Gap Junction Channels Research Articles

Novel Antidepressant Mechanism of Ginsenoside Rg1 in Regulating the Dysfunction of the Glutamatergic System in Astrocytes.

Ginsenoside Rg1, a traditional Chinese medicine monomer, has been shown to have antidepressant effects. We previously found that Rg1 exerts antidepressant effects by improving the gap junction channels (GJCs) dysfunction; however, the downstream mechanisms through which Rg1 ameliorates GJC dysfunction remain unclear. Since hemichannels directly release glutamate, GJC dysfunction decreases the expression levels of glutamate transporters in astrocytes, and glutamatergic system dysfunction plays an essential role in the pathogenesis of depression. The glutamatergic system may be a potential downstream target of Rg1 that exerts antidepressant effects. Therefore, in this study, we aimed to determine the downstream mechanisms by which Rg1 ameliorated GJC dysfunction and exerted its antidepressant effects. Corticosterone (CORT) is used to mimic high glucocorticoid levels in patients with depression in vitro. Primary cortical astrocytes were isolated and phosphorylation of connexin43 (Cx43) as well as the functions of hemichannels, GJCs, and the glutamatergic system were evaluated after drug treatment. Rg1 pretreatment reversed the anomalous activation of Cx43 phosphorylation as well as the dysfunction of hemichannels, GJCs, and the glutamatergic system induced by CORT. These results suggest that Rg1 can ameliorate CORT-induced dysfunction of the glutamatergic system in astrocytes by potentially reducing Cx43 phosphorylation and inhibiting opening of hemichannels, thereby improving GJC dysfunction.

Genome-wide identification of gap junction (connexins and pannexins) genes in black rockfish (Sebastes schlegelii): Evolution and immune response mechanism following challenge

Cell-to-cell communication through gap junction channels is very important to coordinate the functions of cells in all multicellular biological tissues. It allows the direct exchange of ions and small molecules (including second messengers, such as Ca2+, IP3, cyclic nucleotides, and oligonucleotides). In this study, a total of 48 members of the gap junction (GJ) protein family were identified from Sebastes schlegelii. In S. schlegelii, GJ proteins were classified into two types, connexin, and pannexin, and then connexins were divided into five subfamilies. The naming of 48 genes was verified through phylogenetic analysis and syntenic analysis. The connexin proteins contained four transmembrane fragments and two extracellular loops, the lengths of the intracellular loop and C-terminal was quite different, and the C-terminal region was highly variable after post-translational modification. PPI analysis showed that GJs interacted with tight junctions, adhesive junctions, and cell adhesions to form a complex network and participated in cell-cell junction organization, ATP binding, ion channel, voltage-gated conduction, wnt signaling pathway, Fc-γ receptor signaling pathway, and DNA replication. In addition, the S. schlegelii GJ protein was highly expressed in intestinal tissues and remarkably regulated after Edwardsiella tarda and Streptococcus iniae infection. The expression of GJs in intestinal cells of S. schlegelii was significantly regulated by LPS and poly (I:C), which was consistent with the results of intestinal tissue stimulation by pathogens. In conclusion, this study can provide valuable information for further research on the function of S. schlegelii GJ proteins.

Channel-independent function of UNC-9/Innexin in spatial arrangement of GABAergic synapses in C. elegans.

Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in Caenorhabditis elegans, in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.

Early and extensive alterations of glial connexins, distal oligodendrogliopathy type demyelination, and nodal/paranodal pathology are characteristic of multiple system atrophy.

