Progressive cardiomyopathy with intercalated disc disorganization in a rat model of Becker dystrophy.

Becker muscular dystrophy (BMD) is an X-linked disorder due to in-frame mutations in the DMD gene, leading to a less abundant and truncated dystrophin. BMD is less common and severe than Duchenne muscular dystrophy (DMD) as well as less investigated. To accelerate the search for innovative treatments, we developed a rat model of BMD by deleting the exons 45-47 of the Dmd gene. Here, we report a functional and histopathological evaluation of these rats during their first year of life, compared to DMD and control littermates. BMD rats exhibit moderate damage to locomotor and diaphragmatic muscles but suffer from a progressive cardiomyopathy. Single nuclei RNA-seq analysis of cardiac samples revealed shared transcriptomic abnormalities in BMD and DMD rats and highlighted an altered end-addressing of TMEM65 and Connexin-43 at the intercalated disc, along with electrocardiographic abnormalities. Our study documents the natural history of a translational preclinical model of BMD and reports a cellular mechanism for the cardiac dysfunction in BMD and DMD offering opportunities to further investigate the organization role of dystrophin in intercellular communication.

Connexin Expression Is Altered in the Eye Development of Yotari Mice: A Preliminary Study.

This study aimed to explore how Dab1 functional silencing influences the expression patterns of different connexins in the developing yotari (yot) mice eyes as potential determinants of retinogenesis. Using immunofluorescence staining, the protein expression of Dab1, Reelin, and connexin 37, 40, 43, and 45 (Cx37, Cx40, Cx43, and Cx45) in the wild-type (wt) and yot eyes at embryonic days 13.5 and 15.5 (E13.5 and E15.5) were analyzed. Different expression patterns of Cx37 were seen between the wt and yot groups. The highest fluorescence intensity of Cx37 was observed in the yot animals at E15.5. Cx40 had higher expression at the E13.5 when differentiation of retinal layers was still beginning, whereas it decreased at the E15.5 when differentiation was at the advanced stage. Higher expression of Cx43 was found in the yot group at both time points. Cx45 was predominantly expressed at E13.5 in both groups. Our results reveal the altered expression of connexins during retinogenesis in yot mice and their potential involvement in retinal pathology, where they might serve as prospective therapeutic targets.

Insights into the role of connexins and specialized intercellular communication pathways in breast cancer: Mechanisms and applications.

Gap junctions, membrane-based channels comprised of connexin proteins (Cxs), facilitate direct communication among neighbouring cells and between cells and the extracellular space through their hemichannels. The normal human breast expresses various Cxs family proteins, such as Cx43, Cx30, Cx32, Cx46, and Cx26, crucial for proper tissue development and function. These proteins play a significant role in breast cancer development, progression, and therapy response. In primary tumours, there is often a reduction and cytoplasmic mislocalization of Cx43 and Cx26, while metastatic lesions show an upregulation of these and other Cxs. Although existing research predominantly supports the tumour-suppressing role of Cxs in primary carcinomas through channel-dependent and independent functions, controversies persist regarding their involvement in the metastatic process. This review aims to provide an updated perspective on Cxs in human breast cancer, with a specific focus on intrinsic subtypes due to the heterogeneous nature of this disease. Additionally, the manuscript will explore the role of Cxs in immune interactions and novel forms of intercellular communication, such as tunneling nanotubes and extracellular vesicles, within the breast tumour context and tumour microenvironment. Recent findings suggest that Cxs hold potential as therapeutic targets for mitigating metastasis and drug resistance. Furthermore, they may serve as novel biomarkers for cancer prognosis, offering promising avenues for future research and clinical applications.

Cx40 Levels Regulate Hypoxia-Induced Changes in the Migration, Proliferation, and Formation of Gap Junction Plaques in an Extravillous Trophoblast Cell Model.

