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Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions., (Copyright © 2014. Published by Elsevier Masson SAS.)

Decellularized matrices are valuable scaffolds for engineering functional cardiac patches for treating myocardial infarction. However, the lack of quick and efficient electrical coupling between adjacent cells may jeopardize the success of the treatment. To address this issue, we have deposited gold nanoparticles on fibrous decellularized omental matrices and investigated their morphology, conductivity, and degradation. We have shown that cardiac cells engineered within the hybrid scaffolds exhibited elongated and aligned morphology, massive striation, and organized connexin 43 electrical coupling proteins. Finally, we have shown that the hybrid patches demonstrated superior function as compared to pristine patches, including a stronger contraction force, lower excitation threshold, and faster calcium transients.

Granulosa cells (GCs) play an important role during follicle growth and development in preovulatory stage. Moreover, the proteins such as connexins are responsible for formation of protein channel between follicular-cumulus cells and oocyte. This study was aimed to investigate the role of connexin expression in porcine GCs in relation to their cellular distribution and real-time cell proliferation. In the present study, porcine GCs were isolated from the follicles of puberal gilts and then cultured in a real-time cellular analyzer (RTCA) system for 168 h. The expression levels of connexins (Cxs) Cx36, Cx37, Cx40 and Cx43 mRNA were measured by RQ-PCR analysis, and differences in the expression and distribution of Cx30, Cx31, Cx37, Cx43 and Cx45 proteins were analyzed by confocal microscopic visualization. We found higher level of Cx36, Cx37, and Cx43 mRNA expression in GCs at recovery (at 0 h of in vitro culture, IVC) compared to all analyzed time periods of IVC (24, 48, 72, 96, 120, 144 and 168 h; P<0.001). On the other hand, the expression level of Cx40 transcripts was higher after 24 h of IVC compared to 0 h and the other times of IVC (P<0.001). Similarly to mRNAs, the expression levels of Cx31, Cx37 and Cx45 proteins were higher before (0 h) compared to after 168 h of IVC. The expression of Cx30 and Cx43, however, did not vary between the groups. In all, the proteins were distributed throughout the cell membrane rather than in the cytoplasm both before and after IVC. After 24 h of IVC, we observed a significant increase in the proliferation of GCs (log phase). We found differences in the proliferation index between 72-96 and 96- 140 h within the same population of GCs. In conclusion, the decrease in the expression of Cx mRNAs and proteins following IVC could be associated with a breakdown in gap-junction connections (GJCs), and leads to the decreased of their activity, which may be a reason of non-functional existence of connexon in follicular granulosa cells. These data indicated that the differentiation and proliferation of GCs and lutein cells are regulated by distinct mechanisms in pigs.

Excessive recruitment of monocytes and progression of fibrosis are hallmarks of chronic kidney disease (CKD). Recently we reported that the expression of connexin 43 (Cx43) was upregulated in the kidney during experimental nephropathy. To investigate the role of Cx43 in the progression of CKD, we interbred RenTg mice, a genetic model of hypertension-induced CKD, with Cx43+/- mice. The renal cortex of 5-month-old RenTgCx43+/- mice showed a marked decrease of cell adhesion markers leading to reduced monocyte infiltration and interstitial renal fibrosis compared with their littermates. In addition, functional and histological parameters such as albuminuria and glomerulosclerosis were ameliorated in RenTgCx43+/- mice. Interestingly, treatment with Cx43 antisense produced remarkable improvement of renal function and structure in 1-year-old RenTg mice. Similar results were found in Cx43+/- or wild-type mice treated with Cx43 antisense after obstructive nephropathy. Furthermore, in these mice, Cx43 antisense attenuated E-cadherin downregulation and phosphorylation of the transcription factor Sp1 by the ERK pathway resulting in decreased transcription of type I collagen gene. Interestingly, Cx43-specific blocking peptide inhibited monocyte adhesion in activated endothelium and profibrotic pathways in tubular cells. Cx43 was highly increased in biopsies of patients with CKD. Thus, Cx43 may represent a new therapeutic target against the progression of CKD.

Background: Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies., Methods and Results: Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment., (© International & American Associations for Dental Research.)

One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies., (© 2014 Wiley Periodicals, Inc.)

Rationale: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known., Objective: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5., Methods and Results: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling., Conclusions: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Diagnosing arrhythmogenic right ventricular cardiomyopathy (ARVC) is often challenging because no single diagnostic tool is available to detect the disease. We evaluated whether analysis of plakoglobin, N-cadherin, and connexin-43 immunoreactivity can be used as a significant test in diagnosis of ARVC. We selected subjects with suspicion of ARVC (n=22) in patients who underwent endomyocardial biopsy (EMB) in Kyungpook National University Hospital (n=1326). The patients (n=22) were classified into definite ARVC patients (n=17) and borderline ARVC (n=5). We selected control subjects (n=20) who were autopsied and died of non-cardiac disease. Hematoxylin-eosin, Masson's trichrome, and immunohistochemical stains for plakoglobin, N-cadherin, and connexin-43 were used for all specimens. Reduced immunoreactivity of plakoglobin was observed in 13 (76%) of the 17 patients with a definite ARVC and in 4 (80%) of the 5 patients with a borderline ARVC. All subjects displayed no significant reduction of the immunoreactivity for connexin-43 as well as for N-cadherin. Our investigation revealed that the immunohistochemical analysis for plakoglobin had an accuracy of 81%, 76% sensitivity, and 84% specificity in diagnosis of ARVC. Results of our study showed that the immunohistochemical analysis of plakoglobin had a relatively high sensitivity and specificity in ARVC, but immunohistochemistry for plakoglobin alone could not be relied upon as a diagnostic test for ARVC. We confirmed that N-cadherin and connexin-43 had no diagnostic value in ARVC.

Aims: Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light (∼200-300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes. (ii) Using these tools, to map two molecules classically defined as components 'of the desmosome' and 'of the gap junction', and defined their spatial organization., Methods and Results: We built a set-up on a conventional inverted microscope using commercially available optics. Laser illumination, reducing, and oxygen scavenging conditions were used to manipulate the blinking behaviour of individual fluorescent reporters. Movies of blinking fluorophores were reconstructed to generate subdiffraction images at ∼20 nm resolution. With this method, we characterized clusters of connexin43 (Cx43) and of 'the desmosomal protein' plakophilin-2 (PKP2). In about half of Cx43 clusters, we observed overlay of Cx43 and PKP2 at the Cx43 plaque edge. SiRNA-mediated loss of Ankyrin-G expression yielded larger Cx43 clusters, of less regular shape, and larger Cx43-PKP2 subdomains. The Cx43-PKP2 subdomain was validated by a proximity ligation assay (PLA) and by Monte-Carlo simulations indicating an attraction between PKP2 and Cx43., Conclusions: (i) Super-resolution fluorescence microscopy, complemented with Monte-Carlo simulations and PLAs, allows the study of the nanoscale organization of an interactome in cardiomyocytes. (ii) PKP2 and Cx43 share a common hub that permits direct physical interaction. Its relevance to excitability, electrical coupling, and arrhythmogenic right ventricular cardiomyopathy, is discussed.

