Korean red ginseng alleviate depressive disorder by improving astrocyte gap junction function.

ETHNOPHARMACOLOGICAL RELEVANCE: Korean red ginseng (KRG), a processed product of Panax ginseng C. A. Mey, show significant anti-depressive effect in clinic. However, its mechanism is still unclear. AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potential pathogenesis of depression. Therefore, this study's objective is to investigate whether the antidepressant effect of KRG is related to GJIC. MATERIALS AND METHODS: Rat were restraint 8 h every day for 28 consecutive days to prepare depression models, and meanwhile, rats were intragastrically administrated with normal saline, KRG solutions (25, 50 or 100 mg/kg) or fluoxetine (10 mg/kg) 1 h before stress. The behavioral performance was determined by forced swimming test, sucrose preference test and open field test. GJIC was determined by the Lucifer yellow (LY) diffusion distance in prelimb cortex (PLC). In addition, the level of Cx43, one of executors of GJIC, was tested by Western blot. To find out the protective effect of KRG against GJIC dysfunction directly, rats were intracranially injected with carbenoxolone (CBX, blocker of GJIC), and meanwhile normal saline, KRG (100 mg/kg) or fluoxetine (10 mg/kg) was administered daily. The behavioral performance of these rats was detected, and the LY localization injection PLC area was used to detect the gap junction function. RESULTS: Chronic resistant stress (CRS) induced depressive symptoms, as manifested by prolonged immobility time in forced swimming test and decreased sucrose consumption ratio. Administration of KRG alleviated these depressive symptoms significantly. GJIC determination showed that KRG improved the LY diffusion and increased Cx43 level in prefrontal cortex (PFC) significantly, indicated that GJIC dysfunction was alleviated by the treatment of KRG. However, the astrocytes number was also increased by the treatment of KRG, which maybe alleviate depression-like symptoms by increasing the number of astrocytes rather than improving GJIC. Injection of CBX produced depressive symptoms and GJIC dysfunction, as manifested by decreased sucrose consumption ratio and prolonged immobility time in forced swimming test, but no astrocytes number changes, KRG also reversed depressive symptoms and GJIC dysfunction, suggested that the improvement of depressive-like symptoms was improved by GJIC. CONCLUSIONS: KRG alleviate depressive disorder by improving astrocytic gap junction function.

Non-ohmic tissue conduction in cardiac electrophysiology: Upscaling the non-linear voltage-dependent conductance of gap junctions.

Gap junctions are key mediators of intercellular communication in cardiac tissue, and their function is vital to sustaining normal cardiac electrical activity. Conduction through gap junctions strongly depends on the hemichannel arrangement and transjunctional voltage, rendering the intercellular conductance highly non-Ohmic, particularly under steady-state regimes of conduction. Despite this marked non-linear behavior, current tissue-level models of cardiac conduction are rooted in the assumption that gap-junctions conductance is constant (Ohmic), which results in inaccurate predictions of electrical propagation, particularly in the low junctional-coupling regime observed under pathological conditions. In this work, we present a novel non-Ohmic homogenization model (NOHM) of cardiac conduction that is suitable to tissue-scale simulations. Using non-linear homogenization theory, we develop a conductivity model that seamlessly upscales the voltage-dependent conductance of gap junctions, without the need of explicitly modeling gap junctions. The NOHM model allows for the simulation of electrical propagation in tissue-level cardiac domains that accurately resemble that of cell-based microscopic models for a wide range of junctional coupling scenarios, recovering key conduction features at a fraction of the computational complexity. A unique feature of the NOHM model is the possibility of upscaling the response of non-symmetric gap-junction conductance distributions, which result in conduction velocities that strongly depend on the direction of propagation, thus allowing to model the normal and retrograde conduction observed in certain regions of the heart. We envision that the NOHM model will enable organ-level simulations that are informed by sub- and inter-cellular mechanisms, delivering an accurate and predictive in-silico tool for understanding the heart function. Codes are available for download at https://github.com/dehurtado/NonOhmicConduction.

Cerebrospinal Fluid-Contacting Neurons Sense pH Changes and Motion in the Hypothalamus.