The pathological hallmark of multiple system atrophy (MSA) is aberrant accumulation of phosphorylated α-synuclein in oligodendrocytes, forming glial cytoplasmic inclusions (GCIs). Extensive demyelination occurs particularly in the olivopontocerebellar and striatonigral pathways, but its precise mechanism remains elusive. Glial connexins (Cxs), which form gap junction channels between astrocytes and oligodendrocytes, play critical roles in myelin maintenance, and have not been studied in MSA. Therefore, we immunohistochemically investigated glial Cx changes in the cerebellar afferent fibers in 15 autopsied patients with MSA. We classified demyelinating lesions into three stages based on Klüver-Barrera staining: early (Stage I), intermediate (Stage II), and late (Stage III) stages showing subtle, moderate, and severe myelin reduction, respectively. Myelin-associated glycoprotein, but not myelin oligodendrocyte glycoprotein, was preferentially decreased in Stage I, suggesting distal oligodendrogliopathy type demyelination. Accumulation of phosphorylated α-synuclein in oligodendrocytes was frequently seen in Stage I but less frequently observed in Stages II and III. Tubulin polymerization-promoting protein (TPPP/p25α)-positive oligodendrocytes were preserved in Stage I but successively decreased in Stages II and III. Even at Stage I, Cx32 was nearly absent from myelin, despite the relative preservation of other nodal proteins, such as neurofascin, claudin-11/oligodendrocyte-specific protein, and contactin-associated protein 1, which successively decreased in the later stages. Cx32 was re-distributed in the oligodendrocyte cytoplasm and co-localized with GCIs. Cx47 gradually decreased at the oligodendrocyte surface in a stage-dependent manner but was not co-localized with GCIs. Astrocytic Cx43 was down-regulated in Stage I but up-regulated in Stages II and III, reflecting astrogliosis. Cx43/Cx47 gap junctions significantly decreased from Stage I to III. Activated microglia/macrophages and T cells infiltrated in Stage I rather than Stages II and III. Therefore, early and extensive alterations of glial Cxs, particularly Cx32 loss, occur in MSA and may accelerate distal oligodendrogliopathy type demyelination and nodal/paranodal dysfunction through disruption of inter-glial communication.

Ventricular arrhythmias in acute myocardial ischaemia-Focus on the ageing and sex.

Annually, approximately 17 million people die from cardiovascular diseases worldwide, half of them suddenly. The most common direct cause of sudden cardiac death is ventricular arrhythmia triggered by an acute coronary syndrome (ACS). The study summarizes the knowledge of the mechanisms of arrhythmia onset during ACS in humans and in animal models and factors that may influence the susceptibility to life-threatening arrhythmias during ACS with particular focus on the age and sex. The real impact of age and sex on the arrhythmic susceptibility within the setting of acute ischaemia is masked by the fact that ACSs result from coronary artery disease appearing with age much earlier among men than among women. However, results of researches show that in ageing process changes with potential pro-arrhythmic significance, such as increased fibrosis, cardiomyocyte hypertrophy, decrease number of gap junction channels, disturbances of the intracellular Ca2+ signalling or changes in electrophysiological parameters, occur independently of the development of cardiovascular diseases and are more severe in male individuals. A review of the literature also indicates a marked paucity of research in this area in female and elderly individuals. Greater awareness of sex differences in the aging process could help in the development of personalized prevention methods targeting potential pro-arrhythmic factors in patients of both sexes to reduce mortality during the acute phase of myocardial infarction. This is especially important in an era of aging populations in which women will predominate due to their longer lifespan.

A microtubule-connexin-43 regulatory link suppresses arrhythmias and cardiac fibrosis in Duchenne muscular dystrophy mice.

