Fibroblasts in heart scar tissue directly regulate cardiac excitability and arrhythmogenesis.

After heart injury, dead heart muscle is replaced by scar tissue. Fibroblasts can electrically couple with myocytes, and changes in fibroblast membrane potential can lead to myocyte excitability, which suggests that fibroblast-myocyte coupling in scar tissue may be responsible for arrhythmogenesis. However, the physiologic relevance of electrical coupling of myocytes and fibroblasts and its impact on cardiac excitability in vivo have never been demonstrated. We genetically engineered a mouse that expresses the optogenetic cationic channel ChR2 (H134R) exclusively in cardiac fibroblasts. After myocardial infarction, optical stimulation of scar tissue elicited organ-wide cardiac excitation and induced arrhythmias in these animals. Complementing computational modeling with experimental approaches, we showed that gap junctional and ephaptic coupling, in a synergistic yet functionally redundant manner, excited myocytes coupled to fibroblasts.

Connexin 43 participates in atrial electrical remodelling through colocalization with calcium channels in atrial myocytes.

Atrial fibrillation (AF) is associated with atrial conduction disturbances caused by electrical and/or structural remodelling. In the present study, we hypothesized that connexin might interact with the calcium channel through forming a protein complex and, then, participates in the pathogenesis of AF. Western blot and whole-cell patch clamp showed that protein levels of Cav1.2 and connexin 43 (Cx43) and basal ICa,L were decreased in AF subjects compared to sinus rhythm (SR) controls. In cultured atrium-derived myocytes (HL-1 cells), knocking-down of Cx43 or incubation with 30 mmol/L glycyrrhetinic acid significantly inhibited protein levels of Cav1.2 and Cav3.1 and the current density of ICa,L and ICa,T . Incubation with nifedipine or mibefradil decreased the protein level of Cx43 in HL-1 cells. Moreover, Cx43 was colocalized with Cav1.2 and Cav3.1 in atrial myocytes. Therefore, Cx43 might regulate the ICa,L and ICa,T through colocalization with calcium channel subunits in atrial myocytes, representing a potential pathogenic mechanism in AF.

Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability.

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood-brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if BBB integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

Connexin43 hemichannels contribute to working memory and excitatory synaptic transmission of pyramidal neurons in the prefrontal cortex of rats.

The gap junction is essential for the communication between astrocytes and neurons by various connexins. Connexin43 hemichannels (Cx43 HCs), one of important subunits of gap junction protein, is highly expressed in astrocytes. It has been demonstrated that Cx43 HCs is involved in synaptic plasticity and learning and memory. However, whether the role of Cx43 HCs in the prefrontal cortex (PFC), a key brain region mediating cognitive and executive functions including working memory, still remains unclear. Here, we investigate that the role of Cx43 HCs in working memory through pharmacological inhibition of Cx43 HCs in the PFC. Gap26, a specific hemichannels blocker for Cx43 HCs, was bilaterally infused into the prelimbic (PrL) area of the PFC and then spatial working memory was examined in delayed alternation task in T-maze. Furthermore, the effect of Gap26 on synaptic transmission of prefrontal pyramidal neurons was examined using whole-cell patch recording in slice containing PFC. The demonstrate that inhibition of prefrontal cortex Cx43 HCs impairs the working memory and excitatory synaptic transmission of PFC neurons, suggesting that Cx43 HCs in the PFC contributes to working memory and excitatory synaptic transmission of neurons in rats.

Diverse inflammatory threats modulate astrocytes Ca2+ signaling via connexin43 hemichannels in organotypic spinal slices.

Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 ß) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

Activation of Cx43 Hemichannels Induces the Generation of Ca2+ Oscillations in White Adipocytes and Stimulates Lipolysis.

The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels-carbenoxolone, octanol, proadifen and Gap26-as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.

The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension.

BACKGROUND: Recent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. MATERIAL AND METHODS: Our research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. RESULTS: Chronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. CONCLUSION: Our research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

Connexin 43 contributes to phenotypic robustness of the mouse skull.

