Gap Junction Connexin Research Articles

Electron microscopy and functional analysis of recombinant innexin gap junctions

Innexin is a molecular component of invertebrate gap junctions, which have an important role in neural and muscular electrical activity in invertebrates. Although the structure of vertebrate connexin26 was revealed by X-ray crystallography [1], the structure of innexin channels remains poorly understood. To study the structure of innexin gap junction channels, we expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels, and characterized their molecular dimensions and channel permeability using electron microscopy (EM) and a fluorescent dye transfer assay, respectively [2]. Negative-staining and thin-section EM of isolated INX-6 gap junction plaques revealed a loosely packed hexagonal lattice. We performed single particle analysis of purified INX-6 channels with negative-staining and cryo EM. Based on the negative-stain EM images, the class average of the junction form had a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class average of the hemichannels had diameters of up to 140 Å in the presence of detergent micelles. Cryo EM images revealed rotational peaks that could be related to the INX-6 subunits. Structural analysis of the reconstituted INX-6 channels with single particle analysis and electron tomography suggested that the oligomeric number of the INX-6 channel was distinct from that of the dodecameric connexin channel. Dye transfer experiments indicated that the INX-6-GFP-His channels were permeable to 3-kDa and 10-kDa dextran-conjugated tracers. These findings indicate that INX-6 channels have a characteristic oligomer component that differs from that in connexin gap junction channels.

Changes in gap junction expression and function following ischemic injury of spinal cord white matter.

Gap junctions are widely present in spinal cord white matter; however, their role in modulating the dynamics of axonal dysfunction remains largely unexplored. We hypothesized that inhibition of gap junctions reduces the loss of axonal function during oxygen and glucose deprivation (OGD). The functional role of gap junctions was assessed by electrophysiological recordings of compound action potentials (CAPs) in Wistar rat spinal cord slices with the sucrose gap technique. The in vitro slices were subjected to 30-min OGD. Gap junction connexin (Cx) mRNA expression was determined by qPCR and normalized to β-actin. A 30-min OGD resulted in reduction of CAPs to 14.8 ± 4.6% of their pre-OGD amplitude (n = 5). In the presence of gap junction blockers carbenoxolone (Cbx; 100 μM) and 1-octanol (Oct; 300 μM), the CAP reduction in OGD was to only 35.7 ± 5.7% of pre-OGD amplitude in Cbx (n = 9) and to 37.4 ± 8.9% of pre-OGD amplitude in Oct (n = 10). Both drugs also noticeably prolonged the half-decline time of CAP amplitudes in OGD from 6.0 min in no-drug conditions to 9.6 min in the presence of Cbx and to 7.7 min in the presence of Oct, suggesting that blocking gap junctions reduces conduction loss during OGD. With application of Cbx and Oct in the setting of OGD, expression of Cx30 and Cx43 mRNA was downregulated. Our data provide new insights into the role of gap junctions in white matter ischemia and reveal the necessity of a cautious approach in determining detrimental or beneficial effects of gap junction blockade in white matter ischemia.

Amplification of human platelet activation by surface pannexin‐1 channels

BackgroundPannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ∼1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown.ObjectiveTo determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques.MethodsPanx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca2+ influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL−1 apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels.ResultsPANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca2+ influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors.ConclusionsPanx1 is expressed on human platelets and amplifies Ca2+ influx, ATP release and aggregation through the secondary activation of P2X1 receptors. We propose that Panx1 represents a novel target for the management of arterial thrombosis.

Prophase I arrest of mouse oocytes mediated by natriuretic peptide precursor C requires GJA1 (connexin-43) and GJA4 (connexin-37) gap junctions in the antral follicle and cumulus-oocyte complex.

Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMPs) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface.

Role of Connexins and Pannexins in Ischemic Stroke

Synaptic plasticity requires careful synchronization and coordination of neurons and glial cells via various mechanisms of intercellular communication. Among them, are those mediated by i) connexin gap junction channels (GJCs), ii) connexin hemichannels and iii) pannexin channels. Whereas GJCs directly communicate the cytoplasm of contacting cells and coordinate electric and metabolic activities, connexin hemichannels and pannexin channels serve as diffusional pathways for ions and small molecules between the intra- and extracellular compartments. A growing body of evidence has revealed that intercellular communication could be critical in the spread of protective and/or deleterious signals during stroke. Here, we review current findings on the regulation of connexin- and pannexin-based channels in ischemic stroke and how they contribute to cell damage observed in pathology. Depending on intensity of the ischemic event, brain region and connexin subtype expressed, GJCs may provide proper diffusion of energy metabolites and dissipation of toxic substances, whereas, in other circumstances, they could increase damage by spreading toxic molecules. Alternatively, connexin hemichannel and pannexin channel opening may favor the release of neurotoxic substances (e.g., glutamate), but in other cases, they may confer neuroprotection against an ischemic episode by the phenomenon of ischemic preconditioning. Development of new drug modulators using in silico devices for connexin and pannexin-based channels will be crucial for future therapies against stroke.

