RESUMEN
Dysferlinopathy is a genetic human disease caused by mutations in the gene that encodes the dysferlin protein (DYSF). Dysferlin is believed to play a relevant role in cell membrane repair. However, in dysferlin-deficient (blAJ) mice (a model of dysferlinopathies) the recovery of the membrane resealing function by means of the expression of a mini-dysferlin does not arrest progressive muscular damage, suggesting the participation of other unknown pathogenic mechanisms. Here, we show that proteins called connexins 39, 43 and 45 (Cx39, Cx43 and Cx45, respectively) are expressed by blAJ myofibers and form functional hemichannels (Cx HCs) in the sarcolemma. At rest, Cx HCs increased the sarcolemma permeability to small molecules and the intracellular Ca2+ signal. In addition, skeletal muscles of blAJ mice showed lipid accumulation and lack of dysferlin immunoreactivity. As sign of extensive damage and atrophy, muscles of blAJ mice presented elevated numbers of myofibers with internal nuclei, increased number of myofibers with reduced cross-sectional area and elevated creatine kinase activity in serum. In agreement with the extense muscle damage, mice also showed significantly low motor performance. We generated blAJ mice with myofibers deficient in Cx43 and Cx45 expression and found that all above muscle and systemic alterations were absent, indicating that these two Cxs play a critical role in a novel pathogenic mechanism of dysfernolophaties, which is discussed herein. Therefore, Cx HCs could constitute an attractive target for pharmacologic treatment of dyferlinopathies.
Asunto(s)
Conexina 43/genética , Conexinas/genética , Disferlina/genética , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/prevención & control , Miofibrillas/genética , Animales , Calcio/metabolismo , Conexina 43/deficiencia , Conexinas/deficiencia , Creatina Quinasa/sangre , Creatina Quinasa/genética , Modelos Animales de Enfermedad , Disferlina/deficiencia , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Mutación , Miofibrillas/metabolismo , Miofibrillas/patología , Permeabilidad , Condicionamiento Físico Animal , Prueba de Desempeño de Rotación con Aceleración Constante , Sarcolema/metabolismoRESUMEN
PURPOSE: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. METHODS: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. RESULTS: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. CONCLUSIONS: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.
Asunto(s)
Conexina 43/deficiencia , Modelos Animales de Enfermedad , Hígado/irrigación sanguínea , Daño por Reperfusión/metabolismo , Alanina Transaminasa/análisis , Animales , Aspartato Aminotransferasas/análisis , Conexina 43/análisis , Técnicas de Genotipaje , Hígado/patología , Ratones Noqueados , Necrosis , Reacción en Cadena de la Polimerasa , Valores de Referencia , Daño por Reperfusión/patología , Factores de Tiempo , gamma-Glutamiltransferasa/análisisRESUMEN
Abstract Purpose: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. Methods: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. Results: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. Conclusions: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.
Asunto(s)
Animales , Daño por Reperfusión/metabolismo , Conexina 43/deficiencia , Modelos Animales de Enfermedad , Hígado/irrigación sanguínea , Aspartato Aminotransferasas/análisis , Valores de Referencia , Factores de Tiempo , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa , Ratones Noqueados , Conexina 43/análisis , Alanina Transaminasa/análisis , Técnicas de Genotipaje , gamma-Glutamiltransferasa/análisis , Hígado/patología , NecrosisRESUMEN
Many functions of glial cells depend on the formation of selective glial networks mediated by gap junctions formed by members of the connexin family. Olfactory ensheathing cells (OECs) are specialized glia associated with olfactory sensory neuron axons. Like other glia, they form selective networks, however, the connexins that support OEC connectivity in vivo have not been identified. We used an in vivo mouse model to selectively delete candidate connexin genes with temporal control from OECs and address the physiological consequences. Using this model, we effectively abolished the expression of connexin 43 (Cx43) in OECs in both juvenile and adult mice. Cx43-deleted OECs exhibited features consistent with the loss of gap junctions including reduced membrane conductance, largely reduced sensitivity to the gap junction blocker meclofenamic acid and loss of dye coupling. This indicates that Cx43, a typically astrocytic connexin, is the main connexin forming functional channels in OECs. Despite these changes in functional properties, the deletion of Cx43 deletion did not alter the density of OECs. The strategy used here may prove useful to delete other candidate genes to better understand the functional roles of OECs in vivo.
