Cx43 Gap Junction Research Articles

Cellular circadian oscillators in the suprachiasmatic nucleus remain coupled in the absence of connexin-36

In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (∼24h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3–4weeks old) and adult (9–30weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.

Intracellular trafficking pathways of Cx43 gap junction channels

Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Cx43 protein has a short half-life, and rapid synthesis and timely delivery of those proteins to particular subdomains are crucial for the cellular organization of gap junctions and maintenance of intracellular coupling. Impairment in gap junction trafficking contributes to dangerous complications in diseased hearts such as the arrhythmias of sudden cardiac death. Of recent interest are the protein-protein interactions with the Cx43 carboxy-terminus. These interactions have significant impact on the full length Cx43 lifecycle and also contribute to trafficking of Cx43 as well as possibly other functions. We are learning that many of the known non-canonical roles of Cx43 can be attributed to the recently identified six endogenous Cx43 truncated isoforms which are produced by internal translation. In general, alternative translation is a new leading edge for proteome expansion and therapeutic drug development. This review highlights recent mechanisms identified in the trafficking of gap junction channels, involvement of other proteins contributing to the delivery of channels to the cell-cell border, and understanding of possible roles of the newly discovered alternatively translated isoforms in Cx43 biology. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Inhibition of Cx43 mediates protective effects on hypoxic/reoxygenated human neuroblastoma cells

Olfactory ensheathing cells (OECs), a special population of glial cells, are able to synthesise several trophic factors exerting a neuroprotective action and promoting growth and functional recovery in both in vitro and in vivo models. In the present work, we investigated the neuroprotective effects of OEC‐conditioned medium (OEC‐CM) on two different human neuron‐like cell lines, SH‐SY5Y and SK‐N‐SH (neuroblastoma cell lines), under normoxic and hypoxic conditions. In addition, we also focused our attention on the role of connexins (Cxs) in the neuroprotective processes. Our results confirmed OEC‐CM mediated neuroprotection as shown by cell adherence, proliferation and cellular viability analyses. Reduced connexin 43 (Cx43) levels in OEC‐CM compared to unconditioned cells in hypoxic conditions prompted us to investigate the role of Cx43‐Gap junctions (GJs) and Cx43‐hemichannels (HCs) in hypoxic/reoxygenation injury using carbenoxolone (non‐selective GJ inhibitor), ioxynil octanoato (selective Cx43‐GJ inhibitor) and Gap19 (selective Cx43‐HC inhibitor). We found that Cx43‐GJ and Cx43‐HC inhibitors are able to protect SH‐SY5Y and allow to these cultures to overcome the injury. Our findings support the hypothesis that both OEC‐CM and the inhibition of Cx43‐GJs and Cx43‐HCs offer a neuroprotective effect by reducing Cx43‐mediated cell‐to‐cell and cell‐to‐extracellular environment communications.

Connexin43 and Runx2 Interact to Affect Cortical Bone Geometry, Skeletal Development, and Osteoblast and Osteoclast Function.

