Cx43 Gap Research Articles

Abstract 1053: Combination therapy with nonthermal plasma and tirapazamine selectively targets glioblastoma cells and regulates the gap junction phenotype

Abstract Considering the side effects common to most chemotherapeutic agents, a novel approach was developed to selectively treat cancer by targeting the oxidative stress pathway which is induced in many tumor types. Our previous studies demonstrated that nonthermal plasma (NTP) selectively causes apoptotic cell death in melanoma cells while preserving the normal surrounding keratinocytes. The prodrug, tirapazamine, was shown to potentiate the effects of NTP while maintaining selectivity because tirapazamine only activates in hypoxic conditions, an environment common to many tumors. This patented combination therapy has an additive-to-synergistic effect on inducing apoptosis in melanoma. Previous studies have also demonstrated efficacy in a broad range of tumor cells including cervical carcinoma, colon cancer, and pancreatic adenocarcinoma. This report demonstrates for the first time that while NTP alone kills a targeted area of glioblastoma cells and tirapazamine alone kills most of the susceptible cells, the combination of NTP and tirapazamine kills all the cells throughout the tissue culture plate. The mechanism for the increase in the surface area of cell death was identified as the passage of reactive oxygen species (ROS) by intercellular communication through gap junctions. The IC50 dose of tirapazamine required to target several glioblastoma cell lines was less than that for melanoma cells. Through parameter optimization with varied plasma conditions and altered drug dosing, the selective killing of glioblastoma cells was achieved without affecting normal primary astrocytes. Primary astrocytes and glioblastoma cells were co-cultured to observe the effect on the optimal targeting dose of tirapazamine. Glioblastoma cells had an IC50 at hypoxia which was reduced as compared to astrocytes in their normoxia state. The Glioblastoma IC50 values were further decreased when co-cultured with astrocytes. This study documents an increase in the Connexin43 (Cx43) gap junction protein, upon treatment with NTP for both astrocytes and glioblastoma cells. However, treatment with tirapazamine alone ameliorates this effect in glioblastoma cells only and not in astrocytes. The combination therapy of NTP and tirapazamine increased this effect to levels comparable to the original expression levels. The regulation of Cx43 may reduce the metastatic potential of the glioblastoma cells while enabling the combination therapy to show efficacy both between glioblastoma cells and in the context of communication with astrocytes. By this mechanism, the astrocytes may act as conduits to transport ROS between pockets of glioblastoma cells. In summary, the results demonstrate that the novel combination therapy of NTP and tirapazamine is an effective method to selectively target glioblastoma cells with a potential for clinical applications with limited adverse reactions. Citation Format: Matthew Yehl, Andrew Nicolais, Samuel Gerardi, Ahmad Dianat, Olivia Kucharski, Timothy C. Hutcherson, Shoshanna N. Zucker. Combination therapy with nonthermal plasma and tirapazamine selectively targets glioblastoma cells and regulates the gap junction phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1053.

TNNI3K delays atrioventricular conduction and reduces connexin-45 gap junctional coupling

