Age- and myopathy-dependent changes in connexins of normal and cardiomyopathic Syrian hamster ventricular myocardium

The conduction of cardiac action potentials depends on the flow of excitation through gap junctions, which are hexameric protein associations of connexins (Cxs). The major Cx reported in the heart is Cx43, although some Cx40 and Cx45 are also present. There is some evidence for altered Cx content in heart failure. In heart failure, conduction is depressed and slowed conduction may contribute to arrhythmogenesis and (or) the maintenance of arrhythmia. Cx content and distribution were determined in ventricular tissues from normal and cardiomyopathic Syrian hamsters, an animal model of heart failure which has reproducible age-specific cardiomyopathy resulting in heart failure and age-matched controls in three groups: young (3-5 weeks), adult (13-18 weeks), and old (>45 weeks). Frozen, unfixed sections of ventricular tissues were immunofluorescently stained using antibodies against Cx43, Cx40, and Cx45. Cx43 was the predominant Cx detected in all samples. In normal hamsters, Cx43 was localized predominantly at the intercalated disc region, while in myopathic myocytes, it was scattered. In Western blots, Cx43 content of normal hamster hearts was highest in the adult hearts compared with young and old hamster hearts. In contrast, Cx43 content was significantly lower in adult cardiomyopathic hamster hearts compared with all other groups. The alterations of content and distribution of gap junction Cx43 may contribute to diminished conduction, pump function, and arrhythmogenesis in heart failure.     

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.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC

Phosphorylation at unspecified sites is known to regulate the life cycle (assembly, gating, and turnover) of the gap junction protein, Cx43. In this paper, we show that Cx43 is phosphorylated on S365 in cultured cells and heart tissue. Nuclear magnetic resonance structural studies of the C-terminal region of Cx43 with an S365D mutation indicate that it forms a different stable conformation than unphosphorylated wild-type Cx43. Immunolabeling with an antibody specific for Cx43 phosphorylated at S365 shows staining on gap junction structures in heart tissue that is lost upon hypoxia when Cx43 is no longer specifically localized to the intercalated disk. Efficient phosphorylation at S368, an important Cx43 channel regulatory event that increases during ischemia or PKC activation, depends on S365 being unphosphorylated. Thus, phosphorylation at S365 can serve a “gatekeeper” function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance.     

Quiescent mammary epithelial cells have reduced connexin43 but maintain a high level of gap junction intercellular communication

Gap junctions have been implicated in growth control, but it remains unclear whether cells that enter a quiescent state continue to express connexins and maintain a high level of gap junction intercellular communication (GJIC). To this end, MAC-T cells, a bovine mammary epithelial cell line, were serum starved for 48 h to induce a quiescent (G0) state. In quiescent cells, [3H]thymidine incorporation was reduced by 97.3% from serum-fed controls. Western blotting in conjunction with Phosphorlmager analysis revealed up to a 20-fold decrease in the expression of the gap junction protein connexin43 (Cx43 or alpha 1) and a shift toward the unphosphorylated form in quiescent cells. However, cell-to-cell transfer of the gap junction-permeable fluorescent tracer, Lucifer yellow, was only moderately reduced in quiescent cells. In control cells, Cx43 was predominantly perinuclear, although it was also present at sites of cell-cell apposition. In quiescent cells, intracellular labeling for Cx43 decreased without a corresponding reduction at areas of cell-cell contact. Recovery from serum deprivation resulted in increased thymidine incorporation that corresponded with an elevation in Cx43 protein expression and phosphorylation. In parallel studies, MAC-T cells were also induced to enter a quiescent state through contact inhibition. Despite a 20-fold reduction in 5-bromo-2'-deoxyuridine and a substantial reduction in intracellular Cx43, contact inhibited MAC-T cells also maintained gap junctions and GJIC. These experiments demonstrate that the maintenance of dye coupling in quiescent mammary cells is correlated with a redistribution of intracellular stores of Cx43.     

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.     

Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in essentially all tissues. There are 21 connexin genes in the human genome and different tissues express different connexin genes. Most connexins are known to be phosphoproteins. Phosphorylation can regulate connexin assembly into gap junctions, gap junction turnover and channel gating. Given the importance of gap junctions in development, proliferation and carcinogenesis, regulation of gap junction phosphorylation in response to wounding, hypoxia and other tissue insults is proving to be critical for cellular response and return to homeostasis. Connexin43 (Cx43) is the most widely and highly expressed gap junction protein, both in cell culture models and in humans, thus more research has been done on it and more reagents to it are available. In particular, antibodies that can report Cx43 phosphorylation status have been created allowing temporal examination of specific phosphorylation events in vivo. This review is focused on the use of these antibodies in tissue in situ, predominantly looking at Cx43 phosphorylation in brain, heart, endothelium and epithelium with reference to other connexins where data is available. These data allow us to begin to correlate specific phosphorylation events with changes in cell and tissue function. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Connexin43 phosphorylation and cytoprotection in the heart

The fundamental role played by connexins including connexin43 (Cx43) in forming intercellular communication channels (gap junctions), ensuring electrical and metabolic coupling between cells, has long been recognized and extensively investigated. There is also increasing recognition that Cx43, and other connexins, have additional roles, such as the ability to regulate cell proliferation, migration, and cytoprotection. Multiple phosphorylation sites, targets of different signaling pathways, are present at the regulatory, C-terminal domain of Cx43, and contribute to constitutive as well as transient phosphorylation Cx43 patterns, responding to ever-changing environmental stimuli and corresponding cellular needs. The present paper will focus on Cx43 in the heart, and provide an overview of the emerging recognition of a relationship between Cx43, its phosphorylation pattern, and development of resistance to injury. We will also review our recent work regarding the role of an enhanced phosphorylation state of Cx43 in cardioprotection. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Administration of FGF-2 to the heart stimulates connexin-43 phosphorylation at protein kinase C target sites

Fibroblast growth factor-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for 20 min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.     

Administration of FGF-2 to the Heart Stimulates Connexin-43 Phosphorylation at Protein Kinase C Target Sites

Fibroblast growth factor-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for 20 min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.     

Heart and head defects in mice lacking pairs of connexins

Gene ablation studies in mice have revealed roles for gap junction proteins (connexins) in heart development. Of the 20 connexins in vertebrates, four are expressed in developing heart: connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). Although each cardiac connexin has a different pattern of expression, some heart cells coexpress multiple connexins during cardiac morphogenesis. Since different connexins could have overlapping functions, some developmental phenotypes may only become evident when more than one connexin is ablated. In this study, we interbred Cx40(-/-) and Cx43(-/-) mice to generate mice lacking both Cx40 and Cx43. Cx40(-/-)Cx43(-/-) mice die around embryonic day 12.5 (E12.5), much earlier than either Cx40(-/-) or Cx43(-/-) mice, and they exhibit malformed hearts with ventricles that are abnormally rotated, suggesting a looping defect. Some Cx40(-/-)Cx43(-/-) animals also develop head defects characteristic of exencephaly. In addition, we examined mice lacking both Cx40 and Cx37 and found a high incidence of atrial and ventricular septal defects at birth. These results provide further evidence for the importance of gap junctions in embryonic development. Moreover, ablating different pairs of cardiac connexins results in distinct heart defects, suggesting both common and unique functions for Cx40, Cx43, and Cx37 during cardiac morphogenesis.     

Effects of cytochrome P450 inhibitors on agonist-induced Ca2+ responses and production of NO and PGI2 in vascular endothelial cells

The gap junction protein connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43–46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).     

Inhibiting N-cadherin-mediated adhesion affects gap junction communication in isolated rat hearts

Cadherin-mediated adherens junctions is impaired concomitant with a decrease in connexin 43 (Cx43) in diseases or pathological processes. We have investigated the acute effects of adherens junction impairment in isolated rat hearts by introducing Ala-His-Ala-Val-Asp-NH₂ (AHAVD, a synthetic peptide) as a specific inhibitor of N-cadherin. Effect of AHAVD on N-cadherin mediated adhension was analyzed by Cardiomy-ocyte aggregation assay. Laser confocal microscopy showed disrupted cell-cell contacts in cultured neonatal cardiomyocytes co-incubated with 0.2 mM AHAVD. In isolated adult rat hearts, Cx43 was redistributed along the bilateral of cardiomyocytes from the intercalated discs and significant dephosphorylation of Cx43 on serine368 occurred concomitantly with decreased gap junction (GJ) function in dose dependent manner after 1 h perfusion with AHAVD. These results indicate that impairing cad-herin-mediated adhesion by AHAVD rapidly results in Cx43 redistribution and dephosphorylation of serine368, thereby impairing GJ communication function.     

