Gap Junction Protein Research Articles

Peroxisomal dysfunction interferes with odontogenesis and leads to developmentally delayed teeth and defects in distinct dental cells in Pex11b-deficient mice.

Human peroxisomal biogenesis disorders of the Zellweger syndrome spectrum affect skeletal development and induce tooth malformations. Whereas several peroxisomal knockout mouse studies elucidated the pathogenesis of skeletal defects, little information is available on how dental pathologies arise in peroxisomal biogenesis disorder patients. To understand the impact of severe peroxisomal dysfunction on early odontogenesis, here we performed morphometric studies on developing molars of new-born Pex11b knockout mice. Immunofluorescence analysis revealed reduced peroxisome number and mistargeting of the peroxisomal matrix enzyme catalase to the cytoplasm in several dental cell types of the Pex11b knockout animals. We also observed secondary mitochondrial alterations, comprising decreased staining of mitochondrial superoxide dismutase and of complex IV in cells of the developing molar. The peroxisomal defect caused by the PEX11b knockout also decreased the staining of cytokeratin intermediate filaments and of the secretory proteins amelogenin, osteopontin and osteocalcin. Interestingly, the staining of the gap junction protein connexin 43, an important modulator of tissue development, was also decreased, possibly causing the observed cellular disarrangement within the inner enamel epithelium and the odontoblast palisade. Taken together, our results show that the severe phenotype associated with the PEX11b knockout results in a reduction of the number of peroxisomes in dental cells and causes a delay odontogenesis. This adds a new component to the already described symptomatic spectrum induced by severe peroxisomal defects.

Genome-wide identification of gap junction gene family and their expression profiles under low temperature stress in noble scallop Chlamys nobilis

Gap junctions, formed by gap junction proteins (GJ), play crucial roles in cell signaling and immune responses. The structure and function of the GJ from vertebrates (called connexins) have been extensively studied. However, little is known about the proteins forming gap junctions in invertebrates (called innexins). In this study, 14 GJ genes of Chlamys nobilis were identified. GJ proteins are mainly distributed on the plasma membrane, and all proteins are hydrophilic Phylogenetic tree analysis showed that the GJ proteins in C. nobilis were distantly related to those in vertebrates but closely related to those in invertebrates. Conserved motifs analysis of these GJ proteins in C. nobilis identified to have 10 conserved motifs, similar to gap junction proteins in other bivalves. Moreover, expression profiles of CnGJ genes under chronic and acute low temperature stress were also investigated. Results showed that chronic low temperature stress had a significant effect on the expression levels of CnGJ genes, and the expression profiles of CnGJ genes showed significantly variation under acute low temperature stress. All these results indicated that CnGJ genes play important roles in environmental adaptation in scallops. The present study initially elucidated the function of gap junction genes in noble scallop C. nobilis, which provides new insights into the GJ genes in mollusks and will help us better understand their roles in environmental stress in scallops.

Identification and validation of ion channels-related mRNA prognostic signature for glioblastomas.

Glioblastomas (GBM) is a kind of malignant brain tumor with poor prognosis. Identifying new biomarkers is promising for the treatment of GBM. The mRNA-seq and clinical data were obtained from The Cancer Genome Atlas and the Chinese Glioma Genome Atlas databases. The differentially expressed genes were identified using limma R package. The prognosis-related genes were screened out and a risk model was constructed using univariate, least absolute shrinkage and selection operator, and multivariate Cox analysis. Receiver operating characteristic curve was used to assess the efficiency of model. Kaplan-Meier survival curve was applied for the survival analysis. Mutation analysis was conducted using maftools package. The effect of immunotherapy was analyzed according to TIDE score, and the drug sensitivity analysis was performed. The Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis enrichment analyses were performed for the functional analysis. The regulatory network was constructed by STRING and Cytoscape software. RT-qPCR was performed to validate the expression of 3 hub genes in vitro. A risk model was constructed based on 3 ion channels related genes (gap junction protein beta 2 [GJB2], potassium voltage-gated channel subfamily h member 6 [KCNH6], and potassium calcium-activated channel subfamily n member 4 [KCNN4]). The risk score and hub genes were positively correlated with the calcium signaling pathway. Patients were divided into 2 groups based on the risk score calculated by 3 signatures. The infiltration levels of T cell, B lineage, monocytic lineage, and neutrophils were increased in high risk group, while TIDE score was decreased. IC50 of potential drugs for GBM treatment was elevated in the high risk group. Furthermore, GJB2, KCNH6, and KCNN4 were oncogenic, and GJB2 and KCNN4 were upregulated, while KCNH6 was downregulated in high risk group and GBM cells. The regulatory network showed that KCNH6 was targeted by more miRNA and transcription factors and KCNN4 interacted with more drugs. We constructed a three-signature risk model, which could effectively predict the prognosis of GBM development. Besides, KCNH6 and KCNN4 were respectively considered as the targets of molecular targeted treatment and chemotherapy.

