Cx43 Levels Research Articles

Connexin43 Contributes to Alzheimer's Disease by Promoting the Mitochondria-Associated Membrane-Related Autophagy Inhibition.

The perturbed structure and function of mitochondria-associated membranes (MAM), instead of the amyloid cascade, have been gradually proposed to play a basic role in the pathogenesis of Alzheimer's disease (AD). Notably, autophagy inhibition is one of the main mechanisms of MAM dysfunction and plays an important role in neuronal injury. However, the upstream molecular mechanism underlying the MAM dysfunctions remains elusive. Here, we defined an unexpected and critical role of connexin43 (Cx43) in regulating the MAM structure. The expression levels of Cx43 and mitofusin-2 (MFN2, the MAM biomarker) increase significantly in 9-month-old APPswe/PS1dE9 double-transgenic AD model mice, and there is an obvious colocalization relationship. Moreover, both AD mice and cells lacking Cx43 exhibit an evident reduction in the MAM contact sites, which subsequently promotes the conversion from microtubule-associated protein 1 light-chain 3B I (LC3B-I) to LC3B-II via inhibition mTOR-dependent pathway and then initiates the generation of autophagosomes. Autophagosome formation ultimately promotes β-amyloid (Aβ) clearance and attenuates Aβ-associated pathological changes in AD, mainly including astrogliosis and neuronal apoptosis. Our findings not only reveal a previously unrecognized effect of Cx43 on MAM upregulation but also highlight the major player of MAM-induced autophagy inhibition in Cx43-facilitated AD pathogenesis, providing a novel insight into the alternative therapeutic strategies for the early treatment of AD.

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.

Changes in Cx43 and AQP4 Proteins, and the Capture of 3 kDa Dextran in Subpial Astrocytes of the Rat Medial Prefrontal Cortex after Both Sham Surgery and Sciatic Nerve Injury.

A subpopulation of astrocytes on the brain's surface, known as subpial astrocytes, constitutes the "glia limitans superficialis" (GLS), which is an interface between the brain parenchyma and the cerebrospinal fluid (CSF) in the subpial space. Changes in connexin-43 (Cx43) and aquaporin-4 (AQP4) proteins in subpial astrocytes were examined in the medial prefrontal cortex at postoperative day 1, 3, 7, 14, and 21 after sham operation and sciatic nerve compression (SNC). In addition, we tested the altered uptake of TRITC-conjugated 3 kDa dextran by reactive subpial astrocytes. Cellular immunofluorescence (IF) detection and image analysis were used to examine changes in Cx43 and AQP4 protein levels, as well as TRITC-conjugated 3 kDa dextran, in subpial astrocytes. The intensity of Cx43-IF was significantly increased, but AQP4-IF decreased in subpial astrocytes of sham- and SNC-operated rats during all survival periods compared to naïve controls. Similarly, the uptake of 3 kDa dextran in the GLS was reduced following both sham and SNC operations. The results suggest that both sciatic nerve injury and peripheral tissue injury alone can induce changes in subpial astrocytes related to the spread of their reactivity across the cortical surface mediated by increased amounts of gap junctions. At the same time, water transport and solute uptake were impaired in subpial astrocytes.

P163 EMPAGLIFLOZIN AND ITS CARDIOPROTECTIVE EFFECTS IN THE HEART DURING VOLUME OVERLOAD IN AN EXPERIMENTAL MODEL INDUCED BY AORTOCAVAL FISTULA

Mitral regurgitation causes volume overload of the heart, and due to the deregulation of extracellular matrix proteins, the electrical communication between cardiomyocytes provided by connexin channels is disturbed. The aim of this study was to investigate the effect of cardiac volume overload (VO) on its functional parameters, structural remodeling, and connexin43 (Cx43), a protein ensuring electrical synchronization of the heart, and the cardioprotective effect of empagliflozin. Cardiac VO was induced by surgical aortocaval fistula (ACF) in adult, 6-month-old male Wistar rats. Mitral regurgitation and VO developed within four weeks. Subsequently, empagliflozin was administered daily for five weeks at a dose of 10 mg/kg of body weight. Echocardiographic examination showed increased cardiac output and increased Vmax of the aortic valve, decreased ejection fraction and fractional shortening due to ACF-VO. Echocardiographic parameters showed an increase in the diameter of the left ventricle in both systole and diastole, as well as an increase in the volume of the left ventricle at the end of systole and diastole. Empagliflozin normalized the negative parameters in the ACF-VO group. Myocardial protein Cx43 levels were reduced by ACF-VO but normalized by empagliflozin treatment. In addition, empagliflozin suppressed the ACF-VO-induced increase in interstitial collagen detected by van Gieson staining. These findings were consistent with increased protein expression of MMP-2, which is involved in structural remodeling of the extracellular matrix, and was normalized by empagliflozin. Protein kinase C epsilon levels associated with Cx43 and extracellular matrix modulation were reduced by ACF-VO and normalized by empagliflozin. However, protein levels of pro-apoptotic and pro-hypertrophic protein kinase C delta were increased by ACF-VO and normalized by empagliflozin. Protein expression of transforming growth factor beta as well as fibroblast growth factor 21 was increased by ACF-VO and normalized by empagliflozin. The results indicate a cardioprotective effect of empagliflozin due to suppression of myocardial structural remodeling and preservation of Cx43 in an experimental model simulating volume overload. The research was supported by grants APVV-21-0410,VEGA-2/0006/23,VEGA-2/0133/24,VEGA-2/0002/20

