Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

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.

In healthy hearts myocytes are typically coupled to nearest neighbours through gap junctions. Under pathological conditions such as fibrosis, or in scar tissue, or across ablation lines myocytes can uncouple from their neighbours. Electrical conduction may still occur via fibroblasts that not only couple proximal myocytes but can also couple otherwise unconnected regions. We hypothesise that such coupling can alter conduction between myocytes via introduction of delays or by initiation of premature stimuli that can potentially result in reentry or conduction blocks. To test this hypothesis we have developed several 2-cell motifs and investigated the effect of fibroblast mediated electrical coupling between uncoupled myocytes. We have identified various regimes of myocyte behaviour that depend on the strength of gap-junctional conductance, connection topology, and parameters of the myocyte and fibroblast models. These motifs are useful in developing a mechanistic understanding of long-distance coupling on myocyte dynamics and enable the characterisation of interaction between different features such as myocyte and fibroblast properties, coupling strengths and pacing period. They are computationally inexpensive and allow for incorporation of spatial effects such as conduction velocity. They provide a framework for constructing scar tissue boundaries and enable linking of cellular level interactions with scar induced arrhythmia.

Intercellular communication plays a crucial role in cancer, as well as other diseases, such as inflammation, tissue degeneration, and neurological disorders. One of the proteins responsible for this, are connexins (Cxs), which come together to form a hemichannel. When two hemichannels of opposite cells interact with each other, they form a gap junction (GJ) channel, connecting the intracellular space of these cells. They allow the passage of ions, reactive oxygen and nitrogen species (RONS), and signaling molecules from the interior of one cell to another cell, thus playing an essential role in cell growth, differentiation, and homeostasis. The importance of GJs for disease induction and therapy development is becoming more appreciated, especially in the context of oncology. Studies have shown that one of the mechanisms to control the formation and disruption of GJs is mediated by lipid oxidation pathways, but the underlying mechanisms are not well understood. In this study, we performed atomistic molecular dynamics simulations to evaluate how lipid oxidation influences the channel properties of Cx26 hemichannels, such as channel gating and permeability. Our results demonstrate that the Cx26 hemichannel is more compact in the presence of oxidized lipids, decreasing its pore diameter at the extracellular side and increasing it at the amino terminus domains, respectively. The permeability of the Cx26 hemichannel for water and RONS molecules is higher in the presence of oxidized lipids. The latter may facilitate the intracellular accumulation of RONS, possibly increasing oxidative stress in cells. A better understanding of this process will help to enhance the efficacy of oxidative stress-based cancer treatments.

Astrocytes are diverse brain cells that form large networks communicating via gap junctions and chemical transmitters. Despite recent advances, the functions of astrocytic networks in information processing in the brain are not fully understood. In culture, brain slices, and in vivo, astrocytes, and neurons grow in tight association, making it challenging to establish whether signals that spread within astrocytic networks communicate with neuronal groups at distant sites, or whether astrocytes solely respond to their local environments. A multi-electrode array (MEA)-based device called AstroMEA is designed to separate neuronal and astrocytic networks, thus allowing to study the transfer of chemical and/or electrical signals transmitted via astrocytic networks capable of changing neuronal electrical behavior. AstroMEA demonstrates that cortical astrocytic networks can induce a significant upregulation in the firing frequency of neurons in response to a theta-burst charge-balanced biphasic current stimulation (5 pulses of 100 Hz × 10 with 200 ms intervals, 2 s total duration) of a separate neuronal-astrocytic group in the absence of direct neuronal contact. This result corroborates the view of astrocytic networks as a parallel mechanism of signal transmission in the brain that is separate from the neuronal connectome. Translationally, it highlights the importance of astrocytic network protection as a treatment target.

