Search

This study aimed to evaluate if Simvastatin can reduce, and/or prevent, Doxorubicin (Doxo)-induced cardiotoxicity. H9c2 cells were treated with Simvastatin (10 µM) for 4 h and then Doxo (1 µM) was added, and the effects on oxidative stress, calcium homeostasis, and apoptosis were evaluated after 20 h. Furthermore, we evaluated the effects of Simvastatin and Doxo co-treatment on Connexin 43 (Cx43) expression and localization, since this transmembrane protein forming gap junctions is widely involved in cardioprotection. Cytofluorimetric analysis showed that Simvastatin co-treatment significantly reduced Doxo-induced cytosolic and mitochondrial ROS overproduction, apoptosis, and cytochrome c release. Spectrofluorimetric analysis performed by means of Fura2 showed that Simvastatin co-treatment reduced calcium levels stored in mitochondria and restored cytosolic calcium storage. Western blot, immunofluorescence, and cytofluorimetric analyses showed that Simvastatin co-treatment significantly reduced Doxo-induced mitochondrial Cx43 over-expression and significantly increased the membrane levels of Cx43 phosphorylated on Ser368. We hypothesized that the reduced expression of mitochondrial Cx43 could justify the reduced levels of calcium stored in mitochondria and the consequent induction of apoptosis observed in Simvastatin co-treated cells. Moreover, the increased membrane levels of Cx43 phosphorylated on Ser368, which is responsible for the closed conformational state of the gap junction, let us to hypothesize that Simvastatin leads to cell-to-cell communication interruption to block the propagation of Doxo-induced harmful stimuli. Based on these results, we can conclude that Simvastatin could be a good adjuvant in Doxo anticancer therapy. Indeed, we confirmed its antioxidant and antiapoptotic activity, and, above all, we highlighted that Simvastatin interferes with expression and cellular localization of Cx43 that is widely involved in cardioprotection. [ABSTRACT FROM AUTHOR]

Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43−/−), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43−/− mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy. [ABSTRACT FROM AUTHOR]

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43−/−) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/− mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/− mice (cCx43−/− mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2′,7′-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43−/− mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43−/− mice. In cCx43−/− mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43−/− mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43−/− mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts. [ABSTRACT FROM AUTHOR]

Decreased sperm quality is the main cause of male infertility. Studies have found that prenatal dexamethasone exposure (PDE) decreases sperm quality in male offspring after birth, but the mechanism is unclear. Wistar pregnant rats were subcutaneously injected with 0.1, 0.2, and 0.4 mg/kg.d dexamethasone at gestational days 9–20. The testes and sperm of first-generation (F1) offspring were collected, and F1 offspring were mated with wild-type female rats to obtain F2. Compared with the control group, F1 offspring in PDE group had lower sperm count and motility after birth, and the deformity rate increased. F2 fetal rats' body length and weight decreased, and the intrauterine growth retardation rate increased. Meanwhile, PDE decreased the expression of connexin 43 (CX43) in offspring testes, whereas T-box transcription factor 2 (TBX2) promoter region histone 3 lysine 9 acetylation (H3K9ac) level and its expression were increased. Traced back to F1 fetus testes, PDE increased the expression of glucocorticoid receptor (GR) and P300, activated GR protein into the nucleus, and made GR act on the TBX2 promoter region. Further, a series of Sertoli cell interventions confirmed that dexamethasone promoted GR to recruit P300, increased the H3K9ac level of TBX2 promoter region and its expression, and inhibited the expression of CX43. This study confirmed that PDE decreased sperm quality of male offspring, which is related to the epigenetic programming of TBX2/CX43 in the Sertoli cells, provided a theoretical and experimental basis for guiding the rational use of drugs during pregnancy. [ABSTRACT FROM AUTHOR]

Early life stress such as maternal separation (MS), is a major risk factor for developing psychiatric disorders in adulthood. Connexin 43 (CX43), the main type of connexins expressed in astrocytes, has been indicated to participate in depression disorders. Nevertheless, the role of CX43 in MS-induced cognitive impairment and astrocyte dysfunction is unclear. Neonatal C57BL/6 mice were exposed to MS to mimic early life stress. Adeno-associated virus carrying CX43 was inoculated into mice for CX43 overexpression. Sucrose preference test, forced swim test and Morris water maze were performed for evaluating depression-like behaviors and spatial learning and memory of mice in adulthood. Real time quantitative polymerase chain reaction was conducted to detect CX43 mRNA expression in mouse brain. Immunofluorescence staining and western blotting were used for measuring expression levels of astrocytic markers in murine hippocampal dentate gyrus. The results showed that overexpressing CX43 attenuated MS exposure-induced depression-like behaviors and decrease in spatial learning and memory in mice. Upregulating CX43 alleviated MS exposure-induced downregulation of astrocytic markers. Collectively, CX43 overexpression attenuates cognitive deficits and astrocyte dysfunction in mice exposed to MS. [ABSTRACT FROM AUTHOR]

Astrocytes are a major class of glial cell in the central nervous system that have a diverse range of types and functions thought to be based on their anatomical location, morphology and cellular properties. Recent studies highlight that astrocyte dysfunction contributes to the pathogenesis of neurological conditions. However, few studies have described the pattern, distribution and density of astrocytes in the adult human cortex. This study mapped the distribution and density of astrocytes immunolabelled with a range of cytoskeletal and membrane markers in the human frontal cortex. Distinct and overlapping astrocyte populations were determined. The frontal cortex from ten normal control cases (75 ± 9 years) was immunostained with glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase-1 L1 (ALDH1L1), connexin-43 (Cx43), aquaporin-4 (AQP4), and glutamate transporter 1 (GLT-1). All markers labelled populations of astrocytes in the grey and white matter, separate cortical layers, subpial and perivascular regions. All markers were informative for labelling different cellular properties and cellular compartments of astrocytes. ALDH1L1 labelled the largest population of astrocytes, and Cx43-immunopositive astrocytes were found in all cortical layers. AQP4 and GLT-1 labelled distal astrocytic process and end-feet in the same population of astrocytes (98% of GLT-1-immunopositive astrocytes contained AQP4). In contrast, GFAP, the most widely used marker, predominantly labelled astrocytes in superficial cortical layers. This study highlights the diversity of astrocytes in the human cortex, providing a reference map of the distribution of distinct and overlapping astrocyte populations which can be used for comparative purposes in various disease, inflammatory and injury states involving astrocytes. [ABSTRACT FROM AUTHOR]

Synergistic effect of mesenchymal stem cells (MSCs) and graphene monolayer is still a less discussed topic but crucial in the context of creating a graphene skin dressing. Moreover, capturing early changes in the cytoskeleton may prove to be a valuable indicator of cell condition after direct contact with the biomaterial. In this context, here, we investigated the response of human bone marrow mesenchymal stem cells (hBM-MSCs) to graphene monolayer as a scaffold, based on cytoskeleton architecture. Three main components of the cytoskeleton — microfilaments, intermediate filaments and microtubules were characterized as well as analyzed its entire cell morphology (focal adhesions). Based on microscopic analysis and cell area measurements, it can be stated that graphene did not cause a damage or disturbance of any cytoskeleton members and linker proteins (plectin, vinculin) and gap junction protein (connexin 43). hBM-MSCs growing on the graphene monolayer were more spread than cells growing on the glass but without any other significant difference. This suggested that graphene substrate as a monolayer could be a structural reinforcement for MSCs cultivation without affecting their cytoskeleton, which determines cell integrity, connections and migration, all important cell properties in the process of skin wound healing. [ABSTRACT FROM AUTHOR]