Involvement of Connexin43 in the Infrasonic Noise-Induced Glutamate Release by Cultured Astrocytes

Infrasonic noise/infrasound is a type of environmental noise that threatens public health as a nonspecific biological stressor. Glutamate-related excitotoxicity is thought to be responsible for infrasound-induced impairment of learning and memory. In addition to neurons, astrocytes are also capable of releasing glutamate. In the present study, to identify the effect of infrasound on astroglial glutamate release, cultured astrocytes were exposed to infrasound at 16 Hz, 130 dB for different times. We found that infrasound exposure caused a significant increase in glutamate levels in the extracellular fluid. Moreover, blocking the connexin43 (Cx43) hemichannel or gap junction, decreasing the probability of Cx43 being open or inhibiting of Cx43 expression blocked this increase. The results suggest that glutamate release by Cx43 hemichannels/gap junctions is involved in the response of cultured astrocytes to infrasound.

E-cadherin and β-catenin adhesion proteins correlate positively with connexins in colorectal cancer.

The majority of solid cancers present with qualitative and quantitative aberrations of adhesion proteins, including E-cadherin and β-catenin, and connexin (Cx) gap junction proteins, which is consistent with alterations in the expression and location of such proteins in neoplastic cells. Since there are no data on the correlation between adhesion proteins and Cxs in human colorectal cancer (CRC), the aim of the present study was to evaluate the expression and correlation between these proteins. Tissue specimens were obtained from 151 cases of surgically removed colorectal adenocarcinomas. The samples were examined by immunohistochemistry with the use of antibodies against E-cadherin, β-catenin and the three Cxs: Cx26, Cx32 and Cx43. The aberrant expression of the studied adhesion proteins (primarily cytoplasmic for E-cadherin and cytoplasmic and/or nuclear for β-catenin) was observed, whereas only a minority of cases revealed normal membranous distribution of the labeling. The present study is the first in the literature to reveal a correlation between the expression of E-cadherin and β-catenin and the examined Cxs in CRC in humans. The positive correlation between the Cxs, particularly Cx26 and Cx32, and the adhesive proteins occurred in patients without lymph node metastases and in the moderately differentiated tumors (G2). Such a dependency was not observed in the analysis of the correlation between Cx43 and E-cadherin. However, a positive correlation between these proteins was observed in patients with lymph nodes metastases. Additionally, a link between the expression of these adhesion proteins was observed. The present study indicates, for the first time, that the expression of adhesion proteins, E-cadherin and β-catenin, is closely associated with the expression of three studied Cxs in CRC, and that this correlation may improve an understanding of the carcinogenic process in this cancer.

Rapid electrical stimulation causes alterations in cardiac intercellular junction proteins of cardiomyocytes

The intercellular junctions contain two complexes, adhesion junctions (AJ) and connexin (Cx) gap junctions (GJs). GJs provide the pathway for intercellular current flow. AJs mediate normal mechanical coupling and play an important role in the stability of GJs. We investigated the effects of rapid electrical stimulation (RES) on cardiac intercellular junctions, especially β-catenin and Cx43 alterations. We also studied the effects of ANG II receptor blockade on intercellular junction remodeling. Neonatal rats were euthanized by decapitation, and cardiomyocytes were prepared, cultured, and subjected to RES. We used real-time PCR, western blot analysis, and immunohistochemical methods. Conduction properties were examined by an extracellular potential mapping system. Cx43 protein expression in cardiomyocytes was significantly increased after 60 min. β-Catenin expression in the total cell fraction was significantly increased after 30 min. The expression level of β-catenin in the nucleus, which functions as a T cell factor/lymphocyte enhancer binding factor transcriptional activator of Cx43 with its degradation regulated by glycogen synthase kinase-3β, was dramatically increased after 10 min. Conduction velocity was increased significantly by RES for 60 min. Olmesartan prevented most these effects of RES. We showed an increase of phosphorylated glycogen synthase kinase-3β, which is phosphorylated by activated MAPKs and inhibits β-catenin degradation, was attenuated by olmesartan. The changes in β-catenin precede Cx43 GJ remodeling and might play an important role in the formation and stability of GJs. Olmesartan might be a new upstream arrhythmia therapy by modulating intercellular junction remodeling through the β-catenin signaling pathway.

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.

