[Retracted] Tumor suppressor miR­613 induces cisplatin sensitivity in non­small cell lung cancer cells by targeting GJA1.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the colony formation assay data shown in Fig. 2F, the tumor images in Fig. 3A, the "NC" experiment for the Ki67 immunohistochemical staining experiment shown in Fig. 3E and the migration assay data in Fig. 4D were strikingly similar to data appearing in different form in other papers by different authors at different research institutes that had either already been published, or were under consideration for publication at around the same time. Owing to the fact that the contentious data in the above article had already been published prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 23: 385, 2021; DOI: 10.3892/mmr.2021.12024].

N-methyl-D-aspartate Receptor Subunits 2A and 2B Mediate Connexins and Pannexins in the Trigeminal Ganglion Involved in Orofacial Inflammatory Allodynia during Temporomandibular Joint Inflammation.

Temporomandibular joint osteoarthritis (TMJOA) is a severe form of temporomandibular joint disorders (TMD), and orofacial inflammatory allodynia is one of its common symptoms which lacks effective treatment. N-methyl-D-aspartate receptor (NMDAR), particularly its subtypes GluN2A and GluN2B, along with gap junctions (GJs), are key players in the mediation of inflammatory pain. However, the precise regulatory mechanisms of GluN2A, GluN2B, and GJs in orofacial inflammatory allodynia during TMJ inflammation still remain unclear. Here, we established the TMJ inflammation model by injecting Complete Freund's adjuvant (CFA) into the TMJ and used Cre/loxp site-specific recombination system to conditionally knock out (CKO) GluN2A and GluN2B in the trigeminal ganglion (TG). Von-frey test results indicated that CFA-induced mechanical allodynia in the TMJ region was relieved in GluN2A and GluN2B deficient mice. In vivo, CFA significantly up-regulated the expression of GluN2A and GluN2B, Gjb1, Gjb2, Gjc2 and Panx3 in the TG, and GluN2A and GluN2B CKO played different roles in mediating the expression of Gjb1, Gjb2, Gjc2 and Panx3. In vitro, NMDA up-regulated the expression of Gjb1, Gjb2, Gjc2 and Panx3 in satellite glial cells (SGCs) as well as promoted the intercellular communication between SGCs, and GluN2A and GluN2B knocking down (KD) altered the expression and function differently. NMDAR regulated Gjb1 and Panx3 through ERK1/2 pathway, and mediated Gjb2 and Gjc2 through MAPK, PKA, and PKC intracellular signaling pathways. These findings shed light on the distinct functions of GluN2A and GluN2B in mediating peripheral sensitization induced by TMJ inflammation in the TG, offering potential therapeutic targets for managing orofacial inflammatory allodynia.

Effects and mechanisms of puerarin against neuroblastoma: insights from bioinformatics and in vitro experiments.

BACKGROUND: Neuroblastoma, a prevalent solid tumor in children, often manifests with hidden onset sites, rapid growth, and high metastatic potential. The prognosis for children with high-risk neuroblastoma remains poor, highlighting the urgent need for novel prognostic models and therapeutic avenues. In recent years, puerarin, as a kind of small molecule drug extracted from Chinese medicine Pueraria lobata, has demonstrated significant anticancer effects on various cancer cell types. In this study, through bioinformatics analysis and in vitro experiments, the potential and mechanism of puerarin in the treatment of neuroblastoma were investigated, and a prognostic model was established. METHODS: A total of 9 drug-disease related targets were observed by constructing a database of drug targets and disease genes. Besides, GO and KEGG enrichment analysis was performed to explore the potential mechanism of its therapeutic effect. To construct the prognostic model, risk regression analysis and LASSO analysis were carried out for validation. Finally, the prognostic genes were identified. Parachute test and immunofluorescence staining were performed to verify the potential mechanism of puerarin in neuroblastoma treatment. RESULTS: Three prognostic genes, i.e., BIRC5, TIMP2 and CASP9, were identified. In vitro studies verified puerarin's impact on BIRC5, TIMP2, and CASP9 expression, inhibiting proliferation in neuroblastoma SH-SY5Y cells. Puerarin disrupts the cytoskeleton, boosts gap junctional communication, curtailing invasion and migration, and induces mitochondrial damage in SH-SY5Y cells. CONCLUSIONS: Based on network pharmacology and bioinformatics analysis, combined with in vitro experimental verification, puerarin was hereby observed to enhance GJIC in neuroblastoma, destroy cytoskeleton and thus inhibit cell invasion and migration, cause mitochondrial damage of tumor cells, and inhibit cell proliferation. Overall, puerarin, as a natural medicinal compound, does hold potential as a novel therapy for neuroblastoma.

