Cx43 Protein Levels Research Articles

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.

Tamoxifen-induced alterations in the expression of connexin 43 in the chicken ovary

Connexin 43 (Cx43) is a gap junction protein that participates in small molecule exchange between adjacent cells. It is a predominant Cx within the mammalian ovary, where is associated with proper follicle development. The expression and regulation of Cx43 in the chicken ovary is largely unknown. The aim of the present study was to examine the expression of the Cx43 gene (GJA1) and protein as well as the immunolocalization of Cx43 in the laying hen ovary in relation to follicle development, and to examine how tamoxifen (TMX; an estrogen receptor modulator) treatment affects these factors. qRT-PCR and western blotting demonstrated differences in Cx43 mRNA transcript and protein abundances in ovarian white follicles, yellowish follicles, small yellow follicles, and the largest yellow preovulatory follicles (F3–F1). In general, Cx43 was more abundant in hierarchical than prehierarchical follicles and in granulosa cells compared with theca cells. Further, the response to TMX treatment depended on the stage of follicle development and the layer of the follicular wall. Ovarian regression following TMX treatment was accompanied by an increase in Cx43 expression in most ovarian tissues, which may impact the formation and function of Cx43 hemichannels. Overall, our results showed, for the first time, the differences in Cx43 mRNA and protein levels between ovarian follicles, suggesting the potential involvement of this gap junction protein in the regulation of ovarian follicle development and function. In addition, the results indicate a possible role for estradiol in regulation of Cx43 transcription and/or translation in the chicken ovary. Understanding the contribution of Cx43 in mechanisms underlying ovarian follicle development may be of considerable importance for poultry egg production.

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.

CX43 and oxidative stress are the targets of BCB staining to predict the developmental potential of buffalo oocytes.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform invitro maturation, invitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the invitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

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.

Propafenone-induced connexin-43 protein accumulation and intracellular dynamics

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): China Scholarship Council Background Connexin-43 (Cx43) plays a pivotal role in intercellular communication through gap junctions. It is instrumental in propagating electrical signals and allowing ions to flow between neighboring cardiomyocytes. This synchronized electrical activity allows the heart to work as a functional syncytium. Any disruption or malfunction of Cx43 can lead to arrhythmias and other cardiac issues. Understanding the functions and regulation of Cx43 is vital in both basic research and clinical contexts. Propafenone, a class Ic antiarrhythmic drug, has shown promise in rhythm control; however, its precise impact on cardiac cellular physiology, particularly regarding Cx43, remains incompletely understood. Purpose To investigate propafenone’s effect on Cx43 expression, physiology, and underlying mechanisms in cell systems. Methods Used cell lines include human embryonic kidney HEK293 cells transfected with Cx43; differentiated murine embryonic carcinoma EPI7 cells with an epithelioid morphology and visceral endoderm-like END2 cells, endogenously expressing high amounts of functional Cx43. Cx43 protein expression levels were determined by Western blot analysis, whereas immunofluorescence (IF) imaged by a Total Internal Reflection Fluorescence (TIRF) microscope was used to assess the subcellular localization of Cx43 proteins. Dye injections were used to gain insight into the effects of propafenone on Cx43 metabolic coupling. Results and Conclusion Full length Cx43 protein levels were increased after treatment with propafenone and IF microscopy showed an intracellular accumulation of Cx43 protein, both on ectopically and endogenously expressed Cx43. Propafenone did not alter Cx43 half-life, in contrast to the lysosomal inhibitor chloroquine. Finally, gap-junctional coupling was decreased by chronic propafenone treatment. We conclude that the class 1c antiarrhythmic drug propanone increases Cx43 protein expression, resulting in its intracellular accumulation, as a side effect.

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.

Yu Linzhu alleviates primary ovarian insufficiency in a rat model by improving proliferation and energy metabolism of granulosa cells through hif1α/cx43 pathway