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Dutch Research Council (NWO Talent Scheme) Introduction Cardiac conduction delay is the main substrate for triggering arrhythmias. Hence, prolongation of the PR interval on the electrocardiogram (ECG) is a strong predictor of atrial fibrillation, the most common cardiac arrhythmia. In previous research, cardiac troponin I-interacting kinase (TNNI3K) has been identified as a regulator of the PR interval. Various inbred mouse strains showed a correlation between Tnni3k expression levels and PR interval durations. Additionally, transgenic mice overexpressing hTNNI3K presented an extreme PR interval prolongation. Objective This study aims to unravel the mechanism underlying TNNI3K-driven PR interval prolongation. Methods ECG parameters were recorded in mice expressing physiological levels of Tnni3k (congenic), overexpressing hTNNI3K (TNNI3Ktg), or overexpressing kinase-dead hTNNI3K (TNNI3K-KDtg) and were compared to low-Tnni3k expressing DBA/2J control mice. Atrioventricular (AV) conduction was measured in Langendorff-perfused isolated hearts by electrical mapping. Cellular electrophysiology and conductance were measured using the (dual) patch-clamp technique. AV nodal collagen levels were identified by Pico Sirius Red staining. Candidate interactors were identified by immunoprecipitation of transfected HEK293A cell lysates. In stable HeLa-Connexin 45 (Cx45) cell lines expressing TNNI3K, Cx45 protein expression and phosphorylation levels were investigated by Western blot. Localisation of Cx45 was characterised by immunofluorescence. Results At six weeks of age, congenic and TNNI3Ktg mice show a 17% and 35% prolonged PR interval duration, respectively. Nonetheless, the PR interval of TNNI3K-KDtg mice did not change. Electrical mapping experiments on TNNI3Ktg mouse hearts show a corresponding AV conduction delay, which was neither driven by changes in cellular atrial cardiomyocyte electrophysiology nor driven by AV-nodal fibrosis. We next identified Cx45, a highly expressed connexin in the AV node, as a potential TNNI3K interactor. HeLa-Cx45 cells expressing TNNI3K demonstrated a reduced Cx45 conductance compared to controls without changes in kinetics. Moreover, we observed relatively decreased Cx45 phosphorylation and increased Cx45 intracellular accumulation. Conclusion We here present TNNI3K as a kinase-driven modulator of PR interval prolongation and AV conduction delay, which is independent of atrial electrophysiology and AV nodal fibrosis. We further identified Cx45 as a novel interactor of TNNI3K. The presence of TNNI3K reduces Cx45 gap junctional conductance and promotes intracellular Cx45 protein accumulation, which could explain the PR interval prolongation in vivo. Altogether, this study implies a crucial role for TNNI3K in AV nodal conduction.

Exendin-4, a glucagon-like peptide receptor agonist, facilitates osteoblast differentiation via connexin43

To investigate whether exendin-4 (Ex-4), a glucagon-like peptide 1 receptor (GLP-1R) agonist, affects connexin 43 (Cx43) expression in osteoblasts, and determine the specific mechanism underlying Cx43 modulation by Ex-4. Osteoblast-like MC3T3-E1 cells were treated with Ex-4 with or without GLP-1R antagonist. We assessed Cx43 expression using RT-PCR, western blotting, and confocal microscopy; visualized intercellular communication using Lucifer yellow dye transfer assay; evaluated osteoblast differentiation using alkaline phosphatase and Alizarin red S (ARS) staining. Cx43 silencing or overexpression was investigated via RNA-interference or adenovirus infection. The mechanism underlying Cx43 regulation by Ex-4 was determined via treatment with either Src kinase inhibitor, KX2-391, Akt activator, sc79, or inhibitor, LY294002. Ex-4 treatment enhanced Cx43 expression and gap junctional intercellular communication in MC3T3-E1 cells. GLP-1R antagonist pretreatment abrogated the induction of Cx43 expression. Cx43 silencing significantly decreased ARS staining intensity in Ex-4-treated cells, whereas overexpression enhanced cell differentiation. Treatment with KX2-391 reduced both the Ex-4-induced increase of Cx43 expression and p-Akt protein levels. sc79 upregulated Cx43 expression, while LY294002 attenuated Cx43 upregulation by Ex-4. Induced Cx43 expression in osteoblasts via the Src-Akt signaling pathway illustrates the underlying mechanism for promoting osteoblast differentiation by Ex-4.

Regulation of Connexin Gap Junctions and Hemichannels by Calcium and Calcium Binding Protein Calmodulin.

