We used a timed-pregnant rat model to track changes in myometrial contractility during pregnancy and labor and to correlate these changes with upstream signaling events. Myometrium was harvested from CO2-euthanized rats. Although contraction amplitudes increased at 16 and 20 days of pregnancy, contraction incidence and area under the force curve were inhibited, consistent with the myometrial quiescence of pregnancy. The Ca2+ sensitivity of contraction was decreased at 20 days of pregnancy and this was partially reversed in labor. The protein content of h-caldesmon (h-CaD) was increased in pregnancy. A 40-fold increase in the signal from a phospho-CaD antibody specific for phosphorylation at an ERK1/2 site occurred during labor. ERK1/2 activation increased significantly at the onset of labor. Myosin light chain phosphorylation (LC20-P) increased significantly in labor compared with the nonpregnant state. Thus we conclude that the increase in CaD protein content during pregnancy may contribute to a suppression of the contractility of pregnant myometrium. Conversely, CaD phosphorylation, through an ERK1/2-mediated signaling pathway, as well as an increase in basal LC20-P, is suggested to contribute to the reversal of inhibition and promote contraction of the uterus during labor.
- preterm labor
- myosin light chain phosphorylation
preterm labor remains a major cause of perinatal mortality and morbidity, including subsequent long-term disabilities (24). Throughout gestation, it is crucial that the uterus remain quiescent, allowing for fetal maturation in preparation for extrauterine life. At present, the exact molecular mechanisms of myometrial quiescence remain largely unknown. Labor may occur due to a loss of myometrial quiescence or an active increase in uterine contractility, or a combination of both. Several inhibitory factors have been proposed to contribute to the quiescence of the pregnant myometrium. β-Adrenoceptor agonists, calcitonin gene-related peptide, and PGE2 can promote uterine relaxation via their ability to increase the intracellular cAMP levels (29). Other potential mechanisms include caldesmon (CaD), nitric oxide, relaxin receptors, oxytocin effects on the corpus luteum (13), and a paucity of gap junctions (31). The present study focused on CaD and its possible role during pregnancy.
CaD is an actin binding protein that inhibits actomyosin interaction in vitro (33). CaD exists in two isoforms. The high molecular weight form (h-CaD) is restricted to contractile smooth muscle, but the low molecular weight isoform (l-CaD) is more widely distributed. Evidence that CaD is involved in the regulation of smooth muscle contraction through thin filament (i.e., actin mediated) mechanisms has been reported from several groups using different approaches (25, 27). Both synthetic peptide antagonists of CaD (19, 22) and CaD antisense (6) induce sustained elevations of basal contractile tone of vascular smooth muscle, indicating that CaD can play a physiologically significant role in suppression of basal contractility. In vitro motility assays indicate that phosphorylation of CaD by ERK1 reverses the inhibitory effect of CaD on sliding velocity (7). Similarly, the addition of ERK1 to permeabilized canine colonic smooth muscle contracted the muscle (7). However, in permeabilized rabbit vascular smooth muscle, phosphorylation of CaD by constitutively activated ERK2 neither induced contraction nor affected the calcium sensitivity (28). Thus the effects of ERK1/2 on CaD may be isoform specific, tissue, or species specific.
Relatively few studies have been performed in myometrial smooth muscle regarding the possible function of CaD. An increased protein level of CaD in term pregnant human myometrium compared with nonpregnant human myometrium has been reported (39). However, the contractile correlate of the increased CaD level has not been determined and whether CaD's inhibitory action may be reversed by ERK1/2-dependent phosphorylation in a gestation-related manner has not been explored. The purpose of the present study was to determine if an increase in CaD expression might correlate with inhibition in vitro of basal contractility during pregnancy and whether ERK1/2 signaling regulates myometrial contractility and calcium sensitivity during pregnancy.
MATERIALS AND METHODS
Animals and tissue handling.
