Upregulation of connexin43 gap junctions between neointimal smooth muscle cells

Increased expression of connexin43 gap junctions in smooth muscle cells (SMC) is implicated in the response to primary arterial injury and in the early stages of human coronary atherosclerosis, but the relevance of these findings to restenosis is unknown. Here we investigated the expression of connexin43 gap junctions in restenotic aortas of cholesterol-fed double injured rabbits. Immunofluorescence confocal microscopy was used to evaluate temporal and spatial expression patterns and to characterize the major expressing cell type. Parallel studies were conducted by electron microscopy, in situ hybridization and Northern blot analysis. Connexin43 gap junctions- and connexin43 mRNA-expressing cells were abundant in the media of non-injured control aorta. Following primary injury and 6 weeks cholesterol diet, connexin43 gap junctions were found distributed throughout the primary intimal layer; although medial expression was reduced, the overall mRNA expression level remained similar to that of non-injured controls. After secondary injury, no major change in distribution pattern of connexin43 gap junctions occurred up to day 10, when marked neointimal labeling was observed. This overall pattern persisted, though with some diminution, at later stages. On the mRNA level total connexin43 mRNA expression declined to about 40% of control values within 4 days after secondary injury (P < 0.05), but subsequently increased four-fold, attaining levels double that of non-injured controls in the 10-day group (P < 0.005 versus control and 4 days). At later stages mRNA expression levels returned to values similar to those of non-injured controls. At all stages, connexin43 gap junctions were localized to the SMC, not to macrophages. We conclude that the enhanced gap junction formation may contribute to the coordination of the response of SMC after secondary injury, particularly in the early phase of restenosis.     

Voltage-dependent blockade of connexin40 gap junctions by spermine

The effects of spermine and spermidine, endogenous polyamines that block many forms of ion channels, were investigated in homotypic connexin (Cx)-40 gap junctions expressed in N2A cells. Spermine blocked up to 95% of I(j) through homotypic Cx40 gap junctions in a concentration- and transjunctional voltage (V(j))-dependent manner. V(j) was varied from 5 to 50 mV in 5-mV steps and the dissociation constants (K(m)) were determined from spermine concentrations ranging from 10 micro M to 2 mM. The K(m) values ranged from 4.9 mM to 107 micro M for 8.6 < or = V(j) < or = 37.7 mV, within the physiological range of intracellular spermine for V(j) > or = 20 mV. The K(m) values for spermidine were > or = 5 mM. Estimates of the electrical distance (delta) for spermine (z = +4) and spermidine (z = +3) were 0.96 and 0.76 respectively. Cx40 single channel conductance was 129 pS in the presence of 2-mM spermine and channel open probability was significantly reduced in a V(j)-dependent manner. Similar concentrations of spermine did not block I(j) through homotypic Cx43 gap junctions, indicating that spermine selectively blocks Cx40 gap junctions. This is contrary to our previous findings that large tetraalkylammonium ions, also known to block several forms of ion channels, block junctional currents (I(j)) through homotypic connexin Cx40 and Cx43 gap junctions.     

Assembly of connexin43 into gap junctions is regulated differentially by E-cadherin and N-cadherin in rat liver epithelial cells

Cadherins have been thought to facilitate the assembly of connexins (Cxs) into gap junctions (GJs) by enhancing cell-cell contact, however the molecular mechanisms involved in this process have remained unexplored. We examined the assembly of GJs composed of Cx43 in isogenic clones derived from immortalized and nontransformed rat liver epithelial cells that expressed either epithelial cadherin (E-Cad), which curbs the malignant behavior of tumor cells, or neuronal cadherin (N-Cad), which augments the invasive and motile behavior of tumor cells. We found that N-cad expression attenuated the assembly of Cx43 into GJs, whereas E-Cad expression facilitated the assembly. The expression of N-Cad inhibited GJ assembly by causing endocytosis of Cx43 via a nonclathrin-dependent pathway. Knock down of N-Cad by ShRNA restored GJ assembly. When both cadherins were simultaneously expressed in the same cell type, GJ assembly and disassembly occurred concurrently. Our findings demonstrate that E-Cad and N-Cad have opposite effects on the assembly of Cx43 into GJs in rat liver epithelial cells. These findings imply that GJ assembly and disassembly are the down-stream targets of the signaling initiated by E-Cad and N-Cad, respectively, and may provide one possible explanation for the disparate role played by these cadherins in regulating cell motility and invasion during tumor progression and invasion.     

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.     