Ventral Root Boundary Cap Cells of Rat Spinal Cord Contain Connexin-43.

Boundary cap cells are a population of multipotent stem cells that have great potential for the use in the treatment of damaged nervous system. We studied the patterns of distribution of the gap junction protein connexin-43 (Cx43) in boundary cap cells of the ventral root of the spinal cord of rat embryos (E12-E20; n=40). It was found that Cx43 is expressed in ventral boundary cap cells at all stages of its existence during embryogenesis. At the early stages of prenatal development, the cytoplasmic distribution of Cx43 in the boundary cap cells predominates; at the later stages, Cx43-immunopositive punctate structures are identified. These puncta represent gap junction plaques between the cells. It can be assumed that during the early embryogenesis, Cx43 regulates the main histogenetic processes in boundary cap cells and only in the later stages of prenatal development, Cx43-mediated communications are formed between boundary cap cells.

Extracellular Vesicles: A Novel Diagnostic Tool and Potential Therapeutic Approach for Equine Osteoarthritis.

Osteoarthritis (OA) is a chronic progressive degenerative joint disease that affects a significant portion of the equine population and humans worldwide. Current treatment options for equine OA are limited and incompletely curative. Horses provide an excellent large-animal model for studying human OA. Recent advances in the field of regenerative medicine have led to the exploration of extracellular vesicles (EVs)-cargoes of microRNA, proteins, lipids, and nucleic acids-to evaluate their diagnostic value in terms of disease progression and severity, as well as a potential cell-free therapeutic approach for equine OA. EVs transmit molecular signals that influence various biological processes, including the inflammatory response, apoptosis, proliferation, and cell communication. In the present review, we summarize recent advances in the isolation and identification of EVs, the use of their biologically active components as biomarkers, and the distribution of the gap junction protein connexin 43. Moreover, we highlight the role of mesenchymal stem cell-derived EVs as a potential therapeutic tool for equine musculoskeletal disorders. This review aims to provide a comprehensive overview of the current understanding of the pathogenesis, diagnosis, and treatment strategies for OA. In particular, the roles of EVs as biomarkers in synovial fluid, chondrocytes, and plasma for the early detection of equine OA are discussed.

Luminal epithelial cells integrate variable responses to aging into stereotypical changes that underlie breast cancer susceptibility.

Effects from aging in single cells are heterogenous, whereas at the organ- and tissue-levels aging phenotypes tend to appear as stereotypical changes. The mammary epithelium is a bilayer of two major phenotypically and functionally distinct cell lineages: luminal epithelial and myoepithelial cells. Mammary luminal epithelia exhibit substantial stereotypical changes with age that merit attention because these cells are the putative cells-of-origin for breast cancers. We hypothesize that effects from aging that impinge upon maintenance of lineage fidelity increase susceptibility to cancer initiation. We generated and analyzed transcriptomes from primary luminal epithelial and myoepithelial cells from younger <30 (y)ears old and older >55y women. In addition to age-dependent directional changes in gene expression, we observed increased transcriptional variance with age that contributed to genome-wide loss of lineage fidelity. Age-dependent variant responses were common to both lineages, whereas directional changes were almost exclusively detected in luminal epithelia and involved altered regulation of chromatin and genome organizers such as SATB1. Epithelial expression of gap junction protein GJB6 increased with age, and modulation of GJB6 expression in heterochronous co-cultures revealed that it provided a communication conduit from myoepithelial cells that drove directional change in luminal cells. Age-dependent luminal transcriptomes comprised a prominent signal that could be detected in bulk tissue during aging and transition into cancers. A machine learning classifier based on luminal-specific aging distinguished normal from cancer tissue and was highly predictive of breast cancer subtype. We speculate that luminal epithelia are the ultimate site of integration of the variant responses to aging in their surrounding tissue, and that their emergent phenotype both endows cells with the ability to become cancer-cells-of-origin and represents a biosensor that presages cancer susceptibility.

Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild Traumatic Brain Injury.

Connexin43 (Cx43) is the main protein of astrocyte gap junctions, mediating astrocyte coupling of astrocytes into cellular networks, but has also other non-junctional functions. Many pathologies present with altered Cx43 regulation. In this study, we studied Cx43 alterations after mild traumatic brain injury (TBI) in a mouse model. We found that while some astrocyte lost Cx43 expression, a subset of astrocytes experienced an increase in cytoplasmic and hemichannel Cx43. This increase correlated with an increase in phosphorylated Cx43 at serine 368. Cx43 S368A mutant mice, lacking this phosphorylation, exhibited reduced susceptibility to seizures induced by pentylenetetrazol (PTZ). These findings suggest that TBI-induced Cx43 phosphorylation enhances seizure susceptibility.

CSIG-16. A CX43-WNK1-C-MYC SIGNALING AXIS DRIVES GLIOBLASTOMA CANCER STEM CELL SURVIVAL

Abstract Tumors represent a highly dynamic system that relies on coordinated signaling to drive growth and adapt to selective pressures, including those induced by anti-cancer therapies. Given the need for tumors to engage in rapid and coordinated cell-cell communication, mechanisms such as gap junction intracellular communication (GJIC) should be essential. However, the connexin proteins that make up gap junctions have traditionally been considered tumor suppressors based on the frequent loss of GJIC and lower connexin expression in tumor cells compared to non-neoplastic tissue. For this reason, connexin 43 (Cx43) has been proposed to suppress the growth of glioblastoma (GBM), the most common primary malignant brain tumor. However, recent data suggest that this tumor-suppressive effect is context dependent and that connexins can also function in a tumor-promoting role. Using next-generation sequencing techniques, we found Cx43 expressed at high levels in a panel of GBM patient-derived xenograft (PDX) CSCs and that these models rely on Cx43 for their survival and self-renewal. Mechanistically, depletion of Cx43 led to a dramatic loss of c-MYC expression through reduced phosphorylation of its upstream mediator WNK lysine-deficient protein kinase 1 (WNK1). Depletion of WNK1 phenocopied knockdown of Cx43 and reduced MYC protein and mRNA as well as tumor growth in vivo. Together, this work defines a novel signaling axis downstream of gap junction protein expression that promotes tumor growth and cancer stem cell phenotypes in GBM. Due to the difficulty in targeting both Cx43 and MYC, the identification of intermediate targetable signaling nodes may lead to improved therapies for patients with GBM.

Connexin 43 Deficiency Confers Resistance to Immunotherapy in Lung Cancer via Inhibition of the Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes Pathway.

Immune checkpoint inhibitors (ICIs), especially PD-1 inhibitors, are among the first-line therapeutic drugs for the treatment of advanced non-small cell lung cancer (NSCLC). However, most patients are not sensitive to PD-1 inhibitors, and prolonged exposure can lead to acquired resistance. Thus, it is urgent to elucidate the mechanism underlying the resistance of NSCLC to ICIs. Connexin 43 (Cx43) is a gap junction (GJ) protein that is important in therapeutic efficacy to ICIs. In this study, we observed that Cx43 in murine Lewis lung carcinoma (LLC) cells mediated cyclic GMP-AMP (cGAMP) transfer to macrophages. Knockdown of Cx43 reduced T-cell activation, leading to decreased sensitivity of LLC cells to anti-PD-1 therapy. The mechanism might be that knockdown of Cx43 in LLC cells promotes macrophages differentiation into pro-tumour M2 type (TAM), thus activating the STING pathway in macrophages. These findings indicate that downregulation of Cx43 in LLC cells leads to immunotherapy resistance by negatively regulating the cGAS-STING pathway in macrophages. Therefore, Cx43/GJ-mediated signal transmission between lung cancer cells and macrophages provides new insights for increasing immunotherapy sensitivity in NSCLC.

Long-term hybridization in a karst window reveals the genetic basis of eye loss in cavefish.

