Gap-134, a Connexin43 activator, prevents age-related development of ventricular fibrosis in Scn5a+/- mice.

Down-regulation of Connexin43 (Cx43) has often been associated with the development of cardiac fibrosis. We showed previously that Scn5a heterozygous knockout mice (Scn5a+/-), which mimic familial progressive cardiac conduction defect, exhibit an age-dependent decrease of Cx43 expression and phosphorylation concomitantly with activation of TGF-ß pathway and fibrosis development in the myocardium between 45 and 60 weeks of age. The aim of this study was to investigate whether Gap-134 prevents Cx43 down-regulation with age and fibrosis development in Scn5a+/- mice. We observed in 60-week-old Scn5a+/- mouse heart a Cx43 expression and localization remodeling correlated with fibrosis. Chronic administration of a potent and selective gap junction modifier, Gap-134 (danegaptide), between 45 and 60 weeks, increased Cx43 expression and phosphorylation on serine 368 and prevented Cx43 delocalization. Furthermore, we found that Gap-134 prevented fibrosis despite the persistence of the conduction defects and the TGF-ß canonical pathway activation. In conclusion, the present study demonstrates that the age-dependent decrease of Cx43 expression is involved in the ventricular fibrotic process occurring in Scn5a+/- mice. Finally, our study suggests that gap junction modifier, such as Gap-134, could be an effective anti-fibrotic agent in the context of age-dependent fibrosis in progressive cardiac conduction disease.

Influence of the scaffolding protein Zonula Occludens (ZOs) on membrane channels.

Zonula Occludens (ZO) proteins are ubiquitous scaffolding proteins providing the structural basis for the assembly of multiprotein complexes at the cytoplasmic surface of the plasma membrane and linking transmembrane proteins to the filamentous cytoskeleton. They belong to the large family of membrane-associated guanylate kinase (MAGUK)-like proteins comprising a number of subfamilies based on domain content and sequence similarity. ZO proteins were originally described to localize specifically to tight junctions, or Zonulae Occludentes, but this notion was rapidly reconsidered since ZO proteins were found to associate with adherens junctions as well as with gap junctions, particularly with connexin-made intercellular channels, and also with a few other membrane channels. Accumulating evidence reveals that in addition to having passive scaffolding functions in organizing gap junction complexes, including connexins and cytoskeletals, ZO proteins (particularly ZO-1) also actively take part in the dynamic function as well as in the remodeling of junctional complexes in a number of cellular systems. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Gap junctional channels are parts of multiprotein complexes.

Gap junctional channels are a class of membrane channels composed of transmembrane channel-forming integral membrane proteins termed connexins, innexins or pannexins that mediate direct cell-to-cell or cell-to extracellular medium communication in almost all animal tissues. The activity of these channels is tightly regulated, particularly by intramolecular modifications as phosphorylations of proteins and via the formation of multiprotein complexes where pore-forming subunits bind to auxiliary channel subunits and associate with scaffolding proteins that play essential roles in channel localization and activity. Scaffolding proteins link signaling enzymes, substrates, and potential effectors (such as channels) into multiprotein signaling complexes that may be anchored to the cytoskeleton. Protein-protein interactions play essential roles in channel localization and activity and, besides their cell-to-cell channel-forming functions, gap junctional proteins now appear involved in different cellular functions (e.g. transcriptional and cytoskeletal regulations). The present review summarizes the recent progress regarding the proteins capable of interacting with junctional proteins and highlights the function of these protein-protein interactions in cell physiology and aberrant function in diseases. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and functions.

Gap-junction-mediated cell-to-cell communication.

Cells of multicellular organisms need to communicate with each other and have evolved various mechanisms for this purpose, the most direct and quickest of which is through channels that directly connect the cytoplasms of adjacent cells. Such intercellular channels span the two plasma membranes and the intercellular space and result from the docking of two hemichannels. These channels are densely packed into plasma-membrane spatial microdomains termed "gap junctions" and allow cells to exchange ions and small molecules directly. A hemichannel is a hexameric torus of junctional proteins around an aqueous pore. Vertebrates express two families of gap-junction proteins: the well-characterized connexins and the more recently discovered pannexins, the latter being related to invertebrate innexins ("invertebrate connexins"). Some gap-junctional hemichannels also appear to mediate cell-extracellular communication. Communicating junctions play crucial roles in the maintenance of homeostasis, morphogenesis, cell differentiation and growth control in metazoans. Gap-junctional channels are not passive conduits, as previously long regarded, but use "gating" mechanisms to open and close the central pore in response to biological stimuli (e.g. a change in the transjunctional voltage). Their permeability is finely tuned by complex mechanisms that have just begun to be identified. Given their ubiquity and diversity, gap junctions play crucial roles in a plethora of functions and their dysfunctions are involved in a wide range of diseases. However, the exact mechanisms involved remain poorly understood.

