Brain gray matter astroglia-specific connexin 43 ablation attenuates spinal cord inflammatory demyelination.

BACKGROUND: Brain astroglia are activated preceding the onset of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We characterized the effects of brain astroglia on spinal cord inflammation, focusing on astroglial connexin (Cx)43, because we recently reported that Cx43 has a critical role in regulating neuroinflammation. METHODS: Because glutamate aspartate transporter (GLAST)+ astroglia are enriched in the brain gray matter, we generated Cx43fl/fl;GLAST-CreERT2/+ mice that were brain gray matter astroglia-specific Cx43 conditional knockouts (Cx43 icKO). EAE was induced by immunization with myelin oligodendroglia glycoprotein (MOG) 35-55 peptide 10 days after tamoxifen injection. Cx43fl/fl mice were used as controls. RESULTS: Acute and chronic EAE signs were significantly milder in Cx43 icKO mice than in controls whereas splenocyte MOG-specific responses were unaltered. Histologically, Cx43 icKO mice showed significantly less demyelination and fewer CD45+ infiltrating immunocytes, including F4/80+ macrophages, and Iba1+ microglia in the spinal cord than controls. Microarray analysis of the whole cerebellum revealed marked upregulation of anti-inflammatory A2-specific astroglia gene sets in the pre-immunized phase and decreased proinflammatory A1-specific and pan-reactive astroglial gene expression in the onset phase in Cx43 icKO mice compared with controls. Astroglia expressing C3, a representative A1 marker, were significantly decreased in the cerebrum, cerebellum, and spinal cord of Cx43 icKO mice compared with controls in the peak phase. Isolated Cx43 icKO spinal microglia showed more anti-inflammatory and less proinflammatory gene expression than control microglia in the pre-immunized phase. In particular, microglial expression of Ccl2, Ccl5, Ccl7, and Ccl8 in the pre-immunized phase and of Cxcl9 at the peak phase was lower in Cx43 icKO than in controls. Spinal microglia circularity was significantly lower in Cx43 icKO than in controls in the peak phase. Significantly lower interleukin (IL)-6, interferon-γ, and IL-10 levels were present in cerebrospinal fluid from Cx43 icKO mice in the onset phase compared with controls. CONCLUSIONS: The ablation of Cx43 in brain gray matter astroglia attenuates EAE by promoting astroglia toward an anti-inflammatory phenotype and suppressing proinflammatory activation of spinal microglia partly through depressed cerebrospinal fluid proinflammatory cytokine/chemokine levels. Brain astroglial Cx43 might be a novel therapeutic target for MS.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day-night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day-night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals' active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day-night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals' awake, active phase.

Establishment and functional characterization of a murine primary Sertoli cell line deficient of connexin43.

The Sertoli cell (SC) specific connexin43 (Cx43) knockout (SCCx43KO) mouse line is ideal to gain insight into the mechanistic gap junction formation in SC and the seminiferous epithelium. A method for developing primary SC cultures from these mice was established, validated and successfully characterized via polymerase chain reaction, immunohistochemistry, immunofluorescence (IF), and Western blots (WB). It was evident that both knockout (KO) and wild-type (WT) primary cell cultures were similar in morphology. These highly pure SC cultures were subjected to cell proliferation assays indicating no notable proliferation in cultures of both genotypes. Measurements of cell monolayer integrity indicated significant increases in transepithelial electrical resistance and consequently in tight junction expression of the KO cultures. Using semi-quantitative WB and IF, tight junction protein claudin-11 was analyzed. These results support a role for Cx43 in regulating blood-testis barrier (BTB) function, composition, and dynamics in vitro. Thus, the SC deficient Cx43 cell cultures may provide a valuable in vitro tool for a better understanding of the mechanistic role of Cx43 in spermatogenesis and BTB assembly.

Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity.