Some of lipophilic statins have been reported to enhance toxicities induced by antineoplastic agents but the underling mechanism is unclear. The authors investigated the involvement of Cx43-mediated gap junction intercellular communication (GJIC) in the effect of simvastatin on the cellular toxicity induced by etoposide in this study. The results showed that a major component of the cytotoxicity of therapeutic levels of etoposide is mediated by gap junctions composed of connexin 43(Cx43) and simvastatin at the dosage which does not induce cytotoxicity enhances etoposide toxicity by increasing gap junction coupling. The augmentative effect of simvastatin on GJIC was related to the inhibition of PKC-mediated Cx43 phosphorylation at ser368 and subsequent enhancement of Cx43 membrane location induced by the agent. The present study suggests the possibility that upregulation of gap junctions may be utilized to increase the efficacy of anticancer chemotherapies., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Gap junctions play an important role in differentiation of odontoblasts. Gap junction protein, connexin 43 is expressed in odontoblast. However, the detailed localization in odontoblasts has yet to be fully investigated. We investigated the localization of connexin43 in rat odontoblasts immuno-electron microscopically. The rats were transcardially fixed with 1% paraformaldehyde in 0.1M phosphate buffer, and mandibles were decalcified with 10% ethylenediamine tetraacetic acid. Pre-embedding method was carried out for immuno-electron microscopic analysis. Microscopically, gap junctions were localized between bodies of odontoblasts, and between bodies and processes of odontoblasts. The gap junctions were labeled with gold particles that indicated connexin43. These results suggest that gap junctions between odontoblasts are definitely composed of connexin43 in rats, and our methods used in this study is useful to investigate localization of connexin43 immuno-electron microscopically., (Copyright © 2013 Wiley Periodicals, Inc.)

Aims: Recent studies reported slowed conduction velocity (CV) in murine hearts homozygous for the gain-of-function RyR2-P2328S mutation (RyR2(S/S)) and associated this with an increased incidence of atrial and ventricular arrhythmias. The present experiments determined mechanisms contributing to the reduced atrial CV., Methods and Results: The determinants of CV were investigated in murine RyR2(S/S) hearts and compared with those in wild-type (WT) and slow-conducting Scn5a(+/-) hearts. Picrosirius red staining demonstrated increased fibrosis only in Scn5a(+/-) hearts. Immunoblot assays showed similar expressions of Cx43 and Cx40 levels in the three genotypes. In contrast, Nav1.5 expression was reduced in both RyR2(S/S) and Scn5a(+/-) atria. These findings correlated with intracellular microelectrode and loose-patch-clamp studies. Microelectrode measurements showed reduced maximum rates of depolarization in Scn5a(+/-) and RyR2(S/S) atria compared with WT, despite similar diastolic membrane potentials. Loose-patch-clamp measurements demonstrated reduced peak Na(+) currents (INa) in the Scn5a(+/-) and RyR2(S/S) atria relative to WT, with similar normalized current-voltage relationships. In WT atria, reduction in INa could be produced by treatment with high extracellular Ca(2+), caffeine, or cyclopiazonic acid, each expected to produce an acute increase in [Ca(2+)]i., Conclusion: RyR2(S/S) atria show reduced levels of Nav1.5 expression and Na(+) channel function. Reduced Na(+) channel function was also observed in WT atria, following acute increases in [Ca(2+)]i. Taken together, the results suggest that raised [Ca(2+)]i produces both acute and chronic inhibition of Na(+) channel function. These findings may help explain the relationship between altered Ca(2+) homeostasis, CV, and the maintenance of common arrhythmias such as atrial fibrillation.

Background/aims: Primarily, a diet (particularly dietary lipids and vitamins) can reversibly modify intestinal expressions of a few factors like connexin 43 (Cx43), E-cadherin (Cdh1), TP53 and TGFB1 with a special impact on immunity and mutagenesis. Malignant phenotype constitutes a diet-resistant signal streaming with engagement of these molecules which are generated in autonomous ways in colorectal cancer., Methodology: We aimed to compare adhesion proteins: (Cx43) and Cdh1 with TP53 and TGFB1 in colorectal adenocarcinomas. GJA1P1 and Cdh1 with TP53 and TGFB1 were detected with immunohistochemistry in the study of 106 colorectal adenocarcinomas., Results: There was aberrant cytoplasmic expression instead of membranous one of Cx43 and Cdh1 reflecting constitutive destruction of intercellular ties while TP53 showed nuclear expression and TGFB1 accumulated in the cytoplasm. TP53 did not correlate with Cx43 (r=0.083, p=0.397) but correlated with Cdh1 (r=0.199, p=0.041). Cdh1 associated with TGFB1 reaching almost statistical significance (r=0.188, p=0.054), while TGFB1 correlated with Cx43 (r=0.359, p=0.001)., Conclusions: The consequent and constant impairment of cancer intercellular communication seems to engage correlated with each other expressions of Cx43 and TGFB1 in colorectal cancer cells.

Periodontal tissue homeostasis depends on a complex cellular network that conveys cell-cell communication. Gap junctions (GJs), one of the intercellular communication systems, are found between adjacent human periodontal ligament (hPDL) cells; however, the functional GJ coupling between hPDL cells has not yet been elucidated. In this study, we investigated functional gap-junction-mediated intercellular communication in isolated primary hPDL cells. SEM images indicated that the cells were in contact with each other via dendritic processes, and also showed high anti-connexin43 (Cx43) immunoreactivity on these processes. Gap-junctional intercellular communication (GJIC) among hPDL cells was assessed by fluorescence recovery after a photobleaching (FRAP) analysis, which exhibited dye coupling between hPDL cells, and was remarkably down-regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.

Background: We studied the relationships among Cx43, CD105, and VEGF in specimens of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different serum AFP levels with respect to recurrence and metastasis., Methods: Expressions of Cx43, CD105, and VEGF in 234 HCC tissue specimens were examined using tissue microarray and immunohistochemistry. Cx43 mRNA expression was examined in 38 frozen HCC specimens using RT-PCR. Correlations between these expressions and tumor recurrence, metastasis, and prognosis were analyzed using Kaplan-Meier and Cox regression analyses., Results: Cx43 expression correlated with early tumor recurrence (P = 0.001), disease-free survival (P = 0.026), and overall survival (P = 0.000) in patients with serum AFP < 400 ng/ml, but not in those with serum AFP ≥ 400 μg/L. Cx43 expression is an independent predictor of later recurrence and longer overall survival and is inversely correlated with expression of CD105 and VEGF (P = 0.018 and 0.023, respectively), histological differentiation (P = 0.002), vessel tumor embolism (P = 0.029), and focal number (P = 0.017). Immunohistochemistry showed that Cx43 expression in patients with low AFP was lower in patients with distant metastases than in those with no metastasis or those with liver metastasis. Patients with early recurrence expressed less Cx43 mRNA than did those with no recurrence (χ2 = 9.827, P = 0.002)., Conclusions: Cx43 expression can delay early HCC recurrence, metastasis, and poor prognosis after radical hepatectomy in patients with HBV-related HCC and low AFP.

Background & Aims: In cirrhosis, superimposed inflammation often culminates in acute-on-chronic liver failure (ACLF) but the mechanism underlying this increased sensitivity is not clear. Cx43 is a ubiquitous gap junction protein that allows transmission of signals between cells at a much higher rate than the constitutively expressed gap junctions. The aims of the study were to test the hypothesis that inflammation drives the increased expression of hepatic Cx43 and to determine its role by Cx43 inhibition., Methods: Four weeks after bile-duct ligation (BDL) or sham operation, rats were treated with an anti-TNF antibody, or saline; with or without LPS (1mg/kg); given 3h prior to termination. Biochemistry and cytokines were measured in the plasma and hepatic protein expression (NFkB, TNFα, iNOS, 4HNE, Cx26, 32, and 43) and confocal microscopy (Cx26, 32, and 43) were performed. The effect of a Cx43-specific inhibitory peptide was studied in a mouse BDL model., Results: BDL animals administered LPS developed typical features of ACLF but animals administered infliximab were relatively protected. Cx26/32 expression was significantly decreased in BDL animals while Cx43 was significantly increased and increased further following LPS. Infliximab treatment prevented this increase. However, inhibiting Cx43 in BDL mice produced detrimental effects with markedly greater hepatocellular necrosis., Conclusions: The results of this study show for the first time an increased expression of hepatic Cx43 in cirrhosis and ACLF, which was related to the severity of inflammation. This increased Cx43 expression is likely to be an adaptive protective response of the liver to allow better cell-to-cell communication., (Copyright © 2013. Published by Elsevier B.V.)

