Endothelial-specific deletion of connexin40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion.

BACKGROUND: Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. METHODS AND RESULTS: Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. CONCLUSIONS: Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.

OBJECTIVE: The sinoatrial (SA) node consists of a relatively small number of poorly coupled cells. It is not well understood how these pacemaker cells drive the surrounding atrium and at the same time are protected from its hyperpolarizing influence. To explore this issue on a small tissue scale we studied the activation pattern of the mouse SA node region and correlated this pattern with the distribution of different gap junction proteins, connexin (Cx)37, Cx40, Cx43 and Cx45. METHODS AND RESULTS: The mouse SA node was electrophysiologically mapped using a conventional microelectrode technique. The primary pacemaker area was located in the corner between the lateral and medial limb of the crista terminalis. Unifocal pacemaking occurred in a group of pacemaking fibers consisting of 450 cells. In the nodal area transitions of nodal and atrial waveform were observed over small distances ( approximately 100 microm). Correlation between the activation pattern and connexin distribution revealed extensive labeling by anti-Cx45 in the primary and secondary pacemaker area. Within these nodal areas no gradient in Cx45 labeling was found. A sharp transition was found between Cx40- and Cx43-expressing myocytes of the crista terminalis and the Cx45-expressing myocytes of the node. In addition, strands of myocytes labeled for Cx43 and Cx40 protrude into the nodal area. Cx37 labeling was only present between endothelial cells. Furthermore, a band of connective tissue largely separates the nodal from the atrial tissue. CONCLUSIONS: Our results demonstrate strands of Cx43 and Cx40 positive atrial cells protruding into the Cx45 positive nodal area and a band of connective tissue largely separating the nodal and atrial tissue. This organization of the mouse SA node provides a structural substrate that both shields the nodal area from the hyperpolarizing influence of the atrium and allows fast action potential conduction from the nodal area into the surrounding atrium.

Channelopathies: Kir2.1 mutations jeopardize many cell functions.

Andersen's syndrome is caused by mutations in the potassium channel Kir2.1, a major determinant of resting membrane potential. The clinical features of this disease illustrate the importance of a stable resting membrane potential for many cell functions.

Gap junctions in the rabbit sinoatrial node.

In comparison to the cellular basis of pacemaking, the electrical interactions mediating synchronization and conduction in the sinoatrial node are poorly understood. Therefore, we have taken a combined immunohistochemical and electrophysiological approach to characterize gap junctions in the nodal area. We report that the pacemaker myocytes in the center of the rabbit sinoatrial node express the gap junction proteins connexin (Cx)40 and Cx46. In the periphery of the node, strands of pacemaker myocytes expressing Cx43 intermingle with strands expressing Cx40 and Cx46. Biophysical properties of gap junctions in isolated pairs of pacemaker myocytes were recorded under dual voltage clamp with the use of the perforated-patch method. Macroscopic junctional conductance ranged between 0.6 and 25 nS with a mean value of 7.5 nS. The junctional conductance did not show a pronounced sensitivity to the transjunctional potential difference. Single-channel recordings from pairs of pacemaker myocytes revealed populations of single-channel conductances at 133, 202, and 241 pS. With these single-channel conductances, the observed average macroscopic junctional conductance, 7.5 nS, would require only 30-60 open gap junction channels.

Impaired conduction in the bundle branches of mouse hearts lacking the gap junction protein connexin40.

BACKGROUND: Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with Connexin distribution in normal and Cx40-deficient mice hearts. METHODS AND RESULTS: Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine Connexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals. CONCLUSIONS: Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction.

Atrial fibrillation in the goat induces changes in monophasic action potential and mRNA expression of ion channels involved in repolarization.

