Search

Adenoviruses are responsible for a spectrum of pathogenesis including viral myocarditis. The gap junction protein connexin43 (Cx43, gene name GJA1) facilitates rapid propagation of action potentials necessary for each heartbeat. Gap junctions also propagate innate and adaptive antiviral immune responses, but how viruses may target these structures is not understood. Given this immunological role of Cx43, we hypothesized that gap junctions would be targeted during adenovirus type 5 (Ad5) infection. We find reduced Cx43 protein levels due to decreased GJA1 mRNA transcripts dependent upon β-catenin transcriptional activity during Ad5 infection, with early viral protein E4orf1 sufficient to induce β-catenin phosphorylation. Loss of gap junction function occurs prior to reduced Cx43 protein levels with Ad5 infection rapidly inducing Cx43 phosphorylation events consistent with altered gap junction conductance. Direct Cx43 interaction with ZO-1 plays a critical role in gap junction regulation. We find loss of Cx43/ZO-1 complexing during Ad5 infection by co-immunoprecipitation and complementary studies in human induced pluripotent stem cell derived-cardiomyocytes reveal Cx43 gap junction remodeling by reduced ZO-1 complexing. These findings reveal specific targeting of gap junction function by Ad5 leading to loss of intercellular communication which would contribute to dangerous pathological states including arrhythmias in infected hearts., (© 2020 Federation of American Societies for Experimental Biology.)

Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that β1(SCN1B) -mediated adhesion scaffolds trans -activating Na V 1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential β1 localization at the perinexus, where it co-locates with Na V 1.5. Smart patch clamp (SPC) indicated greater sodium current density (I Na ) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, βadp1, potently and selectively inhibited β1-mediated adhesion, in electric cell-substrate impedance sensing studies. βadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal I Na , but not whole cell I Na , in myocyte monolayers. In optical mapping studies, βadp1 precipitated arrhythmogenic conduction slowing. In summary, β1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies., Competing Interests: RV, GH, AA, DH, XW, DK, JS, CC, JJ, LI, ID, JS, JG, SP, RG No competing interests declared, (© 2018, Veeraraghavan et al.)

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., (Copyright © 2016 the American Physiological Society.)

Background: The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin 43 (Cx43, encoded by GJA1) and the truncated, internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. Methods: Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1 + and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 h in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. Results: Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. Conclusions: Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA. [ABSTRACT FROM AUTHOR]

Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with adherens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias. [ABSTRACT FROM AUTHOR]

Introduction: Connexin43 (Cx43, Gja1) gap junctions enable electrical coupling of cardiomyocytes necessary for heart function. Pathological disruption of Cx43 expression or localization results in the arrhythmias of sudden cardiac death. Investigation of Cx43 gap junction biology has focused on transcription, vesicular trafficking, and post translational modifications. The importance of translational regulation however is highlighted by the recent discovery that Cx43 mRNA undergoes internal translation initiation to yield truncated protein isoforms, including the predominant GJA1-20k isoform capable of regulating gap junction formation. We have found that TGF-β signaling, a hallmark of cardiac stressors including hypertrophy, suppresses GJA1-20k expression to limit gap junction formation. The mechanisms regulating Cx43 mRNA internal translation in response to cellular stress are unknown but are critical in harnessing this biology for therapeutic restoration of electrical coupling in diseased hearts. We hypothesize that altered Gja1 -5'UTR usage promotes translation of full length Cx43 over internal translation products resulting in reduced gap junction formation during stress. Methods: Rapid amplification of cDNA ends was used to identify Gja1- 5'UTR isoforms following TGF-β exposure or hypoxia, and Western blotting was used to monitor Gja1 translation. Identified Gja1 -5'UTR variants were transfected into Cx43 knockout cells to assess translational potential. Luciferase assays assessed potential IRES activity within Gja1 mRNA in relation to specific 5'UTR isoforms. Results: TGF-β or hypoxia significantly increase translation of full length Cx43 over Gja1-20k correlating with an increase in truncated Gja1 -5'UTRs. Sequenced UTR isoforms map to genome wide transcription start sites and transfection of Gja1 transcripts with truncated 5'UTRs is sufficient to reduce Gja1-20k translation, phenocopying stress models. Conclusions: Alteration of Gja1 -5'UTR composition represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation to fine tune intercellular communication during stress. [ABSTRACT FROM AUTHOR]