Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.

Connexins 43 and 26 are differentially increased after rat bladder outlet obstruction.

To evaluate the regulation of connexin expression by fluid pressure, we have studied the effects of elevated transmural urine pressure on Connexin43 (Cx43) and Cx26. We chose to focus on these two proteins out of the five connexins (Cx26, 43, 40, 37, and 45) which we found by RT-PCR to be expressed in the rat bladder, since in situ hybridization and immunofluorescence showed that Cx43 is the predominant connexin expressed by smooth muscle cells (SMC), whereas Cx26 is abundantly expressed only in the latter cell type. To evaluate whether these connexins are affected by changes in transmural urine pressure, we used a rat model of bladder outlet obstruction, in which a ligature is placed around the urethra. Under conditions of increased fluid pressure due to urine retention, we observed that the expression of both Cx43 and Cx26 increased at both transcript and protein levels, reaching a maximum 7-9 h after the ligature. Further analysis revealed that these changes were accounted for by a fourfold increase in Cx43 mRNA of SMC but not urothelial cell and by a fivefold increase in Cx26 mRNA of urothelium. Scrape-loading of propidium iodide showed that the latter change was paralleled by a twofold increase in coupling between urothelial cells. The data show that Cx43 and Cx26 are differentially regulated during bladder outlet obstruction and contribute to the response of the bladder wall to increased voiding pressure, possibly to control its elasticity.

Régulation des hémicanaux de connexine 43 : implication dans la cardioprotection contre les lésions ischémiques

La connexine 43 (Cx43) est l’unité protéique de base dans la formation des canaux des jonctions gap (JG) responsables des échanges intercellulaires. Toutefois, elle forme aussi des canaux non-jonctionnels à large conductance, nommés hémicanaux (Hc), qui fournissent un accès entre l’intérieure des cellules et le milieu extracellulaire. Bien qu’ils soient beaucoup moins étudiés que les JG, on estime que les Hc restent normalement à l’état fermé, et ce, grâce à la phosphorylation des connexines qui les forment. Suite à un stress ischémique, les Cx43 se déphosphorylent et entraînent ainsi l’ouverture des Hc de Cx43 (HcCx43), un effet qui compromet la survie des cellules. La protéine kinase C (PKC) est l’enzyme de phosphorylation qui possède le plus grand nombre de sites de phosphorylation sur la Cx43 en comparaison avec les autres kinases. Ses fonctions dépendent de la mise en jeu d’un répertoire d’au moins 12 isoformes distinctes. Dans les cardiomyocytes, les isoformes de PKC participent au développement des réponses adaptées ou mésadaptées au stress ischémique. Malgré que la régulation des canaux de Cx43 par la PKC lors d’une ischémie soit bien documentée, il n’existe pas à l’heure actuelle de connaissances sur les effets fonctionnels spécifiques qu’exercent des différentes isoformes de PKC sur les HcCx43, ni sur la valeur thérapeutique de la modulation de ses derniers. Dans ce contexte, nous avons proposé que les HcCx43 sont régulés sélectivement et différentiellement par les différentes isoformes de PKC et que l’inhibition spécifique de ces hémicanaux peut protéger le coeur lors d’un événement ischémique. Le présent travail comporte trois études qui ont été entreprises spécialement dans le but de valider ces hypothèses. Dans la première étude, nous avons profité de l’expertise du laboratoire du Dr Baroudi dans la dissection des isoformes de PKC pour étudier le rôle fonctionnel de chacune d’elles dans la régulation des HcCx43 en utilisant une gamme unique de peptides synthétiques inhibiteurs et activateurs spécifiques des isoformes de PKC, en combinaison avec la technique du patch-clamp. Nous avons démontré, entre autre, que les HcCx43 sont particulièrement inhibés par l’isoforme PKC epsilon, connue pour son effet cardioprotecteur contre les dommages ischémiques lors d’un préconditionnement ischémique. Dans la deuxième étude, nous avons caractérisé l’effet d’un peptide synthétique mimétique structural de la Cx43 sur la fonction des HcCx43. En plus d’avoir élucidé ces effets sur les propriétés fonctionnelles du canal, nous avons démontré d’une manière directe et indéniable que le peptide Gap26 inhibe et spécifiquement les HcCx43 et que son administration in vitro (cardiomyocytes isolés) et ex vivo (coeur intact) confère à ces modèles expérimentaux une résistance importante contre le stress ischémique. Dans la troisième étude, nous avons investigué pour la première fois in vivo le potentiel de deux peptides uniques mimétiques structuraux de la Cx43, Gap26 et Gap27, dans la cardioprotection contre les lésions ischémiques lorsqu’ils sont administrés à basse dose sous forme d’un bolus intraveineux unique. Nous avons démontré que l’injection de ces peptides avant ou après la survenue de l’ischémie réduit significativement la taille de l’infarctus qui en résulte.En conclusion, l’ensemble de ces résultats révèlent le rôle bénéfique de l’inhibition des HcCx43 lors d’une ischémie et dévoilent un potentiel thérapeutique prometteux des mimétiques structuraux de Cx43 dans la prévention et le traitement de l’infarctus du myocarde.