INTRODUCTION: Sustained atrial fibrillation (AF) is characterized by a marked shortening of the atrial effective refractory period (AERP) and a decrease or reversal of its physiologic adaptation to heart rate. The aim of the present study was to investigate whether the AF-induced changes in AERP in the goat are associated with changes in the atrial monophasic action potential (MAP) and whether an abnormal expression of specific ion channels underlies such changes. METHODS AND RESULTS: Following thoracotomy, MAPs were recorded from the free wall of the right atrium both before induction of AF (control) and after cardioversion of sustained AF (>2 months) in chronically instrumented goats. In control goats, MAP duration at 80% repolarization (MAPD80) shortened (P < 0.01) from 132+/-4 msec during slow pacing (400-msec interval) to 86+/-10 msec during fast pacing (180 msec). After cardioversion of sustained AF, the MAPD80 during slow pacing was as short as 67+/-5 msec (electrical remodeling). Increasing the pacing rate resulted in prolongation (P = 0.02) of the MAPD80 to 91+/-6 msec. Also, MAPD20 (20% repolarization) shortened (P = 0.05) from 32+/-4 msec (400 msec) to 14+/-7 msec (180 msec) in the control goats, whereas it prolonged (P = 0.03) from 20+/-3 msec (400 msec) to 33+/-5 msec (180 msec) in sustained AF. mRNA expression of the L-type Ca2+ channel alpha1c gene and Kv1.5 potassium channel gene, which underlie ICa and IKur, respectively, was reduced in sustained AF compared with sinus rhythm by 32% (P = 0.01) and 45% (P < 0.01), respectively. No significant changes were found in the mRNA levels of the rapid Na+ channel, the Na+/Ca2+ exchanger, or the Kv4.2/4.3 channels responsible for Ito. CONCLUSION: AF-induced electrical remodeling in the goat comprises shortening of MAPD and reversal of its physiologic rate adaptation. Changes in the time course of repolarization of the action potential are associated with changes in mRNA expression of the alpha subunit genes of the L-type Ca2+ channel and the Kv1.5 potassium channel.

Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat.

OBJECTIVE: It has been postulated that high atrial rate induced changes at the level of the gap junctions ('gap junctional remodeling', i.e. changes in distribution, intercellular orientation and expression of gap junction proteins), could be part of the vicious circle of electrophysiologic and structural changes leading to sustained atrial fibrillation (AF). To obtain experimental evidence in favour of such a postulate the timing of this remodeling process was studied in relation to the development of sustained AF in a goat model. METHODS AND RESULTS: Thin sections from the left (LAA) and right atrial appendage (RAA) from goats in sinus rhythm (SR) or AF, induced through programmed endocardial burst pacing for time periods between 0 and 16 weeks, were immunolabeled with antibodies against connexin(Cx)40 and Cx43 and analysed by immunofluorescence and confocal laser scanning microscopy. During SR the distribution pattern for Cx43 was completely homogeneous (LAA and RAA) and for Cx40 mostly homogeneous (LAA: all five goats, RAA: three out of five goats). The distribution pattern for Cx43 remained stable during AF, while the Cx40 distribution pattern became increasingly heterogeneous, both in the LAA and RAA, with increasing duration of pacing. This increase in heterogeneity in Cx40 distribution correlated (Spearman rank order) with an increase in stability of AF and the occurrence of structural changes (myolysis) in atrial myocytes. The Cx40/Cx43 immunofluorescence signal ratio in both the LAA and RAA appeared to be significantly lower in AF (1-16 weeks) as compared to SR (0 weeks); going from 0 to 16 weeks average ratios decreased 54.5% (n=5; P=0.026) in the LAA and 35.8 (n=5; P=0.034) in the RAA. Western blot analyses revealed similar decreases in the total Cx40/Cx43 protein ratio, on average 50.0% (n=5; P=0.008) and 47.8% (n=5; P=0.02) in the LAA and RAA, respectively. No changes were measured in the levels of Cx40 or Cx43 mRNA, as was assessed through RT-PCR. CONCLUSION: The time course of changes in the distribution and content of Cx40 gap junctions as observed during endocardial burst pacing of the goat atrium suggests that Cx40 gap junctional remodeling might be involved in the pathogenesis of sustained atrial fibrillation.

Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation.

