Gap Junctional Intercellular Coupling Research Articles

Elucidating the relationship between arrhythmia and ischemic heterogeneity: an in silico study.

Dialysis causes blood flow defects in the heart that may augment electrophysiological heterogeneity in the form of increased number of ischemic zones in the human left ventricle. We computationally tested whether a larger number of ischemic zones aggravate arrhythmia using a 2D electrophysiological model of the human ventricle.A human ventricle cardiomyocyte model capable of simulating ischemic action potentials was adapted in this study. The cell model was incorporated into a spatial 2D model consisting of known number of ischemic zones. Inter-cellular gap junction coupling within ischemic zones was reduced to simulate slow conduction. Arrhythmia severity was assessed by inducing a re-entry, and quantifying the ensuing breakup and tissue pacing rates.Ischemia elevated the isolated cardiomyocyte's resting potential and reduced its action potential duration. In the absence of ischemic zones, the propensity in the 2D model to induce multiple re-entrant waves was low. The inclusion of ischemic zones provided the substrate for initiation of re-entrant waves leading to fibrillation. Dominant frequency, which measured the highest rate of pacing in the tissue, increased drastically with the inclusion of multiple ischemic zones. Re-entrant wave tip maximum numbers increased from 1 tip (no ischemic zone) to 34 tips when a large number (20) of ischemic zones were included. Computational limiting factors of our platform were identified using software profiling.Clinical significance. Dialysis may promote deleterious arrhythmias by increasing tissue level action potential dispersion.

EHD1 Modulates Cx43 Gap Junction Remodeling Associated With Cardiac Diseases.

Efficient communication between heart cells is vital to ensure the anisotropic propagation of electrical impulses, a function mainly accomplished by gap junctions (GJ) composed of Cx43 (connexin 43). Although the molecular mechanisms remain unclear, altered distribution and function of gap junctions have been associated with acute myocardial infarction and heart failure. A recent proteomic study from our laboratory identified EHD1 (Eps15 [endocytic adaptor epidermal growth factor receptor substrate 15] homology domain-containing protein 1) as a novel interactor of Cx43 in the heart. In the present work, we demonstrate that knockdown of EHD1 impaired the internalization of Cx43, preserving gap junction-intercellular coupling in cardiomyocytes. Interaction of Cx43 with EHD1 was mediated by Eps15 and promoted by phosphorylation and ubiquitination of Cx43. Overexpression of wild-type EHD1 accelerated internalization of Cx43 and exacerbated ischemia-induced lateralization of Cx43 in isolated adult cardiomyocytes. In addition, we show that EHDs associate with Cx43 in human and murine failing hearts. Overall, we identified EHDs as novel regulators of endocytic trafficking of Cx43, participating in the pathological remodeling of gap junctions, paving the way to innovative therapeutic strategies aiming at preserving intercellular communication in the heart.

Connexin 43 coupling in bovine cumulus cells, during the follicular growth phase, and its relationship to in vitro embryo outcomes.

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two-cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.

Induction of Gap Junctional Coupling by Secreted Factor(s) from Mesothelial Cells is Required for the Invasiveness of Endometriosis

Intercellular interactions, including gap junction intercellular coupling (GJIC), have been implicated in many invasive processes (e.g. extravasation, metastasis and oocyte implantation). Yet the role of gap junctions in endometriosis, a major invasive medical problem that affects 6–10% of women worldwide, has not been investigated. Comparisons of the expression profile of endometrial cells (ECs) from endometriosis patients and normal subjects revealed a consistent reduction in the expression levels of connexins (the protein components of gap junctions), including the most abundant isoform, Cx43. This was most notable in stromal cells as the disease progressed, but was also evident in epithelial cells. GJIC of ECs from endometriosis patients was also reduced compared to normal subjects, again most notably in stromal cells. In mimicking the endometriosis process, heterotypic interactions with mesothelial cells induced an increase in GJIC, with stromal cells from endometriosis patients showing the largest response. This enhanced coupling correlated with enhanced trafficking of Cx43 to the cell surface, and enhanced invasiveness through a mesothelial monolayer. Blocking GJIC between endometrial and mesothelial cells using either siRNA or a Cx43 specific peptide blocker dramatically reduced this enhanced invasiveness of endometriosis derived ECs, whether epithelial or stromal in origin. Finally, GJIC induction by mesothelial cells was shown to be mediated by a secreted factor, which is heat resistant and approximately 5–10K is size. This presents a unique opportunity for developing novel therapy for treating endometriosis that would prevent the formation of lesions that cause the pain and incidence of infertility associated with this widespread disease. Currently the only therapies are based on hormonal treatments that only further compromise fertility problems associated with endometriosis (representing 35% of the cases on infertility in the nation).Support or Funding InformationWomen's Health Initiative at UTHSA School of Medicine and CPRIT grants RP150600 and RP160844, and NIH grant UL1 TR001120 for CoresThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epilepsy and astrocyte energy metabolism.