All procedures were approved by our institutional animal care and use committee and complied with the American Physiological Society “Guiding Principles for Research Involving Animals and Human Beings” (2). Sprague-Dawley nonpregnant and timed-pregnant rats (Taconic, Germantown, NY) were euthanized by carbon dioxide inhalation. Delivery was observed to occur on the 22nd or 23rd gestational day. For the collection of in-labor uterine smooth muscle samples, the rat was closely observed and the delivery of the first pup was used as the indication of labor. Excised uteri were immersed immediately into oxygenated Krebs solution at room temperature. The composition of Krebs solution was (in mM) 120 NaCl, 5.9 KCl, 11.5 dextrose, 25 NaHCO3, 1.2 NaH2PO4 · H2O, 1.2 MgCl2 · 6H2O, and 2.5 CaCl2. Approximate 8 × 2 mm (length × width) whole thickness uterine strips oriented parallel to the longitudinal muscle bundles were dissected under a dissection microscope (Olympus VM).
Recording of mechanical responses.
Strips were mounted vertically in 50-ml tissue baths, and tension was recorded with an isometric tension transducer (Grass Instrument Division, West Warwick, RI). Experiments were performed at 37°C as previously described (34). Preparations were allowed to equilibrate for 1 h before initiation of experiments. All myometrial strips were stretched to the optimal length (Lo), defined as the length corresponding to maximal force development with regard to spontaneous contractions (9
Determination of calcium sensitivity in depolarized, intact myometrial strips.
Contractile force from potassium depolarized strips was measured at increasing concentrations of extracellular calcium. KCl (51 mM) was substituted for NaCl on an equimolar basis. Ca2+ -free 51 mM KCl Krebs solution was prepared by the omission of calcium chloride in high-potassium Krebs solution and by the addition of 2 mM EGTA. Forces were normalized to the force induced by 2.5 × 10−3 M CaCl2 for each muscle.
Determination of calcium sensitivity in α-toxin permeabilized myometrial strips.
Thin muscle strips were dissected to have a width of 700 μm and permeabilized with α-toxin by a previously described method (38). α-Toxin from Staphylococcus aureusforms pores that allow the passage of molecules <2–4 kDa across the cell membrane (26). The strips were treated for 30 min at 34°C with 20 μg/ml of α-toxin (List Biological Lab, Campbell, CA) in pCa 6.4 buffer. The calcium ionophore 4-bromo A23187 (10 μM; Calbiochem, La Jolla, CA) was then applied for 20 min at 22°C to deplete calcium from the sarcoplasmic reticulum. The force of contraction was recorded with calcium concentrations clamped at different levels by applying pCa buffers. pCa buffers were prepared by the use of a computer program as previously described (3).
At the end of experiments, muscle strips were quick frozen by immersion in a dry ice-acetone slurry containing 10% TCA and 10 mM DTT. Muscles were stored at −80°C until processed as previously described for protein extraction for Western blotting (18). Protein matched samples (modified Lowry protein assay, DC Protein Assay Kit, Bio-Rad) were subjected to electrophoresis on 10% SDS-polyacrylamide gels, transferred to Immobilon-P membrane (Millipore, Bedford, MA), and then subjected to immunostaining. Blots were visualized with a SuperSignal West Peroxide Solution (Pierce, Rockford, IL). The images were detected with a chemiluminescence imaging screen with a Bio-Rad Molecular Imager phosphor imager and quantified with Multi-Analyst software.
The phospho-p44/42 MAP kinase antibody (Cell Signaling, Beverly, MA; catalog #9101S, 1:1,000) detects ERK1 and ERK2 only when they are catalytically activated by dual phosphorylation at Thr202and Tyr204. The p44/42 MAP kinase antibody (Cell Signaling catalog #9102, 1:1,000) detects total ERK1 and ERK2 MAP kinase protein levels. The CaD polyclonal (1:40,000) antibody was raised against full-length human myometrium CaD and was a gift from K. Mabuchi (Boston Biomedical Research Institute). The monoclonal anti-myosin light chain antibody (catalog M4401, 1:2,500) was a product of Sigma (St. Louis, MO). The anti-phospho CaD antibody was a gift from L. Adam (Bristol Myers Squibb, Princeton, NJ) and was produced against a phosphopeptide containing the CaD sequence surrounding the Ser789 ERK phosphorylation site (5).
Measurements of LC20 phosphorylation.
