Reduced Cx43 Expression Research Articles

Targeting Connexin 43 Prevents Platelet-Derived Growth Factor-BB–Induced Phenotypic Change in Porcine Coronary Artery Smooth Muscle Cells

We previously reported that reducing the expression of the gap junction protein connexin (Cx)43 in mice restricts intimal thickening formation after acute vascular injury by limiting the inflammatory response and the proliferation and migration of smooth muscle cells (SMCs) toward the damaged site. SMC populations isolated from porcine coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening, suggesting that they participate in the restenotic process. Here, we further investigate the relationship between connexin expression and SMC phenotypes using porcine coronary artery SMCs. Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was downregulated and Cx43 was markedly upregulated in stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. In R-SMCs, Cx43 expression was increased and Cx40 was absent. We confirmed that S-SMCs treated with platelet-derived growth factor-BB acquire an R phenotype. This was accompanied by an upregulation of Cx43 and a loss of Cx40. Importantly, platelet-derived growth factor-BB-induced S-to-R phenotypic change was prevented by a reduction of Cx43 expression with antisense, ie, S-SMCs retained their typical elongated appearance and the expression of alpha-smooth muscle actin, a well-known SMC differentiation marker, whereas the expression of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in S-SMCs prevents platelet-derived growth factor-BB-induced S-to-R modulation. This suggests that Cx43 may be an additional target for local delivery strategies aimed at reducing restenosis.

Dose‐dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.

Abstract 1400: Reduced Cx43 Expression Reduces Scar Formation After Coronary Ligation

Gap junctions mediate cell-to-cell electrical coupling required for synchronized cardiac contraction. They also serve as conduits for exchange of small signaling molecules. Reduction in connexin43 (Cx43), the major gap junction protein in ventricular tissue, likely underlies malignant arrhythmias in infarcted hearts. However, little is known about the role of reduced Cx43 expression in infarct healing. This study was performed to test the hypothesis that reduced expression of Cx43 influences cardiac fibroblast function and wound healing after myocardial infarction (MI). Methods: The left anterior coronary artery was ligated in wild-type (WTs) and Cx43-deficient (Cx43 +/− ) mice studied 6 d later. We quantified infarct size by Masson’s trichrome staining; cell proliferation by bromodeoxyuridine staining; matrix metallo-proteinase (MMP) activity in the noninfarcted, border and infarct regions (IZ) by zymography; myofibroblasts by α-smooth muscle actin (αSMA) immunostaining; and collagen deposition by Picrosirius red staining. Results: 6 d post-MI mean infarct size by trichrome staining was not different in Cx43 +/− (46±4%) vs WTs (46±5%). However, significant differences were observed in the Cx43 +/− infarcts. The area of unresorbed necrotic myocardium in the IZ was larger (p<0.01) in Cx43 +/− (40±11%, n=12) vs WTs (17±8%, n=6). Fibroblast proliferation in the IZ was increased (p<0.05) in Cx43 +/− (6.1±0.5%, n=4) vs WTs (4.1±0.7%, n=5), and activated MMP-2 was increased even more (p< 0.05) in the IZ of Cx43 +/− (40±6 relative density units, n=5) vs WTs (29±4 units, n=3) consistent with enhanced fibroblast proliferation. On the other hand, transformation to myofibroblasts in the IZ was diminished as reflected by reduced (p<0.01) αSMA-positive immunostaining in Cx43 +/− (32±16%, n=6) vs WTs (57±5%, n=12), and collagen deposition in the IZ was less (p<0.01) in Cx43 +/− (36±6%, n=6) vs WTs (50±13%, n=8) consistent with fewer myofibroblasts. Conclusion: Reduced Cx43 expression increases fibroblast proliferation and MMP activity, and reduces myofibro-blasts and collagen deposition indicating that remodeling of Cx43 can influence fibroblast function and delay scar formation and cardiac wound healing after MI.

