Connexin Dysfunction Research Articles

Administration of USP7 inhibitor p22077 alleviates Angiotensin II (Ang II)-induced atrial fibrillation in Mice.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is an important contributor to mortality and morbidity. Ubquitin-specific protease 7 (USP7), one of the most abundant ubiquitin-specific proteases (USP), participated in many cellular events, such as cell proliferation, apoptosis, and tumourigenesis. However, its role in AF remains unknown. Here, the mice were treated with Ang II infusion to induce the AF model. Echocardiography was used to measure the atrial diameter. Electrical stimulation was programmed to measure the induction and duration of AF. The changes in atrial remodeling were measured using routine histologic analysis. Here, a significant increase in USP7 expression was observed in Ang II-stimulated atrial cardiomyocytes and atrial tissues, as well as in atrial tissues from patients with AF. The administration of p22077, the inhibitor of USP7, attenuated Ang II-induced inducibility and duration of AF, atrial dilatation, connexin dysfunction, atrial fibrosis, atrial inflammation, and atrial oxidase stress, and then inhibited the progression of AF. Mechanistically, the administration of p22077 alleviated Ang II-induced activation of TGF-β/Smad2, NF-κB/NLRP3, NADPH oxidases (NOX2 and NOX4) signals, the up-regulation of CX43, ox-CaMKII, CaMKII, Kir2.1, and down-regulation of SERCA2a. Together, this study, for the first time, suggests that USP7 is a critical driver of AF and revealing USP7 may present a new target for atrial fibrillation therapeutic strategies.

Cytomembrane Trafficking Pathways of Connexin 26, 30, and 43.

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

Simulation-predicted and -explained inheritance model of pathogenicity confirmed by transgenic mice models

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In this study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal “plugs” aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictive ability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homo-hexameric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable hetero-hexameric channels did not. From the perspective of the conformational energetics of WT and variant Cx26 hexamers, we propose that unaffected carriers could result from a conformational predominance of the WT and pore-shrinkage-incapable hetero-hexamers, while mice with homozygous variants can only harbor an unstable and dysfunctional N-termini-blocking V37M homo-hexamer. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.

Mutations of CX46/CX50 and Cataract Development.

Cataract is a common disease in the aging population. Gap junction has been considered a central component in maintaining homeostasis for preventing cataract formation. Gap junction channels consist of connexin proteins with more than 20 members. Three genes including GJA1, GJA3, and GJA8, that encode protein Cx43 (connexin43), Cx46 (connexin46), and Cx50 (connexin50), respectively, have been identified in human and rodent lens. Cx46 together with Cx50 have been detected in lens fiber cells with high expression, whereas Cx43 is mainly expressed in lens epithelial cells. Disrupted expression of the two connexin proteins Cx46 and Cx50 is directly related to the development of severe cataract in human and mice. In this review article, we describe the main role of Cx46 and Cx50 connexin proteins in the lens and the relationship between mutations of Cx46 or Cx50 and hereditary cataracts. Furthermore, the latest progress in the fundamental research of lens connexin and the mechanism of cataract formation caused by lens connexin dysfunction are summarized. Overall, targeting connexin could be a novel approach for the treatment of cataract.

MiRNA and mRNA Profiling Links Connexin Deficiency to Deafness via Early Oxidative Damage in the Mouse Stria Vascularis.

Pathogenic mutations in the non-syndromic hearing loss and deafness 1 (DFNB1) locus are the primary cause of monogenic inheritance for prelingual hearing loss. To unravel molecular pathways involved in etiopathology and look for early degeneration biomarkers, we used a system biology approach to analyze Cx30−/− mice at an early cochlear post-natal developmental stage. These mice are a DFNB1 mouse model with severely reduced expression levels of two connexins in the inner ear, Cx30, and Cx26. Integrated analysis of miRNA and mRNA expression profiles in the cochleae of Cx30−/− mice at post-natal day 5 revealed the overexpression of five miRNAs (miR-34c, miR-29b, miR-29c, miR-141, and miR-181a) linked to apoptosis, oxidative stress, and cochlear degeneration, which have Sirt1 as a common target of transcriptional and/or post-transcriptional regulation. In young adult Cx30−/− mice (3 months of age), these alterations culminated with blood barrier disruption in the Stria vascularis (SV), which is known to have the highest aerobic metabolic rate of all cochlear structures and whose microvascular alterations contribute to age-related degeneration and progressive decline of auditory function. Our experimental validation of selected targets links hearing acquisition failure in Cx30−/− mice, early oxidative stress, and metabolic dysregulation to the activation of the Sirt1–p53 axis. This is the first integrated analysis of miRNA and mRNA in the cochlea of the Cx30−/− mouse model, providing evidence that connexin downregulation determines a miRNA-mediated response which leads to chronic exhaustion of cochlear antioxidant defense mechanisms and consequent SV dysfunction. Our analyses support the notion that connexin dysfunction intervenes early on during development, causing vascular damage later on in life. This study identifies also early miRNA-mediated biomarkers of hearing impairment, either inherited or age related.

