regulate gap junctional protein trafficking, gap junction assembly, channel gating, and gap junction turnover.14 It is feasible that the phosphorylation state of Cx43 gap junction channels may be altered in dysfunctional labor, which may affect gap junctional communication between myometrial cells; this hypothesis remains to be investigated. In addition, the myometrial cellular environment during labor may be relatively acidic, which may also contribute to a downregulation of gap junctional communication in the uterine muscle. In our study, the onset of labor was not associated with an increase in the level of Cx43 protein as determined by Western analysis, which is consistent with past publications.7,12 In addition, we found that the onset of labor was not associated with a sudden increase in immunodetectable Cx43 gap junctions as determined by immunohistochemistry, which is contradictory to some studies11,12 but consistent with others.21 However, myometrial samples used for analysis in the nonlabor group were ob-
510 Pierce et al
March 2002 Am J Obstet Gynecol
A
B Fig 3. A, Cx43 immunohistochemical staining of rat cardiac muscle used as a positive control. Note that punctate staining representative of gap junction plaques is localized between myocardial cells at the intercalated disk area. (Original magnification 500) B, Cx43 immunohistochemical staining of human myometrial tissue from a term patient (39 weeks’ gestation) not in labor. Note abundance of Cx43 punctate staining between myometrial cells. Very little nonspecific staining occurred with the use of control antibody (not shown). (Original magnification 500)
tained from women who had elective cesarean section at term (mean of 39 weeks’ gestation, Table I), and the presence of Cx43 protein and immunodetectable Cx43 gap junctions may be indicative of imminent labor. Electron microscopy has demonstrated the appearance of gap junctions in human myometrium during late pregnancy before the onset of labor.10,11 In contrast to their findings in the myometrium of some nonlaboring patients at term, Balducci et al11 demonstrated that gap junctions
were not present in preterm (<32 weeks’ gestation) myometrium before labor. The appearance of gap junctions just before the onset of labor may be necessary to prepare the uterus to achieve the coordinated contractions required for successful vaginal delivery.7,10-12 As seen in other studies,7,12 some variability existed between samples using the techniques of immunohistochemistry, immunoblotting, and Northern analysis. For example, in some samples there was greater staining of
Volume 186, Number 3 Am J Obstet Gynecol
tissue sections and larger bands on the immunoblots and Northern blots from the myometrium of patients who were not in labor than of patients who were in labor. Tabb et al7 suggested that the observed variability between patients who were in labor versus those who were not in labor may indicate that muscle from the lower uterine segment obtained during cesarean section may not be representative of the fundal and more muscular regions of the uterus during pregnancy. This view is supported by 2 studies that demonstrated differential expression of Cx4324 and Cx2625 in the upper versus lower segments of the human uterus during pregnancy and labor. It should be noted that all of the myometrial biopsies analyzed in this study were obtained from the lower uterine segment, and care must be taken when interpreting results obtained from only 1 region of the pregnant uterus. Another point of interest is the lack of correlation between Cx43 mRNA and protein expression that occurred in this study. For example, Cx43 mRNA levels increased with the onset of labor, although Cx43 total protein levels (as determined by Western analysis) did not. This is consistent with the results of Chow and Lye,12 who suggested that the discrepancy between Cx43 mRNA and protein expression may be caused by an increased turnover rate of Cx43 protein during trafficking of the Cx43 to the membrane. An alternative explanation may be that this finding may not reflect in vivo conditions; for example, the use of G3PD as an internal loading control may not be appropriate for immunoblotting. In conclusion, dysfunctional labor results neither from aberrant Cx43 mRNA or protein expression nor from a reduction in immunodetectable Cx43 gap junctions. The appearance of gap junctions is likely to be only one of several steps involved in the successful progression of labor. Failure of uterine muscle may be related to the functional capacity of the gap junctions, and not just to their presence. Further study is needed to determine whether contractile dysfunction in labor is associated with disrupted gap junctional communication between myometrial cells. We thank Louis Matej and M. J. DeHart for their technical expertise. REFERENCES
1. Garfield RE, Sims S, Daniel EE. Gap junctions: their presence and necessity in myometrium during parturition. Science 1977; 198:958-60. 2. Garfield RE, Blennerhasset MG, Miller SM. Control of myometrial contractility: role and regulation of gap junctions. Oxf Rev Reprod Biol 1988;10:436-90. 3. Paul DL, Yu K, Bruzzone R, Gimlich RL, Goodenough DA. Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells. Development 1995;121:371-81. 4. Loewenstein WR, Rose B. The cell-cell channel in the control of growth. Sem Cell Biol 1992;3:59-79.