The coupling of osteoblasts and osteocytes by connexin43 (Cx43) gap junctions permits the sharing of second messengers that coordinate bone cell function and cortical bone acquisition. However, details of how Cx43 converts shared second messengers into signals that converge onto essential osteogenic processes are incomplete. Here, we use in vitro and in vivo methods to show that Cx43 and Runx2 functionally interact to regulate osteoblast gene expression and proliferation, ultimately affecting cortical bone properties. Using compound hemizygous mice for the Gja1 (Cx43) and Runx2 genes, we observed a skeletal phenotype not visible in wild-type or singly hemizygous animals. Cortical bone analysis by micro-computed tomography (μCT) revealed that 8-week-old male, compound Gja1+/- Runx2+/- mice have a marked increase in cross-sectional area, endosteal and periosteal bone perimeter, and an increase in porosity compared to controls. These compound Gja1+/- Runx2+/- mice closely approximate the cortical bone phenotypes seen in osteoblast-specific Gja1-conditional knockout models. Furthermore, μCT analysis of skulls revealed an altered interparietal bone geometry in compound hemizygotes. Consistent with this finding, Alizarin red/Alcian blue staining of 2-day-old Gja1+/- Runx2+/- neonates showed a hypomorphic interparietal bone, an exacerbation of the open fontanelles, and a further reduction in the hypoplastic clavicles compared to Runx2+/- neonates. Expression of osteoblast genes, including osteocalcin, osterix, periostin, and Hsp47, was markedly reduced in tibial RNA extracts from compound hemizygous mice, and osteoblasts from compound hemizygous mice exhibited increased proliferative capacity. Further, the reduced osteocalcin expression and hyperproliferative nature of osteoblasts from Cx43 deficient mice was rescued by Runx2 expression. In summary, these findings provide evidence that Cx43 and Runx2 functionally intersect in vivo to regulate cortical bone properties and affect osteoblast differentiation and proliferation, and likely contributes to aspects of the skeletal phenotype of Cx43 conditional knockout mice. © 2017 American Society for Bone and Mineral Research.

Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis.

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

The Sodium Channel Auxiliary Subunit β1 is Structurally Critical for Cardiac Conduction: Evidence from the Single Molecule Scale to the Whole Organ

Electrical impulse propagation in the heart's ventricles is thought to occur by means of direct flow of electric current from cell to cell via gap junctions (GJs) composed of connexin43 (Cx43). We recently demonstrated that cardiac sodium channels (Nav1.5) localized within the perinexus, an intercalated disk (ID) nanodomain adjacent to Cx43 GJ, may enable ephaptic coupling between cardiac myocytes. We hypothesized that the cell adhesion function of β1 may contribute to close intermembrane apposition within these nanodomains and thereby facilitate ephaptic coupling. Super-resolution STochastic Optical Reconstruction Microscopy-based Relative Localization Analysis (STORM-RLA) identified two populations of NaV1.5 within the ID in guinea pig ventricles: One, localized to the perinexus, within 200nm of Cx43 GJs, accounted for 47% of ID-localized NaV1.5 and the other, which co-distributed with N-Cadherin to plicate ID regions, accounted for 29% of ID-localized NaV1.5. These findings were confirmed by immuno-electron microscopy. Confocal microscopy revealed β1 enrichment at the ID, and suggested preferential localization to GJ-enriched interplicate ID regions. STORM-RLA confirmed the preferential localization of β1 to interplicate ID regions: Perinexal nanodomains accounted for 48% of ID-localized β1, whereas N-Cadherin-rich plicate regions accounted for only 8%. In order to test the role of β1 adhesion in the heart, we developed a novel peptide inhibitor of β1 adhesion - βadp1. In electric cell-substrate impedance spectroscopy studies, it inhibited the barrier function in β1-overexpressing 1610 cells but not in native 1610 cells. In guinea pig ventricles, βadp1 (100 µM) compromised the diffusion-resistance of the ID as assessed by perfusion of fixable fluorescent dyes: A 0.5kD molecule preferentially accumulated within IDs in control hearts but not in βadp1-treated hearts. Perinexal intermembrane spacing measured by transmission electron microscopy was increased in βadp1-treated hearts (48 ± 4 µm) compared to untreated hearts (17± 1µm) and those treated with a scrambled control peptide, βadp1-scr (22 ± 1 µm). Notably, neither peptide affected membrane spacing at other ID locations. In optical mapping studies, βadp1 but not βadp1-scr preferentially decreased transverse conduction velocity, increased anisotropy and precipitated spontaneous tachyarrhythmias in a dose-dependent manner. In conclusion, these results suggest a critical role of β1-mediated adhesion in generating close apposition between NaV1.5-rich perinexal membranes and thereby, facilitating ephaptic conduction in the heart. Further, these data highlight β1-mediated adhesion as a target for novel, mechanistically-driven antiarrhythmic therapy.

