Increased co-localization of connexin43 and ZO-1 in dissociated adult myocytes

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium--a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immunoprecipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.     

Increased Co-localization of Connexin43 and ZO-1 in Dissociated Adult Myocytes

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium-a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immuno-precipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.     

Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts

Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.     

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.     

The proteasome regulates the interaction between Cx43 and ZO-1

Gap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO-1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO-1. Overexpression of the PDZ2 domain of ZO-1 and the expression of Cx-43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO-1 and the C-terminus of Cx43. When interaction between Cx43 and ZO-1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non-junctional communication with extracellular environment, contributing to cell and tissue dysfunction.     

ZO-1 determines adherens and gap junction localization at intercalated disks

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.     

Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1

Connexin 43 (Cx43) is a gap junction (GJ) protein widely expressed in mammalian tissues that mediates cell-to-cell coupling. Intercellular channels comprising GJ aggregates form from docking of paired connexons, with one each contributed by apposing cells. Zonula occludens-1 (ZO-1) binds the carboxy terminus of Cx43, and we have previously shown that inhibition of the Cx43/ZO-1 interaction increases GJ size by 48 h. Here we demonstrated that increases in GJ aggregation occur within 2 h (～Cx43 half-life) following disruption of Cx43/ZO-1. Immunoprecipitation and Duolink protein-protein interaction assays indicated that inhibition targets ZO-1 binding with Cx43 in GJs as well as connexons in an adjacent domain that we term the "perinexus." Consistent with GJ size increases being matched by decreases in connexons, inhibition of Cx43/ZO-1 reduced the extent of perinexal interaction, increased the proportion of connexons docked in GJs relative to undocked connexons in the plasma membrane, and increased GJ intercellular communication while concomitantly decreasing hemichannel-mediated membrane permeance in contacting, but not noncontacting, cells. ZO-1 small interfering RNA and overexpression experiments verified that loss and gain of ZO-1 function govern the transition of connexons into GJs. It is concluded that ZO-1 regulates the rate of undocked connexon aggregation into GJs, enabling dynamic partitioning of Cx43 channel function between junctional and proximal nonjunctional domains of plasma membrane.     

Overexpression of cardiac connexin45 increases susceptibility to ventricular tachyarrhythmias in vivo

Electrophysiological remodeling involving gap junctions has been demonstrated in failing hearts and may contribute to intercellular uncoupling, delayed conduction, enhanced arrhythmias, and vulnerability to sudden death in patients with heart failure. Recently, we showed that failing human hearts exhibit marked increases in connexin45 (Cx45) expression in addition to previously documented decreases in connexin43 (Cx43) expression. Each of these changes results in reduced gap junction coupling. The objective of the present study was to examine functional consequences of increased Cx45 in cardiac gap junctions. Transgenic mice with cardiac-selective overexpression of the developmentally downregulated cardiac connexin, connexin45 (Cx45OE mice) were subjected to in vivo electrophysiology studies in which an intracardiac catheter was used to induce ventricular arrhythmias in anesthetized mice, and in which ambulatory ECG monitoring was used to detect spontaneous arrhythmias in unanesthetized mice. Hearts were analyzed by TaqMan RT-PCR, immunostaining, immunoblotting, and echocardiography. Lucifer yellow and neurobiotin dye transfer was used to assess coupling in transgenic and control myocyte cultures. Cx45 mRNA was two orders of magnitude greater in Cx45OE mice. Cx45-immunoreactive signal at gap junctions increased twofold and total Cx45 protein by immunoblotting increased 25% in Cx45OE mice compared with nontransgenic littermate controls. Functionally, Cx45OE mice exhibited more inducible ventricular tachycardia than controls but did not exhibit any other functional or structural derangements as assessed by echocardiography. Ventricular myocytes isolated from Cx45OE mice exhibited diminished intercellular transfer of Lucifer yellow dye and increased transfer of neurobiotin, consistent with altered cell-to-cell communication. Thus increased myocardial expression of Cx45 results in remodeling of intercellular coupling and greater susceptibility to ventricular arrhythmias in vivo.     