Connexin 43 Modulation in Human Chondrocytes, Osteoblasts and Cartilage Explants: Implications for Inflammatory Joint Disorders.

Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 ng/mL Tumor Necrosis Factor alpha (TNFα) and/or 1 ng/mL Interleukin-1 beta (IL-1β), in primary chondrocytes (CH) and osteoblasts (OB). Additionally, we explored the impact of synovial fluids from osteoarthritis patients in CH and cartilage explants, providing a more physio-pathological context. The effect of TNFα on Cx43 expression in cartilage explants was also assessed. TNFα downregulated Cx43 levels both in CH and OB (-73% and -32%, respectively), while IL-1β showed inconclusive effects. The reduction in Cx43 levels was associated with a significant downregulation of the coding gene GJA1 expression in OB only (-65%). The engagement of proteasome in TNFα-induced effects, already known in CH, was also observed in OB. TNFα treatment significantly decreased Cx43 expression also in cartilage explants. Of note, Cx43 expression was halved by synovial fluid in both CH and cartilage explants. This study unveils the regulation of Cx43 in diverse musculoskeletal cell types under various stimuli and in different contexts, providing insights into its modulation in inflammatory joint disorders.

Abstract Mo046: GJA1-20k Restores Connexin-43 Trafficking and β-Catenin Signaling in Myocytes Lacking Desmoplakin

Background: Arrhythmogenic Cardiomyopathy (ACM) is an insidious hereditary heart disease that results in structural and electrical cardiac abnormalities leading to arrhythmogenesis and heart failure. ACM is characterized by cardiomyocyte loss, decreased cellular coupling, and fibrofatty replacement. The disease arises from mutations in desmosomal genes, such as plakophilin (PKP), desmoglein (DSG), and desmoplakin (DSP). The pathogenesis of this disease is linked to dysfunction at the level of several cellular processes and pathways, including Wnt/β-catenin signaling. Recent evidence has shown that GJA1-20k, a truncated isoform of Connexin-43 (Cx43) generated by internal translation, has a therapeutic role in preserving Cx43 trafficking and cell-cell coupling in ACM of DSG origin. This study aims to investigate the role of GJA1-20k in protecting against ACM of DSP origin by evaluating its effect in cell models on Cx43 trafficking and Wnt/β-catenin signaling. Methods: DSP knockdown was performed on HEK cells and mouse neonatal cardiomyocytes. The role of GJA1-20k was determined using imaging, biochemical, and molecular biology techniques. Results: Protein levels of Cx43 and β-catenin were decreased on Western blot (WB) upon DSP knockdown. These findings were corroborated through imaging wherein the signal intensity for Cx43 at cellular junctions and β-catenin at the membrane was significantly decreased compared to the control group. β-catenin signal intensity was also decreased within the nucleus. Upon administration of GJA1-20k, protein levels of Cx43 were normalized on WB. Moreover, the ratio of phosphorylated β-catenin (inactive) to total β-catenin was decreased. Imaging studies identified a significant increase in Cx43 and β-catenin at the membrane in ACM models treated with GJA1-20k. This was also accompanied by an increase in β-catenin signal intensity within the nucleus. A TOP-flash luciferase assay was performed and revealed a significant increase in β-catenin transcriptional activity in the presence of GJA1-20k despite DSP knockdown. Conclusions: These results suggest that GJA1-20k is able to rescue Cx43 trafficking and recover Wnt/β-catenin signaling in cells and cardiomyocytes lacking DSP. The ability of GJA1-20k to restore dysfunctional genetic pathways involved in arrhythmogenesis and fibro-adipogenesis make it an attractive potential candidate for targeted therapy against ACM.

