Cx43 Gene Research Articles

Connexinopathies: a structural and functional glimpse.

Mutations in human connexin (Cx) genes have been related to diseases, which we termed connexinopathies. Such hereditary disorders include nonsyndromic or syndromic deafness (Cx26, Cx30), Charcot Marie Tooth disease (Cx32), occulodentodigital dysplasia and cardiopathies (Cx43), and cataracts (Cx46, Cx50). Despite the clinical phenotypes of connexinopathies have been well documented, their pathogenic molecular determinants remain elusive. The purpose of this work is to identify common/uncommon patterns in channels function among Cx mutations linked to human diseases. To this end, we compiled and discussed the effect of mutations associated to Cx26, Cx32, Cx43, and Cx50 over gap junction channels and hemichannels, highlighting the function of the structural channel domains in which mutations are located and their possible role affecting oligomerization, gating and perm/selectivity processes.

Acute restraint stress provokes sudden cardiac death in normotensive rats and enhances susceptibility to arrhythmogenic effects of adrenaline in spontaneously hypertensive rats.

A high incidence of cardiovascular events and sudden cardiac death (SCD) has been reported following unexpected acute psychosocial stress. The possible pathways by which acute restraint stress (ARS), a kind of acute psychosocial stress, leads to SCD were determined. Using 16-week-old male normotensive Wistar Kyoto rats (WKY, n=24) as controls and spontaneously hypertensive rats (SHR, n=24) as the hypertensive subjects with left ventricular hypertrophy (LVH), we assessed ARS-related incidence of SCD, cardiac and myocardial autonomic nervous system dysfunction, gap junction connexin-43 (Cx43) channel remodeling, and ventricular repolarization abnormality, based on electrocardiography, an adrenaline test, heart rate variability (HRV), and reverse transcriptase polymerase chain reaction analyses. Rats with ARS were introduced into restrainers that allowed head, limb, and tail movement. In normotensive hearts without LVH, ARS induced a higher incidence of SCD attributed to lethal bradycardia, increased cardiac and myocardial sympathetic activation, and gap junction Cx43 channel remodeling, as evidenced by the increases in the ratio of low-frequency and high-frequency powers in HRV, the ratio of myocardial neuropeptide Y (NPY) and acetylcholinesterase (AChE) mRNA expressions, and the up-regulation of LV Cx43 mRNA expression; in hypertensive hearts with LVH, ARS enhanced susceptibility to the malignant arrhythmogenic effects of the adrenaline test (a kind of sympathetic stimuli) accompanied by abnormal ventricular repolarization, as evidenced by increased incidence of ventricular tachycardia and/or ventricular fibrillation during the adrenaline test and prolonged QTc immediately after ARS. ARS may trigger cardiac and myocardial sympathetic predominance, and then induce gap junction Cx43 channel remodeling, finally leading to lethal bradycardia in normotensive WKY. ARS-induced abnormal ventricular repolarization may be responsible for ARS-enhanced susceptibility to sympathetic stimulation in SHR with LVH. Expressions of myocardial NPY, AChE, and Cx43 genes, HRV, QTc and LVH measures showed diagnostic and prognostic potential for predicting ARS-induced SCD.

Circadian expression of connexins in the mouse heart

Significant circadian variations exist in the frequency of cardiac arrhythmia; however, the underlying mechanism is largely unknown. Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is related to the genesis of cardiac arrhythmia. In this study, we investigated if cx40 and cx43 expressed circadianly in the mouse heart using suprachiasmatic nuclei (SCN) lesion and pharmacological autonomic nervous system block mouse. Significant 24-h variations were observed in the expression of cx40 and cx43 in the sham-operated mice. In the SCNX mouse, all genes examined lost circadian rhythm. In the ANSB mouse, the expressions of Bmal1 was dampened significantly but still had circadian rhythm, whereas the two connexin gene expressions lost rhythm. These results suggest that cx40 and cx43 gene expressions have clear circadian rhythm and might be regulated by the central clock in the SCN through the ANS.

