Connexin43 Cx43 Research Articles

BA6b9, a novel sk4 K + channel blocker, attenuates atrial fibrillation substrate in rats with reduced ejection fraction

Abstract Background Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as novel druggable targets for efforts to treat AF. Methods and Findings We examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat post-myocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Interpretation Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Supramolecular self-assembled nanoparticles camouflaged with neutrophil membrane to mitigate myocardial ischemia/reperfusion (I/R) injury

The clinically restored blood supply post myocardial infarction (MI) will break the physiological and metabolic balance of the ischemic myocardial region, leading to a new injury known as myocardial ischemia/reperfusion (I/R) injury. Abundant oxygen brought by the fresh blood would trigger the damaged myocardium to produce excessive reactive oxygen species (ROS) and thereby inflammation along with the recruitment and infiltration of activated neutrophils and macrophages. Inspired by the active targeting process of inflammatory cells, a novel “neutrophil-like” nanoassembly (At@NQA) was prepared by modifying neutrophil membrane fragments onto At@QA NPs containing 1:1 (molar ratio) calixarene molecule (QA) and anti-inflammatory and anti-fibrotic atorvastatin calcium (At). Tail vein injection of the At@NQA could efficiently target the damaged myocardium, inhibit oxidative damage and inflammation, reduce myocardial fibrosis, increase myocardial troponin T (cTnT) and connexin 43 (Cx43) expression, and thus improve cardiac functions effectively.

Guard against internal and external: An antibacterial, anti-inflammation and healing-promoting spray gel based on lyotropic liquid crystals for the treatment of diabetic wound

The diabetic wound is a prevalent and serious complication of diabetes, which easily deteriorates due to susceptibility to infection and difficulty in healing, causing a high risk of amputation and economic burden to patients. Bacterial infection, persistent excessive inflammation, and cellular and angiogenesis disorders are the main reasons for the difficulty of diabetic wound healing. In this study, glycerol monooleate (GMO) was used to prepare lyotropic liquid crystal hydrogel (LLC) containing the natural antimicrobial peptide LL37 and carbenoxolone (CBX) to achieve antibacterial, anti-inflammation, and healing promotion for the treatment of diabetic wounds. The shear-thinning properties of the LLC precursor solution allowed it to be administered in the form of a spray, which perfectly fitted the shape of the wound and transformed into a gel after absorbing wound exudate to act as a wound protective barrier. The faster release of LL37 realized rapid sterilization of wounds, controlled the source of inflammation, and accelerated wound healing. The inflammatory signaling pathway was blocked by the subsequently released CBX, and the spread of the inflammatory response was inhibited and then further weakened. In addition, CBX down-regulated connexin (Cx43) to assist LL37 to promote cell migration and proliferation better. Combined with the pro-angiogenic effect of LL37, the healing of diabetic wounds was significantly accelerated. All these advantages made LL37-CBX-LLC a promising approach for the treatment of chronic diabetic wounds.

Rett syndrome with atrial tachycardia in a girl.

Rett syndrome with atrial tachycardia in a girl.