Dynamics of gap junctions observed in living cells with connexin43-GFP chimeric protein

To study the aggregation of cell-to-cell channels into gap junctions at individual cell-cell contacts, we transfected cells with an expression vector for a chimeric protein composed of the cell-to-cell channel protein connexin43 and a green fluorescent protein. The chimeric channel protein was visualized in the fluorescence microscope and was found to form gap junctions at the cell-cell contacts just like wild-type connexin43. Cells expressing the chimeric protein had functional cell-to-cell channels. Using timelapse videomicroscopy on live cells we observed individual gap junctions over long periods and recorded the time course of aggregation of the chimeric channel protein into gap junctions at newly formed cell-cell contacts. We found that individual small gap junctions were very dynamic, moving about or becoming assembled and disassembled in the course of minutes. Larger gap junctions were more stable than small punctate ones. In control condition, stable new gap junctions were not formed during observation times of 30 min or longer. But at elevated levels of cyclic adenosine monophosphate, the chimeric channel protein began aggregating at new junctions 5-10 minutes after cell-cell contact and continued to concentrate there for at least one hour. Also already established junctions grew in size. The fluorescent chimeric channel protein will be an excellent tool to investigate the regulation of trafficking of connexin from and to the membrane and the mechanism of connexin channel aggregation into gap junctions.     

Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.

Connexin 43 (Cx43alpha1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43alpha1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin-deficient neural crest cells, but the alterations were different from that elicited by Cx43alpha1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43alpha1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency. Based on these findings, we propose a model in which Cx43alpha1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

Association between mitochondria and gap junctions in mammalian myocardial cells

In ventricular myocardial cells of mouse, guinea-pig, dog, and monkey, mitochondria frequently form close associations with gap junctions, the two structures being separated by a space of 20 nm or less. Similar appositions are found in both the mature atria and the developing myocardium of the mouse. The gap junctions assume a variety of configurations with respect to the apposed mitochondria. These include profiles in which the gap junctions conform closely to the contours of mitochondria, as well as profiles in which finger-like sarcolemmal evaginations, composed entirely of gap junctions, extend longitudinally or transversely into an adjoining cell to envelop mitochondria. In mouse ventricular wall, over 40% of the length of gap junctions is juxtaposed to mitochondria, and strands of connecting material are often present in the interspace between the two structures. In addition, in freeze-fracture replicas, portions of mitochondria are found attached to areas of myocardial sarcolemma that contain gap-junctional particles. Since mitochondria are known to sequester Ca²⁺ ion, it is possible that the close association between mitochondria and gap junction may function to buffer the intracellular Ca²⁺ concentration near the gap junctions, and thereby regulate the ionic permeability of the gap junctions.     

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.     

v-Src-mediated phosphorylation of connexin43 on tyrosine disrupts gap junctional communication in mammalian cells

It is not clear how the v-Src oncoprotein disrupts gap junctional communication (GJC) established by connexin43 (Cx43) in mammalian cells. In this study, an experimental system was established to stably express v-Src and wild type (wt) Cx43, or Y247F, Y265F, or Y247F/Y265F Cx43 mutants in a Cx43 knockout (KO) mouse cell line. When co-expressed with v-Src, the levels of phosphotyrosine (pTyr) from Y247F, Y265F, and Y247F/Y265F Cx43 mutants were reduced to approximately 57%, 10%, and 2% of the level of pTyr from wt Cx43, indicating that Y247 and Y265 were phosphorylation targets of v-Src in vivo. These data also implied that phosphorylation of Cx43 at Y265 was required for efficient phosphorylation of Cx43 at Y247. Most importantly, our measurements of GJC demonstrated that, in contrast to the wt Cx43 gap junction channels, the Y247F, Y265F, and Y247F/Y265F Cx43 channels were resistant to the disruption by v-Src. In conclusion, our studies support a model for processive phosphorylation of Cx43 on tyrosine, at the Y265 site followed by the Y247 site, in mediating the disruption of GJC induced by v-Src in mammalian cells.     

Immunolocalization of an extracellular domain of connexin43 in rat heart gap junctions.

Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea-split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs.     

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.     

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Abstract

Gα_q-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gα_q-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions: Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.     

Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Gαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions: Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.     

Differential expression of connexin43 gap junctions in cardiomyocytes isolated from canine thoracic veins.