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.     

Changes in gap junction distribution and connexin expression pattern during human fetal skin development.

Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.     

Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study

Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Connexins 43 (Cx43) are the most widely expressed gap junction proteins. Cellular localization of phosphorylated Cx43 has been implicated in the capacity of gap junctional intercellular communication (GJIC). To follow the functionality of GJIC of different cell types, in monolayer cultures, characterized by different patterns of phosphorylated Cx43, we used a fluorescence recovery after photobleaching (FRAP) technique, and compared two tracers, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). The GJIC capacity was quantified by estimating fluorescence redistribution parameters. The functionality of GJIC was in relation with the staining localization of phosphorylated Cx43 to the cell-cell contact areas, corresponding to gap junctions between contacting cells. GJIC involvement in fluorescence restitution after photobleaching was checked by a gap junction channel inhibition assay. We demonstrated that the choice of the dye did not significantly influence the fluorescence recovery percentages despite a cell line-dependent CFDA release, whereas it had an important impact on fluorescence kinetic profiles. This study reinforces the interest of the gap-FRAP approach to quantify modifications in the functionality of gap junctions and, above all, argues about the limits of CFDA for 3-D future approaches.     

Is the junctional uncoupling elicited in rat ventricular myocytes by some dephosphorylation treatments due to changes in the phosphorylation status of Cx43?

Gap junctions, specialized membrane structures that mediate cell-to-cell communication in almost all animal tissues, are composed of channel-forming integral membrane proteins termed connexins. Most of them, particularly connexin43 (Cx43), the most ubiquitous connexin, the major connexin present in cardiac myocytes, are phosphoproteins. Connexin phosphorylation has been thought to regulate gap junctional protein trafficking, gap junction assembly, channel gating, and turnover. Some connexins, including Cx43, show mobility shifts in gel electrophoresis when cells are exposed to phosphorylating or dephosphorylating treatments. However, after exposure of rat cardiac myocytes to different uncoupling dephosphorylating agents such as H7 or butanedione monoxime, no modification in the Cx43 phosphorylation profile was generally observed. The lack of direct correlation between the inhibition of cell-to-cell communication and changes in the phosphorylation pattern of Cx43 or, conversely, modifications of the latter without modifications of the intercellular coupling degree, suggest that the functional state of junctional channels might rather be determined by regulatory proteins associated with Cx43. The modulation of the activity of junctional channels by protein phosphorylation/dephosphorylation processes very likely requires (as for several other membrane channels) the formation of a multiprotein complex, where pore-forming subunits bind to auxiliary proteins (e.g. scaffolding proteins, enzymes, cytoskeleton elements) that play essential roles in channel localization and activity. Such regulatory proteins, behaving as targets for phosphorylation/dephosphorylation catalysers, might in particular control the open probability of junctional channels. A schematic illustration of the regulation of Cx43-made channels by protein phosphorylation involving a partner phosphoprotein is proposed.Presented at the Biophysical Society Meeting on Ion channels – from Biophysics to disorders, held in May 2003, Rennes, France     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Dephosphorylation agents depress gap junctional communication between rat cardiac cells without modifying the Connexin43 phosphorylation degree.

The functional state of gap junctional channels and the phosphorylation status of Connexine43 (Cx43), the major gap junctional protein in rat heart, were evaluated in primary cultures of neonatal rat cardiomyocytes. H7, able to inhibit a range of serine/threonine protein kinases, progressively reduced gap junctional conductance to approximately 13% of its initial value within 10 min except when protein phosphatase inhibitors were also present. The dephosphorylating agent 2,3-Butanedione monoxime (BDM) produced both a quick and reversible interruption of cell-to-cell communication as well as a parallel slow inhibition of junctional currents. The introduction of a non-hydrolysable ATP analogue (ATPgammaS) in the cytosol delayed the second component, suggesting that it was the consequence of protein dephosphorylation. Western blot analysis reveals 2 forms of Cx43 with different electrophoretic mobilities which correspond to its known phosphorylated and dephosphorylated forms. After exposure of the cells to H7 (1 mmol/l, 1h) or BDM (15 mmol/l, 15 min), no modification in the level of Cx43 phosphorylation was observed. The lack of direct correlation between the inhibition of cell-to-cell communication and changes in the phosphorylation status of Cx43 suggest that the functional state of junctional channels might rather be determined by regulatory proteins associated to Cx43.