In this study we report about the modulation of connexin45 (Cx45) gap junction channel properties by phosphorylation of the connexin molecules through different protein kinases. Phosphorylation of Cx45 was studied in HeLa cells transfected with mouse Cx45 (mCx45). Using Western blotting (WB) and immunocytochemistry, these cells were found exclusively positive for Cx45 and the protein was separated as a doublet of bands with a calculated mass of 46 and 48 kD. After dephosphorylation using calf intestine phosphatase (CIP), the 48 kD band disappeared almost completely leaving a single band at 46 kD. This effect can be prevented by including phosphatase inhibitors during CIP treatment. These results indicate that the 48 kD signal represents a phosphorylated form of Cx45. To investigate the effects of (de)phosphorylation of Cx45 on the conductive properties of gap junction channels built of this connexin, cell pairs were subjected to dual voltage clamp experiments and coupling was determined before and after addition of PMA, 4alpha-PDD, cAMP, cGMP, and pervanadate to the superfusate. 100 nM of the PKC activating phorbol ester PMA increased normalized junctional conductance by 50.9+/-28%. 100 nM of the inactive phorbol ester 4alpha-PDD had no significant effect. Activation of PKA with 1 mM 8-Br-cAMP decreased coupling by 20.9+/-5.7% while 1 mM 8-Br-cGMP (PKG-activation) was ineffective. 100 microM pervanadate, a tyrosine phosphatase inhibitor, reduced coupling by 43.7+/-11.1%. Single channel measurements, under identical phosphorylating conditions, were not significantly different from each other and all frequency histograms exhibited two conductance peaks at approximately 20 and 40 pS. WB analysis revealed, as compared to control conditions, a relative increase of the 48 kD signal upon stimulation with pervanadate (142+/-42%) and 8-Br-cAMP (50+/-23%) whereas neither stimulation with PMA nor 8-Br-cGMP had a significant effect. These experiments show that electrical intercellular conductance via Cx45 gap junction channels is differentially regulated by phosphorylation. However, regulation does not act by changing single channel conductance, but most likely by modulation of the open probability of Cx45 gap junction channels.

Characterization of the rat connexin40 promoter: two Sp1/Sp3 binding sites contribute to transcriptional activation.

OBJECTIVES: The gap junction protein connexin40 (Cx40) is differentially expressed during embryonic development and in adult tissues, for which the molecular basis is unknown. In order to elucidate the molecular mechanisms controlling Cx40 expression, we set out to map and characterize its promoter. METHODS: The transcriptional activity of individual rat Cx40 (rCx40)-derived promoter fragments fused to the luciferase reporter gene was determined by transfection/reporter assays in Cx40-expressing (A7r5, rat smooth muscle embryonic thoracic aorta cells, and BWEM, v-myc transformed rat fetal cardiomyocytes) and Cx40-nonexpressing cells (N2A, mouse neuroblastoma cells). The nature of DNA-protein interactions was investigated by a combination of standard electrophoretic-mobility-shift assays (EMSA) and EMSA/antibody supershift assays. RESULTS: Quantification of luciferase activity in cell lysates revealed that a 235-base-pair fragment, in between map positions -150 and +85 relative to the transcription initiation site, is able to provide for a significant level of transcription in both Cx40-expressing (A7r5, BWEM) and -nonexpressing (N2A) cells. These results indicate that this region contains the basal promoter but is not sufficient to completely determine the endogenous Cx40-expression pattern within these cell types. In search for the responsible transcriptional regulatory element(s), additional segments of the (-150, +85) region were deleted and the remaining fragments were tested for transcriptional activity. These studies established that the regions in between map positions (-96, -71) and (+58, +85) contribute to promoter activity. EMSA with these regions revealed that predominantly two DNA-protein complexes are formed upon incubation with either A7r5, BWEM or N2A nuclear extracts, which could be both inhibited by including excess oligonucleotide containing the Sp1 consensus binding site in the binding reaction. Purified recombinant human Sp1 provided also for a shift in the EMSA using these promoter regions as target fragments. When the DNA-protein complexes formed with nuclear extract were subsequently incubated with either an anti-Sp1 or an anti-Sp3 antibody clear supershifts in the EMSA were obtained, indicating Sp1 and Sp3 binding to both the (-98, -64) and (+53, +87) regions. The introduction of mutations within the core sequence of the putative Sp1/Sp3 binding sites present in these regulatory elements reduced the level of transcriptional activity and abrogated Sp1/Sp3 binding to these sites. CONCLUSION: The results indicate that at least two Sp1/Sp3 binding sites in the rCx40 promoter contribute to the transcriptional activation of its gene in cultured cells.

