Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

Modelling maternal obesity: the effects of a chronic high-fat, high-cholesterol diet on uterine expression of contractile-associated proteins and ex vivo contractile activity during labour in the rat.

Maternal obesity is associated with prolonged and dysfunctional labour and emergency caesarean section, but the mechanisms are unknown. The present study investigated the effects of an adiposity-inducing high-fat, high-cholesterol (HFHC) diet on uterine contractile-associated protein (CAP) expression and ex vivo uterine contractility in term non-labouring (TNL) and term labouring (TL) rats. Female rats were fed either control chow (CON n=20) or HFHC (n=20) diet 6 weeks before conception and during pregnancy. On gestational day 21 (TNL) or day 22 (TL) CON and HFHC (n=10) rats were killed to determine plasma cholesterol, triacylglycerol and progesterone concentrations and collection of myometrium for contractility studies and expression of CAPs caveolin-1 (Cav-1), connexin-43 (CX-43) and it's phosphorylated form (pCX-43), oxytocin receptor (OXTR) and cyclooxygenase-2 (COX-2). HFHC feeding increased visceral fat (P≤0.001), plasma cholesterol (P≤0.001) and triacylglycerol (P=0.039) concentrations. Stage of labour effected uterine expression of CAV-1 (P<0.02), pCX43 and COX-2 (both P<0.03). CAV-1 and pCX43 decreased but COX-2 increased with parturition. Significant diet- and labour-stage interactions were evident for CX-43 and pCX43 (P<0.03 and P<0.004 respectively). CX-43 decreased with TL in HFHC animals but was unaltered in CON. pCX-43 fell with labour in CON but remained high in HFHC. OXTR expression was significantly higher in HFHC compared with CON animals (P<0.03). Progesterone was higher in HFHC rats at term (P<0.014) but fell significantly with labour to similar concentrations as CON. Contractility studies identified synchronous contractions of stable amplitude in lean animals, but unstable asynchronous contractions with obesity. Uterine dose response to oxytocin was blunted during labour in HFHC rats with a log EC50 of -8.84 compared with -10.25 M in CON for integral activity (P<0.05). In conclusion, our adiposity model exhibits adverse effects on contractile activity during labour that can be investigated further to unravel the mechanisms causing uterine dystocia in obese women.

Functional and morphological development of the womb throughout life.

The uterus undergoes changes throughout a woman's life, beginning with her own embryonic development when she is still in the womb, commencing a monthly cycle at the onset of adulthood, and undergoing dramatic changes during pregnancy and parturition. The impact of preterm labour and other perinatal health problems is significant, both in human and financial terms; therefore the study of the physiological and regulatory changes which the uterus undergoes can be of enormous potential benefit. Here we briefly review the current state of knowledge, with an emphasis on the importance of changes in connectivity in the uterine smooth muscle cell network and on recent mathematical modelling work aimed at elucidating the role of spatial heterogeneity in this connected network.

Fine spatiotemporal activity in contracting myometrium revealed by motion-corrected calcium imaging.

Successful childbirth depends on the occurrence of precisely coordinated uterine contractions during labour. Calcium indicator fluorescence imaging is one of the main techniques for investigating the mechanisms governing this physiological process and its pathologies. The effective spatiotemporal resolution of calcium signals is, however, limited by the motion of contracting tissue: structures of interest in the order of microns can move over a hundred times their width during a contraction. The simultaneous changes in local intensity and tissue configuration make motion tracking a non-trivial problem in image analysis and confound many of the standard techniques. This paper presents a method that tracks local motion throughout the tissue and allows for the almost complete removal of motion artefacts. This provides a stabilized calcium signal down to a pixel resolution, which, for the data examined, is in the order of a few microns. As a byproduct of image stabilization, a complete kinematic description of the contraction-relaxation cycle is also obtained. This contains novel information about the mechanical response of the tissue, such as the identification of a characteristic length scale, in the order of 40-50 µm, below which tissue motion is homogeneous. Applied to our data, we illustrate that the method allows for analyses of calcium dynamics in contracting myometrium in unprecedented spatiotemporal detail. Additionally, we use the kinematics of tissue motion to compare calcium signals at the subcellular level and local contractile motion. The computer code used is provided in a freely modifiable form and has potential applicability to in vivo calcium imaging of neural tissue, as well as other smooth muscle tissue.