5-HT4 and 5-HT2 receptors antagonistically influence gap junctional coupling between rat auricular myocytes.

5-hydroxytryptamine-4 (5-HT(4)) receptors have been proposed to contribute to the generation of atrial fibrillation in human atrial myocytes, but it is unclear if these receptors are present in the hearts of small laboratory animals (e.g. rat). In this study, we examined presence and functionality of 5-HT(4) receptors in auricular myocytes of newborn rats and their possible involvement in regulation of gap junctional intercellular communication (GJIC, responsible for the cell-to-cell propagation of the cardiac excitation). Western-blotting assays showed that 5-HT(4) receptors were present and real-time RT-PCR analysis revealed that 5-HT(4b) was the predominant isoform. Serotonin (1 microM) significantly reduced cAMP concentration unless a selective 5-HT(4) inhibitor (GR113808 or ML10375, both 1 microM) was present. Serotonin also reduced the amplitude of L-type calcium currents and influenced the strength of GJIC without modifying the phosphorylation profiles of the different channel-forming proteins or connexins (Cxs), namely Cx40, Cx43 and Cx45. GJIC was markedly increased when serotonin exposure occurred in presence of a 5-HT(4) inhibitor but strongly reduced when 5-HT(2A) and 5-HT(2B) receptors were inhibited, showing that activation of these receptors antagonistically regulated GJIC. The serotoninergic response was completely abolished when 5-HT(4), 5-HT(2A) and 5-HT(2B) were simultaneously inhibited. A 24 h serotonin exposure strongly reduced Cx40 expression whereas Cx45 was less affected and Cx43 still less. In conclusion, this study revealed that 5-HT(4) (mainly 5-HT(4b)), 5-HT(2A) and 5-HT(2B) receptors coexisted in auricular myocytes of newborn rat, that 5-HT(4) activation reduced cAMP concentration, I(Ca)(L) and intercellular coupling whereas 5-HT(2A) or 5-HT(2B) activation conversely enhanced GJIC.

Reciprocal influence of connexins and apical junction proteins on their expressions and functions.

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another.

Epac stimulation induces rapid increases in connexin43 phosphorylation and function without preconditioning effect.

It has been recently shown that beta-adrenergic receptors are able to activate phospholipase C via the cyclic adenosine monophosphate-binding protein Epac. This new interconnection may participate in isoproterenol (Iso)-induced preconditioning. We evaluated here whether Epac could induce PKCepsilon activation and could play a role in ischemic preconditioning through the phosphorylation of connexin43 (Cx43) and changes in gap junctional intercellular communication (GJIC). In cultured rat neonatal cardiomyocytes, we showed that in response to Iso and 8-CPT, a specific Epac activator, PKCepsilon content was increased in particulate fractions of cell lysates independently of protein kinase A (PKA). This was associated with an increased Cx43 phosphorylation. Both Iso and 8-CPT induced an increase in GJIC that was blocked by the PKC inhibitor bisindolylmaleimide. Interestingly, inhibition of PKA partly suppressed both Iso-induced increases in Cx43 phosphorylation and in GJIC. The same PKCepsilon-dependent Cx43 phosphorylation by beta-adrenergic stimulation via Epac was found in adult rat hearts. However, in contrast with Iso that induced a preconditioning effect, perfusion of isolated hearts with 8-CPT prior to ischemia failed to improve the post-ischemia functional recovery. In conclusion, Epac stimulation induces PKCepsilon activation and Cx43 phosphorylation with an increase in GJIC, but Epac activation does not induce preconditioning to ischemia in contrast with beta-adrenergic stimulation.

Aberrant expression and localization of connexin43 and connexin30 in a rat glioma cell line.

