Closure Of Gap Junctions Research Articles

Abstract 660: Injury Induced Connexin 43 Expression Plays A Role In Vascular Endothelial Wound Healing

Vascular surgeries, such as stent placement and coronary artery bypass, damage the endothelial lining of blood vessels. Impaired healing of endothelial damage is a primary contributor to vascular stent and bypass failure, putting patients at risk for myocardial infarction and death. As such, it is necessary to identify regulators of normal endothelial healing and understand how these factors are dysregulated when healing is impaired. Retrospective analysis of RNAseq data from a previous study investigating non-injured vs. regenerating endothelial cells (EC) in mouse aortas revealed a significant increase in the expression of connexin 43 (Cx43) throughout the EC healing process. Although Cx43 functions are important for skin wound healing, the role of connexin 43 in large artery endothelial wound healing has never been investigated. Here, we test the hypothesis that injury-induced increases in Cx43 expression and gap junction functions promote vascular endothelial wound healing. To study Cx43 in the regenerating endothelium in vivo, we developed a novel model of endothelial cell (EC) injury in mouse carotid arteries. In this model, carotids are ligated for a period of one minute, resulting in an EC denudation injury that heals over 24-hours. Western blotting of EC isolated from damaged carotids demonstrated an increase in Cx43 protein in injured mouse EC while immunofluorescent imaging revealed increases in Cx43 staining at EC borders indicative of increased gap junction signaling. Human coronary artery EC scratch assays were also used to study the process of endothelial wound repair in vitro. As Cx43 phosphorylation is the primary regulator of gap junction channel signaling, we used pharmacological strategies (novel peptide treatments) to increase protein kinase C (PKC) phosphorylation at Cx43 serine 368 (Cx43-pS368), associated with gap junction closure and reduced intracellular communication. In cultured EC, increasing Cx43-pS368 resulted in slower wound healing times, suggesting that limiting Cx43 gap junction communication impairs EC wound healing. In conclusion, our data indicate that injury-induced EC upregulation of Cx43 and increases in gap junction intracellular communication may play a key role in EC wound healing responses.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2.

A moderate increase in (55mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. Cx26 hemichannels open in response to moderate elevations of CO2 ( 55mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated of 55mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2 . Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30mm propionate, we found that acidification in the absence of a change in caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). pH-dependent and CO2 -dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the keratitis ichthyosis deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2 -dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction.

PI3k and Stat3: Oncogenes that are Required for Gap Junctional, Intercellular Communication.

Gap junctional, intercellular communication (GJIC) is interrupted in cells transformed by oncogenes such as activated Src. The Src effector, Ras, is required for this effect, so that Ras inhibition restores GJIC in Src-transformed cells. Interestingly, the inhibition of the Src effector phosphatidyl-inositol-3 kinase (PI3k) or Signal Transducer and Activator of Transcription-3 (Stat3) pathways does not restore GJIC. In the contrary, inhibition of PI3k or Stat3 in non-transformed rodent fibroblasts or epithelial cells or certain human lung carcinoma lines with extensive GJIC inhibits communication, while mutational activation of PI3k or Stat3 increases GJIC. Therefore, it appears that oncogenes such as activated Src have a dual role upon GJIC; acting as inhibitors of communication through the Ras pathway, and as activators through activation of PI3k or Stat3. In the presence of high Src activity the inhibitory functions prevail so that the net effect is gap junction closure. PI3k and Stat3 constitute potent survival signals, so that their inhibition in non-transformed cells triggers apoptosis which, in turn, has been independently demonstrated to suppress GJIC. The interruption of gap junctional communication would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. We hypothesize that the GJIC activation by PI3k or Stat3 may be linked to their survival function.

Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis.

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

Age-associated deterioration in follicular fluid induces a decline in bovine oocyte quality.