Dexamethasone-induced insulin resistance is associated with increased connexin 36 mRNA and protein expression in pancreatic rat islets

Augmented glucose-stimulated insulin secretion (GSIS) is an adaptive mechanism exhibited by pancreatic islets from insulin-resistant animal models. Gap junction proteins have been proposed to contribute to islet function. As such, we investigated the expression of connexin 36 (Cx36), connexin 43 (Cx43), and the glucose transporter Glut2 at mRNA and protein levels in pancreatic islets of dexamethasone (DEX)-induced insulin-resistant rats. Study rats received daily injections of DEX (1 mg/kg body mass, i.p.) for 5 days, whereas control rats (CTL) received saline solution. DEX rats exhibited peripheral insulin resistance, as indicated by the significant postabsorptive insulin levels and by the constant rate for glucose disappearance (K-ITT). GSIS was significantly higher in DEX islets (1.8-fold in 16.7 mmol/L glucose vs. CTL, p < 0.05). A significant increase of 2.25-fold in islet area was observed in DEX vs. CTL islets (p < 0.05). Cx36 mRNA expression was significantly augmented, Cx43 diminished, and Glut2 mRNA was unaltered in islets of DEX vs. CTL (p < 0.05). Cx36 protein expression was 1.6-fold higher than that of CTL islets (p < 0.05). Glut2 protein expression was unaltered and Cx43 was not detected at the protein level. We conclude that DEX-induced insulin resistance is accompanied by increased GSIS and this may be associated with increase of Cx36 protein expression.

Efeito do betacaroteno sobre o estresse oxidativo e a expressão de conexina 43 cardíaca

BACKGROUND: Intervention studies have shown an increased mortality in patients who received beta-carotene. However, the mechanisms involved in this phenomenon are still unknown. OBJECTIVE: Evaluate the influence of beta-carotene on oxidative stress and the expression of connexin 43 in rat hearts. METHODS: Wistar rats, weighing approximately 100 g, were allocated in two groups: Control Group (n=30), that received the diet routinely used in our laboratory, and Beta-Carotene Group (n = 28), which received beta-carotene (in crystal form, added and mixed to the diet) at a dose of 500 mg of beta-carotene/kg of diet. The animals received the treatment until they reached 200-250g, when they were sacrificed. Samples of blood, liver and heart were collected to perform Western blotting and immunohistochemistry for connexin 43; morphometric studies, dosages of beta-carotene by high-performance liquid chromatography as well as reduced glutathione, oxidized glutathione and lipids hydroperoxides were performed by biochemical analysis. RESULTS: Beta-carotene was detected only in the liver of Beta-Carotene Group animals (288 ± 94.7 µg/kg). Levels of reduced/oxidized glutathione were higher in the liver and heart of Beta-Carotene Group animals (liver - Control Group: 42.60 ± 1.62; liver - Beta-Carotene Group: 57.40 ± 5.90; p = 0.04; heart: - Control Group: 117.40 ± 1.01; heart - Beta-Carotene Group: 121.81 ± 1.32 nmol/mg protein; p = 0.03). The content of total connexin 43 was larger in Beta-Carotene Group. CONCLUSION: Beta-carotene demonstrated a positive effect, characterized by the increase of intercellular communication and improvement of anti-oxidizing defense system. In this model, mechanism does not explain the increased mortality rate observed with the beta-carotene supplementation in clinical studies. (Arq Bras Cardiol. 2013; [online].ahead print, PP.0-0)