BackgroundYu Linzhu (YLZ) is a classical Chinese traditional formula, which has been used for more than 600 years to regulate menstruation to help pregnancy. However, the mechanism of modern scientific action of YLZ needs to be further studied.MethodsThirty SD female rats were divided into three groups to prepare the blank serum and drug-containing serum, and then using UHPLC-QE-MS to identify the ingredients of YLZ and its drug-containing serum. Twenty-four SD female rats were divided into four groups, except the control group, 4-vinylcyclohexene dicycloxide (VCD) was intraperitoneally injected to establish a primary ovarian insufficiency (POI) model of all groups. Using vaginal smear to show that the estrous cycle of rats was disturbed after modeling, indicates that the POI model was successfully established. The ELISA test was used to measure the follicle-stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH) levels in the serum of rats. HE stain was used to assess the morphology of ovarian tissue. The localization and relative expression levels of CX43 protein were detected by tissue immunofluorescence. Primary ovarian granulosa cells (GCs) were identified by cellular immunofluorescence. CCK8 was used to screen time and concentration of drug-containing serum and evaluate the proliferation effect of YLZ on VCD-induced GCs. ATP kit and Seahorse XFe24 were used to detect energy production and real-time glycolytic metabolism rate of GCs. mRNA and protein expression levels of HIF1α, CX43, PEK, LDH, HK1 were detected by RT-PCR and WB.ResultsUHPLC-QE-MS found 1702 ingredients of YLZ and 80 constituents migrating to blood. YLZ reduced the FSH while increasing the AMH and E2 levels. In ovarian tissues, YLZ improved ovarian morphology, follicle development, and the relative expression of CX43. In vitro studies, we found that YLZ increased the proliferative activity of GCs, ATP levels, glycolytic metabolic rate, HIF1α, CX43, PEK, HK1, LDH mRNA, and protein levels.ConclusionsThe study indicated that YLZ increased the proliferation and glycolytic energy metabolism of GCs to improve follicular development further alleviating ovarian function.

REDUCED CX43 EXPRESSION INDUCES AUTOPHAGY THROUGH ACTIVATION OF THE AMPK-MTOR-ULK1 SIGNALING PATHWAY IN THE COMMON BILE DUCT LIGATION RAT HEART.

Backgrounds: This study aimed to investigate the relationship between Cx43 expression and autophagy mediated by the AMPK-mTOR-Ulk1 signaling pathway in jaundice heart. Methods: In this study, a jaundice model was established in common bile duct ligation (CBDL) rats. Cardiac injury was assessed using various methods including myocardial injury indicators, echocardiography, transmission electron microscopy, hematoxylin and eosin staining, Masson staining, immunohistochemical analyses, and immunofluorescence staining. We investigated the regulatory relationship between Cx43, autophagy, and the AMPK-mTOR-ULK pathway in vivo by administering autophagy agonists (Rapa), autophagy inhibitors (3-MA), and Cx43 inhibitors (Gap 26). In vitro , we observed the relationship between autophagy and the AMPK-mTOR-ULK1 pathway in cells by exposing them to the AMPK inhibitor Compound C and the AMPK activator AICAR. Results: We found that CBDL induced autophagy through the AMPK-mTOR-ULK pathway, leading to the inhibition of myocardial dysfunction. Rapamycin pretreatment with CBDL3d exhibited a protective effect against myocardial injury and promoted autophagy. In contrast, 3-MA had no impact. Pretreatment with rapamycin at CBDL2w enhanced autophagy and aggravated cardiac injury; however, inhibition of autophagy using 3-MA attenuated cardiac injury. Cell viability was enhanced by AMPK inhibitors and inhibited by AMPK agonists. In addition, we observed that increased autophagy led to decreased Cx43 expression, which negatively affected cardiac function. Conclusions: CBDL induces myocardial injury in rats and activates autophagy through the AMPK-mTOR-ULK pathway, resulting in decreased Cx43 protein levels. A moderate increase in early autophagy in CBDL can improve cardiac injury, while late inhibition of autophagy can reduce myocardial injury.

Software BreastAnalyser for the semi-automatic analysis of breast cancer immunohistochemical images

