Inhibition of Inflammatory Changes in Human Myometrial Cells by Cell Penetrating Peptide and Small Molecule Inhibitors of NFκB.

Complications arising from Preterm Birth are the leading causes of neonatal death globally. Current therapeutic strategies to prevent Preterm Birth are yet to demonstrate success in terms of reducing this neonatal disease burden. Upregulation of intracellular inflammatory pathways in uterine cells, including those involving nuclear factor kappa-B (NFκB), have been causally linked to both human term and preterm labor, but the barrier presented by the cell membrane presents an obstacle to interventions aimed at dampening these inflammatory responses. Cell penetrating peptides (CPPs) are novel vectors that can traverse cell membranes without the need for recognition by cell surface receptors and offer the ability to deliver therapeutic cargo internal to cell membranes. Using a human uterine cell culture inflammatory model, this study aimed to test the effectiveness of CPP-cargo delivery to inhibit inflammatory responses, comparing this effect with a small molecule inhibitor (Sc514) that has a similar intracellular target of action within the NFκB pathway (the IKK complex). The CPP Penetratin, conjugated to rhodamine, was able to enter uterine cells within a 60 min timeframe as assessed by live confocal microscopy, this phenomena was not observed with the use of a rhodamine-conjugated inert control peptide (GC(GS)4). Penetratin CPP conjugated to an IKK-inhibitory peptide (Pen-NBD) demonstrated ability to inhibit both the IL1ß-induced expression of the inflammatory protein COX2 and dampen the expression of a bespoke array of inflammatory genes. Truncation of the CPP vector rendered the CPP-cargo conjugate much less effective, demonstrating the importance of careful vector selection. The small molecule inhibitor Sc514 also demonstrated ability to inhibit COX2 protein responses and a broad down-regulatory effect on uterine cell inflammatory gene expression. These results support the further exploration of either CPP-based or small molecular treatment strategies to dampen gestational cell inflammatory responses in the context of preterm birth. The work underlines both the importance of careful selection of CPP vector-cargo combinations and basic testing over a broad time and concentration range to ensure effective responses. Further work should demonstrate the effectiveness of CPP-linked cargos to dampen alternative pathways of inflammation linked to Preterm Birth such as MAP Kinase or AP1.

Depletion of Myofibril-Associated Proteins Using Selective Protein Extraction as a Tool in Cardiac Proteomics.

Muscle tissue poses a particular challenge to proteomic analysis due to a very wide range of protein abundances arising from the dominant expression of myofilament-related proteins. We address this issue by describing proteomic analysis with liquid chromatography-mass spectrometry (LC-MS) and sequential window acquisition of all theoretical mass spectra (SWATH), of guinea pig cardiac tissue prepared in two homogenization buffers: (1) An SDS-based buffer designed to extract "all" tissue proteins and (2) a long-established EDTA-containing buffer thought to preferentially extract non-myofibril-related proteins. We use gene ontology (GO) annotation-based assessment of subcellular localization to indicate if these enriched proteins congregate in the cytoplasm or in organellar lumens. This technique results in the preferential quantitation of less abundant non-myofibrillar proteins and, for future studies, offers the opportunity for more complete analyses of changes in heart tissue protein expression with biological circumstance.

Chemotactic activity of gestational tissues through late pregnancy, term labor, and RU486-induced preterm labor in Guinea pigs.

PROBLEM: Is increased leukocyte chemotactic activity (CA) from gestational tissues necessary for term or preterm labor in guinea pigs? METHOD OF STUDY: Tissue extracts were prepared from pregnant guinea pig decidua-myometrium, cervix, fetal membranes (amniochorion), and placenta during early third trimester (n = 8), term not in labor (TNL, n = 5), and term spontaneous labor (TL, n = 6), RU486-induced preterm labor (PTL, n = 6), or controls (cPTL, n = 5). Leukocyte CA was assessed using a modified Boyden chamber assay. Extract chemokine and maternal progesterone concentrations were quantified by enzyme immunoassay. RESULTS: Only the extracts from amniochorion demonstrated increased CA through late gestation and labor. In contrast, CA was decreased in extracts from amniochorion and cervix from animals after RU486-induced PTL. Maternal progesterone concentrations remained high in all groups. CONCLUSION: Leukocyte CA of intrauterine tissues is increased in term spontaneous labor. However, RU486-induced preterm labor occurs in the absence of increased CA.