Involvement of Hippocampal Astrocytic Connexin-43 in Morphine dependence

Repeated exposure to drugs of abuse can lead to dysregulation of chemical synapses by altering the release and uptake of neurotransmitters. Such alterations in neurotransmission modify synaptic plasticity which causes addictive-like behaviors. Our previous study shed light on the involvement of glial cells in morphine-induced behavioral responses. It has been shown that glial cells play an indispensable role in synaptic transmission through the release of gliotransmitter into and uptake of neurotransmitters from the synaptic cleft. Connexin-43 (Cx43), the dominant Cx protein in astrocytes, is the main component of astrocytic gap junctions and hemichannels. It has a critical role in synaptic efficacy through setting the amount of presynaptic gliotransmitter release in physiological conditions. It is probable that addictive substances affecting gliotransmitters release through the alteration of Cx43 function. In this study, we examined the role of the hippocampal-specific astrocytic connexin (Cx43) in morphine-induced behavioral responses. Male rats received subcutaneous (s.c.) morphine sulfate (10 mg/kg) at an interval of 12 h for 9 days. The animals received microinjection of TAT-Gap19 (inhibitor of Cx43) into the CA1 region before each morning morphine administration. The animals were assessed for morphine dependence by monitoring naloxone hydrochloride precipitated withdrawal somatic signs. Results showed that animals receiving TAT-Gap19 before morphine injection demonstrated a significant reduction in several signs of morphine withdrawal such as Activity, Freezing, Chewing, Ptosis, Defecation, Teeth chattering, Writhing, Penis- licking, Head tremor, Scratching, Sniffing, Rearing, and Diarrhea (One way ANOVA, P < 0.001; P < 0.01; P < 0.05). Our findings suggest that hippocampal Cx43 may be involved in morphine-induced behavioral responses. Therefore, gliotransmitter release by astrocytes seems to be a mechanism which is engaged in addictive-like behaviors.

The Mechanisms Underlying the Protective Action of Selenium Nanoparticles against Ischemia/Reoxygenation Are Mediated by the Activation of the Ca2+ Signaling System of Astrocytes and Reactive Astrogliosis.

In recent years, much attention has been paid to the study of the therapeutic effect of the microelement selenium, its compounds, especially selenium nanoparticles, with a large number of works devoted to their anticancer effects. Studies proving the neuroprotective properties of selenium nanoparticles in various neurodegenerative diseases began to appear only in the last 5 years. Nevertheless, the mechanisms of the neuroprotective action of selenium nanoparticles under conditions of ischemia and reoxygenation remain unexplored, especially for intracellular Ca2+ signaling and neuroglial interactions. This work is devoted to the study of the cytoprotective mechanisms of selenium nanoparticles in the neuroglial networks of the cerebral cortex under conditions of ischemia/reoxygenation. It was shown for the first time that selenium nanoparticles dose-dependently induce the generation of Ca2+ signals selectively in astrocytes obtained from different parts of the brain. The generation of these Ca2+ signals by astrocytes occurs through the release of Ca2+ ions from the endoplasmic reticulum through the IP3 receptor upon activation of the phosphoinositide signaling pathway. An increase in the concentration of cytosolic Ca2+ in astrocytes leads to the opening of connexin Cx43 hemichannels and the release of ATP and lactate into the extracellular medium, which trigger paracrine activation of the astrocytic network through purinergic receptors. Incubation of cerebral cortex cells with selenium nanoparticles suppresses ischemia-induced increase in cytosolic Ca2+ and necrotic cell death. Activation of A2 reactive astrocytes exclusively after ischemia/reoxygenation, a decrease in the expression level of a number of proapoptotic and proinflammatory genes, an increase in lactate release by astrocytes, and suppression of the hyperexcitation of neuronal networks formed the basis of the cytoprotective effect of selenium nanoparticles in our studies.

Recognition of the atrioventricular node anatomical structure: Connection between the retroaortic node and the compact node.

The complex electrophysiological phenomena related to the atrioventricular node (AVN) are due to its complex anatomical structures. Aside from the inferior nodal extension (INE), other node-like tissues, such as the retroaortic node (RN), have been described less extensively and may also share the mechanism of normal conduction and abnormal conduction in AVN re-entrant tachycardia. High-density sections of the entire AVN were obtained from rats and rabbits. Fibrosis was analyzed by Masson's trichrome staining. Connexin (Cx43, Cx40, and Cx45) and ion channel (Nav 1.5, Cav 3.1, and HCN4) proteins were immunohistochemically labeled for the analysis of tissue features. Three-dimensional (3D) reconstruction of the AV junction was performed to clarify the relationships among different structures. The RN expressed the same connexin isoforms as the compact node (CN) and INE. Nav 1.5 labeling was observed at low levels in the CN, RN, and INE, where Cav 3.1 and HCN4 were expressed. The CN connected with the RN in a narrow strip pattern at the start of the CN. The RN presented as a shuttle shape and was the only tissue directly connected with the atrium in the anterior septum. The RN connects with the AVN anatomically, suggesting that direct electrical conduction occurs between them. The entrance of the atria into the AVN is distal to the RN, which may form the fast AVN pathway.