Gene expression, oocyte nuclear maturation and developmental competence of bovine oocytes and embryos produced after in vivo and in vitro heat shock.

Three assays were performed. In assay 1, oocytes harvested during the winter months were subjected to kinetic heat shock by stressing the oocytes at 39.5°C (HS1) or at 40.5°C (HS2) for either 6, 12, 18 or 24 h and then matured at control temperature (38.5°C). The nuclear maturation rates (NMR) of all oocytes were recorded after 24 h. In assay 2, oocytes collected year-round maturated, were implanted via in vitro fertilization (IVF) and developed for 9 days. Gene expression analysis was performed on target genes (Cx43, CDH1, DNMT1, HSPA14) with reference to the two housekeeping genes (GAPDH and SDHA) in embryos. Similarly, in assay 3, genetic analysis was performed on the embryos produced from heat-stressed oocytes (from HS1 and HS2). In assay 1, the duration of heat stress resulted in a significant decline in NMR (P < 0.05) with HS1 for maturated oocytes at 86.4 ± 4.3; 65.5 ± 0.7; 51.3 ± 0.9; 38.1 ± 1.9 and 36.3 ± 0.9, for control, 6 h, 12 h, 18 h and 24 h, respectively. For assays 2 and 3, results demonstrated that DNMT1, Cx43 and HSPA14 were down-regulated in the embryos produced in the warm with respect to the cold months (P < 0.05). A constant up- and down-regulation of DNMT1 and HSPA14 genes were observed in both HS-treated samples. Also, an inconsistent pattern of gene expression was observed in Cx43 and CDH1 genes (P < 0.05). Targeted gene expression was aberrant in embryo development, which can provide evidence on early embryo arrest and slowed embryo development.

Impact of socio-demographic structure of the deaf people communities in prevalence of hereditary hearing loss

Hearing loss caused by environmental or genetic factors concerns more than 10 % of the world population. It leads to disability and considerably reduces the life quality of deaf people. On average, 1 in 1,000 newborns are born deaf, and 50-60 % of cases are due to genetic causes. Nonsyndromic hereditary deafness is a monogenic disease with uniquely high genetic heterogeneity. The prevalence of some forms of genetic deafness varies in different populations and could be determined, as for many other genetic diseases, by the ethnic composition of a population, isolation, founder and «bottleneck» effects, the proportion of consanguineous marriages, and probable heterozygote advantage. It is assumed that high prevalence of hearing loss due to mutations in the GJB2 (Cx26) gene was also influenced by some social factors: a long-standing tradition of assortative marriages between deaf people, combined with growth of their social adaptation and genetic fitness. The start for these events was the breakdown of the deep social isolation of deaf people, which occurred about 300 years ago in Europe, and later in the US, when special schools for the deaf with learning sign language as a common tool for communication were established (linguistic homogamy). Computer simulations and comparative retrospective study showed that over the past 200 years these social processes can have doubled the frequency of deafness in the US caused by the GJB2 gene mutations. Information about the sociodemographic structure of deaf communities in the past is extremely limited by an almost complete lack of relevant archival data. Nevertheless, studies of sociodemographic and medical-genetic characteristics of deaf people’s contemporary communities are important for predicting the prevalence of inherited forms of deafness, as well as for understanding the impact of social factors on the evolutionary processes occurring in human populations.

Electroactive polyurethane/siloxane derived from castor oil as a versatile cardiac patch, part II: HL-1 cytocompatibility and electrical characterizations.