Dilated cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), an inherited degenerative disease of the cardiac and skeletal muscle caused by absence of the protein dystrophin. We showed one hallmark of DMD cardiomyopathy is the dysregulation of cardiac gap junction channel protein connexin-43 (Cx43). Proper Cx43 localization and function at the cardiac intercalated disc (ID) is regulated by post-translational phosphorylation of Cx43-carboxy-terminus residues S325/S328/S330 (pS-Cx43). Concurrently, Cx43 traffics along microtubules (MTs) for targeted delivery to the ID. In DMD hearts, absence of dystrophin results in a hyperdensified and disorganized MT cytoskeleton, yet the link with pS-Cx43 remains unaddressed. To gain insight into the relationship between MTs and pS-Cx43, DMD mice (mdx) and pS-Cx43-deficient (mdxS3A) mice were treated with an inhibitor of MT polymerization, colchicine (Colch). Colch treatment protected mdx, not mdxS3A mice, against Cx43 remodeling, improved MT directionality, and enhanced pS-Cx43/tubulin interaction. Likewise, severe arrhythmias were prevented in isoproterenol-stressed mdx, not mdxS3A mice. Furthermore, MT directionality was improved in pS-Cx43-mimicking mdx (mdxS3E). Mdxutr+/- and mdxutr+/-S3A mice, lacking one copy of dystrophin homolog utrophin, displayed enhanced cardiac fibrosis and reduced lifespan compared with mdxutr+/-S3E; and Colch treatment corrected cardiac fibrosis in mdxutr+/- but not mdxutr+/-S3A. Collectively, the data suggest that improved MT directionality reduces Cx43 remodeling and that pS-Cx43 is necessary and sufficient to regulate MT organization, which plays crucial role in correcting cardiac dysfunction in DMD mice. Thus, identification of novel organizational mechanisms acting on pS-Cx43-MT will help develop novel cardioprotective therapies for DMD cardiomyopathy.NEW & NOTEWORTHY We found that colchicine administration to Cx43-phospho-deficient dystrophic mice fails to protect against Cx43 remodeling. Conversely, Cx43-phospho-mimic dystrophic mice display a normalized MT network. We envision a bidirectional regulation whereby correction of the dystrophic MTs leads to correction of Cx43 remodeling, which in turn leads to further correction of the MTs. Our findings suggest a link between phospho-Cx43 and MTs that provides strong foundations for novel therapeutics in DMD cardiomyopathy.

Brain Energy Metabolism: Astrocytes in Neurodegenerative Diseases.

Astrocytes are the most abundant cells in the brain. They have many important functions in the central nervous system (CNS), including the maintenance of glutamate and ion homeostasis, the elimination of oxidative stress, energy storage in glycogen, tissue repair, regulating synaptic activity by releasing neurotransmitters, and participating in synaptic formation. Astrocytes have special highly ramified structure. Their branches contact with synapses of neurons inwardly, with fine structure and wrapping synapses; their feet contact with blood vessels of brain parenchyma outward, almost wrapping the whole brain. The adjacent astrocytes rarely overlap and communicate with each other through gap junction channels. The ideal location of astrocytes enables them to sense the weak changes of their surroundings and provide the structural basis for the energy supply of neurons. Neurons and astrocytes are closely coupled units of energy metabolism in the brain. Neurons consume a lot of ATPs in the process of neurotransmission. Astrocytes provide metabolic substrates for neurons, maintain high activity of neuron, and facilitate information transmission of neurons. This article reviews the characteristics of glucose metabolism, lipid metabolism, and amino acid metabolism of astrocytes. The metabolic interactions between astrocytes and neurons, astrocytes and microglia were also detailed discussed. Finally, we classified analyzed the role of metabolic disorder of astrocytes in the occurrence and development of neurodegenerative diseases.

Heterotypic Cx43/Cx45 gap junction channel conductance depends on pulse rate and could rescue from arrhythmogenic activity in cardiac tissue