Structure and closure of connexin gap junction channels

Connexin gap junctions comprise assembled channels penetrating two plasma membranes for which gating regulation is associated with a variety of factors, including voltage, pH, Ca(2+), and phosphorylation. Functional studies have established that various parts of the connexin peptides are related to channel closure and electrophysiology studies have provided several working models for channel gating. The corresponding structural models supporting these findings, however, are not sufficient because only small numbers of closed connexin structures have been reported. To fully understand the gating mechanisms, the channels should be visualized in both the open and closed states. Electron crystallography and X-ray crystallography studies recently revealed three-dimensional structures of connexin channels in a couple of states in which the main difference is the conformation of the N-terminal domain, which have helped to clarify the structure in regard to channel closure. Here the closure models for connexin gap junction channels inferred from structural and functional studies are described in the context of each domain of the connexin protein associated with gating modulation.

Roles and regulation of lens epithelial cell connexins

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.

Hyperthermia differently affects connexin43 expression and gap junction permeability in skeletal myoblasts and HeLa cells.

Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK) in hyperthermia-induced changes of connexin43 (Cx43) expression and permeability of Cx43 gap junctions (GJs) in the rabbit skeletal myoblasts (SkMs) and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h) enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC). In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

Progress in gap junctional connexin 43 and myocardial ischemia/reperfusion injury

Background The mechanism of myocardial ischemia/reperfusion (I/R) involves in peroxidation,calcium overload,mitochondria injury,myocardial cells apoptosis and so on.Recent sdudies indicate that the gap junctional(GJ) intercellular communication of myocardial cells may play a role in myocardial I/R injury.Objective This article summarized the relationship between the GJ connexin (Cx) 43 and myocardial I/R injury.Content Cx43 could protected myocardium from I/R injury.The mechanism may be related with calcium overload,mitochondria injury,myocardial cells apoptosis and so on.Trend Researches in future should be taken to further explore of the relation between Cx43 and apoptosis,and strengthen the cilinial applications. Key words: Connexin 43; Gap Junctions; Myocardial reperfusion injury

Abstract 19069: Autoregulation of Connexin43 Gap Junction Formation by Internally Translated Isoforms

During each heartbeat, cell-cell electrical coupling via connexin 43 (Cx43) gap junctions allows billions of individual cardiomyocytes to synchronously contract. The Cx43 protein turns over rapidly, rendering regulation of Cx43 trafficking a critical and continuous cellular need. Altered Cx43 trafficking during heart disease disrupts intercellular coupling and can contribute to the arrhythmias of sudden cardiac death. Better understanding of how Cx43 channels are transported to the cell surface will lead to therapies that can preserve normal electrical coupling of diseased heart muscle. We have identified a novel regulatory mechanism whereby internal translation of the coding sequence of Cx43 ( GJA1 ) mRNA generates N-terminally truncated isoforms that regulate trafficking of full length Cx43 channels. Biochemical analysis of human heart and cell lines reveals at least four additional Cx43 isoforms, with a 20 kDa isoform predominating. We find that in frame AUG codons within GJA1 mRNA represent isoform translation initiation sites and that their ablation arrests trafficking of full-length Cx43. The predominant 20 kDa isoform is sufficient to rescue this trafficking defect when expressed in trans , implicating an essential role as a trafficking chaperone for Cx43. Consistent with these data, expression of Cx43 truncated isoforms is enhanced through inhibition of the PI3K/AKT/mTOR pathway in cardiomyocytes, and concomitantly increases Cx43 gap junction plaque size. Such cap-independent internal translation initiation events may represent a common mechanism for auto-regulation of membrane protein trafficking which is likely altered during stress and, based on these studies, represents a potent target for therapeutic preservation of intercellular coupling in heart disease.

Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5nM or 75nM) and cultured for 4 or 8days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P<0.05) at 4days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P<0.05) but CX43 protein increased (P<0.05) at 4days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P<0.05) but no changes in Bax expression were observed after 4days of DMBA exposures. In contrast, after 8days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia.

Autoregulation of Connexin43 Gap Junction Formation by Internally Translated Isoforms

During each heartbeat, intercellular electrical coupling via connexin43 (Cx43) gap junctions enables synchronous cardiac contraction. In failing hearts, impaired Cx43 trafficking reduces gap junction coupling, resulting in arrhythmias. Here we report that internal translation within Cx43 (GJA1) mRNA occurs, resulting in truncated isoforms that autoregulate Cx43 trafficking. We find that at least four truncated Cx43 isoforms occur in the human heart, with a 20kDa isoform predominating. In-frame AUG codons within GJA1 mRNA are the translation initiation sites and their ablation arrests trafficking of full-length Cx43. The 20kDa isoform is sufficient to rescue this trafficking defect in trans, suggesting it as a trafficking chaperone for Cx43. Limiting cap-dependent translation through inhibition of mTOR enhances truncated isoform expression, increasing Cx43 gap junction size. The results suggest that internal translation is a mechanism of membrane protein autoregulation and a potent target for therapies aimed at restoring normal electrical coupling in diseased hearts.

