Gap Junctional Communication Research Articles

The Polyamine Spermine Potentiates the Propagation of Negatively Charged Molecules through the Astrocytic Syncytium.

The interest in astrocytes, the silent brain cells that accumulate polyamines (PAs), is growing. PAs exert anti-inflammatory, antioxidant, antidepressant, neuroprotective, and other beneficial effects, including increasing longevity in vivo. Unlike neurons, astrocytes are extensively coupled to others via connexin (Cx) gap junctions (GJs). Although there are striking modulatory effects of PAs on neuronal receptors and channels, PA regulation of the astrocytic GJs is not well understood. We studied GJ-propagation using molecules of different (i) electrical charge, (ii) structure, and (iii) molecular weight. Loading single astrocytes with patch pipettes containing membrane-impermeable dyes, we observed that (i) even small molecules do not easily permeate astrocytic GJs, (ii) the ratio of the charge to weight of these molecules is the key determinant of GJ permeation, (iii) the PA spermine (SPM) induced the propagation of negatively charged molecules via GJs, (iv) while no effects were observed on propagation of macromolecules with net-zero charge. The GJ uncoupler carbenoxolone (CBX) blocked such propagation. Taken together, these findings indicate that SPM is essential for astrocytic GJ communication and selectively facilitates intracellular propagation via GJs for negatively charged molecules through glial syncytium.

Resident macrophages of the lung and liver: The guardians of our tissues.

Resident macrophages play a unique role in the maintenance of tissue function. As phagocytes, they are an essential first line defenders against pathogens and much of the initial characterization of these cells was focused on their interaction with viral and bacterial pathogens. However, these cells are increasingly recognized as contributing to more than just host defense. Through cytokine production, receptor engagement and gap junction communication resident macrophages tune tissue inflammatory tone, influence adaptive immune cell phenotype and regulate tissue structure and function. This review highlights resident macrophages in the liver and lung as they hold unique roles in the maintenance of the interface between the circulatory system and the external environment. As such, we detail the developmental origin of these cells, their contribution to host defense and the array of tools these cells use to regulate tissue homeostasis.

Exploration of connexin-43 modulating, multifunctional silver nanocluster-hydrogel system for theranostic management of cancer

Connexin (Cx), a gap junction protein plays an important role in cancer pathophysiology. The enhancement of Cx43, an isoform of Cx, has been reported to improve survival of patients with breast cancer. Since Cxs are expressed in various tissues and organs, their localized modulation is necessary to achieve therapeutic benefits without any side-effects. Therapy combined with diagnostics, i.e. theranostics, is becoming an important facet of cancer management. Thus, considering these two critical aspects, the present study deals with synthesis, bio-imaging, and anti-proliferative effects of a multi-functional chitosan hydrogel incorporating highly fluorescent silver nanoclusters and 4-phenylbutyrate, a histone deacetylase inhibitor for enhancing Cx43 expression. While the remarkable fluorescence of silver nanoclusters enabled cellular uptake and imaging, 4-phenylbutyrate released from the hydrogel specifically upregulated Cx43 expression and induced apoptosis in breast cancer cells. Moreover, the 4-phenylbutyrate released, successfully established gap junction communication between cancer cells as assessed by dye transfer assay. Thus, the current module not only holds potential in cancer theranostics but also for Cx-based cancer therapeutics.

L-Proline improves the cytoplasmic maturation of mouse oocyte by regulating glutathione-related redox homeostasis

Recent studies have shown that l-proline (proline) is an antioxidant to protect cells from oxidative stress in vivo and in vitro. Glutathione (GSH) is a major cellular redox regulator involved in controlling redox balance and is regarded as one of the key indices to predict the cytoplasmic maturation of oocytes. The objectives of this study are to investigate the effect of proline on the developmental potential of mouse oocytes and to determine the role of gap junctional communication (GJC) on intraoocyte GSH concentration during in vitro maturation (IVM). Compared with control (0 mmol/L), 0.5 mmol/L proline supplementation enhanced rates of activated oocytes, 2-cell and 4-cell embryos, and blastocysts. Furthermore, 0.25 and 0.5 mmol/L proline supplementation markedly upregulated mRNA expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) in oocytes and cumulus cells, enhanced GSH concentration in oocytes, and reduced reactive oxygen species (ROS) level in oocytes. Interestingly, carbenoxolone disodium salt (CBX) treatment reduced GSH concentration in oocytes and the rate of early embryo development without proline incubation. Notably, CBX-triggered reduction in the rates of the number of 2-cell and 4-cell embryos and blastocysts were rescued by 0.5 mmol/L proline supplementation. Collectively, these results indicate a novel functional role of proline in oocyte cytoplasmic maturation and regulation of glutathione-related redox homeostasis.