Abstract Introduction Gap junction (GJ) channels, formed of connexin (Cx) proteins, conduit electrical excitation in the heart. It is well established that GJ conductance (gj) depends on transjunctional voltage (Vj), but little is still known about its dynamics during the spread of cardiac excitation. Transitional tissue of atrioventricular (AV) node expresses Cx43 and Cx45, which could form heterotypic GJs, which are highly sensitive to Vj. Purpose Evaluation of heterotypic gj dynamics of Cx43/Cx45 channels during the propagation of excitation in cardiac tissue. Methods The CE-4SM model of cardiac tissue, consisting of discrete cells connected through dynamic GJs, was developed by combining Fenton-Karma equations, which define cardiac excitability (CE), and a 4-state model (4SM) of GJ channel gating, which describes the dependence of gj on Vj and its kinetics. Global optimization methods were applied for 4SM parameter evaluation to fit gj timecourses obtained from electrophysiological recordings. Experiments were performed using dual-whole-cell patch clamp in Novikoff cells, endogenously expressing Cx43, and Hela cells transfected with Cx45. Results We developed a combined cardiac excitability model (CE-4SM), that allowed us to study an interaction between the spread of cardiac excitation and GJ channel voltage. Modeling results shows that during propagation of excitation, the phase difference between each action potential (AP) in adjacent cells, generates two subsequent Vj spikes. First spike causes small decrease of gj, while the second one, a minor increase. These impulses can accumulate into a large overall decay until the impulse rate-dependent steady-state is reached. This phenomenon is caused by variation of delays between APs and consequentially, different durations and amplitudes of Vj spikes developed across GJ channels. Interestingly, such gj decrease was capable of causing drift of spiral wave rotor or termination of fibrillation-like arrhythmia during our modeling experiments. Also, we performed in-vitro experiments to evaluate 4SM model parameters and validate simulation results that showed gj decay at a high pulse rate, which could occur during reentry-like tachycardia. Conclusions Voltage gating of Cx43/Cx45 GJ channels could act as a control circuit, that lowers gj between the transitional cells of AV node at high impulse rate. Presumably, this could help to reduce the conduction or even act as a block of high-rate impulses passing through AV node. Also, such gj decrease could cause a drift of spiral waves and disruption of fibrillation-like processes. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Lithuanian University of Health Sciences, Institute of Cardiology

The Hydrophobic Residues in Amino Terminal Domains of Cx46 and Cx50 Are Important for Their Gap Junction Channel Ion Permeation and Gating.

Lens gap junctions (GJs) formed by Cx46 and Cx50 are important to keep lens transparency. Functional studies on Cx46 and Cx50 GJs showed that the Vj-gating, single channel conductance (γj), gating polarity, and/or channel open stability could be modified by the charged residues in the amino terminal (NT) domain. The role of hydrophobic residues in the NT on GJ properties is not clear. Crystal and cryo-EM GJ structures have been resolved, but the NT domain structure has either not been resolved or has showed very different orientations depending on the component connexins and possibly other experimental conditions, making it difficult to understand the structural basis of the NT in Vj-gating and γj. Here, we generated missense variants in Cx46 and Cx50 NT domains and studied their properties by recombinant expression and dual whole-cell patch clamp experiments on connexin-deficient N2A cells. The NT variants (Cx46 L10I, N13E, A14V, Q15N, and Cx50 I10L, E13N, V14A, N15Q) were all able to form functional GJs with similar coupling%, except Cx46 N13E, which showed a significantly reduced coupling%. The GJs of Cx46 N13E, A14V and Cx50 E13N, N15Q showed a reduced coupling conductance. Vj-gating of all the variant GJs were similar to the corresponding wild-type GJs except Cx46 L10I. The γj of Cx46 N13E, A14V, Cx50 E13N, and N15Q GJs was reduced to 51%, 82%, 87%, and 74%, respectively, as compared to their wild-type γjs. Structural models of Cx46 L10I and A14V predicted steric clashes between these residues and the TM2 residues, which might be partially responsible for our observed changes in GJ properties. To verify the importance of hydrophobic interactions, we generated a variant, Cx50 S89T, which also shows a steric clash and failed to form a functional GJ. Our experimental results and structure models indicate that hydrophobic interactions between the NT and TM2 domain are important for their Vj-gating, γj, and channel open stability in these and possibly other GJs.