Glucocorticoids induced osteonecrosis of the femoral head (GIONFH) is a devastating orthopedic disease. Previous studies suggested that connexin43 is involved in the process of osteogenesis and angiogenesis. However, the role of Cx43 potentiates in the osteogenesis and angiogenesis of bone marrow-derived stromal stem cells (BMSCs) in GIONFH is still not investigated. In this study, BMSCs were isolated and transfected with green fluorescent protein or the fusion gene encoding GFP and Cx43. The osteogenic differentiation of BMSCs were detected after transfected with Cx43. In addition, the migration abilities and angiogenesis of human umbilical vein endothelial cells (HUVECs) were been detected after induced by transfected BMSCs supernatants in vitro. Finally, we established GC-ONFH rat model, then, a certain amount of transfected or controlled BMSCs were injected into the tibia of the rats. Immunohistological staining and micro-CT scanning results showed that the transplanted experiment group had significantly promoted more bone regeneration and vessel volume when compared with the effects of the negative or control groups. This study demonstrated for the first time that the Cx43 overexpression in BMSCs could promote bone regeneration as seen in the osteogenesis and angiogenesis process, suggesting that Cx43 may serve as a therapeutic gene target for GIONFH treatment. [Display omitted] • Cx43 overexpression in BMSCs promotes angiogenesis. • Osteogenic-associated proteins were up-regulated after Cx43 overexpression in BMSCs. • Cx43 overexpression in BMSCs accelerates osteogenesis in a GIONFH rat model. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three‐dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM. [ABSTRACT FROM AUTHOR]

2,3,5,6-Tetramethylpyrazine (TMP) as an active ingredient extracted from a traditional Chinese herbal medicine Ligusticum chuanxiong Hort. has been proved to penetrate blood-brain barrier (BBB) and show neuroprotective effects on cerebral ischemia. However, whether TMP could regulate astrocytic reactivity to facilitate neurovascular restoration in the subacute ischemic stroke needs to be urgently verified. In this research, permanent occlusion of the middle cerebral artery (MCAO) model was conducted and TMP (10, 20, 40 mg/kg) was intraperitoneally administrated to rats once daily for 2 weeks. Neurological function was evaluated by motor deficit score (MDS). Magnetic resonance imaging (MRI) was implemented to analyze tissue injury and cerebral blood flow (CBF). Magnetic resonance angiography (MRA) was applied to exhibit vascular signals. Transmission electron microscopy (TEM) was performed to detect the neurovascular unit (NVU) ultrastructure. Haematoxylin and eosin (HE) staining was utilized to evaluate cerebral histopathological lesions. The neurogenesis, angiogenesis, A1/A2 reactivity, aquaporin 4 (AQP4) and connexin 43 (Cx43) of astrocytes were observed with immunofluorescent staining. Then FGF2/PI3K/AKT signals were measured by western blot. Findings revealed TMP ameliorated neurological functional recovery, preserved NVU integrity, and enhanced endogenous neurogenesis and angiogenesis of rats with subacute ischemia. Shifting A1 to A2 reactivity, suppressing excessive AQP4 and Cx43 expression of astrocytes, and activating FGF2/PI3K/AKT pathway might be potential mechanisms of promoting neurovascular restoration with TMP after ischemic stroke. [ABSTRACT FROM AUTHOR]

BACKGROUND: Connexin 43 is also known as Gja1 protein. Gja1 recombinant lentivirus may improve bladder contractile dysfunction in diabetes. OBJECTIVE: To establish a guinea pig model of diabetic cystopathy, study the way of transurethral infusion of Gja1 recombinant lentiviral drugs, and observe the effect on the expression of connexin 43 protein and mRNA on the surface of detrusor muscle cells in the damaged bladder. METHODS: Eighty healthy guinea pigs of similar age and body mass were selected, 15 of which were randomly selected and conventionally fed and the remaining 65 were given intraperitoneal injection of 1% streptozotocin (200 mg/kg) to establish a guinea pig model of diabetic cystopathy. Urodynamics screening was performed to identify guinea pigs with diabetic cystopathy, which were further randomized into three groups: blank group (n=12), control group (n=12) and experimental group (n=12). The blank group was perfused with 0.2 mL of phosphate buffered saline, the control group was perfused with 0.2 mL of empty recombinant lentivirus, and the experimental group was perfused with 0.2 mL of Gja1 recombinant lentivirus. Three guinea pigs from each group were killed at each observation time point (2, 7, 14, and 28 days) after transfection. Expression and distribution of connexin 43 protein in the bladder were observed by immunohistochemical staining, and the protein and mRNA levels of connexin 43 were detected by western blot and qRT-PCR assays, respectively, RESULTS AND CONCLUSION: After transurethral infusion of Gja1 recombinant lentivirus, the expression levels of connexin 43 protein and mRNA were significantly higher in the experimental group than the blank and control groups (P < 0.01, P < 0.01), and the best expression levels of connexin 43 protein and mRNA were found at 14 days after transfection with Gja1 recombinant lentivirus. There were no significant differences in the expression levels of connexin 43 protein and mRNA between the blank and control groups (P > 0.01). To conclude, Gja1 gene via transurethral perfusion can stably express in bladder tissue and up-regulate the expression of connexin 43 at protein and mRNA levels, which may be beneficial to repair damaged signal transduction and exchange pathways between cells. [ABSTRACT FROM AUTHOR]

The immunoglobulin-like cell adhesion molecule CLMP is a member of the CAR family of cell adhesion proteins and is implicated in human congenital short-bowel syndrome (CSBS). CSBS is a rare but very severe disease for which no cure is currently available. In this review, we compare data from human CSBS patients and a mouse knockout model. These data indicate that CSBS is characterized by a defect in intestinal elongation during embryonic development and impaired peristalsis. The latter is driven by uncoordinated calcium signaling via gap junctions, which is linked to a reduction in connexin43 and 45 levels in the circumferential smooth muscle layer of the intestine. Furthermore, we discuss how mutations in the CLMP gene affect other organs and tissues, including the ureter. Here, the absence of CLMP produces a severe bilateral hydronephrosis—also caused by a reduced level of connexin43 and associated uncoordinated calcium signaling via gap junctions. [ABSTRACT FROM AUTHOR]