Gap junctions are cellular structures which permit direct exchanges of small molecules from cytoplasm to cytoplasm in most of the cells of metazoan organisms. For four decades, it has been observed that the inhibition of this type of intercellular communication is often associated with tumorigenesis. The assumption that loss of homeostasis which characterizes tumor growth could be a consequence of a lack of gap junctional intercellular communication (GJIC) has been reinforced by strategies able to reinduce both GJIC and normalization of the phenotype. So far, no molecular data may explain clearly how gap junctions can regulate cell proliferation. It has been argued that the gap-junction tumor suppressive effect may depend specifically on the connexin type which is expressed. For instance, the transfection of connexin30 (Cx30), a gap junction protein, has been previously associated with a slower growth of rat glioma cells (9L cells). Here, we show that these cells do communicate less compared to the Cx43-expressing parental cells even if the Cx30-transfected cells do express more Cx43. This result was related to the cytoplasmic distribution of Cx43 and a nuclear localization of both the Cx30 and a 20-kDa fragment corresponding to a Cx43 signal. According to these data, it seems that cell growth regulation may depend more on the behavior of connexins than the simple establishment of GJIC.

Transforming growth factor ß receptor inhibition prevents ventricular fibrosis in a mouse model of progressive cardiac conduction disease.

AIMS: Loss-of-function mutations in SCN5A, the gene encoding NaV1.5 channel, have been associated with inherited progressive cardiac conduction disease (PCCD). We have proposed that Scn5a heterozygous knock-out (Scn5a+/-) mice, which are characterized by ventricular fibrotic remodelling with ageing, represent a model for PCCD. Our objectives were to identify the molecular pathway involved in fibrosis development and prevent its activation. METHODS AND RESULTS: Our study shows that myocardial interstitial fibrosis occurred in Scn5a+/- mice only after 45 weeks of age. Fibrosis was triggered by transforming growth factor ß (TGF-ß) pathway activation. Younger Scn5a+/- mice were characterized by a higher connexin 43 expression than wild-type (WT) mice. After the age of 45 weeks, connexin 43 expression decreased in both WT and Scn5a+/- mice, although the decrease was larger in Scn5a+/- mice. Chronic inhibition of cardiac sodium current with flecainide (50 mg/kg/day p.o) in WT mice from the age of 6 weeks to the age of 60 weeks did not lead to TGF-ß pathway activation and fibrosis. Chronic inhibition of TGF-ß receptors with GW788388 (5 mg/kg/day p.o.) in Scn5a+/- mice from the age of 45 weeks to the age of 60 weeks prevented the occurrence of fibrosis. However, current data could not detect reduction in QRS duration with GW788388. CONCLUSION: Myocardial fibrosis secondary to a loss of NaV1.5 is triggered by TGF-ß signalling pathway. Those events are more likely secondary to the decreased NaV1.5 sarcolemmal expression rather than the decreased Na+ current per se. TGF-ß receptor inhibition prevents age-dependent development of ventricular fibrosis in Scn5a+/- mouse.

Astroglial networks promote neuronal coordination.

Astrocytes interact with neurons to regulate network activity. Although the gap junction subunits connexin 30 and connexin 43 mediate the formation of extensive astroglial networks that cover large functional neuronal territories, their role in neuronal synchronization remains unknown. Using connexin 30- and connexin 43-deficient mice, we showed that astroglial networks promoted sustained population bursts in hippocampal slices by setting the basal active state of neurons. Astroglial networks limited excessive neuronal depolarization induced by spontaneous synaptic activity, increased neuronal release probability, and favored the recruitment of neurons during bursting, thus promoting the coordinated activation of neuronal networks. In vivo, this sustained neuronal coordination translated into increased severity of acutely evoked epileptiform events and convulsive behavior. These results revealed that connexin-mediated astroglial networks synchronize bursting of neuronal assemblies, which can exacerbate pathological network activity and associated behavior. Our data thus provide molecular and biophysical evidence predicting selective astroglial gap junction inhibitors as anticonvulsive drugs.

Astroglial gap junctions shape neuronal network activity.

Astrocytes, the third element of the tripartite synapse, are active players in neurotransmission. Up to now, their involvement in neuronal functions has primarily been investigated at the single cell level. However, a key property of astrocytes is that they communicate via extensive networks formed by gap junction channels. Recently, we have shown that this networking modulates the moment to moment basal synaptic transmission and plasticity via the regulation of extracellular potassium and glutamate levels. Here we show that astroglial gap junctional communication also regulates neuronal network activity. We discuss these findings and their implications for brain information processing.