Muscle strips were quick-frozen by immersion in a dry ice-acetone slurry containing 10% TCA and 10 mM DTT. Tissues from nonpregnant,day 20, and in-labor rats were first permeabilized with α-toxin and intracellular Ca2+ concentration ([Ca2+]i) clamped at pCa 7 to prevent complications from the triggering of spontaneous contractions that sometimes occur in intact tissue in response to the freezing solution. Tissues were brought to room temperature in acetone-TCA-DTT, then ground with glass pestles, and washed with ether to remove TCA. Tissues were extracted in a urea sample buffer as previously described (21) and run on 10% polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes and subjected to immunoblot with a specific LC20 antibody (1:1,500, Sigma). Anti-mouse IgG (goat) conjugated with horseradish peroxidase was used as a secondary antibody (1:2,000, Calbiochem). Bands were detected with enhanced chemiluminescence (ECL) (SuperSignal, Pierce) visualized on films and then analyzed by NIH Image or were visualized and analyzed on a Bio-Rad phosphor imager.
Data were analyzed and expressed as means ± SE. The nvalues represent the number of animals used in experiments. Data were compared by using an ANOVA test with probability values ofP < 0.05 considered significant.
Integrated force signals are decreased in pregnant rat myometrium.
Although myometrial quiescence in vivo is a clinical hallmark of normal pregnancy, it has not been clear whether this change in contractility is preserved to any degree in vitro. In looking for an in vitro correlate of myometrial quiescence in vivo, we noted that (Fig.1 A) rat myometrium displays dramatically a decreased incidence of spontaneous contraction in vitro in strips from day 16 and day 20 pregnant rats compared with that from nonpregnant rats (P < 0.001,n = 28, total 92 strips). Conversely, as has been previously reported for rat myometrium (14), the amplitude of spontaneous contractions normalized to tissue dry weight increased and reached a maximal value before the onset of labor at 16 and 20 days of pregnancy (P < 0.001, n = 28, total 80 strips). The net result of changes in contractility was quantitated by integrating the force signal over a 15-min time period (Fig.1 B). The resulting AUC parameter is similar in samples fromday 16 and day 20 of pregnancy compared with that of nonpregnant samples but significantly increased at the onset of labor (P < 0.001).
Calcium sensitivity of pregnant myometrium is decreased.
Casteels and Kuriyama (4) showed that the electrical excitability and action potential spike frequency of rat myometrial smooth muscle is increased in pregnancy; however, net contractile activity as measured by the AUC is decreased. To explain this apparent paradox, we hypothesized that a step in excitation-contraction coupling after the electrical event is altered to inhibit contractility. We speculated that the responsiveness of the contractile apparatus to calcium (Ca sensitivity) might be decreased. As an initial approach, we used 51 mM KCl Krebs solution to depolarize the cell membrane, open calcium channels, and partially equilibrate the extracellular and intracellular calcium levels (16). The forces generated at different extracellular calcium concentrations are expressed as a percentage of the contraction amplitude induced by 51 mM KCl Krebs with 2.5 mM CaCl2. Consistent with the observed decreased contractility, the calcium force curves were rightward shifted in strips from 20-day pregnant rats (Fig.2 A, n = 10). There was a highly significant decrease in calcium sensitivity in the presence of 0.1 and 0.5 mM extracellular Ca for uterine smooth muscle from 20-day pregnant rats (P < 0.001).
We noted that the in vivo length (Lv) of strips from pregnant rats was greater than the Lo for those strips in vitro. Hence, in parallel muscle strips from the same rat, we stretched strips to the Lv as well. However, the calcium force curve was unchanged between Lo and Lv (data not shown).
These findings suggest the presence of an increased calcium threshold for contraction in pregnancy; however, it is also possible that alterations in the conductance of Ca channels or in the activities of Ca pumps could cause the observed difference. Thus, to more directly investigate the Ca sensitivity of the contractile apparatus, we permeabilized myometrial smooth muscle strips with α-toxin and then measured calcium sensitivity with pCa buffers. Interestingly, the myometrium not only exhibits an increased calcium threshold for contraction during pregnancy (i.e., requiring a [Ca2+]i higher than pCa 7.0 for contraction), but calcium sensitivity is also partially restored with the onset of labor (Fig. 2 B, n = 9). Importantly, at pCa 6.4, within the physiological range of [Ca2+]i, uterine samples from laboring rats produce a significantly greater contractile response than that from 20-day pregnant rat myometrial samples (P < 0.05).
h-CaD expression and phosphorylation are increased during pregnancy and labor.