Celastrol exerts antiarrhythmic effects in chronic heart failure via NLRP3/Caspase-1/IL-1β signaling pathway

ObjectivesCelastrol has widespread therapeutic applications in various pathological conditions, including chronic inflammation. Previous studies have demonstrated the potent cardioprotective effects of celastrol. Nevertheless, limited attention has been given to its potential in reducing ventricular arrhythmias (VAs) following myocardial infarction (MI). Hence, this study aimed to elucidate the potential mechanisms underlying the regulatory effects of celastrol on VAs and cardiac electrophysiological parameters in rats after MI. MethodsSprague-Dawley rats were divided at random: the sham, MI, and MI + celastrol groups. The left coronary artery was occluded in the MI and MI + Cel groups. Electrocardiogram, heart rate variability (HRV), ventricular electrophysiological parameters analysis, histology staining of ventricles, Enzyme-linked immunosorbent assay (ELISA), western blotting and Quantitative real-time polymerase chain reaction (qRT-PCR) were performed to elucidate the underlying mechanism of celastrol. Besides, H9c2 cells were subjected to hypoxic conditions to create an in vitro model of MI and then treated with celastrol for 24 hours. Nigericin was used to activate the NLRP3 inflammasome. ResultsCompared with that MI group, cardiac electrophysiology instability was significantly alleviated in the MI + celastrol group. Additionally, celastrol improved HRV, upregulated the levels of Cx43, Kv.4.2, Kv4.3 and Cav1.2, mitigated myocardial fibrosis, and inhibited the NLRP3 inflammasome pathway. In vitro conditions also supported the regulatory effects of celastrol on the NLRP3 inflammasome pathway. ConclusionsCelastrol could alleviate the adverse effects of VAs after MI partially by promoting autonomic nerve remodeling, ventricular electrical reconstruction and ion channel remodeling, and alleviating ventricular fibrosis and inflammatory responses partly by through inhibiting the NLRP3/Caspase-1/IL-1β pathway.

Role of connexin 43 in a rat model of periodontitis-induced renal injury.

This study aims to investigate the role of gap junction mediated by connexin 43 (Cx43) in renal injury induced by periodontitis in rats. Twelve SPF-grade Wistar male rats were divided into a control group and a periodontitis group by using a completely random number table method, with six rats in each group. The control group rats were not treated, while the periodontitis group rats were subjected to wire ligation of the neck of their bilateral maxillary first molars to construct a periodontitis model. After 8 weeks of modeling, the rats were examined for clinical indicators of the periodontium. micro-CT scanning of the maxilla reconstructed its 3D structure and analyzed the absorption of alveolar bone. Histopathological changes in periodontal and renal tissues were detected. MitoSOX red reagent was used to determine reactive oxygen species (ROS) content in renal tissues. A biochemical reagent kit was used to detect serum oxidative stress biomarkers. Real-time fluorescent quantitative-polymerase chain reaction (qRT-PCR) was employed to determine Cx43, nuclear factor kappa-B (NF-κB) , interleukin (IL)-1β, IL-6, BCL2-Associated X (Bax), B-lymphomatoma-2 gene (Bcl-2), and Caspase-3 mRNA were determined. Western blot analysis was used to detect Cx43, NF-κB, IL-1β, Bax, Bcl-2 and Caspase-3 protein. micro-CT 3D reconstruction showed significant bone resorption of the first molar alveolar bone in the periodontitis group rats and decreased height of the alveolar ridge. The distance from the enamel cementum boundary to the top of the alveolar ridge in the periodontitis group was significantly higher than that inthe control group. The histopathological results showed a large number of inflammatory cells that infiltrated the periodontal tissue of the periodontitis group, and the alveolar bone was significantly absorbed. Rats in the periodontitis group also exhibited mild thickening of the glomerular basement membrane, dilation of the Bowman's capsule, and destruction of the brush-like edge of the renal tubules in the renal tissue. The MitoSOX red staining results showed a significant increase in ROS content in the renal tissue of the periodontitis group. The biochemical test results showed that the levels of superoxide dismutase and glutathione in the serum of rats with periodontitis decreased, while that of malondialdehyde increased. The results of qRT-PCR and Western blot showed that the expression levels of Cx43, IL-1β, IL-6, Bax, Caspase-3 mRNA and Cx43, IL-1β, NF-κB, Bax, Caspase-3 proteins in the periodontitis group significantly increased compared with those in the control group, while the expression levels of Bcl-2 mRNA and protein decreased. Periodontitis may activate NF-κB signaling molecules by upregulating the expression of Cx43 in rat kidney tissues, leading to increased levels of inflammation and apoptosis and ultimately inducing kidney injury.