It has been proposed that when gap junctional coupling is reduced in cardiac tissue, action potential propagation can be supported via ephaptic coupling, a mechanism mediated by negative electric potentials occurring in narrow intercellular clefts of intercalated discs (IDs). Recent studies showed that sodium (Na+ ) channels form clusters near gap junction plaques in nanodomains called perinexi, where the ID cleft is even narrower. To examine the electrophysiological relevance of Na+ channel clusters being located in perinexi, we developed a 3D finite element model of two longitudinally abutting cardiomyocytes, with a central Na+ channel cluster on the ID membranes. When this cluster was located in the perinexus of a closely positioned gap junction plaque, varying perinexal width greatly modulated impulse transmission from one cell to the other, with narrow perinexi potentiating ephaptic coupling. This modulation occurred via the interplay of Na+ currents, extracellular potentials in the cleft and patterns of current flow within the cleft. In contrast, when the Na+ channel cluster was located remotely from the gap junction plaque, this modulation by perinexus width largely disappeared. Interestingly, the Na+ current in the ID membrane of the pre-junctional cell switched from inward to outward during excitation, thus contributing ions to the activating channels on the post-junctional ID membrane. In conclusion, these results indicate that the localization of Na+ channel clusters in the perinexi of gap junction plaques is crucial for ephaptic coupling, which is furthermore greatly modulated by perinexal width. These findings are relevant for a comprehensive understanding of cardiac excitation. KEY POINTS: Ephaptic coupling is a cardiac conduction mechanism involving nanoscale-level interactions between the sodium (Na+ ) current and the extracellular potential in narrow intercalated disc clefts. When gap junctional coupling is reduced, ephaptic coupling acts in conjunction with the classical cardiac conduction mechanism based on gap junctional current flow. In intercalated discs, Na+ channels form clusters that are preferentially located in the periphery of gap junction plaques, in nanodomains known as perinexi, but the electrophysiological role of these perinexi has never been examined. In our new 3D finite element model of two cardiac cells abutting each other with their intercalated discs, a Na+ channel cluster located inside a narrowed perinexus facilitated impulse transmission via ephaptic coupling. Our simulations demonstrate the role of narrowed perinexi as privileged sites for ephaptic coupling in pathological situations when gap junctional coupling is decreased.

OBJECTIVE: It is hypothesised that systemic calcitonin delivery with adjunct local platelet-rich fibrin (PRF) therapy is more effective in augmenting osseointegration than calcitonin delivery alone under experimental osteoporosis conditions. The primary objective of the present experiment was to assess the effect of systemic calcitonin delivery with and without adjunct local PRF therapy on osseointegration in ovariectomised osteoporotic rabbits.
METHODS: Thirty female bilaterally ovariectomized rabbits were used. The animals were fed a low-calcium diet to establish a model for osteoporosis. In each animal, 2 implants were bilaterally placed in tibia. The animals were randomly divided equally into 3 groups. In group 1, no treatment was offered (control group). In groups 2 and 3, the animals received intramuscular injections of calcitonin without and with local PRF delivery prior to implant placement, respectively. All animals were euthanised at 12 weeks, and osseointegration was assessed as the gap widths between the bone and implant surface in the cervical, middle and apical third using scanning electron microscopy and energy-dispersive x-ray spectroscopy. The bone-to-implant contact (BIC) was also measured. p < 0.05 was defined as statistically significant.
RESULTS: Gap widths in the cervical (p < 0.001), middle (p < 0.001) and apical third (p < 0.001) were statistically significantly higher in group 1 than groups 2 and 3. Gap widths in the cervical (p < 0.001), middle (p < 0.001) and apical third (p < 0.001) were significantly higher in group 3 than group 2. The mean BIC was statistically significantly higher in the cervical (p < 0.001), middle (p < 0.001) and apical third (p < 0.001) in group 3 compared with groups 2 and 3.
CONCLUSIONS: When used as an adjunct to calcitonin, PRF enhanced osseointegration in an experimental osteoporosis model. However, further well-designed studies with inclusion of additional groups (treatment with PRF alone) are needed.