Brain Disorders and Chemical Pollutants: A Gap Junction Link?

The incidence of brain pathologies has increased during last decades. Better diagnosis (autism spectrum disorders) and longer life expectancy (Parkinson’s disease, Alzheimer’s disease) partly explain this increase, while emerging data suggest pollutant exposures as a possible but still underestimated cause of major brain disorders. Taking into account that the brain parenchyma is rich in gap junctions and that most pollutants inhibit their function; brain disorders might be the consequence of gap-junctional alterations due to long-term exposures to pollutants. In this article, this hypothesis is addressed through three complementary aspects: (1) the gap-junctional organization and connexin expression in brain parenchyma and their function; (2) the effect of major pollutants (pesticides, bisphenol A, phthalates, heavy metals, airborne particles, etc.) on gap-junctional and connexin functions; (3) a description of the major brain disorders categorized as neurodevelopmental (autism spectrum disorders, attention deficit hyperactivity disorders, epilepsy), neurobehavioral (migraines, major depressive disorders), neurodegenerative (Parkinson’s and Alzheimer’s diseases) and cancers (glioma), in which both connexin dysfunction and pollutant involvement have been described. Based on these different aspects, the possible involvement of pollutant-inhibited gap junctions in brain disorders is discussed for prenatal and postnatal exposures.

Connexins in neuromyelitis optica: a link between astrocytopathy and demyelination.

Neuromyelitis optica, a rare neuroinflammatory demyelinating disease of the CNS, is characterized by the presence of specific pathogenic autoantibodies directed against the astrocytic water channel aquaporin 4 (AQP4) and is now considered as an astrocytopathy associated either with complement-dependent astrocyte death or with astrocyte dysfunction. However, the link between astrocyte dysfunction and demyelination remains unclear. We propose glial intercellular communication, supported by connexin hemichannels and gap junctions, to be involved in demyelination process in neuromyelitis optica. Using mature myelinated cultures, we demonstrate that a treatment of 1 h to 48 h with immunoglobulins purified from patients with neuromyelitis optica (NMO-IgG) is responsible for a complement independent demyelination, compared to healthy donors' immunoglobulins (P < 0.001). In parallel, patients' immunoglobulins induce an alteration of connexin expression characterized by a rapid loss of astrocytic connexins at the membrane followed by an increased size of gap junction plaques (+60%; P < 0.01). This was co-observed with connexin dysfunction with gap junction disruption (-57%; P < 0.001) and increased hemichannel opening (+17%; P < 0.001), associated with glutamate release. Blocking connexin 43 hemichannels with a specific peptide was able to prevent demyelination in co-treatment with patients compared to healthy donors' immunoglobulins. By contrast, the blockade of connexin 43 gap junctions with another peptide was detrimental for myelin (myelin density -48%; P < 0.001). Overall, our results suggest that dysregulation of connexins would play a pathogenetic role in neuromyelitis optica. The further identification of mechanisms leading to connexin dysfunction and soluble factors implicated, would provide interesting therapeutic strategies for demyelinating disorders.