Myometrial strips from nonpregnant and pregnant rats were quick-frozen. Protein-matched samples were analyzed by Western blot with CaD and phospho-CaD (p-CaD) antibodies. Band densities were quantitated by using a phosphor imager. Consistent with a previous report where CaD levels were measured in human tissue (39), the CaD protein level is significantly increased during pregnancy in rat myometrium (Fig. 3 B, n = 30). The phospho-CaD antibody used is specific for CaD phosphorylated at an ERK phosphorylation site, Ser789 (5). After normalization for the change in CaD protein levels, about a 40-fold increase in p-CaD immunoreactivity was observed during labor (Fig. 3 C, P < 0.001, n = 30), compared with very minimal phosphorylation of CaD in nonpregnant myometrium (Fig. 3 A).
l-CaD protein levels and phosphorylation levels are unchanged in pregnant rat myometrium.
The l-CaD isoform arises from alternative splicing of exon 4 out of the h-CaD sequence during RNA processing (12). The CaD and phospho-CaD antibodies used here detect both h-CaD and l-CaD. Neither l-CaD protein levels nor phospho l-CaD levels changed during pregnancy in rat myometrium (Fig. 4,n = 30). Shown in Fig. 4 C, the p-l-CaD levels normalized to l-CaD for each sample did not change with pregnancy.
ERK1/2 is activated during labor.
The fact that there is a significant CaD phosphorylation at an ERK1/2 phosphorylation site at the onset of labor suggested that ERK1/2 protein levels and/or ERK1/2 activation levels are increased at this time. ERK1 and ERK2 protein levels were monitored in protein matched samples and were found to be unchanged throughout pregnancy into labor (Fig. 5, n = 24). However, reprobing with phospho-specific ERK antibody indicated that ERK2 was significantly activated only at the onset of labor (Fig.6 C, n = 24,P < 0.01), consistent with the time course of the changes in CaD phosphorylation. Conversely, phospho-ERK1 levels did not significantly change (Fig. 6 B, n = 24,P > 0.13), suggesting that CaD might be a slightly better substrate for ERK2 compared with ERK1 in myometrial cells.
Basal myosin light chain phosphorylation levels do not decrease during myometrial quiescence but do increase during labor.
Smooth muscle contraction can be regulated by both thin filament mechanisms and thick filament mechanisms. Thick filament regulation occurs primarily through phosphorylation of the 20-kDa myosin light chains (LC20-P) (11, 20). Thus both myosin protein levels and LC20-P levels were measured. As is shown in Fig.7 A, the content of myosin as measured by densitometry of LC20 immunoblots in protein-matched samples was unchanged throughout pregnancy and into labor in rat myometrium. Because there is a fixed ratio between the myosin light chain and myosin heavy chain concentrations, this further indicates that the protein levels of myosin in general are unchanged. As shown in Fig.7 B, however, LC20-P levels increased significantly in labor. LC20-P levels were measured at pCa 7.0 in α-toxin-permeabilized samples to ensure that basal levels were measured and not obscured by the spontaneous contractions often triggered by immersion into the freezing solution. A positive control of a 51 mM KCl-stimulated intact in-labor sample is included in Fig. 7 B for comparison. An increase in basal LC20 would tend to increase contractility and, at constant [Ca2+]i, would tend to increase Ca sensitivity. Thus the increase in LC20-P seen in the in labor samples may contribute to the increased contractility seen during labor and also to the relative increase in Ca sensitivity seen at that time.
The main findings of the present study are 1) CaD protein levels increase in the pregnant rat myometrium at a time when the basal [Ca2+]i sensitivity of the muscle decreases; 2) CaD is phosphorylated at an ERK1/2 phosphorylation site when basal Ca sensitivity is increased at the onset of labor; 3) ERK2 activation occurs as parturition becomes imminent; 4) LC20-P increases at the onset of labor.