Dysregulated connexin signaling is implicated in the pathophysiology of pulmonary artery hypertension (PAH). Nicotine affects pulmonary vascular remodeling. However, the potential mechanistic link between connexin signaling and nicotine-induced pulmonary artery remodeling remains unclear. We aimed to investigate the role of connexin 43 (Cx43) in pulmonary artery remodeling in nicotine-administered C57BL/6 J wild-type (WT) and Cx43 heterozygous (Cx43+/−) mice. Hemodynamic parameters and right ventricle pathology were assessed in the mice. Serum biochemical indices of hepatic and renal function were measured. The RT-PCR, immunofluorescence, and western blotting were conducted to evaluate Cx43 mRNA and protein levels. We performed histological staining to identify pulmonary arteries. Wire myography was used to examine contraction and relaxation responses in the pulmonary arteries. Pulmonary vascular permeability was assessed through Evans blue staining. Compared with the WT group, the Cx43+/- group showed lower Cx43 mRNA and protein expression in the pulmonary arteries (P < 0.01). Nicotine treatment significantly increased Cx43 expression (P < 0.01) and induced morphological changes in the pulmonary arteries (P < 0.01). Our findings suggest that Cx43 plays a crucial role in pulmonary artery reactivity and permeability in mice. Furthermore, downregulation of Cx43 expression may contribute to alterations in pulmonary artery structure and function. • Nicotine adversely affects blood lipids and liver and kidney function in mice. • Connexin43 (Cx43) plays a key role in pulmonary artery remodeling. • Cx43 modulates pulmonary artery reactivity and permeability in WT and Cx43+/- mice. • Cx43 affects pulmonary artery structure and function in nicotine-administered mice. [ABSTRACT FROM AUTHOR]

Background: To examine the levels of exosome-derived connexin 43 (Cx43) in plasma and estimate its forecast value in patients with melanoma. Methods: We measured the plasma exosome-derived Cx43 levels in the plasma of 112 melanoma patients and 50 healthy controls. Results: The plasma exosome-derived Cx43 levels in patients with melanoma were substantially downregulated as opposed to the levels in healthy controls (P < 0.001). Kaplan–Meier analysis indicated that overall survival (OS) and disease-free survival (DFS) were poorer in patients with melanoma who exhibited lower levels of plasma exosome-derived Cx43 (both P < 0.001). The levels of plasma exosome-derived Cx43 were considerably elevated in patients with melanoma whose tumor was situated in the skin, tumor size < 10 cm, with Clark level I–III, TNM stages IIb–IV, and had no lymph node metastasis as opposed to patients whose tumor was situated in the viscera or mucosa, tumor size ≥ 10 cm, Clark level IV–V, TNM stages IIb–IV and had lymph node metastasis (all P < 0.05). The receiver operating characteristic (ROC) of plasma exosome-derived Cx43 for forecasting 5-year DFS in patients with melanoma was 0.78 (95% confidence interval (CI): 0.70–0.86), with a specificity of 77.78% and a sensitivity of 81.55%. The ROC of plasma exosome-derived Cx43 for forecasting 5-year OS of patients with melanoma was 0.77 (95% CI: 0.68–0.84), with a specificity of 80.0% and sensitivity of 65.98%. Conclusion: The overall findings indicated that the levels of plasma exosome-derived Cx43 in patients with melanoma were considerably downregulated. It can therefore be inferred that the levels of plasma exosome-derived Cx43 might be a prospective prognostic indicator for 5 5-year OS and 5-year DFS of patients with melanoma. [ABSTRACT FROM AUTHOR]

Background: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. Methods: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), βIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. Results: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVβ3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. Conclusions: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells. [ABSTRACT FROM AUTHOR]

Background: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. Methods: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines' representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. Results: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. Conclusion: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression. [ABSTRACT FROM AUTHOR]

Simple Summary: Skeletal muscles need to be continually active. Physical inactivity, reduced gravity, such as when humans are in space, or aging itself can cause neuromuscular frailty and weakness, causing major diseases. Comprehension of the molecular mechanisms that cause skeletal muscle atrophy can be beneficial to limit such processes and improve human health in different conditions: allowing humans to go into space for a long period, to be inactive for a long period or to age in a better way. When skeletal muscles are chronically inactive, within 5 days and more after 10 days, there is an increase of two molecules, called agrin fragments and neurofilaments, which become highly present in the blood, suggesting neuromuscular instability. The process is accompanied by changes in the membranes of single muscle fibres, the unit that forms our skeletal muscles. They could potentially start to lose important electrolytes and molecules and gradually promote a deleterious process. It has been proved in mice that blocking the appearance of these molecules, called hemichannels, can limit the severity of the loss of muscle mass. Interestingly, these hemichannels also appear during inactivity in humans, opening a possible application for human health and space missions. Human skeletal muscle atrophy and a disproportionate force loss occur within a few days of unloading in space and on Earth, but the underlying mechanisms are not fully understood. Disruption of neuromuscular junction homeostasis has been proposed as one of the possible causes. Here, we investigated the potential mechanisms involved in this neuromuscular disruption induced by a 10-day unilateral lower limb suspension (ULLS) in humans. Specifically, we investigated hemichannels' upregulation, neuromuscular junction and axonal damage, neurotrophins' receptor downregulation and inflammatory transcriptional signatures. Biomarkers were evaluated at local and systemic levels. At the sarcolemmal level, changes were found to be associated with an increased expression of connexin 43 and pannexin-1. Upregulation of the inflammatory transcripts revealed by deep transcriptomics was found after 10 days of ULLS. The destabilisation of the neuromuscular junction was not accompanied by changes in the secretion of the brain-derived neurotrophic factor and neurotrophin-4, while their receptor, BDNF/NT growth factors receptor (TrkB), decreased. Furthermore, at 5 days of ULLS, there was already a significant upregulation of the serum neurofilament light chain concentration, an established clinical biomarker of axonal injury. At 10 days of ULLS, other biomarkers of early denervation processes appeared. Hence, short periods of muscle unloading induce sarcolemmal hemichannels upregulation, inflammatory transcripts upregulation, neuromuscular junction instability and axonal damage. [ABSTRACT FROM AUTHOR]

Background: The Purkinje fibers convey the electrical impulses at much higher speed than the working myocardial cells. Thus, the distribution of the Purkinje network is of paramount importance for the timing and coordination of ventricular activation. The Purkinje fibers are found in the subendocardium of all species of mammals, but some mammals also possess an intramural Purkinje fiber network that provides for relatively instantaneous, burst‐like activation of the entire ventricular wall, and gives rise to an rS configuration in lead II of the ECG. Aim: To relate the topography of the horse heart and the distribution and histology of the conduction system to the pattern of ventricular activation as a mechanism for the unique electrical axis of the equine heart. Methods: The morphology and distribution of the cardiac conduction system was determined by histochemistry. The electrical activity was measured using ECG in the Einthoven and orthogonal configuration. Results: The long axis of the equine heart is close to vertical. Outside the nodal regions the conduction system consisted of Purkinje fibers connected by connexin 43 and long, slender parallel running transitional cells. The Purkinje fiber network extended deep into the ventricular walls. ECGs recorded in an orthogonal configuration revealed a mean electrical axis pointing in a cranial‐to‐left direction indicating ventricular activation in an apex‐to‐base direction. Conclusion: The direction of the mean electrical axis in the equine heart is determined by the architecture of the intramural Purkinje network, rather than being a reflection of ventricular mass. [ABSTRACT FROM AUTHOR]