The tissue-resident macrophages of barrier organs constitute the first line of defence against pathogens at the systemic interface with the ambient environment. In the lung, resident alveolar macrophages (AMs) provide a sentinel function against inhaled pathogens. Bacterial constituents ligate Toll-like receptors (TLRs) on AMs, causing AMs to secrete proinflammatory cytokines that activate alveolar epithelial receptors, leading to recruitment of neutrophils that engulf pathogens. Because the AM-induced response could itself cause tissue injury, it is unclear how AMs modulate the response to prevent injury. Here, using real-time alveolar imaging in situ, we show that a subset of AMs attached to the alveolar wall form connexin 43 (Cx43)-containing gap junction channels with the epithelium. During lipopolysaccharide-induced inflammation, the AMs remained sessile and attached to the alveoli, and they established intercommunication through synchronized Ca(2+) waves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involving Ca(2+)-dependent activation of Akt, because AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the bronchoalveolar lavage. A picture emerges of a novel immunomodulatory process in which a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation.

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation.

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43(-/-) salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43(-/-) samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43(-/-) phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet.

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308.

Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone.

OBJECTIVE: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. APPROACH AND RESULTS: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6(-/-) arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6(-/-) mice were protected against MT elevation in myocardial infarction-induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA-GTP binding, myosin light chain, P42-P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43(+/-) and P2rx7(-/-) mesenteric resistance arteries. CONCLUSIONS: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Connexin defects underlie arrhythmogenic right ventricular cardiomyopathy in a novel mouse model.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) termed a 'disease of the desmosome' is an inherited cardiomyopathy that recently underwent reclassification owing to the identification of left-dominant and biventricular disease forms. Homozygous loss-of-function mutations in the desmosomal component, desmoplakin, are found in patients exhibiting a biventricular form of ARVC; however, no models recapitulate the postnatal hallmarks of the disease as seen in these patients. To gain insights into the homozygous loss-of-function effects of desmoplakin in the heart, we generated cardiomyocyte-specific desmoplakin-deficient mice (DSP-cKO) using ventricular myosin light chain-2-Cre mice. Homozygous DSP-cKO mice are viable but display early ultrastructural defects in desmosomal integrity leading to a cardiomyopathy reminiscent of a biventricular form of ARVC, which includes cell death and fibro-fatty replacement within the ventricle leading to biventricular dysfunction, failure and premature death. DSP-cKO mice also exhibited ventricular arrhythmias that are exacerbated with exercise and catecholamine stimulation. Furthermore, DSP-cKO hearts exhibited right ventricular conduction defects associated with loss of connexin 40 expression and electrical wavefront propagation defects associated with loss of connexin 43 expression. Dose-dependent assessment of the effects of loss of desmoplakin in neonatal ventricular cardiomyocytes revealed primary loss of connexin 43 levels, phosphorylation and function independent of the molecular dissociation of the mechanical junction complex and fibro-fatty manifestation associated with ARVC, suggesting a role for desmoplakin as a primary stabilizer of connexin integrity. In summary, we provide evidence for a novel mouse model, which is reminiscent of the postnatal onset of ARVC while highlighting mechanisms underlying a biventricular form of human ARVC.

Extracellular sodium dependence of the conduction velocity-calcium relationship: evidence of ephaptic self-attenuation.

Our laboratory previously demonstrated that perfusate sodium and potassium concentrations can modulate cardiac conduction velocity (CV) consistent with theoretical predictions of ephaptic coupling (EpC). EpC depends on the ionic currents and intercellular separation in sodium channel rich intercalated disk microdomains like the perinexus. We suggested that perinexal width (WP) correlates with changes in extracellular calcium ([Ca(2+)]o). Here, we test the hypothesis that increasing [Ca(2+)]o reduces WP and increases CV. Mathematical models of EpC also predict that reducing WP can reduce sodium driving force and CV by self-attenuation. Therefore, we further hypothesized that reducing WP and extracellular sodium ([Na(+)]o) will reduce CV consistent with ephaptic self-attenuation. Transmission electron microscopy revealed that increasing [Ca(2+)]o (1 to 3.4 mM) significantly decreased WP Optically mapping wild-type (WT) (100% Cx43) mouse hearts demonstrated that increasing [Ca(2+)]o increases transverse CV during normonatremia (147.3 mM), but slows transverse CV during hyponatremia (120 mM). Additionally, CV in heterozygous (∼50% Cx43) hearts was more sensitive to changes in [Ca(2+)]o relative to WT during normonatremia. During hyponatremia, CV slowed in both WT and heterozygous hearts to the same extent. Importantly, neither [Ca(2+)]o nor [Na(+)]o altered Cx43 expression or phosphorylation determined by Western blotting, or gap junctional resistance determined by electrical impedance spectroscopy. Narrowing WP, by increasing [Ca(2+)]o, increases CV consistent with enhanced EpC between myocytes. Interestingly, during hyponatremia, reducing WP slowed CV, consistent with theoretical predictions of ephaptic self-attenuation. This study suggests that serum ion concentrations may be an important determinant of cardiac disease expression.