During pregnancy, myometrial smooth muscle undergoes profound and largely reversible changes, including stretch-induced hypertrophy designed to prepare for the forceful contractions of labor. However, for the duration of pregnancy, the muscle displays contractile quiescence in vivo, presumably by the alteration of signaling mechanisms. It has not been clear from the existing literature if the contractile quiescence characteristic of the pregnant state persists to any degree in vitro. In the current study we have observed, in vitro, a striking decrease in the contraction incidence in myometrium from 16-day and 20-day pregnant rats. This results in a net suppression of total basal (i.e., in the absence of agonist stimulation) contractility, as measured by the AUC, to the same level as that seen in samples from nonpregnant rats (Fig. 1 C). Thus the decreased AUC is an in vitro correlate of myometrial quiescence. The possibility that mechanical quiescence of pregnant strips was due to nonviability of tissue in vitro was ruled out by the fact that similarly processed strips have comparable contractile amplitudes in response to oxytocin, KCl and PGF2α as do labor samples (23).
A possible mechanism of the decrease in the incidence of contractions in pregnant tissues might be a decrease in electrical excitability. However, Casteels and Kuriyama (4) directly measured electrical activity and membrane excitability in rat myometrium and showed that the excitability actually increases with pregnancy. The dissociation between electrical and mechanical measurements strongly suggests a mechanism(s) of quiescence at a step in excitation-contraction coupling after the electrical event. We postulated that the [Ca2+]i threshold for contraction might increase in pregnant myometrium and, hence, suppress contractility (Fig. 8). In the present study, we monitored the Ca sensitivity of contractile force in two ways, with potassium-depolarized, intact myometrial smooth muscle strips and with α-toxin-permeabilized strips. The use of depolarized strips is indirect but has the advantage of minimal manipulation of the tissue and avoids the possible modification of signaling pathways that might be caused by more destructive approaches. To more directly measure the relationship between intracellular calcium and force, we also used α-toxin permeabilization of myometrium, which allows [Ca2+]i to directly be clamped constant but which also has the disadvantage of disrupting the integrity of the cell membrane. However, α-toxin is not a detergent but forms a hexamer, which functions as a pore of diameter 1–3 nm in the cell membrane. Under controlled conditions, the overall structure of the plasma membrane is left intact and much less “run down” of muscle strips is observed (10). With both methods, the same result was obtained: Ca sensitivity was decreased during pregnancy.
Other techniques for permeabilizing cells, such as Triton X-100 (28), saponin (17), digitonin, and β-escin (15), form larger lesions in the membrane and allow large molecules to pass. Often calmodulin or other signaling molecules need to be added to the bathing solution to compensate for leakage of endogenous proteins from detergent-permeabilized cells. This difference in methodology presumably explains the difference between our results and those previously reported by Izumi et al. (17). These investigators used saponin permeabilization and observed an increase in Ca sensitivity with pregnancy. The difference in the results is quite possibly due to the loss of important gestation-dependent signaling molecules from saponin-treated preparations. It will be of interest in future studies to identify the nature of these putative signaling molecules.
The amplitude of spontaneous baseline contractions (14) and agonist-induced contractions (15) are greater in preparations from pregnant rats compared with nonpregnant rats even when force is normalized to tissue weight or cross-sectional area to compensate for the hypertrophy of the tissues. We reproduced these findings (Fig. 1). This phenomenon is apparently at odds with the known “quiescence” of pregnant myometrium in vivo. However, this may represent a contractile potential that is not used in vivo until labor, when the mechanisms responsible for quiescence are reversed and may be due to an increased availability of calcium from sarcoplasmic reticulum (35).
In the present study, the immunostaining of h-CaD increased three- to fourfold in pregnancy. h-CaD levels did not detectably change during labor. We postulate that the increase in CaD levels leads to the observed increase in the Ca threshold for contraction of this muscle (Fig. 8). Approximately a 40-fold increase in staining with a phospho-h-CaD antibody occurred during labor, compared with very minimal phospho-h-CaD in nonpregnant myometrium. This is the first report of gestation-dependent changes in CaD phosphorylation levels. As mentioned above, CaD inhibits actomyosin interactions and the inhibition can be reversed in vitro under certain conditions by either calmodulin or ERK1/2-mediated phosphorylation of CaD (36, 37). Thus we postulate that CaD phosphorylation may reverse the inhibition of actomyosin interactions by CaD and may contribute to an increased uterine contractility during labor (Fig. 8). An increase in LC20 phosphorylation was also observed in labor; thus changes in basal regulation of both thin filaments and thick filaments contribute to “priming” the system for forceful labor contractions.