Eye loss is a hallmark trait of animals inhabiting perpetual darkness, such as caves. The Mexican tetra ( Astyanax mexicanus) provides an unparalleled model for studying the genetic basis of eye loss. There are two interfertile morphs of the Mexican tetra, sighted surface fish and multiple independently evolved eyeless, blind cavefish populations. Despite decades of research on eye loss in this species, our understanding of the precise genetic basis remains sparse. Here, we focused on the unique Caballo Moro cave, where there is a karst window collapse that introduced sunlight and coexistence of both eyed and eyeless cavefish of similar genetic background. This unique genetic mosaic allowed us to pinpoint coding mutations in Connexin 50 (Cx50), also known as gap junction protein alpha 8b (Gja8b), as critical in the genetic basis of eye loss. CRISPR based knockouts of Cx50 in surface fish result in small or absent eyes as young as 48 hours post-fertilization. Further, we identified similar mutations in Cx50 that alter predicted protein structure among other cave-dwelling fish and even subterranean mammals, indicating a conserved evolutionary mechanism of Cx50 mutations. We introduced a mutation (Cx50-S89K) in mice, which resulted in cataracts, smaller eyes, and smaller lenses. Mutations in Cx50 mimic those identified in human congenital cataracts. We additionally leveraged phenotypic variation in a hybrid cavefish population to demonstrate that eyes provide fish with a metabolic advantage, providing a mechanism by which loss of eyes could be favored by selection. This unique hybridization event allowed us to identify novel alleles that contribute to the convergent evolution of eye loss, providing profound insights into the genetic underpinnings of one of nature's most fascinating adaptive traits.

Protein redistribution in buccal cells as a prognostic marker for arrhythmogenic cardiomyopathy in paediatric patients

Abstract Background Mutations in genes encoding desmosomal proteins [plakoglobin (JUP), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2)] cause complex genetic heart muscle disorders collectively described as arrhythmogenic cardiomyopathies (ACM). Diagnosis and risk stratification, particularly in the paediatric population, remain challenging owing to heterogeneous phenotypic manifestations and reduced genetic penetrance. Immunohistochemical analysis of heart muscle samples has shown that several junctional and signalling molecules are redistributed in patients with ACM, offering diagnostic information. However, the use of this approach in living patients was limited by need for heart samples. Recent studies in adults with ACM showed that the same set of proteins re-localised in the ACM heart also do so in the buccal epithelium. Purpose Given the ease of analysing serial cheek smears, we sought to establish how protein redistribution in buccal cells relates to disease onset and progression in children bearing desmosomal gene mutations. Methods Serial cheek smears from children with desmosomal variants were subjected to immunocytochemistry and confocal microscopy to study the distribution of JUP, DSP and PKP-1 as well as the major gap junction protein connexin43 (Cx43; GJA1) and the major subunit of the NFκB inflammatory pathway; RelA. Results 37 patients Conclusion These data indicate that shifts in junctional/signalling proteins precede the onset of clinical disease. Indeed, in the majority of pre-clinical children, Cx43 and RelA seem to be redistributed first, suggesting that onset of abnormalities in electrical conductivity and inflammatory pathways occur first in the natural disease progression, in agreement with previous clinical observations. Moreover, the data suggest that additional proteins are shifted as the disease progresses. Although further verification is required, this study suggests that analysis of buccal smears may be used as a prognostic/risk stratification test in children with desmosomal variants.

Macrophage-specific lipid nanoparticle therapy blocks the lung's mechanosensitive immunity due to macrophage-epithelial interactions.

The lung's mechanosensitive immune response, which occurs when pulmonary alveoli are overstretched, is a major impediment to ventilation therapy for hypoxemic respiratory failure. The cause is not known. We tested the hypothesis that alveolar stretch causes stretch of alveolar macrophages (AMs), leading to the immune response. In lungs viewed by optical imaging, sessile AMs expressed gap junctional protein connexin-43 (Cx43), and they communicated with the alveolar epithelium through gap junctions. Alveolar hyperinflation increased Ca2+ in the AMs but did not stretch the AMs. The Ca2+ response, and concomitant TNFα secretion by AMs were blocked in mice with AM-specific deletion of Cx43. The AM responses, as also lung injury due to mechanical ventilation at high tidal volume, were inhibited by AM-specific delivery of lipid nanoparticles containing Xestospongin C, which blocked the induced Ca2+ increases. We conclude, Cx43- and Ca2+-dependent AM-epithelial interactions determine the lung's mechanosensitive immunity, providing a basis for therapy for ventilator-induced lung injury.