Aim: Altered expression of connexin 43 (Cx43) has been postulated to be involved in the development and progression of various diseases including erectile dysfunction (ED). The aim of this study was to determine whether distribution and density of the gap junction protein Cx43 are altered in human corpus cavernosum (HCC) tissue samples derived from diabetic or hypogonadal patients with ED compared to those from normal subjects., Methods: HCC tissue sections derived from normal, diabetic and hypogonadal subjects were fixed in 4% formaldehyde, embedded in paraffin and immunostained with a monoclonal mouse anti-rat Cx43 antibody. Cx43 density was expressed as the cumulative number of gap junction plaques per unit area of tissue corrected for number of 4',6-diamidino-2-phenylindole, dihydrochloride-labeled smooth muscle cells (dots per unit area corrected for number of cells)., Results: The distribution of Cx43 plaques in smooth muscle was not affected in tissues derived from diabetic or hypogonadal subjects with ED compared with those from normal subjects. However, the number of Cx43 plaques was significantly reduced in HCC tissues derived from diabetic or hypogonadal subjects (73 ± 8% and 68 ± 11% of normal, respectively), indicating reduced Cx43 gap junctions in diabetic and hypogonadal subjects with ED., Conclusions: Cx43 density in the HCC was diminished in tissue samples derived from diabetic or hypogonadal patients with ED compared to tissue samples from normal non-diabetic subjects. This marked decrease in Cx43 gap junction channels may contribute to attenuated gap junction function and to diminished erectile physiology.

Recent reports have indicated a role of cell-to-cell interactions during gonadal development and functions. Numerous reports indicate that fetal hormonal disruption induces abnormalities in the developing reproductive system and, therefore, may interfere with reproductive functions later in adult life. Hence, this study investigated the effect of androgen deficiency during late prenatal periods on the gap junction-associated connexin 43 (Cx43) and the adherens junction-associated β-catenin expression in the fetal porcine gonads. Thus, pregnant gilts were injected with anti-androgen flutamide (for 7 d, 50 mg/kg BW per day) or corn oil (control groups) starting at 83 (GD90) or 101 (GD108) gestational day. On GD90 and GD108 the fetuses were excised and fetal gonads were obtained. To assess Cx43 and β-catenin expression real-time PCR and immunohistochemistry were performed. In fetal testes, Cx43 was localized between Leydig cells, whereas β-catenin was observed mainly within the seminiferous tubules. In fetal ovaries, Cx43 was detected between interstitial cells and between granulosa cells of forming follicles, whereas β-catenin was found within egg nests, in oocytes' membrane, and in granulosa cells of forming follicles. Immunohistochemistry showed decreased Cx43 and β-catenin expression in fetal gonads from flutamide-treated pigs compared with respective controls. However, the ovaries from animals treated with flutamide on GD108 showed increased Cx43 expression. The changes of Cx43 and β-catenin expression after prenatal flutamide treatment were confirmed at the mRNA level. These findings suggest that androgen deficiency during late gestation may lead to disturbed intercellular interactions in fetal porcine testes affecting testicular functions, as well as impaired follicular formation in fetal ovaries. Our results further signify the role of androgens in the regulation of cell-to-cell interactions within fetal porcine gonads., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: Patients with advanced non-small-cell lung cancer (NSCLC) have poor survival, and platinum-based chemotherapy agents are the standard first-line chemotherapy agents for advanced NSCLC. This study aimed to identify predictive factors associated with the response to chemotherapy and survival in 258 patients with advanced NSCLC treated with platinum-based chemotherapy., Methods: Stage IIIA-IV NSCLC patients diagnosed in Kaifeng second people's hospital (Henan, China) between March 2002 and September 2011 were retrospectively reviewed. All of the patients had received platinum-based chemotherapy, and patients were followed up to date of death or last follow-up to obtain data of response to chemotherapy and survival. Potential prognostic factors such as gender, age, tumor size, tumor type, histologic stage, anemia, calcium levels, ECOG performance status (PS), thrombocytosis, TTF-1, p63, and connexin 43 were analyzed. Response to chemotherapy, overall survival (OS) and progression-free survival (PFS) were calculated by the Kaplan-Meier method and Cox regression model., Results: A univariate analysis indicated that thrombocytosis and connexin 43 were found to be significant prognostic factors (p < 0.001) and ECOG PS, Hb levels, and p63 presented a tendency toward association with survival. Kaplan-Meier survival showed that the mean OS and PFS in chemotherapy responders with connexin 43 ≥ +2 were significantly longer than in chemotherapy responders with connexin 43 ≤ 1+. In contrast, thrombocytosis was associated with increased mortality and resistance to chemotherapy in chemotherapy responders. In addition, all 21 patients of the 5-year OS were from chemotherapy responders with connexin 43 ≥ +2 or non-thrombocytosis., Conclusions: Thrombocytosis and connexin 43 absence may be reliable surrogate markers for the prediction of chemotherapy response and prognosis for patients with advanced NSCLC, and assessment of these factors may identify a population of patients with advanced NSCLC that is likely to have a prolonged life expectancy.

Objective: To study the expression of connexin 43 (Cx43) in various stages of oral carcinogenesis and explore the relation between Cx43 and oral mucous carcinogenesis., Methods: 4-nitroquinoline-1-oxide (4NQO) was used for inducing oral carcinogenesis in SD rats. Tissue samples were obtained from various stages of the disease including normal oral mucosa, precancerous lesions and oral squamous cell carcinoma. Immunohistochemical method was used to determine the expression of Cx43 in various stages of oral carcinogenesis., Results: In the normal rat lingual mucosa, immunohistochemical staining of Cx43 protein was mainly found in the cell membrane, weakly positive in the basal cell layer, increased in stratum spinosum and stratum granulosum, but was negative in the stratum corneum of normal epithelia. Compared with normal epithelia, was significantly decreased in dysplastic and cancerous oral epithelia the staining. The positive rates of Cx43 were respectively 100.00% (10/10), 85.71% (12/14), 66.67% (8/12), 40.00% (4/10), and 33.33% (4/12) in tongue carcinogenesis (in normal, mild, moderate and severe dysplasia, and squamous cell carcinoma tissues). The differences were statistically significant (P<0.05)., Conclusion: Expression level of Cx43 protein was dramatically decreased with the development of rat tongue carcinoma induced by 4NQO, suggesting that abnormal expression of Cx43 protein is involved in oral mucosa carcinogenesis. Decreased Cx43 expression is an early sign of oral mucosa carcinogenesis.

