Protection of cardiac cell-to-cell coupling attenuate myocardial remodeling and proarrhythmia induced by hypertension.

Gap junction connexin channels are important determinants of myocardial conduction and synchronization that is crucial for coordinated heart function. One of the main risk factors for cardiovascular events that results in heart attack, congestive heart failure, stroke as well as sudden arrhythmic death is hypertension. Mislocalization and/or dysfunction of specific connexin-43 channels due to hypertension-induced myocardial remodeling have been implicated in the occurrence of life-threatening arrhythmias and heart failure in both, humans as well as experimental animals. Recent studies suggest that down-regulation of myocardial connexin-43, its abnormal distribution and/or phosphorylation might be implicated in this process. On the other hand, treatment of hypertensive animals with cardioprotective drugs (e.g. statins) or supplementation with non-pharmacological compounds, such as melatonin, omega-3 fatty acids and red palm oil protects from lethal arrhythmias. The antiarrhythmic effects are attributed to the attenuation of myocardial connexin-43 abnormalities associated with preservation of myocardial architecture and improvement of cardiac conduction. Findings uncover novel mechanisms of cardioprotective (antihypertensive and antiarrhythmic) effects of compounds that are used in clinical settings. Well-designed trials are needed to explore the antiarrhythmic potential of these compounds in patients suffering from hypertension.

Anatomical and functional gonadotrope networks in the teleost pituitary.

Mammalian pituitaries exhibit a high degree of intercellular coordination; this enables them to mount large-scale coordinated responses to various physiological stimuli. This type of communication has not been adequately demonstrated in teleost pituitaries, which exhibit direct hypothalamic innervation and expression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in distinct cell types. We found that in two fish species, namely tilapia and zebrafish, LH cells exhibit close cell-cell contacts and form a continuous network throughout the gland. FSH cells were more loosely distributed but maintained some degree of cell-cell contact by virtue of cytoplasmic processes. These anatomical differences also manifest themselves at the functional level as evidenced by the effect of gap-junction uncouplers on gonadotropin release. These substances abolished the LH response to gonadotropin-releasing hormone stimulation but did not affect the FSH response to the same stimuli. Dye transfer between neighboring LH cells provides further evidence for functional coupling. The two gonadotropins were also found to be differently packaged within their corresponding cell types. Our findings highlight the evolutionary origin of pituitary cell networks and demonstrate how the different levels of cell-cell coordination within the LH and FSH cell populations are reflected in their distinct secretion patterns.

Protective effect of grape seed proanthocyanidins extracts on reperfusion arrhythmia in rabbits.

Reperfusion arrhythmia (RA) is one of the main complications which are also an important cause of sudden cardiac death. The aim of this study was to clarify whether grape seed proanthocyanidins extracts (GSPE) were therapeutic agents against RA. The models of cardiac ischemic reperfusion injury were established in rabbits. GSPE (100 mg/kg, and 250 mg/kg body weight/d, respectively) were administered for 3 wk. The incidence rates of arrhythmias before and after reperfusion of each group were recorded, cardiac infarction area and microstructures of cardiac cells of each rabbit were observed, and the expression of connexin 43 (Cx43) was detected by immunohistochemistry. Data were analyzed using the Leica Qwin V3 image analysis system. Reperfusion induced arrhythmia. Ventricular fibrillation (VF) occurred during the early phase of reperfusion after ischemia. Our results showed that GSPE treatment significantly reduced the incidence of VF and the infarction size compared with the model control group. Moreover, the intercalated disks in the model control group showed collapse, displacement and even the formation of cisterns. After being treated by GSPE, the intercalated disks were improved and there were less collapse and displacement. The expression of Cx43 was improved by GSPE treatment, and high dose of GSPE resulted in significant improvement. The study suggests that GSPE has a protective effect on myocardial ischemic reperfusion arrhythmias, which may be mediated by inhibiting the degradation of Cx43 and enhancing gap junctional conductance.

Effect of gap junction uncoupler heptanol on resistance arteries reactivity in experimental models of diabetes, hyperlipemia and hyperlipemia-diabetes.