The Bioactive Phenolic Agents Diaryl Ether CVB2-61 and Diarylheptanoid CVB4-57 as Connexin Hemichannel Blockers.

Inflammation mediators enhance the activity of connexin (Cx) hemichannels, especially in the epithelial and endothelial tissues. As potential release routes for injury signals, such as (oligo)nucleotides, Cx hemichannels may contribute to long-lasting inflammation. Specific inhibition of Cx hemichannels may therefore be a mode of prevention and treatment of long-lasting, chronic sterile inflammation. The activity of Cx hemichannels was analysed in N2A and HeLa cells transfected with human Cx26 and Cx46 as well as in Calu-3 cells, using dye uptake as functional assay. Moreover, the possible impacts of the bioactive phenolic agents CVB2-61 and CVB4-57 on the barrier function of epithelial cells was analysed using Calu-3 cells. Both agents inhibited the dye uptake in N2A cells expressing Cx26 (>5 µM) and Cx46 (>20 µM). In Calu-3 cells, CVB2-61 and CVB4-57 reversibly inhibited the dye uptake at concentrations as low as 5 µM, without affecting the gap junction communication and barrier function, even at concentrations of 20 µM. While CVB2-61 or CVB4-57 maintained a reduced dye uptake in Calu-3 cells, an enhancement of the dye uptake in response to the stimulation of adenosine signalling was still observed after removal of the agents. The report shows that CVB2-61 and CVB4-57 reversibly block Cx hemichannels. Deciphering the mechanisms of the interactions of these agents with Cx hemichannels could allow further development of phenolic compounds to target Cx hemichannels for better and safer treatment of pathologies that involve Cx hemichannels.

TNF-α Plus IL-1β Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1β increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1β treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1β-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell–cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Connexin 32 overexpression increases proliferation, reduces gap junctional intercellular communication, motility and epithelial-to-mesenchymal transition in Hs578T breast cancer cells.

Connexins (Cx) are primary components of gap junctions that selectively allow molecules to be exchanged between adjacent cells, regulating multiple cellular functions. Along with their channel forming functions, connexins play a variety of roles in different stages of tumorigenesis and their roles in tumor initiation and progression is isoform- and tissue-specific. While Cx26 and Cx43 were downregulated during breast tumorigenesis, Cx32 was accumulated in the cytoplasm of the cells in lymph node metastasis of breast cancers and Cx32 was further upregulated in metastasis. Cx32's effect on cell proliferation, gap junctional communication, hemichannel activity, cellular motility and epithelial-to-mesenchymal transition (EMT) were investigated by overexpressing Cx32 in Hs578T and MCF7 breast cancer cells. Additionally, the expression and localization of Cx26 and Cx43 upon Cx32 overexpression were examined by Western blot and immunostaining experiments, respectively. We observed that MCF7 cells had endogenous Cx32 while Hs578T cells did not and when Cx32 was overexpressed in these cells, it caused a significant increase in the percentages of Hs578T cells at the S phase in addition to increasing their proliferation. Further, while Cx32 overexpression did not induce hemichannel activity in either cell, it decreased gap junctional communication between Hs578T cells. Additionally, Cx32 was mainly observed in the cytoplasm in both cells, where it did not form gap junction plaques but Cx32 overexpression reduced Cx43 levels without affecting Cx26. Moreover, migration and invasion potentials of Hs578T and migration in MCF7 were reduced upon Cx32 overexpression. Finally, the protein level of mesenchymal marker N-cadherin decreased while epithelial marker ZO-1 and E-cadherin increased in Hs578T cells. We observed that Cx32 overexpression altered cell proliferation, communication, migration and EMT in Hs578T, suggesting a tumor suppressor role in these cells while it had minor effects on MCF7 cells.