A fluorogenic assay for methylglyoxal.

MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

The impact of maternal obesity on in vivo uterine contractile activity during parturition in the rat.

Maternal obesity is associated with increased risk of prolonged and dysfunctional labor and emergency caesarean section. To elucidate the mechanisms behind the associated uterine dystocia, a translational animal model is required. Our previous work identified that exposure to a high-fat, high-cholesterol (HFHC) diet to induce obesity down-regulates uterine contractile associated protein expression and causes asynchronous contractions ex vivo. This study aims to investigate the impact of maternal obesity on uterine contractile function in vivo using intrauterine telemetry surgery. Virgin female Wistar rats were fed either a control (CON, n = 6) or HFHC (n = 6) diet for 6 weeks prior to conception, and throughout pregnancy. On Day 9 of gestation, a pressure-sensitive catheter was surgically implanted aseptically within the gravid uterus. Following 5 days recovery, intrauterine pressure (IUP) was recorded continuously until delivery of the 5th pup (Day 22). HFHC induced obesity led to a significant 1.5-fold increase in IUP (p = 0.026) and fivefold increase in frequency of contractions (p = 0.013) relative to CON. Determination of the time of labor onset identified that HFHC rats IUP (p = 0.046) increased significantly 8 h prior to 5th pup delivery, which contrasts to CON with no significant increase. Myometrial contractile frequency in HFHC rats significantly increased 12 h prior to delivery of the 5th pup (p = 0.023) compared to only 3 h in CON, providing evidence that labor in HFHC rats was prolonged by 9 h. In conclusion, we have established a translational rat model that will allow us to unravel the mechanism behind uterine dystocia associated with maternal obesity.

Characterization of the tissue-level Ca2+ signals in spontaneously contracting human myometrium.

In the labouring uterus, millions of myocytes forming the complex geometrical structure of myometrium contract in synchrony to increase intrauterine pressure, dilate the cervix and eventually expel the foetus through the birth canal. The mechanisms underlying the precise coordination of contractions in human myometrium are not completely understood. In the present study, we have characterized the spatio-temporal properties of tissue-level [Ca(2+)](i) transients in thin slices of intact human myometrium. We found that the waveform of [Ca(2+)](i) transients and isotonic contractions recorded from thin slices was similar to the waveform of isometric contractions recorded from the larger strips in traditional organ bath experiments, suggesting that the spatio-temporal information obtained from thin slices is representative of the whole tissue. By comparing the time course of [Ca(2+)](i) transients in individual cells to that recorded from the bundles of myocytes we found that the majority of myocytes produce rapidly propagating long-lasting [Ca(2+)](i) transients accompanied by contractions. We also found a small number of cells showing desynchronized [Ca(2+)](i) oscillations that did not trigger contractions. The [Ca(2+)](i) oscillations in these cells were insensitive to nifedipine, but readily inhibited by the T-type Ca(2+) channel inhibitor NNC55-0396. In conclusion, our data suggest that the spread of [Ca(2+)](i) signals in human myometrium is achieved via propagation of long-lasting action potentials. The propagation was fast when action potentials propagated along bundles of myocytes and slower when propagating between the bundles of uterine myocytes.

Calcium signaling in endocardial and epicardial ventricular myocytes from streptozotocin-induced diabetic rats.

AIMS/INTRODUCTION: Abnormalities in Ca2+ signaling have a key role in hemodynamic dysfunction in diabetic heart. The purpose of this study was to explore the effects of streptozotocin (STZ)-induced diabetes on Ca2+ signaling in epicardial (EPI) and endocardial (ENDO) cells of the left ventricle after 5-6 months of STZ injection. MATERIALS AND METHODS: Whole-cell patch clamp was used to measure the L-type Ca2+ channel (LTCC) and Na+ /Ca2+ exchanger currents. Fluorescence photometry techniques were used to measure intracellular free Ca2+ concentration. RESULTS: Although the LTCC current was not significantly altered, the amplitude of Ca2+ transients increased significantly in EPI-STZ and ENDO-STZ compared with controls. Time to peak LTCC current, time to peak Ca2+ transient, time to half decay of LTCC current and time to half decay of Ca2+ transients were not significantly changed in EPI-STZ and ENDO-STZ myocytes compared with controls. The Na+ /Ca2+ exchanger current was significantly smaller in EPI-STZ and in ENDO-STZ compared with controls. CONCLUSIONS: STZ-induced diabetes resulted in an increase in amplitude of Ca2+ transients in EPI and ENDO myocytes that was independent of the LTCC current. Such an effect can be attributed, at least in part, to the dysfunction of the Na+ /Ca2+ exchanger. Additional studies are warranted to improve our understanding of the regional impact of diabetes on Ca2+ signaling, which will facilitate the discovery of new targeted treatments for diabetic cardiomyopathy.