Gap Junction Remodeling in a Novel Engineered Heart Tissue System Cultured under Point Stimulation

Recent advances have shown the importance of electrical stimulation in enhancing maturation and differentiation of engineered heart tissues (EHTs). Conventional electrical maturation protocols pace their EHTs with field stimulation - the tissues are paced simultaneously in a uniform electrical field. This method has been shown to improve cellular alignment, increase contractility, and increase gap junction (Cx43) production. While Cx43 production is increased, the anisotropic ratio is not fully preserved in these culture systems [Trantidou et al, PMID 4459200]. Native cardiac tissue demonstrates an anisotropic propagation of action potHential; in adult rat ventricles the conduction velocity (CV) is 3x faster in the longitudinal direction than the transverse direction [Zimmermann et al, PMID 16582915]. We hypothesize that the loss of anisotropy is due to field stimulation not accurately recapitulating the conditions caused by a depolarizing wave traveling through the tissue. In order to better reproduce native myocardium, we designed a bioreactor which applies a point stimulation at one end of our EHT and allows the wave to propagate down the tissue. We hypothesize that this propagating wave will provide the necessary signals to enhance Cx43 production as well as improve localization of gap junction proteins. Maturity was assessed by the measurement of twitch kinetics, peak force, and conduction velocity. We anticipate that establishing a more realistic expression and subcellular localization of gap junction proteins will improve the utility of future studies involving cell lines from patients with arrhythmogenic cardiomyopathies.

Aberrant Cx43 Expression and Mislocalization in Metastatic Human Melanomas.

At present, it is unclear if melanocytes contain Cx43 gap junctions and whether Cx43 expression is regulated in melanoma onset and progression. To this end, we cultured pure populations of mouse melanocytes and found that they had no detectable Cx43 and exhibited an inability for dye transfer indicating they were devoid of functional gap junctions. Given the evidence that melanomas acquire the expression of other connexin isoforms during tumor progression, we assessed if Cx43 was also expressed and assembled into gap junctions at any stage of human melanoma onset and progression to distant metastases. Nearly all primary melanomas within the epidermis lacked Cx43. In contrast, nodal metastases expressed low levels of Cx43 which was markedly higher in distant metastases that had invaded vital organs. Importantly, in all stages of melanoma progression, Cx43 could be detected in intracellular compartments but was rarely assembled into gap junctions indicative of functional gap junction channels. Overall, these studies suggest that melanocytes do not form Cx43 homocellular gap junctions and even though Cx43 levels increase during melanoma progression, this connexin rarely assembles into gap junction structures.

Myosin VI facilitates connexin 43 gap junction accretion.

ABSTRACTIn this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI–Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI.

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation.

In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1+/- controls, male Gja1-/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and β-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition.

Connexin 43 and Mitochondria in Cardiovascular Health and Disease.

Connexin 43 (Cx43) is the major connexin protein in ventricular cardiomyocytes. Six Cx43 proteins assemble into so-called hemichannels at the sarcolemma and opposing hemichannels form gap junctions, which allow the passage of small molecules and electrical current flow between adjacent cells. Apart from its localization at the plasma membrane, Cx43 is also present in cardiomyocyte mitochondria, where it is important for mitochondrial function in terms of oxygen consumption and potassium fluxes. The expression of gap junctional and mitochondrial Cx43 is altered under several pathophysiological conditions among them are hypertension, hypertrophy, hypercholesterolemia, ischemia/reperfusion injury, post-infarction remodeling, and heart failure. The present review will focus on the role of Cx43 in cardiovascular diseases and will highlight the importance of mitochondrial Cx43 in cardioprotection.

Sufentanil attenuates oxaliplatin cytotoxicity via inhibiting connexin 43‑composed gap junction function.

