Short-term pacing in the mouse alters cardiac expression of connexin43

Background

Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time.

Results

The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias.

Conclusion

Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.     

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.     

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.     

Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells.     

Nonylphenol alters connexin 43 levels and connexin 43 phosphorylation via an inhibition of the p38-mitogen-activated protein kinase pathway

Endocrine-disrupting chemicals are exogenous compounds that mimic or inhibit the action of estrogens or other hormones. Nonylphenol, an environmental contaminant distributed along the St. Lawrence River, has been reported to act as a weak estrogen. Previous studies from our laboratory have shown that rats that were fed fish taken from nonylphenol contaminated sites have altered spermatogenesis and decreased sperm count. The mechanism responsible for this effect is unknown. Gap junctional intercellular communication (GJIC) in the testis is critical for coordinating spermatogenesis. The objectives of the study were to determine the effects of nonylphenol on GJIC and connexin 43 (Cx43) in a murine Sertoli cell line, TM4. Cells were exposed for 24 h to different concentrations (1 to 50 microM) of either nonylphenol or 17beta-estradiol. GJIC was determined using a microinjection approach in which Lucifer yellow was injected directly into a single cell, and GJIC was assessed 3 min postinjection. Nonylphenol exposure decreased GJIC between adjacent cells by almost 80% relative to controls. A significant concentration-dependent reduction in GJIC was observed at nonylphenol concentrations between 1 and 50 microM. Cx43 immunofluorescent staining was reduced at both 10 and 50 microM doses of nonylphenol. Cx43 phosphorylation, as determined by Western blot analysis, was reduced at both 10 and 50 microM concentrations, which may explain, at least in part, the inhibition of GJIC. In contrast, no effect on GJIC or Cx43 protein was observed in cells exposed to 17beta-estradiol at these concentrations. Cx43 has been reported to be phosphorylated via the p38-mitogen-activated protein kinase (MAPK) pathway. P38-MAPK activity was assessed in both control and nonylphenol-exposed cells. A dose-dependent decrease in p38-MAPK activity was observed in nonylphenol-exposed Sertoli cells. Protein kinase C activity was also measured and was not influenced by nonylphenol. These results suggest that nonylphenol inhibits GJIC between Sertoli cells and that this is modulated via nonestrogenic pathways.     

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.     

Differential expression of connexin 43 in mouse mammary cells

In this study we have employed suppressive subtractive hybridization (SSH) analysis to investigate differential gene expression in primary mouse mammary epithelial cells (PMMEC) cultured under mildly apoptotic/quiescent and differentiating conditions. Among a small group of genes whose expression was differentially regulated was connexin 43. In vitro, connexin 43 mRNA and protein were detectable in PMMEC cultured under proliferative or mildly apoptotic conditions. The level of connexin 43 mRNA expression in vivo was also investigated. High levels of expression were found to be associated with the periods of greatest glandular plasticity (pubertal expansion of the mammary tree, early pregnancy and during early involution). Thus, terminally differentiated cells in vivo and in vitro did not express connexin 43 mRNA suggesting that connexin 43 expression, and perhaps facilitated gap junction communication, is associated with undifferentiated progenitor cell populations.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

Reduced atrial connexin43 expression after pediatric heart surgery

Myocardial dysfunction and arrhythmias may be induced by congenital heart defects, but also be the result of heart surgery with cardiopulmonary bypass (CPB), potentially caused by differential expression of connexin40 (Cx40) and connexin43 (Cx43). In 16 pediatric patients undergoing corrective heart surgery, connexin mRNA expression was studied in volume overloaded (VO group, n=8) and not overloaded (NO group, n=8) right atrial myocardium, excised before and after CPB. Additionally, in eight of these patients ventricular specimens were investigated. The atrial Cx43 expression decreased during CPB, which was restricted to the VO group (p=0.008). In contrast, atrial Cx40 mRNA did not change during CPB. In ventricular myocardium compared to atrial mRNA levels, Cx40 was lower (p=0.006) and Cx43 higher (p=0.017) expressed, without significant change during CPB. This study revealed a significant influence of CPB and the underlying heart defect on Cx43 expression.     

Trypanosoma cruzi induces changes in cardiac connexin43 expression

Gap junction proteins (connexins) are required for myocardial function, since they allow intercellular transmission of current carrying ions and signaling molecules. Previous studies demonstrated that rat cardiac myocytes infected with Trypanosoma cruzi lost gap junctional communication and decreased automaticity. We infected mouse cardiac myocytes with trypomastigotes of the Y strain of T. cruzi and observed alterations in connexin43 (Cx43) distribution. One hour post infection Cx43 levels were significantly increased. However, at longer time points post infection there was a significant loss of Cx43 staining in membranes of infected cardiac myocytes. Interestingly, there was also a significant reduction in myocardial Cx43 protein levels during acute infection. These data indicate that T. cruzi infection alters Cx43 expression both in vitro and in vivo. Disruptions in Cx43 may contribute to the pathogenesis of cardiac electrical alterations observed in T. cruzi infection.     