O-223 Latest development of IVM in assisted reproduction

Abstract Latest development of IVM in assisted reproduction In vitro maturation (IVM) is an assisted reproductive technology involving collection of immature cumulus-oocyte complexes at the prophase I stage, which are then matured to the metaphase II stage in vitro. IVM has several advantages over IVF, including negligible risk of ovarian hyperstimulation syndrome, lower medication costs, and patient convenience. Given that IVM can be done within a relatively short time frame without the need for ovarian stimulation, it is particularly useful for fertility preservation in patients with cancer who are unable to delay chemotherapy, or in women with breast cancer for whom exposure to elevated estradiol concentrations may accelerate their disease. The European Society of Human Reproduction and Embryology (ESHRE) Guideline on Female Fertility Preservation published in 2020 suggested IVM as a fertility preservation technique. The Practice Committees of ASRM in 2021 published the document that presented an overview of published evidence supporting the conclusion that IVM should no longer be considered an experimental technique. The potential for wider clinical application of IVM was suggested. Patient populations particularly suited to the use of IVM include women with polycystic ovaries, have higher risk of ovarian hyperstimulation syndrome, and those requiring fertility preservation. During the last two decades, IVM has been utilized for patients with PCOS to reduce the health risks associated with ovarian hyperstimulation syndrome, for fertility preservation, or just as an alternative, more user-friendly approach to ART. The effectiveness of IVM relies on successful synchronization of meiotic and cytoplasmic maturation of oocytes. Efforts to improve the efficacy of IVM have included the use of new IVM culture systems, aimed at enhancing the competence of IVM oocytes. Recently, the use of oocyte prematuration (or pre-IVM) culture systems to prevent spontaneous in vitro maturation processes and maintain cumulus-oocyte gap junctional communication have been described in humans. Over the last five years, a new biphasic IVM culture system has been developed to improve the efficacy of IVM. Recent promising data from this new IVM culture system, called capacitation (CAPA) IVM, showed promising results. CAPA-IVM involves successfully maturing oocytes from small 2–8 mm follicles with no hCG injected prior to oocyte retrieval. Recently, a large RCT compared the efficacy of CAPA IVM and conventional IVF/ICSI in women with PCO showed that CAPA (biphasic) IVM was non-inferior to IVF in term of live birth rate after the first embryo transfer. One of the concerns regarding IVM is the health of babies after utilization of this procedure. Based on currently available data, IVM appears to be safe from a neonatal health and childhood development perspective. There is still room for further development of more efficient IVM protocols (89). Recent knowledge about oocyte physiology and development can be translated into clinical practice to improve the efficacy of these protocols. There are several reasons why all major modern ART centers should have IVM facilities and protocols available. These include the likelihood of wider application of IVM in the future, the requirement to manage indications where IVM is the only option (e.g. fertility preservation, GROS), the increasing need for more patient-friendly ART treatment, and the fact that IVM protocols are improving thanks to advances in knowledge on human follicular and oocyte development.

From Isles of Königsberg to Islets of Langerhans: Examining the Function of the Endocrine Pancreas Through Network Science.

