Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Targeting the Cx26/NANOG/Focal Adhesion Kinase Complex via Cell Penetrating Peptides in Triple Negative Breast Cancers

PurposeTriple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype and has limited treatment options. We previously identified a protein complex unique to TNBC cancer stem cells composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of Cx26 designed to target the complex could attenuate tumor growth in pre-clinical models.Experimental DesignHistological assessment was employed to verify expression of complex members. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Peptides with high affinity were engineered with a cell-penetrating sequence and assessed in functional assays including cell proliferation, self-renewal, and in vivo tumor growth, and downstream signaling changes were measured.ResultsBinding studies revealed that the Cx26 cytoplasmic C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar to micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. An antennapedia cell penetrating peptide sequence was engineered to the Cx26 C-terminal tail and confirmed intracellular localization. The cell-penetrating Cx26 C-terminal tail peptide (aCx26-pep) disrupted self-renewal as assessed by the tumorsphere formation assay and inhibited NANOG target gene expression in TNBC cells but not in luminal mammary epithelial cells. In a pre-clinical setting, aCx26-pep reduced tumor growth and proliferation and induced cell death.ConclusionsWe provide proof-of-concept that a Cx26 peptide-based strategy can inhibit TNBC growth.Translational RelevanceTriple-negative breast cancer (TNBC) is the most treatment-refractory breast cancer subtype and has limited targeted therapy options. TNBC contains a cancer stem cell (CSC) population that underlies growth and therapeutic resistance. We leveraged an aberrant intracellular protein complex in TNBC CSCs containing the gap junction subunit connexin 26 (Cx26), focal adhesion kinase (FAK), and NANOG to develop a peptide-based therapeutic strategy. We show that TNBC can be selectively targeted using this approach. Our findings provide a new strategy for treatment of this patient population by disrupting intracellular Cx26 signaling, which is linked to the CSC and epithelial-to-mesenchymal programs. Our results support the development of therapeutics targeting the Cx26/NANOG/FAK complex via peptide- and small molecule-based approaches, and future efforts will also focus on the development of a combinatorial treatment strategy with FDA-approved chemotherapeutics.One Sentence SummaryA peptide-based targeting strategy for triple-negative breast cancer was developed by targeting connexin 26-associated signaling partners.

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate’s Cochlea

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2, the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

Connexin 26 (GJB2) Gene Mutations Linked With Autosomal Recessive Nonsyndromic Sensorineural Hearing Loss in Iraqi Population.

Objectives: The ARNSHL wasn’t been studied enough in Iraq, this study aimed to detect the prevalence of the three most common mutations of Connexin 26 gene in Iraqi population. This study was conducted in order to detect c.35delG, c.235delC and c.167delT mutations in GJB2 gene, we were employed PCR-RFLP assays.Results: The current case-control study was conducted from January 2018 to January 2020, The study was included 95 deaf patients (55 males and 40 females) their age range between 11-40 years and 21.5 ± 6.3 year (mean ± SD) and 110 healthy control group, their ages range between 10-40 years and 20.1 ± 5.9 year (mean ± SD), these two groups were matched in age and gender. From 95 deaf patients with ARNSHL who were participated in this study, c.35delG was the main frequent mutation encountered with GJB2 gene, The second frequent mutation was c.235delC, None of 95 deaf patients were showed the c.167delT mutation, these variants were not detected in healthy control group which was studied parallel with patients group. Our data conclude that GJB2 c.35delG and c.235delC gene mutations were the main cause of ARNSHL in Iraqi deaf population.

Connexin 26 (GJB2) Gene Mutations Linked With Autosomal Recessive Nonsyndromic Sensorineural Hearing Loss in Iraqi Population

Background:Deafness is a total or partial hearing loss that may appear at any ages with different degrees of severity. Approximately 50% of hearing loss have a genetic origin, among them, the nonsyndromic sensorineural deafness represents about 70% of the cases. From them 80% corresponding to autosomal recessive inheritance deafness. Objective: Autosomal recessive deafness was not been studied enough at molecular level in Iraq, so this study aimed to detect the prevalence of the three most common mutations of Connexin 26 (GJB2) gene in nonsyndromic sensorineural deafness for Iraqi population.Method: The current case-control study was conducted from January 2018 to January 2020 at molecular laboratory in Anatomy and Histology Department/ faculty of Medicine/ Kufa University/Najaf/ Iraq. The study was included 95 deaf patients (55 males and 40 females) their age range between 11-40 years old and 21.5 ± 6.3 year (mean ± SD) and 110 healthy control group, their ages range between 10-40 years old and 20.1 ± 5.9 year (mean ± SD), these two groups were matched in age and gender. In order to detect c.35delG, 235delC and 167delT mutations in GJB2 gene, we were employed the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) technique.Results: From 95 deaf patients with ARNSHL who were participated in this study, the c.35delG was the main frequent mutation encountered with GJB2 gene, among them 35(36.8%) were homozygous, 40(42.1%) were heterozygous and 20 (21.1%) were wild genotypes. The second degree mutation in GJB2 gene was c.235delC mutation, which from the 95 deaf patients, there were 21 (22.1%) carried out homozygous, 33 (34.7%) heterozygous and 42(44.2%) wild genotypes. None of the 95 deaf patients were showed the c.167delT mutation, on the other hand these variants were not detected in healthy control group which was studied parallel with patients group.Conclusion: Our data conclude that the GJB2 c.35delG and c.235delC gene mutations were the main cause of ARNSHL in Iraqi deaf population.