Cardioprotective effects of Empagliflozin in cardiac volume overload-induced by aortocaval fistula in rats

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Slovak Research and Development Agency under the contract no. 21-0410 VEGA grants no. 2/0006/23 Heart failure is characterized by reduction in cardiac output and the occurrence of malignant arrhythmias, thereby contributing significantly to high morbidity and mortality worldwide. Myocardial structural remodelling is a key factor contributing to the development of these life-threatening events and belongs to unsolved clinical issues challenging to investigate in animal models. The aim of our study was to explore the impact of cardiac volume overload (VO) on structural remodelling and electrical coupling protein, connexin43 (Cx43) in the left and right heart ventricles as well as the efficacy of treatment with SGLT2i, Empagliflozin. Aortocaval fistula (ACF) was surgically induced in 6-month-old Wistar rats, to develop VO during 4 weeks, followed by oral administration of Empagliflozin 10 mg/kg/b.w. daily for 5 weeks. At the end of the experiment, biometric parameters were registered and left and right heart tissue samples were snap-frozen and kept at -80°C for western blot analysis and microscopic examination. Findings revealed an increase of heart, left and right ventricular mass due to ACF-VO versus sham controls, which was associated, with eccentric myocardial hypertrophy. Noteworthy, Empagliflozin reduced heart, left and right ventricular mass. Levels of myocardial protein Cx43, that determine electrical coupling among cardiomyocytes, were reduced by ACF-VO, while normalized by treatment with Empagliflozin. Moreover, treatment attenuated ACF-VO-induced increase of interstitial collagen visualized by van Gieson staining. It was in line with the protein expression of MMP-2, which is involved in structural remodeling. The protein levels of PKCε, concerned/associated with modulation of Cx43 and extracellular matrix, were decreased due to ACV-VO but normalized after treatment with Empagliflozin. On the other hand, protein levels of PKCδ, involved in proapoptotic and prohypertrophic pathways, were increased and normalized by Empagliflozin. The changes were more pronounced in the right ventricle compared to the left heart ventricle. In summary, our pilot results point out the benefit of Empagliflozin due to the suppression of myocardial structural remodelling and preservation of Cx43 in the rat ACF-VO model. Furthermore, this Empagliflozin-induced cardioprotection was associated with the normalization of Cx43, MMP-2, and PKC levels independent of glucose control. However, further analyses are needed to reveal the molecular mechanisms of the cardioprotective effects of Empagliflozin.

Hypertension-induced cardiac pro-arrhythmic disorders of connexin-43 and extracellular matrix are attenuated in hairless versus wild-type of spontaneously hypertensive rats

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Development Agency under the contract no. 21-0410 VEGA grants no. 2/0002/20, 2/0006/23 Background Mechanical dysfunction and occurrence of malignant ventricular fibrillation (VF) are hallmarks of hypertensive heart disease and major health issue. Spontaneously hypertensive rats (SHR) that imitates human primary hypertension are prone to develop VF, mainly due to myocardial remodelling associated with disorders of electrical coupling protein connexin-43 (Cx43) and extracellular matrix (ECM) resulting in electrical instability. However, it is not known whether postnatal acclimation of hairless strain of SHR to surrounding temperature may induce cardio-protection. Therefore, we aimed to explore Cx43 and ECM alterations as well as susceptibility to inducible VF in hairless versus wild type SHR. Design and Methods Six-month-old wild type SHR males and females and hairless SHR housed at standard 22°C were examined. Biometric parameters, including electro- and echocardiography were registered. Left ventricular tissue was used for immunolabeling of Cx43 to reveal its topology and for western blotting to determine protein levels of Cx43 and its function modulating protein kinases, PKCε, PKCδ and PKCα as well as for ECM markers, TGF-β, SMAD2/3, MMP-2, collagen-1 and its histological deposition. Susceptibility of the heart to electrically-inducible VF was tested using isolated perfused heart technique. Key results Comparing to wild type males, hairless SHR exhibited less pronounced prolongation of QT and QRS interval as well as an increase of ejection fraction. Myocardial Cx43 and its variant phosphorylated at serine-368, which preserve electrical coupling, were increased and pro-arrhythmic cardiomyocyte Cx43 topology was attenuated in males and females hairless vs wild type SHR. Moreover, the level of β-catenin, which ensures mechano-electrical coupling, was also increased in hairless SHR. On the other hand, collagen-1 and hydroxyproline were lower and the TGF-β1 and SMAD2/3 proteins were suppressed in males hairless vs wild type SHR. Myocardial ECM remodelling was less pronounced in females regardless rat strain. Hairless SHR males were less susceptible to inducible VF compared to wild type SHR, while females were less prone to develop VF regardless the strain. In conclusion, our findings imply that postnatal acclimation of hairless SHR is accompanied by attenuation of hypertension-induced adverse down-regulation of Cx43 and up-regulation of extracellular matrix proteins. It may contribute, at least in part, to the protection from malignant cardiac arrhythmias and mechanical dysfunction.

Elevated calcineurin activity in primary astrocytes leads to the dephosphorylation of connexin 43 in conjunction with increased membrane permeability.

Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.