BACKGROUND: We compared skull shape and variation among genetically modified mice that exhibit different levels of connexin43 (Cx43) channel function, to determine whether Cx43 contributes to craniofacial phenotypic robustness. Specifically, we used two heterozygous mutant mouse models (G60S/+ and I130T/+) that, when compared to their wildtype counterparts, have an ~80% and ~50% reduction in Cx43 function, respectively. RESULTS: Both mutant strains showed significant differences in skull shape compared to wildtype littermates and while these differences were more severe in the G60S/+ mouse, shape differences were localized to similar regions of the skull in both mutants. However, increased skull shape variation was observed in G60S/+ mutants only. Additionally, covariation of skull structures was disrupted in the G60S/+ mutants only, indicating that while a 50% reduction in Cx43 function is sufficient to cause a shift in mean skull shape, the threshold for Cx43 function for disrupting craniofacial phenotypic robustness is lower. CONCLUSIONS: Collectively, our results indicate Cx43 can contribute to phenotypic robustness of the skull through a nonlinear relationship between Cx43 gap junctional function and phenotypic outcomes.

Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle.

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2ß1 Binding in TGFß1-Evoked Renal Tubular Epithelial Cells.

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2ß1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFß1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2ß1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFß1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2ß1 signaling, ahead of establishing Peptide 5 as a potential intervention.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day-night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day-night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals' active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day-night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals' awake, active phase.

Cytoproliferative effect of low dose alpha radiation in human lung cancer cells is associated with connexin 43, caveolin-1, and survivin pathway.

PURPOSE: High LET including alpha radiation-based approaches have been proved as a promising mode for cancer therapy owing to their biophysical and radiobiological advantages compared to photon beams. Studies pertaining to effect of α-radiation on cancer cells are limited to cytotoxic high doses. MATERIALS AND METHODS: In this study, human lung adenocarcinoma (A549) cells were α-irradiated using 241Am α-irradiator and effects of low dose of alpha radiation on these cells was studied under in vitro and in vivo conditions. RESULTS: Clonogenic and other assays showed increased cellular proliferation at lower doses (1.36 and 6.8 cGy) but killing at higher doses (13.6-54.4 cGy). Further studies at low dose of alpha (1.36 cGy) showed increased TGF-ß1 in the conditioned medium (CM) at early time point (24 h) but CM replacement did not affect the clonogenic survival. In these cells, increased phosphorylation of connexin 43 was correlated with decrease in gap-junction communication observed by dye transfer co-culture experiment. A decrease in caveolin-1 but increase in survivin expression was observed in low dose α-irradiated cells. An increase in cyclinD1 and decrease in Bcl-2, the target proteins of survivin, was observed in these cells. Low dose α-irradiated cancer cells transplanted in SCID mice showed significantly higher tumor volume, which was accompanied with an increased fraction of mitotic and PCNA/Ki67 positive cells in these tumor tissues. CONCLUSIONS: Taken together, our results suggest an increase in proliferation and tumor volume at in vitro and in vivo levels, respectively, when A549 cells were irradiated with low dose of α-radiation. These findings may be relevant for a better understanding of radiobiological processes during high LET-based cancer radiotherapy.

A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation.

mRNA-circRNA-miRNAs axes have been characterized in breast cancer, but not as risk-assessment axes for tumor initiation in early-onset breast cancer that is increasing drastically worldwide. To address this gap, we performed circular RNA (circRNA) microarrays and microRNA (miRNA) sequencing on acini of HMT-3522 S1 (S1) breast epithelial risk-progression culture model in 3D and chose an early-stage population miRNome for a validation cohort. Nontumorigenic S1 cells form fully polarized epithelium while pretumorigenic counterparts silenced for gap junction Cx43 (Cx43-KO-S1) lose epithelial polarity, multilayer and mimic premalignant in vivo mammary epithelial morphology. Here, 121 circRNAs and 65 miRNAs were significantly dysregulated in response to Cx43 silencing in cultured epithelia and 15 miRNAs from the patient cohort were involved in epithelial polarity disruption. Focusing on the possible sponging activity of the validated circRNAs to their target miRNAs, we found all miRNAs to be highly enriched in cancer-related pathways and cross-compared their dysregulation to actual miRNA datasets from the cultured epithelia and the patient validation cohort. We present the involvement of gap junction in post-transcriptional axes and reveal Cx43/hsa_circ_0077755/miR-182 as a potential biomarker signature axis for heightened-risk of breast cancer initiation, and that its dysregulation patterns might predict prognosis along breast cancer initiation and progression.

Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake.