In first part of this experiment, biocompatibility of the newly developed electroactive polyurethane/siloxane films containing aniline tetramer moieties was demonstrated with proliferation and differentiation of C2C12 myoblasts. Here we further assessed the cytocompatibility of the prepared samples with HL1-cell line, the electrophysiological properties and the patch clamp recording of the seeded cells over the selected electroactive sample. Presence of electroactive aniline tetramer in the structure of polyurethane/siloxane led to the increased expression of cardiac-specific genes of HL-1 cells involved in muscle contraction and electrical coupling. Our results showed that expression of Cx43, TrpT-2, and SERCA genes was significantly increased in conductive sample compared to tissue culture plate and the corresponding non-conductive analogous. The prepared materials were not only biocompatible in terms of cellular toxicity, but did not alter the intrinsic electrical characteristics of HL-1 cells. Embedding the electroactive moiety into the prepared films improved the properties of these polymeric cardiac construct through the enhanced transmission of electrical signals between the cells. Based on morphological observation, calcium imaging and electrophysiological recordings, we demonstrated the potential applicability of these materials for cardiac tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1398-1407, 2016.

Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue

Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro–cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro–cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P < 0.05). Terminal deoxynucleotidyl transferase–mediated biotinylated deoxyuridine triphosphates nick end-labeling assay demonstrated that apoptosis was absent in FIF, whereas follicles from all other treatments showed positive labeling. Cell proliferation index was higher in isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P < 0.05). Follicle Cx37 messenger RNA levels did not show alterations in any treatment (P > 0.05). Cx37 and Cx43 immunolabeling was localized mainly on granulosa cells and oocytes, respectively. In conclusion, isolation of ovine secondary follicles could be done successfully after vitrification of ovarian tissue, and the basement membrane integrity remained intact after in vitro culture. Although the gene and protein expression of Cx37 did not change after vitrification of ovarian tissue, Cx43 turned out to be altered in secondary follicles after vitrification and in vitro culture.

The audiological analysis in the patients homozygous for the c.-23+1G>A mutation in the GJB2 gene presenting with the loss of hearing in Yakutiya

In the course of previous investigations carried out in the Republic of Sakha (Yakutiya), we have identified the main molecular-genetic factor responsible for the hereditary impairment of hearing among the indigenous population (mostly the Yakuts).The disease was shown to be attributable to the c.-23+1G>A mutation localized in the splice donor site (exon 1) of the GJB2 (Cx26) gene. The present study involved the comprehensive audiological analysis of the patients homozygous for the c.-23+1G>A mutation in the GJB2 gene based on the results of the study of a large sample of the patients residing in Yakutiya. All individuals with the GJB2 genotype c.-23+1G>A/c.-23-1G>A (n=108) at the mean age of 14.32±4.7 years (all ethnic Yakuts)were examined with the use oftonal threshold audiometry for air conduction testing at the frequencies of 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 kHz and bone conduction testing at the frequencies of 0.25, 0.5, 1.0, and 4.0 with a step of 5.0 dB.The results of the ASSR test were used whenever tonal threshold audiometry proved impracticable The data obtained in the study characterize the allelic form of the disease associated with the GJB2 genotype c.-23+1G>A/c.-23-1G>A as the congenital bilateral symmetric (90.1%), sensorineural (90.1%) form of hearing impairment of variable severity (from grade 1 to complete deafness) with the «flat» audiological profile (median slope not more than 5.0 dB in the extended frequency range (EFR) of 0.5, 1.0, 2.0, and 4.0, kHz). It is concluded that the results of the audiological analysis performed in the present study give evidence of relatively homogeneous but variable in terms of severity impairment of hearing in the patients homozygous for the c.-23+1G>A mutation in the GJB2 (Cx26) gene. It may serve as a positive prognostic sign to be used in the development and prescription of hearing aids.

Aberrant Expression of Connexin Isoforms in the Corpus Epididymis of the Adult Rat by Exposure to Estradiol Benzoate or Flutamide at the Weaning Age.