Understanding electrical and chemical transmission in the brain.

The histochemical Falck-Hillarp method for the localization of dopamine (DA), noradrenaline (NA) and serotonin in the central nervous system (CNS) of rodents was introduced in the 1960s. It supported the existence of chemical neurotransmission in the CNS. The monoamine neurons in the lower brain stem formed monosynaptic ascending systems to the telencephalon and diencephalon and monoamine descending systems to the entire spinal cord. The monoamines were early on suggested to operate via synaptic chemical transmission in the CNS. This chemical transmission reduced the impact of electrical transmission. In 1969 and the 1970s indications were obtained that important modes of chemical monoamine communication in the CNS also took place through the extra-synaptic fluid, the extracellular fluid, and long-distance communication in the cerebrospinal fluid involving diffusion and flow of transmitters like DA, NA and serotonin. In 1986, this type of transmission was named volume transmission (VT) by Agnati and Fuxe and their colleagues, also characterized by transmitter varicosity and receptor mismatches. The short and long-distance VT pathways were characterized by volume fraction, tortuosity and clearance. Electrical transmission also exists in the mammalian CNS, but chemical transmission is in dominance. One electrical mode is represented by electrical synapses formed by gap junctions which represent low resistant passages between nerve cells. It allows for a more rapid passage of action potentials between nerve cells compared to chemical transmission. The second mode is based on the ability of synaptic currents to generate electrical fields to modulate chemical transmission. One aim is to understand how chemical transmission can be integrated with electrical transmission and how putative (aquaporin water channel, dopamine D2R and adenosine A2AR) complexes in astrocytes can significancy participate in the clearance of waste products from the glymphatic system. VT may also help accomplish the operation of the acupuncture meridians essential for Chinese medicine in view of the indicated existence of extracellular VT pathways.

Integrative analyses identified gap junction beta-2 as a prognostic biomarker and therapeutic target for breast cancer.

Background: Increasing evidence has shown that connexins are involved in the regulation of tumor development, immune escape, and drug resistance. This study investigated the gene expression patterns, prognostic values, and potential mechanisms of connexins in breast cancer. Methods: We conducted a comprehensive analysis of connexins using public gene and protein expression databases and clinical samples from our institution. Connexin mRNA expressions in breast cancer and matched normal tissues were compared, and multiomics studies were performed. Results: Gap junction beta-2 mRNA was overexpressed in breast cancers of different pathological types and molecular subtypes, and its high expression was associated with poor prognosis. The tumor membrane of the gap junction beta-2 mutated group was positive, and the corresponding protein was expressed. Somatic mutation and copy number variation of gap junction beta-2 are rare in breast cancer. The gap junction beta-2 transcription level in the p110α subunit of the phosphoinositide 3-kinase mutant subgroup was higher than that in the wild-type subgroup. Gap junction beta-2 was associated with the phosphoinositide 3-kinase-Akt signaling pathway, extracellular matrix-receptor interaction, focal adhesion, and proteoglycans in cancer. Furthermore, gap junction beta-2 overexpression may be associated with phosphoinositide 3-kinase and histone deacetylase inhibitor resistance, and its expression level correlated with infiltrating CD8+ T cells, macrophages, neutrophils, and dendritic cells. Conclusions: Gap junction beta-2 may be a promising therapeutic target for targeted therapy and immunotherapy and may be used to predict breast cancer prognosis.

Expression profile and gap-junctional transfer of microRNAs in the bovine cumulus-oocyte complex.