Abstract 604: Targeting Cx43 Prevents Growth Factor-Induced Phenotypic Change in Pig Coronary Artery Smooth Muscle Cells

Percutaneous coronary intervention (PCI) is commonly used to treat atherosclerotic coronary arteries, but its efficacy is limited by restenosis at the site of the intervention. We reported previously that reducing the expression of the gap junction protein connexin43 (Cx43) in mice restricted neointima formation after acute vascular injury by limiting the inflammatory response as well as the proliferation and migration of smooth muscle cells (SMCs) towards the damaged site. SMC populations isolated from the pig coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening suggesting that they participate in the intimal thickening. Here, we further investigate the relationship between connexin expression and SMC phenotype using the distinct types of pig coronary artery SMCs. We show that Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was down-regulated and Cx43 was markedly up-regulated in SMCs of stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. Cx43 expression was increased in R-SMCs and these cells no longer expressed Cx40. When S-SMCs were treated with 10 ng/ml platelet-derived growth factor (PDGF-BB) they acquired a rhomboid phenotype and their migratory activity increased (from 40.3±5.7 to 185.9±27.3 migrating cells; mean±SEM, N=4, P<0.01). These changes were accompanied by an increase in Cx43 and loss of Cx40 expression. Importantly, PDGF-BB-induced phenotypic change of S-SMCs was prevented by reducing Cx43 expression with 100 uM antisense for Cx43. Thus, Cx43 antisense-treated SMCs retained their typical elongated appearance and the expression of some SMC differentiation markers, such as alpha-SM actin, whereas the appearance of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in SMCs prevents growth factor-induced changes towards a deleterious phenotype. Our findings suggest that Cx43 might be an additional target for local delivery strategies aimed at reducing restenosis after PCI.

Relationship of connexin43 expression to phenotypic modulation in cultured human aortic smooth muscle cells

Transition of arterial smooth muscle cells from the contractile to the synthetic phenotype in vivo is associated with up-regulation of the gap-junctional protein, connexin43 (Cx43). However, the role of increased Cx43 expression in relation to the characteristic features of the synthetic phenotype – altered growth, differentiation or synthetic activity – has not previously been defined. In the present study, growth was induced in cultured human aortic smooth muscle cells by treatment with thrombin and with PDGF-bb; growth arrest was induced by serum deprivation and contact inhibition. Alterations in Cx43 expression and gap-junctional communication were analyzed in relation to expression of markers for contractile differentiation and extracellular matrix synthesis. Treatment with thrombin, but not PDGF-bb, led to up-regulation of Cx43 gap junctions, increased synthetic activity yet also enhanced contractile differentiation. Inhibition of growth by deprivation of serum growth factors in sub-confluent cultures had no effect on Cx43 expression or contractile differentiation. Growth arrest by contact inhibition led to progressive reduction in Cx43 expression, in parallel with progressive increase in expression of differentiation markers but no alteration in synthetic activity. Of a range of stimuli examined, only thrombin had the combined effect of increasing Cx43 gap-junction communication, growth and synthesis, yet it also enhanced contractile differentiation. Down-regulation of Cx43 and improved contractile differentiation occurred only when growth arrest was induced through the contact–inhibition pathway, though, in this instance, synthesis remained undiminished. We conclude that Cx43 levels, though having common correlates, are not exclusively linked to the cell phenotype or the state of growth.

Relative Contributions of Connexins 40 and 43 to Atrial Impulse Propagation in Synthetic Strands of Neonatal and Fetal Murine Cardiomyocytes