Maternal age affects the quality of oocytes. The present study examined whether follicular fluid (FF) is a casual factor for age-associated decline in oocyte quality. First, we measured the concentration of advanced glycation end-products (AGE) in FF derived from young (21-45 months; Young-FF) and aged (≥120 months; Aged-FF) cows and found significantly higher concentrations of AGE in Aged-FF than Young-FF. Second, oocytes were collected from ovaries of young or aged cows and cultured in maturation medium containing 10% FF derived from young or aged cows. Regardless of oocyte origin, Aged-FF accelerated nuclear maturation progression and gap junction closure between oocytes and cumulus cells, increased reactive oxygen species (ROS) content and the rate of abnormal fertilisation of oocytes and decreased blastulation rate compared with Young-FF. Furthermore, supplementation of maturation medium with AGE induced similar age-associated events in oocytes derived from young cows, in that AGE accelerated the progression of nuclear maturation, increased ROS content in oocytes, increased the rate of abnormal fertilisation and decreased blastulation rate. In conclusion, maternal aging increased the concentration of AGE in FF, and both AGE and Aged-FF accelerated nuclear maturation and reduced the developmental competence of oocytes.

Gap Junction Regulation and Edema Formation in Neonatal and Adult Hearts

Objectives: In congenital heart surgery, ischemia-induced myocardial edema formation often influences postoperative care, especially after long lasting ischemic periods, since delayed sternal closure is necessary. To evaluate differences in ischemic processes between neonatal and adult hearts we investigated gap junction closure (GJC) and edema formation during normothermic ischemia (35°C) using bioelectrical impedance spectroscopy.

Parameter study of myocardial cell damage with photosensitization reaction under extracellular talaporfin sodium existence

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 333 Conduction block recovery by photosensitization reaction under extracellular talaporfin sodium existence in a cardiomyocyte electrical conduction wire Mariko Kurotsu, Emiyu Ogawa, Tsunenori Arai Graduate School of Science and Technology, Keio University, Japan Westudied theelectrical conduction (EC)block recoverybyphotosensitization reaction (PR) under extracellular talaporfin sodium existence in a novel cardiomyocyte EC wire in vitro. Tachyarrhythmia can be cured by permanent block of abnormal EC. The EC block recovery sometimes occurs in the case of weak damage on myocardium. The pattern cultivation cover glass, which had 60 m width cultivation areas, was used to form the wires. The PR was operated to the wires for 600 s varying 3–120mW/cm2 in 663nm laser irradianceswith 20 g/ml talaporfin sodium. The cardiomyocyte ECwas evaluated by the cross correlation function of a fluorescence brightness of intracellular Ca2+ probe in a wire. In 30 and 60mW/cm2, the EC of 15min after the PR was blocked significantly faster than the reported cardiomyocyte necrosis occurrence timing of the same PR conditions. Despite the EC block probabilities of 15min and 2h after the PR were almost 90% in 60mW/cm2, those in 30mW/cm2 were approximately 90 and 60%, respectively. We think this temporary EC block might be caused by gap junction closure induced by intracellular Ca2+ increase. http://dx.doi.org/10.1016/j.pdpdt.2015.07.036 Light fractionation improves the response to BF-200 ALA-PDT in hairless mouse skin Henriette S. de Bruijn1, Sander Brooks2, Angelique van der Ploeg-van den Heuvel1, Ellen R.M. de Haas2, Dominic J. Robinson1 1 The Centre for Optical Diagnostics and Therapy, Department of Otolaryngology and Head & Neck Surgery, The Netherlands 2 Department of Dermatology, Erasmus MC, Rotterdam, The Netherlands Fractionated illumination significantly enhances the effectiveness of 5-aminolevulinic-acid based photodynamic therapy (ALAPDT) but not of Methyl-AminoLevulinate (MAL). BF-200 ALA is a recently approved novel formulation of 10% ALA in a lecithin-based nanoemulsion. Here we compare the fluorescence kinetics, distribution and PDT induced response in hairless mice using BF-200 ALA, MAL and ALA. Comparable fluorescence kinetics was determined forMAL, ALAandBF-200ALA. Cryo-sectionswere stained for CD31 and co-localization with PpIX fluorescence was significantly stronger after ALA and BF-200 ALA compared to MAL (p<0.01). Intra-vital confocalmicroscopy showed no difference betweenALA and BF-200 ALA at depth in skin. Vascular responses were not different as determined in the skin-fold chamber immediately after PDT or at day 1 in cryosections. Visual skin damage scored up to 14 days post PDT showed an significantly increased response to light fractionation for bothALAandBF-200ALA. BF-200ALAbehaves like ALA in normal mouse skin. While light fractionated ALA-PDT was successfully translated to a treatment modality for human lesions our results suggest that ALA can be replaced by BF-200 ALA, marketed as Ameluz® in Europe, for the treatment of superficial BasalCell-Carcinoma and other skin lesions using light fractionation. http://dx.doi.org/10.1016/j.pdpdt.2015.07.037 Parameter study of myocardial cell damage with photosensitization reaction under extracellular talaporfin sodium existence