Exogenous Cx43 expression decrease cell proliferation rate in rat hepatocarcinoma cells independently of functional gap junction

Background: Gap junction intercellular communication (GJIC) is considered to play a role in the regulation of homeostasis because it regulates important processes, such as cell proliferation and cell differentiation. A reduced or lost GJIC capacity has been observed in solid tumors and studies have demonstrated that GJIC restoration in tumor cells contribute to reversion of the transformed phenotype. This observation supports the idea that restoration of the functional channel is essential in this process. However, in the last years, reports have proposed that just the increase in the expression of specific connexins can contribute to reversion of the malign phenotype in some tumor cells. In the present work, we studied the effects of exogenous Connexin 43 (Cx43) expression on the proliferative behavior and phenotype of rat hepatocarcinoma cells. Results: The exogenous Cx43 did not increase GJIC capacity of transfected cells, but it was critical to decrease the cell proliferation rate as well as reorganization of the actin filaments and cell flattening. We also observed more adhesion capacity to substrate after Cx43 transfection. Conclusion: Cx43 expression leads to a decrease of the growth of the rat hepatocellular carcinoma cells and it contributes to the reversion of the transformed phenotype. These effects were independent of the GJIC and were probably associated with the phosphorylation pattern changes and redistribution of the Cx43 protein.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

A Novel Autosomal Recessive GJA1 Missense Mutation Linked to Craniometaphyseal Dysplasia

Craniometaphyseal dysplasia (CMD) is a rare sclerosing skeletal disorder with progressive hyperostosis of craniofacial bones. CMD can be inherited in an autosomal dominant (AD) trait or occur after de novo mutations in the pyrophosphate transporter ANKH. Although the autosomal recessive (AR)form of CMD had been mapped to 6q21-22 the mutation has been elusive. In this study, we performed whole-exome sequencing for one subject with AR CMD and identified a novel missense mutation (c.716G>A, p.Arg239Gln) in the C-terminus of the gap junction protein alpha-1 (GJA1) coding for connexin 43 (Cx43). We confirmed this mutation in 6 individuals from 3 additional families. The homozygous mutation cosegregated only with affected family members. Connexin 43 is a major component of gap junctions in osteoblasts, osteocytes, osteoclasts and chondrocytes. Gap junctions are responsible for the diffusion of low molecular weight molecules between cells. Mutations in Cx43 cause several dominant and recessive disorders involving developmental abnormalities of bone such as dominant and recessive oculodentodigital dysplasia (ODDD; MIM #164200, 257850) and isolated syndactyly type III (MIM #186100), the characteristic digital anomaly in ODDD. However, characteristic ocular and dental features of ODDD as well as syndactyly are absent in patients with the recessive Arg239Gln Cx43 mutation. Bone remodeling mechanisms disrupted by this novel Cx43 mutation remain to be elucidated.

Four whole-istic aspects of schistosome granuloma biology: fractal arrangement, internal regulation, autopoietic component and closure

This paper centers on some whole-istic organizational and functional aspects of hepatic Schistosoma mansoni granuloma, which is an extremely complex system. First, it structurally develops a collagenic topology, originated bidirectionally from an inward and outward assembly of growth units. Inward growth appears to be originated from myofibroblasts derived from small portal vessel around intravascular entrapped eggs, while outward growth arises from hepatic stellate cells. The auto-assembly of the growth units defines the three-dimensional scaffold of the schistosome granulomas. The granuloma surface irregularity and its border presented fractal dimension equal to 1.58. Second, it is internally regulated by intricate networks of immuneneuroendocrine stimuli orchestrated by leptin and leptin receptors, substance P and Vasoactive intestinal peptide. Third, it can reach the population of ± 40,000 cells and presents an autopoietic component evidenced by internal proliferation (Ki-67+ Cells), and by expression of c-Kit+ Cells, leptin and leptin receptor (Ob-R), granulocyte-colony stimulating factor (G-CSF-R), and erythropoietin (Epo-R) receptors. Fourth, the granulomas cells are intimately connected by pan-cadherins, occludin and connexin-43, building a state of closing (granuloma closure). In conclusion, the granuloma is characterized by transitory stages in such a way that its organized structure emerges as a global property which is greater than the sum of actions of its individual cells and extracellular matrix components.