A proper development of the epididymis during the early postnatal development is required for successful fertility in the adult male. Direct cell-cell communication via connexin (Cx) molecules is a common way of cellular interactions to achieve normal development of a given tissue consisting of different cell types. The present research was attempted to determine the effect of exogenous exposure to estrogenic agonist or antiandrogen at the weaning age on expression of Cx isoforms in the adult corpus epididymis. Male rats were subcutaneously administrated with estradiol benzoate (EB) or flutamide (Flu) at the weaning age. The tissue was collected at 4 months of age. Expressional levels of Cx isoforms were determined by a quantitative real-time PCR. Statistical comparison showed significant increases of Cxs31, 32, 37, 40, and 43 transcript amounts by a treatment of 0.015 mg of EB /kg body weight (BW). A treatment of 1.5 μg of EB /kg BW caused a significant decrease of Cx43 gene expression but increases of Cxs26, 31, 32, 37, and 40 transcript levels. Exposure to 500 mg of Flu/kg BW induced an increase of Cx37 expression but significant decreases of Cxs43 and 45 mRNA levels. Expression of Cx37 was increased by a treatment of 5 mg of Flu/kg BW, while transcript levels of Cxs26, 30.3, 31, 31.1, 32, and 43 were significantly decreased by same treatment. These results demonstrate that exposure to steroidal compounds at the early developmental age alters expression of Cx isoforms in the adult corpus epididymis.

Connexin subtype expression during oral carcinogenesis: A pilot study in patients with oral squamous cell carcinoma.

Gap junctional intercellular communication (GJIC) and connexin (Cx) expression were reported in association with carcinogenesis in various types of tumours. In an earlier histomorphometric study, the protein levels of Cx subtypes 26, 43 and 45 were differentially expressed in oral squamous cell carcinoma (OSCC), corresponding lymph node metastases and dysplasia-free oral mucosa. Moreover, membrane Cx43 acted as an independent prognostic marker in OSCC tissues. This study aimed to confirm the expression of described Cx subtypes at the mRNA level. Hence, a reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of Cx26, Cx43 and Cx45 gene expressions was performed in paired carcinoma and mucosa samples of 15 OSCC patients. Additionally, we assessed the interaction between Cx subtype expression and clinicopathological routine parameters. The RT-qPCR analysis revealed that Cx26 was downregulated in OSCC (P=0.01), while Cx43 was marginally upregulated in cancer tissue (P=0.04). Cx45 was significantly overexpressed in OSCC tissue compared with the intraoral mucosa controls (P<0.01), and remained unchanged at different tumour stages. No significant interactions between differential Cx subtype expression and clinicopathological routine parameters were observed. In conclusion, Cx regulation at the transcriptional level appears to be an early event during the initiation and development of OSCC, and is maintained during further progression. However, the mRNA-protein correlation is variable. This may be indicative of post-transcriptional, translational and degradation regulations being associated with the determination of Cx protein concentration during oral carcinogenesis.

MiR-381 suppresses C/EBPα-dependent Cx43 expression in breast cancer cells.

Cx43 (connexin43) is an enhancer of the metastasis of breast cancer cells. Our previous study identified miR-381 as an indirect suppressor of Cx43 gene expression, with the precise mechanism being not understood. In the present study, using a reporter gene assay, we found that miR-381 suppressed Cx43 gene expression via the promoter region -500/-250. With site-directed gene mutation, we demonstrated that miR-381 could directly bind with the sequences CACUUGUAU in the 3'-UTR so as to inhibit C/EBPα (CCAAT/enhancer-binding protein α) expression. C/EBPα was further identified as a novel transcription factor by binding to a canonic element (AATTGTC) locating at -459/-453in the promoter region of the Cx43 gene. Functionally, we demonstrated that miR-381 suppressed C/EBPα- and Cx43-dependent migration and invasion of breast cancer cells. Finally, we revealed that decreased levels of miR-381 as well as increased expression of C/EBPα and Cx43in the metastatic breast cancer cells and tissues. Therefore we are the first to identify that miR-381 suppresses C/EBPα-dependent Cx43 expression in breast cancer cells. The miR-381-C/EBPα-Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.

X-Linked Hereditary Motor Sensory Neuropathy Type 1 (CMTX1) in a Three-Generation Gelao Chinese Family.