Gap junctions (GJs) are intercellular channels which are composed of the connexin family of proteins that allow electrical and chemical communications and synchronization in tissue ensembles. Evidence suggests that pharmaceutical modulators of these channels may have therapeutic potential or carry undesired liability. In this report, we exogenously expressed human connexin 43 (Cx43, GJA1) and demonstrated functionality in a 96-well flow cytometry assay detecting intercellular transfer of the calcein dye. We have designed a 384-well high-throughput method for detecting the transfer of calcium between HeLa cells expressing Cx43. In this assay, donor cells coexpress Cx43 and the α1A adrenergic Gα-coupled receptor, while recipient cells coexpress Cx43 and the cytoplasmic version of the calcium-sensitive luminescent protein aequorin enhanced by codon optimization (cytoAeq). The two cell populations were mixed, dispensed to 384-well plates, and incubated for 3 h to allow the formation of GJs. Activation of α1A by epinephrine in donor cells led to dose-dependent calcium increases in recipient cells, which were detected by measuring the intensity of aequorin luminescence. The response was dependent on the expression of Cx43 and inhibited by the GJ blocker 18α-glycyrrhetinic acid, suggesting Cx43 GJ-mediated activity. In a parallel experiment with capsaicin and the TrpV1 ion channel in place of phenylephrine and α1A, a similar magnitude of difference in the maximal calcium response was detected in both donor and recipient cells, suggesting that calcium is likely the permeant ion through the GJ. This assay may pave the way for high-throughput screening of GJ modulators for drug discovery.

Background: The existence of myofibroblasts (MFBs) and the role of subendocardial smooth muscle (SSM) layer of human atrial tissue in atrial fibrillation (AF) have not yet been elucidated. We hypothesized that the SSM layer and MFB play some roles in atrial structural remodeling and maintenance of valvular AF in patients who undergo cardiac surgery., Methods: We analyzed immunohistochemical staining of left atrial (LA) appendage tissues taken from 17 patients with AF and 15 patients remaining in sinus rhythm (SR) who underwent cardiac surgery (male 50.0%, 54.1 ± 14.2 years old, valve surgery 87.5%). SSM was quantified by α-smooth muscle actin (α-SMA) stain excluding vascular structure. MFB was defined as α-SMA+ cells with disorganized Connexin 43-positive gap junctions in Sirius red-positive fibrotic area., Results: The SSM layer of atrium was significantly thicker in patients with AF than in those with SR (P=.0091). Patients with SSM layer ≥ 14 μm had a larger LA size (P=.0006) and greater fibrotic area (P=.0094) than those patients whose SSM layer <14 μm. MFBs were found in 7 of 17 (41.2%) patients with AF and 2 of 15 (13.3%) in SR group (P=.0456) in SSM area, colocalized with Periodic Acid-Schiff (PAS) stain-positive glycogen storage cells (95.5%)., Conclusion: SSM layer was closely related to the existence of AF, degrees of atrial remodeling, and fibrosis in patients who underwent open heart surgery. We found that MFB does exist in SSM layer of human atrial tissue co-localized with PAS-positive cells., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Background: The authors wanted to investigate whether the remodeling process in AF regarding gap junction proteins, collagen I, and amyloid may be gender dependent in humans., Methods: In total, 123 patients with sinus rhythm (SR, n = 41) or atrial fibrillation (AF, n = 82) suffering from mitral valve disease undergoing cardiac surgery were included. Of the 123 patients, 66 patients (SR: n = 17, AF: n = 49) were investigated biochemically for the expression of the atrial gap junction proteins connexin40 (Cx40), connexin43 (Cx43) and collagen I and 57 patients (SR: n = 24; AF: n = 33) using histochemical methods for possible amyloid depositions., Results: AF led to increased levels of Cx40, Cx43, and collagen I protein. Regarding Cx40 this upregulation was significantly higher in female than in male patients. For AF-induced changes in collagen or Cx43, there were no significant gender-dependent differences. Amyloid depositions were found with increasing age, but were not significantly related to AF or gender., Conclusions: Remodeling in AF seems to be similar in men and women, with a tendency for women exhibiting somewhat stronger AF-induced changes in Cx40, which is probably a secondary effect because there is nothing known about hormone sensitivity of the Cx40 promoter, and a not significant tendency for higher Cx43 and collagen I., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Colon carcinogenesis is a multistep process and it emanates from a series of molecular and histopathological alterations. Glycyrrhizic acid (GA) is a natural and major pentacyclic triterpenoid glycoside of licorice roots extracts. It has several pharmacological and biological properties such as anti-inflammatory, anti-viral, and anti-cancer. In the present study, we investigated the chemopreventive potential of GA against 1,2-dimethyhydrazine (DMH)-induced precancerous lesions i.e., aberrant crypt foci (ACF) and mucin depleted foci (MDF), and its role in regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of Wistar rats., Methods: Animals were divided into 5 groups. In group III, IV and V, GA was administered at the dose of 15 mg/kg b. wt. orally while in group II, III and IV, DMH was administered subcutaneously in the groin at the dose of 20 mg/kg b.wt once a week for first 5 weeks and animals were euthanized after 9 weeks., Results: GA supplementation suppressed the development of precancerous lesions and it also reduced the infiltration of mast cells, suppressed the immunostaining of Ki-67, NF-kB-p65, COX-2, iNOS and VEGF while enhanced the immunostaining of p53, connexin-43, caspase-9 and cleaved caspase-3. GA treatment significantly attenuated the level of TNF-α and it also reduced the depletion of the mucous layer as well as attenuated the shifting of sialomucin to sulphomucin., Conclusion: Our findings suggest that GA has strong chemopreventive potential against DMH-induced colon carcinogenesis but further studies are warranted to elucidate the precise mechanism of action of GA.

Myocyte apoptosis is considered a major mechanism in the pathogenesis of heart failure. Accordingly, manipulations that inhibit apoptosis are assumed to preserve cardiac function by maintaining myocyte numbers. We tested this assumption by examining the effects of caspase inhibition (CI) on cardiac structure and function in C57BL/6 mouse with pressure overload model induced by transverse aortic constriction (TAC). CI preserved left ventricular (LV) function following TAC compared with the vehicle. TAC increased apoptosis in non-myocytes more than in myocytes and these increases were blunted more in non-myocytes by CI. Total myocyte number, however, did not differ significantly among control and TAC groups and there was no correlation between myocyte number and apoptosis, but there was a strong correlation between myocyte number and an index of myocyte proliferation, Ki67-positive myocytes. Despite comparable pressure gradients, LV hypertrophy was less in the CI group, likely attributable to decreased wall stress. Since changes in myocyte numbers did not account for protection from TAC, several other CI-mediated mechanisms were identified including: (a) lessening of TAC-induced fibrosis, (b) augmentation of isolated myocyte contractility, and (c) increased angiogenesis and Ki67-positive myocytes, which were due almost entirely to the non-myocyte apoptosis, but not myocyte apoptosis, with CI. CI maintained LV function following TAC not by protecting against myocyte loss, but rather by augmenting myocyte contractile function, myocyte proliferation, and angiogenesis resulting in reduced LV wall stress, hypertrophy, and fibrosis.

The sinus node of yak has been studied by the histological methods and transmission electron microscopy. The sinus node artery of yak was also determined by the injection-corrosion casting technique, the angiography, and histological methods. The results showed that the sinus node of yak contained an extensive framework of collagen and two main type cells: pacemaker cells (P cells) and transitional cells (T cells). The P cells had a perinuclear clear zone, contained less myofibrils, and appeared smaller mitochondria than T cells. The T cells were longer and slender than P cells, and had a variety of shapes. At the periphery of sinus node there were many nerve fibers and ganglions. Gap junction did not reveal reaction with anti-connexin43, but it was detected by electron microscopy in the central part of sinus node of yak. The sinus node artery of yak originated from left coronary artery more frequently (98%) than by right (2%). The artery located at the periphery of sinus node. It had an internal elastic membrane throughout its course, and a large nerve bundle was found running in a longitudinal direction., (Copyright © 2012 Wiley Periodicals, Inc.)