Changes in Astroglial Water Flow in the Pre-amyloid Phase of the STZ Model of AD Dementia.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease that is typically sporadic and has a high social and economic cost. We utilized the intracerebroventricular administration of streptozotocin (STZ), an established preclinical model for sporadic AD, to investigate hippocampal astroglial changes during the first 4 weeks post-STZ, a period during which amyloid deposition has yet to occur. Astroglial proteins aquaporin 4 (AQP-4) and connexin-43 (Cx-43) were evaluated, as well as claudins, which are tight junction (TJ) proteins in brain barriers, to try to identify changes in the glymphatic system and brain barrier during the pre-amyloid phase. Glial commitment, glucose hypometabolism and cognitive impairment were characterized during this phase. Astroglial involvement was confirmed by an increase in glial fibrillary acidic protein (GFAP); concurrent proteolysis was also observed, possibly mediated by calpain. Levels of AQP-4 and Cx-43 were elevated in the fourth week post-STZ, possibly accelerating the clearance of extracellular proteins, since these proteins actively participate in the glymphatic system. Moreover, although we did not see a functional disruption of the blood-brain barrier (BBB) at this time, claudin 5 (present in the TJ of the BBB) and claudin 2 (present in the TJ of the blood-cerebrospinal fluid barrier) were reduced. Taken together, data support a role for astrocytes in STZ brain damage, and suggest that astroglial dysfunction accompanies or precedes neuronal damage in AD.

TRV027 REDUCES BLOOD PRESSURE IN SHR AND RESTORES VENTRICULAR HYPERTROPHY BY AFFECTING CONNEXIN 43 PATHWAY

Objective: Hypertension is one of the common causes of cardiovascular diseases. Angiotensin II (AngII) plays a crucial role in hypertension and hypertension-induced cardiac hypertrophy. Whether TRV027, an AngII type 1 receptor biased agonist, could lower blood pressure and treat hypertensive cardiac hypertrophy is unknown. Design and method: Spontaneously hypertensive rats and Wistar-Kyoto rats were selected as the study subjects. Some SHRs were giving TRV027. H9c2 cells were selected as the in vitro study subjects to explore the mechanism of action of TRV027. Results: When compared to the WKY group, the SHR group displayed a pronounced increase in blood pressure. However, after giving subcutaneous pump of TRV027, blood pressure exhibited a marked reduction. Based on the Doppler ultrasound data, prior to the intervention, the SHR group had higher values for LVAWs, LVAWd, LVPWs, and LVPWd when contrasted with the WKY group. Moreover, the left ventricular mass in the SHR group also increased significantly (P<0.05). After the intervention with TRV027 in the SHR group, all the above-mentioned cardiac Doppler parameters were lower than those in the SHR group. HE staining of cardiac sections revealed a slight disarray in myocardial arrangement and significant myocardial hypertrophy in the SHR group. Compared with the WKY group, the level of Cx43 in the heart tissue of the SHR group was decreased, and the phosphorylation of MAPK and ERK2 was increased. After administration of TRV027, the expression levels of these proteins were restored. In the cell intervention, the expression level of Cx43 was significantly decreased, the phosphorylation levels of MAPK and ERK2 were increased, and the levels of MHC and ANP were increased in the AngII intervention group. After the intervention of TRV027 the expressions of Cx43 and MAPK/ERK pathways and MHC and ANP were restored. When Cx43 inhibitor was given to H9C2 cells,along with the decrease of Cx43 expression, the phosphorylation levels of MAPK and ERK2 were increased, while the levels of MHC and ANP were significantly increased. Conclusions: TRV027 lower blood pressure in SHR and ameliorate cardiac hypertrophy induced by hypertension. This effect is likely achieved through the Cx43 pathway.

DISPARATE IMPACT OF PRIMARY HYPERTENSION ON RIGHT VERSUS LEFT HEART VENTRICLES OF MALES AND FEMALES WILD TYPE AND HAIRLESS STRAIN OF SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Poor adherence to lifestyle changes and antihypertensive drugs contributes to increase a risk for malignant cardiac arrhythmias. Hypertension contributes to myocardial structural remodeling, abnormalities in connexin43 (Cx43) which promote electrical instability and mechanical dysfunction. However, it is not known whether changes are confined solely to left heart ventricle (LV) or there is response of right heart ventricle (RV). SHR mimic human primary hypertension and hairless SHR is model of temperature acclimation characterized by increase thermogenesis. Aim of the study was to explore key factors involved in development of malignant arrhythmias and progression to heart failure, in left and right heart ventricles, of males and females wild type (WT) and hairless SHR. Design and method: 6-month-old males and females WT and hairless SHR and normotensive WKY strain of rats were housed at standard 22°C. Biometric parameters, including electro- and echo-cardiographic were registered. LV and RV tissue was used for immuno-labelling of Cx43 to detect its topology and western blotting for determination protein levels of Cx43, PKA, PKC∊, PKCδ and markers of extracellular matrix (ECM) remodelling: TGFβ, SMAD2/3, MMP-2 and collagen-1. Susceptibility of the heart to electrically-inducible VF was tested using isolated perfused heart technique. Results: Comparing to WKY, QT and QRS intervals were prolonged in WT SHR with normalisation in hairless SHR. Hypertension resulted in decrease in Cx43 with its abnormal topology in LV of males and less in hairless SHR but not in females. Cx43 levels and its topology were preserved in RV regardless rat strain and sex. There were no differences in protein levels of ECM-markers when comparing LV and RV in all strains. PKC∊ and PKCδ were increased in RV versus LV in both SHR strains. Hairless SHR males and females were less prone to inducible VF compared to WT SHR. Conclusions: Findings point out that hypertension-induced pro-arrhythmic disorders of Cx43 are related to the left while not to right heart ventricle in both hypertensive strains. There is a benefit of cold acclimation in hypertension that may contribute, to the protection from malignant cardiac arrhythmias and mechanical dysfunction. Supported by Vega2/0002/20,2/0006/23 and APVV21-0410.