Epilepsy is a complex neurological syndrome characterized by neuronal hyperexcitability and sudden, synchronized electrical discharges that can manifest as seizures. It is now increasingly recognized that impaired astrocyte function and energy homeostasis play key roles in the pathogenesis of epilepsy. Excessive neuronal discharges can only happen, if adequate energy sources are made available to neurons. Conversely, energy depletion during seizures is an endogenous mechanism of seizure termination. Astrocytes control neuronal energy homeostasis through neurometabolic coupling. In this review, we will discuss how astrocyte dysfunction in epilepsy leads to distortion of key metabolic and biochemical mechanisms. Dysfunctional glutamate metabolism in astrocytes can directly contribute to neuronal hyperexcitability. Closure of astrocyte intercellular gap junction coupling as observed early during epileptogenesis limits activity-dependent trafficking of energy metabolites, but also impairs clearance of the extracellular space from accumulation of K+ and glutamate. Dysfunctional astrocytes also increase the metabolism of adenosine, a metabolic product of ATP degradation that broadly inhibits energy-consuming processes as an evolutionary adaptation to conserve energy. Due to the critical role of astroglial energy homeostasis in the control of neuronal excitability, metabolic therapeutic approaches that prevent the utilization of glucose might represent a potent antiepileptic strategy. In particular, high fat low carbohydrate "ketogenic diets" as well as inhibitors of glycolysis and lactate metabolism are of growing interest for the therapy of epilepsy.

Computational assessment of the functional role of sinoatrial node exit pathways in the human heart.

AimThe human right atrium and sinoatrial node (SAN) anatomy is complex. Optical mapping experiments suggest that the SAN is functionally insulated from atrial tissue except at discrete SAN-atrial electrical junctions called SAN exit pathways, SEPs. Additionally, histological imaging suggests the presence of a secondary pacemaker close to the SAN. We hypothesise that a) an insulating border-SEP anatomical configuration is related to SAN arrhythmia; and b) a secondary pacemaker, the paranodal area, is an alternate pacemaker but accentuates tachycardia. A 3D electro-anatomical computational model was used to test these hypotheses.MethodsA detailed 3D human SAN electro-anatomical mathematical model was developed based on our previous anatomical reconstruction. Electrical activity was simulated using tissue specific variants of the Fenton-Karma action potential equations. Simulation experiments were designed to deploy this complex electro-anatomical system to assess the roles of border-SEPs and paranodal area by mimicking experimentally observed SAN arrhythmia. Robust and accurate numerical algorithms were implemented for solving the mono domain reaction-diffusion equation implicitly, calculating 3D filament traces, and computing dominant frequency among other quantitative measurements.ResultsA centre to periphery gradient of increasing diffusion was sufficient to permit initiation of pacemaking at the centre of the 3D SAN. Re-entry within the SAN, micro re-entry, was possible by imposing significant SAN fibrosis in the presence of the insulating border. SEPs promoted the micro re-entry to generate more complex SAN-atrial tachycardia. Simulation of macro re-entry, i.e. re-entry around the SAN, was possible by inclusion of atrial fibrosis in the presence of the insulating border. The border shielded the SAN from atrial tachycardia. However, SAN micro-structure intercellular gap junctional coupling and the paranodal area contributed to prolonged atrial fibrillation. Finally, the micro-structure was found to be sufficient to explain shifts of leading pacemaker site location.ConclusionsThe simulations establish a relationship between anatomy and SAN electrical function. Microstructure, in the form of intercellular gap junction coupling, was found to regulate SAN function and arrhythmia.

Connexin43high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular ERK1/2-dependent signaling axis

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43high (DU-145 and MAT-LyLu) and Cx43low prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43high prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43high DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43low AT-2 cells, Cx43low PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43low and Cx43high prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43high prostate cancer and endothelial cells.