Breast cancer is the most diagnosed cancer worldwide and represents the fifth cause of cancer mortality globally. It is a highly heterogeneous disease, that comprises various molecular subtypes, often diagnosed by immunohistochemistry. This technique is widely employed in basic, translational and pathological anatomy research, where it can support the oncological diagnosis, therapeutic decisions and biomarker discovery. Nevertheless, its evaluation is often qualitative, raising the need for accurate quantitation methodologies. We present the software BreastAnalyser, a valuable and reliable tool to automatically measure the area of 3,3’-diaminobenzidine tetrahydrocholoride (DAB)-brown-stained proteins detected by immunohistochemistry. BreastAnalyser also automatically counts cell nuclei and classifies them according to their DAB-brown-staining level. This is performed using sophisticated segmentation algorithms that consider intrinsic image variability and save image normalization time. BreastAnalyser has a clean, friendly and intuitive interface that allows to supervise the quantitations performed by the user, to annotate images and to unify the experts’ criteria. BreastAnalyser was validated in representative human breast cancer immunohistochemistry images detecting various antigens. According to the automatic processing, the DAB-brown area was almost perfectly recognized, being the average difference between true and computer DAB-brown percentage lower than 0.7 points for all sets. The detection of nuclei allowed proper cell density relativization of the brown signal for comparison purposes between the different patients. BreastAnalyser obtained a score of 85.5 using the system usability scale questionnaire, which means that the tool is perceived as excellent by the experts. In the biomedical context, the connexin43 (Cx43) protein was found to be significantly downregulated in human core needle invasive breast cancer samples when compared to normal breast, with a trend to decrease as the subtype malignancy increased. Higher Cx43 protein levels were significantly associated to lower cancer recurrence risk in Oncotype DX-tested luminal B HER2- breast cancer tissues. BreastAnalyser and the annotated images are publically available https://citius.usc.es/transferencia/software/breastanalyser for research purposes.

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.

Stabilization of Cx43 mRNA via RNA-binding protein HuR regulated by polyamines enhances intestinal epithelial barrier function.

Gut barrier dysfunction occurs commonly in patients with critical disorders, leading to the translocation of luminal toxic substances and bacteria to the bloodstream. Connexin 43 (Cx43) acts as a gap junction protein and is crucial for intercellular communication and the diffusion of nutrients. The levels of cellular Cx43 are tightly regulated by multiple factors, including polyamines, but the exact mechanism underlying the control of Cx43 expression remains largely unknown. The RNA-binding protein HuR regulates the stability and translation of target mRNAs and is involved in many aspects of intestinal epithelial pathobiology. Here we show that HuR directly bound to Cx43 mRNA via its 3'-untranslated region in intestinal epithelial cells (IECs) and this interaction enhanced Cx43 expression by stabilizing Cx43 mRNA. Depletion of cellular polyamines inhibited the [HuR/Cx43 mRNA] complex and decreased the level of Cx43 protein by destabilizing its mRNA, but these changes were prevented by ectopic overexpression of HuR. Polyamine depletion caused intestinal epithelial barrier dysfunction, which was reversed by ectopic Cx43 overexpression. Moreover, overexpression of checkpoint kinase 2 in polyamine-deficient cells increased the [HuR/Cx43 mRNA] complex, elevated Cx43 levels, and promoted barrier function. These findings indicate that Cx43 mRNA is a novel target of HuR in IECs and that polyamines regulate Cx43 mRNA stability via HuR, thus playing a critical role in the maintenance of intestinal epithelial barrier function.NEW & NOTEWORTHY The current study shows that polyamines stabilize the Cx43 mRNA via HuR, thus enhancing the function of the Cx43-mediated gap junction. These findings suggest that induced Cx43 by HuR plays a critical role in the process by which polyamines regulate intestinal epithelial barrier.

Insulin regulates gap junction intercellular communication in porcine granulosa cells through modulation of connexin43 protein expression

Gap junction intercellular communication (GJIC) among granulosa cells plays an important role in folliculogenesis, and it is temporal-spatially regulated during follicular development. Connexin (Cx) proteins predominantly form the basal structure of gap junctions in granulosa cells. In our study, immunohistochemical analysis revealed that Cx43 is the most widely expressed connexin in porcine follicles, especially among the large antral follicles. With application of insulin on porcine granulosa cells, we found that insulin significantly facilitated the protein level of Cx43, not mRNA level. This process is dependent on the phosphorylated activities of AKT and Erk since selective AKT and Erk inhibitors, LY294002 and U0126, respectively, hampered the potential of insulin to up-regulate Cx43 protein expression. As a consequence, the insulin-enhanced Cx43-couple GJIC activity in porcine granulosa cells was corresponding attenuated by the administration of LY294002 and U0126. Our findings provide a new insight into the molecular mechanisms by which insulin mediates cell-cell communication in porcine granulosa cells and sheds light on nutrition-reproduction interactions.

Inhibition of Pyk2 Improves Cx43 Intercalated Disc Localization, Infarct Size, and Cardiac Function in Rats With Heart Failure.