Introduction: Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the central nervous system. Dimethyl fumarate (DMF) and monomethyl fumarate (MMF) belong to the disease-modifying drugs in treatment of MS. There is evidence that astrocytes and microglia are involved in MS pathology, but few studies are available about MMF and DMF effects on astrocytes and microglia. The aim of this study was to investigate the effects of MMF and DMF on microglial activation and morphology as well as potential effects on glial viability, Cx43, and AQP4 expressions in different set-ups of an in vitro astrocyte-microglia co-culture model of inflammation. Methods: Primary rat glial co-cultures of astrocytes containing 5% (M5, mimicking "physiological" conditions) or 30% (M30, mimicking "pathological, inflammatory" conditions) of microglia were treated with different concentrations of MMF (0.1, 0.5, and 2 μg/mL) or DMF (1.5, 5, and 15 μM) for 24 h. Viability, proliferation, and cytotoxicity of glial cells were examined using MTT assay. Immunocytochemistry was performed to analyze the microglial phenotypes. Connexin 43 (Cx43) and aquaporin 4 (AQP4) expressions were quantified by immunoblot analysis. Results: Treatment with different concentrations of MMF or DMF for 24 h did not change the glial cell viability in M5 and M30 co-cultures. Microglial phenotypes were not altered by DMF under physiological M5 conditions, but treatment with higher concentration of DMF (15 μM) induced microglial activation under inflammatory M30 conditions. Incubation with different concentrations of MMF had no effects on microglial phenotypes. The Cx43 expression in M5 and M30 co-cultures was not changed significantly by immunoblot analysis after incubation with different concentrations of DMF or MMF for 24 h. The AQP4 expression was significantly increased in M5 co-cultures after incubation with 5 μm DMF. Under the other conditions, AQP4 expression was not affected by DMF or MMF. Discussion: In different set-ups of the astrocyte-microglia co-culture model of inflammation, MMF has not shown significant effects. DMF had only limited effects on microglia phenotypes and AQP4 expression. In summary, mechanisms of action of fumarates probably do not involve direct effects on microglia phenotypes as well as Cx43 and AQP4 expression. [ABSTRACT FROM AUTHOR]

Long cytoplasmic processes of osteocytes orchestrate bone activity by integration of biochemical and mechanical signals and regulate load-induced bone adaptation. Low-Intensity Pulsed Ultrasound (LIPUS) is a clinically used technique for fracture healing that delivers mechanical impulses to the damaged bone tissue in a non-invasive and non-ionizing manner. The mechanism of action of LIPUS is still controversially discussed in the scientific community. In this study, the effect of focused LIPUS (FLIPUS) on the survival of starved MLO-Y4 osteocytes was investigated in vitro. Osteocytes stimulated for 10 min with FLIPUS exhibited extended dendrites, which formed frequent connections to neighboring cells and spanned longer distances. The sonicated cells displayed thick actin bundles and experienced increase in expression of connexin 43 (Cx43) proteins, especially on their dendrites, and E11 glycoprotein, which is responsible for the elongation of cellular cytoplasmic processes. After stimulation, expression of cell growth and survival genes as well as genes related to cell–cell communication was augmented. In addition, cell viability was improved after the sonication, and a decrease in ATP release in the medium was observed. In summary, FLIPUS mitigated apoptosis of starved osteocytes, which is likely related to the formation of the extensive dendritic network that ensured cell survival. [ABSTRACT FROM AUTHOR]

Cell Tissue Res. 2016 Mar. 363 (3): 751 - 63 12 Sosinsky GE, Boassa D, Dermietzel R, Duffy HS, Laird DW, MacVicar B. Pannexin channels are not gap junction hemichannels. Signaling across membranes is vital for tissue and organ function throughout the body. Connexin hemichannels and gap junctions (magenta) as well as pannexin channels (dark blue) are localized to membranes of organelles and the plasma membrane. Gap junctions form when six-connexin oligomers, collectively known as a hemichannel or connexon, bind with another hemichannel on an adjacent cell to make a single nonselective pore that crosses both plasma membranes [[9]]. [Extracted from the article]

Connexin 43 (Cx43) is essential to the function of the vasculature. Cx43 proteins form gap junctions that allow for the exchange of ions and molecules between vascular cells to facilitate cell-to-cell signaling and coordinate vasomotor activity. Cx43 also has intracellular signaling functions that influence vascular cell proliferation and migration. Cx43 is expressed in all vascular cell types, although its expression and function vary by vessel size and location. This includes expression in vascular smooth muscle cells (vSMC), endothelial cells (EC), and pericytes. Cx43 is thought to coordinate homocellular signaling within EC and vSMC. Cx43 gap junctions also function as conduits between different cell types (heterocellular signaling), between EC and vSMC at the myoendothelial junction, and between pericyte and EC in capillaries. Alterations in Cx43 expression, localization, and post-translational modification have been identified in vascular disease states, including atherosclerosis, hypertension, and diabetes. In this review, we discuss the current understanding of Cx43 localization and function in healthy and diseased blood vessels across all vascular beds. [ABSTRACT FROM AUTHOR]

Gap junctions play a role in intercellular communication in the epithelium, muscle and nerve tissues, and many more tissues and organs. These linking regions and the proteins forming them are called connexins (Cx). Recent studies show that cellular homeostatic equilibrium can be influenced by connexins. In this study, the expression of Cx43 and Cx32 in basal cell carcinoma (BCC) and squamous cell carcinomas (SCC) is tried studied. A total of 45 cases, 23 male and 22 female, were included in the study. Twenty of the cases were SCC and twenty-five were BCC. Retrospective data from records were examined and specimens were investigated by immunohistochemistry staining. It was determined that the results of Cx 43 and Cx 32 staining changed according to the tumor type of cases (P = 0,020). It was determined that the results of Cx43 and Cx32 staining did not change according to the sex and age, BCC and SCC subtype, and location. Losses of Cx43 and Cx32 have been shown to be associated with tumor types. We believe that it can be used as a support marker of SCC and BCC differential diagnosis. [ABSTRACT FROM AUTHOR]

Background/Aim: Type 2 diabetes (T2D), characterized by hyperinsulinemia, protects motor neurons against amyotrophic lateral sclerosis (ALS). Type 1 diabetes and a total lack of insulin are associated with increased risk of ALS. Connexin 43 (Cx43), an astrocyte protein, operates as an open pore via which toxic substances from the astrocytes reach motor neurons. Materials and Methods: In the current study, we performed molecular docking of insulin with monomeric Cx31, monomeric Cx43, and hexameric Cx31 to assess whether insulin might affect the pore. Hexameric Cx31 and hexameric Cx43 are transmembrane hemichannels composed of 6 subunits; they bind together to form gap junction intercellular channels. We used the program AutoDock Vina Extended for the molecular docking study. Results: Cx31 shares amino acid and structural similarity to Cx43, and insulin docks to the same position at the N-terminal domain of monomeric Cx31 and monomeric Cx43. Insulin docks within the open hemichannel of hexameric Cx31, potentially blocking it. Molecular dynamics simulation shows that the block is highly stable and may be responsible for the protective effect of T2D on ALS. Conclusion: Insulin, especially intranasal insulin, might be a treatment for ALS. An insulin secretogogue such as oral sulfonylurea or meglitinide might also be of value. [ABSTRACT FROM AUTHOR]