BACKGROUND: Extravillous trophoblasts (EVTs) form stratified columns at the placenta-uterus interface. In the closest part to fetal structures, EVTs have a proliferative phenotype, whereas in the closest part to maternal structures, they present a migratory phenotype. During the placentation process, Connexin 40 (Cx40) participates in both the proliferation and migration of EVTs, which occurs under hypoxia. However, a possible interaction between hypoxia and Cx40 has not yet been established. METHODS: We developed two cellular models, one with "low Cx40" (Jeg-3), which reflected the expression of this protein found in migratory EVTs, and one with "high Cx40" (Jeg-3/hCx40), which reflected the expression of this protein in proliferative cells. We analyzed the migration and proliferation of these cells under normoxic and hypoxic conditions for 24 h. Jeg-3 cells under hypoxia increased their migratory capacity over their proliferative capacity. However, in Jeg-3/hCx40, the opposite effect was induced. On the other hand, hypoxia promoted gap junction (GJ) plaque formation between neighboring Jeg-3 cells. Similarly, the activation of a nitro oxide (NO)/cGMP/PKG-dependent pathway induced an increase in GJ-plaque formation in Jeg-3 cells. CONCLUSIONS: The expression patterns of Cx40 play a crucial role in shaping the responses of EVTs to hypoxia, thereby influencing their migratory or proliferative phenotype. Simultaneously, hypoxia triggers an increase in Cx40 gap junction (GJ) plaque formation through a pathway dependent on NO.

Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes.

Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

The Therapeutic Potential of Targeting the Connexin43 as a New Approach to Reducing Post-surgical Adhesion

Post-surgical peritoneal adhesions are a serious problem causing complications, such as bowel obstruction, infertility, and pain. There are currently no effective ways of preventing post-surgical adhesions. Excess secretion of proinflammatory cytokines and profibrotic molecules by immune cells and adherent fibroblasts are the main mechanism that promotes post-operative fibrotic scars. Although many studies have been conducted on the pathological causes of this disorder, there are still many unknown facts in this matter, so assessment of the role of different molecules in causing inflammation and adhesion can lead to the creation of new treatment methods. Connexins are a group of proteins related to gap junctions that have a role in cell communication and transmitted signaling between adjacent cells. Between different types of connexin protein isoforms, connexin43 is known to be involved in pathological conditions related to inflammation and fibrosis. Recent studies have reported that inhibition of connexin43 has the potential to reduce inflammation and fibrosis by reducing the expression of molecules like α-SMA and plasminogen activator inhibitor (PAI) that are involved in the early stages of adhesion formation. As well as, inhibition of connexin43 may have therapeutic potential as a target to prevent post-surgical peritoneal adhesions.

Connexins in epidermal health and diseases: insights into their mutations, implications, and therapeutic solutions.

The epidermis, the outermost layer of the skin, serves as a protective barrier against external factors. Epidermal differentiation, a tightly regulated process essential for epidermal homeostasis, epidermal barrier formation and skin integrity maintenance, is orchestrated by several players, including signaling molecules, calcium gradient and junctional complexes such as gap junctions (GJs). GJ proteins, known as connexins facilitate cell-to-cell communication between adjacent keratinocytes. Connexins can function as either hemichannels or GJs, depending on their interaction with other connexons from neighboring keratinocytes. These channels enable the transport of metabolites, cAMP, microRNAs, and ions, including Ca2+, across cell membranes. At least ten distinct connexins are expressed within the epidermis and mutations in at least five of them has been linked to various skin disorders. Connexin mutations may cause aberrant channel activity by altering their synthesis, their gating properties, their intracellular trafficking, and the assembly of hemichannels and GJ channels. In addition to mutations, connexin expression is dysregulated in other skin conditions including psoriasis, chronic wound and skin cancers, indicating the crucial role of connexins in skin homeostasis. Current treatment options for conditions with mutant or altered connexins are limited and primarily focus on symptom management. Several therapeutics, including non-peptide chemicals, antibodies, mimetic peptides and allele-specific small interfering RNAs are promising in treating connexin-related skin disorders. Since connexins play crucial roles in maintaining epidermal homeostasis as shown with linkage to a range of skin disorders and cancer, further investigations are warranted to decipher the molecular and cellular alterations within cells due to mutations or altered expression, leading to abnormal proliferation and differentiation. This would also help characterize the roles of each isoform in skin homeostasis, in addition to the development of innovative therapeutic interventions. This review highlights the critical functions of connexins in the epidermis and the association between connexins and skin disorders, and discusses potential therapeutic options.