Computational modeling reveals key contributions of KCNQ and hERG currents to the malleability of uterine action potentials underpinning labor.

The electrical excitability of uterine smooth muscle cells is a key determinant of the contraction of the organ during labor and is manifested by spontaneous, periodic action potentials (APs). Near the end of term, APs vary in shape and size reflecting an ability to change the frequency, duration and amplitude of uterine contractions. A recent mathematical model quantified several ionic features of the electrical excitability in uterine smooth muscle cells. It replicated many of the experimentally recorded uterine AP configurations but its limitations were evident when trying to simulate the long-duration bursting APs characteristic of labor. A computational parameter search suggested that delayed rectifying K(+) currents could be a key model component requiring improvement to produce the longer-lasting bursting APs. Of the delayed rectifying K(+) currents family it is of interest that KCNQ and hERG channels have been reported to be gestationally regulated in the uterus. These currents exhibit features similar to the broadly defined uterine IK1 of the original mathematical model. We thus formulated new quantitative descriptions for several I(KCNQ) and I(hERG). Incorporation of these currents into the uterine cell model enabled simulations of the long-lasting bursting APs. Moreover, we used this modified model to simulate the effects of different contributions of I(KCNQ) and I(hERG) on AP form. Our findings suggest that the alterations in expression of hERG and KCNQ channels can potentially provide a mechanism for fine tuning of AP forms that lends a malleability for changing between plateau-like and long-lasting bursting-type APs as uterine cells prepare for parturition.

Computational modeling of inhibition of voltage-gated Ca channels: identification of different effects on uterine and cardiac action potentials.

The uterus and heart share the important physiological feature whereby contractile activation of the muscle tissue is regulated by the generation of periodic, spontaneous electrical action potentials (APs). Preterm birth arising from premature uterine contractions is a major complication of pregnancy and there remains a need to pursue avenues of research that facilitate the use of drugs, tocolytics, to limit these inappropriate contractions without deleterious actions on cardiac electrical excitation. A novel approach is to make use of mathematical models of uterine and cardiac APs, which incorporate many ionic currents contributing to the AP forms, and test the cell-specific responses to interventions. We have used three such models-of uterine smooth muscle cells (USMC), cardiac sinoatrial node cells (SAN), and ventricular cells-to investigate the relative effects of reducing two important voltage-gated Ca currents-the L-type (ICaL) and T-type (ICaT) Ca currents. Reduction of ICaL (10%) alone, or ICaT (40%) alone, blunted USMC APs with little effect on ventricular APs and only mild effects on SAN activity. Larger reductions in either current further attenuated the USMC APs but with also greater effects on SAN APs. Encouragingly, a combination of ICaL and ICaT reduction did blunt USMC APs as intended with little detriment to APs of either cardiac cell type. Subsequent overlapping maps of ICaL and ICaT inhibition profiles from each model revealed a range of combined reductions of ICaL and ICaT over which an appreciable diminution of USMC APs could be achieved with no deleterious action on cardiac SAN or ventricular APs. This novel approach illustrates the potential for computational biology to inform us of possible uterine and cardiac cell-specific mechanisms. Incorporating such computational approaches in future studies directed at designing new, or repurposing existing, tocolytics will be beneficial for establishing a desired uterine specificity of action.

Recurring patterns in stationary intervals of abdominal uterine electromyograms during gestation.