Background: Connexin 43 (Cx43) has been closely linked to the occurrence and progression of breast cancer. Distant metastasis of breast cancer is aided by the epithelial-mesenchymal transition of circulating tumor cells (CTCs). However, the impact of Cx43 expression on CTCs and the extent of its role in the disease remain unclear. Methods: We determined CTCs in 156 patients, who had breast cancer with a disease course of two or more years. We also measured the expression of Cx43 in the CTCs. The CTCs were detected in the blood of 139 of these patients. These 139 patients were divided into two groups: the Cx43 group and the non-Cx43 group based on their Cx43 expression. Results: Overall, Cx43 expression was found in 83 of the 139 patients (59.7%, 83/139 cases). The two groups significantly differed in terms of the number of mixed biphenotypic type CTCs and the total number of CTCs (P

Gap junctions, which are mainly composed of connexin units, play an indispensable role in cell morphogenesis, proliferation, migration, adhesion and differentiation of osteoblast lineage cells, and thus mediate bone development, homeostasis and disease occurrence. Platelet-derived growth factor-AA (PDGF-AA) is proved to have a great influence on osteoblast cell lines and is widely applied in the field of bone defect and wound healing. However, the role of PDGF-AA on gap junction formation in the osteoblast lineage remains elusive. In the current study, we aimed to investigate the impact of PDGF-AA on gap junction formation and cell-to-cell communication in the osteoblast lineage and explore its underlying biomechanism. We first found that PDGF-AA promoted cell proliferation and thus increased gap junction formations in living primary osteoblasts and MC3T3-E1 cells through scrape loading and dye transfer (SL/DT) assay. We then confirmed that PDGF-AA enhanced gap junction formations through up-regulation of connexin 43 (Cx43). We next detected the activation of p-Akt signaling in primary osteoblasts and MC3T3-E1 cells that were induced by PDGF-AA. Through inhibitory experiments, we further confirmed that PDGF-AA-mediated gap junction formation occurred via the activation of PI3K/Akt signaling. Taking together, our results provided evidences that PDGF-AA promoted gap junction formation in the osteoblast lineage through p-Akt signaling, which helped to understand the role of PDGF-AA in bone regeneration and diseases.

In the field of human in vitro fertilization (IVF), selecting the best oocyte for freezing or embryo for transfer remains an important focus of clinical practice. Although several techniques are and have been used for this goal, results have generally not been favorable and/or are invasive such that damage to some embryos occurs, resulting in a reduced number of healthy births. Therefore, the search continues for non-invasive oocyte and embryo quality markers that signal the development of high-quality embryos. Multiple studies indicate the important positive effects of retinoic acid (RA) on oocyte maturation and function. We previously showed that a high follicular fluid (FF) RA concentration at the time of oocyte retrieval in IVF protocols was associated with oocytes, giving rise to the highest quality embryos, and that cumulus granulosa cells (CGCs) are the primary source of follicle RA synthesis. Data also demonstrated that connexin-43 (Cx43), the main connexin that forms gap junctions in CGCs, is regulated by RA and that RA induces a rapid increase in gap junction communication. Here, we hypothesize that CGC RA plays a causal role in oocyte competency through its action on Cx43 and, as such, may serve as a biomarker of oocyte competence. Multiple studies have demonstrated the requirement for Cx43 in CGCs for the normal progression of folliculogenesis, and that the increased expression of this connexin is linked to the improved developmental competence of the oocyte. The data have shown that RA can up-regulate gap junction intercellular communication (GJIC) in the cumulus–oocyte complex via a non-genomic mechanism that results in the dephosphorylation of Cx43 and enhanced GJIC. Recognizing the positive role played by gap junctions in CGCs in oocyte development and the regulation of Cx43 by RA, the findings have highlighted the possibility that CGC RA levels may serve as a non-invasive indicator for selecting high-quality oocytes for IVF procedures. In addition, the data suggest that the manipulation of Cx43 with retinoid compounds could provide new pharmacological approaches to improve IVF outcomes in cases of failed implantation, recurrent miscarriage, or in certain diseases that are characterized by reduced fecundity, such as endometriosis.

Loss of function of the Retinoblastoma gene (RB1) due to mutations is commonly seen in human osteosarcomas. One of the Rb1 gene functions is to facilitate cell fate from mesenchymal stem cells to osteoblasts and prevent adipocyte differentiations. In this study, we demonstrate that a stable reduction of Rb1 expression (RbKD) in murine osteoblasts causes them to express higher levels of PPAR-ɣ and other adipocyte-specific transcription factors while retaining high expression of osteoblast-specific transcription factors, Runx2/Cbfa1 and SP7/Osterix. Inhibition of gap junctional intercellular communication (GJIC) in osteoblasts is another mechanism that causes osteoblasts to transdifferentiate to adipocytes. We found that preosteoblasts exposed to osteoblast differentiating media (DP media) increased GJIC. RbKD cells showed reduced GJIC along with a reduction in expression of Cx43, the protein that mediates GJIC. Other membrane associated adhesion protein Cadherin 11 (Cad11) was also decreased. Since PPAR-ɣ is increased with Rb1 loss, we wondered if the reduction of this transcription factor would reverse the changes observed. Reduction of PPAR-ɣ in control osteoblasts slightly increased bone-specific expression and reduced adipocytic expression as expected along with an increase in Cad11 and Cx43 expression. GJIC remained high and was unaffected by a reduction in PPAR-ɣ in control cells. Knockdown of PPAR-ɣ in RbKD cells reduced adipocyte gene expression, while osteoblast-specific expression showed improvement. Cx43, Cad11 and GJIC remained unaffected by PPAR-ɣ reduction. Our observations suggest that increased PPAR-ɣ that happens with Rb1 loss only affects osteoblast-adipocyte-specific gene expression but does not completely reverse Cx43 gene expression or GJIC. Therefore, these effects may represent independent events triggered by Rb1loss and/or the differentiation process.

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has shown promise in preclinical models of myocardial infarction, but graft myocardium exhibits incomplete host–graft electromechanical integration and a propensity for pro-arrhythmic behavior. Perhaps contributing to this situation, hPSC-CM grafts show low expression of connexin 43 (Cx43), the major gap junction (GJ) protein, in ventricular myocardia. We hypothesized that Cx43 expression and function could be rescued by engineering Cx43 in hPSC-CMs with a series of phosphatase-resistant mutations at three casein kinase 1 phosphorylation sites (Cx43-S3E) that have been previously reported to stabilize Cx43 GJs and reduce arrhythmias in transgenic mice. However, contrary to our predictions, transgenic Cx43-S3E hPSC-CMs exhibited reduced Cx43 expression relative to wild-type cells, both at baseline and following ischemic challenge. Cx43-S3E hPSC-CMs showed correspondingly slower conduction velocities, increased automaticity, and differential expression of other connexin isoforms and various genes involved in cardiac excitation–contraction coupling. Cx43-S3E hPSC-CMs also had phosphorylation marks associated with Cx43 GJ internalization, a finding that may account for their impaired GJ localization. Taken collectively, our data indicate that the Cx43-S3E mutation behaves differently in hPSC-CMs than in adult mouse ventricular myocytes and that multiple biological factors likely need to be addressed synchronously to ensure proper Cx43 expression, localization, and function.