Comprehensive strategies for the treatment of colorectal cancer (CRC) have become increasingly important. One of the most important factors is pain relief. Therefore, patients with CRC are concurrently treated with analgesics and chemotherapeutic agents; however, the effects of analgesics on the therapeutic activity of chemotherapeutic agents remain largely unknown. The present study investigated the effects of three widely used analgesics in clinics: Fentanyl, remifentanil and sufentanil, on the cytotoxicity of oxaliplatin, a commonly used chemotherapeutic agent for CRC. Furthermore, the underlying mechanisms of those effects in association with connexin 43 (Cx43)‑composed gap junction (GJ) function were analyzed. The Lovo, Colo320, HCT116 and HT29 human CRC cell lines, with or without Cx43 expression, were used to examine the effects of the three analgesics on the cytotoxicity of oxaliplatin. The results demonstrated that in the cell lines expressing Cx43 (Lovo and Colo320), the cytotoxicity of oxaliplatin was attenuated and Cx43 GJ function was inhibited. Sufentanil, not fentanyl or remifentanil, inhibited Cx43 GJ function effectively, and reduced the cytotoxicity of oxaliplatin. In contrast, these effects were not observed in the other two colon cancer cell lines not expressing Cx43 (HCT116 and HT29). These results suggested that alternation of Cx43 GJ function may regulate the cytotoxicity of oxaliplatin in regard to CRC. Furthermore, sufentanil, not fentanyl or remifentanil, suppressed the cytotoxicity of oxaliplatin through inhibition of Cx43 GJ function. These results may be beneficial for the treatment of CRC and reduction of treatment resistance.

A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43–Ezrin Interaction Disruptors Using IncuCyte ZOOM

Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin–protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.

GJA1 gene variations in sudden unexplained nocturnal death syndrome in the Chinese Han population

Sudden unexplained nocturnal death syndrome (SUNDS) is a conundrum to both forensic pathologists and physicians, more than 80% of which the molecular pathogenesis remains unclear. Reported studies on both clinical and genetic phenotypes suggest SUNDS is related to congenital and acquired arrhythmias. Recent researches have linked the mutations of gene gap junction alpha 1 (GJA1) with arrhythmogenic cardiac disorders. In the present study, we investigate the potential correlation between GJA1 gene variations and the occurrence of SUNDS. Genomic DNA was extracted from the blood samples of both 124 sporadic SUNDS patients and 125 healthy controls to screen GJA1 gene for candidate variants using polymerase chain reaction (PCR) and direct DNA sequencing. One novel homozygous variant c.169C>T and one heterozygous SNP c.624C>T (rs530633057) were determined in 124 SUNDS cases (one case for each detected variant) and none of the 125 healthy controls. Base C>T transition at nucleotide position 169 led to termination of protein production after glutamine (Q) at codon 57 which is very likely to result in decreased expression of Cx43 gap junction channels and cause arrhythmic sudden death. This is the first report of GJA1 gene variations in SUNDS in the Chinese Han population, which suggests a novel susceptibility gene for Chinese SUNDS.

Mitochondria Transfer from Hematopoietic Stem and Progenitor Cells to Pdgfrα+/Sca-1-/CD48dim BM Stromal Cells Via CX43 Gap Junctions and AMPK Signaling Inversely Regulate ROS Generation in Both Cell Populations