Mitochondrial fission controls astrocyte morphogenesis and organization in the cortex.

Dysfunctional mitochondrial dynamics are a hallmark of devastating neurodevelopmental disorders such as childhood refractory epilepsy. However, the role of glial mitochondria in proper brain development is not well understood. We show that astrocyte mitochondria undergo extensive fission while populating astrocyte distal branches during postnatal cortical development. Loss of mitochondrial fission regulator, Dynamin-related protein 1 (Drp1), decreases mitochondrial localization to distal astrocyte processes, and this mitochondrial mislocalization reduces astrocyte morphological complexity. Functionally, astrocyte-specific conditional deletion of Drp1 induces astrocyte reactivity and disrupts astrocyte organization in the cortex. These morphological and organizational deficits are accompanied by loss of astrocytic gap junction protein Connexin 43. These findings uncover a crucial role for mitochondrial fission in coordinating astrocytic morphogenesis and organization, revealing the regulation of astrocytic mitochondria dynamics as a critical step in neurodevelopment.

Regulation of the gap junction interplay during postnatal development in the rat epididymis.

During postnatal development of the rat epididymis, a change in the expression of gap junction proteins, or connexins (Cxs), occurs, in which Gjb2 (Cx26) and Gja1 (Cx43) levels in the proximal epididymis are decreased, while Gjb1 (Cx32), Gjb4 (Cx30.3) and Gjb5 (Cx31.1) levels increase. The mechanism(s) responsible for the switch in Cx expression is unknown. The aim of this study is to identify the mechanisms responsible for the decrease in GJB2 protein levels and the increase in other Cxs during postnatal development. Results indicate that decreased Gjb2 expression for 48h does not alter the expression of other Cxs in RCE-1 principal cells, suggesting a lack of compensatory expression. Sequence analysis of both Gjb2 and Gjb1 promoters identified common multiple response elements to steroid hormones. Using RCE-1 cells, we observed that dexamethasone increased Gjb2 mRNA levels by twofold after 48h, while estradiol had no effect. Orchidectomy in rats resulted in a significant increase in GJB2 and decreased GJB1 in the caput and corpus epididymidis. Changes in Cxs protein levels were prevented by testosterone in orchidectomized rats. Similar results were observed in the prostate, another androgen-receptive organ. LNCaP cells, which are androgen-responsive, showed that exogenous dihydrotestosterone (DHT) decreased Gjb2 mRNA levels by approximately 50% concomitant with a 1.5-fold increase in Gjb1 levels. Using a GJB1 promoter construct we showed that DHT could induce transactivation of the luciferase transgene, while transactivation of two GJB2 promoters were unaltered. Results indicate that androgens and glucocorticoids regulate the expression of epididymal Cxs.

GAP JUNCTION FUNCTION IS ESSENTIAL FOR SURVIVAL OF ACUTE LYMPHOBLASTIC LEUKEMIA CELLS.

Acute lymphoblastic leukemia has an intimate physical relationship with nonmalignant bone marrow stromal cells. We have recently demonstrated that stromal cells contribute to the survival of leukemia cells and that there is a bidirectional transfer of intracellular material between them. Understanding the mechanisms of stromal support of leukemia may provide insights into new therapies. To test the hypothesis that gap junctions are formed between acute lymphoblastic leukemia cells and nonmalignant stromal cells, and that gap junction function is essential for the survival of leukemia cells. We employed a well-characterized in vitro model of human bone marrow stromal cells and primary human B lymphoblastic leukemia cells and measured leukemia cell survival in coculture using flow cytometry. We measured the effects of gap junction antagonist peptides, carbenoxolone (a drug known to interfere with the gap junction function), and several leukemia chemotherapy drugs including methotrexate upon leukemia cell survival. We demonstrated that stromal cells need to be alive and metabolically active to keep leukemia cells alive. Physical contact between stromal and leukemia cells leads to an increase in gap junction proteins in leukemia cells. Gap junction inhibitory peptides impaired leukemia cell survival as did carbenoxolone, a nonpeptide inhibitor of the gap junction function. Stromal cell survival was not affected. We observed a very modest enhancement of methotrexate antileukemia activity by low-dose carbenoxolone but no significant interactions with dexamethasone, vincristine, mercaptopurine, or doxorubicin. These studies demonstrate that acute lymphoblastic cell survival is impaired by interference with the gap junction function. The development of drugs targeting gap junctions may provide a novel approach to the therapy of acute lymphoblastic leukemia.