Islets of Langerhans are multicellular microorgans located in the pancreas that play a central role in whole-body energy homeostasis. Through secretion of insulin and other hormones they regulate postprandial storage and interprandial usage of energy-rich nutrients. In these clusters of hormone-secreting endocrine cells, intricate cell-cell communication is essential for proper function. Electrical coupling between the insulin-secreting beta cells through gap junctions composed of connexin36 is particularly important, as it provides the required, most important, basis for coordinated responses of the beta cell population. The increasing evidence that gap-junctional communication and its modulation are vital to well-regulated secretion of insulin has stimulated immense interest in how subpopulations of heterogeneous beta cells are functionally arranged throughout the islets and how they mediate intercellular signals. In the last decade, several novel techniques have been proposed to assess cooperation between cells in islets, including the prosperous combination of multicellular imaging and network science. In the present contribution, we review recent advances related to the application of complex network approaches to uncover the functional connectivity patterns among cells within the islets. We first provide an accessible introduction to the basic principles of network theory, enumerating the measures characterizing the intercellular interactions and quantifying the functional integration and segregation of a multicellular system. Then we describe methodological approaches to construct functional beta cell networks, point out possible pitfalls, and specify the functional implications of beta cell network examinations. We continue by highlighting the recent findings obtained through advanced multicellular imaging techniques supported by network-based analyses, giving special emphasis to the current developments in both mouse and human islets, as well as outlining challenges offered by the multilayer network formalism in exploring the collective activity of islet cell populations. Finally, we emphasize that the combination of these imaging techniques and network-based analyses does not only represent an innovative concept that can be used to describe and interpret the physiology of islets, but also provides fertile ground for delineating normal from pathological function and for quantifying the changes in islet communication networks associated with the development of diabetes mellitus.

Anchored-PKA regulates connexin-43 gap junction communication in the heart

Cardiac connexin-43 (Cx43) forms gap junction (GJ) channels at the intercalated discs (ID) of cardiomyocytes ensuring anisotropic action potential (AP) propagation. This process is essential for sequential and coordinated contraction of the heart. Stimulation of β-adrenergic receptors activates protein kinase A (PKA), which in turn increases electrical conduction velocity. Myocardial infarction (MI) is characterized by a prolonged ischemia leading to irreversible ventricular cardiomyocytes death and impaired cardiac function. To compensate this loss, chronic activation of PKA signaling is established. In addition, post-MI, Cx43 subcellular localization is altered affecting GJ communication and AP propagation leading to uncoordinated contractions and fatal arrhythmias. Considering the key role of Cx43 and PKA signaling in AP propagation, we investigated the potential regulation of GJ communication by PKA under cardiac physiological condition and post-MI. First, we assessed Cx43 phosphorylation with PKA specific inhibition and with a peptide displacing PKA anchoring from A-Kinase Anchoring Protein (AKAPs). Then, we identified the ezrin as the AKAP mediated the regulation of Cx43 phosphorylation by immunoprecipitation, immunofluorescence and siRNA mediated knockdown experiments. We assessed by functional GAP-FLIP experiments the key role of ezrin-PKA complex in the regulation of intercellular communication. Finally, we generated a left anterior descending coronary artery model of MI in rats to evaluate by proximity ligation assay and western blot experiments the Cx43-ezrin-PKA complex localization and expression. We point that ezrin forms a supramolecular complex with PKA and Cx43 at the ID of cardiomyocytes. Ezrin regulates Cx43 phosphorylation and GJ communication in cardiomyocytes. Interestingly, post-MI we noticed a significant reduction in Cx43 level and phosphorylation associated with a decrease in proximity with ezrin at the ID. Our data identified a cardiac PKA-ezrin-Cx43 complex regulating Cx43 phosphorylation, and therefore, GJ communication. In addition, we reported a loss of proximity between ezrin and Cx43 post-MI. We proposed that this may explain partly an impaired AP propagation leading to uncoordinated contractions post MI.

Dynamics of Connexin 43 Down Modulation in Human Articular Chondrocytes Stimulated by Tumor Necrosis Factor Alpha.

Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (−46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-β1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-β1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.