Orthologs at the Base of the Olfactores Clade.

Tunicate orthologs in the human genome comprise just 84 genes of the 19,872 protein-coding genes and 23 of the 16,528 non-coding genes, yet they stand at the base of the Olfactores clade, which radiated to generate thousands of tunicate and vertebrate species. What were the powerful drivers among these genes that enabled this process? Many of these orthologs are present in gene families. We discuss the biological role of each family and the orthologs' quantitative contribution to the family. Most important was the evolution of a second type of cadherin. This, a Type II cadherin, had the property of detaching the cell containing that cadherin from cells that expressed the Type I class. The set of such Type II cadherins could now detach and move away from their Type I neighbours, a process which would eventually evolve into the formation of the neural crest, "the fourth germ layer", providing a wide range of possibilities for further evolutionary invention. A second important contribution were key additions to the broad development of the muscle and nerve protein and visual perception toolkits. These developments in mobility and vision provided the basis for the development of the efficient predatory capabilities of the Vertebrata.

Ibrutinib Contributes to Atrial Arrhythmia through the Autophagic Degradation of Connexins by Inhibiting the PI3K-AKT-mTOR Signaling Pathway.

BACKGROUND: Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced AF remains incompletely elucidated. METHODS: We investigated the proportion of ibrutinib-treated CLL patients with new-onset AF. Optical mapping was conducted to reveal the proarrhythmic effect of ibrutinib on HL-1 cells. Fluorescence staining and western blot were used to compare connexins 43 and 40 expression in ibrutinib-treated and control groups. To identify autophagy phenotypes, we used western blot to detect autophagy-related proteins, transmission electron microscopy to picture autophagosomes, and transfected mCherry-GFP-LC3 virus to label autophagosomes and lysosomes. Hydroxychloroquine as an autophagy inhibitor was administered to rescue ibrutinib-induced Cx43 and Cx40 degradation. RESULTS: About 2.67% of patients developed atrial arrhythmias after ibrutinib administration. HL-1 cells treated with ibrutinib exhibited diminished conduction velocity and a higher incidence of reentry-like arrhythmias compared to controls. Cx43 and Cx40 expression reduced along with autophagy markers increased in HL-1 cells treated with ibrutinib. Inhibiting autophagy upregulated Cx43 and Cx40. CONCLUSIONS: The off-target effect of ibrutinib on the PI3K-AKT-mTOR signaling pathway caused connexin degradation and atrial arrhythmia via promoting autophagy. CLINICAL TRIAL REGISTRATION: ChiCTR2100046062, https://clin.larvol.com/trial-detail/ChiCTR2100046062.

Astroglial gap junctions strengthen hippocampal network activity by sustaining afterhyperpolarization via KCNQ channels.

Throughout the brain, astrocytes form networks mediated by gap junction channels that promote the activity of neuronal ensembles. Although their inputs on neuronal information processing are well established, how molecular gap junction channels shape neuronal network patterns remains unclear. Here, using astroglial connexin-deficient mice, in which astrocytes are disconnected and neuronal bursting patterns are abnormal, we show that astrocyte networks strengthen bursting activity via dynamic regulation of extracellular potassium levels, independently of glutamate homeostasis or metabolic support. Using a facilitation-depression model, we identify neuronal afterhyperpolarization as the key parameter underlying bursting pattern regulation by extracellular potassium in mice with disconnected astrocytes. We confirm this prediction experimentally and reveal that astroglial network control of extracellular potassium sustains neuronal afterhyperpolarization via KCNQ voltage-gated K+ channels. Altogether, these data delineate how astroglial gap junctions mechanistically strengthen neuronal population bursts and point to approaches for controlling aberrant activity in neurological diseases.

Understanding the Role of Connexins in Hepatocellular Carcinoma: Molecular and Prognostic Implications.