Neuronal Panx1 drives peripheral sensitization in experimental plantar inflammatory pain

BackgroundThe channel-forming protein Pannexin1 (Panx1) has been implicated in both human studies and animal models of chronic pain, but the underlying mechanisms remain incompletely understood.MethodsWild-type (WT, n = 24), global Panx1 KO (n = 24), neuron-specific Panx1 KO (n = 20), and glia-specific Panx1 KO (n = 20) mice were used in this study at Albert Einstein College of Medicine. The von Frey test was used to quantify pain sensitivity in these mice following complete Freund’s adjuvant (CFA) injection (7, 14, and 21 d). The qRT-PCR was employed to measure mRNA levels of Panx1, Panx2, Panx3, Cx43, Calhm1, and β-catenin. Laser scanning confocal microscopy imaging, Sholl analysis, and electrophysiology were utilized to evaluate the impact of Panx1 on neuronal excitability and morphology in Neuro2a and dorsal root ganglion neurons (DRGNs) in which Panx1 expression or function was manipulated. Ethidium bromide (EtBr) dye uptake assay and calcium imaging were employed to investigate the role of Panx1 in adenosine triphosphate (ATP) sensitivity. β-galactosidase (β-gal) staining was applied to determine the relative cellular expression levels of Panx1 in trigeminal ganglia (TG) and DRG of transgenic mice.ResultsGlobal or neuron-specific Panx1 deletion markedly decreased pain thresholds after CFA stimuli (7, 14, and 21 d; P < 0.01 vs. WT group), indicating that Panx1 was positively correlated with pain sensitivity. In Neuro2a, global Panx1 deletion dramatically reduced neurite extension and inward currents compared to the WT group (P < 0.05), revealing that Panx1 enhanced neurogenesis and excitability. Similarly, global Panx1 deletion significantly suppressed Wnt/β-catenin dependent DRG neurogenesis following 5 d of nerve growth factor (NGF) treatment (P < 0.01 vs. WT group). Moreover, Panx1 channels enhanced DRG neuron response to ATP after CFA injection (P < 0.01 vs. Panx1 KO group). Furthermore, ATP release increased Ca2+ responses in DRGNs and satellite glial cells surrounding them following 7 d of CFA treatment (P < 0.01 vs. Panx1 KO group), suggesting that Panx1 in glia also impacts exaggerated neuronal excitability. Interestingly, neuron-specific Panx1 deletion was found to markedly reduce differentiation in cultured DRGNs, as evidenced by stunted neurite outgrowth (P < 0.05 vs. Panx1 KO group; P < 0.01 vs. WT group or GFAP-Cre group), blunted activation of Wnt/β-catenin signaling (P < 0.01 vs. WT, Panx1 KO and GFAP-Cre groups), and diminished cell excitability (P < 0.01 vs. GFAP-Cre group) and response to ATP stimulation (P < 0.01 vs. WT group). Analysis of β-gal staining showed that cellular expression levels of Panx1 in neurons are significantly higher (2.5-fold increase) in the DRG than in the TG.ConclusionsThe present study revealed that neuronal Panx1 is a prominent driver of peripheral sensitivity in the setting of inflammatory pain through cell-autonomous effects on neuronal excitability. This hyperexcitability dependence on neuronal Panx1 contrasts with inflammatory orofacial pain, where similar studies revealed a prominent role for glial Panx1. The apparent differences in Panx1 expression in neuronal and non-neuronal TG and DRG cells are likely responsible for the distinct impact of these cell types in the two pain models.