In this report, we describe a three-generation family (the Gelao nationality, a minority ethnic group from Guizhou Province in the southwest China) with one affected member with Charcot-Marie-Tooth neuropathy X type 1 (CMTX1) in each generation. The three affected members carrying the R164W mutation in the Cx32 gene had different clinical symptoms. The proband, a 13-year-old boy presented recurrent episodes of transient central nervous system symptoms and concomitant transient diffuse white matter lesions on magnetic resonance imaging. His grandfather had the peripheral neurological presentations with later onset in the fourth decade, characterized by slowly progressive weakness of the distal muscles, atrophy, and foot deformities. But no sensory loss was observed. The proband's 38-year-old mother denied any neurological symptoms. The examination was normal except for pes cavus and diminished deep tendon reflexes in her lower limbs bilaterally. Genetic sequencing revealed the proband and his grandfather had a hemizygous mutation (p.164R > W) of CJB1 gene, and his mother had R164W heterozygous mutation. Our three cases denied symptoms of sensory disturbances, the sensory examination including touch, pin prick, and temperature sensation showed no obvious abnormalities. Thus, further investigation is needed to improve our understanding of the Cx32 protein function in the nervous system.

EFFECT OF BOVINE SERUM ADDED TO MATURATION MEDIUM ON IN VITRO-MATURATION AND SOME GENES EXPRESSIONS OF BUFFALO OOCYTES

The effect of supplementing TCM-199 culture medium with bovine sera on buffalo’s oocytes maturation in vitro and expression of some related genes were studied. A total 1149 immature buffalo oocytes were randomly divided into three groups, G1( n=315) were cultured in medium with no serum supplement(control), G2( n=531) were cultured in medium supplemented with 10% Fetal calf serum (FCS) and G3(n= 303) were cultured in medium supplemented with 10% Estrous cow serum (ECS). After maturation, oocytes in each group were classified according to its shape and expansion into 2 subgroups, good (GO) and fair (FO) expanded oocytes. Expression of GAPDH, CX43, IGF II and β- Actin genes were studied in each subgroup. Results revealed that G3 and G2 showed high maturation rate (P<0.01 & 0.05 respectively) compared to G1(control) and ECS was superior (P<0.05) than FCS. The qualitative expression of genes under study showed no difference among the studied groups, except CX43 gene expression was missed in FO in G1 concluding that addition of bovine sera especially estrus cow serum enhance in vitro maturation of the buffalo oocytes and CX4 gene expression and further studies is needed to enhance the expression of CX43 gene which might help in increasing maturation rate.

Effects of silver nanoparticles on neonatal testis development in mice.

BackgroundMetal nanoparticles (MNPs) play an important role in consumer products. An increasing use of MNPs has raised concerns about potential risks for human health. Therefore, in vivo tests of MNPs are urgently required. Using mice as a model animal, the aim of the present study was designed to investigate the effect of biologically synthesized silver nanoparticles (AgNPs) on spermatogenesis in neonatal mice.MethodsAgNPs were synthesized using Bacillus funiculus. The prepared nanoparticles were characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, Fourier transform-infrared spectroscopy, and transmission electron microscopy. The prepared AgNPs were used to investigate testis development in neonatal mice. Institute of Cancer Research neonatal male mice were used in all experiments and were treated with different doses (0, 1, and 5 mg/kg) of AgNPs five times (interval of 3 days from postnatal day [PND] 8–21) by abdominal subcutaneous injection.ResultsThe results showed that the sperm abnormalities such as quality and quantity were significantly increased by the synthesized AgNPs. The diameter of the convoluted tubules shrank significantly in mice treated with AgNPs on PND28 and PND42. The results of reverse transcription-quantitative polymerase chain reaction indicated that the E1f1ay, Gsta4, and Fdx1 genes were up-regulated, and the Amh, Cx43, and Claudin-11 genes were down-regulated in response to AgNPs exposure on PND28; however, these genes recovered at PND60. AgNPs had no effect on the recombination levels of chromosomes in germ cells.ConclusionThese results demonstrated the adverse effects of AgNPs on the male reproductive tract, particularly spermatogenesis and the quality of sperm. This study suggests that the development of nanomaterials should be safer and non-toxic to the living organisms and the potential reprotoxicity of AgNPs should be investigated more carefully.