Pierce et al 511
5. Beyer EC, Goodenough DA, Paul DL. The connexins: a family of related gap junction proteins. In: Hertzberg EL, Johnson R, editors. Gap junction. New York: Alan R. Liss; 1988. p.167-75. 6. Beyer EC, Paul DL, Goodenough DA. Connexin 43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 1987;105:2621-9. 7. Tabb T, Thilander G, Grover A, Hertzberg E, Garfield R. An immunochemical and immunocytologic study of the increase in myometrial gap junctions (and connexin 43) in rats and humans during pregnancy. Am J Obstet Gynecol 1992;167:559-67. 8. Miyoshi H, Boyle MB, MacKay LB, Garfield RE. Gap junction currents in cultured muscle cells from human myometrium. Am J Obstet Gynecol 1998;178:588-93. 9. Makowski L, Caspar DLD, Phillips WC, Goodenough DA. Gap junction structures. II. Analysis of the x-ray diffraction data. J Cell Biol 1977;74:629-45. 10. Garfield RE, Hayashi, RH. Appearance of gap junctions in the myometrium of women during labor. Am J Obstet Gynecol 1981;140:254-60. 11. Balducci J, Risek B, Gilula NB, Hand A, Egan JFX, Vintzileos AM. Gap junction formation in human myometrium: a key to preterm labor? Am J Obstet Gynecol 1993;168:1609-15. 12. Chow L, Lye SJ. Expression of the gap junction protein connexin43 is increased in the human myometrium toward term and with the onset of labor. Am J Obstet Gynecol 1994;170:788-95. 13. Hendrix EM, Mao SJ, Everson W, Larsen WJ. Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor. Mol Reprod Dev 1992;33:27-38. 14. Laird DW, Castillo M, Kasprzak L. Gap junction turnover, intracellular trafficking, and phosphorylation of connexin 43 in brefeldin A–treated rat mammary tumor cells. J Cell Biol 1995;131:1193-203. 15. Piersanti M, Lye SJ. Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos. Endocrinology 1995;136:3571-8. 16. Geimonen E, Jiang W, Ali M, Fishman GI, Garfield RE, Andersen J. Activation of protein kinase C in human uterine smooth muscle induces connexin-43 gene transcription through an AP-1 site in the promoter sequence. J Biol Chem 1996;271:23667-74. 17. Echetebu CO, Ali M, Izban MG, MacKay L, Garfield RE. Localization of regulatory protein binding sites in the proximal region of human myometrial connexin 43 gene. Mol Hum Reprod 1999;5:757-66. 18. McElvy SS, Miodovnik M, Myatt L, Khoury J, Siddiqi TA. Is human myometrial sampling at the time of cesarean delivery safe? Am J Obstet Gynecol 2000;183:1583-6. 19. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. 20. Norwitz ER, Robinson JN, Challis JRG. Current concepts: the control of labor. N Engl J Med 1999;341:660-6. 21. Rezapour M, Kilarski WM, Severs NJ, Gourdie RG, Rothery S, Backstrom T, et al. Quantitative immunoconfocal analysis of human myometrial gap junction connexin 43 in relation to steroid hormone concentrations at term labour. Hum Reprod 1997;12:159-66. 22. Warn-Cramer BJ, Cottrell GT, Burt JM, Lau AF. Regulation of connexin-43 gap junctional communication by mitogenactivated protein kinase. J Biol Chem 1998;273:9188-96. 23. Morley GE, Ek-Vitorin JF, Taffet SM, Delmar M. Structure of connexin 43 and its regulation by pHi. J Cardiovasc Electrophysiol 1997;8:939-51. 24. Sparey C, Robson SC, Bailey J, Lyall F, Europe-Finner GN. The differential expression of myometrial connexin-43, cyclooxygenase-1 and –2, and Gsα proteins in the upper and lower segments of the human uterus during pregnancy and labor. J Clin Endocrinol Metab 1999;84:1705-10. 25. Ciray HN, Fu X, Olovsson M, Ahlsen G, Shuman C, Lindblom B, et al. Presence and localization of connexins 43 and 26 in cell cultures derived from myometrial tissues from nonpregnant and pregnant women and from leiomyomas. Am J Obstet Gynecol 2000;182:926-30.
Key words: Connexin 43, gap junction, myometrium, labor, dysfunctional labor
Gap junctions are thought to be one of several essential components for effective labor and delivery.1,2 Gap junctions mediate cell-to-cell communication, maintain normal cell function, and play an important role in embryonic development and possibly growth control.3,4 They also provide low-resistance pathways between smooth muscle cells, thereby increasing their electrical coupling to allow increased coordination of contractile activity. Connexins are a family of gap junction proteins that are named for their molecular weights, with connexin 43 (molecular weight, 43 kD) being the major gap junction protein present in myometrium.5-8 Gap junctions are formed when a connexon (hemichannel) from 1 cell pairs with a symmetrically opposed connexon from an adjacent cell, forming a channel that permits the passage of
From the Division of Maternal-Fetal Medicine,a the Department of Pathology,b and the Department of Clinical Investigation,c Madigan Army Medical Center, and the Cancer Research Center of Hawaii, University of Hawaii.d Supported by the Department of the Air Force Clinical Investigations, grant No. SGO 2000-0015H. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Air Force, the Department of the Army, or the Department of Defense. Received for publication June 14, 2001; revised October 1, 2001; accepted October 18, 2001. Reprint requests: Brian T. Pierce, MD, Department of Obstetrics and Gynecology, Darnall Army Community Hospital, 36000 Darnall Loop, Fort Hood, TX 76544-5063. Copyright 2002, Mosby, Inc. All rights reserved. 0002-9378/2002 $35.00 + 0 6/1/121108 doi:10.1067/mob.2002.121108
504