The E3 ubiquitin ligase c-Cbl mediates integrin ß1 ubiquitination during dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by dilatation of the ventricular chambers and impaired myocardial contractility. The results of our previous study indicated that a deficiency in matricellular cartilage oligomeric matrix protein (COMP) led to spontaneous and progressive DCM in mice via the ubiquitination/degradation of integrin ß1. However, the specific ubiquitin enzyme involved in degradation of integrin ß1 and the pathogenesis of DCM remain elusive. We first compared gene expression profiles in hearts from 3-month-old wild type and COMP-/- mice using microarray analysis. Among the E3 ubiquitin ligases upregulated in COMP-/- hearts, c-Cbl silencing rescued the ubiquitination/degradation of integrin ß1, myofilament loss, apoptosis and connexin-43 deficiency in cardiomyocytes due to the silencing of COMP. Furthermore, c-Cbl silencing by intramyocardial injections of siRNA into 1-month-old COMP-/- mice ameliorated spontaneous DCM in vivo, as evidenced by the inhibition of the dilation of ventricular chambers, impaired ejection fraction and myofilament loss. A subsequent cellular ubiquitination assay revealed that overexpression of c-Cbl induced ubiquitination of integrin ß1, whereas the G306E mutation in c-Cbl, which prevented the binding of c-Cbl to its substrates, had no effect on integrin ß1 ubiquitination, indicating that c-Cbl directly caused the ubiquitination of integrin ß1 in the hearts. In conclusion, our results demonstrate that c-Cbl mediates the ubiquitination/degradation of integrin ß1, which leads to COMP deficiency-induced DCM.

De novo expression of connexin hemichannels in denervated fast skeletal muscles leads to atrophy.

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.

Absence of connexin43 and connexin45 does not disturb pre- and peri-implantation development.

Gap junctional intercellular communication is assumed to play an important role during pre- and peri-implantation development. In this study, we eliminated connexin43 (Cx43) and connexin45 (Cx45), major gap junctional proteins in the pre- and peri-implantation embryo. We generated Cx43 -/- Cx45 -/- embryos by Cx43 +/- Cx45 +/- intercrossing, because mice deficient in Cx43 (Cx43 -/-) exhibit perinatal lethality and those deficient in Cx45 (Cx45 -/-) exhibit early embryonic lethality. Wild-type, Cx43 -/-, Cx45 -/-, and Cx43 -/- Cx45 -/- blastocysts all showed similar outgrowths in in vitro culture. Moreover, Cx43 -/- Cx45 -/- embryos were obtained at the expected Mendelian ratio up to embryonic day 9.5, when the Cx45 -/- mutation proved lethal. The Cx43 -/- Cx45 -/- embryos seemed to have no additional developmental abnormalities in comparison with the single knockout strains. Thus, pre- and peri-implantation development does not require Cx43 and Cx45. Other gap junctional proteins are expressed around these stages and these may compensate for the lack of Cx43 and Cx45.

Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice.

BACKGROUND & AIMS: In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca(2+) responses) in glia, but it is not clear how this process affects intestinal function. We investigated whether Ca(2+)-mediated responses in enteric glia are required to maintain gastrointestinal function. METHODS: We used in situ Ca(2+) imaging to monitor glial Ca(2+) responses, which were manipulated with pharmacologic agents or via glia-specific disruption of the gene encoding connexin-43 (Cx43) (hGFAP::CreER(T2+/-)/Cx43(f/f) mice). Gastrointestinal function was assessed based on pellet output, total gut transit, colonic bead expulsion, and muscle tension recordings. Proteins were localized and quantified by immunohistochemistry, immunoblot, and reverse transcription polymerase chain reaction analyses. RESULTS: Ca(2+) responses in enteric glia of mice were mediated by Cx43 hemichannels. Cx43 immunoreactivity was confined to enteric glia within the myenteric plexus of the mouse colon; the Cx43 inhibitors carbenoxolone and 43Gap26 inhibited the ability of enteric glia to propagate Ca(2+) responses. In vivo attenuation of Ca(2+) responses in the enteric glial network slowed gut transit overall and delayed colonic transit--these changes are also observed during normal aging. Altered motility with increasing age was associated with reduced glial Ca(2+)-mediated responses and changes in glial expression of Cx43 messenger RNA and protein. CONCLUSIONS: Ca(2+)-mediated responses in enteric glia regulate gastrointestinal function in mice. Altered intercellular signaling between enteric glia and neurons might contribute to motility disorders.