CSF-contacting (CSF-c) cells are present in the walls of the brain ventricles and the central canal of the spinal cord and found throughout the vertebrate phylum. We recently identified ciliated somatostatin-/GABA-expressing CSF-c neurons in the lamprey spinal cord that act as pH sensors as well as mechanoreceptors. In the same neuron, acidic and alkaline responses are mediated through ASIC3-like and PKD2L1 channels, respectively. Here, we investigate the functional properties of the ciliated somatostatin-/GABA-positive CSF-c neurons in the hypothalamus by performing whole-cell recordings in hypothalamic slices. Depolarizing current pulses readily evoked action potentials, but hypothalamic CSF-c neurons had no or a very low level of spontaneous activity at pH 7.4. They responded, however, with membrane potential depolarization and trains of action potentials to small deviations in pH in both the acidic and alkaline direction. Like in spinal CSF-c neurons, the acidic response in hypothalamic cells is mediated via ASIC3-like channels. In contrast, the alkaline response appears to depend on connexin hemichannels, not on PKD2L1 channels. We also show that hypothalamic CSF-c neurons respond to mechanical stimulation induced by fluid movements along the wall of the third ventricle, a response mediated via ASIC3-like channels. The hypothalamic CSF-c neurons extend their processes dorsally, ventrally, and laterally, but as yet, the effects exerted on hypothalamic circuits are unknown. With similar neurons being present in rodents, the pH- and mechanosensing ability of hypothalamic CSF-c neurons is most likely conserved throughout vertebrate phylogeny.SIGNIFICANCE STATEMENT CSF-contacting neurons are present in all vertebrates and are located mainly in the hypothalamic area and the spinal cord. Here, we report that the somatostatin-/GABA-expressing CSF-c neurons in the lamprey hypothalamus sense bidirectional deviations in the extracellular pH and do so via different molecular mechanisms. They also serve as mechanoreceptors. The hypothalamic CSF-c neurons have extensive axonal ramifications and may decrease the level of motor activity via release of somatostatin. In conclusion, hypothalamic somatostatin-/GABA-expressing CSF-c neurons, as well as their spinal counterpart, represent a novel homeostatic mechanism designed to sense any deviation from physiological pH and thus constitute a feedback regulatory system intrinsic to the CNS, possibly serving a protective role from damage caused by changes in pH.

Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone.

BACKGROUND: Glia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown. METHODS: Primary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry. RESULTS: We found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane's Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane. CONCLUSIONS: We propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

Ginsenoside Rg1 alleviates corticosterone-induced dysfunction of gap junctions in astrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenoside Rg1 (Rg1), one of the major bioactive ingredients of Panax ginseng C. A. Mey, has neuroprotective effects in animal models of depression, but the mechanism underlying these effects is still largely unknown AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potentially novel pathogenic mechanism for depression. Thus, we investigated that whether antidepressant-like effects of Rg1 were related to GJIC. MATERIALS AND METHODS: Primary rat prefrontal cortical and hippocampal astrocytes cultures were treated with 50µM CORT for 24h to induce gap junction damage. Rg1 (0.1, 1, or 10µM) or fluoxetine (1µM) was added 1h prior to CORT treatment. A scrape loading and dye transfer assay was performed to identify the functional capacity of gap junctions. Western blot was used to detect the expression and phosphorylation of connexin43 (Cx43), the major component of gap junctions. RESULTS: Treatment of primary astrocytes with CORT for 24h inhibited GJIC, decreased total Cx43 expression, and increased the phosphorylation of Cx43 at serine368 in a dose-dependent manner. Pre-treatment with 1µM and 10µM Rg1 significantly improved GJIC in CORT-treated astrocytes from the prefrontal cortex and hippocampus, respectively, and this was accompanied by upregulation of Cx43 expression and downregulation of Cx43 phosphorylation. CONCLUSION: These findings provide the first evidence indicating that Rg1 can alleviate CORT-induced gap junction dysfunction, which may have clinical significance in the treatment of depression.

The Potential Role of Gap Junctional Plasticity in the Regulation of State.

Electrical synapses are the functional correlate of gap junctions and allow transmission of small molecules and electrical current between coupled neurons. Instead of static pores, electrical synapses are actually plastic, similar to chemical synapses. In the thalamocortical system, gap junctions couple inhibitory neurons that are similar in their biochemical profile, morphology, and electrophysiological properties. We postulate that electrical synaptic plasticity among inhibitory neurons directly interacts with the switching between different firing patterns in a state-dependent and type-dependent manner. In neuronal networks, electrical synapses may function as a modifiable resonance feedback system that enables stable oscillations. Furthermore, the plasticity of electrical synapses may play an important role in regulation of state, synchrony, and rhythmogenesis in the mammalian thalamocortical system, similar to chemical synaptic plasticity. Based on their plasticity, rich diversity, and specificity, electrical synapses are thus likely to participate in the control of consciousness and attention.