MicroRNAs (miRNA) are important regulators of oocyte maturation, playing a key role in modulating gene expression both in a temporal- and spatial-specific manner. These small non-coding RNAs are involved in important processes during oocyte maturation, acting as messengers between the oocyte and its surrounding cumulus cells. Despite its significance, the bidirectional communication mechanism is still unknown. To test miRNA communication between oocyte and surrounding cumulus cells through the gap junctions the gap junctions were either blocked with carbenoxolone or not. MiRNA sequencing of oocytes at 1, 6, and 22 h of in vitro maturation was then performed. Among the differentially expressed miRNAs, bta-miR-21-5p, a regulator of cumulus cell viability and oocyte maturation, was the only previously known miRNA. Furthermore, by labeling a bta-miR-21-5p mimic with FAM, crossing of this miRNA through the gap junctions within the cumulus-oocyte complex could be visualized and internalization in the oocyte was confirmed by RT-qPCR. In conclusion, this study provides, for the first time, evidence that miRNA communication within the bovine cumulus-oocyte complex is enabled through the gap junctional network.

Protector Role of Cx30.2 in Pancreatic ß-Cell against Glucotoxicity-Induced Apoptosis.

Glucotoxicity may exert its deleterious effects on pancreatic ß-cell function via a myriad of mechanisms, leading to impaired insulin secretion and, eventually, type 2 diabetes. ß-cell communication requires gap junction channels to be present among these cells. Gap junctions are constituted by transmembrane proteins of the connexins (Cxs) family. Two Cx genes have been identified in ß cells, Cx36 and Cx30.2. We have found evidence that the glucose concentration on its own is sufficient to regulate Cx30.2 gene expression in mouse islets. In this work, we examine the involvement of the Cx30.2 protein in the survival of ß cells (RIN-m5F). METHODS: RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. RESULTS: High glucose did not induce apoptosis in RIN-m5F ß cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. CONCLUSION: The data suggest that the upregulation of Cx30.2 protects ß cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders.

Cx40 Levels Regulate Hypoxia-Induced Changes in the Migration, Proliferation, and Formation of Gap Junction Plaques in an Extravillous Trophoblast Cell Model.

BACKGROUND: Extravillous trophoblasts (EVTs) form stratified columns at the placenta-uterus interface. In the closest part to fetal structures, EVTs have a proliferative phenotype, whereas in the closest part to maternal structures, they present a migratory phenotype. During the placentation process, Connexin 40 (Cx40) participates in both the proliferation and migration of EVTs, which occurs under hypoxia. However, a possible interaction between hypoxia and Cx40 has not yet been established. METHODS: We developed two cellular models, one with "low Cx40" (Jeg-3), which reflected the expression of this protein found in migratory EVTs, and one with "high Cx40" (Jeg-3/hCx40), which reflected the expression of this protein in proliferative cells. We analyzed the migration and proliferation of these cells under normoxic and hypoxic conditions for 24 h. Jeg-3 cells under hypoxia increased their migratory capacity over their proliferative capacity. However, in Jeg-3/hCx40, the opposite effect was induced. On the other hand, hypoxia promoted gap junction (GJ) plaque formation between neighboring Jeg-3 cells. Similarly, the activation of a nitro oxide (NO)/cGMP/PKG-dependent pathway induced an increase in GJ-plaque formation in Jeg-3 cells. CONCLUSIONS: The expression patterns of Cx40 play a crucial role in shaping the responses of EVTs to hypoxia, thereby influencing their migratory or proliferative phenotype. Simultaneously, hypoxia triggers an increase in Cx40 gap junction (GJ) plaque formation through a pathway dependent on NO.

Differential Regulation of Hemichannels and Gap Junction Channels by RhoA GTPase and Actin Cytoskeleton: A Comparative Analysis of Cx43 and Cx26.