Abdominal uterine electromyograms (uEMG) studies have focused on uterine contractions to describe the evolution of uterine activity and preterm birth (PTB) prediction. Stationary, non-contracting uEMG has not been studied. The aim of the study was to investigate the recurring patterns in stationary uEMG, their relationship with gestation age and PTB, and PTB predictivity. A public database of 300 (38 PTB) three-channel (S1-S3) uEMG recordings of 30 min, collected between 22 and 35 weeks' gestation, was used. Motion and labour contraction-free intervals in uEMG were identified as 5-min weak-sense stationarity intervals in 268 (34 PTB) recordings. Sample entropy (SampEn), percentage recurrence (PR), percentage determinism (PD), entropy (ER), and maximum length (L MAX) of recurrence were calculated and analysed according to the time to delivery and PTB. Random time series were generated by random shuffle (RS) of actual data. Recurrence was present in actual data (p<0.001) but not RS. In S3, PR (p<0.005), PD (p<0.01), ER (p<0.005), and L MAX (p<0.05) were higher, and SampEn lower (p<0.005) in PTB. Recurrence indices increased (all p<0.001) and SampEn decreased (p<0.01) with decreasing time to delivery, suggesting increasingly regular and recurring patterns with gestation progression. All indices predicted PTB with AUC≥0.62 (p<0.05). Recurring patterns in stationary non-contracting uEMG were associated with time to delivery but were relatively poor predictors of PTB.

A computational model of the ionic currents, Ca2+ dynamics and action potentials underlying contraction of isolated uterine smooth muscle.

Uterine contractions during labor are discretely regulated by rhythmic action potentials (AP) of varying duration and form that serve to determine calcium-dependent force production. We have employed a computational biology approach to develop a fuller understanding of the complexity of excitation-contraction (E-C) coupling of uterine smooth muscle cells (USMC). Our overall aim is to establish a mathematical platform of sufficient biophysical detail to quantitatively describe known uterine E-C coupling parameters and thereby inform future empirical investigations of physiological and pathophysiological mechanisms governing normal and dysfunctional labors. From published and unpublished data we construct mathematical models for fourteen ionic currents of USMCs: Ca2+ currents (L- and T-type), Na+ current, an hyperpolarization-activated current, three voltage-gated K+ currents, two Ca2+-activated K+ current, Ca2+-activated Cl current, non-specific cation current, Na+-Ca2+ exchanger, Na+-K+ pump and background current. The magnitudes and kinetics of each current system in a spindle shaped single cell with a specified surface area:volume ratio is described by differential equations, in terms of maximal conductances, electrochemical gradient, voltage-dependent activation/inactivation gating variables and temporal changes in intracellular Ca2+ computed from known Ca2+ fluxes. These quantifications are validated by the reconstruction of the individual experimental ionic currents obtained under voltage-clamp. Phasic contraction is modeled in relation to the time constant of changing [Ca2+]i. This integrated model is validated by its reconstruction of the different USMC AP configurations (spikes, plateau and bursts of spikes), the change from bursting to plateau type AP produced by estradiol and of simultaneous experimental recordings of spontaneous AP, [Ca2+]i and phasic force. In summary, our advanced mathematical model provides a powerful tool to investigate the physiological ionic mechanisms underlying the genesis of uterine electrical E-C coupling of labor and parturition. This will furnish the evolution of descriptive and predictive quantitative models of myometrial electrogenesis at the whole cell and tissue levels.

Three-dimensional electron microscopic reconstruction of intracellular organellar arrangements in vascular smooth muscle--further evidence of nanospaces and contacts.

The sarcoplasmic reticulum (SR) of smooth muscle is crucial for appropriate regulation of Ca(2+) signalling. In visceral and vascular smooth muscles the SR is known to periodically lie in close register, within a few nanometres, to the plasma membrane. Recent work has focussed on reconstructions of the ultrastructural arrangement of this so-called peripheral SR that may be important for the genesis of phenomena such as Ca(2+) sparks. Here, we turn our attention to vascular smooth muscle and explore the 3-dimensional (3D) ultrastructural positioning of SR found deeper in the cell that is involved in the propagation of Ca(2+) waves. We use digital reconstruction and volume rendering of serial electron microscopic sections from isolated resistance arteries, pressurized in vitro to mimic cellular geometric conformations anticipated in vivo, to map SR positioning. We confirm that these central portions of SR are in close register with mitochondria and the nucleus with all three organelles tightly enveloped by a myofilament/cytoskeletal lattice. Nanospacings between the SR and individual mitochondria are visible and in three dimensions as the SR contorts to accommodate these organelles. Direct connection of the SR and nuclear membranes is confirmed. Such 3D positioning of centrally located SR further informs us of its likely role in the manifestation of spatiotemporal Ca(2+) dynamics: signal encoding may be facilitated by spatially directed release of Ca(2+) to influence several processes crucial to vascular smooth muscle and resistance artery function including myofilament activation by Ca(2+) waves, mitochondrial respiration and gene transcription.