Conduction block recovery by photosensitization reaction under extracellular talaporfin sodium existence in a cardiomyocyte electrical conduction wire

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 333 Conduction block recovery by photosensitization reaction under extracellular talaporfin sodium existence in a cardiomyocyte electrical conduction wire Mariko Kurotsu, Emiyu Ogawa, Tsunenori Arai Graduate School of Science and Technology, Keio University, Japan Westudied theelectrical conduction (EC)block recoverybyphotosensitization reaction (PR) under extracellular talaporfin sodium existence in a novel cardiomyocyte EC wire in vitro. Tachyarrhythmia can be cured by permanent block of abnormal EC. The EC block recovery sometimes occurs in the case of weak damage on myocardium. The pattern cultivation cover glass, which had 60 m width cultivation areas, was used to form the wires. The PR was operated to the wires for 600 s varying 3–120mW/cm2 in 663nm laser irradianceswith 20 g/ml talaporfin sodium. The cardiomyocyte ECwas evaluated by the cross correlation function of a fluorescence brightness of intracellular Ca2+ probe in a wire. In 30 and 60mW/cm2, the EC of 15min after the PR was blocked significantly faster than the reported cardiomyocyte necrosis occurrence timing of the same PR conditions. Despite the EC block probabilities of 15min and 2h after the PR were almost 90% in 60mW/cm2, those in 30mW/cm2 were approximately 90 and 60%, respectively. We think this temporary EC block might be caused by gap junction closure induced by intracellular Ca2+ increase. http://dx.doi.org/10.1016/j.pdpdt.2015.07.036 Light fractionation improves the response to BF-200 ALA-PDT in hairless mouse skin Henriette S. de Bruijn1, Sander Brooks2, Angelique van der Ploeg-van den Heuvel1, Ellen R.M. de Haas2, Dominic J. Robinson1 1 The Centre for Optical Diagnostics and Therapy, Department of Otolaryngology and Head & Neck Surgery, The Netherlands 2 Department of Dermatology, Erasmus MC, Rotterdam, The Netherlands Fractionated illumination significantly enhances the effectiveness of 5-aminolevulinic-acid based photodynamic therapy (ALAPDT) but not of Methyl-AminoLevulinate (MAL). BF-200 ALA is a recently approved novel formulation of 10% ALA in a lecithin-based nanoemulsion. Here we compare the fluorescence kinetics, distribution and PDT induced response in hairless mice using BF-200 ALA, MAL and ALA. Comparable fluorescence kinetics was determined forMAL, ALAandBF-200ALA. Cryo-sectionswere stained for CD31 and co-localization with PpIX fluorescence was significantly stronger after ALA and BF-200 ALA compared to MAL (p<0.01). Intra-vital confocalmicroscopy showed no difference betweenALA and BF-200 ALA at depth in skin. Vascular responses were not different as determined in the skin-fold chamber immediately after PDT or at day 1 in cryosections. Visual skin damage scored up to 14 days post PDT showed an significantly increased response to light fractionation for bothALAandBF-200ALA. BF-200ALAbehaves like ALA in normal mouse skin. While light fractionated ALA-PDT was successfully translated to a treatment modality for human lesions our results suggest that ALA can be replaced by BF-200 ALA, marketed as Ameluz® in Europe, for the treatment of superficial BasalCell-Carcinoma and other skin lesions using light fractionation. http://dx.doi.org/10.1016/j.pdpdt.2015.07.037 Parameter study of myocardial cell damage with photosensitization reaction under extracellular talaporfin sodium existence