Dysfunction of astrocytic connexins 30 and 43 in the medial prefrontal cortex and hippocampus mediates depressive-like behaviours

Astrocytic connexin dysfunction is closely associated with synaptic impairment and contributes to the pathological development of depressive-like behaviours. However, little is known about the expression of connexins in astrocytes from different brain regions, or how tissue specific connexin expression affects local neuronal activity. Here, we established a mouse model of chronic social defeated stress (CSDS), from which we isolated astrocytes from the medial prefrontal cortex (mPFC), hippocampus, amygdala, and ventral tegmental area (VTA). Expression profiling was then performed for connexins Cx26, Cx30, and Cx43. Expression of Cx30 and Cx43 was significantly decreased in mPFC and hippocampus of CSDS mice and was strongly associated with decreases in neuronal activity. Furthermore, overexpression of Cx30 and Cx43 in the mPFC and hippocampus increased neuronal activity and inhibited depressive-like behaviours; while suppression of Cx30 and Cx43 in normal mice was sufficient to reduce neuronal activity and induced depressive-like behaviours. Taken togetner, aberrant expression of astrocytic Cx30 and Cx43 in the mPFC and hippocampus significantly affects brian region-specific neuronal activity and drives depressive-like behaviours. These observations provide novel insights into the role of astrocyte gene expression in stress-induced depressive-like behaviours.

GW26-e2378 Noninvasive Vagus Nerve Stimulation Breaks the Vicious Cycles between Connexin Dysfunction and Early Atrial Electrical Remodeling

GW26-e2378 Noninvasive Vagus Nerve Stimulation Breaks the Vicious Cycles between Connexin Dysfunction and Early Atrial Electrical Remodeling

Connexins and gap junctions in the inner ear--it's not just about K⁺ recycling.

Normal development, function and repair of the sensory epithelia in the inner ear are all dependent on gap junctional intercellular communication. Mutations in the connexin genes GJB2 and GJB6 (encoding CX26 and CX30) result in syndromic and non-syndromic deafness via various mechanisms. Clinical vestibular defects, however, are harder to connect with connexin dysfunction. Cx26 and Cx30 proteins are widely expressed in the epithelial and connective tissues of the cochlea, where they may form homomeric or heteromeric gap junction channels in a cell-specific and spatiotemporally complex fashion. Despite the study of mutant channels and animal models for both recessive and dominant autosomal deafness, it is still unclear why gap junctions are essential for auditory function, and why Cx26 and Cx30 do not compensate for each other in vivo. Cx26 appears to be essential for normal development of the auditory sensory epithelium, but may be dispensable during normal hearing. Cx30 appears to be essential for normal repair following sensory cell loss. The specific modes of intercellular signalling mediated by inner ear gap junction channels remain undetermined, but they are hypothesised to play essential roles in the maintenance of ionic and metabolic homeostasis in the inner ear. Recent studies have highlighted involvement of gap junctions in the transfer of essential second messengers between the non-sensory cells, and have proposed roles for hemichannels in normal hearing. Here, we summarise the current knowledge about the molecular and functional properties of inner ear gap junctions, and about tissue pathologies associated with connexin mutations.

Connexins and Atrial Fibrillation

Atrial fibrillation (AF) is an extremely common arrhythmia with important consequences and presently suboptimal therapeutic options.1 A great deal of research has been performed to understand the detailed mechanisms of AF, with the hope that a better appreciation of the fundamental determinants of arrhythmogenesis will lead to the development of novel, more successful treatment possibilities.2 Article see p 216 One aspect of AF pathophysiology that has elicited great interest has been changes in gap junction/connexin physiology. An important role for altered gap junction function and the potential importance of gap junctions as a therapeutic target for AF were first emphasized by Spach and Starmer over 15 years ago.3 Since that time, our understanding of gap junction physiology, biophysics, and molecular biology has increased enormously,4,5 as have studies on their role in AF pathogenesis and management.2 Gap junctions contain transmembrane ion-channel proteins called connexins (Figure,panel A). Connexons (containing 6 connexin molecules each) in the gap junctions of adjacent cardiomyocytes line up and attach, transferring ions or molecules <1 kDa freely between cells, coupling them electrically (Figure, panel B). Cardiac tissues express a variety of connexins, including connexin (Cx)40, Cx43, Cx45, Cx30.2/31.9, and Cx37,5 with all but Cx37 present in cardiomyocytes and the most important connexins in atrial tissue being Cx40 and Cx43. There is evidence that posttranslational modification, particularly connexin phosphorylation, is important in governing connexin localization and function.6 Figure. A , Connexons contain 6 connexins each, arranged in a circle. Connexins …

Effects of Chronic Gap Junction Conduction–Enhancing Antiarrhythmic Peptide GAP-134 Administration on Experimental Atrial Fibrillation in Dogs