ions and small molecules (<1000-1700 D) between neighboring cells.9 Each connexon consists of an oligomer of 6 connexin proteins that form the central aqueous pore.9 During most of pregnancy, gap junction plaques (clusters of numerous gap junctions) are undetectable or are present in very low numbers as measured by electron microscopy or immunohistochemistry.7,10-12 This is consistent with the presence of poorly coordinated, lowamplitude contractions (Braxton Hicks) during pregnancy. The onset of labor is associated with a dramatic increase in the number and size of the plaques, an increase in the electrical conductivity of the myometrium, and the development of spontaneous, well-coordinated labor contractions.7,10-12 Correlating to the appearance of gap junction plaques, connexin 43 (Cx43) messenger RNA (mRNA) levels increase in the human myometrium toward term and are highest during delivery.12 Effective labor may require the synthesis and assembly of Cx43 into functional gap junctions at the myometrial cell surface.1,2 The exact mechanisms that regulate the synthesis and assembly of gap junctions in the human myometrium are not known. Hendrix et al13 found in rats that Cx43 was synthesized several days before labor but accumulated within the cytoplasm until parturition, when it was rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These Cx43-positive gap junctions disappeared within 24 hours after delivery. Cx43 phosphorylation may regulate gap junctional protein trafficking, gap junction assembly, channel gating, and gap junction turnover.14 Laird et al14 demonstrated in rat mammary tumor cells that Cx43 is transiently phosphorylated in the endoplas-
Volume 186, Number 3 Am J Obstet Gynecol
mic reticulum/Golgi apparatus, where it is available for the assembly of new gap junction channels. The authors also showed that the phosphorylation of Cx43 is necessary for the trafficking of Cx43 to the membrane and that the rapid turnover of gap junction plaques correlates with dephosphorylated forms of Cx43.14 Studies that investigated the transcriptional regulation of the human Cx43 gene identified activator protein 1 and specificity protein 1 consensus sites in the promoter sequence of the Cx43 gene.15-17 Upregulation of Cx43 gene expression appears to involve induction of the transcription factors c-jun and c-fos (which bind to the activator protein 1 site on the Cx43 promoter) and specificity protein 1 (which binds to the specificity protein 1 site) after protein kinase C activation.15-17 In this study, we explored the hypothesis that aberrant Cx43 expression, which may lead to decreased gap junction formation and disrupted gap junctional communication, may be responsible, at least in part, for the lack of high amplitude, well-coordinated contractions occurring in patients with dysfunctional labor. The goal of the present investigation was to determine whether Cx43 is differentially expressed in normal versus dysfunctional labor. Material and methods Subjects. The human subjects protocol for this study was reviewed and approved by the institutional review board of Madigan Army Medical Center. Written informed consent was obtained from all subjects after they were consented for, but before cesarean section was performed. Study patients included all women undergoing cesarean section at >35 weeks’ gestation. Patients with chorioamnionitis were excluded from this study. Clinical information for each patient was recorded and included maternal age, gestational age, birth weight, length of labor, cervical dilation, Montevideo units for the final 10 minutes of available tracing, number of contractions in the final 10 minutes of available tracing, and presence and duration of rupture of membranes. For purposes of analysis, study patients were divided into the following 3 groups on the basis of indication for cesarean section and the characteristics of the first stage of labor: 1. No labor (elective) cesarean section group: cesarean section before the onset of labor 2. Dysfunctional labor group: laboring patients undergoing cesarean section with the labor course classified as failure to progress, protracted active phase, or secondary arrest of dilation 3. Normal labor group: laboring patients undergoing cesarean section for indications unrelated to the first stage of labor characteristics. These included nonreassuring fetal heart rate tracing, desired repeat cesarean section presenting in labor, and contraindications for vaginal delivery presenting in labor.
Pierce et al 505
Failure to progress was defined as the inability to progress past latent phase labor, despite adequate uterine contractions and with or without pitocin augmentation. Secondary arrest of dilation was defined as cessation of cervical dilation for a period of 2 hours, despite adequate uterine contractions and with or without pitocin augmentation. Nonreassuring fetal heart rate tracing was defined as a fetal heart rate tracing demonstrating characteristics necessitating operative intervention as determined by the attending staff obstetrician. Contraindications for vaginal delivery included prior classical cesarean section and suspected fetal macrosomia in a pregnancy complicated by diabetes mellitus. Montevideo units were determined by internal uterine pressure catheter (IUPC) and defined as the summation of the amplitude of each uterine contraction minus the baseline in a 10-minute period. Average contraction strength was defined as Montevideo units divided by the number of contractions in a 10-minute period. The use of IUPCs was determined by the attending physician and was not a criterion for study enrollment, although all (11 of 11) patients with dysfunctional labor and 4 of 7 patients with normal labor had IUPCs. The final 10 minutes of available IUPC tracing was used to calculate Montevideo units and average contraction strength for study purposes. Tissue collection. Myometrial biopsies (approximately 2 cm 1 cm 1 cm) from the lower uterine segment were excised from the superior middle margin of the uterine incision during cesarean section after delivery of the fetus and placenta. Human myometrial sampling at cesarean delivery has been shown not to increase overall maternal morbidity in the presence or absence of labor.18 Tissue samples were washed in physiologic saline solution