Atrial tissue expresses both connexin 40 (Cx40) and 43 (Cx43) proteins. To assess the relative roles of Cx40 and Cx43 in atrial electrical propagation, we synthesized cultured strands of atrial myocytes derived from mice with genetic deficiency in Cx40 or Cx43 expression and measured propagation velocity (PV) by high-resolution optical mapping of voltage-sensitive dye fluorescence. The amount of Cx40 and/or Cx43 in gap junctions was measured by immunohistochemistry and total or sarcolemmal Cx43 or Cx40 protein by immunoblotting. Progressive genetic reduction in Cx43 expression decreased PV from 34+/-6 cm/sec in Cx43(+/+) to 30+/-8 cm/sec in Cx43(+/-) and 19+/-11 cm/sec in Cx43(-/-) cultures. Concomitantly, the cell area occupied by Cx40 immunosignal in gap junctions decreased from 2.0+/-1.6% in Cx43(+/+) to 1.7+/-0.5% in Cx43(+/-) and 1.0+/-0.2% in Cx43(-/-) strands. In contrast, progressive genetic reduction in Cx40 expression increased PV from 30+/-2 cm/sec in Cx40(+/+) to 40+/-7 cm/sec in Cx40(+/-) and 45+/-10 cm/sec in Cx40(-/-) cultures. Concomitantly, the cell area occupied by Cx43 immunosignal in gap junctions increased from 1.2+/-0.9% in Cx40(+/+) to 2.8+/-1.4% in Cx40(+/-) and 3.1+/-0.6% in Cx40(-/-) cultures. In accordance with the immunostaining results, immunoblots of the Triton X-100-insoluble fraction revealed an increase of Cx43 in gap junctions in extracts from Cx40-ablated atria, whereas total cellular Cx43 remained unchanged. Our results suggest that the relative abundance of Cx43 and Cx40 is an important determinant of atrial impulse propagation in neonatal hearts, whereby dominance of Cx40 decreases and dominance of Cx43 increases local propagation velocity.

Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis

RationaleGap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition.MethodsWe investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF).ResultsHere we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF.ConclusionThese results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

Reduced Connexin43 Expression Limits Neointima Formation After Balloon Distension Injury in Hypercholesterolemic Mice

Reducing the expression of the gap junction protein connexin43 (Cx43) inhibits the progression of atherosclerosis, a chronic inflammatory disease. Furthermore, acute vascular injury induced by percutaneous coronary interventions is associated with increased Cx43 expression in neointimal smooth muscle cells (SMCs). However, the relevance of Cx43 after acute vascular injury remains unclear. To investigate whether reducing Cx43 expression would affect neointima formation in vivo, we subjected hypercholesterolemic Cx43+/- LDL receptor-deficient (LDLR-/-) mice and Cx43+/+LDLR-/- control littermates to carotid balloon distension injury, which induced marked endothelial denudation and activation of medial SMCs. We observed decreased macrophage infiltration in Cx43+/-LDLR-/- mice 7 days after injury. Similarly, peritoneal macrophages isolated from Cx43+/-LDLR-/- mice showed reduced migration in vitro compared with Cx43+/+LDLR-/- macrophages. Interestingly, Cx43+/-LDLR-/- macrophages also displayed decreased chemotactic activity for SMCs. In addition, we observed less SMC infiltration and proliferation in Cx43+/-LDLR-/- mice 7 and 14 days after balloon angioplasty. Likewise, Cx43+/-LDLR-/- SMCs showed decreased proliferation and migration in vitro compared with Cx43+/+LDLR-/- cells. All these events resulted in a reduction of neointimal thickening after vascular injury in Cx43+/-LDLR-/- mice. The present study shows for the first time that reducing Cx43 limits neointima formation after acute vascular injury by decreasing the inflammatory response and reducing SMC migration and proliferation. Thus, decreasing Cx43 expression may offer a novel therapeutic strategy for reducing restenosis after percutaneous coronary intervention.

Transition from Preinvasive Carcinoma In Situ to Seminoma Is Accompanied by a Reduction of Connexin 43 Expression in Sertoli Cells and Germ Cells

Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap functional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semi quantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

Regional reduction in excitability and connexin 43 expression in areas of conduction disturbance in canine ventricles with chronic rapid pacing and ameroid constrictor