Connexins are the structural components of gap junctions and hemichannels that mediate the communication and exchange of small molecules between cells, and between the intracellular and extracellular environment, respectively. Connexin (Cx) 46 is predominately expressed in lens fiber cells, where they function in maintaining the homeostasis and transparency of the lens. Cx46 mutations are associated with impairment of channel function, which results in the development of congenital cataracts. Cx46 gap junctions and hemichannels are closely regulated by multiple mechanisms. Key regulators of Cx46 channel function include Ca2+ and calmodulin (CaM). Ca2+ plays an essential role in lens homeostasis, and its dysregulation causes cataracts. Ca2+ associated CaM is a well-established inhibitor of gap junction coupling. Recent studies suggest that elevated intracellular Ca2+ activates Cx hemichannels in lens fiber cells and Cx46 directly interacts with CaM. A Cx46 site mutation (Cx46-G143R), which is associated with congenital Coppock cataracts, shows an increased Cx46-CaM interaction and this interaction is insensitive to Ca2+, given that depletion of Ca2+ reduces the interaction between CaM and wild-type Cx46. Moreover, inhibition of CaM function greatly reduces the hemichannel activity in the Cx46 G143R mutant. These research findings suggest a new regulatory mechanism by which enhanced association of Cx46 with CaM leads to the increase in hemichannel activity and dysregulation may lead to cataract development. In this review, we will first discuss the involvement of Ca2+/CaM in lens homeostasis and pathology, and follow by providing a general overview of Ca2+/CaM in the regulation of Cx46 gap junctions. We discuss the most recent studies concerning the molecular mechanism of Ca2+/CaM in regulating Cx46 hemichannels. Finally, we will offer perspectives of the impacts of Ca2+/CaM and dysregulation on Cx46 channels and vice versa.

The amino terminal domain plays an important role in transjunctional voltage-dependent gating kinetics of Cx45 gap junctions

Gap junction (GJ) channels formed by Cx45 exist in nodal cells in the heart where the action potential propagation is the slowest. The cellular mechanisms of slow propagation speed (or longer junctional delay) in nodal cells could be a combination of several factors, including lack of voltage-gated sodium channels, smaller cell size, and a lower GJ coupling conductance of Cx45. Compared to other cardiac GJs, Cx45 GJs possess not only the lowest unitary channel conductance, but also the highest extent and the fastest kinetics of the transjunctional voltage-dependent gating (Vj-gating) together with a slow recovery. These unique gating properties could make Cx45 GJs more vulnerable for dynamic uncoupling to a much lower coupling level, especially when junctional delay is lengthened and/or the heart rate is elevated. The molecular mechanisms determining the Vj-gating properties of Cx45 (a connexin belongs to γ group) GJs have not been studied. Previous functional studies on the amino terminal (NT) domain chimeras or point variants of other connexins belong to α or β group showed that their NT domains played an important role in determining their Vj-gating properties. The crystal and cryo-electron microscope structures of homologous connexin GJs showed that the NT domain lines the GJ pore, a position that could serve a role in Vj-sensing and gating. We hypothesize that the residues in the NT domain of Cx45 are important for its Vj-gating properties. Protein sequence alignment of human Cx45 NT domain with the connexins in the α and β groups revealed that the second and the eighth residues in Cx45 are different from most of these connexins. We generated a total of 14 variants on these two residues and studied their ability to form functional GJs and their Vj-gating properties in model cells. Our results revealed an important role of these two residues on fast Vj-gating kinetics and formation of morphological and functional GJ channels. In contrast, no Vj-gating change was observed on a GFP tagged Cx45 at its carboxyl terminus.

Connexin/gap junctions and cytoskeletons synergistically control lens fiber cell morphogenesis and metabolism

We have investigated the underlying mechanisms for how a loss of the Gja3 gene (encoding Cx46) leads to variable nuclear cataracts between the C57BL/6J (B6), 129SvJae (129) and B6/129 strain backgrounds, and determined how the genetic variances of periaxin (encoding a cytoskeletal scaffold protein) and CP49 (encoding an intermediate filament protein) modulate the cataract formation. We hypothesize that the Cx46 gap junctions and cytoskeletons synergistically support lens transparency by not only controlling the fiber cell morphogenesis during development but also regulating the exchanges of metabolites between fiber cells to maintain lens homeostasis. Using genetic manipulation and confocal imaging analysis, we have found that both B6‐CP49 and B6‐periaxin inhibit the severity of nuclear cataracts and that the 129‐periaxin proteins are predominantly colocalized with F‐actin and associated with abnormal protrusions on fiber cell surface. The degree of disrupted inner fiber cell surface topology is correlated with the severity of nuclear cataract formation. Metabolite profiling has revealed changes of antioxidants, glycolysis pathway intermediates and lipids compositions in Gja3 knockout (KO) lenses of different strain backgrounds. The Gja3 KO lenses have significant lower levels of glutathione (GSH) than wild‐type controls. Combined effects of gap junctions, CP49‐intermediate filament and periaxin‐mediated cytoskeleton synergistically regulate the interdigitation structures and metabolism of lens inner fiber cells, which modulate lens transparency, homeostasis and cataractogenesis.Support or Funding InformationSupported by EY013849 grant from the National Eye Institute

Connexin 50-R205G Mutation Perturbs Lens Epithelial Cell Proliferation and Differentiation.

