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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 Ca(2+) signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca(2+) fluxes whereas low-quality embryos caused a heightened and prolonged Ca(2+) response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.
Stress responsive signaling coordinated by nuclear factor erythroid 2-related factor 2 (Nrf2) provides an adaptive response for protection of cells against toxic insults, oxidative stress and ...metabolic dysfunction. Nrf2 regulates a battery of protective genes by binding to regulatory antioxidant response elements (AREs). The aim of this study was to examine how Nrf2 signals cell stress status and regulates transcription to maintain homeostasis.
In live cell microscopy we observed that Nrf2 undergoes autonomous translocational frequency-modulated oscillations between cytoplasm and nucleus. Oscillations occurred in quiescence and when cells were stimulated at physiological levels of activators, they decrease in period and amplitude and then evoke a cytoprotective transcriptional response. We propose a mechanism whereby oscillations are produced by negative feedback involving successive de-phosphorylation and phosphorylation steps. Nrf2 was inactivated in the nucleus and reactivated on return to the cytoplasm. Increased frequency of Nrf2 on return to the cytoplasm with increased reactivation or refresh-rate under stress conditions activated the transcriptional response mediating cytoprotective effects. The serine/threonine-protein phosphatase PGAM5, member of the Nrf2 interactome, was a key regulatory component.
We found that Nrf2 is activated in cells without change in total cellular Nrf2 protein concentration. Regulation of ARE-linked protective gene transcription occurs rather through translocational oscillations of Nrf2. We discovered cytoplasmic refresh rate of Nrf2 is important in maintaining and regulating the transcriptional response and links stress challenge to increased cytoplasmic surveillance. We found silencing and inhibition of PGAM5 provides potent activation of Nrf2.
Frequency modulated translocational oscillations of Nrf2 mediate the ARE-linked cytoprotective transcriptional response.
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.
This is the first study to investigate the effect of dietary induced adiposity upon uterine contractile activity in vivo. Uterine contractile activity was measured by surgically implanting a pressure sensitive telemeter probe into the gravid uterus using aseptic techniques. Using this surgical technique, it was identified that feeding a high‐fat, high‐cholesterol diet to induce adiposity significantly prolonged labor compared to lean control fed animals.
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 Ca2+ 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.
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.
This is the first report to investigate the effects of streptozotocin‐induced diabetes on L‐type Ca2+ current simultaneously measured with Ca2+ transients in epicardial and endocardial left ventricular myocytes isolated from rats 5–6 months after the induction of diabetes. Streptozotocin‐induced diabetes causes a progressive increase in the amplitude of endocardial and epicardial myocyte Ca2+ transients that is independent of the L‐type Ca2+ current. The effect of streptozotocin‐induced diabetes on Ca2+ handling might be attributed, at least in part, to the dysfunction of the Na+/Ca2+ exchanger.
Abstract Calcium-activated potassium channels are important in a variety of smooth muscles, contributing to excitability and contractility. In the myometrium previous work has focussed on the large ...conductance channels (BK), and the role of small conductance channels (SK) has received scant attention, despite the finding that over-expression of an SK channel isoform (SK3) results in uterine dysfunction and delayed parturition. This study therefore characterises the expression of the three SK channel isoforms (SK1–3) in rat myometrium throughout pregnancy and investigates their effect on cytosolic Ca and force and compares this with that of BK channels. Consistent expression of all SK isoform transcripts and clear immunostaining of SK1–3 was found. Inhibition of SK1–3 channels (apamin, scyllatoxin) significantly inhibited outward current, caused membrane depolarisation and elicited action potentials in previously quiescent cells. Apamin or scyllatoxin increased the amplitude of Ca and force in spontaneously contracting myometrial strips throughout gestation. The functional effect of SK inhibition was larger than that of BK channel inhibition. Thus we show for the first time that SK1–3 channels are expressed and translated throughout pregnancy and contribute to outward current, regulate membrane potential and hence Ca signals in pregnant rat myometrium. They contribute more to quiescence that BK channels.
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