Short-term depression of gap junctional coupling in reticular thalamic neurons of absence epileptic rats.

KEY POINTS: Gap junctional electrical coupling between neurons of the reticular thalamic nucleus (RTN) is critical for hypersynchrony in the thalamo-cortical network. This study investigates the role of electrical coupling in pathological rhythmogenesis in RTN neurons in a rat model of absence epilepsy. Rhythmic activation resulted in a Ca(2+) -dependent short-term depression (STD) of electrical coupling between pairs of RTN neurons in epileptic rats, but not in RTN of a non-epileptic control strain. Pharmacological blockade of gap junctions in RTN in vivo induced a depression of seizure activity. The STD of electrical coupling represents a mechanism of Ca(2+) homeostasis in RTN aimed to counteract excessive synchronization. ABSTRACT: Neurons in the reticular thalamic nucleus (RTN) are coupled by electrical synapses, which play a major role in regulating synchronous activity. This study investigates electrical coupling in RTN neurons from a rat model of childhood absence epilepsy, genetic absence epilepsy rats from Strasbourg (GAERS), compared with a non-epileptic control (NEC) strain, to assess the impact on pathophysiological rhythmogenesis. Whole-cell recordings were obtained from pairs of RTN neurons of GAERS and NEC in vitro. Coupling was determined by injection of hyperpolarizing current steps in one cell and monitoring evoked voltage responses in both activated and coupled cell. The coupling coefficient (cc) was compared under resting condition, during pharmacological interventions and repeated activation using a series of current injections. The effect of gap junctional coupling on seizure expression was investigated by application of gap junctional blockers into RTN of GAERS in vivo. At resting conditions, cc did not differ between GAERS and NEC. During repeated activation, cc declined in GAERS but not in NEC. This depression in cc was restored within 25 s and was prevented by intracellular presence of BAPTA in the activated but not in the coupled cell. Local application of gap junctional blockers into RTN of GAERS in vivo resulted in a decrease of spike wave discharge (SWD) activity. Repeated activation results in a short-term depression (STD) of gap junctional coupling in RTN neurons of GAERS, depending on intracellular Ca(2+) mechanisms in the activated cell. As blockage of gap junctions in vivo results in a decrease of SWD activity, the STD observed in GAERS is considered a compensatory mechanism, aimed to dampen SWD activity.

Gap junctions in developing thalamic and neocortical neuronal networks.

The presence of direct, cytoplasmatic, communication between neurons in the brain of vertebrates has been demonstrated a long time ago. These gap junctions have been characterized in many brain areas in terms of subunit composition, biophysical properties, neuronal connectivity patterns, and developmental regulation. Although interesting findings emerged, showing that different subunits are specifically regulated during development, or that excitatory and inhibitory neuronal networks exhibit various electrical connectivity patterns, gap junctions did not receive much further interest. Originally, it was believed that gap junctions represent simple passageways for electrical and biochemical coordination early in development. Today, we know that gap junction connectivity is tightly regulated, following independent developmental patterns for excitatory and inhibitory networks. Electrical connections are important for many specific functions of neurons, and are, for example, required for the development of neuronal stimulus tuning in the visual system. Here, we integrate the available data on neuronal connectivity and gap junction properties, as well as the most recent findings concerning the functional implications of electrical connections in the developing thalamus and neocortex.

Spatial profiles of electrical mismatch determine vulnerability to conduction failure across a host-donor cell interface.