Gold nanoparticle-mediated (GNOME) laser perforation: a new method for a high-throughput analysis of gap junction intercellular coupling.

The present report evaluates the advantages of using the gold nanoparticle-mediated laser perforation (GNOME LP) technique as a computer-controlled cell optoperforation to introduce Lucifer yellow (LY) into cells in order to analyze the gap junction coupling in cell monolayers. To permeabilize GM-7373 endothelial cells grown in a 24 multiwell plate with GNOME LP, a laser beam of 88 μm in diameter was applied in the presence of gold nanoparticles and LY. After 10 min to allow dye uptake and diffusion through gap junctions, we observed a LY-positive cell band of 179 ± 8 μm width. The presence of the gap junction channel blocker carbenoxolone during the optoperforation reduced the LY-positive band to 95 ± 6 μm. Additionally, a forskolin-related enhancement of gap junction coupling, recently found using the scrape loading technique, was also observed using GNOME LP. Further, an automatic cell imaging and a subsequent semi-automatic quantification of the images using a java-based ImageJ-plugin were performed in a high-throughput sequence. Moreover, the GNOME LP was used on cells such as RBE4 rat brain endothelial cells, which cannot be mechanically scraped as well as on three-dimensionally cultivated cells, opening the possibility to implement the GNOME LP technique for analysis of gap junction coupling in tissues. We conclude that the GNOME LP technique allows a high-throughput automated analysis of gap junction coupling in cells. Moreover this non-invasive technique could be used on monolayers that do not support mechanical scraping as well as on cells in tissue allowing an in vivo/ex vivo analysis of gap junction coupling.

Effects of isoprenaline on endothelial connexins and angiogenesis in a human endothelial cell culture system.

Downregulation of endothelial connexins has been shown to result in impaired angiogenesis. Isoprenaline is known to upregulate Cx43 in cardiomyocytes. Effects of isoprenaline on endothelial connexins are unknown. We wanted to investigate whether isoprenaline might induce upregulation of connexins Cx37, Cx40, or Cx43 in human endothelial cells and whether it may promote angiogenesis. Human umbilical vein endothelial cells (HUVECs) were cultured until confluence (5days) and subsequently seeded in Matrigel in vitro angiogenesis assays for 18h. During the entire cell culture and angiogenesis period, cells were treated with vehicle or isoprenaline (100nM). Finally, the resulting angiogenetic network was investigated (immuno)histologically. Moreover, expression of Cx37, Cx40, and Cx43 was determined by Western blot. In addition, we measured functional intercellular gap junction coupling by dye injection using patch clamp technique. Isoprenaline resulted in significantly enhanced expression of endothelial Cx43 and to a lower degree of Cx40 and Cx37. The number of coupling cells was significantly increased. Regarding angiogenesis, we observed significantly enhanced formation of branches and a higher complexity of the tube networks with more branches/length. Isoprenaline increases endothelial connexin expression and intercellular coupling and promotes tube formation.

Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia

Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca2+]i) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca2+]i oscillations followed by larger and sustained [Ca2+]i increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca2+]i increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca2+]i increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca2+]i increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage during the acute phase of ischemia.

Relevance of gap junctions and large pore channels in traumatic brain injury

In case of traumatic brain injury (TBI), occurrence of central nervous tissue damage is frequently aligned with local modulations of neuronal and glial gap junction channel expression levels. The degree of gap junctional protein expression and intercellular coupling efficiency, as well as hemichannel function has substantially impact on the course of trauma recovery and outcome. During TBI, gap junctions are especially involved in the intercellular molecule trafficking on repair of blood vessels and the regulation of vasomotor tone. Furthermore, gliosis and astrocytic swelling due to mechanical strain injury point out the consequences of derailed gap junction communication. This review addresses the outstanding role of gap junction channels in TBI pathophysiology and links the current state of results to applied clinical procedures as well as perspectives in acute and long-term treatment options.

Ketone bodies upregulate endothelial connexin 43 (Cx43) gap junctions

Ketosis occurs as a metabolic consequence of negative energy balance in post-calving lactating dairy cows. Metabolism of free fatty acids, released from adipose tissue, generates excessive amounts of acetoacetate (AcAc), β-hydroxybutyrate (BHB), and acetone (Ac) in the liver, which are released into the blood. The effects of ketone bodies on endothelial cells include increased rates of portal vein and liver blood flow and decreased cytokine secretion in response to both bacterial and viral infections. The aim of the current study was to understand the effects of AcAc, BHB and Ac, on expression of connexin 43 (Cx43) and gap junctional intercellular coupling (GJIC) in bovine aortic endothelial cells (BAECs).Confocal microscopy, Western blotting, and real-time quantitative RT-PCR indicated that Cx43 mRNA and protein expression increased after endothelial cell exposure to ketone bodies and that this was accompanied by upregulation of GJIC and cell migration. These effects were most obvious when BAECs were treated with a combination of the three ketones. Ketone bodies were shown to activate ERK and p38 MAPK as early as 3h after treatment and an ERK inhibitor (PD98059) or p38 MAPK inhibitor (SB203580) were found to antagonise the ketone-induced increase in Cx43 protein expression. Thus, ketone bodies up-regulate Cx43 expression and GJIC in BAECs via activation of ERK and p38 MAPK.

A Dominant Loss-of-Function Gja1 (Connexin43) Mutation Alters Decidual Angiogenesis and Placental Orientation in Mice.

The gap junction protein connexin43 (CX43) is expressed in the uterine stroma and in mice is required for appropriate angiogenesis during decidualization. Previous work had shown that loss of CX43 from the uterine stroma causes growth of the implanting embryo to arrest in conjunction with reduced production of angiogenic factors and restricted growth of new decidual blood vessels. The objective of the present study was to characterize placental development in mice carrying the dominant loss-of-function Gja1Jrt allele which, rather than eliminating CX43 function, alters it by substituting serine for glycine at residue 60 (CX43G60S) in the first extracellular loop. This mutation restricts but does not eliminate CX43-based gap junctional intercellular coupling and mutant females exhibit > 50% reduction in litter size. Matings were designed so that placental development could be analyzed in mutant vs. wildtype females into which embryos of both genotypes were implanting, with the genotype of each embryo being determined by PCR. Expression of genes involved in angiogenesis was analyzed by qRT-PCR. Histological and immunohistochemical analysis revealed abundant and enlarged sinusoids combined with an ectoplacental cone with disturbed orientation in the invasion route. This phenomenon leads to placentas with broadened invasion fronts or with laterally shifted orientation into the maternal compartment. This phenotype is independent of the implanting embryo's genotype and thus is hypothesized to be due to the changes in maternal decidual vessel development. The morphological changes are correlated with significantly (P < 0.05) increased levels of transcripts encoding HOXA10 and VEGF, both of which are known to play essential roles in decidualization. In contrast, previous work had revealed HOXA10 and VEGF to be down-regulated in implantation sites lacking CX43. The present findings confirm the important role played by CX43 in stromal angiogenesis and placental development but indicate that a mutant form of CX43 can have adverse effects that would not have been predicted from analysis of mice lacking this protein. Supported by the Deutsche Forschungsgemeinschaft and Canadian Institutes of Health Research.

Prostaglandin E2 maintains mouse ESC undifferentiated state through regulation of connexin31, connexin43 and connexin45 expression: Involvement of glycogen synthase kinase 3β/β‐catenin

Although previous reports have examined the function of prostaglandin E₂ (PGE₂) on gap junctions and undifferentiated stem cells, its effects on the reciprocal action of connexin (Cx) isoforms and undifferentiation in embryonic stem cells (ESCs) are unclear. Therefore, we investigated the role of PGE₂ on Cx isoforms and maintenance of mouse ESC undifferentiated state. We have analysed 10 Cx genes, but found nine of them. PGE₂ (50 μM) stimulated Cx31, Cx32, Cx40, Cx43 and Cx45 mRNA expression. Amongst them, PGE₂ maximally stimulated the Cx43 mRNA expression and gap junction inter-cellular coupling. Therefore, we investigated the effect of PGE₂ on Cx43 expression. PGE₂ activated cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation. In addition, treatments of adenylate cyclase activators increased Cx43 expression, but not PI3K/Akt phosphorylation. PGE₂ also inactivated GSK-3β and stimulated active-β-catenin. Furthermore, a ChiP assay demonstrated the association of β-catenin with the Cx26 (as control) and Cx43 promoter. Finally, down-regulation of PGE₂-induced Cx isoforms by AH 6809, Cx31-, Cx43-, Cx45 small interfering (si)RNA and 18α-glycyrrhetinic acid decreased levels of undifferentiated markers of ESCs, including Oct4, FoxD3, Sox2 and SSEA-1, but Nanog did not be down-regulated by Cx43 siRNA. PGE₂ stimulates Cx isoforms via GSK-3β/β-catenin via EP2-receptor-dependent cAMP/PKA and PI3K/Akt in mouse ESCs, thereby partially contributing to the maintenance of their undifferentiated state.

Contractility in Type III Cochlear Fibrocytes Is Dependent on Non-muscle Myosin II and Intercellular Gap Junctional Coupling

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

Chemical Connexin Impairment in the Developing Gonad Associated with Offspring Infertility

A dramatical decline in human male reproductive function has been reported for the past 20 years. Many recent epidemiological, clinical and experimental findings suggest that the reproductive dysfunction could result from prenatal and neonatal chemical compound exposure. Even if numerous studies argue for a relationship between male infertility and environmental and/or occupational exposure, the molecular mechanisms by which these anti-reproductive compounds act are still unclear. Recent findings showed that a family of transmembranous proteins, connexins, regulates numerous physiological functions involved in the development such as cell proliferation, differentiation, migration and apoptosis. In the testis and the ovary, connexins are known to be essential for the establishment and the maintenance of spermatogenesis in males and oogenesis and folliculogenesis in females. Moreover, mutation of connexin genes leads to several developmental human diseases (myelin-related diseases, hearing loss, congenital cataract, skin disorders or more complex syndromes such as the oculodendrodigital dysplasia....) and altered connexin expression, trafficking and degradation are often associated with the tumoral process. We propose, in the present work, to give an overview of connexin expression and intercellular gap junction coupling during development: in preimplantation, implantation and postimplantation embryos. Moreover, we underline the impact of maternal chemical exposure on connexin expression during fetal gonad development and we link this effect to future offspring fertility.

Testicular connexin 43, a precocious molecular target for the effect of environmental toxicants on male fertility

Many recent epidemiological, clinical and experimental findings support the hypothesis that environmental toxicants are responsible for the increasing male reproductive disorders (congenital malformations, declining sperm counts and testicular cancer) over the past 20 years. It has also been reported that exposure to these toxicants, during critical periods of development (fetal and neonatal), represents a more considerable risk for animals and humans than exposure during adulthood. However, the molecular targets for these chemicals have not been clearly identified. Recent studies showed that a family of transmembranous proteins, named connexins, regulates numerous physiological processes involved in testicular development and function, such as Sertoli and germ cell proliferation, differentiation, germ cell migration and apoptosis. In the testis, knockout strategy revealed that connexin 43, the predominant connexin in this organ, is essential for spermatogenesis. In addition, there is evidence that many environmental toxicants could alter testicular connexin 43 by dysregulation of numerous mechanisms controlling its function. In the present work, we propose first to give an overview of connexin expression and intercellular gap junction coupling in the developing fetal and neonatal testes. Second, we underline the impact of maternally chemical exposure on connexin 43 expression in the perinatal developing testis. Lastly, we attempt to link this precocious effect to male offspring fertility.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His–Purkinje network

Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Fenofibrate attenuates contact-stimulated cell motility and gap junctional coupling in DU-145 human prostate cancer cell populations

In the present study, we investigated the effects of fenofibrate on the invasive potential of DU-145 human prostate cancer cells in the context of gap junctional intercellular coupling and the formation of reactive oxygen species. Time-lapse analyses of cell motility, accompanied by tests of cell viability, membrane microviscosity, reactive oxygen species accumulation and the function of gap junctional protein connexin 43 were performed in monolayer cultures of DU-145 cells following fenofibrate administration. Fenofibrate inhibited the motility of DU-145 cells and attenuated gap junctional intercellular coupling in a manner independent of its effects on cell viability, PPARα activation and cell membrane micro-viscosity. Instead, N-acetyl-L-cysteine, a scavenger of reactive oxygen species, restored cell motility and gap junctional coupling in fenofibrate-treated DU-145 cell populations. These data indicate that two parameters crucial for cancer cell metastatic potential, i.e. cell motility and gap junctional coupling, are inhibited by fenofibrate. Thus, fenofibrate affects prostate cancer cell invasion via an orchestrated action on versatile cancer cell properties determining this process. A novel mechanism of anti-invasive activity of fenofibrate, which depends on its interference with cell motility and the function of gap junctions regulated by reactive oxygen species, is suggested.

DU-145 prostate carcinoma cells that selectively transmigrate narrow obstacles express elevated levels of Cx43

The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the “leading front” formation during cancer invasion.