Multiple sclerosis (MS) is a neurodegenerative disease marked by chronic neuroinflammation thought to be mediated by the inflammasome pathway. Connexin 43 (Cx43) hemichannels contribute to the activation of the inflammasome through the release of adenosine triphosphate (ATP) inflammasome activation signals. The objective of the study was to evaluate if the Cx43 hemichannel blocker, tonabersat, is effective in modulating the inflammatory response and reducing disability in the myelin oligodendrocyte glycoprotein 35–55-induced experimental autoimmune encephalomyelitis (MOG35–55 EAE) model of MS. Here, we show that the Cx43 hemichannel blocking drug, tonabersat, significantly reduced expression of neuroinflammatory markers for microglial activation (ionized calcium-binding adapter molecule 1 (Iba1)) and astrogliosis (glial fibrillary acidic protein (GFAP)) while preserving myelin basic protein (MBP) expression levels in the corpus callosum, motor cortex, and striatum regions of the brain in MOG35–55 EAE mice. Reduced NOD-like receptor protein 3 (NLRP3) inflammasome complex assembly and Caspase-1 activation confirmed the drug’s mode of action. MOG35–55 EAE mice showed clinical signs of MS, but MOG35–55 EAE mice treated with tonabersat retained behavior closer to normal. These data suggest that clinical trial phase IIb-ready tonabersat may merit further investigation as a promising candidate for MS treatment.

Juvenile traumatic brain injury (jTBI) is the leading cause of death and disability for children and adolescents worldwide, but there are no pharmacological treatments available. Aquaporin 4 (AQP4), an astrocytic perivascular protein, is increased after jTBI, and inhibition of its expression with small interference RNA mitigates edema formation and reduces the number of reactive astrocytes after jTBI. Due to the physical proximity of AQP4 and gap junctions, coregulation of AQP4 and connexin 43 (Cx43) expressions, and the possibility of water diffusion via gap junctions, we decided to address the potential role of astrocytic gap junctions in jTBI pathophysiology. We evaluated the role of Cx43 in the spread of the secondary injuries via the astrocyte network, such as edema formation associated with blood–brain barrier dysfunctions, astrogliosis, and behavioral outcome. We observed that Cx43 was altered after jTBI with increased expression in the perilesional cortex and in the hippocampus at several days post injury. In a second set of experiments, cortical injection of small interference RNA against Cx43 decreased Cx43 protein expression, improved motor function recovery, and decreased astrogliosis but did not result in differences in edema formation as measured via T2-weighted imaging or diffusion-weighted imaging at 1 day or 3 days. Based on our findings, we can speculate that while decreasing Cx43 has beneficial roles, it likely does not contribute to the spread of edema early after jTBI.

This document is a corrigendum published in the journal Frontiers in Pharmacology. It corrects errors in a previously published article titled "Ginsenoside Rg1 ameliorates neuroinflammation via suppression of connexin43 ubiquitination to attenuate depression." The errors are related to Figure 7 and a paragraph in the Results section. The corrected information is provided, and the authors state that these corrections do not change the scientific conclusions of the article. The corrigendum also includes information about the authors, the journal's open access policy, and a note about the opinions expressed in the article. [Extracted from the article]

Collective cell migration mediates multiple tissue morphogenesis processes. Yet how multi-dimensional mesenchymal cell movements are coordinated remains mostly unknown. Here we report that coordinated mesenchymal cell migration during chicken feather elongation is accompanied by dynamic changes of bioelectric currents. Transcriptome profiling and functional assays implicate contributions from functional voltage-gated Ca2+ channels (VGCCs), Connexin-43 based gap junctions, and Ca2+ release activated Ca2+ (CRAC) channels. 4-Dimensional Ca2+ imaging reveals that the Sonic hedgehog-responsive mesenchymal cells display synchronized Ca2+ oscillations, which expand progressively in area during feather elongation. Inhibiting VGCCs, gap junctions, or Sonic hedgehog signaling alters the mesenchymal Ca2+ landscape, cell movement patterns and feather bud elongation. Ca2+ oscillations induced by cyclic activation of opto-cCRAC channels enhance feather bud elongation. Functional disruption experiments and promoter analysis implicate synergistic Hedgehog and WNT/β-Catenin signaling in activating Connexin-43 expression, establishing gap junction networks synchronizing the Ca2+ profile among cells, thereby coordinating cell movement patterns.

BackgroundGenome-wide association studies conducted on QRS duration, an electrocardiographic measurement associated with heart failure and sudden cardiac death, have led to novel biological insights into cardiac function. However, the variants identified fall predominantly in non-coding regions and their underlying mechanisms remain unclear.ResultsHere, we identify putative functional coding variation associated with changes in the QRS interval duration by combining Illumina HumanExome BeadChip genotype data from 77,898 participants of European ancestry and 7695 of African descent in our discovery cohort, followed by replication in 111,874 individuals of European ancestry from the UK Biobank and deCODE cohorts. We identify ten novel loci, seven within coding regions, including ADAMTS6, significantly associated with QRS duration in gene-based analyses. ADAMTS6 encodes a secreted metalloprotease of currently unknown function. In vitro validation analysis shows that the QRS-associated variants lead to impaired ADAMTS6 secretion and loss-of function analysis in mice demonstrates a previously unappreciated role for ADAMTS6 in connexin 43 gap junction expression, which is essential for myocardial conduction.ConclusionsOur approach identifies novel coding and non-coding variants underlying ventricular depolarization and provides a possible mechanism for the ADAMTS6-associated conduction changes.

Background: Connexin 43 (Cx43), the predominant gap junction protein in hearts, is modified by specific (de)phosphorylation events under physiological and pathological states to affect myocardium function and structure. Previously we found that deficiency in Cx43 S282 phosphorylation could impair intercellular communication and contribute to cardiomyocyte apoptosis by activating p38 mitogen-activated protein kinase (p38 MAPK)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway, which is involved in myocardium injury in ischemia/reperfusion (I/R) heart. In addition, mutant at Cx43 S282 substituted with alanine heterozygous mice (S282A+/−) exhibited different degrees of ventricular arrhythmias and only some underwent myocardium apoptosis. In this study, we aimed to investigate the role of Cx43 pS282 in different cardiac pathological phenotypes. Methods: We examined cardiac function, structure, and relevant protein expression in S282A+/− mice (aged 2, 10 and 30 weeks) by electrocardiograph, echocardiography, histological staining, and co-immunoprecipitation followed by Western blot. Intraperitoneal isoprenaline injection and I/R surgery were applied in S282A+/− mice as external stimulus. 2,3,5-triphenyltetrazolium chloride staining was used for myocardium infarction evaluation. Results: Adult S282A+/− mice (aged 10 and 30 weeks) still exhibited spontaneous arrhythmia. Unlike neonatal stage (aged around 2 weeks), no apoptosis-related manifestations and the activation of p38 MAPK-Fas-FADD apoptotic pathway were observed in adult S282A+/− hearts. S282A+/− neonatal mice with cardiomyocytes apoptosis exhibited more than 60% dephosphorylation at Cx43 S282 than WT mice, while less than 40% S282 dephosphorylation were found in adult S282A+/− mice. In addition, although S282A+/− mice displayed normal cardiac function, they were highly susceptible to isoproterenol-induced ECG alternans and prone to cardiac injury and deaths upon I/R attack. Conclusions: These results reinforce that Cx43 S282 dephosphorylation acts as a susceptibility factor in regulating cardiomyocyte survival and cardiac electrical homeostasis in basal conditions and contributes to myocardium injury in the setting of I/R. Cx43 S282 phosphorylation was competent to induce spontaneous arrhythmias, cardiomyocyte apoptosis and deaths based on the degree of S282 dephosphorylation.