BACKGROUND: Electrophysiological mismatch between host cardiomyocytes and donor cells can directly affect the electrical safety of cardiac cell therapies; however, the ability to study host-donor interactions at the microscopic scale in situ is severely limited. We systematically explored how action potential (AP) differences between cardiomyocytes and other excitable cells modulate vulnerability to conduction failure in vitro. METHODS AND RESULTS: AP propagation was optically mapped at 75 µm resolution in micropatterned strands (n=152) in which host neonatal rat ventricular myocytes (AP duration=153.2±2.3 ms, conduction velocity=22.3±0.3 cm/s) seamlessly interfaced with genetically engineered excitable donor cells expressing inward rectifier potassium (Kir2.1) and cardiac sodium (Na(v)1.5) channels with either weak (conduction velocity=3.1±0.1 cm/s) or strong (conduction velocity=22.1±0.4 cm/s) electrical coupling. Selective prolongation of engineered donor cell AP duration (31.9-139.1 ms) by low-dose BaCl2 generated a wide range of host-donor repolarization time (RT) profiles with maximum gradients (∇RT(max)) of 5.5 to 257 ms/mm. During programmed stimulation of donor cells, the vulnerable time window for conduction block across the host-donor interface most strongly correlated with ∇RT(max). Compared with well-coupled donor cells, the interface composed of poorly coupled cells significantly shortened the RT profile width by 19.7% and increased ∇RT(max) and vulnerable time window by 22.2% and 19%, respectively. Flattening the RT profile by perfusion of 50 µmol/L BaCl2 eliminated coupling-induced differences in vulnerability to block. CONCLUSIONS: Our results quantify how the degree of electrical mismatch across a cardiomyocyte-donor cell interface affects vulnerability to conduction block, with important implications for the design of safe cardiac cell and gene therapies.

Relationship between gap-junctional conductance and conduction velocity in mammalian myocardium.

BACKGROUND: Gap junction resistivity, R(j), has been proposed as a key determinant of conduction velocity (CV). However, studies in connexin-gene knockout mice demonstrated significant CV slowing only with near-complete connexin deletion, and these findings led to the concept of a significant redundancy of myocardial gap junctions. We challenged this prevailing concept and addressed the hypothesis that there is a continuous relationship between R(j) and CV, each independently measured in human and guinea-pig myocardium. METHODS AND RESULTS: R(j) and CV were directly measured by oil-gap impedance and microelectrode techniques in human left ventricular myocardium from patients with hypertrophic cardiomyopathy and in guinea-pig atrial and ventricular myocardium before and during pharmacological uncoupling with 20-µmol/L carbenoxolone. There was a continuous relationship between R(j) and CV in human and guinea-pig myocardium, pre- and post-carbenoxolone (r(2)=0.946; P<0.01). In guinea-pig left ventricle, left atrium, and right atrium, carbenoxolone increased R(j) by 28±9%, 26±16%, and 25±14% and slowed CV by 17±3%, 23±8%, and 11±4% respectively (all P<0.05 versus control). As a clinically accessible measure of local microscopic myocardial conduction slowing in vivo in the intact human heart, carbenoxolone prolonged electrogram duration in the right atrium (39.7±4.2 to 42.3±4.3 ms; P=0.01) and right ventricle (48.1±2.5 to 53.3±5.3 ms; P<0.01). CONCLUSIONS: There is a continuous relationship between R(j) and CV that is consistent between cardiac chambers and across species, indicating that naturally occurring variations in cellular coupling can account for variations in CV, and that the concept that there is massive redundancy of coupling is not tenable.

Gingko biloba extracts protect auditory hair cells from cisplatin-induced ototoxicity by inhibiting perturbation of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) may play an important role in the hearing process. Cisplatin is an anticancer drug that causes hearing loss and Gingko biloba extracts (EGb 761) have been used as an antioxidant and enhancer for GJIC. The purpose of this study was to examine the efficiency of EGb 761 in protecting against cisplatin-induced apoptosis and disturbance of GJIC. House Ear Institute-Organ of Corti 1 auditory cells were cultured and treated with cisplatin (50 µM) and EGb (300 µg/ml) for 24h, and then analyzed by immunocytochemistry (Annexin V/propidium iodide) and Western blots. GJIC was evaluated by scrape-loading dye transfer (SLDT). Basal turn organ of Corti (oC) explants from neonatal (p3) rats were exposed to cisplatin (1-10 µM) and EGb (50-400 µg/ml). The number of intact hair cells was counted by co-labeling with phalloidin and MyoVIIa. EGb prevented cisplatin-induced apoptosis in immunostaining and decreased caspase 3 and poly-ADP-ribose polymerase bands, which were increased in cisplatin-treated cells in Western blots. EGb prevented abnormal intracellular locations of connexin (Cx) 26, 30, 31, and 43 in cells treated with cisplatin and increased quantities of Cx bands. EGb also prevented cisplatin-induced disturbance of GJIC in SLDT. In oC explants, EGb significantly prevented hair cell damage induced by cisplatin. In animal studies, EGb significantly prevented cisplatin-induced hearing loss across 16 and 32 kHz. These results show that cisplatin induces ototoxicity including hearing loss as well as down-regulation of GJIC and inhibition of Cxs in auditory cells. EGb prevents hearing loss in cisplatin-treated rats by inhibiting down-regulation of Cx expression and GJIC. The disturbance of GJIC or Cx expression may be one of the important mechanisms of cisplatin-induced ototoxicity.