and divided into thirds for Northern analysis, Western analysis, and immunohistochemistry. For Northern and Western analysis, tissue samples were wrapped in foil, snap-frozen in liquid nitrogen or in an isopentane cryobath, and stored at –70°C until further processing. For immunohistochemistry experiments, tissue samples were embedded in ornithine carbamyl transferase, snap-frozen in isopentane, and stored at –70°C before sectioning and staining. Rat heart was prepared in a similar fashion as the human myometrial tissue and was used as a positive control in each experiment performed. Northern analysis. Total cellular RNA was isolated from the myometrium and rat heart using the method of Chomczynski and Sacchi.19 Frozen tissues were placed into 3 mL of Solution D19 in a 50-mL tube on dry ice and were minced with a scalpel. Samples were then homogenized at room temperature with a Polytron hand-held tissue homogenizer. (model pT1200C Brinkman Instruments, Inc, Westbury, NY) and allowed to sit at room temperature for 15 minutes. One-tenth volume of 2 mol/L (2 molar = 2 mol/L) sodium acetate (pH 4.0), one-fifth volume of chloroform-isoamyl alcohol mixture (48:1), and
506 Pierce et al
an equal volume of diethyl pyrocarbonate–treated watersaturated phenol was added to each homogenate with thorough mixing by inversion after the addition of each reagent. The final suspension was shaken vigorously, transferred to a 15-mL tube, and cooled on ice for 20 minutes. Samples were centrifuged at 4°C for 20 minutes at 9,500 rpm in a Beckman GS-15R tabletop centrifuge. (Beckman Coulter, Inc, Fullerton, Calif) The aqueous layer was removed and placed into 1.5-mL microcentrifuge tubes in 600-µL aliquots. An equal volume of isopropanol was added, and samples were either frozen overnight at –70°C or on dry ice for 15 minutes. Samples were centrifuged at 15,300 rpm for 20 minutes, and the resulting RNA pellets were dissolved in 600 µL of Solution D, precipitated again with an equal volume of isopropanol either overnight at –70°C or on dry ice for 15 minutes, centrifuged for 20 minutes, and washed in 80% ice cold ethanol. RNA pellets were centrifuged for 10 minutes, resuspended in 75 µL of diethyl pyrocarbonate–treated water, and quantitated using a ultraviolet spectrophotometer. Total RNA (30 µg/lane) was denatured, resolved in a 1.2% agarose-formaldehyde gel, transferred to a GeneScreen (NEN, Boston, Mass) membrane in 10X saline sodium citrate, and ultraviolet light cross-linked to the membrane. Probes consisted of complementary DNAs (cDNAs) for Cx436 and 18S ribosomal RNA (Ambion, Inc., Austin, Tex) as an internal loading standard. Probes were labeled by random priming (Amersham, Arlington Heights, Ill) with [α-32P] deoxycytidine triphosphate (3,000 Ci/mmol, Amersham, Arlington Heights, Ill) and the Klenow fragment of Escherichia coli DNA polymerase. Unincorporated counts were removed using STE push columns (Stratagene, La Jolla, Calif). Prehybridization and hybridization were performed at 55°C using the SuperTM Hybridization Buffer System (DNA Technologies, Inc., Rockville, MD) following the manufacturer’s instructions. Blots probed with Cx43 were washed 3 5 minutes in 2 saline sodium citrate at room temperature before images were obtained with a BioRad GS-505 Molecular Imager System (Bio-Rad Laboratories, Hercules, Calif). Blots that were stripped and reprobed with the 18S cDNA were washed in the buffers supplied in the SuperTM Hybridization Buffer System kit (DNA Technologies, Inc., Rockville, Md) following the manufacturer’s instructions. Densitometry was used to measure the relative quantity of Cx43 mRNA present in the samples (calculated as Cx43:18S ratio). Western analysis. Myometrial samples and rat heart were homogenized on ice in RIPA lysis buffer (10 mmol/L Tris, pH 7.2/150 mmol/L NaCl/1% sodium deoxycholate/1% Triton X-100/0.1% sodium dodecyl sulfate/1 mmol/L polymethylsulfonyl fluoride/10 mmol/L NaF) containing protease inhibitors. One protease inhibitor tablet (Roche, Palo Alto, Calif) was added
March 2002 Am J Obstet Gynecol
per 10 mL of lysis buffer, and 100 µL of protease inhibitor mix (Sigma, St. Louis, Mo) was added per 40 mL of lysis buffer. Lysis buffer was added to tissue samples at a ratio of 1 g tissue:20 mL lysis buffer (approximate weight of each tissue sample for protein extraction was 200 mg). Homogenates were incubated on ice for 15 to 20 minutes and centrifuged at 14,000 rpm for 10 minutes, and the supernatants containing extracted protein were collected. An aliquot of the supernatant was removed for protein determination using the Lowry protein assay using bovine serum albumin (BSA) as a standard. Aliquots of the protein extracts were frozen at –70°C and saved for Western analysis. Thirty µg of protein from each sample was diluted in 2 loading buffer (20% glycerol/20% sodium dodecyl sulfate (SDS) /0.1% bromophenol blue/0.1 mol/L Tris-HCl, pH 6.8/10% β-mercaptoethanol). Samples were heated at 90°C for 2 minutes, electrophoresed in a 10% polyacrylamide gel with a 6% stacking gel, and transferred for 2 hours at 200 mA (4°C) onto a polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, Mass). The membrane was blocked overnight in 5% nonfat milk/phosphate-buffered saline solution/0.1% Tween20/1% goat serum at 4°C before a 1-hour incubation with a rabbit polyclonal antiserum (diluted 1:1,000) directed against the last 15 amino acids of the Cx43 carboxyl terminal tail (residues 368-382). The membrane was incubated with a peroxidase-conjugated goat anti-rabbit immunoglobin G (IgG) (1:25,000 dilution; Pierce, Rockford, Ill) as the secondary antibody, washed, and then incubated with a mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (G3PD; Chemicon, Temecula, Calif) antibody used as an internal loading standard (1:1,000 dilution). The final incubation step involved a