29 research outputs found

    Modelling the Effects of Nifedipine on Ventricular and Myometrial Cells of Pregnant Rats

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    In this study we have used computational models to investigate the effects of nifedipine on two different cell types; the rat ventricular cell and the rat myometrial cell. Nifedipine is a calcium-channel blocker commonly used by health services around the world to treat both cardiovascular conditions (such as high blood pressure) and as a tocolytic to treat pre-term birth. The latter usage is prohibited in pregnant patients with pre-existing heart conditions. By applying discrete blocks to the L-Type calcium channels in each cell model we were able to simulate the presence of nifedipine at varying concentrations. Using the electrical and ionic responses to blocking these channels as indicators, we have been able to quantify and describe the effects of nifedipine in each cell type and compare them qualitatively. Although any level of block will reduce the maximum level of intracellular calcium in the myometrial cell, a 60% block or higher is required to produce a change in the morphology of the calcium transient. It remains to be shown if the dose required to achieve this could result in a patient with a pre-existing heart condition experiencing hypotension or other pathological cardiac conditions during labor, if nifedipine is used as a tocolytic

    Myometrial cyclic AMP function

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    Background Uncovering the processes that drive labour onset is essential to reduce the adverse consequences of dysfunctional labour. Myometrial cAMP signalling is upregulated during pregnancy promoting uterine quiescence. Changes in its components and effectors have been identified at the onset of term labour. Preterm labour (PTL) treatments targeting this pathway have limited effectiveness and serious maternal effects. In this study, real-time FRET imaging was used to investigate compartmentalised cAMP signals at distinct cellular sites. Methods Myometrial biopsies were obtained from women at term or in distinct causes of PTL. Tissues were processed for mRNA and protein extraction or cell isolation. Primary myometrial cells (HPMCs) and an hTERT-HM cell line expressed either a cytosolic (EPAC-SH187) or plasmalemma (AKAP79-CUTie) genetically encoded FRET sensor. The florescence emission changes were monitored following isoproterenol and PGE2 treatment to determine intracellular cAMP concentrations. Results Differences in cAMP signalling components were detected in PTL compared to term with variations in effector predominance and an associated increase in OTR expression in twin-PTL. Stimulus-specific subcellular compartmentalisation of cAMP was identified in both cell types with differential regulation by phosphodiesterases (PDEs). Significant disparities were detected in the amplitude, kinetics, and regulation of cAMP signals between the two cell types. For the HPMCs, a prolonged time in culture was associated with a reduction in PDE activity and altered cell phenotype. Conclusion The cAMP signalling system is influential in the final pathway of labour, primarily regulating OTR expression. This study established the technique of FRET imaging in human myometrial cells, determining the cell model of choice and culture conditions to explore localised cAMP signalling. The findings provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium and pave the way for unravelling the details of how this fundamental pathway operates and its role in pregnancy and labour.Open Acces

    Intimations on the Development of Effective Treatment for the Prevention of Preterm Birth: β3 Adrenergic Receptor Signaling in the Human Myometrium

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    Preterm birth before the thirty-seventh week of pregnancy is the leading cause of infant morbidity and mortality in the US. Infants born prematurely can spend weeks or months in the hospital, costing upwards of thirty billion a year in the US alone. Treatments for patients in labor preterm are ineffective and none are FDA approved. The β3 adrenergic receptor (β3AR) has been shown to be present in various tissues, including the human myometrium. Using contractile studies, stimulation of the β3AR has been shown to mediate relaxation. Although some pathways have been described, the mechanism(s) underlying β3AR-mediated relaxation in the myometrium are incompletely known. In other smooth muscles, the β3AR has been shown to mediate relaxation via nitric oxide (NO)-guanylyl cyclase-cGMP signaling. However, NO-mediated relaxation of the myometrium is cGMP-independent. Our studies showed that β3AR agonist, mirabegron, can be used to mediate relaxation in the human myometrium. The mechanisms associated with this relaxation are revealed to be increased production of NO in myometrial endothelial cells, stimulation of Ca2+ activated K+ channel (BKCa), and down regulation of the contractile associated protein connexin 43 (Cx43). The mechanosensitive channel Piezo1 was identified in the myometrium and showed to promote relaxation using similar mechanisms involving NO and BKCa. Our studies showed that NO mediated S-nitrosation of Cx43 promote a hemichannel state over a gap junction state. Together this research indicates the potential of β3AR as a target for developing tocolytic strategies involving combination therapy with mechanisms that have additive effects