Panax notoginseng saponins enhances the cytotoxicity of cisplatin via increasing gap junction intercellular communication.

Panax Notoginseng Saponins (PNS) have been well known to have anti-tumor activity and enhance cytotoxicity of some cancer chemotherapy agents, but the mechanisms underlying these effects are still unknown. This study investigates the effect of PNS on cytotoxicity of cisplatin and the relationship between this effect and the modulation of gap junctions (GJ) function by PNS in a transfected cell line. The cytotoxicity of cisplatin (0.25-1 µg/mL) was increased in the presence of GJ. Inhibition of gap junction by either GJ blocker or interception of Connexin (Cx) expression decreased the cytotoxicity of cisplatin. Increasing GJ function enhanced cytotoxicity of cisplatin, only in the cells with functional GJ. PNS (50-200 µg/mL) significantly enhanced cisplatin cytotoxicity, but this effect required functional gap junctions between the cells. Exposure of the cells to PNS (50-200 µg/mL) for 4 h leads to a significant enhance in dye coupling of GJ in a dose-dependent manner. These results suggest that PNS increases the cytotoxicity of cisplatin through enhancement of GJ activity.

Remodeling of mechanical junctions and of microtubule-associated proteins accompany cardiac connexin43 lateralization.

BACKGROUND: Desmosomes and adherens junctions provide mechanical continuity between cardiac cells, whereas gap junctions allow for cell-cell electrical/metabolic coupling. These structures reside at the cardiac intercalated disc (ID). Also at the ID is the voltage-gated sodium channel (VGSC) complex. Functional interactions between desmosomes, gap junctions, and VGSC have been demonstrated. Separate studies show, under various conditions, reduced presence of gap junctions at the ID and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction "lateralization"). OBJECTIVE: To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling. METHODS: Adult sheep were subjected to right ventricular pressure overload (pulmonary hypertension). Tissue was analyzed by quantitative confocal microscopy and by transmission electron microscopy. Ionic currents were measured using conventional patch clamp. RESULT: Quantitative confocal microscopy demonstrated lateralization of immunoreactive junctional molecules. Desmosomes and gap junctions in lateral membranes were demonstrable by electron microscopy. Cx43/desmosomal remodeling was accompanied by lateralization of 2 microtubule-associated proteins relevant for Cx43 trafficking: EB1 and kinesin protein Kif5b. In contrast, molecules of the VGSC failed to reorganize in plaques discernable by confocal microscopy. Patch-clamp studies demonstrated change in amplitude and kinetics of sodium current and a small reduction in electrical coupling between cells. CONCLUSIONS: Cx43 lateralization is part of a complex remodeling that includes mechanical and gap junctions but may exclude components of the VGSC. We speculate that lateralization results from redirectionality of microtubule-mediated forward trafficking. Remodeling of junctional complexes may preserve electrical synchrony under conditions that disrupt ID integrity.

Peripheral and central inputs shape network dynamics in the developing visual cortex in vivo.

Spontaneous network activity constitutes a central theme during the development of neuronal circuitry [1, 2]. Before the onset of vision, retinal neurons generate waves of spontaneous activity that are relayed along the ascending visual pathway [3, 4] and shape activity patterns in these regions [5, 6]. The spatiotemporal nature of retinal waves is required to establish precise functional maps in higher visual areas, and their disruption results in enlarged axonal projection areas (e.g., [7-10]). However, how retinal inputs shape network dynamics in the visual cortex on the cellular level is unknown. Using in vivo two-photon calcium imaging, we identified two independently occurring patterns of network activity in the mouse primary visual cortex (V1) before and at the onset of vision. Acute manipulations of spontaneous retinal activity revealed that one type of network activity largely originated in the retina and was characterized by low synchronicity (L-) events. In addition, we identified a type of high synchronicity (H-) events that required gap junction signaling but were independent of retinal input. Moreover, the patterns differed in wave progression and developmental profile. Our data suggest that different activity patterns have complementary functions during the formation of synaptic circuits in the developing visual cortex.