Mouse Models of SCN5A-Related Cardiac Arrhythmias.

Mutations of SCN5A gene, which encodes the α-subunit of the voltage-gated Na(+) channel Na(V)1.5, underlie hereditary cardiac arrhythmic syndromes such as the type 3 long QT syndrome, cardiac conduction diseases, the Brugada syndrome, the sick sinus syndrome, a trial standstill, and numerous overlap syndromes. Patch-clamp studies in heterologous expression systems have provided important information to understand the genotype-phenotype relationships of these diseases. However, they could not clarify how SCN5A mutations can be responsible for such a large spectrum of diseases, for the late age of onset or the progressiveness of some of these diseases and for the overlapping syndromes. Genetically modified mice rapidly appeared as promising tools for understanding the pathophysiological mechanisms of cardiac SCN5A-related arrhythmic syndromes and several mouse models have been established. This review presents the results obtained on these models that, for most of them, recapitulate the clinical phenotypes of the patients. This includes two models knocked out for Nav1.5 ß1 and ß3 auxiliary subunits that are also discussed. Despite their own limitations that we point out, the mouse models still appear as powerful tools to elucidate the pathophysiological mechanisms of SCN5A-related diseases and offer the opportunity to investigate the secondary cellular consequences of SCN5A mutations such as the expression remodeling of other genes. This points out the potential role of these genes in the overall human phenotype. Finally, they constitute useful tools for addressing the role of genetic and environmental modifiers on cardiac electrical activity.

RhoA GTPase and F-actin dynamically regulate the permeability of Cx43-made channels in rat cardiac myocytes.

Gap junctions are clusters of transmembrane channels allowing a passive diffusion of ions and small molecules between adjacent cells. Connexin43, the main channel-forming protein expressed in ventricular myocytes, can associate with zonula occludens-1, a scaffolding protein linked to the actin cytoskeleton and to signal transduction molecules. The possible influence of Rho GTPases, major regulators of cellular junctions and of the actin cytoskeleton, in the modulation of gap junctional intercellular communication (GJIC) was examined. The activation of RhoA by cytoxic necrotizing factor 1 markedly enhanced GJIC, whereas its specific inhibition by the Clostridium botulinum C3 exoenzyme significantly reduced it. RhoA activity affects GJIC without major cellular redistribution of junctional plaques or changes in the Cx43 phosphorylation pattern. As these GTPases frequently act via the cortical cytoskeleton, the importance of F-actin in the modulation of GJIC was investigated by means of agents interfering with actin polymerization. Cytoskeleton stabilization by phalloidin slowed down the kinetics of channel rundown in the absence of ATP, whereas its disruption by cytochalasin D rapidly and markedly reduced GJIC despite ATP presence. Cytoskeleton stabilization by phalloidin markedly reduced the consequences of RhoA activation or inactivation. This mechanism appears to be the first described capable to both up- or down-regulate GJIC through RhoA activation or, conversely, inhibition. The inhibition of Rho downstream kinase effectors had no effect on GJIC. The present results provide further insight into the gating and regulation of junctional channels and identify a new downstream target for the small G-protein RhoA.

The connexin turnover, an important modulating factor of the level of cell-to-cell junctional communication: comparison with other integral membrane proteins.

The constituent proteins of gap junctions, called "connexins" (Cxs) in chordates, are generally renewed several times a day, in approximately the same rate range as many other integral plasma membrane proteins and the proteins of other channels, other intercellular junctions or different membrane receptors. This permanent renewal turns on a fine-tuned balance among various processes, such as gene transcription, mRNA stability and processing, protein synthesis and oligomerization, posttranslational modifications, transport to the plasma membrane, anchoring to the cytoskeleton, connexon aggregation and docking, regulation of endocytosis and controlled degradations of the proteins. Subtle changes at one or some of these steps would represent an exquisite level of regulation that extends beyond the rapid channel opening and closure events associated with channel gating; membrane channels and receptors are constantly able to answer to physiological requirements to either up- or downregulate their activity. The Cx turnover rate thereby appears to be a key component in the regulation of any protein, particularly of gap junctional proteins. However, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation remain poorly understood.