Anatoly Shmygol

Vimentin-Positive, c-KIT-Negative Interstitial Cells in Human and Rat Uterus: A Role in Pacemaking?

Abstract The mechanism underlying spontaneous pacemaker potential in the uterus is not clearly understood. Several spontaneously active smooth muscles have interstitial cells of Cajal (ICCs) or ICC-like cells. We therefore examined cells from freshly dispersed uterine muscle strips (from pregnant human and rat myometrium) and in situ uterine preparations to determine the cell types present. Both preparations revealed numerous ICC-like cells; they were multipolar, with spider-like projections and enlarged central regions. These cells were readily distinguished from uterine myocytes by their morphology and ultrastructure, i.e., no myofilaments, numerous mitochondria, caveolae, and filaments. In addition, the ICC-like cells were noncontractile. These cells were negative to c-kit, a classic marker for ICCs. They stained positive for the intermediate filament, vimentin, a marker for cells of mesenchymal origin but not differentiated myocytes. The ICC-like cells had a more or less stable resting membrane potential of −58 ± 7 mV compared with smooth-muscle cells, −65 ± 13 mV, and produced outward current in response to voltage clamp pulses. However, in contrast with uterine myocytes, inward currents were not observed. This is the first description of ICC-like cells in myometrium and their role in the uterus is discussed, as possible inhibitors of intrinsic smooth-muscle activity.

The Physiological Basis of Uterine Contractility: A Short Review

In this review we discuss our current understanding of the cellular basis of uterine contractility, highlighting those areas requiring further study. It is clear that the basic processes of excitation‐contraction coupling lie within the myometrial cell, and that these may be modified by agonists. Pacemaker acitivity, however, remains a mystery. The contribution of extracellular calcium entry to contraction is shown to be vital, whilst the role of the sarcoplasmic reticulum remains controversial. Much current experimental focus is on pathways controlling and regulating contraction, and we discuss sensitisation mechanisms and question their role in intact uterine preparations.

Uterine Selection of Human Embryos at Implantation

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca2+ signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca2+ fluxes whereas low-quality embryos caused a heightened and prolonged Ca2+ response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.

Depletion of membrane cholesterol eliminates the Ca2+‐activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes

Changes in membrane cholesterol content have potent effects on cell signalling and contractility in rat myometrium and other smooth muscles. We have previously shown that depletion of cholesterol with methyl‐β‐cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca2+ signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19–21 rats showed that outward K+ current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol‐saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K+ channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca2+‐sensitive K+ channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca2+ level ([Ca2+]i transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca2+ signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol‐rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca2+ signalling and uterine contractility.

Multiple mechanisms involved in oxytocin-induced modulation of myometrial contractility

AbstractOxytocin is a small peptide hormone with multiple sites of action in human body. It regulates a large number of reproduction-related processes in all species. Particularly important is its ability to stimulate uterine contractility. This is achieved by multiple mechanisms involving sarcoplasmic reticulum Ca2+ release and sensitization of the contractile apparatus to Ca2+. In this paper, we review the data published by us and other groups on oxytocin-induced modulation of uterine contractility. We conclude that sensitization of contractile apparatus to Ca2+ is the most relevant physiological effect of oxytocin on human myometrium.

Ca2+ entry, efflux and release in smooth muscle

Control of smooth muscle is vital for health. The major route to contraction is a rise in intracellular [Ca2+], determined by the entry and efflux of Ca2+ and release and re-uptake into the sarcoplasmic reticulum (SR). We review these processes in myometrium, to better understand excitation-contraction coupling and develop strategies for preventing problematic labours. The main mechanism of elevating [Ca2+] is voltage-gated L-type channels, due to pacemaker activity, which can be modulated by agonists. The rise of [Ca2+] produces Ca-calmodulin and activates MLCK. This phosphorylates myosin and force results. Without Ca2+ entry uterine contraction fails. The Na/Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) remove Ca2+, with contributions of 30% and 70% respectively. Studies with PMCA-4 knockout mice show that it contributes to reducing [Ca2+] and relaxation. The SR contributes to relaxation by vectorially releasing Ca2+ to the efflux pathways, and thereby increasing their rates. Agonists binding produces IP3 which can release Ca from the SR but inhibition of SR Ca2+ release increases contractions and Ca2+ transients. It is suggested that SR Ca2+ targets K+ channels on the surface membrane and thereby feedback to inhibit excitability and contraction.

Characterization of the molecular and electrophysiological properties of the T‐type calcium channel in human myometrium

Rises in intracellular calcium are essential for contraction of human myometrial smooth muscle (HMSM) and hence parturition. The T‐type calcium channel may play a role in this process. The aim was to investigate the role of the T‐type calcium channel in HMSM by characterizing mRNA expression, protein localization, electrophysiological properties and function of the channel subunits Cav3.1(α1G), Cav3.2(α1H), and Cav3.3(α1I). QRT‐PCR, immunohistochemistry, electrophysiology and invitro contractility were performed on human myometrial samples from term, preterm, labour and not in labour. QRT‐PCR analysis of Cav3.1, Cav3.2 and Cav3.3 demonstrated expression of Cav3.1 and Cav3.2 with no significant change (P > 0.05) associated with gestation or labour status. Immunohistochemistry localized Cav3.1 to myometrial and vascular smooth muscle cells whilst Cav3.2 localized to vascular endothelial cells and invading leucocytes. Voltage clamp studies demonstrated a T‐type current in 55% of cells. Nickel block of T‐type current was voltage sensitive (IC50 of 118.57 ± 68.9 μm at −30 mV). Activation and inactivation curves of ICa currents in cells expressing T‐type channels overlapped demonstrating steady state window currents at the resting membrane potential of myometrium at term. Current clamp analysis demonstrated that hyperpolarizing pulses to a membrane potential greater than −80 mV elicited rebound calcium spikes that were blocked reversibly by 100 μm nickel. Contractility studies demonstrated a reversible decrease in contraction frequency during application of 100 μm nickel (P < 0.05). We conclude that the primary T‐type subunit expressed in some MSMCs is Cav3.1. We found that application of 100 μm nickel to spontaneously contracting human myometrium reversibly slows contraction frequency.

A new technique for simultaneous and in situ measurements of Ca2+ signals in arteriolar smooth muscle and endothelial cells

We report here the first local and global Ca(2+) measurements made from in situ terminal arterioles. The advantages of the method are that there is minimal disturbance to the vessels, which retain their relationship to the tissue they are supplying (rat ureter) and the small size of vessel that can be studied. Good loading with the Ca(2+) indicator, Fluo-4 was obtained, and confocal sectioning through the tissue enabled vascular smooth muscle and endothelial cells to be clearly seen, along with red blood cells, nerve endings and the ureteric smooth muscle cells. We find the terminal arterioles to be extremely active, both spontaneously and in response to nor-adrenaline stimulation, with Ca(2+) sparks occurring in the vascular myocytes and Ca(2+) puffs in the endothelial cells. Even under resting conditions, endothelial cells produced oscillations and waves, which could pass from cell to cell, whereas the vascular myocytes only produced waves in response to agonist stimulation, and with no increase in the frequency of Ca(2+) sparks, and no spread from cell to cell. We compare our data to those obtained in dissected intact vessels and single cells. We conclude that this approach is a convenient and useful method for studying inter- and intracellular Ca(2+) signalling events and communication between cell types, particularly in very small vessels.

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

Abstract:  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 Ca2+ concentration ([Ca2+]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 [Ca2+]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.

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post‐partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome‐wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper‐polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long‐lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility.