Distribution Of Cx43 Research Articles

Expression and cellular distribution of Cdk4 and Cx43 is related to porcine cumulus cells real-time proliferation

Expression and cellular distribution of Cdk4 and Cx43 is related to porcine cumulus cells real-time proliferation

Melatonin attenuates hypertension-related proarrhythmic myocardial maladaptation of connexin-43 and propensity of the heart to lethal arrhythmias

We hypothesized that the pineal hormone melatonin, which exhibits cardioprotective effects, might affect myocardial expression of cell-to-cell electrical coupling protein connexin-43 (Cx43) and protein kinase C (PKC) signaling, and hence, the propensity of the heart to lethal ventricular fibrillation (VF). Spontaneously hypertensive (SHR) and normotensive Wistar rats fed a standard rat chow received melatonin (40 μg/mL in drinking water during the night) for 5 weeks, and were compared with untreated rats. Melatonin significantly reduced blood pressure and normalized triglycerides in SHR, whereas it decreased body mass and adiposity in Wistar rats. Compared with healthy rats, the threshold to induce sustained VF was significantly lower in SHR (18.3 ± 2.6 compared with 29.2 ± 5 mA; p < 0.05) and increased in melatonin-treated SHR and Wistar rats to 33.0 ± 4 and 32.5 ± 4 mA. Melatonin attenuated abnormal myocardial Cx43 distribution in SHR, and upregulated Cx43 mRNA, total Cx43 protein, and its functional phosphorylated forms in SHR, and to a lesser extent, in Wistar rat hearts. Moreover, melatonin suppressed myocardial proapoptotic PKCδ expression and increased cardioprotective PKCε expression in both SHR and Wistar rats. Our findings indicate that melatonin protects against lethal arrhythmias at least in part via upregulation of myocardial Cx43 and modulation of PKC-related cardioprotective signaling.

Smoking is Associated with Remodeling of Gap Junction in the Rat Heart: Smoker's Paradox Explanation?

In a previous study utilizing the rat model, exposure to tobacco smoke for 5 weeks increased survival after AMI, despite similar age and infarct size between the smokers and nonsmokers, and absence of reperfusion. Thus, this study aimed to analyze the effects of exposure to tobacco smoke on intensity, distribution or phosphorylation of connexin 43 in the rat heart. Wistar rats weighing 100 g were randomly allocated into 2 groups: 1) CONTROL (n = 25); 2) Exposed to tobacco smoke (ETS), n = 23. After 5 weeks, left ventricular morphometric analysis, immunohistochemistry and western blotting for connexin 43 (Cx43) were performed. Collagen volume fraction, cross-sectional areas, and ventricular weight were not statistically different between control and ETS. ETS showed lower stain intensity of Cx43 at intercalated disks ( 2.32 ± 0.19; ETS: 1.73 ± 0.18; p = 0.04). The distribution of CX43 at intercalated disks did not differ between the groups ( 3.73 ± 0.12; ETS: 3.20 ± 0.17; p = 0.18). ETS rats showed higher levels of dephosphorylated form of Cx43 (CONTROL: 0.45 ± 0.11; ETS: 0.90 ± 0.11; p = 0.03). On the other hand, total Cx43 did not differ between control and ETS groups ( 0.75 ± 0.19; ETS: 0.93 ± 0.27; p = 0.58). Exposure to tobacco smoke resulted in cardiac gap junction remodeling, characterized by alterations in the quantity and phosphorylation of the Cx43, in rats hearts. This finding could explain the smoker's paradox observed in some studies.

Density and distribution of connexin 43 in corpus cavernosum tissue from diabetic and hypogonadal patients with erectile dysfunction.

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. 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). 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. 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.

Androgen Signaling Disruption during Fetal and Postnatal Development Affects Androgen Receptor and Connexin 43 Expression and Distribution in Adult Boar Prostate

To date, limited knowledge exists regarding the role of the androgen signaling during specific periods of development in the regulation of androgen receptor (AR) and connexin 43 (Cx43) in adult prostate. Therefore, in this study we examined mRNA and protein expression, and tissue distribution of AR and Cx43 in adult boar prostates following fetal (GD20), neonatal (PD2), and prepubertal (PD90) exposure to an antiandrogen flutamide (50 mg/kg bw). In GD20 and PD2 males we found the reduction of the luminal compartment, inflammatory changes, decreased AR and increased Cx43 expression, and altered localization of both proteins. Moreover, enhanced apoptosis and reduced proliferation were detected in the prostates of these animals. In PD90 males the alterations were less evident, except that Cx43 expression was markedly upregulated. The results presented herein indicate that in boar androgen action during early fetal and neonatal periods plays a key role in the maintenance of normal phenotype and functions of prostatic cells at adulthood. Furthermore, we demonstrated that modulation of Cx43 expression in the prostate could serve as a sensitive marker of hormonal disruption during different developmental stages.

Photoperiod-Dependent Effects of 4-tert-Octylphenol on Adherens and Gap Junction Proteins in Bank Vole Seminiferous Tubules

In the present study we evaluated in vivo and in vitro effects of 4-tert-octylphenol (OP) on the expression and distribution of adherens and gap junction proteins, N-cadherin, β-catenin, and connexin 43 (Cx43), in testes of seasonally breeding rodents, bank voles. We found that in bank vole testes expression and distribution of N-cadherin, β-catenin, and Cx43 were photoperiod dependent. Long-term treatment with OP (200 mg/kg b.w.) resulted in the reduction of junction proteins expressions (P < 0.05, P < 0.01) and their delocalization in the testes of males kept in long photoperiod, whereas in short-day animals slight increase of Cx43 (P < 0.05), N-cadherin, and β-catenin (statistically nonsignificant) levels was observed. Effects of OP appeared to be independent of FSH and were maintained during in vitro organ culture, indicating that OP acts directly on adherens and gap junction proteins in the testes. An experiment performed using an antiestrogen ICI 182,780 demonstrated that the biological effects of OP on β-catenin and Cx43 involve an estrogen receptor-mediated response. Taken together, in bank vole organization of adherens and gap junctions and their susceptibility to OP are related to the length of photoperiod. Alterations in cadherin/catenin and Cx43-based junction may partially result from activation of estrogen receptor α and/or β signaling pathway.

Abstract 18558: Characterization of Cx43-PKP2 Interaction by Direct Stochastic Optical Reconstruction Microscopy

The cardiac intercalated disc is composed of 3 structures involved in the electrical and mechanical coupling of the cardiomyocytes: gap junctions, desmosomes and adherens junctions. Several studies suggest that these structures interact at the intercalated disc. In particular a crosstalk between the desmosomal and gap junction proteins PKP2 and Cx43 has been demonstrated. To characterize the proximity of these two proteins we used a Duolink assay. With this method, we demonstrated that Cx43 and PKP2 reside in the periphery of the adult cardiac intercalated disc at distances &lt;40 nm. To further investigate this interaction we performed direct stochastic optical reconstruction microscopy (dSTORM) experiments. This new approach is a valuable methodology for studying the cellular architecture at the nanoscale. The dSTORM uses standard fluorescent dyes that are controlled by the laser excitation intensity and the imaging buffer conditions can be induced to switch on and off (“blink”). For these experiments, 2.000 images were recorded in a stack, each of these images showing a different subset of the fluorescent dyes active. The analysis of these images (the localization of each blink) gave a reconstructed super-resolution image with a resolution of 10nm. The images obtained in isolated neonatal cardiomyocytes showed that 43.5% of the Cx43 spots/clusters analysed (n=200 of 5 experiments) are an average of 11.38 nm apart from PKP2 (SE= 1.67), 62% of them being in contact (0nm). In conclusion, these two techniques allow us to visualize the interaction and distribution of Cx43 and PKP2 at the nanoscale in the intercalated disc and suggest that a fraction of PKP2 molecules interact directly with Cx43.

DYNAMIC ALTERATIONS OF CONNEXIN 43, MATRIX METALLOPROTEINASE-2 AND TISSUE INHIBITOR OF MATRIX METALLOPROTEINASE-2 DURING VENTRICULAR FIBRILLATION IN DOGS

ObjectivesWe aimed to investigate the dynamic alterations of cardiac connexin 43 (Cx 43), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the setting of different ventricular fibrillation (VF)...

REMODELLING OF CONNEXIN 43 IN ATRIAL MYOCARDIUM OF PATIENTS WITH ATRIAL FIBRILLATION

ObjectivesRemodelling of connexins was found accompanying with atrial fibrillation. The aim of the study is to investigate whether it is the remodelling of connexin 43 (Cx43) plays an important role...

Reversible atrial gap junction remodeling during hypoxia/reoxygenation and ischemia: a possible arrhythmogenic substrate for atrial fibrillation

Alteration of cardiomyocyte gap-junctions and component connexins (Cx) has been suggested to contribute to the development of atrial fibrillation (AF), including postoperative AF. We tested different possible stimuli, such as hypoxia and ischemia, influencing Cx43 and Cx40 expression and distribution in cultured atrial cells (HL-1) and reversibility of these processes after reoxygenation. Western-blot analysis and immunostaining using anti-Cx43, anti-Cx40 and anti-zonula occludens polyclonal antibodies were performed. HL-1 cells exposed to hypoxia for 24 and 48 h showed a reduction of Cx43 protein levels by 75% and 90% respectively (p < 0.001). During reoxygenation following 24 h of hypoxia, Cx43 levels increased to reach the basal level within 48 h, while they remained at low level during reoxygenation following 48 h of hypoxia. Furthermore, atrial cardiomyocytes subjected to simulated ischemia (SI) were incubated in normoxic and hypoxic conditions for 3, 6, 9, 12 h. Atrial cardiomyocytes subjected to SI in addition to normoxia showed a progressive reduction of Cx43 levels beginning from 3 h. During SI and hypoxia, atrial Cx43 levels showed an initial decrease after 3 h with a subsequent rescue beginning from 6 h of exposure (p = 0.001). Hypoxia and ischemia per se downregulate Cx43 protein expression in atrial cardiomyocytes, but protein downregulation is reversible, depending on hypoxia duration and the association of the two triggers. These alterations characterize several conditions and might contribute to the generation of an arrhythmogenic substrate leading to AF onset and/or maintenance.

The expression and distribution of Connexin43 are further disrupted in no-reflow region of a swine model

Introduction and objectivesIschemia and ischemia/reperfusion can dephosphorylate and redistribute Connexin 43 (Cx43). But it is unknown whether no-reflow phenomenon has an effect on the expression and distribution of Cx43 after...

Essential role of connecxin 43 in ischaemic postconditioning during myocardial ischaemia-reperfusion

BackgroundRecent study show that ischaemic postconditioning (IPC) may provide a new therapy for reperfusion after myocardial infarction. To evaluate the cardio protection of IPC during myocardial ischaemia-reperfusion by measuring the...

CONNEXIN 43 EXPRESSION IN THE HUMAN COCHLEA: AN IMMUNOHISTOCHEMISTRY STUDY

BackgroundMutations in the gene GJA1 which encodes gap junction protein connexin 43 (Cx43) have been linked to nonsyndromic prelingual deafness in humans. The protein forms gap junction channels (GJChs), creating intercellular pathways for ions and small molecules – such as second messengers (IP3, cAMP, cGMP, and others), diverse metabolites (e.g. glucose, amino acids, glutathione, ATP) and neuroprotectant (e.g. adenosine) – to reach neighboring cells. Cx43 in glial cells of the central nervous system (CNS) and in satellite glial cells of the peripheral nociceptive sensory ganglia has been studied for its structure as well as function in signal transduction and neuron protection. Distribution of Cx43 in the human cochlea is an important unresolved issue.Material and MethodsFor the first time we analysed, using immunofluorescence and confocal immunofluorescence, the expression and distribution of Cx43 in 5 normal fresh human cochleae obtained at surgery for removal of giant posterior cranial fossa meningioma.ResultsIn the lateral wall of the cochlea, Cx43 was expressed mainly in the basal cell layer of the stria vascularis and the fibrocytes of the spiral ligament. In the organ of Corti, Cx43 antibody strongly stained both inner and outer pillar cells, the covering layer cells of the basilar membrane and the supporting cells underneath outer hair cells. In the spiral ganglion, Cx43 expression was located in the satellite glial cells (SGCs) surrounding type I neurons.ConclusionsCx43 expression was found in human cochleae including the lateral wall, organ of Corti and spiral ganglion satellite cells. Potential roles played by GJChs built up by Cx43 in maintaining homeostasis of the cochlea and protection of neurons are speculated.

No-reflow disrupts the expression and distribution of Connexin 43 in a swine model

Introduction and objectivesIschemia and ischemia/reperfusion can dephosphorylate and redistribute Connexin 43 (Cx43). But it is unknown whether no-reflow phenomenon has an effect on the expression and distribution of Cx43 after acute infarction and reperfusion. Methods21 open-chest pigs were divided into three groups. Left anterior descending artery (LAD) occlusion for 90min before 180min of reperfusion was made in ischemia/reperfusion group. The pigs in ischemia groups were either subjected to LAD ligation for 90min or for 270min. No-reflow and risk regions were determined pathologically by dye staining. Cx43 expression was measured by western blotting and quantitative RT-PCR analysis. Cx43 spatial distribution was shown by immunofluorescence examination. ResultsThe content of phosphorylated and mRNA of Cx43 were higher in reflow region than in the no-reflow or sustained ischemic region. The distribution of Cx43 was also altered in no-reflow region. ConclusionsThere are some differences in synthesis, expression and distribution of myocardial Cx43 at microvascular level after ischemia/reperfusion. Cx43 is partially rephosphorylated with reperfusion only in the reflow myocardium.

Effect of antiarrhythmic peptide on ventricular arrhythmia induced by lysophosphatidic acid

To investigate the effect and potential mechanism of lysophosphatidic acid (LPA) and antiarrhythmic peptide (AAP10) on rabbit ventricular arrhythmia. Twenty-four rabbits were randomly divided into three groups (n = 8 each): control group, LPA group and AAP10 + LPA group. Using arterially perfused rabbit ventricular wedge preparations, transmural ECG and action potentials from both endocardium and epicardium were simultaneously recorded in the whole process of all experiments with two separate floating microeletrodes. The incidence of ventricular arrhythmia post S1S2 stimulation was recorded. Protein levels of nonphosphorylated Cx43 and total Cx43 were evaluated by Western blot. The distribution of nonphosphorylated Cx43 was observed by confocal immunofluorescence microscopy. Compared with the control group, the QT interval, endocardial action potential duration, transmural repolarization dispersion (TDR) and incidence of ventricular arrhythmia were significantly increased and nonphosphorylated Cx43 expression was significantly upregulated in the LPA group. Compared with the LPA group, cotreatment with AAP10 can reduce the QT interval, endocardial action potential duration, TDR and incidence of ventricular arrhythmia (25.0% vs 62.5%, P < 0.01) and downregulate nonphosphorylated Cx43. LPA could promote the arrhythmia possibly by upregulating nonphosphorylated Cx43 and subsequent gap junction transmission inhibition. Gap junction enhancer AAP10 could attenuate the pro-arrhythmic effect of LPA probably by downregulating myocardial nonphosphorylated Cx43 expression.

Inhibition of connexin43 dephosphorylation is involved in protective effects of diltiazem on cardiac function during hypoxic injury.

Connexin43 (Cx43), a gap junction protein, mediates cell-cell communication via electrical and chemical coupling. Ischemic stress of the cardiac muscle interrupts intercellular communication by changing the distribution and phosphorylation status of Cx43. This may be a factor contributing to reentrant arrhythmia. The calcium channel blocker diltiazem is known for its protective and anti-arrhythmogenic effect in ischemic heart disease. In this study, we assess the effect of diltiazem pretreatment upon ischemia-induced phosphorylation change of Cx43. Langendorff preparations of isolated Wistar rat hearts were performed. After stabilization, hearts were treated with (D+) or without diltiazem (D⁻), then subjected to hypoxia-reoxygenation. After perfusion, the left ventricle was prepared for immunocytochemistry and immunoblot analysis. During perfusion, left ventricular function was better in the D+ group than the D⁻ group. Immunostaining of the heart indicated that dephosphorylated Cx43 (dpCx43) signal was increased after hypoxic perfusion, and this finding was confirmed by immunoblot data. The quantitative area analysis of dpCx43 using the immunohistochemical approach showed that the dpCx43-positive area was enlarged, as the hypoxic perfusion time was longer, and it was reduced by pretreatment of diltiazem. There was a negative correlation between the dpCx43 area and %RPP (rate-pressure product), calculated by heart rate and contraction force. Pretreatment of diltiazem could protect the heart against hypoxia-reoxygenation injury by attenuation of dephosphorylation of Cx43. The anti-arrhythmic mechanism of diltiazem may include the preservation of phosphorylation status of Cx43 after hypoxia-reoxygenation injury.

CX43 expression, phosphorylation, and distribution in the normal and autoimmune orchitic testis with a look at gap junctions joining germ cell to germ cell

Spermatogenesis requires connexin 43 (Cx43).This study examines normal gene transcription, translation, and phosphorylation of Cx43 to define its role on germ cell growth and Sertoli cell's differentiation, and identifies abnormalities arising from spontaneous autoimmune orchitis (AIO) in mink, a seasonal breeder and a natural model for autoimmunity. Northern blot analysis detected 2.8- and a 3.7-kb Cx43 mRNA bands in seminiferous tubule-enriched fractions. Cx43 mRNA increased in seminiferous tubule-enriched fractions throughout development and then seasonally with the completion of spermatogenesis. Cx43 protein levels increased transiently during the colonization of the tubules by the early-stage spermatocytes. Cx43 phosphorylated (PCx43) and nonphosphorylated (NPCx43) in Ser368 decreased during the periods of completion of meiosis and Sertoli cell differentiation, while Cx43 mRNA remained elevated throughout. PCx43 labeled chiefly the plasma membrane except by stage VII when vesicles were also labeled in Sertoli cells. Vesicles and lysosomes in Sertoli cells and the Golgi apparatus in the round spermatids were NPCx43 positive. A decrease in Cx43 gene expression was matched by a Cx43 protein increase in the early, not the late, phase of AIO. Total Cx43 and PCx43 decreased with the advance of orchitis. The study makes a novel finding of gap junctions connecting germ cells. The data indicate that Cx43 protein expression and phosphorylation in Ser368 are stage-specific events that may locally influence the acquisition of meiotic competence and the Sertoli cell differentiation in normal testis. AIO modifies Cx43 levels, suggesting changes in Cx43-mediated intercommunication and spermatogenic activity in response to cytokines imbalances in Sertoli cells.

E0226 Effects of bone marrow mesenchymal stem cells on electrophysiological function in rats with myocardial infarction

ObjectiveConcerns that intramyocardial delivery of immature cells could cause potentially life-threatening ventricular arrhythmias have been repeatedly raised. The aim of this study is to investigate the electrophysiological and arrhythmogenic effects...

Changes in myocardial connexin 43 during ventricular fibrillation

To observe changes in connexin 43 (Cx43) after ventricular fibrillation (VF) and the effects of rotigaptide (ZP123) on Cx43. Thirty domestic pigs were randomly assigned to three groups (10 in each group): sham group, model group and ZP123 group. VF was induced by an 80 V AC transthoracic shock for 5 seconds with the use of subcutaneous needles. Before the induction of VF, animals in ZP123 group were administered with ZP123 (1 μg/kg bolus+10 μg×kg(-1)×h(-1) dissolved in 50 ml normal saline and pumped for 15 minutes ). Those in model group received 50 ml normal saline pumped for 15 minutes. For pigs in sham group VF was not induced and no fluid was given. After 8 minutes of VF, animals were euthanized and myocardial tissues were harvested along the long axis of each left ventricular free wall. Immunofluorescence combined with laser scanning confocal microscope was used to detect the distribution of Cx43. Western blotting was used for quantitative determination of Cx43 protein expression. Immunofluorescence signals for Cx43 in sham group were strong and regularly distributed. In model group, Cx43 signals were weak and distributed in heterogeneity, while in ZP123 group, Cx43 signals were enhanced and their distribution were much more orderly. Compared with sham group, the percentage area and the optical densities (A value) of Cx43 fluorescence signals and Cx43 protein expression were significantly decreased in model group [the percentage area: (0.64±0.36)% vs.(1.27±0.19)%, A value: 15 201± 2 613 vs. 30 634±4 975, Cx43 protein expression: 0.72±0.08 vs. 0.97±0.07, all P<0.05]. The level of Cx43 expression in ZP 123 group [the percentage area (0.96±0.16)%, A value 22 100±4 404, Cx43 protein expression 0.82±0.04] was much higher than model group (all P<0.05). During VF, down-regulation of myocardial Cx43 expression occurred, which could be attenuated by administration of ZP123.

E0077 Effect of pulsed alternating microcurrentstimulation on communication junction function of cocultured rat mesenchymal stem cells and cardiac muscle cells

ObjectiveThis study was to investigate the effect of pulsed alternating micro-current (PAMC) stimulation on the communication junction function of co-cultured rat mesenchymal stem cells (MSCs) and cardiac muscle cells by...