Mechanism-based targeting of cardiac arrhythmias by phytochemicals and medicinal herbs: A comprehensive review of preclinical and clinical evidence.

Cardiac arrhythmias, characterized by an irregular heartbeat, are associated with high mortality and morbidity. Because of the narrow therapeutic window of antiarrhythmic drugs (AADs), the management of arrhythmia is still challenging. Therefore, searching for new safe, and effective therapeutic options is unavoidable. In this study, the antiarrhythmic effects of medicinal plants and their active constituents were systematically reviewed to introduce some possible candidates for mechanism-based targeting of cardiac arrhythmias. PubMed, Embase, and Cochrane library were searched from inception to June 2021 to find the plant extracts, phytochemicals, and multi-component herbal preparations with antiarrhythmic activities. From 7337 identified results, 57 original studies consisting of 49 preclinical and eight clinical studies were finally included. Three plant extracts, eight multi-component herbal preparations, and 26 phytochemicals were found to have antiarrhythmic effects mostly mediated by affecting K+ channels, followed by modulating Ca2+ channels, upstream target pathways, Nav channels, gap junction channels, and autonomic receptors. The most investigated medicinal plants were Rhodiola crenulata and Vitis vinifera. Resveratrol, Oxymatrine, and Curcumin were the most studied phytochemicals found to have multiple mechanisms of antiarrhythmic action. This review emphasized the importance of research on the cardioprotective effect of medicinal plants and their bioactive compounds to guide the future development of new AADs. The most prevalent limitation of the studies was their unqualified methodology. Thus, future well-designed experimental and clinical studies are necessary to provide more reliable evidence.

Molecular and Morphological Characteristics of the De-Obstructed Rat Urinary Bladder-An Update.

Many patients with outlet obstruction secondary to prostatic enlargement have lower urinary tract symptoms (LUTSs) and an increased frequency of micturition. The standard treatment is transurethral resection of the prostate (TURP), which alleviates obstruction and symptoms. However, after TURP, 20–40 percent of patients continue to experience LUTSs. The aim of the present study in rats was to identify the mechanisms that do not normalize after the removal of the obstruction and that could explain the persisting symptoms. We had microarray data from control, obstructed, and de-obstructed female rat bladders, which made it possible to study 14,553 mRNA expressions. We also had a bank of electron micrographs from similar detrusors. Microarrays: There were significant differences between the control and obstructed bladders for 1111 mRNAs. The obstructed and de-obstructed bladders differed significantly for 1059 mRNAs. The controls and the de-obstructed bladders differed significantly for 798 mRNAs. We observed many mRNAs that were increased in the obstructed bladder and then decreased to control levels after de-obstruction, and many mRNAs that were decreased in the obstructed bladder and then increased following de-obstruction. mRNAs that were significantly higher or lower in the de-obstructed bladder than in the control bladder were also found. Ultrastructure: The detrusor cells in the obstructed bladders had cross-sectional areas that were much larger than those in the controls. The control cells had smooth outlines and similar cross-sectional areas. The de-obstructed detrusor cells had larger cross-sectional areas than the controls, as well as corrugated surfaces. The cell areas varied, suggesting that the shrinkage of the de-obstructed cells was not even. We did not find any points of contact of the gap junction plaque type between the detrusor cells. There were abundant finger-like processes between the detrusor cells in the obstructed and in de-obstructed bladders, which were only occasionally found in the control detrusors. They are the only possible localization for gap junction channels. The de-obstructed rat bladder is not an organ with properties intermediate between those of the control and obstructed bladders. Instead, de-obstructed bladders have gene expressions, morphologies, and functional properties of the individual cells and their organization, which make them distinctly different from both control and obstructed bladders.

Loss of fiber cell communication may contribute to the development of cataracts of many different etiologies.