Expression of gap junctional connexin proteins in ovine fetal ovaries: effects of maternal diet.

Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been shown that several environmental factors, including maternal diet, may affect fetal organ growth and function. To determine whether maternal diet affects expression of Cx26, Cx32, Cx37, and Cx43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 d before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from the ASe and HSe groups was fed a restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of Cxs followed by image analysis. All four Cxs were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary, and antral follicles; in granulosa and theca layers of secondary and antral follicles; stroma; and blood vessels. Cx32 was in oocytes, granulosa, and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression, Cx26 in granulosa layer of antral follicles was decreased (P < 0.01) by HSe in the M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P < 0.05) by the M diet with HSe. Thus, Cxs may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance of maternal diet in fetal growth and development.

Atrial remodeling and the substrate for atrial fibrillation in rat hearts with elevated afterload.

BACKGROUND: Although arterial hypertension and left ventricular hypertrophy are considered good epidemiological indicators of the risk of atrial fibrillation (AF) in patients, the link between elevated afterload and AF remains unclear. We investigated atrial remodeling and the substrate for arrhythmia in a surgical model of elevated afterload in rats. METHODS AND RESULTS: Male Wistar rats (aged 3-4 weeks) were anesthetized and subjected to either partial stenosis of the ascending aorta (AoB) or sham operation (Sham). Experiments were performed on excised hearts 8, 14, and 20 weeks after surgery. Unipolar electrograms were recorded from the left atrial epicardial surface of perfused hearts using a 5×5 electrode array. Cryosections of left atrial tissue were retained for histological and immunocytochemical analyses. Compared to Sham, AoB hearts showed marked left atrial hypertrophy and fibrosis at 14 and 20 weeks postsurgery. The incidence and duration of pacing-induced AF was increased in hearts from AoB rats at 20 weeks postsurgery. The substrate for arrhythmia was associated with reduced vectorial conduction velocity and greater inhomogeneity in conduction but without changes in effective refractory period. Left atrial expression of the gap junction protein, connexin43, was markedly reduced in AoB compared with Sham hearts. CONCLUSIONS: Using a small-animal model, we demonstrate that elevated afterload in the absence of systemic hypertension results in increased inducibility of AF and left atrial remodeling involving fibrosis, altered atrial connexin43 expression, and marked conduction abnormalities.

Responses of a bursting pacemaker to excitation reveal spatial segregation between bursting and spiking mechanisms.

Central pattern generators (CPGs) frequently include bursting neurons that serve as pacemakers for rhythm generation. Phase resetting curves (PRCs) can provide insight into mechanisms underlying phase locking in such circuits. PRCs were constructed for a pacemaker bursting complex in the pyloric circuit in the stomatogastric ganglion of the lobster and crab. This complex is comprised of the Anterior Burster (AB) neuron and two Pyloric Dilator (PD) neurons that are all electrically coupled. Artificial excitatory synaptic conductance pulses of different strengths and durations were injected into one of the AB or PD somata using the Dynamic Clamp. Previously, we characterized the inhibitory PRCs by assuming a single slow process that enabled synaptic inputs to trigger switches between an up state in which spiking occurs and a down state in which it does not. Excitation produced five different PRC shapes, which could not be explained with such a simple model. A separate dendritic compartment was required to separate the mechanism that generates the up and down phases of the bursting envelope (1) from synaptic inputs applied at the soma, (2) from axonal spike generation and (3) from a slow process with a slower time scale than burst generation. This study reveals that due to the nonlinear properties and compartmentalization of ionic channels, the response to excitation is more complex than inhibition.

[Effects of Chinese herbal compound for supplementing qi and activating blood circulation on actin, Cx43 expressions and gap junctional intercellular communication functions of myocardial cells in patients with Coxsackie virus B 3 viral myocarditis].