Objectives: Borderline left ventricle is the left ventricular morphology at the favorable end of the hypoplastic left heart syndrome. In contrast to the severe end, it is suitable for biventricular repair. Wondering whether it is possible to identify cases suitable for biventricular repair from a developmental viewpoint, we investigated the myocardial histology of borderline and severely hypoplastic left ventricles., Methods: Postmortem specimens of neonatal, unoperated human hearts with severe hypoplastic left heart syndrome and borderline left ventricle were compared with normal specimens and hearts from patients with transposition of the great arteries. After tissue sampling of the lateral walls of both ventricles, immunohistochemical and immunofluorescence stainings against cardiac troponin I, N-cadherin, and connexin 43, important for proper cardiac differentiation, were done., Results: All severely hypoplastic left hearts (7/7) and most borderline left ventricle hearts (4/6) showed reduced sarcomeric expressions of troponin I in left and right ventricles. N-cadherin and connexin 43 expressions were reduced in intercalated disks. The remaining borderline left ventricle hearts (2/6) were histologically closer to control hearts., Conclusions: Four of 6 borderline left ventricle hearts showed myocardial histopathology similar to the severely hypoplastic left hearts. The remainder were similar to normal hearts. Our results and knowledge regarding the role of epicardial-derived cells in myocardial differentiation lead us to postulate that an abnormal epicardial-myocardial interaction could explain the observed histopathology. Defining the histopathologic severity with preoperative myocardial biopsy samples of hearts with borderline left ventricle might provide a diagnostic tool for preoperative decision making., (Copyright © 2012 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

The objective of this research was to study the mechanisms of opening of the blood-prostate barrier and increased permeability of prostate tissue induced by microbubble cavitation. Thirty-five rabbits were randomly divided into four study groups: (1) control group and groups exposed to (2) microbubble alone, (3) ultrasound alone, or (4) combined intervention (ultrasound + microbubble group). Evans blue (EB) tracer was used to gauge the changes of permeability of prostate tissue. Furthermore, light and electron microscopy analyses were conducted, as well as the western blot analysis of expression of gap junction (Cx43) protein. We observed that EB concentration in prostate tissue was significantly greater in the ultrasound + microbubble group compared with either intervention alone (p < 0.05, both comparisons). Furthermore, light microscopy of tissue samples from animals exposed to ultrasound + microbubble showed epithelial cell disarrangement, loss of interstitial structure, and thickness of fibrous stroma. In line with these findings, electron microscopy analysis demonstrated widening of cell gaps and broken cell connections, as well as more dense lysosomes and secretary granules, and mitochondrial swelling. These changes were absent in the animals exposed to microbubble or ultrasound alone. Finally, only combined treatment with microbubble or ultrasound significantly elevated expression of Cx43 (p < 0.05 vs. control group). In conclusion, increases of permeability of prostate tissue by acoustic cavitation appear to involve opening of tight junctions, widening of intracellular spaces, changes in the structure of acinar cell membrane, enhancement of vesicular transport, and loosening of fibrous stroma. Increased expression of cell gap junction protein will help to restore normal connections between cells and the blood-prostate barrier after the treatment.

Glioblastoma cells are characterized by high proliferation and invasive capacities. Tumor development has been associated with a decrease of gap-junctional intercellular communication, but the concrete involvement of gap junction proteins, connexins, remains elusive since they are also suspected to promote cell invasion. In order to better understand how connexins control the glioma cell phenotype, we studied the consequences of inhibiting the intrinsic expression of the major astrocytic connexin, Connexin43, in human U251 glioblastoma cells by the shRNA strategy. The induced down-regulation of Cx43 expression has various effects on the U251 cells such as increased clonogenicity, angiogenesis and decreased adhesion on specific extracellular matrix proteins. We demonstrate that the invasion capacity measured in vitro and ex vivo correlates with Cx43 expression level. For the first time in a cancer cell context, our work demonstrates that Cx43 cofractionates, colocalizes and coimmunoprecipitates with a lipid raft marker, caveolin-1 and that this interaction is inversely correlated to the level of Cx43. This localization of Cx43 in these lipid raft microdomains regulates both homo- and heterocellular gap junctional communications (respectively between U251 cells, or between U251 cells and astrocytes). Moreover, the adhesive and invasive capacities are not dependent, in our model, on Cav-1 expression level. Our results tend to show that heterocellular gap junctional communication between cancer and stroma cells may affect the behavior of the tumor cells. Altogether, our data demonstrate that Cx43 controls the tumor phenotype of glioblastoma U251 cells and in particular, invasion capacity, through its localization in lipid rafts containing Cav-1., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Several arrhythmogenic mechanisms have been inferred from animal heart failure models. However, the translation of these hypotheses is difficult because of the lack of functional human data. We aimed to investigate the electrophysiological substrate for arrhythmia in human end-stage nonischemic cardiomyopathy., Methods and Results: We optically mapped the coronary-perfused left ventricular wedge preparations from human hearts with end-stage nonischemic cardiomyopathy (heart failure, n=10) and nonfailing hearts (NF, n=10). Molecular remodeling was studied with immunostaining, Western blotting, and histological analyses. Heart failure produced heterogeneous prolongation of action potential duration resulting in the decrease of transmural action potential duration dispersion (64 ± 12 ms versus 129 ± 15 ms in NF, P<0.005). In the failing hearts, transmural activation was significantly slowed from the endocardium (39 ± 3 cm/s versus 49 ± 2 cm/s in NF, P=0.008) to the epicardium (28 ± 3 cm/s versus 40 ± 2 cm/s in NF, P=0.008). Conduction slowing was likely due to connexin 43 (Cx43) downregulation, decreased colocalization of Cx43 with N-cadherin (40 ± 2% versus 52 ± 5% in NF, P=0.02), and an altered distribution of phosphorylated Cx43 isoforms by the upregulation of the dephosphorylated Cx43 in both the subendocardium and subepicardium layers. Failing hearts further demonstrated spatially discordant conduction velocity alternans which resulted in nonuniform propagation discontinuities and wave breaks conditioned by strands of increased interstitial fibrosis (fibrous tissue content in heart failure 16.4 ± 7.7 versus 9.9 ± 1.4% in NF, P=0.02)., Conclusions: Conduction disorder resulting from the anisotropic downregulation of Cx43 expression, the reduction of Cx43 phosphorylation, and increased fibrosis is likely to be a critical component of arrhythmogenic substrate in patients with nonischemic cardiomyopathy.