Identification of miR-200a as a novel suppressor of connexin 43 in breast cancer cells.

Both miRNAs (miRs) and connexin 43 (Cx43) were important regulators of the metastasis of breast cancer, whereas the miRs regulating Cx43 expression in breast cancer cells were still obscure. In the present study, we scanned and found miR-1, miR-206, miR-200a, miR-381, miR-23a/b and miR-186 were functional suppressors of human Cx43 mRNA and protein expression. Specially, we demonstrated that only miR-200a could directly target the 3'-untranslated region (3'-UTR) of human Cx43 gene. Functionally, overexpression of Cx43 in MCF cells potentiated the migration activity, whereas additional miR-200a treatment notably prevented this effect. Finally, we demonstrated that decreased levels of miR-200a and elevated expression of Cx43 in the metastatic breast cancer tissues compared with the primary ones. Thus, we are the first to identify miR-200a as a novel and direct suppressor of human Cx43, indicating that miR200a/Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.

Conductive interpenetrating networks of polypyrrole and polycaprolactone encourage electrophysiological development of cardiac cells

Conductive and electroactive polymers have the potential to enhance engineered cardiac tissue function. In this study, an interpenetrating network of the electrically-conductive polymer polypyrrole (PPy) was grown within a matrix of flexible polycaprolactone (PCL) and evaluated as a platform for directing the formation of functional cardiac cell sheets. PCL films were either treated with sodium hydroxide to render them more hydrophilic and enhance cell adhesion or rendered electroactive with PPy grown via chemical polymerization yielding PPy–PCL that had a resistivity of 1.0±0.4kΩcm, which is similar to native cardiac tissue. Both PCL and PPy–PCL films supported cardiomyocyte attachment; increasing the duration of PCL pre-treatment with NaOH resulted in higher numbers of adherent cardiomyocytes per unit area, generating cell densities which were more similar to those on PPy–PCL films (1568±126cellsmm−2, 2880±439cellsmm−2, 3623±456cellsmm−2 for PCL with 0, 24, 48h of NaOH pretreatment, respectively; 2434±166cellsmm−2 for PPy–PCL). When cardiomyocytes were cultured on the electrically-conductive PPy–PCL, more cells were observed to have peripheral localization of the gap junction protein connexin-43 (Cx43) as compared to cells on NaOH-treated PCL (60.3±4.3% vs. 46.6±5.7%). Cx43 gene expression remained unchanged between materials. Importantly, the velocity of calcium wave propagation was faster and calcium transient duration was shorter for cardiomyocyte monolayers on PPy–PCL (1612±143μm/s, 910±63ms) relative to cells on PCL (1129±247μm/s, 1130±20ms). In summary, PPy–PCL has demonstrated suitability as an electrically-conductive substrate for culture of cardiomyocytes, yielding enhanced functional properties; results encourage further development of conductive substrates for use in differentiation of stem cell-derived cardiomyocytes and cardiac tissue engineering applications. Statement of SignificanceCurrent conductive materials for use in cardiac regeneration are limited by cytotoxicity or cost in implementation. In this manuscript, we demonstrate for the first time the application of a biocompatible, conductive polypyrrole–polycaprolactone film as a platform for culturing cardiomyocytes for cardiac regeneration. This study shows that the novel conductive film is capable of enhancing cell–cell communication through the formation of connexin-43, leading to higher velocities for calcium wave propagation and reduced calcium transient durations among cultured cardiomyocyte monolayers. Furthermore, it was demonstrated that chemical modification of polycaprolactone through alkaline-mediated hydrolysis increased overall cardiomyocyte adhesion. The results of this study provide insight into how cardiomyocytes interact with conductive substrates and will inform future research efforts to enhance the functional properties of cardiomyocytes, which is critical for their use in pharmaceutical testing and cell therapy.