Heart failure causes changes in Cx43 (Connexin43) regulation that are associated with arrhythmic heart disease. Pyk2 (proline-rich tyrosine kinase 2) is activated in cardiomyopathies and phosphorylates Cx43 to decrease intercellular communication. This study was designed to determine if Pyk2 inhibition improves cardiac function in a myocardial infarction (MI)-induced heart failure model in rats. MI (ligation of left anterior descending artery) rats were treated with the Pyk2 inhibitor PF4618433. Hemodynamic and structural parameters were monitored in Sham (n=5), MI-vehicle (n=5), and MI-PF4618433 (n=8) groups. Heart tissues were collected after 6 weeks to assess Pyk2 and Cx43 protein level and localization. PF4618433 produced no observed adverse effects and inhibited ventricular Pyk2. PF4618433 reduced the MI infarct size from 34% to 17% (P=0.007). PF4618433 improved stroke volume (P=0.031) and cardiac output (P=0.009) in comparison to MI-vehicle with values similar to the Sham group. PF4618433 also led to an increase in the ejection fraction (P=0.002) and fractional shortening (P=0.006) when compared with the MI-vehicle (32% and 35% improvement, respectively) yet were lower in comparison with the Sham group. Pyk2 inhibition decreased Cx43 tyrosine phosphorylation (P=0.043) and maintained Cx43 at the intercalated disc in the distal ventricle 6 weeks post-MI. Unlike other attempts to decrease Cx43 remodeling after MI-induced heart failure, inhibition of Pyk2 activity maintained Cx43 at the intercalated disc. This may have aided in the reduced infarct size (acute time frame) and improved cardiac function (chronic time frame). Additionally, we provide evidence that Pyk2 is activated following MI in human left ventricle, implicating a novel potential target for therapy in patients with heart failure.

The effect of different doses of nonylphenol on the blood-testicular barrier integrity, hormone level, and DNA damage in the testes of rats.

Determining the molecular characteristics of the damage caused by NP exposure in the testis is very important for understanding the source of the damage and developing treatment methods accordingly. Therefore, in this study, it is aimed to evaluate the toxic effects that different doses of NP may cause in the testis, including blood-testicular barrier integrity and sperm DNA damage. For this purpose, 50 adult male Wistar albino rats were used in the study. Low, medium, and high-dose NP groups and the corn oil group were formed. After NP administration at determined doses for 15 days, the testis tissue taken under anesthesia was fixed in formaldehyde. Paraffin blocks were embedded using the routine histological tissue follow-up method. Histopathological and immunohistochemical analyses were performed by taking 5μm thick sections from paraffin blocks. The other testicular tissue was taken for the Western blot, Elisa, and comet methods, and the findings of sperm DNA analysis and the blood-testicular barrier were examined. NP caused the seminiferous epithelium to be disorganized and have significantly fewer cells in the testes of rats in different dose NP-induced groups. Compared with the control group, mTOR, Cx43, SCF, and HSP70 protein levels were decreased, while the expression of MMP-9 levels was increased in the different NP dose groups. Furthermore, tissue testosterone and inhibin B levels and SF-1 immunoreactivity intensity gradually decreased depending on the dose increase of NP. DNA damage of testicular tissues were increased in NP groups depending on NP dose. These results suggest that it is evident that NP, a commonly used industrial chemical, is an endocrine disrupting chemical (EDC) with estrogenic activity exerting adverse effects on health and that urgent measures are needed regarding the use.

NON-PHARMACOLOGICAL INTERVENTIONS UP-REGULATE MYOCARDIAL CONNEXIN-43 AND DECREASE A RISK OF MALIGNANT ARRHYTHMIAS IN EXPERIMENTAL MODEL OF ESSENTIAL HYPERTENSION

