Spatial heterogeneity enhances and modulates excitability in a mathematical model of the myometrium

The muscular layer of the uterus (myometrium) undergoes profound changes in global excitability prior to parturition. Here, a mathematical model of the myocyte network is developed to investigate the hypothesis that spatial heterogeneity is essential to the transition from local to global excitation which the myometrium undergoes just prior to birth. Each myometrial smooth muscle cell is represented by an element with FitzHugh–Nagumo dynamics. The cells are coupled through resistors that represent gap junctions. Spatial heterogeneity is introduced by means of stochastic variation in coupling strengths, with parameters derived from physiological data. Numerical simulations indicate that even modest increases in the heterogeneity of the system can amplify the ability of locally applied stimuli to elicit global excitation. Moreover, in networks driven by a pacemaker cell, global oscillations of excitation are impeded in fully connected and strongly coupled networks. The ability of a locally stimulated cell or pacemaker cell to excite the network is shown to be strongly dependent on the local spatial correlation structure of the couplings. In summary, spatial heterogeneity is a key factor in enhancing and modulating global excitability

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

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 profles 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

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 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

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 L-type Ca2+ channel (LTCC) and Na+/Ca2+ exchanger (NCX) currents. Fluorescence photometry techniques were used to measure intracellular free Ca2+ concentration [Ca2+]i. Results: Although LTCC current was not significantly altered, the amplitude (AMP) of Ca2+ transients increased significantly in EPI-STZ and ENDO-STZ compared to controls. Time to peak (TPK) LTCC current, TPK Ca2+ transient, time to half (THALF) decay of LTCC current and THALF decay of Ca2+ transients were not significantly changed in EPI-STZ and ENDO-STZ myocytes compared to controls. NCX current was significantly smaller in EPI-STZ and in ENDO-STZ compared to controls. Conclusions: STZ-induced diabetes resulted in an increase in AMP of Ca2+ transients in EPI and ENDO myocyte that was independent of LTCC current. Such an effect can be attributed, at least in part, to the dysfunction of NCX. 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

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

© 2020 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd 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

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

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

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

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

The information that connects genotypes and phenotypes is essentially embedded in research articles written in natural language. To facilitate access to this knowledge, we constructed a framework for the curation of the scientific literature studying the molecular mechanisms that control leaf growth and development in Arabidopsis thaliana (Arabidopsis). Standard structured statements, called relations, were designed to capture diverse data types, including phenotypes and gene expression linked to genotype description, growth conditions, genetic and molecular interactions, and details about molecular entities. Relations were then annotated from the literature, defining the relevant terms according to standard biomedical ontologies. This curation process was supported by a dedicated graphical user interface, called Leaf Knowtator. A total of 283 primary research articles were curated by a community of annotators, yielding 9947 relations monitored for consistency and over 12,500 references to Arabidopsis genes. This information was converted into a relational database (KnownLeaf) and merged with other public Arabidopsis resources relative to transcriptional networks, protein–protein interaction, gene co-expression, and additional molecular annotations. Within KnownLeaf, leaf phenotype data can be searched together with molecular data originating either from this curation initiative or from external public resources. Finally, we built a network (LeafNet) with a portion of the KnownLeaf database content to graphically represent the leaf phenotype relations in a molecular context, offering an intuitive starting point for knowledge mining. Literature curation efforts such as ours provide high quality structured information accessible to computational analysis, and thereby to a wide range of applications