peroxidase-conjugated goat anti-mouse IgG secondary antibody (Chemicon, Temecula, Calif) at 1:25,000 dilution. Immunodetection of the Cx43 and G3PD protein was performed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, Ill) following the manufacturer’s instructions. Densitometry was used to measure the relative quantity of Cx43 protein present in the samples (calculated as Cx43:G3PD ratio). Immunohistochemistry. Frozen myometrial tissues and rat heart were cut into 5 µm sections using a cryostat at –20°C. Sections were collected onto Biocare Kling-On HIER slides (Biocare Medical, Walnut Creek, Calif) and allowed to dry for 15 to 20 minutes at room temperature. Tissue sections were fixed in 100% acetone (4°C) for 10 minutes and dried completely before storage at –70°C. Slides were post-fixed in 100% acetone (4°C) for 10 minutes and transferred to 70% acetone (4°C) and 50% acetone (4°C) for 30 seconds each. Slides were rinsed briefly in tap water and then in wash buffer (tris buffered saline solution with surfactant; Biocare Medical, Walnut Creek, Calif). Immunostaining occurred with the use of the Dako
Pierce et al 507
Volume 186, Number 3 Am J Obstet Gynecol
Table I. Patient demographics and labor characteristics (mean value ± SD) No labor n Age (y) Gestational age (wk) Birth weight (g) Dilation (cm) Nulliparity Length of labor (h)
10 29.0 ± 7.3 38.8 ± 1.4 3446.1 ± 69.3 0-1 10% N/A
Dysfunctional labor 11 25.5 ± 6.9 39.4 ± 1.5 3689.9 ± 799.4 6-7 91% 28.3 ± 10.2
Normal labor 7 30.0 ± 2.3 37.7 ± 1.3 3302.9 ± 656.4 4-5 0% 10.6 ± 6.8
P value
NS* NS* NS* <.0001* <.0001+ .002‡
NS, Not significant. *Analysis of variance. +χ2 analysis. ‡Student t test.
EnVision+ System (Dako Corporation, Carpinteria, Calif). Briefly, endogenous peroxidase activity was quenched by incubating the sections with a 3% hydrogen peroxide solution for 5 minutes. Sections were rinsed in wash buffer and were incubated for 60 minutes at room temperature with a rabbit polyclonal anti-Cx43 antibody applied at a dilution of 1:200 (Zymed Laboratories, Inc., San Francisco, Calif). Sections were rinsed in wash buffer and incubated with a biotin-free, horseradish peroxidase-labeled polymer conjugated to goat anti-rabbit secondary antibodies (Dako Corporation) for 30 minutes. Rinsed sections were then incubated for 5 minutes with a 3,3’-diaminobenzidine chromogen-substrate to form a colored reaction product. Slides were counterstained with Mayer’s hematoxylin (Dako Corporation), dehydrated, and permanently mounted. A positive control of rat heart was used in each experiment performed. Negative controls were achieved by applying normal rabbit serum (normal rabbit immunoglobulin fraction, Dako Corporation) diluted 1:8,000 instead of the anti-Cx43 primary antibody. Each tissue examined possessed its own negative control slide. For the immunohistochemical analysis, the whole area of the specimens was read at objective magnifications from 10 to 40. Two to 4 tissue sections per specimen were scored for intensity of Cx43 staining independently by 2 investigators (Brian T. Pierce, MD, and Lisa M. Pierce, DSc) blinded to the clinical group from which the tissue came. Staining intensity was scored as absent or faint (+), mild (++), moderate (+++), and dense (++++). Statistical analysis. Data were analyzed with the use of the computer statistical program Statview for Windows (version 4.57; SAS, Cary, NC). The Mann-Whitney U test was used to assess differences in mRNA and protein expression among the different groups of patients and to assess differences in Cx43 mRNA levels, Cx43 protein levels, frequency of uterine contractions, Montevideo units, average contraction strength, length of labor, and cervical dilation compared with degree of Cx43 immunohistochemical staining (absent/mild versus moderate/dense). Analysis of variance, the Student t
test, and χ2 analysis were used to assess demographic differences in labor characteristics among the groups. Simple regression analysis was used to assess for correlation between mRNA expression or protein expression and cervical dilation, length of labor, Montevideo units, frequency of uterine contractions, and average strength of uterine contractions. The relationship between Cx43 staining intensity and labor status was analyzed with the χ2 test. Statistical significance was determined by P < .05. Results Patient demographic information and labor characteristics are listed in Table I. Patients in the various categories differed with respect to cervical dilation, parity, and length of labor (P < .05; Table I). Cx43 mRNA levels. Twenty-eight myometrial specimens were available for Cx43 mRNA determination by Northern analysis. These included 10 patients who were not in labor, 11 patients with dysfunctional labor, and 7 patients with normal labor. The Cx43 cDNA probe hybridized to a transcript of approximately 3.3 kb in size in both the rat heart and human myometrium, similar to that reported in previous studies (Fig 1, A).11,12 Cx43 mRNA expression per labor classification is presented in Fig 1, B. Expression of Cx43 mRNA increased during labor (mean ± SE: mRNA [Cx43:18S ratio] = 0.123 ± 0.031 [normal and dysfunctional labor combined] versus 0.041 ± 0.006 [no labor], P < .05). mRNA analysis by subgroup. There was no difference in mean Cx43 mRNA expression between patients with dysfunctional labor versus patients with normal labor (P > .05). Cx43 mRNA expression was elevated in patients with dysfunctional labor compared with patients not in labor (mean ± SE: mRNA [Cx43:18S ratio] = 0.131 ± 0.047 [dysfunctional labor] versus 0.041 ± 0.006 [no labor], P = .038). Cx43 mRNA expression also tended to be elevated in patients with normal labor compared with patients who were not in labor (mean ± SE: mRNA [Cx43:18S ratio] = 0.109 ± 0.036 [normal labor] versus 0.041 ± 0.006 [no labor], P =.051).