It has been suggested that altered connexin43 (Cx43) expression may be associated with reentrant ventricular arrhythmias in chronic myocardial infarction. A similar possibility was investigated in canine hearts without necrotic scar, which were subjected to chronic rapid pacing (CRP) at 240 beats/min for 4 weeks and progressive obliteration of the left coronary circumflex artery (LCx) by an ameroid constrictor. Electrophysiologic alterations identified by mapping the LCx territory (191 unipolar recordings) included activation delay, ST segment potential elevation and reduced maximum negative slope (−dV/dtmax) in the activation complex of unipolar electrograms. The LCx was divided into areas displaying depressed conduction (LCx1: 0.07±0.04 m/s,mean±SD) and milder depression (LCx2: 0.23 ±0.01 m/s). Cx43 measured by Western blot in tissue sampled from LCx1 was significantly reduced (62±63%) but not in LCx2 (39±87%) with reference to the left anterior descending artery territory (100%). Moreover, -dV/dtmax was significantly depressed (5.3±3.6 mV/ms) and ST segment potential was significantly elevated (17.7±17 mV) in LCx1 in comparison with LCx2 (10±3.9 mV/ms and −1.1±1.9 mV, respectively). The data indicate that conduction disturbances are induced by reduced Cx43 expression in conjunction with depressed excitability in viable energy-stressed myocardium. Supported by the Canadian Institutes of Health Research and Quebec Heart Foundation.

Mechanisms underlying conduction slowing and arrhythmogenesis in nonischemic dilated cardiomyopathy.

Heart Failure (HF) is associated with an increased risk of sudden death caused by ventricular tachyarrhythmias. Recent studies have implicated repolarization abnormalities and, in particular, exaggerated heterogeneity of transmural repolarization in the genesis of polymorphic ventricular tachycardia in a canine model of nonischemic dilated cardiomyopathy. The presence and degree to which conduction abnormalities play a role in arrhythmogenesis in this model are uncertain. HF was produced in dogs by rapid RV-pacing for 3 to 4 weeks. High-resolution optical action potentials were recorded from epicardial and endocardial surfaces of arterially perfused canine wedge preparations isolated from LV and RV of normal and failing dogs. Cellular and molecular determinants of conduction were investigated using patch-clamp recordings, Western blot analysis, and immunocytochemistry. HF was associated with marked prolongation (by 33%) of the QRS duration of the volume conducted electrocardiogram and significant (>20%) slowing of epicardial and endocardial conduction velocities (CV) in both LV and RV. Cx43 expression was reduced by >40% in epicardial and endocardial layers of the LV, but was unchanged in the RV of failing hearts. Despite greater epicardial than endocardial Cx43 expression, epicardial CV was consistently slower (P<0.01). Immunocytochemical analysis revealed predominant colocalization of Cx43 with N-cadherin in normal versus failing samples, because Cx43 was redistributed from the intercalated disk to lateral cell borders in failing tissue. Moreover, a significant (P<0.05) increase in hypophosphorylated Cx43 was detected in the LV and RV of failing hearts. Action potential upstroke velocities in isolated ventricular myocytes from normal and failing hearts were not different (P=0.8, not significant), and Masson trichrome staining revealed no significant change in fibrosis content in HF. Nonischemic dilated cardiomyopathy is associated with significant slowing of CV that was not directly related to reduced Cx43 expression. Changes in phosphorylation and localization of Cx43 may contribute to gap-junction dysfunction, CV slowing, and arrhythmias in HF.

Altered connexin43 expression produces arrhythmia substrate in heart failure.