Interaction of arsenic with gap junction protein connexin 43 alters gap junctional intercellular communication

Chronic exposure to Arsenic pollution in ground water is one of the largest environmental health disasters in the world. The toxicity of trivalent Arsenicals primarily happens due to its interaction with sulfhydryl groups in proteins. Arsenic binding to the protein can change the conformation of the protein and alter its interactions with other proteins leading to tissue damage. Therefore, much importance has been given to the studies of Arsenic bound proteins, for the purpose of understanding the origins of toxicity and to explore therapeutics. Here we study the dynamic effect of Arsenic on Connexin 43 (Cx43), a protein that forms the gap junctions, whose alteration deeply perturbs the cell-to-cell communication vital for maintaining tissue homeostasis. In silico molecular modelling and in vitro studies comparing Arsenic treated and untreated conditions show distinct results. Gap junction communication is severely disrupted by Arsenic due to reduced availability of unaltered Cx43 in the membrane bound form. In silico and Inductively Coupled Plasma Mass Spectrometry studies revealed the interaction of Arsenic to the Cx43 preferably occurs through surface exposed cysteines, thereby capping the thiol groups that form disulfide bonds in the tertiary structure. This leads to disruption of Cx43 oligomerization, and altered Cx43 is incompetent for transportation to the membrane surface, often forming aggregates primarily localizing in the endoplasmic reticulum. Loss of functional Cx43 on the cell surface have a deleterious effect on cellular homeostasis leading to selective vulnerability to cell death and tissue damage.     

Targeting connexin43 expression accelerates the rate of wound repair

The repair of tissue damage is a key survival process in all organisms and involves the coordinated activation of several cell types. Cell-cell communication is clearly fundamental to this process, and a great deal is known about extracellular communication within the wound site via cytokines. Here we show that direct cell-cell communication through connexin 43 (Cx43) gap junction channels also plays a major role in the wound healing process. In two different wound healing models, incisional and excisional skin lesions, we show that a single topical application of Cx43 antisense gel brings about a transient downregulation of Cx43 protein levels, and this results in a dramatic increase in the rate of wound closure. Cx43 knockdown reduces inflammation, seen both macroscopically, as a reduction in swelling, redness, and wound gape, and microscopically, as a significant decrease in neutrophil numbers in the tissue around the wound. One long-term consequence of the improved rate of healing is a significant reduction in the extent of granulation tissue deposition and the subsequent formation of a smaller, less distorted, scar. This approach is likely to have widespread therapeutic applications in other injured tissues and opens up new avenues of research into improving the wound healing process.     

Regulated expression of the X. tropicalis connexin43 promoter

The spatio-temporal expression pattern of the connexin43 gene during Xenopus development has been described (Van der Heyden et al. 2001). To further investigate the regulation and function of connexin43 (Cx43) in amphibians, we have isolated the gene from Xenopus tropicalis (Xt) and determined its structure. The X. tropicalis Cx43 gene displays the typical two exon-one intron connexin configuration, where the first exon is non-coding. The predicted amino acid sequence of the XtCx43 protein is highly homologous to that of X. laevis, chicken and mammals. Expression of XtCx43 cDNA in N2A cells results in gap-junction plaque formation. Promoter activity of a 3.5 kb upstream region of the X. tropicalis Cx43 gene, including exon 1, mimics endogenous timing of expression after injection of reporter constructs in X. laevis embryos.     

Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells.

We examined the expression and function of gap junctions in two rat osteoblastic cell lines, ROS 17/2.8 and UMR 106-01. The pattern of expression of gap junction proteins in these two cell lines was distinct: ROS cells expressed only connexin43 on their cell surface, while UMR expressed predominantly connexin45. Immunoprecipitation and RNA blot analysis confirmed the relative quantitation of these connexins. Microinjected ROS cells passed Lucifer yellow to many neighboring cells, but UMR cells were poorly coupled by this criterion. Nevertheless, both UMR and ROS cells were electrically coupled, as characterized by the double whole cell patch-clamp technique. These studies suggested that Cx43 in ROS cells mediated cell-cell coupling for both small ions and larger molecules, but Cx45 in UMR cells allowed passage only of small ions. To demonstrate that the expression of different connexins alone accounted for the lack of dye coupling in UMR cells, we assessed dye coupling in UMR cells transfected with either Cx43 or Cx45. The UMR/Cx43 transfectants were highly dye coupled compared with the untransfected UMR cells, but the UMR/Cx45 transfectants demonstrated no increase in dye transfer. These data demonstrate that different gap junction proteins create channels with different molecular permeabilities; they suggest that different connexins permit different types of signalling between cells.     

MIR-206 regulates connexin43 expression during skeletal muscle development

Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3′-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development.