Sertoli cells are essential in development of a functional testis. During puberty, Sertoli cell maturation can be characterized by a number of markers, including anti-Müllerian hormone (AMH) and its receptor (AMHR2), androgen receptor (AR), cyclin-dependent kinase inhibitor (CDKN1B), and connexin 43 (Cx43). In the present study, immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) were used to characterize changes in expression of AMH, AMHR2, AR, CDKN1B, and Cx43 in prepubertal, postpubertal, and adult equine testes. During puberty, AMH expression decreased, and expression of AR as well as CDKN1B increased in Sertoli cells coinciding with the period of Sertoli cell maturation, arrest of cell proliferation, and presumptive AMH regulation by testosterone. Expression of AMHR2 appeared to decrease in Sertoli cells and increase in Leydig cells during pubertal maturation of the equine testis. In addition, expression and distribution of Cx43 changed during puberty in the stallion, suggesting a role for Cx43 in Sertoli cell signaling and maturation, hormone secretion, and blood-testis barrier formation. We concluded that Sertoli cell maturation during puberty in the stallion was accompanied by a reduced expression of AMH and its receptor, arrest of cell proliferation, increased expression of AR, and organization of gap-junctional communication., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Interstitial cells of Cajal (ICC) have been identified in urinary bladder of several species, but their presence in mice remains uncertain. Meanwhile, dozens of reports indicate that dysregulation of connexin 43 plays an important role in bladder overactivity, but its localization has not been clearly defined, with reports of expression in either the smooth muscle or in myofibroblasts. We recently identified a population of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) positive cells that resemble ICC and are distinct from smooth muscle, fibroblasts, myofibroblasts and neurons. Thus we sought to define more clearly the molecular signature of ICC and in doing so resolve some of these uncertainties., Principle Findings: Immunofluorescent localization revealed that NTPDase2-positive cells lie closely adjacent to smooth muscle but are separate from them. NTPDase2 positive cells exhibited co-localization with the widely accepted ICC marker - c-kit. They were further shown to co-localize with other ICC markers CD34 and Ano1, but not with mast cell marker tryptase. Significantly, they show convincing co-localization with connexin 43, which was not present in smooth muscle. The identity of these cells as ICC was further confirmed by the presence of three mesenchymal markers - vimentin, desmin, and PDGFβ receptor, which indicates their mesenchymal origin. Finally, we observed for the first time, the presence of merlin/neurofibromin 2 in ICC. Normally considered a neuronal protein, the presence of merlin suggests ICC in bladder may have a role in neurotransmission., Conclusions: NTPDase2 positive cells in mice bladder are ICC, which can be defined by the presence of c-Kit, CD34, Ano1, NTPDase2, connexin 43, vimentin, desmin, PDGFβ receptor and merlin/NF2. These data establish a definitive molecular expression profile, which can be used to assist in explorations of their functional roles, and the presence of NTPDase2 suggests that purinergic signaling plays a role in regulation of ICC function.

The morphological and functional heterogeneity of solid tumour cells can be observed in cancer cell lines cultured in vitro. We have combined analyses of microclones developed from single cells with micropore transmigration assays to demonstrate the co-existence of cellular subsets differing in morphology and motile activity, as well as Cx43 (connexin 43) and N-cadherin expression within lung carcinoma A549 populations. 'Fibroblastoid' cells, characterized by high motility, polarized morphology and plasmalemmal localization of Cx43, displayed the strongest aptitude for transmigration through narrow obstacles. Due to high mitotic activity, they maintain the whole population but can also give rise to a sub-population of quiescent and immobile 'epithelioid' cells. Our observations indicate that phenotypic transitions between the fibroblastoid and epithelioid phenotype account for the heterogeneity of metastable A549 cell populations., (© The Author(s) Journal compilation © 2012 Portland Press Limited)

Introduction: Amenorrhea following spinal cord injury (SCI) has been well documented. There has been little research on the underlying molecular mechanisms and therapeutics., Aim: The purpose of the present study was to investigate the effect of GSNO in ameliorating SCI-induced amenorrhea through affecting the expression of CX43, NFkB, and ERβ protein., Methods: SCI was induced in female SD rats at the T9-T10 level. Estrous stage was determined by vaginal smear. GSNO (50 µg/kg body weight) was gavage fed daily. Animals were sacrificed on day 7 and 14 post SCI. Ovaries were fixed for histological and biochemical studies. Expression levels of ERβ, CX-43, and NFkB were analyzed by Western blot and immunofluorescence., Main Outcome Measures: GSNO hastens resumption of the estrous cycle following SCI-induced transient arrest., Results: Resumption of estrous cycle was hastened by GSNO. Atretic and degenerating follicles seen in the ovary of SCI rats on day 14 post-SCI were decreased in GSNO treated animals. The increased CX43 expression observed with SCI ovary was decreased by GSNO. ERβ expression decreased significantly on day 7 and 14 post-SCI and was restored with GSNO treatment. Following SCI, NFkB expression was increased in the ovarian follicles and the expression was reduced with GSNO administration. The number of terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate (UTP) nick end labeling positive follicular and luteal cells was increased after SCI. GSNO-treated animals had significantly fewer apoptotic cells in the ovary., Conclusion: SCI-induced amenorrhea is accompanied by an increase in CX43 expression and a decrease in ERβ expression. SCI animals treated with GSNO resumed the estrous cycle significantly earlier. These results indicate a potential therapeutic value for GSNO in treating amenorrhea among SCI patients., (© 2011 International Society for Sexual Medicine.)

Background: The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2(-/-)) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection., Methodology: Rhesus monkeys and C57BL/6 and Nos2(-/-) mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2(+) cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue., Results: Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2(+) cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2(-/-) mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue., Conclusion: T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas' heart disease.

Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.

Objective: To study the clinical significance of interstitial cell of Cajal (ICC) in spontaneous neonatal gastric perforation by examining the expression of c-kit and Cx43 in neonates with this disorder., Methods: The gastric specimens of 19 cases of neonatal gastric perforation from 2001 to 2010 and 8 cases of accidental death without digestive tract malformations (control) were collected. Immunohistochemical staining was employed to examine the expression of c-kit and Cx43 (immunomarkers of ICCs) in gastric tissues., Results: The muscular layer of the stomach wall became thinner or deficient in the gastric perforation group. C-kit and Cx43 positive cells in gastric tissues decreased significantly in the gastric perforation group compared with those in the control group (P＜0.01)., Conclusions: The development of spontaneous neonatal gastric perforation is associated with the decreased quantity of ICCs and damaged gap junction structure of the stomach wall.

One of the central challenges in cardiac tissue engineering is the control of the assembly and organization of functional cardiac tissue. Maintenance of a three-dimensional tissue architecture is key to myocardial function in vivo, and a variety of studies hint that provision of topological cues within scaffolds can facilitate the engineering of functional myocardial tissue by promoting this architecture. To explore this possibility in an isolated and well-defined fashion, we have designed scaffolds of polydimethylsiloxane (PDMS) with microtopographic pillars ("micropegs") to provide cells with defined structures with which to interact in three dimensions. We show that these surfaces permit HL-1 cardiomyocytes to grow, form myofibrillar structures and cell-cell adhesions, and beat spontaneously. Additionally, the cells and their nuclei interact with the full length of the micropegs, indicating that the micropegs promote a three-dimensional cytoarchitecture in the context of a two-dimensional scaffold. We also show that the number of cells interacting with a micropeg can be controlled by manipulating incubation time, micropeg spatial arrangement, or micropeg diameter. Western blots reveal that the expression of the junctional markers N-cadherin and connexin 43 is upregulated in the presence of specific arrangements of micropegs, suggesting that micropegs can enhance cardiomyocyte function. Together, these data show that microtopography can be used to provide three-dimensional adhesion and control the assembly of functional cardiac tissue on a two-dimensional surface., (This journal is © The Royal Society of Chemistry 2011)