The Expression, Phosphorylation, and Localization of Connexin 43 and Gap-Junctional Intercellular Communication during the Establishment of a Synchronized Contraction of Cultured Neonatal Rat Cardiac Myocytes

We analyzed the expression, phosphorylation, and localization of the major cardiac gap-junction protein connexin 43 (Cx43) during the establishment of a synchronized contraction in confluent monolayers of primary cultured neonatal rat cardiac myocytes, combined with a functional assay of gap junctions by the microinjection-dye transfer method. Monitoring of the beating rate and synchronization by Fotonic Sensor showed that at Day 1 of culture cardiac myocytes contracted spontaneously but irregularly, that the contractile rate increased with culture time, and that a synchronized contraction was gradually formed. At Day 7, the confluent cells exhibited synchronous contraction with a relatively constant rate (125 ± 20 beats/min). Cardiac myocytes expressed a large amount of Cx43 mRNA even at Day 1 and maintained the expression until at least Day 7. Immunofluorescence of Cx43 showed that the localization of Cx43-positive spots was mostly restricted to cell-cell contacts between myocytes and that few Cx43-positive spots were present between myocytes and fibroblasts or between fibroblasts. The amount of Cx43 protein, the proportion of phosphorylated forms to the nonphosphorylated one, and the number and total area of Cx43-positive spots increased with culture time. Gap-junctional intercellular communication measured by dye transfer assay was also increased with culture time and correlated well with the number and total area of Cx43-positive spots. Our systematic study suggests that a concerted action of the expression, phosphorylation, and localization of Cx43 and gap-junctional intercellular communication plays a major role in the reestablishment of synchronous beating of cultured neonatal rat cardiac myocytes.     

Connexin 43 interacts with zona occludens-1 and -2 proteins in a cell cycle stage-specific manner

Gap junction channels play an important role in cell growth control, secretion and embryonic development. Gap junctional communication and channel assembly can be regulated by protein-protein interaction with kinases and phosphatases. We have utilized tandem mass spectrometry (MS/MS) sequence analysis as a screen to identify proteins from cell lysates that interact with the C-terminal cytoplasmic region of connexin 43 (Cx43). MS/MS analysis of tryptic fragments yielded several proteins including zona occludens-1 (ZO-1), a structural protein previously identified to interact with Cx43, and ZO-2, a potential novel interacting partner. We confirmed the interaction of ZO-2 with Cx43 by using a combination of fusion protein "pull down," co-immunoprecipitation, and co-localization experiments. We show that the C-terminal region of Cx43 is necessary for interaction with the PDZ2 domain of ZO-2. Far Western analysis revealed that ZO-2 can directly bind to Cx43 independent of other interacting partners. Immunofluorescence studies indicate that both ZO-1 and ZO-2 can co-localize with Cx43 within the plasma membrane at apparent gap junctional structures. We examined Cx43 interaction with ZO-1 and ZO-2 at different stages of the cell cycle and found that Cx43 had a strong preference for interaction with ZO-1 during G0, whereas ZO-2 interaction occurred approximately equally during G0 and S phases. Since essentially all of the Cx43 in G0 cells is assembled into Triton X-100-resistant junctions, Cx43-ZO-1 interaction may contribute to their stability.     

A proposed role for ZO-1 in targeting connexin 43 gap junctions to the endocytic pathway

Gap junctions are intercellular channels organized in plaque that directly link adjacent cells. Connexins (Cx), the constitutive proteins of gap junctions are associated with several partner proteins (cytoskeletal, anchoring) which could participate in plaque formation and degradation. Coimmunoprecipitation and indirect immunofluorescence analyses showed that ZO-1, a tight junction-associated protein, was linked to Cx43 in the testis. By using gamma-hexachlorocyclohexane (HCH), known to induce gap junction endocytosis, we demonstrated that endocytosis increased Cx43/ZO-1 association within the cytoplasm of treated Sertoli cells. In control cells, the two proteins were present, as expected, at the plasma membrane level, but poorly colocalized. The increased intracytoplasmic Cx43/ZO-1 complex was associated with a shift towards increased levels of Cx43 P1 and P2 isoforms. The HCH induced Cx43 hyperphosphorylation was abolished by the ERK inhibitor PD98059 suggesting that this effect could be mediated through activation of the ERK pathway. These data strongly support a novel role for ZO-1 in the turnover of Cx43 during gap junction plaque endocytosis.     

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.     

Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.     

Structural changes in the carboxyl terminus of the gap junction protein connexin43 indicates signaling between binding domains for c-Src and zonula occludens-1

Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of connexin-associating proteins. In particular, c-Src can disrupt the connexin43 (Cx43)-zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. The binding sites for ZO-1 and c-Src correspond to widely separated Cx43 domains (approximately 100 residues apart); however, little is known about the structural modifications that may allow information to be transferred over this distance. Here, we have characterized the structure of the connexin43 carboxyl-terminal domain (Cx43CT) to assess its ability to interact with domains from ZO-1 and c-Src. NMR data indicate that the Cx43CT exists primarily as an elongated random coil, with two regions of alpha-helical structure. NMR titration experiments determined that the ZO-1 PDZ-2 domain affected the last 19 Cx43CT residues, a region larger than that reported to be required for Cx43CT-ZO-1 binding. The c-Src SH3 domain affected Cx43CT residues Lys-264-Lys-287, Ser-306-Glu-316, His-331-Phe-337, Leu-356-Val-359, and Ala-367-Ser-372. Only region Lys-264-Lys-287 contains the residues previously reported to act as an SH3 binding domain. The specificity of these interactions was verified by peptide competition experiments. Finally, we demonstrated that the SH3 domain could partially displace the Cx43CT-PDZ-2 complex. These studies represent the first structural characterization of a connexin domain when integrated in a multimolecular complex. Furthermore, we demonstrate that the structural characteristics of a disordered Cx43CT are advantageous for signaling between different binding partners that may be important in describing the mechanism of channel closure or internalization in response to pathophysiological stimuli.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre+;Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre+;Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus

Gap junctions (GJs) are aggregates of channels that provide for direct cytoplasmic connection between cells. Importantly, this connection is thought responsible for cell-to-cell transfer of the cardiac action potential. The GJ channels of ventricular myocytes are composed of connexin43 (Cx43). Interaction of Cx43 with zonula occludens-1 (ZO-1) is localized not only at the GJ plaque, but also to the region surrounding the GJ, the perinexus. Cx43 in the perinexus is not detectable by immunofluorescence, yet localization of Cx43/ZO-1 interaction to this region indicated the presence of Cx43. Therefore, we hypothesized that Cx43 occurs in the perinexus at a lower concentration per unit membrane than in the GJ itself, making it difficult to visualize. To overcome this, the Duolink protein–protein interaction assay was used to detect Cx43. Duolink labeling of cardiomyocytes localized Cx43 to the perinexus. Quantification demonstrated that signal in the perinexus was lower than in the GJ but significantly higher than in nonjunctional regions. Additionally, Duolink of Triton X-100-extracted cultures suggested that perinexal Cx43 is nonjunctional. Importantly, the voltage gated sodium channel Na_v1.5, which is responsible for initiation of the action potential, was found to interact with perinexal Cx43 but not with ZO-1. This work provides a detailed characterization of the structure of the perinexus at the GJ edge and indicates that one of its potential functions in the heart may be in facilitating conduction of action potential.     

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.     

Role of catenins in the development of gap junctions in rat cardiomyocytes

Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane-associated guanylate kinase protein, zonula occludens-1 (ZO-1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO-1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of alpha-catenin, beta-catenin, ZO-1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca(2+) (<5 microM) medium, then shifted to 1.8 mM Ca(2+) medium (Ca(2+) switch). Diffuse cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca(2+) medium. Staining of alpha-catenin, beta-catenin, and ZO-1 was detected at the plasma membrane of cell-cell contact sites 10 min after Ca(2+) switch, whereas Cx43 staining was first detected, colocalized with ZO-1 at the plasma membrane, 30 min after Ca(2+) switch. Distinct junctional and extensive cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen 2 h after Ca(2+) switch. Immunoprecipitation of Triton X-100 cardiomyocyte extracts using anti-beta-catenin antibodies showed that beta-catenin was associated with alpha-catenin, ZO-1, and Cx43 at 2 h after Ca(2+) switch. Intracellular application of antisera against alpha-catenin, beta-catenin, or ZO-1 by electroporation of cardiomyocytes cultured in low Ca(2+) medium inhibited the redistribution of Cx43 to the plasma membrane following Ca(2+) switch. These results suggest the formation of a catenin-ZO-1-Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO-1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions.     

Basic fibroblast growth factor stimulates connexin-43 expression and intercellular communication of cardiac fibroblasts

Gap junctions (GJ) are membrane specializations responsible for intercellular communication and for ensuring electrical and/or metabolic coupling between cells. They are composed of connexins, a family of related proteins. Connexin-43 (Cx43) is a major connexin of the rat heart, expressed by myocytes as well as non-muscle cells. In this communication we have examined expression of Cx43 by cardiac fibroblasts and regulation of its expression by an endogenous mitogen, basic fibroblast growth factor (bFGF). Recombinant human bFGF, administered to cultured cells which had been maintained in 0.5% serum for 48 h, induced dose-dependent and statistically significant increases in Cx43 mRNA as well as protein accumulation, at 6 h after addition. Intercellular communication was also increased at 6 h but not 30 min after bFGF treatment, as assessed using a scrape-loading protocol. It is concluded that the bFGF-induced stimulation of Cx43 expression caused increased coupling between cardiac fibroblasts. This would be of importance in injured myocardium, the increased bFGF content of which might stimulate electrical coupling involving fibroblasts of the scar tissue.