Mechanosensitive Responses of Resident Alveolar

BackgroundThe extent of micromechanical coupling between resident alveolar macrophages (AMs) and the surrounding tissue niche remains unclear. Lung overexpansion due to therapeutic mechanical ventilation imposes distortional stresses on the alveolar epithelium (AE), causing acute lung injury (ALI). Sessile AMs, lying adjacent to the AE, are causally implicated in ALI. However, it is not clear whether micromechanical coupling with the AE induces proinflammatory distortional stresses in sessile AMs. We addressed this question in live mouse lungs by real‐time confocal microscopy (RCM).MethodsFor RCM, we inflated and blood‐perfused mouse lungs at physiological pressures. To detect cytosolic Ca2+ (cCa2+), we microinfused fluo‐4 by alveolar micropuncture. To detect sessile AMs, we microinfused fluorophore‐conjugated anti‐Siglec‐F and anti‐CD11c Abs. To induce transient lung overexpansion, we increased the alveolar pressure from 5 to 15 cmH2O for 15 seconds. We determined alveolar diameter in terms of fluo‐4 fluorescence, which marked the epithelial margins. We determined cell diameter of sessile AMs in terms of Siglec‐F fluorescence, which marked the cell boundary. We detected gap junctional communication (GJC) by fluorescence recovery after photobleaching (FRAP).ResultsSessile AMs were exclusively located in non‐distending microniches of the alveolus and therefore, did not stretch during lung expansion. Nevertheless, a subset of AMs responded to lung overexpansion by transiently increasing cCa2+. The cCa2+ increase initiated in 5 min, reaching peak at two‐times baseline in 20 min, then subsiding to baseline after a further 20 min. Concomitantly, in AE adjacent to AMs, there was marked increase in cCa2+ oscillations, but no increase in mean cCa2+. In lungs with AM‐specific Cx43 deletion, the transient cCa2+ response was blocked in AMs, but not in adjoining AE. FRAP revealed GJC only in cCa2+‐responsive AMs. In paired comparisons, AMs lacking GJC failed to show expansion‐induced Ca2+ responses (p<0.05).ConclusionsWe report the unexpected finding that sessile AMs were exclusively located in immobile microniches of alveoli and therefore did not stretch during lung expansion. Nevertheless, following expansion there was Cx43‐mediated Ca2+communication with the AE, causing cCa2+ increases in sessile AMs. Although further studies are required to evaluate the potentially proinflammatory responses induced by this Ca2+ response, we propose Cx43 of sessile AMs might be a therapeutic target to mitigate lung expansion‐induced ALI.

Flavonoid Nobiletin Attenuates Cyclophosphamide-Induced Cystitis in Mice through Mechanisms That Involve Inhibition of IL-1β Induced Connexin 43 Upregulation and Gap Junction Communication in Urothelial Cells.

Bladder inflammatory diseases cause various urinary symptoms, such as urinary frequency and painful urination, that impair quality of life. In this study, we used a mouse model of cyclophosphamide (CYP)-induced bladder inflammation and immortalized human urothelial (TRT-HU1) cells to explore the preventive potential of nobiletin (NOB), a polymethoxylated flavone enriched in citrus fruit peel, and investigate its mechanism of action in the bladder. Prophylaxis with PMF90 (60% NOB) attenuated the development of bladder inflammation and urinary symptoms in CYP-treated mice. PMF90 also reduced the upregulation of connexin 43 (Cx43), a major component of gap junction channels, in the bladder mucosa of CYP-treated mice. Stimulation of TRT-HU1 cells with the pro-inflammatory cytokine IL-1β increased Cx43 mRNA and protein expression and enhanced gap junction coupling—responses that were prevented by pre-treatment with NOB. In urothelium-specific Cx43 knockout (uCx43KO) mice, macroscopic signs of bladder inflammation and changes in voiding behavior induced by CYP treatment were significantly attenuated when compared to controls. These findings indicate the participation of urothelial Cx43 in the development of bladder inflammation and urinary symptoms in CYP-treated mice and provide pre-clinical evidence for the preventive potential of NOB through its anti-inflammatory effects on IL-1β signaling and urothelial Cx43 expression.

Neuroimmunomodulatory balance between pro and anti‐inflammatory cytokines regulate m‐CoV induced alteration of gap junction protein Cx43