Aims: The intercalated disc (ID) contains two complexes, the adhesion junction (AJ) and the gap junction (GJ). We studied ID remodelling and its potential role in arrhythmogenesis and investigated the effects of olmesartan on ID remodelling during development of heart failure (HF) in UM-X7.1 cardiomyopathic hamsters., Methods and Results: The UM-X7.1 hamsters showed left ventricular (LV) hypertrophy by the age of 10-15 weeks and a moderate impairment in LV contractility at 20 weeks. At age 10-15 weeks, 10-20% of the hamsters died suddenly without HF, and ventricular tachycardia (VT)/ventricular fibrillation (VF) was induced in ∼30% of hamsters. Electron microscopy showed that density linking cell-to-cell adhesion was irregular and unclearly defined, and filamentous structures attached to electron-dense components were arranged in disorder. Western blotting showed that the total cellular expression level of β-catenin was decreased, and expression of nuclear β-catenin, which functions as a T-cell factor/lymphocyte enhancer binding factor transcriptional activator, was also remarkably decreased. At age 20 weeks, LV connexin43 expression showed a remarkable decrease, and the VT/VF induction rate was ∼90%. In UM-X7.1 hamsters, olmesartan improved abnormal ID ultrastructural changes, attenuated the decrease of total cellular and nuclear β-catenin expression, decreased VT/VF induction, and improved survival rate., Conclusion: These results suggest that changes in AJ protein precede connexin43 GJ alterations, and ID remodelling might contribute to arrhythmogenesis during the development of HF. Angiotensin receptor blockade might be a new therapy for lethal ventricular arrhythmia by modulating both AJ and GJ remodelling.

Background: Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors with variable malignant potential. Connexin-43 (Cx43) is the commonest gap-junction protein and has been frequently investigated in oncology. Our aim was to establish the immunohistochemical expression of Cx43 in relation to GIST location, size, Ki67 index, tumor grade and follow-up.?, Materials and Methods: The study included postoperative samples of 46 patients treated for GIST in the 1999-2010 time frame. Complete clinical workup was available for 38 patients (82.6%); total surgical resection was carried out in 32 (84.2%) patients, while 13 (34.2%) patients underwent chemotherapy. Median follow-up was 40.7 months (range, 1-134). ?, Results: The calculated incidence of GIST in our setting was 11.5 per million. Cx43 was expressed in 43/46 (93.5%) GIST cases, with a significant difference between stomach- and small intestine-derived tumors (p=0.006). Ki67 was 10% on average (range, 1-22) and was not correlated with tumor location (p=0.194). Cx43 did not show significance with regard to tumor size (p=0.264) or higher tumor grade (p=0.658), as opposed to Ki67, which significantly correlated with both (p=0.0048 and p<0.001, respectively). Cx43 and Ki67 were not significantly correlated (p=0.708). Ki67 correlated with time to recurrence (p=0.022). Ki67 >11% was taken as the indication to start imatinib chemotherapy (sensitivity 61.5%, specificity 92.0%, p=0.022). Ten (66.7%) of 15 patients with long-term (>5 years) follow-up were in remission.?, Conclusion: Cx43 was frequently expressed in GISTs regardless of tumor site. However, no significant relationships to histopathological parameters suggestive for prognosis were found. Further investigations might clarify the roles of Cx43 in GIST oncogenesis.

Aims: Pre-treatment with dietary ω3 polyunsaturated fatty acids (ω3-PUFA) has been reported to reduce the incidence of new-onset atrial fibrillation (AF) following cardiac surgery. In a canine cardiac surgery model, we evaluated the impact of dietary ω3-PUFA on atrial electrophysiological properties, inflammatory markers, the atrial endothelin-1 (ET-1) system, and the expression and distribution of connexin 43., Methods and Results: Adult mongrel dogs received either normal chow (NC, n = 11) or chow supplemented with fish oil (FO, 0.6 g ω3-PUFA/kg/day, n = 9) for 3 weeks before surgery. A left thoracotomy was performed, and the left atrial appendage (LAA) was excised. Atrial pacing/recording wires were placed, and the pericardium/chest was closed. The atrial ratio of ω6/ω3 lipids decreased from 15-20 in NC to 2-3 in FO. FO treatment lowered pre-surgical and stabilized post-surgical arachidonate levels. Peak neutrophil to lymphocyte ratio was lower and decayed faster in FO-treated animals. Extensive inflammatory cell infiltration was present in NC atria, but was reduced in FO-treated dogs. FO-treated animals had lower post-surgical atrial expression of inducible nitric oxide synthase (iNOS) and reduced plasma ET-1. Expression of ET-1 and inositol trisphosphate receptor type-2 proteins in the LAA was also reduced. FO treatment prolonged post-operative atrial effective refractory period, slowed heart rate, and enhanced heart rate variability. Importantly, AF (>30 s) was inducible in four of six NC dogs, but no FO dogs., Conclusion: Dietary FO attenuated AF inducibility following cardiac surgery by modulating autonomic tone and heart rate. FO also reduced atrial inflammation, iNOS, and ET-1 expression.

The aim of this study was to quantify the number and size of connexin43 (Cx43) gap junctions in the circular and longitudinal layers of myometrium of the non-pregnant pig. We developed a novel approach to measure the mean surface area (s), numerical density (N(v)) and surface density (S(v)) of gap junctions using confocal microscopy and stereological analysis. Immunolabelled Cx43 gap junctions were measured in the subendometrial and deep regions of the circular layer and in the longitudinal layer of the myometrium of pre-pubertal pig and mature pig at pre-ovulatory and secretory stages of the oestrous cycle. In the circular subendometrial region, all investigated stereological parameters of Cx43 gap junctions (s, N(v) and S(v)) were significantly higher (p<0.05) than those of the circular deep region and the longitudinal layer in all three groups of animals. These results indicate the large-scale heterogeneity of the number and size of Cx43 gap junctions across the myometrium in non-pregnant pig and emphasize the existence of functional diversity among myometrial cells., (© 2009 Blackwell Verlag GmbH.)

Using immunohistochemistry (IHC), this study aimed to evaluate the regional distribution pattern of some biologically active proteins in the epididymis of Egyptian water buffalo and to determine the structural-functional relationships of the different epididymal structures. Wax-embedded sections from different regions of the epididymal duct from adult, clinically healthy, buffalo bulls were used. Primary antibodies against angiotensin converting enzyme (ACE), S-100, galactosyltransferase (GalTase), alpha smooth muscle actin (α-SMA), connexin 43 (Cx43) and vascular endothelial growth factor (VEGF) were used for immunohistochemical studies. The results showed that, in addition to the well-known principal and basal cells, the epididymal epithelium, similar to that of other species, possessed apical cells and intraepithelial leukocytes. IHC showed that, with the exception of VEGF which reacted negatively, all antibodies used displayed variable reactivity in the different epididymal structures. Apical cells expressed a strong reaction with ACE along the entire length of the duct. The principal cells in the caput epididymis exhibited a distinct reactivity with S-100 and GalTase. The peritubular muscular coat displayed a marked immunostaining for α-SMA and for Cx43. In conclusion these findings showed a regional-specific distribution pattern, distinct from that in bovine bulls. Some potential functional capacities, especially absorptive and secretory ones, are discussed in relation to the different epididymal regions., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

This paper describes a microfluidic-based assay capable of measuring gap-junction mediated dye diffusion in cultured cells. The technique exploits multistream laminar flow to selectively expose cells to different environments, enabling continuous loading of cells in one compartment while monitoring, in real time, dye diffusion into cells of a neighboring compartment. A simple one-dimensional diffusion model fit to the data extracted the diffusion coefficient of four different dyes, 5-(6)-carboxyfluorescein, 5-chloromethylfluorescein, Oregon green 488 carboxylic acid, and calcein. Different inhibitors were assayed for their ability to reduce dye coupling. The chip can screen multiple inhibitors in parallel in the same cell preparation, demonstrating its potential for high throughput. The technique provides a convenient method to measure gap junction mediated diffusion and a screen for drugs that affect gap junction communication.