Gap junctions in cardiovascular disease.

Connexins, the protein molecules forming gap junction channels, are reduced in number or redistributed from intercalated disks to lateral cell borders in a variety of cardiac diseases. This "gap junction remodeling" is considered to be arrhythmogenic. Using a simple model of human ventricular myocardium, we found that quantitative remodeling data extracted from the literature gave rise to only small to moderate changes in conduction velocity and the anisotropy ratio. Especially for longitudinal conduction, cytoplasmic resistivity (and thus cellular geometry) is much more important than commonly realized. None of the remodeling data gave rise to slow conduction on the order of a few centimeters per second.

The length dependency of calcium activated contractions in the femoral artery smooth muscle studied with different methods of skinning.

The relationship between the calcium concentration and the isometric tension obtained with different techniques of skinning provides information on the biochemical events of contraction in vascular smooth muscle. Muscle preparations of the rabbit femoral artery were skinned with triton X-100, saponin, beta-escin and alpha-toxin and the relationship between the calcium concentration and isometric tension was determined at different preparation lengths. We determined the calcium sensitivity as a function of muscle length with different techniques of skinning. At a pCa of 6.0, triton X-100 skinned smooth muscle of the femoral artery generated 50% of the maximal tension. In alpha-toxin skinned preparations, this calcium sensitivity was shifted to a pCa of 5.6. The sensitivity of the saponin and 3-escin skinned preparations were in between those of the triton X-100 and the alpha-toxin skinned preparations. The cooperativity of the regulation of contraction varied among the differently skinned preparations between 3 (alpha-toxin) and 6 (triton X-100). The relationships between the calcium concentration and the isometric tension of the differently skinned preparations up to the optimal length for tension generation did not exhibit any length dependency. The length tension relationship, obtained from the maximal response at the highest calcium concentration is in line with that from other studies. The presence of intracellular proteins and membranes affects the regulation of contraction in smooth muscle of the femoral artery.

Human connexin40 gap junction channels are modulated by cAMP.

OBJECTIVE: Gap junction channels provide for direct electrical coupling between cells, and play an important role in homeostasis and electrical coupling. One of the proteins that form gap junctions, Connexin40 (Cx40), shows restricted expression in the body, and is found in blood vessels and in the atrium and conduction system of the heart. We have investigated whether gap junction channels formed of Cx40 are modulated by protein-kinase-A-mediated phosphorylation. METHODS: A communication-deficient human hepatoma cell line (SKHep1) was stably transfected with human Cx40 cDNA and the properties of Cx40 gap junctions channels and their modulation by cAMP were analyzed using immunocytochemistry, Western blotting, dual patch clamp, and dye coupling. RESULTS: Administration of 1 mM 8-Br-cAMP resulted in a mobility shift of Cx40 protein on western blot and increased macroscopic gap junctional conductance between cell pairs by 46.2 +/- 12.0% (mean +/- S.E.M., n = 8). Under control conditions, single channel experiments revealed three single channel conductances around 30, 80 and 120 pS. When cAMP was added, channel conductances of 46 and 120 pS were observed. In monolayers, cAMP also increased the permeability of Cx40 gap junction channels for Lucifer Yellow by 58%. CONCLUSIONS: Macroscopic conductance and permeability of Cx40 gap junctions is strongly increased by cAMP and may play a role in the regulation of intercellular communication in the heart and vasculature.

Effects of anisotropy on the development of cardiac arrhythmias associated with focal activity.

The anisotropy that normally exists in the myocardium may be either enhanced in peri-infarction zones by loss of lateral cell connections or reduced by redistribution of gap junctions. To test how the degree of anisotropy affects the development of ectopic focal activity, we carried out computer simulations in which a model of an ectopic focus is incorporated as the central element of a two-dimensional sheet of ventricular cells. At low values of intercellular coupling conductance (Gc), the focus region is spontaneously active, but the limited intercellular current flow inhibits propagation. At high Gc, automaticity is suppressed by the loading effects of the surrounding cells. At intermediate Gc, the ectopic activity may propagate into the sheet. In the case of isotropic coupling, the minimum size of the focus region for propagation to occur (in terms of number of collaborating cells within the focus) is as small as approximately ten cells, and this number decreases with increasing anisotropy. Thus, the presence of anisotropy facilitates the development of ectopic focal activity. We conclude that the remodeling that occurs in peri-infarction zones may create a substrate that either facilitates (enhanced anisotropy) or inhibits (reduced anisotropy) the development of cardiac arrhythmias associated with ectopic focal activity.