Biphasic Effect of Basic Fibroblast Growth Factor on Anterior Pituitary Folliculostellate TtT/GF Cell Coupling, and Connexin 43 Expression and Phosphorylation.

Basic fibroblast growth factor (bFGF) is a mitogenic and differentiating cytokine. In the anterior pituitary, folliculostellate (FS) cells constitute the major source of bFGF. bFGF affects endocrine cell proliferation and secretion in the anterior pituitary. In addition, bFGF increases its own expression by acting directly on FS cells. FS cell Cx43-mediated gap junction intercellular communication allows the establishment of an intrapituitary network for the transmission of information. In the present study, we assessed how bFGF regulates FS cell coupling. Time course studies were carried out on the FS cell line TtT/GF. Short-term bFGF treatment induced a transient cell uncoupling and the phosphorylation in Ser368 of membrane-bound Cx43 without modifying Cx43 levels. We demonstrated the involvement of the protein kinase C (PKC) isoform α in the phosphorylation of Cx43 in S368. Moreover, we showed that bFGF induced PKCα activation by stimulating its expression, phosphorylation and association with the plasma membrane. The long-term incubation with bFGF increased TtT/GF cell coupling, total Cx43 levels and Cx43 accumulation at the cell membrane of cytoplasmic projections. The Cx43 level increase was a result of the stimulation of Cx43 gene transcription as mediated by the extracellular-regulated kinase 1/2 signalling pathway. Taken together, the data show that bFGF modulates TtT/GF cell coupling by activating different pathways that lead to opposite effects on Cx43 phosphorylation and expression depending on the duration of the exposure of the cells to bFGF. A short-term bFGF exposure reduces cell-to-cell communication as a mean of desynchronising FS cells. By contrast, long-term exposure to bFGF enhances cell-to-cell communication and facilitates coordination among FS cells.

Abstract 5449: Transfection of connexin 43 enhances sunitinib-induced apoptosis in malignant mesothelioma cells possibly via its interaction with Bax

Abstract Introduction: Malignant mesothelioma (MM) is an aggressive tumor arising from mesothelial cells. MM is an increasing tumor worldwide with poor prognosis due to its chemo and radioresistant characteristics. We are aiming to enhance chemosensitivity of MM, and focus our attention on connexin (Cx), which is a component of intercellular channel called gap junction (GJ). Various evidences suggest that cellular physiology is maintained via GJ function. However, in cancer cells, GJ function is often aberrant, associating with the decreased Cx expression. Previously, we have shown that the sensitivity of MM cells to cisplatin was enhanced by increasing expression of Cx43, which is the major Cx subtype in mesothelial cells. Recently, it is suggested that MM is a highly angiogenic tumor and vascular endothelial growth factor (VEGF) signaling pathway plays a key role in pathogenesis of MM. Therefore, tyrosine kinase inhibitors (TKIs) are thought to be new prominent therapeutic agents for this tumor; however, they displayed a limited therapeutic activity in MM patients. Hence, the restoration of Cx expression would be beneficial to treatments with sunitinib (SU), a representative TKI, to exert an antiproliferative effect in MM patients. The present study aims to evaluate the effect of Cx43 transfection on the cytotoxicity of SU in MM cells. Methods: We used H28, a human MM cell line, to transfect Cx43 gene. The transfected cell was named H28-T. Cell viability was assessed by MTT assay. SubG1 population (suggested apoptotic cell population) was measured by flow cytometry. Protein interaction and expression level were analyzed by western blotting after immunoprecipitation. Results: At first, sensitivity to SU was compared between H28 and H28-T. Cell viability of H28-T was significantly lower than that of H28 in the presence of 0.38-10 μM SU. Moreover, the rate of SubG1 population was significantly higher in H28-T than in H28 in the presence of 10 μM SU. Next, the mechanism of enhanced cytotoxicity of SU was investigated using H28-T. To examine the involvement of GJ function, the combined treatment with SU and 18β-glycyrrhetinic acid, a GJ inhibitor, was performed; however, cell viability was not altered as compared to SU treatment alone. The interaction between Cx43 and Bax, a proapoptotic factor, was then investigated. It was shown that Bax was immunoprecipitated with an anti-Cx43 antibody. Protein expression of Bax was higher in H28-T than in H28. Furthermore, a cleaved (active) form of Bax was detected by treatment with 10 μM SU only in H28-T. Conclusion: These findings indicate that Cx43 directly interacts with Bax, and this interaction may be involved in enhancement of SU-induced apoptosis in MM cells through a GJ function-independent mechanism. Citation Format: Miaki Uzu, Hiromi Sato, Tatsuro Kashiba, Takuya Fujiwara, Yukihiro Shibata, Katsunori Yamaura, Akihiro Hisaka. Transfection of connexin 43 enhances sunitinib-induced apoptosis in malignant mesothelioma cells possibly via its interaction with Bax. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5449. doi:10.1158/1538-7445.AM2015-5449