Connexins (Cxs) are transmembrane proteins that assemble into gap junction channels (GJCs) and hemichannels (HCs). Previous researches support the involvement of Rho GTPases and actin microfilaments in the trafficking of Cxs, formation of GJCs plaques, and regulation of channel activity. Nonetheless, it remains uncertain whether distinct types of Cxs HCs and GJCs respond differently to Rho GTPases or changes in actin polymerization/depolymerization dynamics. Our investigation revealed that inhibiting RhoA, a small GTPase that controls actin polymerization, or disrupting actin microfilaments with cytochalasin B (Cyto-B), resulted in reduced GJCs plaque size at appositional membranes and increased transport of HCs to non-appositional plasma membrane regions. Notably, these effects were consistent across different Cx types, since Cx26 and Cx43 exhibited similar responses, despite having distinct trafficking routes to the plasma membrane. Functional assessments showed that RhoA inhibition and actin depolymerization decreased the activity of Cx43 GJCs while significantly increasing HC activity. However, the functional status of GJCs and HCs composed of Cx26 remained unaffected. These results support the hypothesis that RhoA, through its control of the actin cytoskeleton, facilitates the transport of HCs to appositional cell membranes for GJCs formation while simultaneously limiting the positioning of free HCs at non-appositional cell membranes, independently of Cx type. This dynamic regulation promotes intercellular communications and reduces non-selective plasma membrane permeability through a Cx-type dependent mechanism, whereby the activity of Cx43 HCs and GJCs are differentially affected but Cx26 channels remain unchanged.

The Therapeutic Potential of Targeting the Connexin43 as a New Approach to Reducing Post-Surgical Adhesion.

Post-surgical peritoneal adhesions are a serious problem causing complications, such as bowel obstruction, infertility, and pain. There are currently no effective ways of preventing post-surgical adhesions. Excess secretion of proinflammatory cytokines and profibrotic molecules by immune cells and adherent fibroblasts is the main mechanism thatpromotes post-operative fibrotic scars. Although many studies have been conducted on the pathological causes of this disorder, there are still many unknown facts in this matter, so assessment of the role of different molecules in causing inflammation and adhesion can lead to the creation of new treatment methods. Connexins are a group of proteins related to gap junctions that have a role in cell communication and transmitted signaling between adjacent cells. Between different types of connexin protein isoforms, connexin43 is known to be involved in pathological conditions related to inflammation and fibrosis. Recent studies have reported that inhibition of connexin43 has the potential to reduce inflammation and fibrosis by reducing the expression of molecules like α-SMA and plasminogen activator inhibitor (PAI) that are involved in the early stages of adhesion formation. Further, inhibition of connexin43 may have therapeutic potential as a target to prevent post-surgical peritoneal adhesions.

Deficient gap junction coupling of two common hearing loss-related variants in GJB2.

Objectives: To explore the functional consequences of two common variants, p.V37I and c.299-300delAT in hearing loss associated gene GJB2. Methods: Connexin 26 expression and gap junctional permeability were studied in HEK 293T cells transfected with plasmids expressing GJB2 wild-type, p.V37I, or c.299-300delAT CX26 proteins with fluorescent tags. Functional analyses of different GJB2 haplotypes were performed to fully assess the alteration of ionic and small-molecule coupling. Results: The p.V37I protein was localized at the plasma membrane, but failed to effectively transport intercellular propidium iodide or Ca2+ efficiently, indicating impairment of both biochemical and ionic coupling. The presence of GJB2 p.V37I appeared to increase the sensitivity of cells to H2O2 treatment. In contrast, the known variant c.299-300delAT protein was not transported to the cell membrane and could not form gap junctions, instead being confined to the cytoplasm. Ionic and biochemical coupling was defective in c.299-300delAT-transfected cells. Conclusion: The p.V37I and c.299-300delAT GJB2 mutations resulted in deficient gap junction-mediated coupling. Environmental factors may impact the functional consequences of GJB2 p.V37I. These results may inspire the development of molecular therapies targeting GJB2 mutations for hearing loss.

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

Cardiac Fibroblasts Enhance MMP2 Activity to Suppress Gap Junction Function in Cardiomyocytes.