Possible mechanisms involved in the protective effect of lutein against cyclosporine-induced testicular damage in rats

Oxidative stress and aberrant inflammatory response have important implications in cyclosporin-induced reproductive functions. Previous studies have shown that agents with antioxidant and anti-inflammatory activities might be beneficial in reversing cyclosporin-induced reproductive impairment. Lutein is a naturally occurring compound with antioxidant and anti-inflammatory properties. However, the effect of lutein against cyclosporin-induced reproductive impairment remains in complete. Hence, we investigated the protective effect of lutein, specifically focusing on the role of nuclear factor erythroid 2 related factor-2 (Nrf2)/heme-oxygenase-1 (HO-1)/connexin-43 (Cx-43) upregulation system against cyclosporine-induced reproductive impairment. Six male Wistar rats were allotted into 5 groups and given daily gavage of cyclosporine (40 mg/kg) and/or lutein (30 mg/kg) for four (4) weeks or in combination, respectively. The testicular antioxidant scaffolds: superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), add to sulfhydryl (T-SH), non-protein sulfhydryl (NP-SH), glutathione reductase (GR), glutathione-S -transferase (GST), glutathione peroxidase (GSH-Px), thiobarbituric acid reactive substances (TBARS), myeloperoxidase (MPO), testicular proinflammatory cytokines, apoptotic related protein, nucleic acids, sialic acid, testicular proton pump ATPase, stress responsive protein, BTB-related protein and total protein levels in the testes were assayed thereafter. Cyclosporin significantly increased NOX-1, TNF-α, IL-1β, MPO, caspase-3 and -9 levels, which were reversed by lutein. Lutein reversed cyclosporin-induced decreases in Nrf2, HO-1, BCL-2, cytochrome C, with corresponding increase in CAT, SOD, GSH, T-SH, NP-SH, GST, GR, GSH-Px, and Cx-43 levels compared to cyclosporin groups. Lutein also abates cyclosporin-induced alterations Na + -K + -ATPase activities. Our findings showed that lutein's protective effect against cyclosporin-induced reproductive impairment might be associated with mechanisms linked to its antioxidant, anti-apoptotic, and anti-inflammatory properties, notably through up-regulation of Nrf2/HO-1/Cx-43 signaling and down-regulation of NOX-1 signaling.

Oxidative stress and inflammation cause auditory system damage via glial cell activation and dysregulated expression of gap junction proteins in an experimental model of styrene-induced oto/neurotoxicity

BackgroundRedox imbalance and inflammation have been proposed as the principal mechanisms of damage in the auditory system, resulting in functional alterations and hearing loss. Microglia and astrocytes play a crucial role in mediating oxidative/inflammatory injury in the central nervous system; however, the role of glial cells in the auditory damage is still elusive.ObjectivesHere we investigated glial-mediated responses to toxic injury in peripheral and central structures of the auditory pathway, i.e., the cochlea and the auditory cortex (ACx), in rats exposed to styrene, a volatile compound with well-known oto/neurotoxic properties.MethodsMale adult Wistar rats were treated with styrene (400 mg/kg daily for 3 weeks, 5/days a week). Electrophysiological, morphological, immunofluorescence and molecular analyses were performed in both the cochlea and the ACx to evaluate the mechanisms underlying styrene-induced oto/neurotoxicity in the auditory system.ResultsWe showed that the oto/neurotoxic insult induced by styrene increases oxidative stress in both cochlea and ACx. This was associated with macrophages and glial cell activation, increased expression of inflammatory markers (i.e., pro-inflammatory cytokines and chemokine receptors) and alterations in connexin (Cxs) and pannexin (Panx) expression, likely responsible for dysregulation of the microglia/astrocyte network. Specifically, we found downregulation of Cx26 and Cx30 in the cochlea, and high level of Cx43 and Panx1 in the ACx.ConclusionsCollectively, our results provide novel evidence on the role of immune and glial cell activation in the oxidative/inflammatory damage induced by styrene in the auditory system at both peripheral and central levels, also involving alterations of gap junction networks. Our data suggest that targeting glial cells and connexin/pannexin expression might be useful to attenuate oxidative/inflammatory damage in the auditory system.

Zuogui Pills improve testicular development of offspring rats exposed to prenatal stress via Cx43