508 Pierce et al
A
March 2002 Am J Obstet Gynecol
B Fig 1. A, Representative Northern blot demonstrating Cx43 mRNA expression in nonlaboring and laboring patients. Lane R = rat heart positive control; lanes 7, 8 = no labor; lane 3 = normal labor; lanes 1, 2, 4-6 = dysfunctional labor. B, Cx43 mRNA expression per clinical group. Values presented as mean Cx43:18S ratio + SE.
Cx43 protein levels. Twenty-six myometrial specimens were available for Cx43 protein determination by Western analysis. These included 10 patients who were not in labor, 10 patients with dysfunctional labor, and 6 patients with normal labor. The anti-Cx43 antibody detected a band of approximately 43 kD in size in both the rat heart and human myometrium (Fig 2, A). The anti-G3PD antibody, used as an internal loading standard, detected a polypeptide of approximately 36 kD in size in both the rat heart and human myometrium (Fig 2, A). Cx43 protein expression per labor classification is presented in Fig 2, B. There was no difference in Cx43 protein expression between patients in labor versus patients not in labor (mean ± SE: protein [Cx43:G3PD ratio] = 0.880 ± 0.181 [normal and dysfunctional labor combined] versus 1.123 ± 0.182 [no labor], P > .05). Protein analysis by subgroup. No significant differences in mean Cx43 protein levels existed between patients with dysfunctional labor versus patients with normal labor, patients with dysfunctional labor versus patients not in labor, or for patients with normal labor versus patients not in labor (P > .05 for all). No associations were observed between Cx43 mRNA or protein expression and length of labor, cervical dilation, Montevideo units, frequency of uterine contractions, or average contraction strength. The lack of association occurred for all patients analyzed together and when broken down by subgroup. Cx43 mRNA expression did not correlate with Cx43 protein expression overall or when analyzed with respect to the clinical grouping. Immunohistochemistry. Twenty-eight myometrial specimens were available for localization of Cx43 protein by immunohistochemistry. These included 10 patients who were not in labor, 11 patients with dysfunctional labor, and 7 patients with normal labor. As expected, the antiCx43 antibody specifically stained the intercalated disk regions between rat myocardial cells (Fig 3, A). In human
Table II. Patient labor characteristics and degree of Cx43 immunohistochemical staining
Absent/mild Moderate/dense
No labor
Normal labor
Dysfunctional labor
4 6
5 2
5 6
myometrium, this antibody also stained punctate regions between cells representative of gap junction plaques (Fig 3, B). Very little background staining occurred with the use of the control antibody (normal rabbit serum) in all rat heart and human myometrial tissue specimens, demonstrating the specificity of the anti-Cx43 antibody (not shown). The extent of immunohistochemical staining per group is presented in Table II. There was no difference among the various clinical groups with regard to degree of staining (χ2 = 1.77, P > .05). There also was no difference in mean Cx43 mRNA levels or mean Cx43 protein levels between tissues graded as absent/mild compared with those graded as moderate/dense (P > .05). Similarly, intensity of Cx43 immunostaining was not associated with frequency of uterine contractions, Montevideo units, average contraction strength, length of labor, or cervical dilation (P > .05). Comment The exact mechanisms of labor, both normal and abnormal, remain unknown. The regulation of uterine activity during pregnancy can be divided into 4 distinct physiologic phases (quiescence, activation, stimulation, and involution) that are influenced by several stimulatory and inhibitory factors.20 Cx43 is one of several contractionassociated proteins involved in myometrial activation. Patch-clamp experiments demonstrated that Cx43 is the major gap junction protein in the human myometrium.8
Pierce et al 509
Volume 186, Number 3 Am J Obstet Gynecol
B
A
Fig 2. A, Representative Western blot demonstrating Cx43 protein expression in nonlaboring and laboring patients. Lane R = rat heart positive control; lanes 1, 2, 7-9, 12 = no labor; lanes 6, 11 = normal labor; lanes 3-5, 10, 13 = dysfunctional labor. B, Cx43 protein expression per clinical group. Values presented as mean Cx43:G3PD ratio + SE.