Protection of a Ceramide Synthase 2 Null Mouse from Drug-induced Liver Injury: ROLE OF GAP JUNCTION DYSFUNCTION AND CONNEXIN 32 MISLOCALIZATION

Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as d-galactosamine, CCl4, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.

Cardiac ß -catenin Alterations Play an Important Role in Gap Junction Remodeling of Cardiomyocytes Exposed to Rapid Electrical Stimulation

The intercalated disc (ID) contains two complexes, adhesion junction (AJ) and connexin (Cx) gap junction (GJ). GJs provide the pathway for intercellular current flow, and AJ mediates normal mechanical coupling and plays an important role in the stability of GJs.

Alterations in cardiac beta-catenin precede connexin gap junction remodeling in cardiomyocytes exposed to rapid electrical stimulation

The Intercalated Disc (ID) contains two complexes, Adhesion Junction (AJ) and Connexin (Cx) Gap Junction (GJ). GJs provide the pathway for intercellular current flow, enabling coordinated action potential propagation and contraction. AJ mediates normal mechanical coupling between cardiomyocytes and plays an important role in the stability of GJ. Purpose: The purpose of this study was to study ID remodeling, especially β-catenin, which is one of the component proteins of AJ, and Cx43 alterations caused by Rapid Electrical Stimulation (RES) in cardiomyocytes. In addition, we investigated the effects of olmesartan, Angiotensin II receptor blockade on ID remodeling. Methods: Cultured neonatal rat ventricular myocytes were subjected to RES at 3.0 Hz for up to 60 min. We used real-time PCR, western blotting, and immunohistochemical methods to investigate expressions. Conduction properties of the cultured ventricular myocytes were examined by extracellular potential mapping by using a specifically designed multi-electrode array system. Results: Cx43 protein expression in cardiomyocytes was significantly increased (x 1.5 vs 0 min. p<0.01) after 60 min RES. Beta-catenin expression in total cell fraction was also significantly increased (x 1.5 vs 0 min. p<0.05) after 30 min. Interestingly, the expression level of β-catenin at nucleus, which functions as T Cell Factor/Lymphocyte Enhancer binding Factor (LEF/TCF) transcriptional activator of Cx43 and its degradation is modulated by glycogen synthase kinase 3β(GSK3β), was dramatically increased (x 1.4 vs 0 min. p<0.05) after 10 min. Conduction velocity was increased significantly by RES for 60min (25% increase). Treatment of cardiomyocytes with olmesartan prevented most these effects of RES. Moreover, we showed that an increase of phospholylated GSK3β, which is phosphorylated by activated mitogen-activated protein kinases due to RES and inhibits β-catenin degradations, was attenuated by olmesartan. Conclusions: The changes in AJ protein β-catenin precede Cx43 GJ remodeling and might play an important role in the formation and stability of GJs during RES exposure to cardiomyocytes. Olmesartan might be a new upstream arrhythmia therapy by modulating both AJ and GJ remodeling through β-catenin signaling pathway.

Silencing of desmoplakin decreases connexin43/Nav1.5 expression and sodium current in HL-1 cardiomyocytes

Desmosomes and gap junctions are situated in the intercalated disks of cardiac muscle and maintain the integrity of mechanical coupling and electrical impulse conduction between cells. The desmosomal plakin protein, desmoplakin (DSP), also plays a crucial role in the stability of these interconnected components as well as gap junction connexin proteins. In addition to cell‑to‑cell junctions, other molecules, including voltage‑gated sodium channels (Nav1.5) are present in the intercalated disk and support the contraction of cardiac muscle. Mutations in genes encoding desmosome proteins may result in fatal arrhythmias, including arrhythmogenic right ventricular cardiomyopathy (ARVC). Therefore, the aim of the present study was to determine whether the presence of DSP is necessary for the normal function and localization of gap junction protein connexin43 (Cx43) and Nav1.5. To examine this hypothesis, RNA interference was utilized to knock down the expression of DSP in HL‑1 cells and the content, distribution and function of Cx43 and Nav1.5 was assessed. Western blotting and flow cytometry experiments revealed that Cx43 and Nav1.5 expression decreased following DSP silencing. In addition, immunofluorescence studies demonstrated that a loss of DSP expression led to an abnormal distribution of Cx43 and Nav1.5, while scrape‑loading dye/transfer revealed a decrease in dye transfer in DSP siRNA‑treated cells. The sodium current was also recorded by the whole‑cell patch clamp technique. The results indicated that DSP suppression decreased sodium current and slowed conduction velocity in cultured cells. The present study indicates that impaired mechanical coupling largely affects electrical synchrony, further uncovering the pathogenesis of ARVC.