We investigated the phenotypic features of cardiomyocytes, including the gap junctions, in the myocardial sleeve of thoracic veins. Single cardiomyocytes, isolated from the canine pulmonary veins (PV) and superior vena cava (SVC) using digestive enzymes, were examined by immunoconfocal microscopy using antisera against connexin43 (Cx43), Cx40, and other cell markers. The results showed that isolated cardiomyocytes displayed rod shapes of various sizes, ranging from <50 microm to >200 microm in length, and all the cells expressed alpha-actinin and vinculin. Gap junctions made of various amounts of Cx43 and Cx40 were found at the cell borders. These two connexins were extensively co-localized. Comparison between the thoracic veins showed that cells of the SVC contained more Cx43 gap junctions (total Cx43 gap junctions area per cell surface area, 4.0 +/- 0.2% vs 1.5 +/- 0.2%; p<0.01). In addition, for single-nucleus cells, those from the PV were longer (103.7 +/- 3.6 vs 85.0 +/- 3.1 microm; p<0.01) but narrower (14.4 +/- 0.5 vs 16.9 +/- 0.9 microm; p<0.01). In conclusion, canine thoracic veins contain cardiomyocytes with differences in shape and gap junctions, suggesting that the electrical conduction properties may be different between the thoracic veins.     

Expression of connexin43 gap functions between cultured vascular smooth muscle cells is dependent upon phenotype

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.     

Selective effect of PDGF on connexin43 versus connexin40 comprised gap junction channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g(j) and gamma(j), respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g(j) in Cx40/Cx40 pairs, but decreased g(j) in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g(j) suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g(j) involved a decrease in both gamma(j) and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

An essential role for connexin43 gap junctions in mouse coronary artery development

Connexin43 knockout mice die neonatally from conotruncal heart malformation and outflow obstruction. Previous studies have indicated the involvement of neural crest perturbations in these cardiac anomalies. We provide evidence for the involvement of another extracardiac cell population, the proepicardial cells. These cells give rise to the vascular smooth muscle cells of the coronary arteries and cardiac fibroblasts in the heart. We have observed the abnormal presence of fibroblast and vascular smooth muscle cells in the infundibular pouches of the connexin43 knockout mouse heart. In addition, the connexin43 knockout mice exhibit a variety of coronary artery patterning defects previously described for neural crest-ablated chick embryos, such as anomalous origin of the coronary arteries, absent left or right coronary artery, and accessory coronary arteries. However, we show that proepicardial cells also express connexin43 gap junctions abundantly. The proepicardial cells are functionally well coupled, and this coupling is significantly reduced with the loss of connexin43 function. Further analysis revealed an elevation in the speed of cell locomotion and cell proliferation rate in the connexin43-deficient proepicardial cells. A parallel analysis of proepicardial cells in transgenic mice with dominant negative inhibition of connexin43 targeted only to neural crest cells showed none of these coupling, proliferation or migration changes. These mice exhibit outflow obstruction, but no infundibular pouches. Together these findings indicate an important role for connexin43 in coronary artery patterning, a role that probably involves the proepicardial and cardiac neural crest cells. We discuss the potential involvement of connexin43 in human cardiovascular anomalies involving the coronary arteries.     

Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40

Gap junction channels provide the basis for the electrical syncytial properties of the heart as a communicating electrical network. Cardiac gap junction channels are predominantly composed of connexin 40 or connexin 43. The conductance of these channels (g(j)) can be regulated pharmacologically: substances which activate protein kinase C, protein kinase A or protein kinase G may alter Cx43 gap junction conductance. However, for PKC, this seems to be subtype specific. Thus, antiarrhythmic peptides can enhance g(j) via activation of PKCepsilon, while FGF-2 reduces g(j) via PKCepsilon. Lipophilic drugs can uncouple the channels. Besides an acute regulation of g(j), the expression of the cardiac connexins can also be regulated. A decrease in Cx43 with a concomitant increase in Cx40 has been found in end-stage failing hearts, while in renovascular hypertension, an increase in Cx43 has been described. Mediators like endothelin-1, angiotensin-II, TGF-beta, VEGF, and cAMP have been shown to increase Cx43. Interestingly, endothelin-1 and angiotensin-II increased Cx43 but did not affect Cx40 expression. In contrast, in humans suffering from atrial fibrillation (AF), the content in Cx40 can be enhanced while Cx43 was unaltered, although in several other studies, other changes of the cardiac connexins were found, which might be related to the type of AF. Regarding the role of calcium, the content in both Cx40 and Cx43 was decreased in cultured neonatal rat cardiomyocytes after 24 h administration of 100 nM verapamil. Thus, gap junctional channels can be affected pharmacologically either acutely by modulating gap junction conductance or chronically by altering gap junction protein expression. Interestingly, it appears that the expression of Cx43 and Cx40 can be differentially regulated.