Recently, we found that repolarization heterogeneities between subepicardial and midmyocardial cells can form a substrate for reentrant ventricular arrhythmias in failing myocardium. We hypothesized that the mechanism responsible for maintaining transmural action potential duration heterogeneities in heart failure is related to intercellular uncoupling from downregulation of cardiac gap junction protein connexin43 (Cx43). With the use of the canine model of pacing-induced heart failure, left ventricles were sectioned to expose the transmural surface (n = 5). To determine whether heterogeneous Cx43 expression influenced electrophysiological function, high-resolution transmural optical mapping of the arterially perfused canine wedge preparation was used to measure conduction velocity (theta(TM)), effective transmural space constant (lambda(TM)), and transmural gradients of action potential duration (APD). Absolute Cx43 expression in failing myocardium, quantified by confocal immunofluorescence, was uniformly reduced (by 40 +/- 3%, P < 0.01) compared with control. Relative Cx43 expression was heterogeneously distributed and lower (by 32 +/- 18%, P < 0.05) in the subepicardium compared with deeper layers. Reduced Cx43 expression in heart failure was associated with significant reductions in intercellular coupling between transmural muscle layers, as evidenced by reduced theta(TM) (by 18.9 +/- 4.9%) and lambda(TM) (by 17.2 +/- 1.4%; P < 0.01) compared with control. Heterogeneous transmural distribution of Cx43 in failing myocardium was associated with lower subepicardial theta(TM) (by 12 +/- 10%) and lambda(TM) (by 13 +/- 7%), compared with deeper transmural layers (P < 0.05). APD dispersion was greatest in failing myocardium, and the largest transmural APD gradients were consistently found in regions exhibiting lowest relative Cx43 expression. These data demonstrate that reduced Cx43 expression produces uncoupling between transmural muscle layers leading to slowed conduction and marked dispersion of repolarization between epicardial and deeper myocardial layers. Therefore, Cx43 expression patterns can potentially contribute to an arrhythmic substrate in failing myocardium.

Increased Apoptosis and Inflammation after Focal Brain Ischemia in Mice Lacking Connexin43 in Astrocytes

Astrocytes secrete cytokines and neurotrophic factors to neurons, consistent with a neurosupportive role for astrocytes. However, in ischemic or metabolic insults, the function of astrocytic gap junctions composed mainly from connexin43 (Cx43) remains controversial. We have previously shown that heterozygous Cx43 null mice subjected to middle cerebral artery occlusion exhibited significantly enhanced stroke volume and apoptosis compared to wild-type mice. In this study, we used mice in which the human GFAP promoter-driven cre transgene deletes the floxed Cx43 gene in astrocytes, excluding the effects from reduced Cx43 expression in many other cell types as well as astrocytes. We induced focal brain ischemia in mice lacking Cx43 in astrocytes [Cre(+)] and control littermates [Cre(-)]. Cre(+) mice showed a significantly increased stroke volume and enhanced apoptosis, detected by terminal dUTP nick-end labeling and caspase-3 immunostaining, compared to Cre(-) mice. Inflammatory response assessed by the microglial marker CD11b was amplified in the penumbra of Cre(+) mice compared to that of Cre(-) mice. Our results suggest that astrocytic gap junctions could be important for the regulation of neuronal apoptosis and the inflammatory response after stroke. These findings support the view that astrocytes play a critical role in neuroprotection during ischemic insults.

High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes.

To investigate the role of the gap junction protein, connexin-43 (Cx43) in the maintenance of retinal vascular homeostasis in diabetic retinopathy. In human retinal pericytes (HRPs) and bovine retinal pericytes (BRPs) grown for 7 days in normal (5 mM) or high (30 mM)-glucose medium, the Cx43 protein level was determined by Western blot analysis. Parallel experiments were performed in HRPs to determine the Cx43 mRNA level by RT-PCR, the distribution and localization of Cx43 protein by immunostaining, and gap junction intercellular communication (GJIC) activity by a scrape-loading dye transfer technique. Distribution and localization of Cx43 protein was also determined in pericyte-endothelial cell cocultures. Western blot analysis of the Cx43 protein level in HRPs and BRPs indicated reduced Cx43 expression in the high-glucose condition (69.1% +/- 17% of control, P = 0.004; 62.3% +/- 19% of control, P = 0.001, respectively). The Cx43 mRNA level in HRPs grown in high-glucose medium also showed significant reduction (71.4% +/- 16.8% of control, P = 0.02). The relative number of Cx43 plaques indicative of Cx43 localization at specific sites of contact between adjacent cells showed significant reduction in the high-glucose condition (61% +/- 10% of control, P = 0.002); similarly, a significant reduction in the number of plaques was observed in cocultures grown in high-glucose medium compared with those in normal medium (59.4% +/- 29% of control, P = 0.001). Cells with reduced Cx43 expression showed significantly reduced transfer of lucifer yellow (61% +/- 13% of control, P = 0.001; r = 0.9). High-glucose-induced downregulation of Cx43 expression and inhibition of GJIC in retinal pericytes may play a role in the disruption of vascular homeostasis in diabetic retinopathy.