    An investigation into the combination of nifedipine with potassium channel openers as potential tocolytic therapy for preterm labour; and a novel potassium channel blocker as potential therapy for post-partum haemorrhage

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    Background Preterm labour and post-partum haemorrhage are leading causes of pregnancy morbidity and mortality. Previous work identified potassium channels expressed in myometrium and hypothesized modulation of channels with greater expression in MSMC than VSMC will influence contractility and avoid cardiovascular effects. By combining calcium channel blockers with potassium channel openers an enhanced tocolytic effect is anticipated. VU590 inhibits Kir 7.1 and it was hypothesised would elicit a contractile effect with therapeutic potential for post-partum haemorrhage. Aim To determine the effect of select potassium channel openers and a specific potassium channel blocker in myometrial contractility. Methods Human and murine myometrial strips were used in contractility organ bath experiments. Select combined doses were tested in myometrial small arteries using wire myography. Western blotting was carried out to determine the gestational and labour-state expression of potassium channels in human myometrium and myometrial small arteries. Results Pinacidil demonstrated a relaxatory effect on both myometrial and vascular smooth muscle. Riluzole reduced contractility alone and greater inhibition in combination with nifedipine than nifedipine alone. Riluzole appeared to have a mild effect on myometrial arteries. Kir 7.1 showed a trend of diminished expression by gestation and was downregulated in term and preterm labour states. VU590 elicited a significant increase contractility characterised by a prolonged contraction phase of up to 6.7±1.9 hrs (VU590 10 µM). A gestational-dependent effect was seen on murine myometrium. Conclusion The combination of nifedipine with potassium channel openers has a more potent effect on reducing contractility than either compound alone. Riluzole combined with nifedipine warrants further investigation for potential tocolytic therapy. VU590 augments spontaneous contractions profoundly in human myometrium in vitro and could have potential therapeutic benefits in the treatment of postpartum haemorrhage

    Parturition, oxytocin, inflammation, myocyte damage and obesity; A study of myometrium and haematological parameters in human pregnancy and labour at term