PurposeTo investigate the underlying mechanisms for how the mouse Cx50-R205G point mutation, a homologue of the human Cx50-R198W mutation that is linked to cataract-microcornea syndrome, affects proper lens growth and fiber cell differentiation to lead to severe lens phenotypes.MethodsEdU labeling, immunostaining, confocal imaging analysis, and primary lens epithelial cell culture were performed to characterize the lens epithelial cell (LEC) proliferation and fiber cell differentiation in wild-type and Cx50-R205G mutant lenses in vivo and in vitro.ResultsThe Cx50-R205G mutation severely disrupts the lens size and transparency. Heterozygous and homozygous Cx50-R205G mutant and Cx50 knockout lenses all show decreased central epithelium proliferation while only the homozygous Cx50-R205G mutant lenses display obviously decreased proliferating LECs in the germinative zone of neonatal lenses. Cultured Cx50-R205G lens epithelial cells reveal predominantly reduced Cx50 gap junction staining but no change of the endoplasmic reticulum stress marker BiP. The heterozygous Cx50-R205G lens fibers show moderately disrupted Cx50 and Cx46 gap junctions while the homozygous Cx50-R205G lens fibers have drastically reduced Cx50 and Cx46 gap junctions with severely altered fiber cell shape in vivo.ConclusionsThe Cx50-R205G mutation inhibits both central and equatorial lens epithelial cell proliferation to cause small lenses. This mutation also disrupts the assembly and functions of both Cx50 and Cx46 gap junctions in lens fibers to alter fiber cell differentiation and shape to lead to severe lens phenotypes.

Mutation-targeted siRNA therapy for connexin 26-associated keratitis-ichthyosis-deafness syndrome

Background & Aim Keratitis-ichthyosis-deafness (KID) syndrome is a severe, currently untreatable condition characterized by ocular, auditory and cutaneous abnormalities, with major complications from infection to skin cancer. 86% of cases are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in gene GJB2, encoding gap junction protein connexin 26 (Cx26), which alters gating properties of Cx26 channels and gap junction intercellular communication in a dominant manner. There is a pressing need to develop new treatments for the condition. We hypothesized that design and optimization of a mutant-allele-specific siRNA (AS-siRNA) should rescue the cellular phenotype in patient epidermal keratinocytes, and as such could form the basis of a future gene therapy. Methods, Results & Conclusion To that end, a KID syndrome cell line (KID-KC) was established from primary keratinocytes of a KID syndrome patient with heterozygous p.D50N mutation, which displayed impaired gap junction intercellular communication and hyperactive hemichannels, confirmed by dye transfer, patch clamp and neurobiotin uptake assays. In KID-KCs, treatment with AS-siRNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wildtype allele. This resulted in correction of both gap junction intercellular communication and hemichannel activity. Furthermore, AS-siRNA treatment caused only low-level off-target effects in KID-KCs, as detected by genome-wide RNA sequencing. Our data provide an important proof-of-concept for the potential use of AS-siRNA in treating KID syndrome, and in other genetic conditions due to dominant mutations.