Although l-CaD (also called nonmuscle CaD) is widely expressed, its functional role has not yet been elucidated. Transient transfection of l-CaD prevented myosin II-dependent cell contractility in fibroblasts (8). In animal studies, overexpression of l-CaD is found in hypertrophied bladder smooth muscle in the rabbit during urinary outflow obstruction and has been suggested to be a molecular marker for the pathology (30). In the current study, we observed that there were no gestation-dependent changes in l-CaD protein levels or phosphorylation levels in pregnancy-induced hypertrophy of rat myometrium (Fig. 4). Possibly, the physiological hypertrophy associated with pregnancy differs mechanistically from the pathological hypertrophy associated with the overexpression of l-CaD in the bladder.
The phospho-CaD antibody used in this study is specific for the ERK1/2 phosphorylation site at Ser789 (5), implicating ERK1/2 in regulation of myometrial contractility. Although the identities of all phosphorylation sites in CaD have not been elucidated yet, approximately two-thirds of the total amount of phosphate in CaD is incorporated into the Ser759 and Ser789 ERK1/2 phosphorylation sites (1). Phosphorylation at Ser759 of CaD has been reported to be almost undetectable in mammalian smooth muscle (5). Because of the low level of phosphorylation at this site, it is unlikely that CaD function is altered in smooth muscle tissue through reversible phosphorylation at Ser759.
Interestingly, we find that only ERK2 is significantly phosphorylated and activated at the onset of labor (Fig. 6 C), a time when we also observed an increase in phosphorylation of h-CaD at the Ser789 site. Future studies will be needed to determine whether ERK2 has a preference for the Ser789 site or whether signaling pathways specifically target ERK2 more than ERK1 to CaD in myometrial cells. In a previous paper, Ruzycky (32) reported that ERK1/2 activity increased from day 15 today 20 of pregnancy in rat myometrium. Immediately before parturition, ERK1/2 activity declined sharply (32). However, this study used an indirect assay for ERK1/2 activity involving membrane subcellular fractions, and the samples were not quick-frozen to preserve the native phosphorylation status. Thus methodological differences may explain the difference in results.
The basal LC20-P levels increase slightly in labor. Compared with muscles that are fully activated by 51 mM KCl depolarization of intact tissue, the changes in basal LC20-P are relatively small, but the increase in LC20-P seen at a constant pCa 7.0 in α-toxin-permeabilized muscle from in-labor pregnant rats may contribute to the observed relative increase in Ca sensitivity seen at this time and may, furthermore, “prime” the muscle for a robust contractile response to the many hormonal and paracrine factors present during labor.
In summary, we conclude that the increase in h-CaD protein content during pregnancy may contribute to a suppression of the contractility of the pregnant myometrium by raising the calcium threshold for contraction. Conversely, h-CaD phosphorylation, through an ERK2-mediated signaling pathway, and also a slight increase in basal LC20-P is suggested to partially reverse the inhibition and contribute to a restored calcium sensitivity and increased basal uterine contractility during labor. We emphasize that these changes in basal activity of the contractile apparatus that we have observed in vitro will be a subset of the total number of multiple changes that occur in the in vivo setting in the presence of numerous paracrine and hormonal influences.
The authors thank Dr. K. Mabuchi (BBRI) for the gift of CaD antibody and Dr. L. Adam (Bristol Myers Squibb, Princeton, NJ) for the gift of the phospho-CaD antibody. We also thank M. DeMont for expert assistance in the preparation of the manuscript and C. Gallant for skilled technical assistance.
This research was supported by National Institutes of Health Grants HL-31704 and HL-42293 to K. G. Morgan and GM-07592 to Y. Li.
Address for reprint requests and other correspondence: Y. Li, Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
October 3, 2002;10.1152/ajpregu.00290.2002
- Copyright © 2003 the American Physiological Society