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    The process of parturition resulting in the delivery of a newborn is a fundamental event ensuring survival of the species. In humans, the main clinical problems of parturition include activation of the process too early or too late resulting in the delivery of pre-term and post-term infants, both with their own implications for future health for the mother and baby. Additionally, where parturition systems are not activated correctly, dysfunctional labour with the resulting need for caesarean delivery (CS), in addition to atonic post-partum haemorrhage can also ensue. Overall, in the UK up to 40% of pregnancies are affected by one of these problems. However, the exact processes involved in the initiation and maintenance of parturition in the human are not fully understood. With such an important event, influences are most likely to be multi-factorial, with hormonal, mechanical, inflammatory, biochemical and maternal environmental factors playing a part. The aims of this thesis were to investigate influences on parturition in human pregnancy. Firstly, the myometrial transcriptional effects of long term exposure to the uterotonic oxytocin (OT) were examined. Further investigation of the myometrial and maternal peripheral response to uterine contractions in-vitro and in-vivo was also made with particular reference to the role of inflammation and myocyte damage. Additionally, the influence of maternal factors, particularly obesity, on the myometrial in-vitro contractile function and response to OT was studied. Initially, 150 gene arrays were produced using the Illumina platform. The samples were derived from myometrium taken at pre-labour CS which subsequently underwent functional contractility experiments in an organ bath. Five drug environments were studied, namely OT, acetic acid (OT vehicle), ML7(a tocolytic acting via inhibition of myosin light chain kinase), ML7 & OT and finally DMSO (ML7 vehicle). Additionally, five time-points of 0, 1, 2, 4, and 6 hours after drug addition were used, resulting in 5 samples for each drug and time combination. The results indicated that despite a clear enhancement of myometrial contractile activity by OT, this functional response does not appear to be mediated by cellular transcription. However, there was a clear contraction and time dependent transcriptional wave, with overrepresentation of genes associated with inflammation and cellular damage/apoptosis, and down-regulation of pathways concerning cellular metabolism. These findings were confirmed by QPCR on further myometrial samples undergoing additional in-vitro functional studies. In addition to the temporal and contractile association with the inflammatory response, our data suggest inflammation occurs in response to myocyte cellular damage regardless of mode of damage e.g. contractile or chemically induced. This was demonstrated by inflammatory upregulation in myometrium exposed to the tocolytic agents nifedipine and ritodrine, which is not seen in response to ML7. Additionally, the myometrial inflammatory response was enhanced by the infective agent LPS. However, contrary to other proposals, the enhanced inflammatory response of the myometrium did not alter or promote the in-vitro contractile ability of the myometrium or its response to OT. This myometrial transcriptional data therefore suggests that the inflammatory response of labour is associated with contraction, chemical or infection induced myometrial cellular damage, but would not be considered necessary for a contractile response. Our in-vivo study of peripheral changes in the maternal circulation again supported our in-vitro myometrial data. Data showed that the effect of pregnancy at term was limited to increased white cell count driven by a neutrophilia, with no suggestion of leukocyte priming prior to labour. Additionally, term pregnancy is associated with an increase in CRP, an increase in GCSF (corresponding with the neutrophilia) in addition to suppression of the chemokines CCL11 and CCL22. Subsequently, we found that repeated blood samples taken at 2 hourly intervals during term labour induced dramatic changes in inflammatory cells and inflammatory mediators in the maternal circulation. Importantly, these changes occur in a co-ordinated time and contraction dependent manner, with the degree of inflammation associated with the length of time in labour and the degree of myocyte damage as measured by circulating CK and Mb. Our study of the influence of maternal factors on myometrial contractile ability and response to OT examined in-vitro myometrial contractility of 609 myometrial strips from 85 women. We demonstrated that maternal obesity does not impair spontaneous or OT induced myometrial contractions in-vitro. Furthermore, maternal age, ethnicity, parity, previous caesarean delivery,gestation at delivery and birthweight do not influence in-vitro myometrial spontaneous or OT induced contractile activity. This therefore suggests that the observed implication of these maternal and infant factors on parturition in-vivo (high rates of induction of labour, high rates of intrapartum caesarean delivery and post partum haemorrhage) cannot be explained by an effect on myometrial contraction per se. This therefore merits further investigation as to alternative mechanisms to ultimately promote and effective, uncomplicated and safe labour and vaginal delivery for at risk mothers. In summary, this thesis provides evidence that the myometrial contractions of human labour, whether spontaneous or OT induced are capable of inducing a temporal wave of transcriptional changes associated with the processes of inflammation, cellular damage/apoptosis with inhibition of cellular metabolic processes. In addition, maternal peripheral circulating factors mirror the myometrial transcriptional changes. These changes are highly comparable with those seen in response to exercising skeletal muscle, and in this model have been shown to play an important role in muscle repair and remodelling after exercise. Therefore, we would suggest that the inflammatory reaction typically associated with human labour occurs as a non-specific response to contraction induced cellular damage and may play a role in postpartum repair and remodelling of the uterus

    Investigating the role of anoctamin 1 in the calcium dependent depolarisation of the myometrium