The sleeping brain regulates to the edge of chaos

One of the most intriguing ideas in complexity theory is the notion that some systems can organize dynamically to a point critically poised between order and disorder, hovering at the so-called “edge of chaos”. It has been proposed that the computational performance of neural networks is optimized when close to the order–disorder phase transition. In this presentation we explore the novel hypothesis that the human brain may be operating at the edge of chaos during slow-wave sleep (SWS), the deepest phase of NREM (non-rapid-eye-movement) sleep. We build on an existing continuum model of the cortex [1] to incorporate known changes in specific neurotransmitter concentrations—GABA increase with simultaneous acetylcholine (ACh) decrease—during descent from wake into natural SWS [2]. The GABA boost is modeled as an anesthetic-like prolongation of the inhibitory postsyaptic potential (IPSP) paired with a restriction of gap-junction connectivity, while ACh suppression reduces resting cell voltage but enhances excitatory synaptic efficiency. Our model is able to produce a plausible sequence of time-series for EEG progression through the stages of NREM sleep (see Figure ​Figure11). Figure 1 Model-generated sleep electrocorticograms for descent from wake to deep NREM. Model predictions are the excitatory soma voltage recorded at one point on a 120×120 cortical grid after filtering with a 0.5-Hz high-pass filter. These sleep-induced neurotransmitter changes can have profound effects on cortical stability: alterations in inhibitory gap-junction connectivity controls a pattern-forming Turing instability, and manipulations of IPSP duration can lead to Hopf temporal oscillations which, in a pathological limit, can lead to whole-of-cortex seizure. We argue that normal brain function requires a balance between Turing and Hopf instabilities, and that descent into deep sleep entails a rebalancing in favor the Hopf instability. Model simulations predict that the spatiotemporal patterns for NREM sleep stages-1 to -4 are chaotic, showing exponential trajectory divergence from closely similar starting conditions. In contrast, the seizure state is highly ordered and non-chaotic. Since most sleepers do not proceed to seizure, we posit the existence of a protective mechanism that regulates the naturally sleeping brain so that it remains close to—but does not cross—the disorder/order boundary during deepest sleep. There is clinical evidence that high cortical activity is associated with closure of gap-junctions [3]. This has motivated a learning rule that regulates the gap-junction conductivity based on the spatial covariance of inhibitory firing-rate activity across the two-dimensional cortical grid. We find that this rule enables the cortex to regulate its slow-wave dynamics from chaotic to marginally-ordered, and that regulation failure typically leads to seizure onset.

Uptake of Betaine into Mouse Cumulus-Oocyte Complexes via the SLC7A6 Isoform of y+L Transporter1

Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.

Pregnancy programming and preeclampsia: identifying a human endothelial model to study pregnancy-adapted endothelial function and endothelial adaptive failure in preeclamptic subjects.

We have previously reported that the increase in vasodilator production in an ovine model pregnancy is underpinned by an increase in connexin 43 (Cx43) gap junction function, so allowing more uterine artery endothelial cells to produce a more sustained Ca(2+) burst response to agonist stimulation. Since activation of endothelial nitric oxide synthase (eNOS) requires elevated [Ca(2+)]i, it follows that the direct result of enhanced bursting in turn is an increase in nitric oxide (NO) production per cell from more cells, and for a longer period of time. Preeclampsia (PE) is associated with endothelial vasodilatory dysfunction, and the endocrine profile of women with PE includes an increase in a number of factors found in wound sites. The common action of these growth factors and cytokines in wound sites is to mediate Cx43 dysfunction through kinase phosphorylation and closure. Translational studies are now needed to establish if inhibitory phosphorylation of Cx43 in human endothelium is the cause of endothelial dysfunction in PE subjects and if so, to identify the kinase pathways best targeted for therapy in PE subjects. Consistent with this we have already shown endothelial Ca(2+) and NO responses of human umbilical vein from normal subjects are similar to that of ovine pregnant uterine artery, and that those same responses in cords from PE subjects are blunted to levels more typical of nonpregnant uterine artery. In this review we summarize the further evidence that growth factors and cytokines may indeed mediate endothelial dysfunction in PE subjects through closure of Cx43 gap junctions. We also consider how we may clinically translate our studies to humans by using intact umbilical vein and isolated HUVEC in primary culture for an initial screen of drugs to prevent deleterious Cx43 phosphorylation, with the ultimate goal of reversing PE-related endothelial dysfunction.

Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation

The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging.

Light‐ and dopamine‐regulated receptive field plasticity in primate horizontal cells

Center-surround antagonistic receptive fields (CSARFs) are building blocks for spatial vision and contrast perception. Retinal horizontal cells (HCs) are the first lateral elements along the visual pathway, and are thought to contribute to receptive field surrounds of higher order neurons. Primate HC receptive fields have not been found to change with light, and dopaminergic modulation has not been investigated. Recording intracellularly from HCs in dark-adapted macaque retina, we found that H1-HCs had large receptive fields (λ = 1,158 ± 137 μm) that were reduced by background light (-45%), gap junction closure (-53%), and D1 dopamine receptor activation (-48%). Tracer coupling was modulated in a correlative manner, suggesting that coupling resistance plays a dominant role in receptive field formation under low light conditions. The D1 antagonist SCH23390 increased the size of receptive fields (+13%), suggesting tonic dopamine release in the dark. Because light elevates dopamine release in primate retina, our results support a dopaminergic role in post-receptoral light adaptation by decreasing HC receptive field diameters, which influences the center-surround receptive field organization of higher-order neurons and thereby spatial contrast sensitivity.

Role of Integrative Signaling Through Gap Junctions in Toxicology

Gap junctional intercellular communication (GJIC) plays a central role in coordinating signal-transduction pathways that control gene expression inside of cells with those of neighboring cells in maintaining the homeostasis of a tissue. The normal homeostatic set point of gap junctions within tissues is in an open state, and although transient closure of gap junctions in response to mitogenic effectors is normal, chronic closure of channels by continuous exposure to environmental and food-borne contaminants can result in adverse health effects such as cancer, teratogenesis, reproductive dysfunction, neuropathies, and cardiac arrhythmias. GJIC is the primary means of integrating signal transduction pathways controlling gene expression between contiguous cells. Thus, bioassay systems that can measure GJIC offer a central, more biosystems approach to assessing the potential for toxicants to epigenetically alter gene expression.

Epidermal growth factor receptor kinase activity is required for gap junction closure and for part of the decrease in ovarian follicle cGMP in response to LH

The meiotic cell cycle in mouse oocytes is arrested in prophase, and then restarted when LH acts on the surrounding granulosa cells. The granulosa cells keep meiosis arrested by providing a source of cGMP that diffuses into the oocyte through gap junctions, and LH restarts the cell cycle by closing the junctions and by decreasing granulosa cell cGMP, thus lowering oocyte cGMP. Epidermal growth factor receptor (EGFR) activation is an essential step in triggering LH-induced meiotic resumption, but its relationship to the cGMP decrease in the follicle is incompletely understood, and its possible function in causing gap junction closure has not been investigated. Here, we use EGFR agonists (epiregulin and amphiregulin) and an EGFR kinase inhibitor (AG1478) to study the function of the EGFR in the signaling pathways leading to the release of oocytes from prophase arrest. Our results indicate that the EGFR kinase contributes to LH-induced meiotic resumption in two different ways. First, it is required for gap junction closure. Second, it is required for an essential component of the decrease in follicle cGMP. Our data show that the EGFR kinase-dependent component of the cGMP decrease is required for LH-induced meiotic resumption, but they also indicate that an as yet unidentified pathway accounts for a large part of the cGMP decrease.