Effects of temperature on transjunctional voltage-dependent gating kinetics in Cx45 and Cx40 gap junction channels

Gap junctions (GJs) are intercellular channels directly linking neighbouring cells and are dodecamers of connexins. In the human heart, connexin40 (Cx40), Cx43, and Cx45 are expressed in different regions of the heart forming GJs ensuring rapid propagation of action potentials in the myocardium. Two of these connexins, Cx40 and Cx45, formed functional GJs with prominent transjunctional voltage-dependent gating (Vj-gating), which can be a mechanism to down regulate coupling conductance (Gj). It is not clear the effects of temperature on Vj-gating properties. We expressed Cx40 or Cx45 in N2A cells to study the Vj-gating extent, the kinetics of deactivation, and the recovery time course from deactivation at 22 °C, 28 °C, and 32 °C. Dynamic uncoupling between cell pairs were evaluated at different temperatures, junctional delays, and/or repeating frequencies. Cx40 or Cx45 GJs showed little changes in the extent of Vj-gating, but in both cases with a faster deactivation kinetics at high temperatures. The recovery from deactivation was faster at higher temperatures for Cx45 GJs, but not for Cx40 GJs. Cx45 GJs, but not Cx40 GJs, were dynamically uncoupled when sufficient junctional delays and/or repeating frequency in all tested temperatures. Gap junction specific dynamic uncoupling could play an important role in regulating action potential propagation speed in Cx45 enriched nodal cells in the heart.

Rutaecarpine Prevents High Glucose-induced Cx37 Gap Junction Dysfunction in Human Umbilical Vein Endothelial Cells

Rutaecarpine Prevents High Glucose-induced Cx37 Gap Junction Dysfunction in Human Umbilical Vein Endothelial Cells

Abstract 21374: Adenovirus Targets Cardiac Gap Junctions to Facilitate Viral Replication

Myocarditis is a leading cause of sudden cardiac death in young adults, accounting for 20% of cases under 40 years of age in the United States. Adenovirus is a major etiological agent in this disease yet much remains unknown regarding pathogenesis and predisposition to infection. During adenoviral infection, viral proteins uncouple cell cycle checkpoints for viral DNA genome replication to occur. Connexin43 (Cx43) gap junctions play an essential role in propagation of action potentials to precipitate each heartbeat. Cx43 has also been implicated in cell cycle regulation, leading us to hypothesize that adenovirus targets Cx43 to facilitate viral replication, resulting in arrhythmogenic alterations in cardiac intercellular communication. We find reduced Cx43 expression at the mRNA and protein level within 24 hours of infection with adenovirus type 5 (Ad5) in human epithelial cells. Studies in human induced pluripotent stem cell-derived cardiomyocytes reveal alterations in calcium transient propagation and reduced Cx43 protein levels. Growth factor signaling is activated by early Ad5 proteins and is known to negatively regulate gap junction coupling. We performed genetic screens and have identified the viral protein E4-ORF1 as sufficient in suppression of Cx43 expression and modulation of cardiac transcription factors. siRNA-mediated knockdown of Cx43 protein enhances viral genome replication while transient overexpression of Cx43 significantly inhibits late viral protein synthesis and genome replication. Finally, using dominant-negative trafficking-dead and closed-channel Cx43 mutants, we conclude that inhibition of Ad5 replication by Cx43 is gap junction dependent. Together, these data provide novel insight into molecular mechanisms underlying arrhythmogenic viral myocarditis in addition to understanding the role of Cx43 in adenoviral pathogenesis. Revealing viral mechanisms of gap junction dissolution will contribute not just to the development of novel antiviral therapeutics, but will also provide insight into fundamental Cx43 biology relevant to other forms of heart disease.

Abstract 376: Acute Modulation of Sodium Channel Auxiliary Subunit β1-mediated Adhesion: a Novel Antiarrhythmic Strategy