A computational model of excitation and contraction in uterine myocytes from the pregnant rat.

Aberrant uterine myometrial activities in humans are major health issues. However, the cellular and tissue mechanism(s) that maintain the uterine myometrium at rest during gestation, and that initiate and maintain long-lasting uterine contractions during delivery are incompletely understood. In this study we construct a computational model for describing the electrical activity (simple and complex action potentials), intracellular calcium dynamics and mechanical contractions of isolated uterine myocytes from the pregnant rat. The model reproduces variant types of action potentials - from spikes with a smooth plateau, to spikes with an oscillatory plateau, to bursts of spikes - that are seen during late gestation under different physiological conditions. The effects of the hormones oestradiol (via reductions in calcium and potassium selective channel conductance), oxytocin (via an increase in intracellular calcium release) and the tocolytic nifedipine (via a block of L-type calcium channels currents) on action potentials and contractions are also reproduced, which quantitatively match to experimental data. All of these results validated the cell model development. In conclusion, the developed model provides a computational platform for further investigations of the ionic mechanism underlying the genesis and control of electrical and mechanical activities in the rat uterine myocytes.

Author Correction: Maternal obesity-induced decreases in plasma, hepatic and uterine polyunsaturated fatty acids during labour is reversed through improved nutrition at conception.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Maternal obesity-induced decreases in plasma, hepatic and uterine polyunsaturated fatty acids during labour is reversed through improved nutrition at conception.

Maternal obesity is associated with prolonged and dysfunctional labour, potentially through decreased synthesis of prostaglandins that stimulate myometrial contractions. We assessed the impact of maternal obesity on concentrations of precursor fatty acids (FA) for prostaglandin synthesis and whether any changes could be reversed by improved nutrition post-conception. Wistar rats were fed control (CON) or High-Fat, High-cholesterol (HFHC) diets 6 weeks before mating. At conception half the dams switched diets providing 4 dietary groups: (1) CON, (2) HFHC, (3) CON-HFHC or (4) HFHC-CON. During parturition rats were euthanized and FA composition of plasma, liver and uterus determined. Visceral fat was doubled in rats exposed to the HFHC diet prior to and/or during pregnancy compared to CON. HFHC diet increased MUFAs but decreased omega-3 and omega-6 PUFAs in plasma and liver. Uterine omega-3 FA concentrations were halved in HFHC versus CON rats, but all other FAs were similar. Switching from HFHC to CON diet at conception restored all FA profiles to those seen in CON rats. The increased MUFA and decreased PUFA concentrations in obese HFHC dams may contribute to aberrant prostaglandin synthesis and dysfunctional myometrial activity and it may be possible to reverse these changes, and potentially improve labour outcomes, by improving nutrition at conception.

Control of uterine Ca2+ by membrane voltage: toward understanding the excitation-contraction coupling in human myometrium.

Myometrial contractility is a complex and dynamic physiological process that changes substantially during pregnancy and culminates in childbirth. Uterine contractions are initiated by transient rises in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), which in turn are triggered and controlled by myometrial action potentials. The sequence of events between the action potential generation and the contraction initiation is referred to as excitation-contraction coupling. Hormones and other physiologically active substances affect myometrial contractility by modulating different steps in the excitation-contraction coupling process. It is therefore imperative that we understand that process to understand the regulation of myometrial contractility. The complex action potentials generated by human myometrium result from the activity of many ion channels, transporters, and pumps. Two types of myometrial action potential waveform have been described in the literature: a plateau type and a spike type. Parameters of the myometrial [Ca(2+)](i) transients and contractions differ depending on the type of action potential that triggers them. Some aspects of the excitation-contraction coupling are unique to human myometrium and cannot be found in animal models; some others are common between many species. This article reviews the current state and discusses future directions of physiological research on human myometrial excitation-contraction coupling.