Cardiac conduction abnormalities in mice lacking the gap junction protein connexin40.

INTRODUCTION: The gap junction protein connexin40 (Cx40) normally is expressed in the murine atrial myocardium and ventricular conduction system. In mice lacking Cx40, several changes in the surface ECG have been described. In this study, we analyzed cardiac conduction in more detail. METHODS AND RESULTS: In open chest mice under urethane anesthesia, epicardial electrodes were used to determine a number of atrial and ventricular pacing parameters. The corrected sinus node recovery time was significantly longer in Cx40-/- mice than in Cx40+/+ mice (44.4 +/- 7.2 msec vs 35.5 +/- 8.0 msec). In addition, the Wenckebach period was longer in Cx40-/- mice compared with the wild type (84.6 +/- 5.4 msec vs 78.8 +/- 3.6 msec), with the AV node probably limiting AV conduction in both cases. Whereas arrhythmias could not be induced by ventricular burst pacing in any of the mice, atrial burst pacing induced atrial tachyarrhythmias in 5 of 10 Cx40-/- mice, but not in any of 9 Cx40+/+ mice. Conduction velocities were measured in vivo using an array of unipolar recording electrodes. Ventricular conduction velocity did not differ between the groups, but atrial conduction velocity was reduced by 30% in Cx40-/- mice compared with the wild type. Heterozygous Cx40+/- mice did not differ significantly from the wild type in any respect. CONCLUSION: These findings indicate that in the atria and the AV conduction system, Cx40 is an important determinant of conduction.

The effect of length on the sensitivity to phenylephrine and calcium in intact and skinned vascular smooth muscle.

The length dependency of the sensitivity to activators of the smooth muscle of different blood vessels is not yet fully understood. Muscle preparations of the aorta, the femoral artery and the portal vein of the rabbit were investigated for the length dependency of the sensitivity to phenylephrine and calcium in both intact and triton X-100 skinned preparations. For intact smooth muscles we found that at increased preparation length, the sensitivity of contraction was increased. The femoral artery showed the largest effect and the portal vein the smallest. In the skinned preparations of the three preparations the calcium sensitivity was not dependent on the preparation length. We conclude that the changes of the sensitivity in intact preparations are not caused by changes of the calcium sensitivity of the contractile proteins.

Selective inhibition of gap junction channel activity by synthetic peptides.

1. The aim of this study was to inhibit specifically one type of gap junction channel in cells expressing multiple connexins (Cx) using synthetic oligopeptides. 2. A7r5 cells (an aortic smooth muscle cell line expressing Cx40 and Cx43) were incubated overnight with synthetic oligopeptides (P180-195) corresponding to a segment of the second extracellular loop of Cx43. This segment is different in sequence from the corresponding location in Cx40. 3. P180-195 (500 microM) decreased cell-to-cell coupling as assessed by dye coupling and dual whole-cell voltage clamp. The decrease in permeability and junctional conductance was caused by selective inhibition of Cx43 gap junction channels. In contrast, overnight incubation of A7r5 cells with oligopeptides corresponding to a segment of the intracellular cytoplasmic tail of Cx43 was without effect. 4. These results indicate that oligopeptides P180-195 may interact with the extracellular domain of the Cx43 protein, thereby possibly mimicking connexin-connexin binding. This apparently inhibits Cx43 channel activity without disturbing the activity of Cx40 channels. 5. Experiments with oligopeptides corresponding to the equivalent part of the second extracellular loop of Cx40 (P177-192) pointed towards a selective inhibition of Cx40 channel activity. 6. Competition assays using synthetic oligopeptides may help to resolve the regulatory properties of gap junction channels in primary cells expressing multiple Cx.