Our finding that Cx43 mRNA expression increases with the onset of labor is consistent with a prior study investigating Cx43 mRNA levels in the human myometrium12 and suggests a role for Cx43 in labor initiation. The results from this study suggest that ineffective labor does not appear to be caused by aberrant expression of Cx43 mRNA or protein or by a reduction in immunodetectable Cx43 gap junctions. We found no significant difference between mRNA levels, protein levels, or Cx43 immunostaining in the myometrium of women in normal labor compared with those with dysfunctional labor. Similar results were obtained in another study21 that used quantitative immunoconfocal analysis to investigate Cx43 expression in term patients who were not in labor, those with normal labor, or those with oxytocinresistant dystocia; the authors found no difference in immunodetectable Cx43 gap junctions among the 3 patient groups. Our data are further supported by the results of Garfield and Hayashi10 who found that the failure to progress in labor was not caused by an absence of gap junctions as determined by electron microscopy, which remains the most accurate method to visualize gap junctions between myometrial cells. It should be noted that the technique of immunohistochemistry used in this study could not rule out the possibility that the Cx43 protein associated with the myometrial cell membranes (Fig 3, B) may actually be internal to the cell and may not be associated with plaques of organized gap junctions. The results from our study and these 2 others10,21 suggest that the failure of the uterine muscle to successfully expel the fetus and placenta during delivery may be related to the functional capacity of the gap junctions, not just their presence. Consistent with this hypothesis is the fact that our study revealed no increased frequency of uterine contractions, Montevideo units, or average contraction strength with increased intensity of Cx43 immunostaining. Alternatively, it is quite possible that
factors unrelated to myometrial contractions, such as cephalopelvic disproportion and fetal malposition, are involved in dysfunctional labor. This may explain the lack of significant differences in Cx43 expression observed among the patient groups in this study. Although no significant difference was observed in the level of immunodetectable Cx43 gap junctions in the myometrium of women with dysfunctional labor compared with women with normal labor, we could not determine by the techniques used in this study whether a difference existed in the functional capacity of the gap junctions observed in these patients. Traditional methods to examine gap junctional communication to determine the existence of functional channels include electrophysiologic techniques, microinjection of fluorescent molecules, and scrape loading/dye transfer techniques.8,22 Gap junctional communication is regulated by the phosphorylation of Cx43 protein as well as by pH.14,22,23 Cx43 phosphorylation may regulate gap junctional protein trafficking, gap junction assembly, channel gating, and gap junction turnover.14 It is feasible that the phosphorylation state of Cx43 gap junction channels may be altered in dysfunctional labor, which may affect gap junctional communication between myometrial cells; this hypothesis remains to be investigated. In addition, the myometrial cellular environment during labor may be relatively acidic, which may also contribute to a downregulation of gap junctional communication in the uterine muscle. In our study, the onset of labor was not associated with an increase in the level of Cx43 protein as determined by Western analysis, which is consistent with past publications.7,12 In addition, we found that the onset of labor was not associated with a sudden increase in immunodetectable Cx43 gap junctions as determined by immunohistochemistry, which is contradictory to some studies11,12 but consistent with others.21 However, myometrial samples used for analysis in the nonlabor group were ob-
510 Pierce et al
March 2002 Am J Obstet Gynecol
A
B Fig 3. A, Cx43 immunohistochemical staining of rat cardiac muscle used as a positive control. Note that punctate staining representative of gap junction plaques is localized between myocardial cells at the intercalated disk area. (Original magnification 500) B, Cx43 immunohistochemical staining of human myometrial tissue from a term patient (39 weeks’ gestation) not in labor. Note abundance of Cx43 punctate staining between myometrial cells. Very little nonspecific staining occurred with the use of control antibody (not shown). (Original magnification 500)
tained from women who had elective cesarean section at term (mean of 39 weeks’ gestation, Table I), and the presence of Cx43 protein and immunodetectable Cx43 gap junctions may be indicative of imminent labor. Electron microscopy has demonstrated the appearance of gap junctions in human myometrium during late pregnancy before the onset of labor.10,11 In contrast to their findings in the myometrium of some nonlaboring patients at term, Balducci et al11 demonstrated that gap junctions
were not present in preterm (<32 weeks’ gestation) myometrium before labor. The appearance of gap junctions just before the onset of labor may be necessary to prepare the uterus to achieve the coordinated contractions required for successful vaginal delivery.7,10-12 As seen in other studies,7,12 some variability existed between samples using the techniques of immunohistochemistry, immunoblotting, and Northern analysis. For example, in some samples there was greater staining of
Volume 186, Number 3 Am J Obstet Gynecol
tissue sections and larger bands on the immunoblots and Northern blots from the myometrium of patients who were not in labor than of patients who were in labor. Tabb et al7 suggested that the observed variability between patients who were in labor versus those who were not in labor may indicate that muscle from the lower uterine segment obtained during cesarean section may not be representative of the fundal and more muscular regions of the uterus during pregnancy. This view is supported by 2 studies that demonstrated differential expression of Cx4324 and Cx2625 in the upper versus lower segments of the human uterus during pregnancy and labor. It should be noted that all of the myometrial biopsies analyzed in this study were obtained from the lower uterine segment, and care must be taken when interpreting results obtained from only 1 region of the pregnant uterus. Another point of interest is the lack of correlation between Cx43 mRNA and protein expression that occurred in this study. For example, Cx43 mRNA levels increased with the onset of labor, although Cx43 total protein levels (as determined by Western analysis) did not. This is consistent with the results of Chow and Lye,12 who suggested that the discrepancy between Cx43 mRNA and protein expression may be caused by an increased turnover rate of Cx43 protein during trafficking of the Cx43 to the membrane. An alternative explanation may be that this finding may not reflect in vivo conditions; for example, the use of G3PD as an internal loading control may not be appropriate for immunoblotting. In conclusion, dysfunctional labor results neither from aberrant Cx43 mRNA or protein expression nor from a reduction in immunodetectable Cx43 gap junctions. The appearance of gap junctions is likely to be only one of several steps involved in the successful progression of labor. Failure of uterine muscle may be related to the functional capacity of the gap junctions, and not just to their presence. Further study is needed to determine whether contractile dysfunction in labor is associated with disrupted gap junctional communication between myometrial cells. We thank Louis Matej and M. J. DeHart for their technical expertise. REFERENCES
1. Garfield RE, Sims S, Daniel EE. Gap junctions: their presence and necessity in myometrium during parturition. Science 1977; 198:958-60. 2. Garfield RE, Blennerhasset MG, Miller SM. Control of myometrial contractility: role and regulation of gap junctions. Oxf Rev Reprod Biol 1988;10:436-90. 3. Paul DL, Yu K, Bruzzone R, Gimlich RL, Goodenough DA. Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells. Development 1995;121:371-81. 4. Loewenstein WR, Rose B. The cell-cell channel in the control of growth. Sem Cell Biol 1992;3:59-79.