The metabolic syndrome, which comprises obesity and diabetes, is a major public health problem and the awareness of energy homeostasis control remains an important worldwide issue. The energy balance is finely regulated by the central nervous system (CNS), notably through neuronal networks, located in the hypothalamus and the dorsal vagal complex (DVC), which integrate nutritional, humoral and nervous information from the periphery. The glial cells' contribution to these processes emerged few year ago. However, its underlying mechanism remains unclear. Glial connexin 43 hemichannels (Cx43 HCs) enable direct exchange with the extracellular space and can regulate neuronal network activity. In the present study, we sought to determine the possible involvement of glial Cx43 HCs in energy balance regulation. We here show that Cx43 is strongly expressed in the hypothalamus and DVC and is associated with glial cells. Remarkably, we observed a close apposition of Cx43 with synaptic elements in both the hypothalamus and DVC. Moreover, the expression of hypothalamic Cx43 mRNA and protein is modulated in response to fasting and diet-induced obesity. Functionally, we found that Cx43 HCs are largely open in the arcuate nucleus (ARC) from acute mice hypothalamic slices under basal condition, and significantly inhibited by TAT-GAP19, a mimetic peptide that specifically blocks Cx43 HCs activity. Moreover, intracerebroventricular (i.c.v.) TAT-GAP19 injection strongly decreased food intake, without further alteration of glycaemia, energy expenditures or locomotor activity. Using the immediate early gene c-Fos expression, we found that i.c.v. TAT-GAP19 injection induced neuronal activation in hypothalamic and brainstem nuclei dedicated to food intake regulation. Altogether, these results suggest a tonic delivery of orexigenic molecules associated with glial Cx43 HCs activity and a possible modulation of this tonus during fasting and obesity.

Myocardial infarction affects Cx43 content of extracellular vesicles secreted by cardiomyocytes.

Ischemic heart disease has been associated with an impairment on intercellular communication mediated by both gap junctions and extracellular vesicles. We have previously shown that connexin 43 (Cx43), the main ventricular gap junction protein, assembles into channels at the extracellular vesicle surface, mediating the release of vesicle content into target cells. Here, using a comprehensive strategy that included cell-based approaches, animal models and human patients, we demonstrate that myocardial ischemia impairs the secretion of Cx43 into circulating, intracardiac and cardiomyocyte-derived vesicles. In addition, we show that ubiquitin signals Cx43 release in basal conditions but appears to be dispensable during ischemia, suggesting an interplay between ischemia-induced Cx43 degradation and secretion. Overall, this study constitutes a step forward for the characterization of the signals and molecular players underlying vesicle protein sorting, with strong implications on long-range intercellular communication, paving the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders.

Comparative Analysis of Cx31 and Cx43 in Differentiation-Competent Rodent Keratinocytes.

When considering connexin expression and regulation, the epidermis of the skin is one of the most complex tissues found in mammals even though it largely contains a single cell type, the keratinocyte. In the rodent epidermis, up to 9 connexin family members have been detected at the mRNA level. Many of these connexins are temporally and spatially regulated in coordination with keratinocyte progenitor cell differentiation and migration from the stratum basale to form the stratum spinosum and stratum granulosum layers before finally forming the stratum corneum. Cx43 is the principal connexin found in basal keratinocytes and to a lesser degree found in keratinocytes that have begun to differentiate where Cx26, Cx30 and Cx31 become prevalent. Here we show that the CRISPR-Cas9 ablation of Cx43 reduces overall gap junction coupling in monolayer cultures of rat epidermal keratinocytes (REKs) and dysregulates the differentiation of REKs when grown in organotypic cultures. Natively found in differentiated keratinocytes, Cx31 readily assembles into gap junctions when expressed in REKs where it can extensively co-assemble into the same gap junctions with co-expressed Cx30. Time-lapse imaging indicated that many Cx31 gap junctions are mobile within the plasma membrane undergoing both fusion and fission events. Finally, the persistence of pre-existing Cx31 gap junctions in the presence of the protein trafficking blocker, brefeldin A, is longer than that found for Cx43 gap junctions indicating that it has a distinctly different life expectancy in REKs. Collectively, this study highlights the importance of Cx43 in rodent keratinocyte differentiation and suggests that Cx31 acquires life-cycle properties that are distinct from Cx43.

A Working Hypothesis Regarding Identical Pathomechanisms between Clinical Efficacy and Adverse Reaction of Clozapine via the Activation of Connexin43.