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-α blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

Gap junction reduction in cardiomyocytes following transforming growth factor-β treatment and Trypanosoma cruzi infection

Gap junction connexin-43 (Cx43) molecules are responsible for electrical impulse conduction in the heart and are affected by transforming growth factor-β (TGF-β). This cytokine increases during Trypanosoma cruzi infection, modulating fibrosis and the parasite cell cycle. We studied Cx43 expression in cardiomyocytes exposed or not to TGF-β T. cruzi, or SB-431542, an inhibitor of TGF-β receptor type I (ALK-5). Cx43 expression was also examined in hearts with dilated cardiopathy from chronic Chagas disease patients, in which TGF-β signalling had been shown previously to be highly activated. We demonstrated that TGF-β treatment induced disorganised gap junctions in non-infected cardiomyocytes, leading to a punctate, diffuse and non-uniform Cx43 staining. A similar pattern was detected in T. cruzi-infected cardiomyocytes concomitant with high TGF-β secretion. Both results were reversed if the cells were incubated with SB-431542. Similar tests were performed using human chronic chagasic patients and we confirmed a down-regulation of Cx43 expression, an altered distribution of plaques in the heart and a significant reduction in the number and length of Cx43 plaques, which correlated negatively with cardiomegaly. We conclude that elevated TGF-β levels during T. cruzi infection promote heart fibrosis and disorganise gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia that characterise severe cardiopathy in Chagas disease.

Severity of chronic experimental Chagas' heart disease parallels tumour necrosis factor and nitric oxide levels in the serum: models of mild and severe disease

Heart tissue inflammation, progressive fibrosis and electrocardiographic alterations occur in approximately 30% of patients infected by Trypanosoma cruzi, 10-30 years after infection. Further, plasma levels of tumour necrosis factor (TNF) and nitric oxide (NO) are associated with the degree of heart dysfunction in chronic chagasic cardiomyopathy (CCC). Thus, our aim was to establish experimental models that mimic a range of parasitological, pathological and cardiac alterations described in patients with chronic Chagas’ heart disease and evaluate whether heart disease severity was associated with increased TNF and NO levels in the serum. Our results show that C3H/He mice chronically infected with the Colombian T. cruzi strain have more severe cardiac parasitism and inflammation than C57BL/6 mice. In addition, connexin 43 disorganisation and fibronectin deposition in the heart tissue, increased levels of creatine kinase cardiac MB isoenzyme activity in the serum and more severe electrical abnormalities were observed in T. cruzi-infected C3H/He mice compared to C57BL/6 mice. Therefore, T. cruzi-infected C3H/He and C57BL/6 mice represent severe and mild models of CCC, respectively. Moreover, the CCC severity paralleled the TNF and NO levels in the serum. Therefore, these models are appropriate for studying the pathophysiology and biomarkers of CCC progression, as well as for testing therapeutic agents for patients with Chagas’ heart disease.

Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells.

Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Hypertension increases connexin43 in a tissue-specific manner.

BACKGROUND: Connexin43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in the contractility of the vascular wall and in the electrical coupling of cardiac myocytes. The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart. METHODS AND RESULTS: Rats were made hypertensive after one renal artery was clipped (two kidney, one-clip renal model) or after the administration of deoxycorticosterone and salt (DOCA-salt model). After 4 weeks, all rats showed a similar increase in intra-arterial mean blood pressure and in the thickness of both the aortic wall and the heart. Northern blot analysis of aorta mRNA and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 in aorta as the control animals. In contrast, no difference in Cx43 mRNA or in the immunolabeled protein was observed in heart. CONCLUSIONS: The results show that rats exhibiting a similar degree of blood pressure elevation, as the result of different mechanisms, feature a comparable increase in Cx43 gene expression, which was observed in the aortic but not in the cardiac muscle. These data suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.