Bone adapts to changes in the physical environment by modulating remodeling through bone resorption and formation to maintain optimal bone mass. As the most abundant connexin subtype in bone tissue, connexin 43 (Cx43)-forming hemichannels are highly responsive to mechanical stimulation by permitting the exchange of small molecules (

Major depressive disorder (MDD) is a leading chronic mental illness worldwide, characterized by anhedonia, pessimism and even suicidal thoughts. Connexin 43 (Cx43), mainly distributed in astrocytes of the brain, is by far the most widely and ubiquitously expressed connexin in almost all vital organs. Cx43 forms gap junction channels in the brain, which mediate energy exchange and effectively maintain physiological homeostasis. Increasing evidence suggests the crucial role of Cx43 in the pathogenesis of MDD. Neuroinflammation is one of the most common pathological features of the central nervous system dysfunctions. Inflammatory factors are abnormally elevated in patients with depression and are closely related to nearly all links of depression. After activating the inflammatory pathway in the brain, the release and uptake of glutamate and adenosine triphosphate, through Cx43 in the synaptic cleft, would be affected. In this review, we have summarized the association between Cx43 and neuroinflammation, the cornerstones linking inflammation and depression, and Cx43 abnormalities in depression. We also discuss the significant association of Cx43 in inflammation and depression, which will help to explore new antidepressant drug targets. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR), a microvascular complication of diabetes, is associated with pronounced inflammation arising from the activation of a nucleotide-binding and oligomerization domain-like receptor (NLR) protein 3 (NLRP3) inflammasome. Cell culture models have shown that a connexin43 hemichannel blocker can prevent inflammasome activation in DR. The aim of this study was to evaluate the ocular safety and efficacy of tonabersat, an orally bioavailable connexin43 hemichannel blocker, to protect against DR signs in an inflammatory non-obese diabetic (NOD) DR mouse model. For retina safety studies, tonabersat was applied to retinal pigment epithelial (ARPE-19) cells or given orally to control NOD mice in the absence of any other stimuli. For efficacy studies, either tonabersat or a vehicle was given orally to the inflammatory NOD mouse model two hours before an intravitreal injection of pro-inflammatory cytokines, interleukin-1 beta, and tumour necrosis factor-alpha. Fundus and optical coherence tomography images were acquired at the baseline as well as at 2- and 7-day timepoints to assess microvascular abnormalities and sub-retinal fluid accumulation. Retinal inflammation and inflammasome activation were also assessed using immunohistochemistry. Tonabersat did not have any effect on ARPE-19 cells or control NOD mouse retinas in the absence of other stimuli. However, the tonabersat treatment in the inflammatory NOD mice significantly reduced macrovascular abnormalities, hyperreflective foci, sub-retinal fluid accumulation, vascular leak, inflammation, and inflammasome activation. These findings suggest that tonabersat may be a safe and effective treatment for DR. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) is a highly dynamic structure controlling the proper functioning of heart muscle. ECM remodeling with enhanced collagen deposition due to hemodynamic overload impairs cardiomyocyte adhesion and electrical coupling that contributes to cardiac mechanical dysfunction and arrhythmias. We aimed to explore ECM and connexin-43 (Cx43) signaling pathways in hemodynamically overloaded rat heart as well as the possible implication of angiotensin (1-7) (Ang (1-7)) to prevent/attenuate adverse myocardial remodeling. Male 8-week-old, normotensive Hannover Spraque–Dawley rats (HSD), hypertensive (mRen-2)27 transgenic rats (TGR) and Ang (1-7) transgenic rats (TGR(A1-7)3292) underwent aortocaval fistula (ACF) to produce volume overload. Five weeks later, biometric and heart tissue analyses were performed. Cardiac hypertrophy in response to volume overload was significantly less pronounced in TGR(A1-7)3292 compared to HSD rats. Moreover, a marker of fibrosis hydroxyproline was increased in both ventricles of volume-overloaded TGR while it was reduced in the Ang (1-7) right heart ventricle. The protein level and activity of MMP-2 were reduced in both ventricles of volume-overloaded TGR/TGR(A1-7)3292 compared to HSD. SMAD2/3 protein levels were decreased in the right ventricle of TGR(A1-7)3292 compared to HSD/TGR in response to volume overload. In parallel, Cx43 and pCx43 implicated in electrical coupling were increased in TGR(A1-7)3292 versus HSD/TGR. It can be concluded that Ang (1-7) exhibits cardio-protective and anti-fibrotic potential in conditions of cardiac volume overload. [ABSTRACT FROM AUTHOR]

Injury to the central nervous system (CNS) provokes an inflammatory reaction and secondary damage that result in further tissue damage and destruction of neurons away from the injury site. Upon injury, expression of connexin 43 (Cx43), a gap junction protein, upregulates and is responsible for the spread and amplification of cell death signals through these gap junctions. In this study, we hypothesise that the downregulation of Cx43 by scaffold-mediated controlled delivery of antisense oligodeoxynucleotide (asODN), would minimise secondary injuries and cell death, and thereby support tissue regeneration after nerve injuries. Specifically, using spinal cord injury (SCI) as a proof-of-principle, we utilised a fibre-hydrogel scaffold for sustained delivery of Cx43asODN, while providing synergistic topographical cues to guide axonal ingrowth. Correspondingly, scaffolds loaded with Cx43asODN, in the presence of NT-3, suppressed Cx43 up-regulation after complete transection SCI in rats. These scaffolds facilitated the sustained release of Cx43asODN for up to 25 days. Importantly, asODN treatment preserved neurons around the injury site, promoted axonal extension, decreased glial scarring, and reduced microglial activation after SCI. Our results suggest that implantation of such scaffold-mediated asODN delivery platform could serve as an effective alternative SCI therapeutic approach. [ABSTRACT FROM AUTHOR]

Simple Summary: Connexins are proteins which comprise gap junctions in cells. These junctions can directly connect neighboring cells and the cell interior with the extracellular microenvironment and thus they act as tissue integrators. Alterations in connexin regulation can lead to unfavorable shifts in the tissue adhesive context thus eradicating the constraints of the normal tissue microenvironment, triggering (or enhancing) cell motility. This review tries to examine the role of connexins in orchestrating the tumor microenvironment and hence their role in malignancy. Today's research on the processes of carcinogenesis and the vital activity of tumor tissues implies more attention be paid to constituents of the tumor microenvironment and their interactions. These interactions between cells in the tumor microenvironment can be mediated via different types of protein junctions. Connexins are one of the major contributors to intercellular communication. They form the gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc., between neighboring tumor cells as well as between tumor and stromal cells. Connexin hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, connexins have been reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. The pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization, and functionality as well as their channel assembly and non-channel functions. In this review, we have summarized the data on the contribution of connexins to the formation of the tumor microenvironment and to cancer initiation and progression. [ABSTRACT FROM AUTHOR]