Peptidic Connexin43 Therapeutics in Cardiac Reparative Medicine.

Connexin (Cx43)-formed channels have been linked to cardiac arrhythmias and diseases of the heart associated with myocardial tissue loss and fibrosis. These pathologies include ischemic heart disease, ischemia-reperfusion injury, heart failure, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and Duchenne muscular dystrophy. A number of Cx43 mimetic peptides have been reported as therapeutic candidates for targeting disease processes linked to Cx43, including some that have advanced to clinical testing in humans. These peptides include Cx43 sequences based on the extracellular loop domains (e.g., Gap26, Gap 27, and Peptide5), cytoplasmic-loop domain (Gap19 and L2), and cytoplasmic carboxyl-terminal domain (e.g., JM2, Cx43tat, CycliCX, and the alphaCT family of peptides) of this transmembrane protein. Additionally, RYYN peptides binding to the Cx43 carboxyl-terminus have been described. In this review, we survey preclinical and clinical data available on short mimetic peptides based on, or directly targeting, Cx43, with focus on their potential for treating heart disease. We also discuss problems that have caused reluctance within the pharmaceutical industry to translate peptidic therapeutics to the clinic, even when supporting preclinical data is strong. These issues include those associated with the administration, stability in vivo, and tissue penetration of peptide-based therapeutics. Finally, we discuss novel drug delivery technologies including nanoparticles, exosomes, and other nanovesicular carriers that could transform the clinical and commercial viability of Cx43-targeting peptides in treatment of heart disease, stroke, cancer, and other indications requiring oral or parenteral administration. Some of these newly emerging approaches to drug delivery may provide a path to overcoming pitfalls associated with the drugging of peptide therapeutics.

Connexins as therapeutic targets in neurological and neuropsychiatric disorders

Astrocytes represent the reticular part of the central nervous system; gap junctions formed by connexins Cx43, Cx30- and Cx26 provide for homocellular astrocyte-astrocyte coupling, whereas connexins Cx30, Cx32, Cx43, and Cx47 connect astrocytes and oligodendrocytes. Astroglial networks are anatomically and functionally segregated being homologous to neuronal ensembles. Connexons, gap junctions and hemichannels (unpaired connexons) are affected in various neuropathologies from neuropsychiatric to neurodegenerative diseases. Manipulation of astrocytic connexins modulates the size and outreach of astroglial syncytia thus affecting astroglial homeostatic support. Modulation of astrocytic connexin significantly modifies pharmacological profile of many CNS drugs, which represents an innovative therapeutic approach for CNS disorders; this approach is now actively tested in pre-clinical and clinical studies. Wide combination of connexin modulators with CNS drugs open new promising perspectives for fundamental studies and therapeutic strategies.

Blockade of Hemichannels Normalizes the Differentiation Fate of Myoblasts and Features of Skeletal Muscles from Dysferlin-Deficient Mice.