Mind the gap: Engineering resilient gap junctions

Gap junctions are indispensible for intercellular communication between cardiac myocytes. They enable the heart to function as a syncytium and warrant maintenance of total cardiac activation, repolarization and contraction. 1 Jongsma H.J. Wilders R. Gap junctions in cardiovascular disease. Circ Res. 2000; : 1193-1197 Crossref PubMed Scopus (321) Google Scholar The heart's ability to sustain life even after myocardial infarction can be attributed to these intercellular channels, which allow the cardiac impulse to find alternative routes to cause contraction, and subsequent ejection of blood into the circulation, even if slightly less efficient than before. During myocardial ischemia, closure of gap junctions may help to (metabolically and electrically) isolate the diseased myocardium from the healthy surrounding myocardium (leading to “healing over” of the ECG signs of ischemia). Design and characterization of the first peptidomimetic molecule that prevents acidification-induced closure of cardiac gap junctionsHeart RhythmVol. 7Issue 10PreviewGap junctions are potential targets for pharmacologic intervention. We previously developed a series of peptide sequences that prevent closure of connexin43 (Cx43) channels, bind to cardiac Cx43, and prevent acidification-induced uncoupling of cardiac gap junctions. Full-Text PDF

Detection of (Reversible) Myocardial Ischemic Injury by Means of Electrical Bioimpedance

The scope of this paper was to determine whether ischemic and reperfusion damage in cardiac surgery can be detected by measurement of electrical bioimpedance (EBI). Conventional pacing wires were replaced by pacing wires with sputtered iridium coating in order to reduce polarization associated with two-electrode impedance measurements. A custom-built bioimpedance analyzer (Osypka Medical GmbH, Berlin, Germany) measured the real part of impedance Re(Z) and the phase (ϕ) at three frequencies (1, 10, and 1000 kHz) and determined an extracellular space index (EZRI) as the quotient of Re(Z) at 1000 kHz and Re(Z) at 1 kHz. Our study included six patients (conventional coronary artery bypass graft, age 68.1 ± 8.3 years) subject to routine cardioplegic ischemia and reperfusion. Preischemic bioimpedance measurements were not impaired by interference of the beating heart. Intraischemically, bioimpedance at 1 kHz and phase at 10 kHz increased until opening of a bypass graft, which is probably induced by closure of gap junctions and cell swelling processes. After cross clamping, EZRI slowly decreased as an effect of mild cell swelling. After ischemia, values returned almost to baseline measurements, indicating sufficient reperfusion processes. Measurement of EBI correlates with myocardial ischemic injury and is applicable in a two-electrode setup providing low-polarization pacing wires.

Design and characterization of the first peptidomimetic molecule that prevents acidification-induced closure of cardiac gap junctions

Gap junctions are potential targets for pharmacologic intervention. We previously developed a series of peptide sequences that prevent closure of connexin43 (Cx43) channels, bind to cardiac Cx43, and prevent acidification-induced uncoupling of cardiac gap junctions. The purpose of this study was to identify and validate the minimum core active structure in peptides containing an RR-N/Q-Y motif. Based on that information, we sought to generate a peptidomimetic molecule that acts on the chemical regulation of Cx43 channels. Experiments were based on a combination of biochemical, spectroscopic, and electrophysiologic techniques as well as molecular modeling of active pharmacophores with Cx43 activity. Molecular modeling analysis indicated that the functional elements of the side chains in the motif RRXY form a triangular structure. Experimental data revealed that compounds containing such a structure bind to Cx43 and prevent Cx43 chemical gating. These results provided us with the first platform for drug design targeted to the carboxyl terminal of Cx43. Using that platform, we designed and validated a peptidomimetic compound (ZP2519; molecular weight 619 Da) that prevented octanol-induced uncoupling of Cx43 channels and pH gating of cardiac gap junctions. Structure-based drug design can be applied to the development of pharmacophores that act directly on Cx43. Small molecules containing these pharmacophores can serve as tools to determine the role of gap junction regulation in the control of cardiac rhythm. Future studies will determine whether these compounds can function as pharmacologic agents for the treatment of a selected subset of cardiac arrhythmias.