Modulation of reactive oxygen species (ROS) levels in hematopoietic stem cells (HSC) is crucial to control HSC quiescence and blood formation. High ROS levels are required for leukocyte formation while low ROS levels are essential to maintain HSC quiescence. However, regulation of ROS content in HSC is poorly understood. Adhesion interactions between HSC and their bone marrow (BM) stromal cells (BMSC) via CXCL12/CXCR4 maintain HSC in a quiescence non-motile state, protecting them from 5-FU chemotherapy insult (Sugiyama, Immunity, 2006). Surface CXCL12 expression by BMSC is dependent on connexin-43 (Cx43) gap junctions mediated cell contact (Schajnovitz, Nat. Immunol., 2011) and BM hematopoietic stem and progenitor cells (HSPC) survive and eliminate excess ROS levels post 5-FU chemotherapy treatment, by transferring ROS to BMSC in a Cx43 dependent manner (Taniguchi, PNAS 2012).Here, we report that ROS content of BM HSPC inversely correlates with ROS levels in adjacent BMSC. Administration of the pro inflammatory cytokine G-CSF results in decreased HSPC Cx43 expression, elevated ROS levels and increased glucose uptake. Conversely, in the BM stromal microenvironment, G-CSF administration generated lower ROS level and reduced glucose uptake. Up-regulation of BM Sphingosine 1-Phosphate (S1P), a downstream target of G-CSF required for ROS production in HSPC, reduced stromal ROS content and proliferation. Accordingly, mice with reduced BM S1P levels (S1Plow) have lower BM content of HSPC, accompanied by reduced ROS, glucose uptake and lactate production in these cells. More importantly, BM from S1Plow mice has a 3 fold increased frequency of primitive ROSlow/ EPCR+ long-term repopulating cells, as evident by immunophenotypic analysis and long-term competitive repopulation assays. Concomitantly, S1Plow mice have increased content of BMSC with higher ROS levels and glucose uptake, leading to higher BM content of colony-forming unit fibroblasts. Our results reveal a dynamic and inverse metabolic relationship between BM HSC and the stroma microenvironment. We hypothesized that the opposite metabolic state of HSPC and BMSC is due to mitochondrial transfer between the two populations. Therefore, we created chimeric mice by transplanting mitochondria labeled GFP (mito-GFP) HSPC to wild type (WT) mice and detected 88% of the host BMSC to contain donor-derived mitochondria, indicating the existence of mitochondria transfer from hematopoietic cells to BMSC in vivo. This transfer is bidirectional, albeit at a lesser degree, as determined in reverse chimeric mice where up to 26% of the donor-derived HSPCs acquired recipient mitochondria. Mitochondrial transfer can be recapitulated also in vitro in an overnight co-culture system of mito-GFP HSPC and primary BMSC, resulting in mitochondrial transfer and increased ROS content in a subpopulation of osteogenic BM PDGFRα+/ Sca-1-/CD48dim stromal cells. Mitochondrial transfer is cell contact dependent and mediated by Cx43 gap junctions. In vitro co-culture of mito-GFPHSPC from Cx43 deficient (KO) mice with WT or Cx43 KO BMSC reduced 50% mitochondrial transfer to PDGFRα+/Sca-1-/CD48dim stromal cells. Contrarily, the mitochondrial transfer from WT HSPC to Cx43 KO stromal cells was not affected, revealing that Cx43 expression on HSPC, but not on BM stromal cells, is specifically required for mitochondrial transfer. Interestingly, in vitro inhibition of AMP-activated protein kinase (AMPK), a crucial metabolic regulator, dramatically increased mitochondrial transfer from HSPC to BMSC. Administration of the AMPK inhibitor BML in vivo increased ROS content of PDGFRα+/Sca-1- BMSC while decreasing it in HSPC, further suggesting that AMPK inhibition regulates mitochondrial transfer and ROS production. Our results imply that mitochondria are scavenged by the BM osteogenic microenvironment to prevent excessive ROS levels in the HSC pool and in parallel to activate bone formation. Altogether, we have discovered a dynamic, inverse metabolic state between BM HSPC and their supporting stromal microenvironment during quiescence, proliferationand differentiation of these two populations. Thus, blood cell production and bone generation take place at the expense of the other. This metabolic seesaw is mediated by mitochondrial transfer from HSPC to osteogenic BM stroma in a HSPC Cx43 gap-junction dependent manner and regulated through AMPK signaling. DisclosuresNo relevant conflicts of interest to declare.