Connexins, a family of tetraspan membrane proteins forming intercellular channels localized in gap junctions, play a pivotal role at the different stages of tumor progression presenting both pro- and anti-tumorigenic effects. Considering the potential role of connexins as tumor suppressors through multiple channel-independent mechanisms, their loss of expression may be associated with tumorigenic activity, while it is hypothesized that connexins favor the clonal expansion of tumor cells and promote cell migration, invasion, and proliferation, affecting metastasis and chemoresistance in some cases. Hepatocellular carcinoma (HCC), characterized by unfavorable prognosis and limited responsiveness to current therapeutic strategies, has been linked to gap junction proteins as tumorigenic factors with prognostic value. Notably, several members of connexins have emerged as promising markers for assessing the progression and aggressiveness of HCC, as well as the chemosensitivity and radiosensitivity of hepatocellular tumor cells. Our review sheds light on the multifaceted role of connexins in HCC pathogenesis, offering valuable insights on recent advances in determining their prognostic and therapeutic potential.

Cardiovascular outcomes and molecular targets for the cardiac effects of Sodium-Glucose Cotransporter 2 Inhibitors: A systematic review.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i), a new class of glucose-lowering drugs traditionally used to control blood glucose levels in patients with type 2 diabetes mellitus, have been proven to reduce major adverse cardiovascular events, including cardiovascular death, in patients with heart failure irrespective of ejection fraction and independently of the hypoglycemic effect. Because of their favorable effects on the kidney and cardiovascular outcomes, their use has been expanded in all patients with any combination of diabetes mellitus type 2, chronic kidney disease and heart failure. Although mechanisms explaining the effects of these drugs on the cardiovascular system are not well understood, their effectiveness in all these conditions suggests that they act at the intersection of the metabolic, renal and cardiac axes, thus disrupting maladaptive vicious cycles while contrasting direct organ damage. In this systematic review we provide a state of the art of the randomized controlled trials investigating the effect of SGLT2i on cardiovascular outcomes in patients with chronic kidney disease and/or heart failure irrespective of ejection fraction and diabetes. We also discuss the molecular targets and signaling pathways potentially explaining the cardiac effects of these pharmacological agents, from a clinical and experimental perspective.

Expression and Function of Connexins in Extracellular Vesicles.

Extracellular vesicles (EVs) are recognized as major vehicles for exchange of information across distant cells and tissues, which have been extensively explored for diagnosis and therapeutic purposes. The presence of multiple connexin (Cx) proteins has been described in EVs, where they might facilitate EV-cell communication. However, quantitative changes in Cx levels and functional assessment of Cx channels have only been established for Cx43. In present work, we provide a detailed description of the protocols we have optimized to assess the expression and permeability of Cx43 channels in EVs derived from cultured cells and human peripheral blood. Particularly, we include some modifications to improve quantitative analysis of EV-Cx43 by enzyme-linked immunosorbent assay (ELISA) and assessment of channel functionality by sucrose-density gradient ultracentrifugation, which can be easily adapted to other Cx family members, leveraging the development of diagnostic and therapeutic applications based on Cx-containing EVs.

Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

Protocol for the Study of Connexin and DNA Interactions.

Connexins (Cxs) are transmembrane proteins which form hemichannels and gap junction channels at the plasma membrane. These channels allow the exchange of ions and molecules between the intra- and extracellular space and between cytoplasm of adjacent cells, respectively. The channel function of Cx assemblies has been extensively studied; however, "noncanonical" functions have emerged in the last few decades and have capture the attentions of many researchers, including the role of some Cxs as gene modulators or transcription factors. In this chapter, we describe a protocol to study the interaction of Cx46 with DNA in HeLa cells. These methods can facilitate understanding the role of Cxs in physiological processes and pathological mechanisms, including, for example, the contribution of Cx46 in maintaining stemness of glioma cancer stem cells.

Gap junctions in polycystic ovary syndrome: Implications for follicular arrest.

Gap junctions are specialized intercellular conduits that provide a direct pathway between neighboring cells, which are involved in numerous physiological processes, such as cellular differentiation, cell growth, and metabolic coordination. The effect of gap junctional hemichannels in folliculogenesis is particularly obvious, and the down-regulation of connexins is related to abnormal follicle growth. Polycystic ovary syndrome (PCOS) is a ubiquitous endocrine disorder of the reproductive system, affecting the fertility of adult women due to anovulation. Exciting evidence shows that gap junction is involved in the pathological process related to PCOS and affects the development of follicles in women with PCOS. In this review, we examine the expression of connexins in follicular cells of PCOS and figure out whether such communication could have consequences for PCOS women. While along with results from clinical and related animal studies, we summarize the mechanism of connexins involved in the pathogenesis of PCOS.