Signaling networks in somatic cells and oocytes activated during ovulation

The molecular and structural changes in the follicle triggered by the gonadotropin luteinizing hormone (LH) at ovulation require complex interactions between somatic cells and the oocytes. It is well established that the signals originating from the oocytes play an essential role in orchestrating the growth and development of the follicle. Conversely, the exact contribution of local signals in the two compartments to ovulation is less clear. LH causes oocyte meiotic resumption, cumulus expansion, and follicle rupture through direct activation of granulosa cells, but also indirectly by acting on cumulus cells and the oocyte. Recent studies from our and other laboratories have identified some of the components of the signaling network activated at this critical transition. The oocyte meiotic arrest is maintained through the activity of GPCR and adenylyl cyclases endogenous to the oocyte and by diffusion of signals through gap junction connections with the soma. It is possible that cAMP, and more recently, cGMP, diffuses through the gap junction network from somatic cells to the oocyte to maintain meiotic arrest. Recent evidence suggests that the biochemical mechanism by which cGMP maintains meiotic arrest is likely through regulation of the oocyte PDE and therefore, indirectly cAMP. The exact series of events regulating reentry of the oocyte into the cell cycle are still not completely understood, but it is widely accepted that a decrease in cAMP is the primary signal. This decrease in cAMP is caused by an activation of the oocyte PDE but also by closure of the gap junctions. The regulatory mechanisms in the soma that lead to activation of the PDE in the oocytes and gap junction closure are the focus of intense investigation. Several signaling pathways converge on these two final events. LH initially activates cAMP signaling but then the stimulus branches into a number of contiguous and interacting pathways. Activation of the EGF network is one of these pathways and accumulating evidence strongly indicates that this is a component critical for oocyte maturation, cumulus expansion, and follicle rupture. Because these regulatory mechanisms are conserved in rodents and in humans, a better understanding of these signaling networks activated during ovulation will provide new pharmacological opportunities for the manipulation of fertility.

Calcium-Calmodulin Regulation of Connexin43 Involves a Cytoplasmic Loop Domain

Connexin43 (Cx43) is widely expressed throughout the mammalian body and is the predominant gap junction protein in the ventricular myocardium. Cx43 was recently reported to contain a calmodulin (CaM) binding site on its cytoplasmic loop (CL) domain near its third transmembrane domain (Zhou et al., JBC 282: 35005-17, 2007). Intracellular calcium (Cai)-dependent regulation of cardiac and Cx43 gap junctions has also been reported, but the function of this putative CaM-binding site has never been directly examined. In dual whole cell patch clamp experiments, murine neuro2a (N2a)-Cx43 cell gap junctional conductance (gj) declined by 95% within 10 min (n=3) during bath perfusion with 1 μM ionomycin + 1.8 mM external Ca2+ (Cao). Cx43 gj declined by only 20% in nominally zero Cao (n=5). This Ca2+-dependent uncoupling was demonstrated to be CaM-dependent by acute (10-15 min) pretreatment with 2 μM calmidazolium, wherein Cx43 gj declined by < 10% within 10 min (n=4). To directly test for the involvement of the Cx43 amino acid residue #136-158 domain in this Ca2+/CaM-dependent gap junction uncoupling process, 1 μM peptides were added to both whole cell patch pipettes and the 1 μM ionomycin/1.8 mM Cao perfusion experiments were repeated. The Cx43 #136-158 sequence mimetic peptide (Kd(CaM) = 860 nM) effectively prevented the Cx43 gj decline (< 3%, n=4) whereas a scrambled sequence peptide control failed to prevent the Ca2+-induced rundown of Cx43 gj (<90%, n=3). These data unequivocally demonstrate that influx of external Ca2+ induces closure of Cx43 gap junctions in a CaM-dependent process involving the Cx43 residue #136-158 CL domain. This process has significant implications for the modulation of cardiac gj by Cai and the “healing-over” of infarcted myocardium. Supported by NIH grants GM62999 & EY-05684 to JJY and HL-042220 to RDV.