Mouse hepatitis virus (MHV), a murine β‐CoV, is an experimental model used to understand the viral‐induced acute neuroinflammation and chronic progressive demyelination, which are the characteristic hallmarks of the human neurological disease Multiple Sclerosis. MHV induced neuroinflammation provides an excellent cause‐effective platform to understand how the virus can initiate neuroinflammation by causing direct neuroglial cell dystrophy, leading to chronic progressive myelin damage with or without concurrent axonal loss. Previous studies have postulated that a significant nexus between host proteins is also involved in regulating host response to viral replication. One such protein is a transmembrane protein connexin 43 (Cx43) belonging to the gap junction family. Cx43 is one of the most abundant gap junction proteins in astrocytes responsible for metabolic coupling with other CNS cells to maintain homeostasis. MHV infection in mice results in a significant decrease in the expression of Cx43 during the acute stage (day 5‐6) of MHV infection when viral titer reaches its peak and the brain presents with acute encephalitis characterized by perivascular cuffing and microglial nodule formation. Surprisingly, Cx43 returns to normal levels as infectious viral particles go below the detection limit by day 10 p.i. which marks the transition of innate immune responses to adaptive immunity. Interestingly, this reduced expression of Cx43 at Day 5 accords with increased pro‐inflammatory cytokines, oxidative stress, and activation of UPR pathway proteins. This study investigated the transcriptional regulation of TNF‐α, Il‐6, IL‐10, IL‐1β and TGF‐β in the alteration of Cx43 expression and trafficking during day 5/6 and day 10 post MHV infection. TNF‐α, IL‐10, IL‐6,IL‐1β are the major inflammatory cytokines upregulated during the acute stage of MHV infection (5‐6 p.i.). Additionally, TNF‐α and IL‐1β are known to be negative regulators of Cx43 expression and could be attributed to the decrease in Cx43 observed in our studies. Furthermore, our studies also revealed that in MHV infection, apart from the very high expression of IL‐10 another anti‐inflammatory cytokine TGF‐β expression increases when proinflammatory milieu of cytokines shifts towards the anti‐inflammatory condition in the inflamed brain. As TGF‐β is a positive regulator of Cx43, it may be responsible for the restoration of Cx43 expression and its trafficking to the cell surface resulting in functional gap junctional communication. This re‐establishment of intercellular communication maybe essential to bring back the homeostasis in the inflamed brain. The balance between pro‐inflammatory cytokines and the anti‐inflammatory mediators may regulate the host response to counteract the viral infection by altering the gap junctional communication. This study thus presents crucial evidence to support the nexus between immune inflammatory mediators and host regulatory responders in MHV induced neuroinflammation.

Adenovirus increases arrhythmia susceptibility during acute cardiac infection

Myocarditis is responsible for 42% of sudden cardiac death in young adults, yet mechanisms underlying virally induced arrhythmia remain elusive. Adenovirus is a leading etiological agent of myocarditis but species‐specificity has limited development of animal disease models. Normal electrical impulse propagation in the heart is achieved through intercellular coupling via gap junctions, composed primarily of connexin43 (Cx43). Changes in Cx43 expression, localization, and/or function underlie the arrhythmias of sudden cardiac death. Given that gap junctions also propagate innate and adaptive antiviral immune responses, we hypothesized that adenovirus would target Cx43 to facilitate replication. Our prior work has demonstrated that Cx43 expression and function are indeed reduced rapidly during adenoviral infection limiting gap junction communication in human epithelial and cardiac cells. We are now employing a recently described cardiotropic strain of mouse adenovirus, MAdV‐3, to develop our work into whole animal studies and investigate virally‐induced alterations in cardiac electrophysiology and the molecular mechanisms of the resulting arrhythmogenic substrate. Adult mice were inoculated with MAdV‐3 and sampled after 7 days to model acute cardiac infection. We find MAdV‐3 viral genomes are specifically enriched in heart tissue, confirming cardiotropism. No cardiomyopathy was apparent by echocardiography or histopathology, consistent with human acute myocarditis patients. We find reductions in cardiac ion channel and connexin mRNA transcript levels after infection, and at the protein level, Cx43 is phosphorylated at residues known to reduce function. Ex vivo optical mapping experiments illustrate decreased conduction velocity in infected hearts. Turning to primary adult mouse cardiomyocytes, we detect prolonged action potential duration and impaired K+ current in infected cells by patch clamping. Confocal and super‐resolution localization microscopy of infected cardiac tissue and isolated cardiomyocytes reveals remodeling of gap junctions with alterations in complexing with scaffolding proteins and other ion channels at the cell‐cell junction. Finally, employing human iPSC‐derived cardiomyocytes and human adenovirus type‐5, we find increased Cx43 phosphorylation and, using optical mapping, perturbation of intercellular coupling during infection, just as we see with MAdV‐3. Our data demonstrate that reduced cellular coupling and ion channel function during adenoviral infection generates an arrhythmogenic substrate prior to an appreciable immune response or cardiomyopathy development. MAdV‐3 infection therefore provides a novel model of cardiac infection and myocarditis and provides physiologically relevant insight into mechanisms of virally‐induced arrhythmias.