Cardiac impulse propagation is thought to occur by direct cell to cell flow of current via connexin43 (Cx43) gap junctions (GJs). We recently demonstrated that cardiac sodium channels (Na v 1.5) localized within the perinexus, an intercalated disk (ID) nanodomain adjacent to Cx43 GJ, may enable ephaptic coupling between cardiac myocytes. We hypothesized that β1-mediated adhesion may closely approximate membranes within ID nanodomains, facilitating ephaptic coupling. Super-resolution STochastic Optical Reconstruction Microscopy-based Relative Localization Analysis (STORM-RLA) and immuno-electron microscopy identified two Na V 1.5 populations within the ID in guinea pig ventricles (GPVs): A perinexal population, accounting for 47% of ID-localized Na V 1.5 and a plicate population, co-distributing with N-Cadherin, accounting for 29%. β1 was preferentially localized to the perinexus (48% of ID-localized β1) over N-Cadherin-rich plicate regions (8%). βadp1, a novel peptide inhibitor of β1 adhesion, selectively and dose-dependently inhibited barrier function in β1-overexpressing 1610 cells in electric cell-substrate impedance spectroscopy studies. Neither βadp1 nor a scrambled control peptide (βadp1-scr) affected I Na or action potentials in isolated GPV myocytes. However, βadp1 reduced peak current recorded from Na V 1.5 clusters adjacent Cx43-EGFP at cell-cell contacts using scanning ion conductance microscopy-guided patch clamp. In GPVs, βadp1 (100 μM) compromised the diffusion-resistance of the ID as assessed by perfusion of fixable fluorescent dyes. βadp1 (48±4 μm) but not βadp1-scr (22±1 μm) increased perinexal intermembrane spacing compared to control GPVs (17±1μm). Optical mapping revealed that βadp1 but not βadp1-scr slowed conduction in GPVs and iPSC-derived cardiomyocyte monolayers. Importantly, in GPVs, βadp1 increased conduction anisotropy and precipitated spontaneous tachyarrhythmias in a dose-dependent manner. Thus, β1-mediated adhesion generates close apposition between Na V 1.5-rich perinexal membranes, facilitating ephaptic conduction in the heart. Importantly, β1-mediated adhesion may be a target for novel, mechanistically-driven antiarrhythmic therapy.

Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes

Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 μM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state.

Complementary expression and phosphorylation of Cx46 and Cx50 during development and following gene deletion in mouse and in normal and orchitic mink testes.

Gap junction-mediated communication helps synchronize interconnected Sertoli cell activities. Besides, coordination of germ cell and Sertoli cell activities depends on gap junction-mediated Sertoli cell-germ cell communication. This report assesses mechanisms underlying the regulation of connexin 46 (Cx46) and Cx50 in mouse testis and those accompanying a "natural" seasonal and a pathological arrest of spermatogenesis, resulting from autoimmune orchitis (AIO) in mink. Furthermore, the impact of deleting Cx46 or Cx50 on the expression, phosphorylation of junction proteins, and spermatogenesis is evaluated. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. During the mink active spermatogenic phase, Cx50 became phosphorylated and localized to the site of the blood-testis barrier. By contrast, Cx46 was dephosphorylated and associated with annular junctions, suggesting phosphorylation/dephosphorylation of Cx46 and Cx50 involvement in the barrier dynamics. Cx46-positive annular junctions in contact with lipid droplets were found. Cx46 and Cx50 expression and localization were altered in mink with AIO. The deletion of Cx46 or Cx50 impacted on other connexin expression and phosphorylation and differently affected tight and adhering junction protein expression. The level of apoptosis, determined by ELISA, and a number of Apostain-labeled spermatocytes and spermatids/tubules were higher in mice lacking Cx46 (Cx46-/-) than wild-type and Cx50-/- mice, arguing for life-sustaining Cx46 gap junction-mediated exchanges in late-stage germ cells secluded from the blood by the barrier. The data show that expression and phosphorylation of Cx46 and Cx50 are complementary in seminiferous tubules.

Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells.

The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance.

Connexin 43 Communication Channels in Follicular Dendritic Cell Development and in Follicular Lymphomas

Follicular dendritic cells (FDC) show homo- and heterocellular metabolic coupling through connexin 43 (Cx43) gap junctions and support B cell selection and maturation in germinal centers. In follicular lymphomas B cells escape apoptosis while FDC develop abnormally. Here we tested Cx43 channels in reactive FDC development and follicular lymphomas. In culture, the treatment of FDC-B cell clusters (resembling to “ex vivo” germinal centers) with Gap27 peptide, mimicking the 2nd extracellular loop of Cx43 protein, significantly impaired FDC-B cell cluster formation and cell survival. In untreated cultures of intact clusters, cell proliferation showed a moderate reduction. In tissues, Cx43 protein levels run parallel with the density of FDC both in reactive germinal centers and in malformed follicles of follicular lymphomas and showed strong upregulation in newly generated and/or degrading bi-/multinuclear FDC of rudimentary processes. However, the inverse correlation between Cx43 expression and B cell proliferation seen in reactive germinal centers was not detected in follicular lymphomas. Furthermore, Cx43 levels were not associated with either lymphoma grade or bone marrow involvement. Our results suggest that Cx43 channels are critical in FDC and “ex vivo” germinal center development and in the persistence of FDC in follicular lymphomas but do not affect tumor progression.