Circ_0049979 ameliorates myocardial infarction through improving Cx43-mediated endothelial functions

Endothelial injury is a fundamental pathogenesis of coronary atherosclerotic heart disease (CHD). Circular RNAs (circRNAs) are important post-transcriptional regulators in many human major diseases, including CHD. The aim of the present study was to explore the role of circ_0049979, a novel identified circRNA from ANO8 gene locus, in endothelial injury during CHD. We found that expression of circ_0049979 was reduced by ox-LDL treatment in HUVECs in a dose-dependent manner. Loss- and gain-of-function experiments demonstrated that knockdown of circ_0049979 decreased the capacities of proliferation, migration and tube formation in normal HUVECs. While, overexpression of circ_0049979 improved these capacities in both normal and ox-LDL-incubated HUVECs. Then, the online bioinformatic tool Circinteractome was used to predicted the target miRNAs of circ_0049979, and miR-653 was selected as the candidate. We demonstrated that miR-653 directly interacted with and was negatively regulated by circ_0049979, and played a negative role in regulating proliferation, migration and tube formation of HUVECs. In terms of the mechanism, miR-653 post-transcriptionally suppressed the expression of the gap junction protein 43 (Cx43), a key protein of endothelial tight junction. Finally, we verified that overexpression of circ_0049979 was able to alleviate plaque formation, lipid deposition, and endothelial cell apoptosis, as well as myocardial infarction, in coronary atherosclerotic mice in vivo. In conclusion, circ_0049979 plays a protective role in coronary atherosclerotic myocardial infarction by improving miR-653/Cx43-mediated endothelial functions.

Morphological, electrophysiological, and molecular alterations in foetal noncompacted cardiomyopathy induced by disruption of ROCK signalling.

Left ventricular noncompaction cardiomyopathy is associated with heart failure, arrhythmia, and sudden cardiac death. The developmental mechanism underpinning noncompaction in the adult heart is still not fully understood, with lack of trabeculae compaction, hypertrabeculation, and loss of proliferation cited as possible causes. To study this, we utilised a mouse model of aberrant Rho kinase (ROCK) signalling in cardiomyocytes, which led to a noncompaction phenotype during embryogenesis, and monitored how this progressed after birth and into adulthood. The cause of the early noncompaction at E15.5 was attributed to a decrease in proliferation in the developing ventricular wall. By E18.5, the phenotype became patchy, with regions of noncompaction interspersed with thick compacted areas of ventricular wall. To study how this altered myoarchitecture of the heart influenced impulse propagation in the developing and adult heart, we used histology with immunohistochemistry for gap junction protein expression, optical mapping, and electrocardiography. At the prenatal stages, a clear reduction in left ventricular wall thickness, accompanied by abnormal conduction of the ectopically paced beat in that area, was observed in mutant hearts. This correlated with increased expression of connexin-40 and connexin-43 in noncompacted trabeculae. In postnatal stages, left ventricular noncompaction was resolved, but the right ventricular wall remained structurally abnormal through to adulthood with cardiomyocyte hypertrophy and retention of myocardial crypts. Thus, this is a novel model of self-correcting embryonic hypertrabeculation cardiomyopathy, but it highlights that remodelling potential differs between the left and right ventricles. We conclude that disruption of ROCK signalling induces both morphological and electrophysiological changes that evolve over time, highlighting the link between myocyte proliferation and noncompaction phenotypes and electrophysiological differentiation.

A 3D hydrogel scaffold with adjustable interlayer spacing for the formation of compact myocardial tissue

AbstractThe design and optimization of three‐dimensional tissue scaffolds have presented a persistent challenge for myocardial tissue engineering. While current scaffolds have made strides in mimicking in vivo three‐dimensional environments, the presence of interlayer spacing has limited the formation of densely packed tissue, impeding cell‐to‐cell connections. We proposed a novel dynamic three‐dimensional myocardial tissue engineering scaffold with adjustable interlayer spacing, aiming to improve the uniformity of cell distribution and facilitate effective cell communication. The device was composed of hydrogel scaffolds that were fabricated with poly (ethylene glycol) diacrylate (PEGDA) and flexible elastomer actuators that composed of polyurethane acrylate (PUA). Human induced pluripotent stem cell‐derived cardiomyocytes mixed with gelatin methacryloyl (GelMA) were seeded onto the multi‐layered scaffold with initial spacing of 100–500 μm. Experimental results indicated the seeding efficiency was maximized at an initial spacing of 400 μm. By decreasing the interlayer spacing, cell growth morphology and gap junction protein expression was improved. This work demonstrated the effect of interlayer spacing modulation to the tissue density and provided a potential approach for cultivation of compact and multi‐layered myocardial tissue.