This study aims to reveal the mechanism of prenatal stress in affecting the testicular development of offspring rats and the intervention effects of Zuogui Pills via connexin 43(Cx43). Forty pregnant SD rats were randomized into a blank control group, a mo-del group, a high-dose(18.9 g·kg~(-1)) Zuogui Pills group, a low-dose(9.45 g·kg~(-1)) Zuogui Pills group, and a vitamin E(1.44 mg·kg~(-1)) group. The other groups except the blank control group was subjected to chronic unpredictable mild stress for the modeling of prenatal stress. The model was evaluated by sucrose preference test, open field test, and enzyme-linked immunosorbent assay(ELISA) of the glucocorticoid level. ELISA was employed to measure the thyroxine 4(T4), testosterone(T), and follicle-stimulating hormone(FSH) levels to assess kidney deficiency. Hematoxylin-eosin(HE) staining was employed to evaluate the status of testicular germ cells. An automatic sperm analyzer was used to measure the sperm quality. Immunofluorescence double staining was employed to detect the expression of Cx43 and follicle-stimulating hormone receptor(FSHR) in the testes of offspring rats. The mRNA and protein levels of Cx43, FSHR, phosphatidylinositol 3-kinase(PI3K), and protein kinase B(Akt) were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot, respectively. Prenatal stress induced testicular development disorders in offspring rats. The HE staining results showed that on the day of birth, the model group had reduced seminiferous tubules in the testes, elevated FSH level in the serum, and lowered Cx43 level in the testicular tissue. Male offspring rats of 60 days old had reduced testicular spermatogenic function, decreased sperm quality, elevated FSH level and lowered T level in the serum, and down-regulated protein and mRNA levels of Cx43, FSHR, PI3K, and Akt in the testicular tissue. Zuogui Pills alleviated the abnormal development and dysfunction of testicles in the offspring rats caused by prenatal stress. In summary, Zuogui Pills may weaken the effects of prenatal stress on testicular development and spermatogenic function of offspring rats by activating the PI3K/Akt pathway to regulate Cx43 expression in the testicular tissue.

Multiple exposures to low-dose ionizing radiation induced the initiation and progression of pro-atherosclerotic phenotypes in mice and vascular endothelial cell damage.

This study aimed to investigate the effects of low-dose radiation on the abdominal aorta of mice and vascular endothelial cells. Wild-type and tumor-bearing mice were exposed to 15 sessions of low-dose irradiation, resulting in cumulative radiation doses of 187.5, 375, and 750 mGy. The effect on the cardiovascular system was assessed. Immunohistochemistry analyzed protein expressions of PAPP-A, CD62, P65, and COX-2 in the abdominal aorta. Microarray technology, Gene Ontology analysis, and pathway enrichment analysis evaluated gene expression changes in endothelial cells exposed to 375 mGy X-ray. Cell viability was assessed using the Cell Counting Kit 8 assay. Immunofluorescence staining measured γ-H2AX levels, and real-time polymerase chain reaction quantified mRNA levels of interleukin-6 (IL-6), ICAM-1, and Cx43. Hematoxylin and eosin staining revealed thickening of the inner membranes and irregular arrangement of smooth muscle cells in the media membrane at 375 and 750 mGy. Inflammation was observed in the inner membranes at 750 mGy, with a clear inflammatory response in the hearts of tumor-bearing mice. Immunohistochemistry indicated increased levels of PAPP-A, P65, and COX-2 post-irradiation. Microarray analysis showed 425 up-regulated and 235 down-regulated genes, associated with processes like endothelial cell-cell adhesion, IL-6, and NF-κB signaling. Cell Counting Kit 8 assay results indicated inhibited viability at 750 mGy in EA.hy926 cells. Immunofluorescence staining demonstrated a dose-dependent increase in γ-H2AX foci. Reverse transcription quantitative PCR results showed increased expression of IL6, ICAM-1, and Cx43 in EA.hy926 cells post 750 mGy X-ray exposure. Repeated low-dose ionizing radiation exposures triggered the development of pro-atherosclerotic phenotypes in mice and damage to vascular endothelial cells.

Bee gomogenat enhances the healing process of diabetic wounds by orchestrating the connexin-pannexin gap junction proteins in streptozotocin-induced diabetic mice

Delay in wound healing remains one of diabetes's worse side effects, which increases mortality. The proposed study sought to scrutinize the implications of bee gomogenat (BG) on diabetic's wound closure in a streptozotocin-(STZ)-enhanced type-1 diabetes model’s rodents. We used 3 different mice groups: group 1 non-diabetic rodents "serving as control", group 2 diabetic rodents, and group3 BG-treated diabetic rodents. We noticed that diabetic rodents experience a delayed wound closure, which emerged as a significant (*P < 0.05) decline in the deposition of collagen as compared to control non-diabetic animals. We noticed that diabetic rodents have a delayed wound closure characterized by a significant (*P < 0.05) decrease in the CD31 expression (indicator for wound angiogenesis and neovascularization) and an apparent elevation in the expression of such markers of inflammation as MCP-1 and HSP-70 as compared to control animals. Moreover, diabetic animals displayed a significant (*P < 0.05) increase in the expression of gap junction proteins Cx43 and a significant decrease in the expression of Panx3 in the wounded skin tissues when compared to the controls. Intriguingly, topical application with BG on the diabetic wounded skin tissues contributes to a significant (#P < 0.05) enhancing in the collagen deposition, up-regulating the level of CD31 expression and a significant (#P < 0.05) down-regulation in the MCP-1 and HSP-70 expressions as compared to diabetic non-treated animals. The expression's levels of Cx43 and Panx3 were significantly (#P < 0.05) retrieved in diabetic rodents after BG treatment. Taken together, our findings showed for the first time that BG promotes the recovering process and accelerated the closure of diabetic related wounds.