The Therapeutic Effect and the Possible Mechanism of C-Phycocyanin in Lipopolysaccharide and Seawater-Induced Acute Lung Injury

BackgroundSeawater drowning-induced acute lung injury (ALI) is a severe clinical condition characterized by increased alveolar-capillary permeability, excessive inflammatory response, and refractory hypoxemia. C-phycocyanin (C-PC), a biliprotein found in blue-green algae such as spirulina platensis, is widely used in the food and dietary nutritional supplement fields due to its beneficial pharmacological effects. Previous studies have revealed that C-PC has anti-inflammatory, antioxidant, and anti-apoptotic activities.PurposeTherefore, this study investigated the protective effect and underlying mechanisms of C-PC on lipopolysaccharide (LPS) and seawater (SW) induced ALI (SW and LPS-induced ALI).MethodsAn SW and LPS mouse model of ALI mice was established through intratracheal administration of 5mg/kg LPS and 25% SW. Different doses of C-PC (100, 200 and 400 mg/kg) were administered by intraperitoneal injection for seven days. In addition, gap junction communication in RAW264.7 and MLE-12 cells was determined following stimulation with 25% SW and 10 μg/ml LPS after treatment with C-PC (120 μg/ml). Moreover, the arterial partial pressure of oxygen, lung wet/dry weight ratios, total protein content and MPO levels in the bronchoalveolar lavage fluid (BALF), and the histopathologic and ultrastructure staining of the lung tissues were determined. The oxidative stress index, levels of the pro-inflammatory mediators, epithelial cell viability and apoptosis, and the regulatory effect of C-PC on the NF-κB/NLRP3 axis were investigated.ResultsThe results showed that C-PC significantly alleviated pathological damages, suppressed oxidative stress, inflammation and apoptosis, and enhanced the viability of epithelial cells in the lung tissues. Furthermore, C-PC was shown to inhibit activation of the NF-κB/NLRP3 pathway and the formation of the NLRP3 inflammasome complex.ConclusionsIn conclusion, C-PC shows promising therapeutic value in SW and LPS-induced ALI/ARDS, providing new insight into ALI/ARDS treatment.

L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9.

This study was designed to investigate the effect of different concentrations of L-cysteine supplementation into the maturation medium on the oocyte nuclear maturation, cumulus cell expansion, ultrastructure of the oocytes and the expression of oocyte-derived growth factors BMP-15, GDF-9 and CB-1 genes. Cumulus oocyte complexes (COCs) were collected from cow's ovaries obtained from abattoir and incubated at 38.5°C in maturation media supplemented with 0, 0.6, 0.8 or 1mM L-cysteine in 5% CO2 under humidified air for 24hr. We found that a significantly higher percentage of oocytes progressed to metaphase II stage in the in vitro maturation (IVM) medium supplemented with L-cysteine, particularly 0.8mM group, compared with untreated control oocytes. Additionally, L-cysteine treatment significantly increased the number of expanded COCs and the degree of expansion of individual COCs. Results of RT-qPCR showed significant increase in expression levels of BMP-15 and GDF-9 in L-cysteine-treated groups compared with control one. Electron microgram showed improvement of cytoplasmic maturation regarding ultrastructure of the oocytes and oocyte-cumulus cell gap junction communication in all L-cysteine-treated groups especially 0.8mM L-cysteine-treated one. In conclusion, supplementation of IVM medium with a potential anti-oxidant, L-cysteine can effectively improve in vitro oocytes cytoplasmic and nuclear maturation via activation of oocyte maturation related BMP-15 and GDF-9 genes in bovine oocytes, benefiting the extended researches about the potential applications of L-cysteine in mammalian breeding technologies.

Correlations Between the Expression of Stromal Cell Activation Related Biomarkers, L-NGFR, Phospho-ERK1-2 and CXCL12, and Primary Myelofibrosis Progression.