Cataract-causing mutation of human connexin 46 impairs gap junction, but increases hemichannel function and cell death.

Connexin channels play a critical role in maintaining metabolic homeostasis and transparency of the lens. Mutations in connexin genes are linked to congenital cataracts in humans. The G143R missense mutation on connexin (Cx) 46 was recently reported to be associated with congenital Coppock cataracts. Here, we showed that the G143R mutation decreased Cx46 gap junctional coupling in a dominant negative manner; however, it significantly increased gap junctional plaques. The G143R mutant also increased hemichannel activity, inversely correlated with the level of Cx46 protein on the cell surface. The interaction between cytoplasmic loop domain and C-terminus has been shown to be involved in gating of connexin channels. Interestingly, the G143R mutation enhanced the interaction between intracellular loop and Cx46. Furthermore, this mutation decreased cell viability and the resistance of the cells to oxidative stress, primarily due to the increased hemichannel function. Together, these results suggest that mutation of this highly conserved residue on the cytoplasmic loop domain of Cx46 enhances its interaction with the C-terminus, resulting in a reduction of gap junction channel function, but increased hemichannel function. This combination leads to the development of human congenital cataracts.

Erratum: Interfering amino terminal peptides and functional implications for heteromeric gap junction formation

Connexin43 (Cx43) is widely expressed in many different tissues of the human body. In cells of some organs, Cx43 is co-expressed with other connexins (Cx), including Cx46 and Cx50 in lens, Cx40 in atrium, Purkinje fibers, and the blood vessel wall, Cx45 in heart, and Cx37 in the ovary. Interactions with the co-expressed connexins may have profound functional implications. The abilities of Cx37, Cx45, Cx46, and Cx50 to function in heteromeric gap junction combinations with Cx43 are well documented. Different studies disagree regarding the ability of Cx43 and Cx40 to produce functional heteromeric gap junctions with each other. We review previous studies regarding the heteromeric interactions of Cx43. The possibility of negative functional interactions between the cytoplasmic pore-forming amino-terminal (NT) domains of these connexins was assessed using pentameric connexin sequence-specific NT domain [interfering NT (iNT)] peptides applied to cells expressing homomeric Cx40, Cx37, Cx45, Cx46, and Cx50 gap junctions. A Cx43 iNT peptide corresponding to amino acids 9-13 (Ac-KLLDK-NH2) specifically inhibited the electrical coupling of Cx40 gap junctions in a transjunctional voltage (V j)-dependent manner without affecting the function of homologous Cx37, Cx46, Cx50, and Cx45 gap junctions. A Cx40 iNT (Ac-EFLEE-OH) peptide counteracted the V j-dependent block of Cx40 gap junctions, whereas a similarly charged Cx50 iNT (Ac-EEVNE-OH) peptide did not, suggesting that these NT domain interactions are not solely based on electrostatics. These data are consistent with functional Cx43 heteromeric gap junction formation with Cx37, Cx45, Cx46, and Cx50 and suggest that Cx40 uniquely experiences functional suppressive interactions with a Cx43 NT domain sequence. These findings present unique functional implications about the heteromeric interactions between Cx43 and Cx40 that may influence cardiac conduction in atrial myocardium and the specialized conduction system.