Abnormal intercellular communication caused by connexin dysfunction may contribute to atrial fibrillation (AF). The present study assessed the effect of the gap junction conduction-enhancing antiarrhythmic peptide GAP-134 on AF inducibility and maintenance in a dog model of atrial cardiomyopathy. Twenty-four dogs subject to simultaneous atrioventricular pacing (220 bpm for 14 days) were randomly assigned to placebo treatment (PACED-CTRL; 12 dogs) or oral GAP-134 (2.9 mg/kg BID; PACED-GAP-134; 12 dogs) starting on day 0. UNPACED-CTRL (4 dogs) and UNPACED-GAP-134 (4 dogs) served as additional control groups. Change in left atrial (LA) systolic area from baseline to 14 days was calculated using transoesophageal echocardiography. At 14 days, animals underwent an open-chest electrophysiological study. PACED-CTRL dogs (versus UNPACED-CTRL) had a shorter estimated LA wavelength (8.0+/-1.4 versus 24.4+/-2.5 cm, P<0.05) and a greater AF vulnerability (mean AF duration, 1588+/-329 versus 25+/-34 seconds, P<0.05). Oral GAP-134 had no effect on AF vulnerability in UNPACED dogs. Compared with PACED-CTRL dogs, PACED-GAP-134 dogs had a longer estimated LA wavelength (10.2+/-2.8 versus 8.0+/-1.4 cm, respectively, P<0.05). Oral GAP-134 did not significantly reduce AF inducibility or maintenance in the entire group of 24 PACED dogs; in a subgroup of dogs (n=11) with less than 100% increase in LA systolic area, oral GAP-134 reduced AF induction from 100% to 40% and mean AF duration from 1737+/-120 to 615+/-280 seconds (P<0.05). Oral GAP-134 reduces pacing-induced decrease in LA wavelength and appears to attenuate AF vulnerability in dogs with less atrial mechanical remodeling. Gap junction modulation may affect AF in some circumstances.

Gap junctions and connexins in the inner ear: their roles in homeostasis and deafness

Mutations in GJB2 and GJB6, the genes encoding the gap-junction proteins connexin 26 and connexin 30, are the most common cause of autosomal recessive nonsyndromic deafness in many populations across the world. In this review, we discuss current ideas about the roles of gap junctions in the inner ear and the implications of connexin mutations on auditory function. In recent years, a complex picture of the roles of gap junctions in cochlear physiology emerged. Rather than being mere conduits for the circulation of potassium ions in the inner ear, gap junctions have been implicated in intercellular signaling among nonsensory cells and may be involved in the maintenance of the endothelial barrier in the stria vascularis. Studies of mutant channels and mouse models for connexin-related deafness have provided valuable insights into some of the mechanisms by which connexin dysfunction causes cochlear degeneration. They have also identified potential therapeutic interventions for specific connexin mutations, such as the restoration of normal connexin 26 protein levels in GJB6-associated deafness. Despite recent advances, a better understanding of the complexity of gap-junctional communication in the inner ear and the structure-function relationships of connexin proteins is required for the development of mechanism-based treatments of connexin-associated hearing loss.

Abstract 1087: Effects of Oral Gap Junction Conduction-Enhancing Antiarrhythmic Peptide GAP-134 on Experimental Atrial Fibrillation in Dogs

Background: Abnormal intercellular communication caused by connexin dysfunction may promote atrial fibrillation (AF). Objective: To assess the effect of the gap junction conduction-enhancing antiarrhythmic peptide GAP-134 on AF inducibility and maintenance in a new dog model of atrial cardiomyopathy. Methods and Results: Twenty four dogs underwent simultaneous atrioventricular pacing (2 weeks at 220 bpm, atrioventricular delay 0 ms), and were randomly assigned to placebo treatment (PACED-PLACEBO; 12 dogs) or oral GAP-134 (PACED-GAP 134; 12 dogs) (starting at day 0). Percent change in left atrial systolic area (Δ% LASA) from baseline to 2 weeks was calculated using trans-esophageal echocardiography. At 2 weeks, animals underwent an open chest electrophysiological study; conduction velocity (CV) when pacing at 150ms cycle length (CL), effective refractory periods (ERP) and AF vulnerability were measured. The mean plasma concentration of GAP-134 was 557 ± 239 nmol/L. GAP-134 increased CV (395.1 ± 63.2 vs 307.8 ± 54.6 mm/s, p&lt;0.01), and shortened ERP at 200ms CL (104.0 ± 8.6 vs 112.8 ± 11.5 ms, P&lt;0.05). GAP-134 significantly reduced AF inducibility [% burst attempts inducing AF] and maintenance [mean AF duration, number of episodes &gt;10min] in dogs with less than 100% ΔLASA (n=5). In dogs with more structural remodeling (ΔLASA ≥100%, n=7), CV increased but AF inducibility was unaffected. Conclusions: Oral GAP-134 prevents CV slowing in a dog model of atrial cardiomyopathy, but attenuates AF inducibility and maintenance only in dogs with less mechanical remodeling.