Dysferlinopathies are muscle dystrophies caused by mutations in the gene encoding dysferlin, a relevant protein for membrane repair and trafficking. These diseases are untreatable, possibly due to the poor knowledge of relevant molecular targets. Previously, we have shown that human myofibers from patient biopsies as well as myotubes derived from immortalized human myoblasts carrying a mutated form of dysferlin express connexin proteins, but their relevance in myoblasts fate and function remained unknown. In the present work, we found that numerous myoblasts bearing a mutated dysferlin when induced to acquire myogenic commitment express PPARγ, revealing adipogenic instead of myogenic commitment. These cell cultures presented many mononucleated cells with fat accumulation and within 48 h of differentiation formed fewer multinucleated cells. In contrast, dysferlin deficient myoblasts treated with boldine, a connexin hemichannels blocker, neither expressed PPARγ, nor accumulated fat and formed similar amount of multinucleated cells as wild type precursor cells. We recently demonstrated that myofibers of skeletal muscles from blAJ mice (an animal model of dysferlinopathies) express three connexins (Cx39, Cx43, and Cx45) that form functional hemichannels (HCs) in the sarcolemma. In symptomatic blAJ mice, we now show that eight-week treatment with a daily dose of boldine showed a progressive recovery of motor activity reaching normality. At the end of this treatment, skeletal muscles were comparable to those of wild type mice and presented normal CK activity in serum. Myofibers of boldine-treated blAJ mice also showed strong dysferlin-like immunoreactivity. These findings reveal that muscle dysfunction results from a pathophysiologic mechanism triggered by mutated dysferlin and downstream connexin hemichannels expressed de novo lead to a drastic reduction of myogenesis and favor muscle damage. Thus, boldine could represent a therapeutic opportunity to treat dysfernilopathies.

Uncoupling of the Astrocyte Syncytium Differentially Affects AQP4 Isoforms.

The water channel protein aquaporin-4 (AQP4) and the gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) are astrocytic proteins critically involved in brain water and ion homeostasis. While AQP4 is mainly involved in water flux across the astrocytic endfeet membranes, astrocytic gap junctions provide syncytial coupling allowing intercellular exchange of water, ions, and other molecules. We have previously shown that mice with targeted deletion of Aqp4 display enhanced gap junctional coupling between astrocytes. Here, we investigate whether uncoupling of the astrocytic syncytium by deletion of the astrocytic connexins Cx43 and Cx30 affects AQP4 membrane localization and expression. By using quantitative immunogold cytochemistry, we show that deletion of astrocytic connexins leads to a substantial reduction of perivascular AQP4, concomitant with a down-regulation of total AQP4 protein and mRNA. Isoform expression analysis shows that while the level of the predominant AQP4 M23 isoform is reduced in Cx43/Cx30 double deficient hippocampal astrocytes, the levels of M1, and the alternative translation AQP4ex isoform protein levels are increased. These findings reveal a complex interdependence between AQP4 and connexins, which are both significantly involved in homeostatic functions and astrogliopathologies.

Rat caval vein myocardium undergoes changes in conduction characteristics during postnatal ontogenesis.

The electrophysiological properties of the superior vena cava (SVC) myocardium, which is considered a minor source of atrial arrhythmias, were studied in this study during postnatal development. Conduction properties were investigated in spontaneously active and electrically paced SVC preparations obtained from 7-60-day-old male Wistar rats using optical mapping and microelectrode techniques. The presence of high-conductance connexin 43 (Cx43) was evaluated in SVC cross-sections using immunofluorescence. It was found that SVC myocardium is excitable, electrically coupled with the atrial tissue, and conducts excitation waves at all stages of postnatal development. However, the conduction velocity (CV) of excitation and action potential (AP) upstroke velocity in SVC were significantly lower in neonatal than in adult animals and increased with postnatal maturation. Connexins Cx43 were identified in both neonatal and adult rat SVC myocardium; however, the abundance of Cx43 was significantly less in neonates. The gap junction uncoupler octanol affected conduction more profound in the neonatal than in adult SVC. We demonstrated for the first time that the conduction characteristics of SVC myocardium change from a slow-conduction (nodal) to a high-conduction (working) phenotype during postnatal ontogenesis. An age-related CV increase may occur due to changes of AP characteristics, electrical coupling, and Cx43 presence in SVC cardiomyocyte membranes. Observed changes may contribute to the low proarrhythmicity of adult caval vein cardiac tissue, while pre- or postnatal developmental abnormalities that delay the establishment of the working conduction phenotype may facilitate SVC proarrhythmia.

The liver injury following ischemia and reperfusion is worse in experimental knockout heterozygote mouse model for expression of connexin 431.

Purpose:To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury.Methods:We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology.Results:The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group.Conclusions:This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.