The lens is an avascular organ that is supported by an internal circulation of water and solutes. This circulation is driven by ion pumps, channels and transporters in epithelial cells and by ion channels in fiber cells and is maintained by fiber-fiber and fiber-epithelial cell communication. Gap junctional intercellular channels formed of connexin46 and connexin50 are critical components of this circulation as demonstrated by studies of connexin null mice and connexin mutant mice. Moreover, connexin mutants are one of the most common causes of autosomal dominant congenital cataracts. However, alterations of the lens circulation and coupling between lens fiber cells are much more prevalent, beyond the connexin mutant lenses. Intercellular coupling and levels of connexins are decreased with aging. Gap junction-mediated intercellular communication decreases in mice expressing mutant forms of several different lens proteins and in some mouse models of lens protein damage. These observations suggest that disruption of ionic homeostasis due to reduction of the lens circulation is a common component of the development of many different types of cataracts. The decrease in the lens circulation often reflects low levels of lens fiber cell connexins and/or functional gap junction channels.

Temporal signals drive the emergence of multicellular information networks

Coordinated responses to environmental stimuli are critical for multicellular organisms. To overcome the obstacles of cell-to-cell heterogeneity and noisy signaling dynamics within individual cells, cells must effectively exchange information with peers. However, the dynamics and mechanisms of collective information transfer driven by external signals are poorly understood. Here we investigate the calcium dynamics of neuronal cells that form confluent monolayers and respond to cyclic ATP stimuli in microfluidic devices. Using Granger inference to reconstruct the underlying causal relations between the cells, we find that the cells self-organize into spatially decentralized and temporally stationary networks to support information transfer via gap junction channels. The connectivity of the causal networks depends on the temporal profile of the external stimuli, where short periods, or long periods with small duty fractions, lead to reduced connectivity and fractured network topology. We build a theoretical model based on communicating excitable units that reproduces our observations. The model further predicts that connectivity of the causal network is maximal at an optimal communication strength, which is confirmed by the experiments. Together, our results show that information transfer between neuronal cells is externally regulated by the temporal profile of the stimuli and internally regulated by cell-cell communication.

TNF-α Plus IL-1β Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1β increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1β treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1β-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell–cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Alpha4 contributes to the dysfunction of the pancreatic beta cell under metabolic stress

The current study examined the roles of Alpha4, a non-canonical subunit of protein phosphatase 2A, in the regulation of acute (insulin secretion) and chronic (cell dysfunction) effects of glucose in pancreatic beta cells. Alpha4 is expressed in human islets, rat islets and INS-1832/13 cells. Incubation of INS-1832/13 cells and rat islets with high glucose (HG) significantly increased the expression of Alpha4. C2-Ceramide, a biologically active sphingolipid, also increased the expression of Alpha4 in INS-1832/13 cells and rat islets. Subcellular distribution studies of Alpha4 in low glucose (LG) and HG exposed INS-1832/13 cells revealed that it is predominantly cytosolic, and its expression is significantly increased in the non-nuclear/cytosolic fractions in cells exposed to HG. siRNA-mediated knockdown of Alpha4 exerted minimal effects on glucose- or KCl-induced insulin secretion. siRNA-mediated deletion of Alpha4 significantly increased p38MAPK and JNK1/2 phosphorylation under LG conditions, comparable to the degree seen under HG conditions. Paradoxically, a significant potentiation of HG-induced p38MAPK and JNK2 phosphorylation was noted following Alpha4 deletion. HG-induced CHOP expression (ER stress marker) and caspase-3 activation were markedly attenuated in cells following Alpha4 knockdown. Deletion of Alpha4 in INS-1832/13 cells prevented HG-induced loss in the expression of Connexin36, a gap junction channel protein, which has been implicated in normal beta cell function. Lastly, depletion of endogenous Alpha4 significantly reduced HG-induced cell death in INS-1832/13 cells. Based on these findings we conclude that Alpha4 contributes to HG-induced metabolic dysfunction of the islet beta cell.