Th1 cells downregulate connexin 43 gap junctions in astrocytes via microglial activation.

We previously reported early and extensive loss of astrocytic connexin 43 (Cx43) in acute demyelinating lesions of multiple sclerosis (MS) patients. Because it is widely accepted that autoimmune T cells initiate MS lesions, we hypothesized that infiltrating T cells affect Cx43 expression in astrocytes, which contributes to MS lesion formation. Primary mixed glial cell cultures were prepared from newborn mouse brains, and microglia were isolated by anti-CD11b antibody-conjugated magnetic beads. Next, we prepared astrocyte-rich cultures and astrocyte/microglia-mixed cultures. Treatment of primary mixed glial cell cultures with interferon (IFN) γ, interleukin (IL)-4, or IL-17 showed that only IFNγ or IL-17 at high concentrations reduced Cx43 protein levels. Upon treatment of astrocyte-rich cultures and astrocyte/microglia-mixed cultures with IFNγ, Cx43 mRNA/protein levels and the function of gap junctions were reduced only in astrocyte/microglia-mixed cultures. IFNγ-treated microglia-conditioned media and IL-1β, which was markedly increased in IFNγ-treated microglia-conditioned media, reduced Cx43 protein levels in astrocyte-rich cultures. Finally, we confirmed that Th1 cell-conditioned medium decreased Cx43 protein levels in mixed glial cell cultures. These findings suggest that Th1 cell-derived IFNγ activates microglia to release IL-1β that reduces Cx43 gap junctions in astrocytes. Thus, Th1-dominant inflammatory states disrupt astrocytic intercellular communication and may exacerbate MS.

Abstract 13129: Acute Inhibition of Sodium Channel Beta Subunit (β1) -mediated Adhesion is Highly Proarrhythmic

Introduction: The sodium channel auxiliary subunit and cell adhesion molecule β1 (SCN1b) is enriched at cardiomyocyte intercalated disks (ID) along with the pore-forming subunit Na V 1.5. We recently demonstrated that the perinexus, an Na V 1.5-rich ID nanodomain located near connexin43 (Cx43) gap junctions (GJ), is important for cell-to-cell electrical coupling. Disrupting close membrane apposition within the perinexus was highly proarrhythmic. Hypothesis: β1-mediated adhesion is key to close membrane apposition within the perinexus; therefore, inhibiting β1-mediated adhesion may disrupt perinexal structure, impair conduction and precipitate arrhythmias. Methods and Results: Na V 1.5 and β1 localization within ID nanodomains was quantitatively assessed from super-resolution STochastic Optical Reconstruction Microscopy (STORM) images of en face IDs from adult guinea pig ventricles. Na V 1.5 and β1 were located 63±15 and 75±25 nm from Cx43 GJ; 31% of both Na V 1.5 and β1 localized to GJ perinexi. β1-mediated adhesion was acutely inhibited by βadp1, a novel peptide mimetic of β1’s putative extracellular adhesion domain. βadp1 decreased cellular adhesion as assessed by Electric Cell-Substrate Impedance Spectroscopy (ECIS) in β1-overexpressing 1610 cells in a dose-dependent manner but not in native 1610 cells. Perinexal intermembrane spacing as measured by Transmission Electron Microscopy (TEM) increased 3-fold from 17±1 nm in vehicle controls (0.25% DMSO) to 49±5 nm (p<0.05) in 100 μM βadp1-treated hearts; however, intermembrane spacing at other ID locations was unaltered (22±1 vs. 24±1 nm). Optical mapping revealed preferential slowing of transverse conduction (8.0±0.7 vs. 16.5±1.2 cm/s, p<0.05) and increased anisotropy (3.9±0.2 vs. 2.6±0.2, p<0.05) in βadp1-treated hearts relative to vehicle controls. Further, 3 of 4 βadp1-treated hearts exhibited spontaneous ventricular tachycardias. Conclusions: These data support a role for β1-mediated adhesion in maintaining close apposition between Na V 1.5-rich perinexal membranes. Importantly, acute inhibition of β1-mediated adhesion resulted in disruption of close membrane apposition within the GJ perinexus and precipitated severe conduction slowing and spontaneous arrhythmias.