Interfering amino terminal peptides and functional implications for heteromeric gap junction formation

Connexin43 (Cx43) is widely expressed in many different tissues of the human body. In cells of some organs, Cx43 is co-expressed with other connexins (Cx), including Cx46 and Cx50 in lens, Cx40 in atrium, Purkinje fibers, and the blood vessel wall, Cx45 in heart, and Cx37 in the ovary. Interactions with the co-expressed connexins may have profound functional implications. The abilities of Cx37, Cx45, Cx46, and Cx50 to function in heteromeric gap junction combinations with Cx43 are well documented. Different studies disagree regarding the ability of Cx43 and Cx40 to produce functional heteromeric gap junctions with each other. We review previous studies regarding the heteromeric interactions of Cx43. The possibility of negative functional interactions between the cytoplasmic pore-forming amino-terminal (NT) domains of these connexins was assessed using pentameric connexin sequence-specific NT domain [interfering NT (iNT)] peptides applied to cells expressing homomeric Cx40, Cx37, Cx45, Cx46, and Cx50 gap junctions. A Cx43 iNT peptide corresponding to amino acids 9–13 (Ac-KLLDK-NH2) specifically inhibited the electrical coupling of Cx40 gap junctions in a transjunctional voltage (Vj)-dependent manner without affecting the function of homologous Cx37, Cx46, Cx50, and Cx45 gap junctions. A Cx40 iNT (Ac-EFLEE-OH) peptide counteracted the Vj-dependent block of Cx40 gap junctions, whereas a similarly charged Cx50 iNT (Ac-EEVNE-OH) peptide did not, suggesting that these NT domain interactions are not solely based on electrostatics. These data are consistent with functional Cx43 heteromeric gap junction formation with Cx37, Cx45, Cx46, and Cx50 and suggest that Cx40 uniquely experiences functional suppressive interactions with a Cx43 NT domain sequence. These findings present unique functional implications about the heteromeric interactions between Cx43 and Cx40 that may influence cardiac conduction in atrial myocardium and the specialized conduction system.

Abstract 194: Increasing Expression of Connexin43 in Bone Marrow Stem Cells Enhances Their Protective Effects on the Infarcted Heart

Introduction: Coupling of stem cells (SCs) injected into the infarcted heart to endogenous cells that survived injury may enhance SC survival and engraftment and improve their protective effects. Connexin43 (Cx43) gap junctions are some of the first proteins expressed by differentiating SCs transplanted into the heart after myocardial infarction (MI), suggesting that cell coupling is critical for SC survival and differentiation. Hypothesis: Overexpression of Cx43 in SCs will enhance their ability to engraft, differentiate, and improve cardiac function and survival after transplant into the infarcted heart. Methods: Adult mouse SCs isolated from compact bone were sorted for c-kit surface marker using magnetic beads. SCs were transduced with PLL3.7 lentivirus encoding the Cx43 gene (GJA1) at MOI=300 to produce overexpression, which was confirmed by Western analysis on SC lysates. MI was induced in 12-week-old male C57BL/6 mice by ligating the left anterior descending coronary artery, and 40,000 SCs in 20 uL saline were administered immediately after MI via intramyocardial injection. A total of 69 animals were used: 18 received Cx43 SCs, 30 received non-transduced SCs (NTSC), 11 received only saline, and 10 underwent sham surgery. Echocardiography was performed 1 and 2 weeks post-MI. Results: Animals receiving Cx43 SCs had improved survival (90.0%) versus NTSC (76.7%) and saline controls (70.0%). The Cx43 SC group displayed improved ejection fraction versus NTSC and saline controls at 1 week (48.7% v. 43.3%, p=0.04; 30.1%, p<0.0001) and 2 weeks post-MI (45.7% v. 40.4%, p=0.08; 26.8%, p=0.0004). Similar improvements in fractional shortening, stroke volume, and cardiac output were observed. Administration of Cx43 SC and NTSC attenuated remodeling with no difference between the two groups. Both decreased end-diastolic and systolic volumes, increased thickness of the infarct-related anterior wall, and decreased heart weight (HW), HW/body weight and HW/tibia length ratios at 1 and 2 weeks post-MI. Conclusion: All SCs improved survival and cardiac function, and attenuated remodeling. Transplantation of Cx43 SCs improved survival and cardiac function even more than NTSCs, suggesting that Cx43 may play a crucial role in engraftment and differentiation.