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice.

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.

Downregulation of connexin 43 expression by high glucose reduces gap junction activity in microvascular endothelial cells.

Impairment of retinal vascular homeostasis is associated with the development and progression of diabetic retinopathy involving gap junction intercellular communication (GJIC) activity. The principal gap junction protein of intercellular communication, connexin, was investigated to determine the effects of high glucose concentrations on the expression of endothelial-specific connexins (Cx37, Cx40, and Cx43), connexin phosphorylation pattern, and GJIC activity. Rat microvascular endothelial (RME) cells grown in high (30 mmol/l)-glucose medium for 9 days had reduced Cx43 expression: Cx43 mRNA (68 +/- 13% of control; P = 0.019, n = 5) and protein (55.6 +/- 16% of control; P = 0.003, n = 5) levels were reduced; however, Cx37 and Cx40 expression was not affected. Using alkaline phosphatase and Western blot analyses, we identified three forms of Cx43: a nonphosphorylated form (P0) and two phosphorylated forms (P1 and P2). Expression of all three forms was decreased in cells grown in high-glucose medium: PO, 73 +/- 15% of control (P = 0.04); P1, 57 +/- 16% of control (P = 0.01); and P2, 42 +/- 22% of control (P = 0.006). Using immunofluorescence microscopy, we observed Cx43 localization at specific sites of contact (plaques) between adjacent cells. In cells grown in high-glucose medium, we observed reduced plaque counts (63 +/- 6% of control; P = 0.009) and decreased intensity of Cx43 immunofluorescence compared with cells grown in normal medium. Furthermore, using scrape load dye transfer (SLDT) technique, we found that these cells exhibited reduced GJIC activity (60% of control; P = 0.01, n = 5). The reduction in GJIC activity correlated with the decreased Cx43 protein levels (r = 0.9). These results indicate that high glucose concentrations inhibited GJIC activity by reducing Cx43 synthesis in RME cells. Impaired intercellular communication may contribute to breakdown of homeostatic balance in diabetic microangiopathy.

Connexin43 null mutation increases infarct size after stroke

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein connexin43 (Cx43) and provide a substrate for formation of a functional syncytium implicated in the spatial buffering capacity of astrocytes. To study the function of gap junctions in the brain, we used heterozygous Cx43 null mice, which exhibit reduced Cx43 expression. Western blot analysis showed a reduction in the level of Cx43 protein and GJIC in astrocytes cultured from heterozygote mice. The level of Cx43 is reduced in the adult heterozygote cerebrum to 40% of that present in the wild-type. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild-type and heterozygote mice after focal ischemia. In our model of focal stroke, the middle cerebral artery was occluded at two points, above and below the rhinal fissure. Four days after surgery, mice were killed, the brains were sectioned and analyzed. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared with wild-type (14.4 +/- 1.4 mm(3) vs. 7.7 +/- 0.82 mm(3), P < 0.002). These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection after ischemic injury.

High resolution optical mapping reveals conduction slowing in connexin43 deficient mice.