Anesthetics and Cell-Cell Communication: Potential Ca2+-Calmodulin Role in Gap Junction Channel Gating by Heptanol, Halothane and Isoflurane.

Cell–cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca2+ channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics. In our publication, we concluded that neither cytosolic Ca2+i nor pHi were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca2+ concentration ([Ca2+]i) and consequential activation of calmodulin linked to gap junction proteins.

Heterogeneities in Ventricular Conduction Following Treatment with Heptanol: A Multi-Electrode Array Study in Langendorff-Perfused Mouse Hearts.

Background: Previous studies have associated slowed ventricular conduction with the arrhythmogenesis mediated by the gap junction and sodium channel inhibitor heptanol in mouse hearts. However, they did not study the propagation patterns that might contribute to the arrhythmic substrate. This study used a multi-electrode array mapping technique to further investigate different conduction abnormalities in Langendorff-perfused mouse hearts exposed to 0.1 or 2 mM heptanol. Methods: Recordings were made from the left ventricular epicardium using multi-electrode arrays in spontaneously beating hearts during right ventricular 8 Hz pacing or S1S2 pacing. Results: In spontaneously beating hearts, heptanol at 0.1 and 2 mM significantly reduced the heart rate from 314 ± 25 to 189 ± 24 and 157 ± 7 bpm, respectively (ANOVA, p < 0.05 and p < 0.001). During regular 8 Hz pacing, the mean LATs were increased by 0.1 and 2 mM heptanol from 7.1 ± 2.2 ms to 19.9 ± 5.0 ms (p < 0.05) and 18.4 ± 5.7 ms (p < 0.05). The standard deviation of the mean LATs was increased from 2.5 ± 0.8 ms to 10.3 ± 4.0 ms and 8.0 ± 2.5 ms (p < 0.05), and the median of phase differences was increased from 1.7 ± 1.1 ms to 13.9 ± 7.8 ms and 12.1 ± 5.0 ms by 0.1 and 2 mM heptanol (p < 0.05). P5 took a value of 0.2 ± 0.1 ms and was not significantly altered by heptanol at 0.1 or 2 mM (1.1 ± 0.9 ms and 0.9 ± 0.5 ms, p > 0.05). P50 was increased from 7.3 ± 2.7 ms to 24.0 ± 12.0 ms by 0.1 mM heptanol and then to 22.5 ± 7.5 ms by 2 mM heptanol (p < 0.05). P95 was increased from 1.7 ± 1.1 ms to 13.9 ± 7.8 ms by 0.1 mM heptanol and to 12.1 ± 5.0 ms by 2 mM heptanol (p < 0.05). These changes led to increases in the absolute inhomogeneity in conduction (P5–95) from 7.1 ± 2.6 ms to 31.4 ± 11.3 ms, 2 mM: 21.6 ± 7.2 ms, respectively (p < 0.05). The inhomogeneity index (P5–95/P50) was significantly reduced from 3.7 ± 1.2 to 3.1 ± 0.8 by 0.1 mM and then to 3.3 ± 0.9 by 2 mM heptanol (p < 0.05). Conclusion: Increased activation latencies, reduced CVs, and the increased inhomogeneity index of conduction were associated with both spontaneous and induced ventricular arrhythmias.

Connexin and Pannexin Large-Pore Channels in Microcirculation and Neurovascular Coupling Function.

Microcirculation homeostasis depends on several channels permeable to ions and/or small molecules that facilitate the regulation of the vasomotor tone, hyperpermeability, the blood–brain barrier, and the neurovascular coupling function. Connexin (Cxs) and Pannexin (Panxs) large-pore channel proteins are implicated in several aspects of vascular physiology. The permeation of ions (i.e., Ca2+) and key metabolites (ATP, prostaglandins, D-serine, etc.) through Cxs (i.e., gap junction channels or hemichannels) and Panxs proteins plays a vital role in intercellular communication and maintaining vascular homeostasis. Therefore, dysregulation or genetic pathologies associated with these channels promote deleterious tissue consequences. This review provides an overview of current knowledge concerning the physiological role of these large-pore molecule channels in microcirculation (arterioles, capillaries, venules) and in the neurovascular coupling function.