Pierce et al 511
5. Beyer EC, Goodenough DA, Paul DL. The connexins: a family of related gap junction proteins. In: Hertzberg EL, Johnson R, editors. Gap junction. New York: Alan R. Liss; 1988. p.167-75. 6. Beyer EC, Paul DL, Goodenough DA. Connexin 43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 1987;105:2621-9. 7. Tabb T, Thilander G, Grover A, Hertzberg E, Garfield R. An immunochemical and immunocytologic study of the increase in myometrial gap junctions (and connexin 43) in rats and humans during pregnancy. Am J Obstet Gynecol 1992;167:559-67. 8. Miyoshi H, Boyle MB, MacKay LB, Garfield RE. Gap junction currents in cultured muscle cells from human myometrium. Am J Obstet Gynecol 1998;178:588-93. 9. Makowski L, Caspar DLD, Phillips WC, Goodenough DA. Gap junction structures. II. Analysis of the x-ray diffraction data. J Cell Biol 1977;74:629-45. 10. Garfield RE, Hayashi, RH. Appearance of gap junctions in the myometrium of women during labor. Am J Obstet Gynecol 1981;140:254-60. 11. Balducci J, Risek B, Gilula NB, Hand A, Egan JFX, Vintzileos AM. Gap junction formation in human myometrium: a key to preterm labor? Am J Obstet Gynecol 1993;168:1609-15. 12. Chow L, Lye SJ. Expression of the gap junction protein connexin43 is increased in the human myometrium toward term and with the onset of labor. Am J Obstet Gynecol 1994;170:788-95. 13. Hendrix EM, Mao SJ, Everson W, Larsen WJ. Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor. Mol Reprod Dev 1992;33:27-38. 14. Laird DW, Castillo M, Kasprzak L. Gap junction turnover, intracellular trafficking, and phosphorylation of connexin 43 in brefeldin A–treated rat mammary tumor cells. J Cell Biol 1995;131:1193-203. 15. Piersanti M, Lye SJ. Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos. Endocrinology 1995;136:3571-8. 16. Geimonen E, Jiang W, Ali M, Fishman GI, Garfield RE, Andersen J. Activation of protein kinase C in human uterine smooth muscle induces connexin-43 gene transcription through an AP-1 site in the promoter sequence. J Biol Chem 1996;271:23667-74. 17. Echetebu CO, Ali M, Izban MG, MacKay L, Garfield RE. Localization of regulatory protein binding sites in the proximal region of human myometrial connexin 43 gene. Mol Hum Reprod 1999;5:757-66. 18. McElvy SS, Miodovnik M, Myatt L, Khoury J, Siddiqi TA. Is human myometrial sampling at the time of cesarean delivery safe? Am J Obstet Gynecol 2000;183:1583-6. 19. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. 20. Norwitz ER, Robinson JN, Challis JRG. Current concepts: the control of labor. N Engl J Med 1999;341:660-6. 21. Rezapour M, Kilarski WM, Severs NJ, Gourdie RG, Rothery S, Backstrom T, et al. Quantitative immunoconfocal analysis of human myometrial gap junction connexin 43 in relation to steroid hormone concentrations at term labour. Hum Reprod 1997;12:159-66. 22. Warn-Cramer BJ, Cottrell GT, Burt JM, Lau AF. Regulation of connexin-43 gap junctional communication by mitogenactivated protein kinase. J Biol Chem 1998;273:9188-96. 23. Morley GE, Ek-Vitorin JF, Taffet SM, Delmar M. Structure of connexin 43 and its regulation by pHi. J Cardiovasc Electrophysiol 1997;8:939-51. 24. Sparey C, Robson SC, Bailey J, Lyall F, Europe-Finner GN. The differential expression of myometrial connexin-43, cyclooxygenase-1 and –2, and Gsα proteins in the upper and lower segments of the human uterus during pregnancy and labor. J Clin Endocrinol Metab 1999;84:1705-10. 25. Ciray HN, Fu X, Olovsson M, Ahlsen G, Shuman C, Lindblom B, et al. Presence and localization of connexins 43 and 26 in cell cultures derived from myometrial tissues from nonpregnant and pregnant women and from leiomyomas. Am J Obstet Gynecol 2000;182:926-30.