Bifurcation analysis of genetically engineered pacemaking in mammalian heart.

Genetically engineered pacemaking in ventricular cells has been achieved by down-regulation of the time independent inward rectifying current (I(K1)), or insertion of the hyperpolarisation-activated funny current (I(f)). We analyse the membrane system (i.e. ionic concentrations clamped) of an epicardial Luo-Rudy dynamic cell model using continuation algorithms with the maximum conductance (g) of I(K1) and I(f) as bifurcation parameters. Pacemaker activity can be induced either via Hopf or homoclinic bifurcations. As g(K1) is decreased by approximately 74%, autorhythmicity emerged via a homoclinic bifurcation, i.e., the periodicity first appear with infinitely large periods. In contrast, the insertion of g(f) induced periodicity via a subcritical Hopf bifurcation at g(f) approximately 0.25 mSmicroF(-1). Stable autorhythmic action potentials occurred at g(f) > 0.329 mSmicroF(-1).

The virtual ventricular wall: a tool for exploring cardiac propagation and arrhythmogenesis.

Methods for the experimental and clinical investigation of cardiac arrhythmias are limited to inferring propagation within the myocardium, from surface measurements, or from electrodes at a few sites within the cardiac wall. Biophysically and anatomically detailed computational models of cardiac tissues offer a powerful way for studying the electrical propagation processes and arrhythmias within the virtual heart. We use virtual tissues to study and visualise the effects of patho- and physiological conditions, and pharmacological interventions on transmural propagation in the virtual ventricular walls. Class III drug actions are quantitatively explained by changes induced in the transmural dispersion of action potential duration. We illustrate the automated construction of a virtual anisotropic ventricle from Diffusion Tensor MRI for individual hearts, and use it to explore mechanisms leading to ventricular fibrillation. The virtual ventricular wall provides an effective tool for exploring, evaluating and visualising processes during the initiation and maintenance of ventricular arrhythmias.

Polymorphisms in the myocilin promoter unrelated to the risk and severity of primary open-angle glaucoma.

PURPOSE: To investigate the proximal 2.5 kb promoter in the myocilin (MYOC) gene for mutations in Chinese patients with primary open-angle glaucoma (POAG). PATIENTS AND METHODS: We screened for sequence alterations in the MYOC promoter in 88 unrelated Chinese patients with POAG and 94 unrelated individuals without glaucoma, aged 50 years or above, as control subjects. In addition, the specific MYOC.mt1 polymorphism was determined in a total of 212 POAG patients and 221 control subjects. The relationships between POAG phenotype and the identified polymorphisms were studied by univariate analysis, multivariable logistic regression analysis, and haplotype analysis. RESULTS: All polymorphisms identified in this study followed Hardy-Weinberg equilibrium (P > 0.12) both in POAG patients and controls. Both univariate and multivariable logistic regression analyses showed no polymorphism that was significantly associated with the risk of POAG, P > 0.08 and P > 0.044 respectively. Haplotype analysis further indicated no association of MYOC promoter polymorphisms with the susceptibility for POAG (P > 0.1). On the other hand, there was no difference of POAG phenotypes among different genotypes of MYOC.mt1 (P > 0.31). CONCLUSIONS: In this study on the Chinese population, polymorphisms in the MYOC promoter are not related to the risk of POAG. There is no association between the MYOC.mt1 promoter polymorphism with the severity of POAG.