Clozapine (CLZ) is an approved antipsychotic agent for the medication of treatment-resistant schizophrenia but is also well known as one of the most toxic antipsychotics. Recently, the World Health Organization's (WHO) global database (VigiBase) reported the relative lethality of severe adverse reactions of CLZ. Agranulocytosis is the most famous adverse CLZ reaction but is of lesser lethality compared with the other adverse drug reactions of CLZ. Unexpectedly, VigiBase indicated that the prevalence and relative lethality of pneumonia, cardiotoxicity, and seizures associated with CLZ were more serious than that of agranulocytosis. Therefore, haematological monitoring in CLZ patients monitoring system provided success in the prevention of lethal adverse events from CLZ-induced agranulocytosis. Hereafter, psychiatrists must amend the CLZ patients monitoring system to protect patients with treatment-resistant schizophrenia from severe adverse CLZ reactions, such as pneumonia, cardiotoxicity, and seizures, according to the clinical evidence and pathophysiology. In this review, we discuss the mechanisms of clinical efficacy and the adverse reactions of CLZ based on the accumulating pharmacodynamic findings of CLZ, including tripartite synaptic transmission, and we propose suggestions for amending the monitoring and medication of adverse CLZ reactions associated with pneumonia, cardiotoxicity, and seizures.

Cx43 promotes SHF-DPCs proliferation in the hair follicle of Albas cashmere goats from anagen to telogen.

Connexin 43 (Cx43), known to form gap junction transmembrane channels between the cytoplasm of two adjacent cells, plays a key role in physiological functions, such as regulating cell growth, differentiation, and maintaining tissue homeostasis. Cashmere goat is an important farm animal that provides cashmere, which was produced by secondary hair follicles (SHF), for human consumption; however, there is no report about the role of Cx43 on the growth and development of SHF in cashmere goat. In this study, we investigated the effect of Cx43 on proliferation secondary hair follicle dermal papilla cells (SHF-DPCs) in Albas cashmere goat. In SHF-DPCs, Cx43 overexpression promoted cell proliferation and upregulated the expression of IGF-1, whereas Cx43 knockdown was associated with the opposite effects. These results suggested that Cx43 may promote cell proliferation by inducing IGF-1. Overall, our research not only contributes to a better understanding of the mechanism of the growth and development of SHF in cashmere goat, but also shed light on cashmere quality control in the future.

Apocynin Treatment Prevents Cardiac Connexin 43 Hemichannels Hyperactivity by Reducing Nitroso-Redox Stress in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal disease that causes cardiomyopathy and is associated with oxidative stress. In the heart, oxidative stress interferes with the location of connexin 43 (Cx43) to the intercalated discs causing its lateralization to the plasma membrane where Cx43 forms hemichannels. We tested the hypothesis that in DMD cardiomyopathy, increased oxidative stress is associated with the formation and activation of Cx43 hemichannels. For this, we used mdx mice as a DMD model and evaluated cardiac function, nitroso-redox changes and Cx43 hemichannels permeability. Mdx hearts presented increased NADPH oxidase-derived oxidative stress and increased Cx43 S-nitrosylation compared to controls. These redox changes were associated with increased Cx43 lateralization, decreased cardiac contractility and increased arrhythmic events. Pharmacological inhibition of NADPH oxidase using apocynin (one month) reduced systemic oxidative stress and reversed the aforementioned changes towards normal, except Cx43 lateralization. Opening of Cx43 hemichannels was blocked by apocynin treatment and by acute hemichannel blockade with carbenoxolone. NADPH oxidase inhibition also prevented the occurrence of apoptosis in mdx hearts and reversed the ventricular remodeling. These results show that NADPH oxidase activity in DMD is associated with S-nitrosylation and opening of Cx43 hemichannels. These changes lead to apoptosis and cardiac dysfunction and were prevented by NADPH oxidase inhibition.

Intercellular communication and ion channels in neuropathic pain chronicization.

BACKGROUND: Neuropathic pain is caused by primary lesion or dysfunction of either peripheral or central nervous system. Due to its complex pathogenesis, often related to a number of comorbidities, such as cancer, neurodegenerative and neurovascular diseases, neuropathic pain still represents an unmet clinical need, lacking long-term effective treatment and complex case-by-case approach. AIM AND METHODS: We analyzed the recent literature on the role of selective voltage-sensitive sodium, calcium and potassium permeable channels and non-selective gap junctions (GJs) and hemichannels (HCs) in establishing and maintaining chronic neuropathic conditions. We finally focussed our review on the role of extracellular microenvironment modifications induced by resident glial cells and on the recent advances in cell-to-cell and cell-to-extracellular environment communication in chronic neuropathies. CONCLUSION: In this review, we provide an update on the current knowledge of neuropathy chronicization processes with a focus on both neuronal and glial ion channels, as well as on channel-mediated intercellular communication.