Connexin30 and Connexin43 show a time-of-day dependent expression in the mouse suprachiasmatic nucleus and modulate rhythmic locomotor activity in the context of chronodisruption

BackgroundThe astroglial connexins Cx30 and Cx43 contribute to many important CNS functions including cognitive behaviour, motoric capacity and regulation of the sleep-wake cycle. The sleep wake cycle, is controlled by the circadian system. The central circadian rhythm generator resides in the suprachiasmatic nucleus (SCN). SCN neurons are tightly coupled in order to generate a coherent circadian rhythm. The SCN receives excitatory glutamatergic input from the retina which mediates entrainment of the circadian system to the environmental light-dark cycle. Connexins play an important role in electric coupling of SCN neurons and astrocytic-neuronal signalling that regulates rhythmic SCN neuronal activity. However, little is known about the regulation of Cx30 and Cx43 expression in the SCN, and the role of these connexins in light entrainment of the circadian system and in circadian rhythm generation.MethodsWe analysed time-of-day dependent as well as circadian expression of Cx30 and Cx43 mRNA and protein in the mouse SCN by means of qPCR and immunohistochemistry. Moreover, we analysed rhythmic spontaneous locomotor activity in mice with a targeted deletion of Cx30 and astrocyte specific deletion of Cx43 (DKO) in different light regimes by means of on-cage infrared detectors.ResultsFluctuation of Cx30 protein expression is strongly dependent on the light-dark cycle whereas fluctuation of Cx43 protein expression persisted in constant darkness. DKO mice entrained to the light-dark cycle. However, re-entrainment after a phase delay was slightly impaired in DKO mice. Surprisingly, DKO mice were more resilient to chronodisruption.ConclusionCircadian fluctuation of Cx30 and Cx43 protein expression in the SCN is differently regulated. Cx30 and astroglial Cx43 play a role in rhythm stability and re-entrainment under challenging conditions.

Dysfunction of astrocytic connexins 30 and 43 in the medial prefrontal cortex and hippocampus mediates depressive-like behaviours

Astrocytic connexin dysfunction is closely associated with synaptic impairment and contributes to the pathological development of depressive-like behaviours. However, little is known about the expression of connexins in astrocytes from different brain regions, or how tissue specific connexin expression affects local neuronal activity. Here, we established a mouse model of chronic social defeated stress (CSDS), from which we isolated astrocytes from the medial prefrontal cortex (mPFC), hippocampus, amygdala, and ventral tegmental area (VTA). Expression profiling was then performed for connexins Cx26, Cx30, and Cx43. Expression of Cx30 and Cx43 was significantly decreased in mPFC and hippocampus of CSDS mice and was strongly associated with decreases in neuronal activity. Furthermore, overexpression of Cx30 and Cx43 in the mPFC and hippocampus increased neuronal activity and inhibited depressive-like behaviours; while suppression of Cx30 and Cx43 in normal mice was sufficient to reduce neuronal activity and induced depressive-like behaviours. Taken togetner, aberrant expression of astrocytic Cx30 and Cx43 in the mPFC and hippocampus significantly affects brian region-specific neuronal activity and drives depressive-like behaviours. These observations provide novel insights into the role of astrocyte gene expression in stress-induced depressive-like behaviours.

Modulation of gap junctional intercellular communication in HaCaT cells by xenobiotics