In myelofibrosis, pathologically enhanced extracellular matrix production due to aberrant cytokine signalling and clonal megakaryocyte functions result(s) in impaired hemopoiesis. Disease progression is still determined by detecting reticulin and collagen fibrosis with Gomori’s silver impregnation. Here, we tested whether the expression growth related biomarkers L-NGFR/CD271, phospho-ERK1-2 and CXCL12 can be linked to the functional activation of bone marrow stromal cells during primary myelofibrosis progression. Immunoscores for all tested biomarkers showed varying strength of positive statistical correlation with the silver impregnation based myelofibrosis grades. The intimate relationship between spindle shaped stromal cells positive for all three markers and aberrant megakaryocytes was likely to reflect their functional cooperation. L-NGFR reaction was restricted to bone marrow stromal cells and revealed the whole length of their processes. Also, L-NGFR positive cells showed the most intersections, the best statistical correlations with myelofibrosis grades and the strongest interrater agreements. CXCL12 reaction highlighted stromal cell bodies and a weak extracellular staining in line with its constitutive release. Phospho-ERK1-2 reaction showed a similar pattern to CXCL12 in stromal cells with an additional nuclear staining in agreement with its role as a transcription factor. Both p-ERK1-2 and CXCL12 were also expressed at a moderate level in sinus endothelial cells. Connexin 43 gap junction communication channels, known to be required for CXCL12 release to maintain stem cell niche, were also expressed progressively in the myelofibrotic stromal network as a support of compartmental functions. Our results suggest that, diverse growth related pathways are activated in the functionally coupled bone marrow stromal cells during myelofibrosis progression. L-NGFR expression can be a useful biological marker of stromal cell activation which deserves diagnostic consideration for complementing Gomori’s silver impregnation.

The Role of GJD2(Cx36) in Refractive Error Development.

Refractive errors are common eye disorders characterized by a mismatch between the focal power of the eye and its axial length. An increased axial length is a common cause of the refractive error myopia (nearsightedness). The substantial increase in myopia prevalence over the last decades has raised public health concerns because myopia can lead to severe ocular complications later in life. Genomewide association studies (GWAS) have made considerable contributions to the understanding of the genetic architecture of refractive errors. Among the hundreds of genetic variants identified, common variants near the gap junction delta-2 (GJD2) gene have consistently been reported as one of the top hits. GJD2 encodes the connexin 36 (Cx36) protein, which forms gap junction channels and is highly expressed in the neural retina. In this review, we provide current evidence that links GJD2(Cx36) to the development of myopia. We summarize the gap junctional communication in the eye and the specific role of GJD2(Cx36) in retinal processing of visual signals. Finally, we discuss the pathways involving dopamine and gap junction phosphorylation and coupling as potential mechanisms that may explain the role of GJD2(Cx36) in refractive error development.

Activation of the unfolded protein response by Connexin47 mutations associated with Pelizaeus-Merzbacher-like disease

Pelizaeus-Merzbacher-like disease type 1 (PMLD1) is a hypomyelinating disorder arising in patients with mutations in GJC2, encoding Connexin47 (Cx47). PMLD1 causes nystagmus, cerebellar ataxia, spasticity and changes in CNS white matter detected by MRI. At least one mutation (p.I33M) yields a much milder phenotype, spastic paraplegia type 44 (SPG44). Cx47 contributes to gap junction communication channels between oligodendrocytes (OLs), the myelinating cells in the central nervous system (CNS), and between OLs and astrocytes. Prior studies in cell lines have shown that PMLD1 mutants such as p.P87S display defective protein trafficking, intracellular retention in the ER and loss-of-function. Here we show that when expressed in primary OLs, three PMLD1 associated mutants (p.P87S, p.Y269D and p.M283T) show ER retention of Cx47 and evidence of activation of the cellular stress (unfolded protein response, UPR) and apoptotic pathways. On the other hand, the milder SPG44 associated mutation p.I33M shows a wild-type-like subcellular distribution and no activation of the UPR or apoptotic pathways. These studies provide new insight into a potential element of toxic gain of function underlying the mechanism of PMLD1 that should help guide future therapeutic approaches.