Connexin 43 (Cx43) is the predominant gap junction protein within porcine ovary and is required for proper follicle and corpus luteum (CL) development. Recent research suggests maternally or neonatally mediated effects of antiandrogens on reproductive function during adulthood, notably those dependent on gap junctional communication. The current study was conducted to determine whether late gestational or neonatal exposure to the antiandrogen flutamide influences Cx43 gene expression in the adult porcine ovary. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 posnatally. After animals reached sexual maturity, the ovaries were collected from treated and nontreated (control) pigs. Expression of Cx43 mRNA and protein was determined for preantral and antral follicles and for CLs. In addition, 3β-hydroxysteroid dehydrogenase (3β-HSD) expression and progesterone concentration were determined for luteal tissues. In preantral follicles, Cx43 mRNA was down-regulated (P < 0.01) following maternal and neonatal flutamide exposure. In large antral follicles, Cx43 mRNA was up-regulated (P < 0.01) after neonatal flutamide administration. Immunofluorescence showed that Cx43 expression decreased (P < 0.001) in preantral follicles and increased (P < 0.001) in large antral follicles following flutamide exposure. In luteal tissues, Cx43 and 3β-HSD expression and progesterone concentration decreased (P < 0.01) after postnatal flutamide treatment. Overall, these results suggest the involvement of androgens in the regulation of Cx43 expression in pig ovary. Moreover, alteration of Cx43 expression by the administration of flutamide during particular prenatal and neonatal time periods may affect porcine follicle development, as well as CL formation and function., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Connexins (Cx) are membrane proteins able to influence cell trophoblast responses, such as proliferation, differentiation, migration and invasiveness. Likewise, glucocorticoids are also known to modulate many factors involved in implantation, including trophoblast gap-junction intercellular communication, although their influence on pregnancy is controversial. In order to investigate the effects of betamethasone, a synthetic glucocorticoid, on Cx and glucocorticoid receptor (GR) expression and localisation, as well as on cell proliferation, the extravillous trophoblast-derived HTR-8/SVneo cell line was used as a model. The results, confirmed by means of immunofluorescence, demonstrate that betamethasone selectively modifies GR and Cx expression, enhancing the GRα isoform without affecting GRβ, and inhibiting Cx40 expression whilst increasing that of Cx43 and Cx45. Furthermore, betamethasone was shown to exert an inhibitory action on cell proliferation. In this model the abortion drug RU-486 (mifepristone), reported to be a GR antagonist, did not counteract this effect of betamethasone. On the contrary, it induced responses similar to those of the hormone. Knowing that RU-486 is also a potent progesterone-receptor antagonist, the effect of progesterone alone and in combination with the drug on Cx expression and cell proliferation was then tested. Progesterone showed the same effect as betamethasone on Cx expression, but it did not affect proliferation. Based on these results, neither the abortion effects of RU-486 nor the protective action of betamethasone and progesterone are exerted by modulation of Cx. RU-486 did not antagonise the progesterone effect, suggesting that its abortive action does not involve alteration of trophoblast Cx expression.

Stem cells transplanted to the ischemic myocardium usually encounter massive cell death within a few days of therapy. Hypoxic preconditioning (HPC) is currently employed as a strategy to prepare stem cells for increased survival and engraftment in the heart. However, HPC of stem cells has provided varying results, supposedly due to the differences in the oxygen concentration, duration of exposure, and passage conditions. In the present study, we determined the effect of HPC on rat mesenchymal stem cells (MSCs) exposed to 0.5% oxygen concentration for 24, 48, or 72 h. We evaluated the expression of prosurvival, proangiogenic, and functional markers such as hypoxia-inducible factor-1α, VEGF, phosphorylated Akt, survivin, p21, cytochrome c, caspase-3, caspase-7, CXCR4, and c-Met. MSCs exposed to 24-h hypoxia showed reduced apoptosis on being subjected to severe hypoxic conditions. They also had significantly higher levels of prosurvival, proangiogenic, and prodifferentiation proteins when compared with longer exposure (72 h). Cells taken directly from the cryopreserved state did not respond effectively to the 24-h HPC as those that were cultured under normoxia before HPC. Cells cultured under normoxia before HPC showed decreased apoptosis, enhanced expression of connexin-43, cardiac myosin heavy chain, and CD31. The preconditioned cells were able to differentiate into the cardiovascular lineage. The results suggest that MSCs cultured under normoxia before 24-h HPC are in a state of optimal expression of prosurvival, proangiogenic, and functional proteins that may increase the survival and engraftment in the infarct heart. These results could provide further insights into optimal preparation of MSCs which would greatly influence the effectiveness of cell therapy in vivo.

Gap junctions are composed of a family of evolutionarily conserved integral plasma membrane proteins termed connexins. The aims of the research reported here were (1) to evaluate the Cx43 protein and mRNA of both low histological grade and high histological grade astrocyte tumors; (2) to evaluate if the immunohistochemistry of the Cx43 protein in astrocytomas could be correlated to histological grade, to proliferative activity (Ki67/Mib1-index) and to immunolabelling of the epidermal growth factor receptor (EGFR); (3) to evaluate if the reduction in levels of the Cx43 protein correlates with reduction in levels of the Cx43 mRNA. This study showed that the immunohistochemical labelling of Cx43 is reduced in grade III and grade IV. Decreased gap junction-mediated intercellular communication corresponds to decreased Cx43 and increased cellular proliferation rates, as measured by immunolabelling of the Ki67 nuclear antigen. This study demonstrated also the persistence in high grade astrocytomas of elevated levels of Cx43 mRNA. The reduced levels of Cx43 protein should not to be ascribed to a reduced genetic transcription, but to an alteration of the post-transcriptional mechanisms such as the regulation of its synthesis and/or the intracellular transport to membrane sites., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

Objective: Restoration of myocardial blood flow after ischemia triggers an inflammatory response involving toll-like receptors. Toll-like receptor 2 deficiency is associated with a reduced infarct size after myocardial ischemia and reperfusion. Because a marked mortality was observed in C3HeN wild-type mice, which was absent in TLR2 mice, we tested whether cardiac arrhythmias are the underlying pathology and aimed to elucidate how toll-like receptor 2 ligation might prevent lethal arrhythmias., Design: Experimental animal model., Setting: University hospital research laboratory., Subjects: Male C3HeN mice., Interventions: Myocardial ischemia and reperfusion was surgically induced by ligation of the left anterior descending coronary artery for 20 mins followed by 24 hrs of reperfusion. Electrocardiography was continuously recorded during the observation period through an implantable telemetry transmitter to detect cardiac arrhythmias during reperfusion., Measurements and Main Results: Toll-like receptor 2 expression was associated with a 51% mortality rate (23 of 45 mice died) after myocardial ischemia and reperfusion. Absence of toll-like receptor 2 improved survival toward 100% (17 of 17 mice survived). Electrocardiography diagnostics in conscious animals and histologic analysis revealed that absence of toll-like receptor 2 signaling prevented the formation of pathologic heart rate turbulence after myocardial ischemia and reperfusion and modulated the density of connexin 43-positive gap junctions in the ischemic area compared with wild-type hearts, indicating arrhythmia as the cause underlying the observed mortality., Conclusions: The results presented here indicate toll-like receptor 2 as a novel target for the prevention of lethal arrhythmic complications after myocardial ischemia and reperfusion.