Connexins as Precocious Markers and Molecular Targets for Chemical and Pharmacological Agents in Carcinogenesis

Gap junctions, intercellular channels structured by the connexin protein family, have been implicated in the control of cell homeostasis, proliferation, differentiation and death. A loss of the gap junction intercellular communication and/or connexin dysfunction are typical features of cancer per se and have been associated with the effect of many carcinogens. Indeed, many early human neoplasia of various organs and human tumor cell lines exhibit deficient connexin-mediated communication expression mainly related, in a large number of observations, with an aberrant cytoplasmic localization of this membranous protein. Restoration of normal phenotype in transformed cells by restoration of exogenous connexin gave rise to the concept that connexins may act as tumor suppressors. However, the mechanisms by which connexins mediate such a tumor suppressor effect are multiple. They may result from: formation of functional channels; hemichannels or are directly associated with connexin expression. In addition, the literature shows that they may be dependent upon the cell type and the connexin type. In the present review, we analyze all these aspects of connexin/gap junction involvement in the carcinogenesis process, in human cancers and discuss the possibility of using connexins as potential anti-oncogenic targets for cancer chemoprevention and/or chemotherapy.

Model-Dependent Effects of the Gap Junction Conduction–Enhancing Antiarrhythmic Peptide Rotigaptide (ZP123) on Experimental Atrial Fibrillation in Dogs

Abnormal intercellular communication caused by connexin dysfunction may be involved in atrial fibrillation (AF). The present study assessed the effect of the gap junctional conduction-enhancing peptide rotigaptide on AF maintenance in substrates that result from congestive heart failure induced by 2-week ventricular tachypacing (240 bpm), atrial tachypacing (ATP; 400 bpm for 3 to 6 weeks), and isolated atrial myocardial ischemia. Electrophysiological study and epicardial mapping were performed before and after rotigaptide administration in dogs with ATP and congestive heart failure, as well as in similarly instrumented sham dogs that were not tachypaced. For atrial myocardial ischemia, dogs administered rotigaptide before myocardial ischemia were compared with no-drug myocardial ischemia controls. ATP significantly shortened the atrial effective refractory period (P=0.003) and increased AF duration (P=0.008), with AF lasting >3 hours in all 6-week ATP animals. Rotigaptide increased conduction velocity in ATP dogs slightly but significantly (P=0.04) and did not affect the effective refractory period, AF duration, or atrial vulnerability. In dogs with congestive heart failure, rotigaptide also slightly increased conduction velocity (P=0.046) but failed to prevent AF promotion. Rotigaptide had no statistically significant effects in sham dogs. Myocardial ischemia alone increased AF duration and impaired conduction (based on conduction velocity across the ischemic border and indices of conduction heterogeneity). Rotigaptide prevented myocardial ischemia-induced conduction slowing and AF duration increases. Rotigaptide improves conduction in various AF models but suppresses AF only for the acute ischemia substrate. These results define the atrial antiarrhythmic profile of a mechanistically novel antiarrhythmic drug and suggest that gap junction dysfunction may be more important in ischemic AF than in ATP remodeling or congestive heart failure substrates.

Disruption of gap junctions in toxicity and carcinogenicity.

Although the specific role of connexin-mediated gap junctional intercellular communication in the control of cell homeostasis, proliferation, and death are still not clear, several lines of evidence support these roles. The disturbance of this communication, through multiple mechanisms, may in the short term be a protective mechanism to limit the spread of toxicity in a tissue following chemical or radiation damage. However, sustained downregulation confers a loss of tumor-suppressive action. Consequently, connexin dysfunction has been associated with both the action of many carcinogens and being a feature of cancer per se. Connexins offer not only a target for cancer chemoprevention but also for exploitation in chemotherapy through the "bystander" effect.