GJA1 reverses arsenic-induced EMT via modulating MAPK/ERK signaling pathway

Arsenic is known as a well-established human carcinogen. Gap Junction Protein Alpha 1 (GJA1) is a multifunction protein that forms gap junction channels and is important for intercellular communication. Recently, its aberrant expression has been shown to associate with cancer recurrence and metastatic spread. However, whether GJA1 plays a role in arsenic carcinogenesis remains unknown. Here, we demonstrated that chronic exposure of human bronchial epithelial BEAS-2B cells to sodium arsenite promoted epithelial-mesenchymal transition (EMT) via increasing the expression of EMT inducer S100A4 and activation of MAPK/ERK signaling. In vitro and in vivo experiments showed that chronic exposure to sodium arsenite reduced GJA1 expression. Forced expression of GJA1 inhibited sodium arsenite-induced EMT via suppressing MAPK/ERK signaling whereas GJA1 knockdown produced an opposite effect. Intriguingly, chronic exposure to sodium arsenite increased autophagy flux. Inhibition of autophagy by pharmacological intervention or genetic deletion of autophagy core gene Beclin-1 upregulated GJA1 expression. These results suggested that GJA1 restrained the carcinogenic effect of sodium arsenite by limiting MAPK/ERK signaling, and GJA1 expression was decreased by arsenic-activated autophagy. In addition, interventions directed at enhancing the level or functional activity of GJA1 could be of preventive and therapeutic interest.

Anchored-PKA regulates connexin-43 gap junction communication in the heart

Cardiac connexin-43 (Cx43) forms gap junction (GJ) channels at the intercalated discs (ID) of cardiomyocytes ensuring anisotropic action potential (AP) propagation. This process is essential for sequential and coordinated contraction of the heart. Stimulation of β-adrenergic receptors activates protein kinase A (PKA), which in turn increases electrical conduction velocity. Myocardial infarction (MI) is characterized by a prolonged ischemia leading to irreversible ventricular cardiomyocytes death and impaired cardiac function. To compensate this loss, chronic activation of PKA signaling is established. In addition, post-MI, Cx43 subcellular localization is altered affecting GJ communication and AP propagation leading to uncoordinated contractions and fatal arrhythmias. Considering the key role of Cx43 and PKA signaling in AP propagation, we investigated the potential regulation of GJ communication by PKA under cardiac physiological condition and post-MI. First, we assessed Cx43 phosphorylation with PKA specific inhibition and with a peptide displacing PKA anchoring from A-Kinase Anchoring Protein (AKAPs). Then, we identified the ezrin as the AKAP mediated the regulation of Cx43 phosphorylation by immunoprecipitation, immunofluorescence and siRNA mediated knockdown experiments. We assessed by functional GAP-FLIP experiments the key role of ezrin-PKA complex in the regulation of intercellular communication. Finally, we generated a left anterior descending coronary artery model of MI in rats to evaluate by proximity ligation assay and western blot experiments the Cx43-ezrin-PKA complex localization and expression. We point that ezrin forms a supramolecular complex with PKA and Cx43 at the ID of cardiomyocytes. Ezrin regulates Cx43 phosphorylation and GJ communication in cardiomyocytes. Interestingly, post-MI we noticed a significant reduction in Cx43 level and phosphorylation associated with a decrease in proximity with ezrin at the ID. Our data identified a cardiac PKA-ezrin-Cx43 complex regulating Cx43 phosphorylation, and therefore, GJ communication. In addition, we reported a loss of proximity between ezrin and Cx43 post-MI. We proposed that this may explain partly an impaired AP propagation leading to uncoordinated contractions post MI.