Cx43 and mechanotransduction in bone.

Bone adaptation to changes in mechanical stimuli occurs by adjusting bone formation and resorption by osteoblasts and osteoclasts, to maintain optimal bone mass. Osteocytes coordinate the actions of these cells on the bone surface by sensing mechanical forces and producing cytokines that increase or prevent osteoblast and osteoclast differentiation and function. Channels formed by connexins (Cxs) and, in particular, connexin 43 (Cx43) in osteoblasts and osteocytes are central part of this mechanism to control bone mass. Cx43 hemichannels are opened by fluid flow and mediate the anti-apoptotic effect of mechanical stimulation in vitro, suggesting that Cx43 participates in mechanotransduction. However, mice lacking Cx43 in osteoblasts and/or osteocytes show an increased anabolic response to loading and decreased catabolic response to unloading. This evidence suggests that Cx43 channels expressed in osteoblastic cells are not required for the response to mechanical stimulation, but mediate the consequence of lack thereof. The molecular basis of these unexpected responses to mechanical stimulation is currently under investigation.

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells.

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21(cip1.) and p16(INK4a), and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Loss of αT-catenin alters the hybrid adhering junctions in the heart and leads to dilated cardiomyopathy and ventricular arrhythmia following acute ischemia.

It is generally accepted that the intercalated disc (ICD) required for mechano-electrical coupling in the heart consists of three distinct junctional complexes: adherens junctions, desmosomes and gap junctions. However, recent morphological and molecular data indicate a mixing of adherens junctional and desmosomal components, resulting in a 'hybrid adhering junction' or 'area composita'. The α-catenin family member αT-catenin, part of the N-cadherin-catenin adhesion complex in the heart, is the only α-catenin that interacts with the desmosomal protein plakophilin-2 (PKP2). Thus, it has been postulated that αT-catenin might serve as a molecular integrator of the two adhesion complexes in the area composita. To investigate the role of αT-catenin in the heart, gene targeting technology was used to delete the Ctnna3 gene, encoding αT-catenin, in the mouse. The αT-catenin-null mice are viable and fertile; however, the animals exhibit progressive cardiomyopathy. Adherens junctional and desmosomal proteins were unaffected by loss of αT-catenin, with the exception of the desmosomal protein PKP2. Immunogold labeling at the ICD demonstrated in the αT-catenin-null heart a preferential reduction of PKP2 at the area composita compared with the desmosome. Furthermore, gap junction protein Cx43 was reduced at the ICD, including its colocalization with N-cadherin. Gap junction remodeling in αT-catenin-knockout hearts was associated with an increased incidence of ventricular arrhythmias after acute ischemia. This novel animal model demonstrates for the first time how perturbation in αT-catenin can affect both PKP2 and Cx43 and thereby highlights the importance of understanding the crosstalk between the junctional proteins of the ICD and its implications for arrhythmogenic cardiomyopathy.

Blastocyst implantation failure relates to impaired translational machinery gene expression.

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43(del/del)). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43(del/del) oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43(del/del) oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF.

Evidence for the role of connexin 43-mediated intercellular communication in the process of intracortical bone resorption via osteocytic osteolysis.