Objective: Available data suggest that meta-inflammation and oxidative stress as well as endogenous deficiency of omega-3 and melatonin may be involved in hypertension-induced myocardial abnormalities that increase propensity to cardiac arrhythmias. We aimed to explore whether supplementation with omega-3 and melatonin as well as cold acclimation may affect arrhythmogenicity of rats suffering from essential hypertension. We focused on myocardial connexin-43(Cx43) channel protein, which ensures cardiac cell-cell coupling for electrical signal propagation, thereby is crucial factor impacting susceptibility of the heart malignant arrhythmias. Design and method: Experiments were performed using adult, male, spontaneously hypertensive rats (SHR, wild type), hairless SHR (mutant strain) and normotensive Wistar rats. SHR were treated for two-month with omega-3 (Omacor,200mg/day) or melatonin (400ug/day) and compared with untreated rats. Hairless SHR adapted to cold were compared with wild type SHR and normotensive Wistar rats. Biometrical parameters were registered and left ventricular heart tissue was taken for analysis of Cx43 and PKC↙, which via phosphorylation affects Cx43 channel function. Isolated perfused heart was used for the examination of its susceptibility to electrically-induced sustained ventricular fibrillation (VF). Results: Supplementation with omega-3 or melatonin and cold acclimation did not affect significantly blood pressure as well as heart or left ventricular weights of SHR or hairless SHR. However, the electrical threshold to induce VF was significantly lower (∼20mA) in non-treated compared with treated SHR (∼35mA) as well as in wild type SHR compared to hairless SHR (∼40mA). Antiarrhythmic effect was associated with increased protein levels of Cx43 and its functional phosphorylated forms in left ventricles of treated SHR and hairless SHR compared to untreated wild type SHR. Moreover, pro-arrhythmic myocardial Cx43 distribution on lateral sides of the cardiomyocytes was attenuated by treatment or cold acclimation. In parallel, there was an increase of PKC↙ expression that may enhance cardio-protective Cx43 phosphorylation. Conclusions: In conclusion, findings indicate that non-pharmacological interventions can protect hypertensive rat heart from malignant arrhythmias, at least in part, by targeting myocardial Cx43. It is challenging to pay attention to non-pharmacological approaches in humans suffering from primary hypertension.

The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes.

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.

Lead-induced cardiomyocytes apoptosis by inhibiting gap junction intercellular communication via modulating the PKCα/Cx43 signaling pathway

ObjectiveThe aim of this study was to investigate the regulatory mechanism of Pb regulates gap junction intercellular communication to induced apoptosis in H9c2 cells. MethodsH9c2 cell line is used as the research object in this study, and treated with different concentrations of Pb acetate. Subsequently, Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. The levels of lactate dehydrogenase (LDH), aspartate transaminase (AST) and creatine kinase-MB (CK-MB) in the supernatants were measured using respective commercial enzyme-linked immune sorbent assay (ELISA) kits. Western blot was used to detect the expression of apoptosis-related protein in H9c2 cells in each group. Quantitative RT-PCR Analysis Total RNA was extracted from frozen H9c2 cells using Trizol reagent, the PKCα and Cx43 in the supernatant of H9c2 cells was determined by the BCA protein detection kit. ResultsH9c2 cells increased release of cardiac enzymes (LDH, AST, and CK-MB) and decreased cell survival rate, and the Cx43, p-Cx43, PKCα and p-PKCα protein levels showed a dose-dependent decrease after Pb treatment. PKCα was activated with PMA, the relative expression level of Cx43 protein increased significantly, the expression of Bcl-2 increased and Bax and Cyt-c decreased compared with Pb exposure group, and the myocardial enzymes (LDH, AST, and CK-MB) in cell culture supernatant decreased compared with Pb exposure group, indicating that the degree of cell damage was alleviated. Results showed that Pb inhibited PKCα activity, decreased the expression of total Cx43 and P-Cx43 protein, and aggravated myocardial injury. ConclusionsPb decrease gap junction intercellular communication, which induce apoptosis in H9c2 cells by inhibiting the PKCα/Cx43 signaling pathway.

MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

Down-regulation of connexin 43 contributes to structure and function of pulmonary artery in nicotine-administered mice

Dysregulated connexin signaling is implicated in the pathophysiology of pulmonary artery hypertension (PAH). Nicotine affects pulmonary vascular remodeling. However, the potential mechanistic link between connexin signaling and nicotine-induced pulmonary artery remodeling remains unclear. We aimed to investigate the role of connexin 43 (Cx43) in pulmonary artery remodeling in nicotine-administered C57BL/6 J wild-type (WT) and Cx43 heterozygous (Cx43+/−) mice. Hemodynamic parameters and right ventricle pathology were assessed in the mice. Serum biochemical indices of hepatic and renal function were measured. The RT-PCR, immunofluorescence, and western blotting were conducted to evaluate Cx43 mRNA and protein levels. We performed histological staining to identify pulmonary arteries. Wire myography was used to examine contraction and relaxation responses in the pulmonary arteries. Pulmonary vascular permeability was assessed through Evans blue staining. Compared with the WT group, the Cx43+/- group showed lower Cx43 mRNA and protein expression in the pulmonary arteries (P < 0.01). Nicotine treatment significantly increased Cx43 expression (P < 0.01) and induced morphological changes in the pulmonary arteries (P < 0.01). Our findings suggest that Cx43 plays a crucial role in pulmonary artery reactivity and permeability in mice. Furthermore, downregulation of Cx43 expression may contribute to alterations in pulmonary artery structure and function.