OBJECTIVE: To observe the effect of Chinese herbal compound for supplementing qi and activating blood circulation (CHC) on the gap junctional intercellular communication (GJIC) function of myocardial cells in patients with Coxsackie virus B 3 (CVB3) viral myocarditis. METHODS: Expressions of actin and connexin43 (Cx43) in myocardial cells of patients arranged in three groups (the normal control group, the viral infected group and the CHC treated group) were detected by immunohistochemical method; the fluorescence photobleaching recovery rate of cells was detected by laser scanning confocal microscope. RESULTS: As compared with the viral infected group, the expressions of actin and Cx43 were increased and the GJIC function was improved in the CHC treated group. CONCLUSION: CHC could antagonize viral injury on skeleton protein, and repair the structure of gap junction channel to improve the GJIC function of myocardial cells after being attacked by CVB3.

Epigallocatechin-3 gallate, a green tea catechin, attenuated the downregulation of the cardiac gap junction induced by high glucose in neonatal rat cardiomyocytes.

AIMS: The remodeling of cardiac gap junctions has been postulated to contribute to the arrhythmias in a diabetic heart. Epigallocatechin-3 gallate (EGCG), a green tea catechin, has recently been recognized for its protection in cardiovascular disease. This study investigated the effect of EGCG on the possible remodeling of gap junctions under high glucose in cultured neonatal rat cardiomyocytes. METHODS: Cardiomyocytes pre-incubated with high glucose (30mM) were co-treated by EGCG. The expression of Connexin43 (Cx43), Cx40 and Cx45 were determined by Western blot and real-time RT-PCR. The function of cells coupling was evaluated by scrape loading dye transfer study. The Mitogen-activated protein kinases (MAPK) were quantified by Western blot. RESULTS: The protein expression of Cx43 was reduced by high glucose (30mM, 72h). Addition of EGCG to high glucose treated cardiomyocytes attenuated the Cx43 reduction in a dose- and time-dependent manner and also recovered the reduced function of cells coupling. The mRNA or protein level of Cx40 and Cx45 showed no significant change by high glucose (30mM, 72h) or EGCG co-treatment (40microM, 24h). Nor did the Cx43 mRNA level. EGCG (40muM) activated the time-dependent phosphorylated Erk, JNK and p38 MAPK. The p38 MAPK inhibitor SB203580 (10microM), however, attenuated the protective effect of EGCG. CONCLUSION: EGCG could attenuate the downregulation of gap junction induced by high glucose in cultured neonatal rat cardiomyocytes. The p38 MAPK pathway was partly involved in this effect of EGCG.

Gap junction heterogeneity as mechanism for electrophysiologically distinct properties across the ventricular wall.

Gap junctions are critical to maintaining synchronized impulse propagation and repolarization. Heterogeneous expression of the principal ventricular gap junction protein connexin43 (Cx43) is associated with action potential duration (APD) dispersion across the anterior ventricular wall. Little is known about Cx43 expression patterns and their disparate impact on regional electrophysiology throughout the heart. We aimed to determine whether the anterior and posterior regions of the heart are electrophysiologically distinct. Multisegment, high-resolution optical mapping was performed in canine wedge preparations harvested separately from the anterior left ventricle (aLV; n = 8) and posterior left ventricle (pLV; n = 8). Transmural APD dispersion was significantly greater on the aLV than the pLV (45 +/- 13 vs. 26 +/- 8.0 ms; P < 0.05). Conduction velocity dispersion was also significantly higher (P < 0.05) across the aLV (39 +/- 7%) than the pLV (16 +/- 3%). Carbenoxolone perfusion significantly enhanced APD and conduction velocity dispersion on the aLV (by 1.53-fold and 1.36-fold, respectively), but not the pLV (by 1.27-fold and 1.2-fold, respectively), and produced a 4.2-fold increase in susceptibility to inducible arrhythmias in the aLV. Confocal immunofluorescence microscopy revealed significantly (P < 0.05) greater transmural dispersion of Cx43 expression on the aLV (44 +/- 10%) compared with the pLV wall (8.3 +/- 0.7%), suggesting that regional expression of Cx43 expression patterns may account for regional electrophysiological differences. Computer simulations affirmed that localized uncoupling at the epicardial-midmyocardial interface is sufficient to produce APD gradients observed on the aLV. These data demonstrate that the aLV and pLV differ importantly with respect to their electrophysiological properties and Cx43 expression patterns. Furthermore, local underexpression of Cx43 is closely associated with transmural electrophysiological heterogeneity on the aLV. Therefore, regional and transmural heterogeneous Cx43 expression patterns may be an important mechanism underlying arrhythmia susceptibility, particularly in disease states where gap junction expression is altered.