BACKGROUND: Connexin 43 (Cx43) is the predominant gap junction protein in bone. Mice with a bone-specific deletion of Cx43 (cKO) have an osteopenic cortical phenotype. In a recent study, we demonstrated that cKO mice are resistant to bone loss induced by hindlimb suspension (HLS), an animal model of skeletal unloading. This protective effect occurred primarily as a result of lower osteoclast-mediated bone resorption. Interestingly, we also documented a significant increase in cortical osteocyte apoptosis and reduced sclerostin production. In the present study, we investigated whether osteocytic osteolysis - bone resorption by osteocytes within lacunae - is induced by HLS and the potential effect of Cx43 deficiency on this process during unloading. METHODS: 6-month-old male Cx43 cKO or wild-type (WT) mice were subjected to three weeks of HLS (Suspended) or normal loading conditions (Control) (n = 5/group). Lacunar morphology and tartrate-resistant acid phosphatase (TRACP) staining were assessed on sections of femur cortical bone. Experimental groups were compared via two-way ANOVA. RESULTS: Empty lacunae were 26% larger in cKO-Control vs. WT-Control (p < 0.05), while there was no difference in the size of empty lacunae between Control and Suspended within WT or cKO (p > 0.05). Similarly, there was a trend (p = 0.06) for 11% larger lacunae containing viable osteocytes for cKO-Control vs. WT-Control, with no apparent effect of loading condition. There was no difference in the proportion of TRACP + cells between WT-Control and cKO-Control (p > 0.05); however, WT-Suspended mice had 246% more TRACP + osteocytes compared WT-Control mice (p < 0.05). There was no difference in TRACP staining between cKO-Control and cKO-Suspended (p > 0.05). CONCLUSIONS: Prior to undergoing apoptosis, osteocytes in cKO mice may be actively resorbing their respective lacunae via the process of osteocytic osteolysis. Interestingly, the proportion of TRACP + osteocytes increased dramatically following unloading of WT mice, an effect that was not observed in cKO mice subjected to HLS. The results of the present study provide initial evidence that osteocytic osteolysis is occurring in cortical bone in response to mechanical unloading. Furthermore, Cx43 deficiency appears to protect against osteocytic osteolysis in a manner similar to the inhibition of unloading-induced osteoclast activation that we have documented previously.

Activation of RISK and SAFE pathways is not involved in the effects of Cx43 deficiency on tolerance to ischemia-reperfusion injury and preconditioning protection.

Connexin 43 (Cx43) deficiency increases myocardial tolerance to ischemia-reperfusion injury and abolishes preconditioning protection. It is not known whether modifications in baseline signaling through protective RISK or SAFE pathways or in response to preconditioning may contribute to these effects. To answer this question we used Cx43(Cre-ER(T)/fl) mice, in which Cx43 expression is abolished after 4-hydroxytamoxifen (4-OHT) administration. Isolated hearts from Cx43(Cre-ER(T)/fl) mice, or from Cx43(fl/fl) controls, treated with vehicle or 4-OHT, were submitted to global ischemia (40 min) and reperfusion. Cx43 deficiency was associated with reduced infarct size after ischemia-reperfusion (11.17 ± 3.25 % vs. 65.04 ± 3.79, 59.31 ± 5.36 and 65.40 ± 4.91, in Cx43(fl/fl) animals treated with vehicle, Cx43(fl/fl) mice treated with 4-OHT, and Cx43(Cre-ER(T)/fl) mice treated with vehicle, respectively, n = 8-9, p < 0.001). However, the ratio phosphorylated/total protein expression for Akt, ERK-1/2, GSK3ß and STAT3 was not increased in normoxic samples from animals lacking Cx43. Instead, a reduction in the phosphorylation state of GSK3ß was observed in Cx43-deficient mice (ratio: 0.15 ± 0.02 vs. 0.56 ± 0.11, 0.77 ± 0.15, and 0.46 ± 0.14, respectively, n = 5-6, p < 0.01). Furthermore, ischemic preconditioning (IPC, 4 cycles of 3.5 min of ischemia and 5 min of reperfusion) increased phosphorylation of ERK-1/2, GSK3ß, and STAT3 in all hearts without differences between groups (n = 5-6, p < 0.05), although Cx43 deficient mice were not protected by either IPC or pharmacological preconditioning with diazoxide. Our data demonstrate that modification of RISK and SAFE signaling does not contribute to the role of Cx43 in the increased tolerance to myocardial ischemia-reperfusion injury and in preconditioning protection.