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    The myometrial smooth muscle of the uterus is responsible for force generation during labour, leading to successful delivery and expulsion of the foetus. This contraction occurs as a result of electrical signalling, and the firing of the myometrial action potential. Development of a more detailed understanding the mechanisms that regulate electrical excitability in the myometrium may allow for the identification of new strategies to manage preterm birth and prolonged labour clinically. The calcium activated chloride channel (CaCC) anoctamin 1 (ANO1) has been suggested to play an important role in promoting myometrial contractility, though its potential mechanism is unclear. In this work, a novel model of uterine excitability has been developed to allow investigation of these processes in silico. Simulations run using this model suggest that ANO1 may play a role in initiating the myometrial action potential and coupling the electrical signalling to chemical signalling via oxytocin. ANO1 may also play a role in stabilising and prolonging the action potential. These hypotheses are investigated experimentally using multiple model systems. Cultured immortalised uterine myocytes and intact human tissue were investigated using specific pharmacological inhibition of ANO1 with the novel, potent pharmacological agent Ani9. Experiments using the Cre-Lox system to achieve inducible smooth muscle specific deletion of ANO1 were also carried out. These experiments observed no evidence of ANO1 promoting uterine contractility, suggesting that previously reported findings were the result of non-specific actions of inhibitors. Transcriptional analysis of cultured myocytes and myometrial tissue suggests ANO6 as a potential alternative candidate for forming a CaCC in the myometrium. Simulations also suggest potential roles for the canonical transient receptor protein channel (TRPC6) in initiating the action potential in response to diacyl glycerol generation, and potassium channels such as TWIK-related Potassium Channel 1 (TREK1) in mechano-sensing and regulation of the duration of the myometrial action potential. Plasticity of the myometrial conductome is also considered as a mechanism that underlies the heterogeneity in electrical behaviours between cells, and may dynamically modulate the electrical properties of the cell over physiologically relevant timescales to regulate the action potential. These potential electrophysiological mechanisms are suggested as potential avenues for further investigatio

    The new technique for accurate estimation of the spinal cord circuitry:recording reflex responses of large motor unit populations

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    We propose and validate a non-invasive method that enables accurate detection of the discharge times of a relatively large number of motor units during excitatory and inhibitory reflex stimulations. HDsEMG and intramuscular EMG (iEMG) were recorded from the tibialis anterior muscle during ankle dorsiflexions performed at 5%, 10%, and 20% of the maximum voluntary contraction (MVC) force, in 9 healthy subjects. The tibial nerve (inhibitory reflex) and the peroneal nerve (excitatory reflex) were stimulated with constant current stimuli. In total, 416 motor units were identified from the automatic decomposition of the HDsEMG. The iEMG was decomposed using a state-of-the-art decomposition tool and provided 84 motor units (average of two recording sites). The reflex responses of the detected motor units were analyzed using the peri-stimulus time histogram (PSTH) and the peri-stimulus frequencygram (PSF). The reflex responses of the common motor units identified concurrently from the HDsEMG and the iEMG signals showed an average disagreement (the difference between number of observed spikes in each bin relative to the mean) of 8.2±2.2% (5% MVC), 6.8±1.0% (10% MVC), and 7.5±2.2% (20% MVC), for reflex inhibition, and 6.5±4.1%, 12.0±1.8%, 13.9±2.4%, for reflex excitation. There was no significant difference between the characteristics of the reflex responses, such as latency, amplitude and duration, for the motor units identified by both techniques. Finally, reflex responses could be identified at higher force (four of the nine subjects performed contraction up to 50% MVC) using HDsEMG but not iEMG, because of the difficulty in decomposing the iEMG at high forces. In conclusion, single motor unit reflex responses can be estimated accurately and non-invasively in relatively large populations of motor units using HDsEMG. This non-invasive approach may enable a more thorough investigation of the synaptic input distribution on active motor units at various force levels

    Management of hypertension in pregnancy — prevention, diagnosis, treatment and long-term prognosis. A position statement of the Polish Society of Hypertension, Polish Cardiac Society and Polish Society of Gynaecologists and Obstetricians

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    ADDITIONAL INFORMATION This article has been co‑published in Kardiologia Polska (doi:10.33963/KP.14904), Arterial Hypertension (doi:10.5603/AH.a2019.0011), and Ginekologia Polska (doi:10.5603/GP.2019.0074). The articles in Kardiologia Polska, Arterial Hypertension, and Ginekologia Polska are identical except for minor stylistic and spelling differences in keeping with each journal’s style. Any citation can be used when citing this article
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