Asunto(s)
Conexina 43/fisiología , Uniones Comunicantes/fisiología , Técnicas de Inactivación de Genes , Neuroglía/fisiología , Bulbo Olfatorio/citología , Envejecimiento/metabolismo , Animales , Conexina 43/deficiencia , Conexina 43/genética , Cruzamientos Genéticos , Femenino , Uniones Comunicantes/efectos de los fármacos , Genes Reporteros , Genes Sintéticos , Integrasas/genética , Masculino , Ácido Meclofenámico/farmacología , Ratones , Ratones Noqueados , Proteína Proteolipídica de la Mielina/genética , Bulbo Olfatorio/metabolismo , Técnicas de Placa-Clamp , Tamoxifeno/farmacologíaRESUMEN
BACKGROUND: Cellular channels composed of connexin 43 are known to act as key players in the life cycle of the skin and consequently to underlie skin repair. OBJECTIVE: This study was specifically set up to investigate the suite of molecular mechanisms driven by connexin 43-based channels on wound healing. METHODS: To this end, a battery of parameters, including re-epithelialization, neovascularization, collagen deposition and extracellular matrix remodeling, was monitored over time during experimentally induced skin repair in heterozygous connexin 43 knockout mice. RESULTS: It was found that connexin 43 deficiency accelerates re-epithelialization and wound closure, increases proliferation and activation of dermal fibroblasts, and enhances the expression of extracellular matrix remodeling mediators. CONCLUSION: These data substantiate the notion that connexin 43 may represent an interesting therapeutic target in dermal wound healing.
Asunto(s)
Colágeno/metabolismo , Conexina 43/deficiencia , Matriz Extracelular/metabolismo , Repitelización/fisiología , Animales , Proliferación Celular , Conexina 43/genética , Fibroblastos/fisiología , Heterocigoto , Masculino , Ratones , Ratones Noqueados , Neovascularización FisiológicaRESUMEN
Denervation of skeletal muscles induces atrophy, preceded by changes in sarcolemma permeability of causes not yet completely understood. Here, we show that denervation-induced Evans blue dye uptake in vivo of fast, but not slow, myofibers was acutely inhibited by connexin (Cx) hemichannel/pannexin1 (Panx1) channel and purinergic ionotropic P2X7 receptor (P2X7R) blockers. Denervated myofibers showed up-regulation of Panx1 and de novo expression of Cx39, Cx43, and Cx45 hemichannels as well as P2X7Rs and transient receptor potential subfamily V, member 2, channels, all of which are permeable to small molecules. The sarcolemma of freshly isolated WT myofibers from denervated muscles also showed high hemichannel-mediated permeability that was slightly reduced by blockade of Panx1 channels or the lack of Panx1 expression, but was completely inhibited by Cx hemichannel or P2X7R blockers, as well as by degradation of extracellular ATP. However, inhibition of transient receptor potential subfamily V, member 2, channels had no significant effect on membrane permeability. Moreover, activation of the transcription factor NFκB and higher mRNA levels of proinflammatory cytokines (TNF-α and IL-1ß) were found in denervated WT but not Cx43/Cx45-deficient muscles. The atrophy observed after 7 d of denervation was drastically reduced in Cx43/Cx45-deficient but not Panx1-deficient muscles. Therefore, expression of Cx hemichannels and P2X7R promotes a feed-forward mechanism activated by extracellular ATP, most likely released through hemichannels, that activates the inflammasome. Consequently, Cx hemichannels are potential targets for new therapeutic agents to prevent or reduce muscle atrophy induced by denervation of diverse etiologies.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Conexinas/metabolismo , Desnervación , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Sarcolema/metabolismo , Análisis de Varianza , Animales , Conexina 43/deficiencia , Azul de Evans/metabolismo , Masculino , Microscopía Fluorescente , Músculo Esquelético/inervación , Ratas , Ratas Sprague-DawleyRESUMEN
GJA1 gene (Connexin43, also known as Cx43) is the most abundant gap junction protein isoform in animal cells and is associated with bone development in embryos. The objective of the present work was to evaluate in vivo osteal development in GJA1-deficient fetal mice through determination of the histological and molecular alterations induced by partial or total deletion of the GJA1 gene. Heterozygous C57BL/6 mice (HT) harboring a null mutation of the GJA1 gene were mated, and pregnant females were submitted to euthanasia and Caesarean section from 12.5 to 19.5 days post coitum (dpc). HT (GJA1(+/-)) and homozygous (GJA1(-/- )) knockout (KO) mutants and wild-type (WT) fetuses were identified by polymerase chain reaction (PCR), and development curves were constructed on the basis of fetus weight and crown-rump length. Histopathological, histochemical, and real-time PCR analyses were performed in order to assess the expression of markers associated with bone development, namely, osteocalcin, osteopontin, alkaline phosphatase, RUNX2, GJA1, GJC1 (Cx45), and GJA3 (Cx46). HT and KO fetuses exhibited delays in the differentiation of osteoblasts and, consequently, in bone development in comparison with the WT group. Additionally, less deposition of mineralized and osteoid matrix was observed in GJA1-deficient fetuses. Bone development in KO fetuses was delayed through the moment of birth, but in HT animals the delay only extended until 17.5 dpc, following which development was normalized. The expression of genes coding for osteocalcin, osteopontin, alkaline phosphatise, and RUNX2 were also delayed in GJA1-deficient fetuses. Animals that exhibited a lower expression of GJA1 presented delayed expression of the GJC1 and GJA3 genes and their corresponding protein products in the bone tissue. The results of the present study contribute to our understanding of the function of GJA1 during bone development and suggest that GJC1 could play a role in restoring intercellular communication in GJA1-deficient mice.