Organotypic heart slices from mice might provide a promising in vitro model for cardiac research because of the vast availability of genetically modified specimens, combined with the unrestricted feasibility of experimental interventions. However, murine heart slices undergo rapid degeneration in culture. Therefore, we developed optimal conditions to preserve their structure and function in culture. Mouse ventricular heart samples were transversely cut into 300 µm thick slices. Slices were then cultured under various conditions of diastolic preload, systolic compliance and medium agitation. Continuous stimulation was performed either by optical stimulation or by electrical field stimulation. Contractility was continuously measured, and cellular survival, structure and gene expression were analyzed. Significant improvements in viability and function were achieved by elastic fixation with the appropriate diastolic preload and the rapid shaking of a ß-mercaptoethanol-supplemented medium. At 1 Hz pacing, mouse heart slices maintained stable contractility for up to 48 h under optogenetic pacing and for one week under electrical pacing. In cultured slices, the native myofibril structure was well preserved, and the mRNAs of myosin light chain, titin and connexin 43 were constantly expressed. Conclusions: Adult murine heart slices can be preserved for one week and provide a new opportunity to study cardiac functions. [ABSTRACT FROM AUTHOR]

Gap-junction-forming connexins are exquisitely regulated by post-translational modifications (PTMs). In particular, the PTM of connexin 43 (Cx43), a tumor suppressor protein, regulates its turnover and activity. Here, we investigated the interaction of Cx43 with the ubiquitin-related modifier 1 (URM-1) protein and its impact on tumor progression in two breast cancer cell lines, highly metastatic triple-negative MDA-MB-231 and luminal breast cancer MCF-7 cell lines. To evaluate the subsequent modulation of Cx43 levels, URM-1 was downregulated in these cells. The transcriptional levels of epithelial-to-mesenchymal transition (EMT) markers and the metastatic phenotype were assessed. We demonstrated that Cx43 co-localizes and interacts with URM-1, and URMylated Cx43 was accentuated upon cellular stress. The significant upregulation of small ubiquitin-like modifier-1 (SUMO-1) was also observed. In cells with downregulated URM-1, Cx43 expression significantly decreased, and SUMOylation by SUMO-1 was affected. The concomitant expression of EMT markers increased, leading to increased proliferation, migration, and invasion potential. Inversely, the upregulation of URM-1 increased Cx43 expression and reversed EMT-induced processes, underpinning a role for this PTM in the observed phenotypes. This study proposes that the URMylation of Cx43 in breast cancer cells regulates its tumor suppression properties and contributes to breast cancer cell malignancy. [ABSTRACT FROM AUTHOR]

Mutations in the GJA1 gene that encodes connexin43 (Cx43) cause several rare genetic disorders, including diseases affecting the epidermis. Here, we examined the in vitro functional consequences of a Cx43 mutation, Cx43-G38E, linked to a novel human phenotype of hypotrichosis, follicular keratosis and hyperostosis. We found that Cx43-G38E was efficiently translated in Xenopus oocytes and localized to gap junction plaques in transfected HeLa cells. Cx43-G38E formed functional gap junction channels with the same efficiency as wild-type Cx43 in Xenopus oocytes, although voltage gating of the gap junction channels was altered. Notably, Cx43-G38E significantly increased membrane current flow through the formation of active hemichannels when compared to wild-type Cx43. These data demonstrate the association of increased hemichannel activity to a connexin mutation linked to a skeletal-cutaneous phenotype, suggesting that augmented hemichannel activity could play a role in skin and skeletal disorders caused by human Cx43 mutations. [ABSTRACT FROM AUTHOR]

Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Acupuncture & Tuina Science is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Gap junctional intercellular communication (GJIC) is considered a key biological mechanism to maintain homeostasis in cell differentiation and growth. In addition, as another major signaling pathway associated with cell proliferation and differentiation, Wnt/β-catenin signaling appears to trigger several cellular responses against injury. The purpose of the present study was to investigate the effects of a known toxic agent, benzo[a]pyrene (BaP), on the regulation and interaction between GJIC and Wnt/β-catenin signaling. BaP treatment resulted in GJIC inhibition and decreases the major GJIC protein connexin 43 (Cx43) in WB-F344 rat liver epithelial cells. We also found BaP-mediated downregulation of Wnt/β-catenin signaling related to the PI3K-Akt pathway. To identify the relationship between GJIC and Wnt/β-catenin signaling, we treated WB-F344 cells with the Wnt agonist CHIR99021 and found that it inhibited GJIC while causing a significant reduction in Cx43 expression at both the mRNA and protein levels, through the repression of promoter activity. This Wnt agonist-mediated GJIC inhibition was confirmed using a small interfering RNA directed against the Wnt antagonist Dact2, indicating that Wnt/β-catenin signaling negatively regulates GJIC. Despite the inverse correlation between Wnt/β-catenin signaling and Cx43 promoter activation as indicated by downregulation of β-catenin nuclear translocation and upregulation of Cx43 promoter activation involving HNF3β, BaP treatment decreased the Cx43 protein expression, which was associated with protein degradation, possibly through protein kinase C activation. In conclusion, our results revealed the mechanism of BaP-induced inhibition of GJIC and Wnt/β-catenin signaling. More importantly, linking Wnt/β-catenin signaling to Cx protein expression will have profound implications in understanding the relationships among different major signaling pathways associated with cell proliferation and differentiation in toxicity. [ABSTRACT FROM AUTHOR]

Gap junctions (GJs), which are composed of connexins (Cxs), provide channels for direct information exchange between cells. Cx expression has a strong spatial specificity; however, its influence on cell behavior and information exchange between cells cannot be ignored. A variety of factors in organisms can modulate Cxs and subsequently trigger a series of responses that have important effects on cellular behavior. The expression and function of Cxs and the number and function of GJs are in dynamic change. Cxs have been characterized as tumor suppressors in the past, but recent studies have highlighted the critical roles of Cxs and GJs in cancer pathogenesis. The complex mechanism underlying Cx and GJ involvement in cancer development is a major obstacle to the evolution of therapy targeting Cxs. In this paper, we review the post-translational modifications of Cxs, the interactions of Cxs with several chaperone proteins, and the effects of Cxs and GJs on cancer. EUAGovitD21XQu5xc5u6gf Video Abstract [ABSTRACT FROM AUTHOR]

Background: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvβ3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. Methods: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). Results: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. Conclusions: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis. [ABSTRACT FROM AUTHOR]

Neuropathic pain (NP) is a chronic disease caused by damage to the peripheral or central nervous system. Connexin 43 (Cx43), the primary connexin expressed by astrocytes, has been reported to be significantly increased in NP. However, the roles and mechanisms of Cx43 in the development and maintenance of NP remain largely unknown, while microglia activation has been commonly regarded as a key factor of NP. In the present study, we found that Cx43 deletion significantly ameliorated spared nerve injury (SNI)-induced NP and suppressed SNI induced c-Fos expression in the spinal cord. Notably, Cx43 deletion led to much less SNI-induced microglia activation in the spinal cord. These results suggest that astrocyte Cx43 may play a significant role in regulating microglial activation and NP. • Cx43 deletion ameliorated spared nerve injury (SNI)-induced neuropathic pain. • Cx43 deletion suppressed SNI-induced microglia activation in the spinal cord. • Cx43 deletion did not affect SNI-induced astrocyte activation and microglia recruitment. [ABSTRACT FROM AUTHOR]