Although it is crucial to promptly restore blood perfusion to revive the ischemic myocardium, reperfusion itself can paradoxically contribute to the electrical instability and arrhythmias of the myocardium. Several studies have revealed that cardiac fibroblasts can impact cardiac electrophysiology through various mechanisms including the deposition of extracellular matrix, release of chemical mediators, and direct electrical coupling with myocytes. Previously, we have shown that hypoxia/reoxygenation (H/R)-treated rat fibroblasts conditional medium (H/R-FCM) could decrease the spontaneous beating frequency of rat neonatal cardiomyocytes and downregulate the expression of gap junction proteins. However, the specific mechanism by which H/R-FCM affects the gap junctions requires further investigation. H/R-FCM was obtained by culturing confluent rat cardiac fibroblasts (RCF) for 4 h under hypoxic conditions. Gap junction function, hemichannel activity, and expression of Cx43 were examined upon treatment with H/R-FCM. Gelatin zymography was performed to detect matrix metalloproteinase (MMP) activity in the conditioned medium. The effect of H/R-FCM and MMP2 inhibitors on cardiac electrophysiology and arrhythmias was investigated with an isolated rat ischemia/reperfusion (I/R) model. H/R-FCM treatment impaired gap junction function, downregulated Cx43 expression, and increased hemichannel activity in rat cardiomyocytes (H9c2). The adverse effect of H/R-FCM on gap junction, which was confirmed by the cardiomyocyte H/R model, was involved in the activation of MMP2. MMP2 inhibition could partially attenuate the detrimental effects of I/R on myocardial electrophysiological indices and arrhythmia susceptibility. Our study indicates that inhibition of MMP2 may be a promising therapeutic target for the treatment of reperfusion arrhythmia.

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2.

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

Effect of hydrogen peroxide on lens transparency, intracellular pH, gap junction coupling, hydrostatic pressure and membrane water permeability.

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H2O2) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H2O2 in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H2O2 on lens physiology. We investigated transparency, intracellular pH (pHi), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 µM H2O2; the results were compared with control lenses incubated in the saline without H2O2. There was a significant decrease in lens transparency in H2O2-treated lenses. In control lenses, pHi decreases from ∼7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H2O2 for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was ∼2-fold higher at the junction between the differentiating and mature fiber cells and ∼1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H2O2-exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H2O2 caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H2O2 could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts.

Calcium Regulation of Connexin Hemichannels.

Connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells are of paramount importance for intercellular communication. In physiological conditions, HCs can form gap junction (GJ) channels, providing a direct diffusive path between neighbouring cells. In addition, unpaired HCs provide conduits for the exchange of solutes between the cytoplasm and the extracellular milieu, including messenger molecules involved in paracrine signalling. The synergistic action of membrane potential and Ca2+ ions controls the gating of the large and relatively unselective pore of connexin HCs. The four orders of magnitude difference in gating sensitivity to the extracellular ([Ca2+]e) and the cytosolic ([Ca2+]c) Ca2+ concentrations suggests that at least two different Ca2+ sensors may exist. While [Ca2+]e acts as a spatial modulator of the HC opening, which is most likely dependent on the cell layer, compartment, and organ, [Ca2+]c triggers HC opening and the release of extracellular bursts of messenger molecules. Such molecules include ATP, cAMP, glutamate, NAD+, glutathione, D-serine, and prostaglandins. Lost or abnormal HC regulation by Ca2+ has been associated with several diseases, including deafness, keratitis ichthyosis, palmoplantar keratoderma, Charcot-Marie-Tooth neuropathy, oculodentodigital dysplasia, and congenital cataracts. The fact that both an increased and a decreased Ca2+ sensitivity has been linked to pathological conditions suggests that Ca2+ in healthy cells finely tunes the normal HC function. Overall, further investigation is needed to clarify the structural and chemical modifications of connexin HCs during [Ca2+]e and [Ca2+]c variations. A molecular model that accounts for changes in both Ca2+ and the transmembrane voltage will undoubtedly enhance our interpretation of the experimental results and pave the way for developing therapeutic compounds targeting specific HC dysfunctions.

Gap-junction-mediated bioelectric signaling required for slow muscle development and function in zebrafish.

Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and in vivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development.

Spontaneous Repolarization Alternans Causes VT/VF Rearrest That Is Suppressed by Preserving Gap Junctions.