Hepsin as a potential therapeutic target for alleviating acetaminophen-induced hepatotoxicity via gap-junction regulation and oxidative stress modulation

Acetaminophen (APAP) overdose is a leading cause of drug-induced liver damage, highlighting the limitations of current emergency treatments that primarily involve administering the glutathione precursor N-acetylcysteine and supportive therapy. This study highlights the essential protective role of the type II transmembrane serine protease (TTSP), hepsin, in mitigating acetaminophen-induced liver injury, particularly through its regulation of gap junction (GJ) abundance in response to reactive oxygen stress in the liver. We previously reported that reduced levels of activated hepatocyte growth factor and the c-Met receptor tyrosine kinase—both of which are vital for maintaining cellular redox balance—combined with increased expression of GJ proteins in hepsin-deficient mice. Here, we show that hepsin deficiency in mice exacerbates acetaminophen toxicity compared to wild-type mice, leading to more severe liver pathology, elevated oxidative stress, and greater mortality within 6 h after exposure. Administering hepsin had a protective effect in both mouse models, reducing hepatotoxicity by modulating GJ abundance. Additionally, transcriptome analysis and a functional GJ inhibitor have highlighted hepsin's mechanism for managing oxidative stress. Combining hepsin with relatively low doses of N-acetylcysteine had a synergistic effect that was more efficacious than high-dose N-acetylcysteine alone. Our results illustrate the crucial role of hepsin in modulating the abundance of hepatic GJs and reducing oxidative stress, thereby offering early protection against acetaminophen-induced hepatotoxicity and a new, combination approach. Emerging as a promising therapeutic target, hepsin holds potential for combination therapy with N-acetylcysteine, paving the way for novel approaches in managing drug-induced liver injury.Graphical 1. Hepsin−/− mice exhibit exacerbated APAP toxicity, resulting in more severe liver damage, elevated oxidative stress, and higher mortality.2. Hepsin is crucial in protecting against APAP-induced liver injury by regulating gap junctions and reducing oxidative stress.3. Combining hepsin with low doses of N-acetylcysteine provides greater protection against APAP-induced hepatotoxicity than high-dose NAC alone.

Neurotropic Murine β-Coronavirus Infection Causes Differential Expression of Connexin 47 in Oligodendrocyte Subpopulations Associated with Demyelination.

Gap junctions (GJs) play a crucial role in the survival of oligodendrocytes and myelination of the central nervous system (CNS). In this study, we investigated the spatiotemporal changes in the expression of oligodendroglial GJ protein connexin 47 (Cx47), its primary astroglial coupling partner, Cx43, and their association with demyelination following intracerebral infection with mouse hepatitis virus (MHV). Neurotropic strains of MHV, a β-coronavirus, induce an acute encephalomyelitis followed by a chronic demyelinating disease that shares similarities with the human disease multiple sclerosis (MS). Our results reveal that Cx47 GJs are persistently lost in mature oligodendrocytes, not only in demyelinating lesions but also in surrounding normal appearing white and gray matter areas, following an initial loss of astroglial Cx43 GJs during acute infection. At later stages after viral clearance, astroglial Cx43 GJs re-emerge but mature oligodendrocytes fail to fully re-establish GJs with astrocytes due to lack of Cx47 GJ expression. In contrast, at this later demyelinating stage, the increased oligodendrocyte precursor cells appear to exhibit Cx47 GJs. Our findings further highlight varying degrees of demyelination in distinct spinal cord regions, with the thoracic cord showing the most pronounced demyelination. The regional difference in demyelination correlates well with dynamic changes in the proportion of different oligodendrocyte lineage cells exhibiting differential Cx47 GJ expression, suggesting an important mechanism of progressive demyelination even after viral clearance.