Asunto(s)
Conexina 43/deficiencia , Osteoblastos/citología , Osteogénesis/fisiología , Análisis de Varianza , Animales , Biomarcadores/metabolismo , Diferenciación Celular/genética , Conexina 43/genética , Conexina 43/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Largo Cráneo-Cadera , Femenino , Peso Fetal/genética , Uniones Comunicantes/genética , Eliminación de Gen , Histocitoquímica , Ratones , Ratones Noqueados , Osteoblastos/metabolismo , Osteogénesis/genética , Embarazo , Costillas/químicaRESUMEN
The mechanisms involved in Alzheimer's disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-ß peptide (Aß) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aß. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aß(25-35) increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aß. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aß(25-35)-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aß(25-35)-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aß leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aß.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Muerte Celular/fisiología , Neuroglía/fisiología , Neuronas/patología , Fragmentos de Péptidos/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Conexina 43/antagonistas & inhibidores , Conexina 43/deficiencia , Conexina 43/metabolismo , Ácido Glutámico/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/fisiologíaRESUMEN
Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-ß peptide (Aß) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3 h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aß-treated microglia (CM-Aß). The effects of CM-Aß were mimicked by TNF-α and IL-1ß and were abrogated by neutralizing TNF-α with soluble receptor and IL-1ß with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aß made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aß induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia.
Asunto(s)
Adenosina Trifosfato/metabolismo , Astrocitos/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Ácido Glutámico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Adenosina Trifosfato/farmacología , Péptidos beta-Amiloides/farmacología , Animales , Animales Recién Nacidos , Astrocitos/química , Astrocitos/efectos de los fármacos , Biotinilación/métodos , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Células Cultivadas , Corteza Cerebral/citología , Técnicas de Cocultivo/métodos , Conexina 43/deficiencia , Conexinas/farmacología , Medios de Cultivo Condicionados/farmacología , Femenino , Fluoresceínas , Ácido Glutámico/farmacología , Interleucina-1beta/metabolismo , Lantano/farmacología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/farmacología , Proteínas del Tejido Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuropéptidos/farmacología , Oxígeno/farmacología , Fragmentos de Péptidos/farmacología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4) -induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis.
Asunto(s)
Quimiocina CCL4/toxicidad , Conexina 43/deficiencia , Cirrosis Hepática/inducido químicamente , Hígado/patología , Patología Molecular , Animales , RatonesRESUMEN
Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.
Asunto(s)
Colágeno/metabolismo , Conexina 43/deficiencia , Granuloma/patología , Hepatopatías/patología , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/patología , Animales , Recuento de Células , Proliferación Celular , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente Indirecta , Silenciador del Gen , Granuloma/metabolismo , Granuloma/parasitología , Hepatopatías/metabolismo , Hepatopatías/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antígeno Nuclear de Célula en Proliferación/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esquistosomiasis mansoni/metabolismoRESUMEN
Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-gamma (INF-gamma) or tumor necrosis factor-alpha (TNF-alpha) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-gamma plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-alpha antibody, suggesting the release and autocrine action of TNF-alpha. Treatment with INF-gamma plus TNF-alpha also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell-cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18 alpha-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-gamma plus LPS or INF-gamma plus TNF-alpha. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.