BACKGROUND: Ventricular tachycardia (VT)/ventricular fibrillation (VF) rearrest after successful resuscitation is common, and survival is poor. A mechanism of VT/VF, as demonstrated in ex vivo studies, is when repolarization alternans becomes spatially discordant (DIS ALT), which can be enhanced by impaired gap junctions (GJs). However, in vivo spontaneous DIS ALT-induced VT/VF has never been demonstrated, and the effects of GJ on DIS ALT and VT/VF rearrest are unknown. OBJECTIVES: This study aimed to determine whether spontaneous VT/VF rearrest induced by DIS ALT occurs in vivo, and if it can be suppressed by preserving Cx43-mediated GJ coupling and/or connectivity. METHODS: We used an in vivo porcine model of resuscitation from ischemia-induced cardiac arrest combined with ex vivo optical mapping in porcine left ventricular wedge preparations. RESULTS: In vivo, DIS ALT frequently preceded VT/VF and paralleled its incidence at normal (37°C, n = 9) and mild hypothermia (33°C, n = 8) temperatures. Maintaining GJs in vivo with rotigaptide (n = 10) reduced DIS ALT and VT/VF incidence, especially during mild hypothermia, by 90% and 60%, respectively (P < 0.001; P < 0.013). Ex vivo, both rotigaptide (n = 5) and αCT11 (n = 7), a Cx43 mimetic peptide that promotes GJ connectivity, significantly reduced DIS ALT by 60% and 100%, respectively (P < 0.05; P < 0.005), and this reduction was associated with reduced intrinsic heterogeneities of action potential duration rather than changes in conduction velocity restitution. CONCLUSIONS: These results provide the strongest in vivo evidence to date suggesting a causal relationship between spontaneous DIS ALT and VT/VF in a clinically realistic scenario. Furthermore, our results suggest that preserving GJs during resuscitation can suppress VT/VF rearrest.

Structural basis for pathogenic variants of GJB2 and hearing levels of patients with hearing loss.

OBJECTIVES: The crystal structure of the six protomers of gap junction protein beta 2 (GJB2) enables prediction of the effect(s) of an amino acid substitution, thereby facilitating investigation of molecular pathogenesis of missense variants of GJB2. This study mainly focused on R143W variant that causes hearing loss, and investigated the relationship between amino acid substitution and 3-D structural changes in GJB2. METHODS: Patients with nonsyndromic hearing loss who appeared to have two GJB2 pathogenic variants, including the R143W variant, were investigated. Because the X-ray crystal structure of the six protomers of the GJB2 protein is known, R143W and structurally related variants of GJB2 were modeled using this crystal structure as a template. The wild-type crystal structure and the variant computer-aided model were observed and the differences in molecular interactions within the two were analyzed. RESULTS: The predicted structure demonstrated that the hydrogen bond between R143 and N206 was important for the stability of the protomer structure. From this prediction, R143W related N206S and N206T variants showed loss of the hydrogen bond. CONCLUSION: Investigation of the genotypes and clinical data in patients carrying the R143W variant on an allele indicated that severity of hearing loss depends largely on the levels of dysfunction of the pathogenic variant on the allele, whereas a patient with the homozygous R143W variant demonstrated profound hearing loss. We concluded that these hearing impairments may be due to destabilization of the protomer structure of GJB2 caused by the R143W variant.

Establishing a 3D-cultured system based on alginate-hydrogel embedding benefits the in vitro maturation of porcine Oocytes.

The in vitro maturation (IVM) quality of oocytes is directly related to the subsequent developmental potential of embryos and a fundamental of in vitro embryo production. However, conventional IVM methods fail to maintain the gap-junction intercellular communication (GJIC) between cumulus-oocyte complexes (COCs), which leads to insufficient oocyte maturation. Herein, we investigated the effects of three different three-dimensional (3D) culture methods on oocyte development in vitro, optimized of the alginate-hydrogel embedding method, and assessed the effects of the alginate-hydrogel embedding method on subsequent embryonic developmental potential of oocytes after IVM and parthenogenetic activation (PA). The results showed that Matrigel embedding and alginate-hydrogel embedding benefited the embryonic developmental potential of oocytes after IVM and PA. With the further optimization of alginate-hydrogel embedding, including crosslinking and decrosslinking of parameters, we established a 3D culture system that can significantly increase oocyte maturation and the blastocyst rate of embryos after PA (27.2 ± 1.5 vs 36.7 ± 2.8, P < 0.05). This 3D culture system produced oocytes with markedly increased mitochondrial intensity and membrane potential, which reduced the abnormalities of spindle formation and cortical granule distribution. The alginate-hydrogel embedding system can also remarkably enhance the GJIC between COCs. In summary, based on alginate-hydrogel embedding, we established a 3D culture system that can improve the IVM quality of porcine oocytes, possibly by enhancing GJIC.