Gap Junction Channel Regulation: A Tale of Two Gates-Voltage Sensitivity of the Chemical Gate and Chemical Sensitivity of the Fast Voltage Gate.

Gap junction channels are regulated by gates sensitive to cytosolic acidification and trans-junctional voltage (Vj). We propose that the chemical gate is a calmodulin (CaM) lobe. The fast-Vj gate is made primarily by the connexin's NH2-terminus domain (NT). The chemical gate closes the channel slowly and completely, while the fast-Vj gate closes the channel rapidly but incompletely. The chemical gate closes with increased cytosolic calcium concentration [Ca2+]i and with Vj gradients at Vj's negative side. In contrast, the fast-Vj gate closes at the positive or negative side of Vj depending on the connexin (Cx) type. Cxs with positively charged NT close at Vj's negative side, while those with negatively charged NT close at Vj's positive side. Cytosolic acidification alters in opposite ways the sensitivity of the fast-Vj gate: it increases the Vj sensitivity of negative gaters and decreases that of positive gaters. While the fast-Vj gate closes and opens instantaneously, the chemical gate often shows fluctuations, likely to reflect the shifting of the gate (CaM's N-lobe) in and out of the channel's pore.

Cellular communication among smooth muscle cells: The role of membrane potential via connexins.

Communication via action potentials among neurons has been extensively studied. However, effective communication without action potentials is ubiquitous in biological systems, yet it has received much less attention in comparison. Multi-cellular communication among smooth muscles is crucial for regulating blood flow, for example. Understanding the mechanism of this non-action potential communication is critical in many cases, like synchronization of cellular activity, under normal and pathological conditions. In this paper, we employ a multi-scale asymptotic method to derive a macroscopic homogenized bidomain model from the microscopic electro-neutral (EN) model. This is achieved by considering different diffusion coefficients and incorporating nonlinear interface conditions. Subsequently, the homogenized macroscopic model is used to investigate communication in multi-cellular tissues. Our computational simulations reveal that the membrane potential of syncytia, formed by interconnected cells via connexins, plays a crucial role in propagating oscillations from one region to another, providing an effective means for fast cellular communication. Statement of Significance: In this study, we investigated cellular communication and ion transport in vascular smooth muscle cells, shedding light on their mechanisms under normal and abnormal conditions. Our research highlights the potential of mathematical models in understanding complex biological systems. We developed effective macroscale electro-neutral bi-domain ion transport models and examined their behavior in response to different stimuli. Our findings revealed the crucial role of connexinmediated membrane potential changes and demonstrated the effectiveness of cellular communication through syncytium membranes. Despite some limitations, our study provides valuable insights into these processes and emphasizes the importance of mathematical modeling in unraveling the complexities of cellular communication and ion transport.

Gap Junctions or Hemichannel-Dependent and Independent Roles of Connexins in Fibrosis, Epithelial-Mesenchymal Transitions, and Wound Healing.

Fibrosis initially appears as a normal response to damage, where activated fibroblasts produce large amounts of the extracellular matrix (ECM) during the wound healing process to assist in the repair of injured tissue. However, the excessive accumulation of the ECM, unresolved by remodeling mechanisms, leads to organ dysfunction. Connexins, a family of transmembrane channel proteins, are widely recognized for their major roles in fibrosis, the epithelial-mesenchymal transition (EMT), and wound healing. Efforts have been made in recent years to identify novel mediators and targets for this regulation. Connexins form gap junctions and hemichannels, mediating communications between neighboring cells and inside and outside of cells, respectively. Recent evidence suggests that connexins, beyond forming channels, possess channel-independent functions in fibrosis, the EMT, and wound healing. One crucial channel-independent function is their role as the primary functional component for cell adhesion. Other channel-independent functions of connexins involve their roles in mitochondria and exosomes. This review summarizes the latest advances in the channel-dependent and independent roles of connexins in fibrosis, the EMT, and wound healing, with a particular focus on eye diseases, emphasizing their potential as novel, promising therapeutic targets.