The hippocampus plays a key role in memory formation and learning. According to the concept of active systems memory consolidation, transiently stored memory traces are transferred from the hippocampus into the neocortex for permanent storage. This phenomenon relies on hippocampal network oscillations, particularly sharp wave ripples [SPW-Rs). In this process prior saved data in the hippocampus may be reactivated. Recent investigations reveal that several neurotransmitters and neuromodulators including norepinephrine, acetylcholine, serotonin, etc., suppress SPW-Rs activity in rodents\' hippocampal slices. This suppression of SPW-Rs may depend on various presynaptic and postsynaptic parameters including decrease in calcium influx, hyperpolarization/depolarization and alteration in gap junctions\' function in pyramidal cells. In this study, we demonstrate the impact of calcium influx and gap junctions on pyramidal cells for the modulation of SPW-Rs in a computational model of CA1.We used,SPW-Rs model with some modifications. SPW-Rs are simulated with gradual reduction of calcium and with decreasing conductance through gap junctions in PCs. Both, with calcium reduction as well as with conductance reduction through gap junctions, SPW-Rs are suppressed. Both effects add up synergistically in combination.

The synchronization of astrocytes via gap junctions (GJ) is a crucial mechanism in epileptic conditions, contributing to the synchronization of the neuronal networks. Little is known about the endogenous response of GJ in genetic absence epileptic animal models. We evaluated and quantified astrocyte GJ protein connexin (Cx) 30 and 43 in the somatosensory cortex (SSCx), ventrobasal (VB), centromedian (CM), lateral geniculate (LGN) and thalamic reticular (TRN) nuclei of thalamus of genetic absence epilepsy rats from Strasbourg (GAERS), Wistar albino glaxo rats from Rijswijk (WAG/Rij) and control Wistar animals using immunohistochemistry and Western Blot. The Cx30 and Cx43 immunopositive astrocytes per unit area were quantified for each region of the three animal strains. Furthermore, Cx30 and Cx43 Western Blot was applied to the tissue samples from the same regions of the three strain. The number of Cx30 immunopositive astrocytes showed significant increase in both GAERS and WAG/Rij compared to control Wistar in all brain regions studied except LGN of WAG/Rij animals. Furthermore, Cx43 in both GAERS and WAG/Rij showed significant increase in SSCx, VB and TRN. The protein expression was increased in both Cx30 and Cx43 in the two epileptic strains compared to control Wistar animals. The significant increase in the astrocytic GJ proteins Cx30 and Cx43 and the differences in the co-expression of Cx30 and Cx43 in the genetically absence epileptic strains compared to control Wistar animals may suggest that astrocytic Cx\'s may be involved in the mechanism of absence epilepsy. Increased number of astrocytic Cx\'s in GAERS and WAG/Rij may represent a compensatory response of the thalamocortical circuitry to the absence seizures or may be related to the production and/or development of absence seizures.

The principal function of the ventricular conduction system is rapid electrical activation of the ventricles. The aim of this study is to conduct a morphometric study to pinpoint the morphological parameters that define cardiac conduction cells, allowing us to distinguish them from other cells. Five male horse hearts and five male dog hearts were used in the study. The hearts were fixed in a 5% formaldehyde solution. Histological sections of 5 μm thickness were acquired and stained with hematoxylin-eosin and Masson\'s trichrome and cardiac conduction cells and their junctions were identified by desmin, connexin 40 and a PAS method. We found statistically significant differences in cardiac conduction fibers density and thickness, which was much higher in horses than in dogs (p = 0.000 for both values). By comparing the measured parameters of the cells in both species, we determined that cardiac conduction cells area and diameters were greater in horses than in dogs (p = 0.000 for all values). In dogs there are more junctions (30.8%) than in horses (26.1%), a statistically significant difference (p = 0.041). Our findings regarding the cardiac conduction fibers distribution in the animal species studied becomes new knowledge that contributes to the morphological study of this component of the cardiac conduction system and also makes it possible to locate exactly the site with the highest density of cardiac conduction fibers as a contribution to the cardiological study of these structures that lead to the prevention of ventricular arrhythmias and the identification of their treatment site.