Mice with conditional deletion of Cx26 exhibit no vestibular phenotype despite secondary loss of Cx30 in the vestibular end organs

Connexins are components of gap junctions which facilitate transfer of small molecules between cells. One member of the connexin family, Connexin 26 (Cx26), is prevalent in gap junctions in sensory epithelia of the inner ear. Mutations of GJB2, the gene encoding Cx26, cause significant hearing loss in humans. The vestibular system, however, does not usually show significant functional deficits in humans with this mutation. Mouse models for loss of Cx26 function demonstrate hearing loss and cochlear pathology but the extent of vestibular dysfunction and organ pathology are less well characterized. To understand the vestibular effects of Cx26 mutations, we evaluated vestibular function and histology of the vestibular sensory epithelia in a conditional knockout (CKO) mouse with Cx26 loss of function. Transgenic C57BL/6 mice, in which cre-Sox10 drives excision of the Cx26 gene from non-sensory cells flanking the sensory epithelium of the inner ear (Gjb2-CKO), were compared to age-matched wild types. Animals were sacrificed at ages between 4 and 40 weeks and their cochlear and vestibular sensory organs harvested for histological examination. Cx26 immunoreactivity was prominent in the peripheral vestibular system and the cochlea of wild type mice, but absent in the Gjb2-CKO specimens. The hair cell population in the cochleae of the Gjb2-CKO mice was severely depleted but in the vestibular organs it was intact, despite absence of Cx26 expression. The vestibular organs appeared normal at the latest time point examined, 40 weeks. To determine whether compensation by another connexin explains survival of the normal vestibular sensory epithelium, we evaluated the presence of Cx30 in the Gjb2-CKO mouse. We found that Cx30 labeling was normal in the cochlea, but it was decreased or absent in the vestibular system. The vestibular phenotype of the mutants was not different from wild-types as determined by time on the rotarod, head stability tests and physiological responses to vestibular stimulation. Thus presence of Cx30 in the cochlea does not compensate for Cx26 loss, and the absence of both connexins from vestibular sensory epithelia is no more injurious than the absence of one of them. Further studies to uncover the physiological foundation for this difference between the cochlea and the vestibular organs may help in designing treatments for GJB2 mutations.

Diseases associated with leaky hemichannels.

Hemichannels (HCs) and gap junction channels (GJCs) formed by protein subunits called connexins (Cxs) are major pathways for intercellular communication. While HCs connect the intracellular compartment with the extracellular milieu, GJCs allow the interchange of molecules between cytoplasm of two contacting cells. Under physiological conditions, HCs are mostly closed, but they can open under certain stimuli allowing the release of autocrine and paracrine molecules. Moreover, some pathological conditions, like ischemia or other inflammation conditions, significantly increase HCs activity. In addition, some mutations in Cx genes associated with human diseases, such as deafness or cataracts, lead to the formation of more active HCs or “leaky HCs.” In this article we will revise cellular and molecular mechanisms underlying the appearance of leaky HCs, and the consequences of their expression in different cellular systems and animal models, in seeking a common pattern or pathological mechanism of disease.