Gap junctions are clusters of transmembrane proteins which enable direct communication between adjacent cells. Gap junctions allow intercellular diffusion of small molecules like metabolites (such as glucose), second messengers (such as cAMP, ATP) and ions (such as Mg2+, K+). That process, called gap junctional intercellular communication (GJIC), allows coordination of many essential functions in multicellular organisms, such as directed cell differentiation, cell growth, cellular and tissue homeostasis. Moreover, dysfunctional GJIC has been linked to a variety of human diseases.The final aim of this project is to elucidate whether estrogenic acting chemicals play a role in GJIC. As a first step, two industrial chemicals, bisphenol A (BPA) and 4‐nonylphenol (4‐NP), were selected and assessed as modulators of GJIC in the human epidermal keratinocyte line HaCaT.To study GJIC of HaCaT cells, the scrape loading/dye transfer (SL/DT) assay was applied to HaCaT cultures grown at standard conditions with serial dilution treatments of either BPA or 4‐NP versus ‐a vehicle treatment (&lt; 1% ethanol) for 24 hours. The SL/DT assay entails cutting a cell monolayer with a scalpel to allow for the incorporation of a fluorescent dye (Lucifer Yellow) into the ‘scraped’ cells. The dye transfer via opened gap junctions into adjacent cells is then investigated by fluorescence microscopy and image analysis. The number of Lucifer Yellow positive cells is a quantitative descriptor of GJIC activity in treated cells versus untreated cells. In addition to the results provided by the SL/DT functional assay, quantitative PCR was performed in parallel to assess the mRNA levels for GJIC protein connexins (Cx26, Cx36, and Cx43).The results of the SL/DT assays showed that both BPA and 4‐NP reduce GJIC in the human epidermal keratinocyte line in a dose range of 10 down to 0.1 μM. Currently qPCR experiments are being conducted to elucidate whether this is due to closed gap junctions or to modulation in expression of connexin encoding genes.With this research, these properties alongside qPCR may be relevant to answer the question of whether the observed modulation of GJIC in skin cells is due to an estrogenic activity. Furthermore, a better understanding of GJIC modulation may clarify the role of GJIC in disease and make it a future target for pharmacological, and particularly, dermatopharmacological approaches.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of Dedifferentiation of Adult Human Retinal Pigment Epithelial Cells in vitro. Morphological and Molecular Genetic Analysis

Changes in morphology and molecular genetic profile of human retinal pigment epithelial (RPE) cells in vitro were studied when exposed to the basic fibroblast growth factor (bFGF). The cell morphology was estimated by cell area and perimeter, spreading and polarization coefficients. It was demonstrated that the number of elongated (fibroblast-like) cells and the number of cells, which size was less than in the control increased in 48 h after adding the factor to the culture. At the same time, the cell proliferative activity decreased (according to MTT test). Immunocytochemical analysis demonstrated a decrease in the staining for connexin Cx43 and an increase in the intensity of staining for protein the Otx2 neuroepithelium protein. Simultaneously, the number of nestin-positive cells and βIII-tubulin positive cells increased. Using quantitative real-time PCR method, an increase in mRNA expression of the KLF4, OCT4, NANOG, OTX2, and NES and decrease in mRNA expression of the MITF and KRT18 were detected in RPE cells treated with bFGF; this indicates enhancement of cell dedifferentiation. These data are confirmed by a decrease in the COL1A1 mRNA expression indicating a decrease in synthetic cell activity. The results indicate that single (short-time) effect by bFGF is sufficient to activate the mechanism, which decreases the cell differentiation level toward neuroepithelium.

Heterotypic docking compatibility of human connexin37 with other vascular connexins

Human vascular connexins (Cx37, Cx40, Cx43, and Cx45) can form various types of gap junction channels to synchronize vasodilation/constriction to control local circulation. Most of our knowledge on heterotypic gap junctions of these vascular connexins was from studies on rodent connexins. In human vasculature, the same four homolog connexins exist, but whether these human connexins can form heterotypic GJs as those of rodents have not been fully studied. Here we used in vitro expression system to study the coupling status and GJ channel properties of human heterotypic Cx37/Cx40, Cx37/Cx43, and Cx37/Cx45 GJs. Our results showed that Cx37/Cx43 and Cx37/Cx45 GJs, but not Cx37/Cx40 GJs, were functional and each with unique rectifying channel properties. The failure of docking between Cx37 and Cx40 could be rescued by designed Cx40 variants. Characterization of the heterotypic Cx37/Cx43 and Cx37/Cx45 GJs may help us in understanding the intercellular communication at the myoendothelial junction.