Analysis of mice with genetically altered expression of cardiac connexins can provide insights into the role of individual gap junction channel proteins in cell-to-cell communication, impulse propagation, and arrhythmias. However, conflicting results have been reported regarding conduction velocity slowing in mice heterozygous for a null mutation in the gene encoding connexin43 (Cx43). High-resolution optical mapping was used to record action potentials from 256 sites, simultaneously, on the ventricular surface of Langendorff perfused hearts from 15 heterozygous (Cx43+/-) and 8 wildtype (Cx43+/+) mice (controls). A sensitive method for measuring epicardial conduction velocity was developed to minimize confounding influences of subepicardial breakthrough and virtual electrode effects. Epicardial conduction velocity was significantly slower (23 to 35%, P<0.01) in Cx43+/- mice compared to wildtype. There was no change in conduction patterns or anisotropic ratio (Cx43+/- 1.54+/-0.33; Cx43+/+ 1.57+/-0.17) suggesting that Cx43 expression was reduced uniformly throughout myocardium. The magnitude of reductions in conduction velocity and Cx43 protein expression (45%) were similar in mice in which the null allele occurred in a pure C57BL/6J genetic background versus a mixed (C57BL/6J X 129) background. Action potential duration did not differ between mice of different genotypes. A approximately 50% reduction of Cx43 expression causes significant conduction velocity slowing in the Cx43+/- mouse heart. The apparent lack of conduction velocity changes reported in previous studies may be related to technical factors rather than variations in genetic background. High-resolution optical mapping is a powerful tool for investigating molecular determinants of propagation and arrhythmias in genetically engineered mice.

Reduced levels of connexin43 in cervical dysplasia: inducible expression in a cervical carcinoma cell line decreases neoplastic potential with implications for tumor progression

Loss of gap junctional intercellular communication (GJIC) has been linked to aberrant proliferation and an enhanced neoplastic phenotype. Many human tumors, including the cervical carcinoma line HeLa, have been reported to be deficient in expression of the gap junction protein connexin43 (Cx43) and GJIC. To determine if this is an early event in carcinogenesis, we utilized immunohistochemistry to screen a series of cervical biopsy samples and demonstrated a major reduction in Cx43 expression in dysplastic regions compared to normal epithelia. To determine whether this loss influences the neoplastic behavior of cervical carcinoma cells, we have constructed HeLa cell lines in which Cx43 expression can be induced in response to doxycycline. This approach allows for the discrimination of Cx43-mediated effects from those due to pre-existing clonal heterogeneity. Cx43 induction in these cells led to assembly of functional junctions but did not alter growth control in vitro as measured by logarithmic growth, saturation density or focus formation when in co-culture with growth-controlled fibroblasts. However, Cx43 induction decreased two indices of neoplasia: it reduced anchorage-independent growth and attenuated the growth rate of tumor xenografts. These results indicate that established HeLa cell lines are unresponsive to Cx43-mediated signals which are thought to mediate growth control of non-transformed cells, however, Cx43 expression can still reduce aspects of the neoplastic phenotype of these cells, indicating that loss of connexin signaling in dysplastic cells may contribute to their neoplastic progression.

Voltage-gated Na+ channel activity and connexin expression in Cx43-deficient cardiac myocytes.

Dynamic interplay between active and passive electrical properties of cardiac myocytes is based on interrelationships between various channels responsible for depolarizing and repolarizing ionic currents and intercellular conductances. Mice with targeted disruption of the connexin43 (Cx43) gene have hearts completely devoid of Cx43, the principal gap junctional protein expressed in mammalian hearts. To determine whether cardiac myocytes that develop in an abnormal environment of reduced intercellular coupling have altered active membrane properties, we studied whole cell action potentials, Na+ channel currents, and Na+ channel expression and distribution via immunoblotting and confocal immunofluorescence in neonatal ventricular myocytes isolated from Cx43 wild-type, heterozygous, and homozygous null hearts. Action potential morphology, peak Na+ current, activation and inactivation kinetics, and Na+ channel protein expression and distribution were not different among myocytes isolated from wild-type, heterozygous, or null hearts. Active membrane properties and Na+ channel activity were completely normal in Cx43-deficient myocytes isolated from hearts that have been shown to exhibit markedly reduced Cx43 expression, gap junction number, and epicardial conduction delay. Despite a genetic inability to produce Cx43 and a developmental history that culminates in marked gross cardiac morphologic abnormalities, premature death, and myocardial inexcitability ex vivo, cardiac Na+ channel distribution and function appear to be normal in Cx43 null hearts. Although intimate structural and functional interrelationships have been described between ion channels and gap junction channels, expression and function of Na+ channels is not affected by the absence of Cx43.