Regulation of oxytocin receptors and oxytocin receptor signaling.

The oxytocin (OT) -oxytocin receptor (OTR) system plays a key role in many aspects of mammalian reproduction as well as several other physiological processes such as bond pairing and cardiovascular homeostasis. To manifest these diverse physiological roles, the transcription and expression of the OTR is tightly regulated within reproductive, cardiovascular, and neuronal tissues. The expression of the OTR within the mammalian uterus is regulated during gestation with a peak at the day of delivery. The control of this dramatic increase in expression is mediated in rodent species by a combination of stretch, classical steroid hormone stimulation, and repression. In the human uterus events are less clear, although a prominent role for inflammatory-related rapid-response genes and novel transcription factors such as hMafF (human homologue of chicken musculoaponeurotic fibrosarcoma oncogene family protein F) have been put forward. In the uterus the potent uterotonic actions of OT are mediated by the OTR through G-protein activation to stimulate phospholipase C activity. The activated OTR increases contraction frequency and increases force by sensitizing the contractile apparatus of the myocytes to calcium. This review summarizes the current knowledge of the complex regulation of OTR transcription in the myometrium and the intracellular signaling mechanisms through which OT mediates its potent stimulatory effects.

Preterm labour. Myometrial function in prematurity.

The primary function of the uterus during gestation is to harbour the growing conceptus in a largely quiescent environment. Upon maturation of the fetus to a point sufficient for extrauterine survival, the uterus must remodel itself sufficiently to generate forceful contractions during labour. During preterm delivery, the process of remodelling of the myometrium occurs early due to a number of different causes, although the underlying basis for myometrial contraction remains the same. This review summarises the anatomical, physiological and molecular basis for contraction. We describe the fibre structure of the human uterus and how this relates to the spread of electrical excitation during a contraction. The process of excitation within a single myometrial cell is described, as well as how this relates to contraction. We then focus on how excitation-contraction coupling is modulated by intercellular communication, pharmacomechanical-coupling and hormonal milieu. Lastly, we consider the actions of the commonly accepted uterine agonists oxytocin, prostaglandin F(2alpha), and prostaglandin E(2), and the tocolytic ritodrine.

Progesterone-Dependent Induction of Phospholipase C-Related Catalytically Inactive Protein 1 (PRIP-1) in Decidualizing Human Endometrial Stromal Cells.

Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca(2+) release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors.

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes.

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.

Single-cell mechanics and calcium signalling in organotypic slices of human myometrium.

Elucidation of cellular mechanisms regulating myometrial contractility is crucial for improvement in management of many obstetric abnormalities, such as premature delivery, uterine dystocia and post-partum haemorrhage. Myometrial contractions are triggered by periodic synchronous rises in intracellular calcium concentration ([Ca(2+)]i) elicited by spontaneously generated action potentials propagating throughout the entire myometrium. During labour, hormones like oxytocin and prostaglandins potentiate uterine contractions by increasing their duration, strength and frequency. The most informative approach to studying the mechanisms underlying hormonal modulation of uterine contractility is to record [Ca(2+)]i responses to hormones in intact myometrial samples that have not been subjected to enzymatic treatment for cell isolation or cell culture conditions. However, the spatio-temporal resolution of such recording is limited due to the motion artifacts occurring in contracting tissue. Here we describe the application of our newly developed motion correction algorithm to investigate the [Ca(2+)]i dynamics in control and oxytocin stimulated slices of human myometrium on a cellular level. We present evidence that oxytocin induces asynchronous [Ca(2+)]i oscillations in individual myocytes within intact myometrium which are similar to those observed in cultured cells. The oscillations occur between synchronous action potential-driven [Ca(2+)]i transients but appear to be unrelated to contractions. Furthermore, the oxytocin-triggered [Ca(2+)]i oscillations wane within 30-50min of hormone application, while the action potential induced [Ca(2+)]i transients remain augmented. We conclude that oxytocin-induced [Ca(2+)]i oscillations are not relevant to the acute regulation of myometrial contractility but may play a role in longer-term regulatory processes, for example, by triggering gene expression.