InVitro Models of GJB2-Related Hearing Loss Recapitulate Ca2+ Transients via a Gap Junction Characteristic of Developing Cochlea.

SummaryMutation of the Gap Junction Beta 2 gene (GJB2) encoding connexin 26 (CX26) is the most frequent cause of hereditary deafness worldwide and accounts for up to 50% of non-syndromic sensorineural hearing loss cases in some populations. Therefore, cochlear CX26-gap junction plaque (GJP)-forming cells such as cochlear supporting cells are thought to be the most important therapeutic target for the treatment of hereditary deafness. The differentiation of pluripotent stem cells into cochlear CX26-GJP-forming cells has not been reported. Here, we detail the development of a novel strategy to differentiate induced pluripotent stem cells into functional CX26-GJP-forming cells that exhibit spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of the developing cochlea. Furthermore, these cells from CX26-deficient mice recapitulated the drastic disruption of GJPs, the primary pathology of GJB2-related hearing loss. These in vitro models should be useful for establishing inner-ear cell therapies and drug screening that target GJB2-related hearing loss.

Gap junction composed of connexin43 modulates 5‑fluorouracil, oxaliplatin and irinotecan resistance on colorectal cancers.

Chemotherapy is one of the most commonly used therapeutic strategies for metastatic colon cancer. However, the development of resistance to chemotherapeutic agents limits their application in clinical use. The underlying mechanisms of this resistance development require further elucidation. The current study investigated the effects of connexin43 (Cx43) gap junctions on 5‑fluorouracil (5‑FU), oxaliplatin and irinotecan in colon cancer cells. Three different methods were used to manipulate Cx43 gap junction function: i)Cell culture at different densities; ii) pretreatment with a Cx43 specific inhibitor or enhancer; and iii)Cx43 gene knock‑down. Results indicated that the cell toxicity of 5‑FU, oxaliplatin and irinotecan was cell density‑dependent, which was mediated by gap junctions. Downregulation of Cx43 gap junction functioning attenuated 5‑FU, oxaliplatin and irinotecan toxicity in colon cancer cells, which was increased in cells treated with a Cx43 gap junction function enhancer. Thus, the results of the present study suggest that resistance to 5‑FU, oxaliplatin and irinotecan in colon cancer cells was relative to Cx43 expression loss as cancer developed, which may indicate a novel basis for therapeutic strategy development to combat drug resistance in numerous cell types, in addition to colon cancer cells.

Connexin 43 Mediates White Adipose Tissue Beiging by Facilitating the Propagation of Sympathetic Neuronal Signals

"Beige" adipocytes reside in white adipose tissue (WAT) and dissipate energy as heat. Several studies have shown that cold temperature can activate pro-opiomelanocortin-expressing (POMC) neurons and increase sympathetic neuronal tone to regulate WAT beiging. WAT, however, is traditionally known to be sparsely innervated. Details regarding the neuronal innervation and, more importantly, the propagation of the signal within the population of "beige" adipocytes are sparse. Here, we demonstrate that beige adipocytes display an increased cell-to-cell coupling via connexin 43 (Cx43) gap junction channels. Blocking of Cx43 channels by 18α-glycyrrhetinic acid decreases POMC-activation-induced adipose tissue beiging. Adipocyte-specific deletion of Cx43 reduces WAT beiging to a level similar to that observed in denervated fat pads. In contrast, overexpression of Cx43 is sufficient to promote beiging even with mild cold stimuli. These data reveal the importance of cell-to-cell communication, effective in cold-induced WAT beiging, for the propagation of limited neuronal inputs in adipose tissue.