The understanding of the involvement of the gap junctions (GJ) in the vascular reactivity is an ongoing effort. In this study we questioned on impact of pathologies such as diabetes, hyperlipemia, and simultaneous hyperlipemia-diabetes on GJ involvement in the contractile/relaxant response of the mesenteric resistance arteries. To this purpose, four groups of Golden Syrian hamsters were used: (i) diabetics (D), injected by streptozotocin, (ii) hyperlipemics (H), fed the standard chow of the species supplemented with 3% cholesterol and 15% butter, (iii) simultaneously hyperlipemic-diabetics (HD), and (iv) controls (C), age-matched normal healthy animals. At 24 weeks after the beginning of the experiment, the vascular reactivity of the resistance arteries was measured by the myograph technique in the presence/absence of 1 mM Heptanol (Hep) and of vasoconstrictors and vasodilators. The results showed that: (i) in pathological conditions 1 mM Hep significantly impaired the constrictor response of the hamster resistance arteries to both 10(-5) M NA (noradrenaline, agonist of alpha(1)-adrenoceptors) and 64.1 mM K+ (potassium ion, the major intracellular cation). The impairment occur in the group range: HD < H < D < C being the highest at the simultaneous insult of hyperlipemia and diabetes; (ii) independently of the pathological condition, 1 mM Hep abolishes both endothelium-dependent and independent relaxation of the hamster resistance arteries. At 1 mM Hep we noticed a reversible effect on endothelium-dependent relaxation that may be partially restored (in normal) in the presence of L-arginine. It is hoped that these results may contribute to understanding of the involvement of GJ in vascular pathology/dysfunction.

Connexon connexions in the thalamocortical system.

Electrical synapses are composed of gap junction channels that interconnect neurons. They occur throughout the mammalian brain, although this has been appreciated only recently. Gap junction channels, which are made of proteins called connexins, allow ionic current and small organic molecules to pass directly between cells, usually with symmetrical ease. Here we review evidence that electrical synapses are a major feature of the inhibitory circuitry in the thalamocortical system. In the neocortex, pairs of neighboring inhibitory interneurons are often electrically coupled, and these electrical connections are remarkably specific. To date, there is evidence that five distinct subtypes of inhibitory interneurons in the cortex make electrical interconnections selectively with interneurons of the same subtype. Excitatory neurons (i.e., pyramidal and spiny stellate cells) of the mature cortex do not appear to make electrical synapses. Within the thalamus, electrical coupling is observed in the reticular nucleus, which is composed entirely of GABAergic neurons. Some pairs of inhibitory neurons in the cortex and reticular thalamus have mixed synaptic connections: chemical (GABAergic) inhibitory synapses operating in parallel with electrical synapses. Inhibitory neurons of the thalamus and cortex express the gap junction protein connexin 36 (C x 36), and knocking out its gene abolishes nearly all of their electrical synapses. The electrical synapses of the thalamocortical system are strong enough to mediate robust interactions between inhibitory neurons. When pairs or groups of electrically coupled cells are excited by synaptic input, receptor agonists, or injected current, they typically display strong synchrony of both subthreshold voltage fluctuations and spikes. For example, activating metabotropic glutamate receptors on coupled pairs of cortical interneurons or on thalamic reticular neurons can induce rhythmic action potentials that are synchronized with millisecond precision. Electrical synapses offer a uniquely fast, bidirectional mechanism for coordinating local neural activity. Their widespread distribution in the thalamocortical system suggests that they serve myriad functions. We are far from a complete understanding of those functions, but recent experiments suggest that electrical synapses help to coordinate the temporal and spatial features of various forms of neural activity.

Sexually dimorphic hormonal regulation of the gap junction protein, CX43, in rats and altered female reproductive function in CX43+/- mice.

Astrocytic gap junctional communication is important in steroid hormone regulation of reproductive processes at the level of the hypothalamus, including estrous cyclicity and sexual behavior. We examined the effects of estradiol and progesterone on the abundance of the gap junctional protein, connexin 43 (CX43), which is highly expressed in astrocytes. Gonadectomized rats received hormone treatments that induce maximal sexual behavior and gonadotropin surges in females (estrogen for 48 h followed by progesterone, estrogen alone or progesterone alone). Control animals received vehicle (oil) injections. In the female rat preoptic area (POA), containing the gonadotropin-releasing hormone (GnRH) cell bodies, treatment with estrogen, progesterone or estrogen + progesterone significantly increased CX43 protein levels in immunoblots. In contrast, estrogen + progesterone significantly decreased CX43 levels in the male rat POA. This sexually dimorphic hormonal regulation of CX43 was not evident in the hypothalamus, which contains primarily GnRH nerve terminals. Treatment with estrogen + progesterone significantly decreased CX43 levels in both the male and female hypothalamus. To examine the role of CX43 in female reproductive function, we studied heterozygous female CX43 (CX43+/-) mice. Most mutant mice did not show normal estrous cycles. In addition, when compared to wild type females, CX43+/- mice had reduced lordosis behavior. These data suggest that hypothalamic CX43 expression is regulated by steroid hormones in a brain-region-specific and sexually dimorphic manner. Therefore, gap junctional communication in the POA and hypothalamus may be a factor regulating the estrous cycle and sexual behavior in female rodents.

Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling.

BACKGROUND: Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions. Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling. METHODS AND RESULTS: High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (>80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation. Transients from adjacent myotubes displayed different excitation thresholds, indicating an absence of coupling between cells, consistent with skeletal muscle biology. In contrast, simultaneous transduction of HDFs with MyoD and connexin43-encoding vectors resulted in the appearance of transients in adjacent myotubes with identical thresholds, indicative of electrical coupling. Notably, dye transfer studies confirmed gap junctional intercellular communication. CONCLUSIONS: Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. These observations strengthen the prospect of developing gene-based strategies for repairing cardiac conduction defects.

Reverse structural and gap-junctional remodeling after prolonged atrial fibrillation in the goat.

BACKGROUND: Prolonged atrial fibrillation (AF) results in electrical, structural, and gap-junctional remodeling. We examined the reversibility of the changes in (ultra)structure and gap junctions. METHODS AND RESULTS: Four groups of goats were used: (1) sinus rhythm (SR), (2) 4 months' AF (4 mo AF), (3) 2 months' SR after 4 mo AF (2 mo post-AF), and (4) 4 months' SR after 4 mo AF (4 mo post-AF). Atria were characterized electrophysiologically, (ultra)structure was studied by light and electron microscopy, and structural and gap-junctional protein expression was studied by immunohistochemistry or Western blotting. The atrial effective refractory period had completely returned to normal values 2 mo post-AF. Induced AF episodes still lasted for minutes at 2 and 4 mo post-AF, compared with seconds in the SR group. Structural abnormalities were still present at 2 and 4 mo post-AF, although to a lesser extent. The increased atrial myocyte diameter was back to normal at 4 mo post-AF. The number of myocytes with severe myolysis had almost normalized 4 mo post-AF, whereas myocytes with mild myolysis remained significantly increased. Extracellular matrix area fraction after 4 mo AF was similar to SR. However, the extracellular matrix fraction per myocyte had increased after 4 mo AF and remained higher post-AF. Changes in expression of structural proteins were partially restored post-AF. The reduction of connexin 40 that was observed during AF was completely reversed at 4 mo post-AF. CONCLUSIONS: Recovery from structural remodeling after 4 mo AF is a slow process and is still incomplete 4 mo post-AF. Several months post-AF, the duration of AF episodes is still prolonged (minutes).

Chemopreventive effect of orange oil on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine in rats: an ultrastructural study.

Orange peel oil is used extensively as an approved flavour enhancer in fruit drinks, carbonated beverages and as a scenting agent in soaps and cosmetics. Limonene, which is a monocyclic monoterpene is present in orange peel oil from 90 to 95% (w/w). Monoterpenes have been shown to be very effective chemopreventive agents against several rodent tumors and are currently in clinical trials. However, not much information is available regarding the ultrastructural changes associated with the chemopreventive effects of the monoterpenes. The effect of orange oil on the suppression of preneoplastic hepatic lesions during N-nitrosodiethylamine (DEN) induced hepatocarcinogenesis was studied electron microscopically. Rats were administered 200 ppm DEN through drinking water for a period of 1 month. After an interval of 2 weeks, the animals were administered orange oil by gavage for a period of 5 1/2 months. The chemopreventive effect of orange oil was monitored on the basis of liver weight profile, histological pattern by light microscopy and ultrastructural alterations by electronmicroscopy. Orange oil administration following DEN treatment showed decreased liver weights, increased intercellular gap junctional complexes, cell density and polarity when compared with only the DEN treated rats. In the present study chemopreventive effect of orange oil on DEN-induced hepatic preneoplasia in rats which is associated with the restoration of the normal phenotype and upregulation of junctional complexes has been demonstrated.

Characterization of alpha-smooth muscle actin positive cells in mineralized human dental pulp cultures.

In response to injury, pulp precursor cells can differentiate into odontoblast-like cells that produce reparative dentine. In culture, pulp cells form mineralizing nodules, but the characteristics of the cells involved in this process are still not fully known. Human pulp cells for culture were obtained from coronal pulp isolated from non-erupted molars, and were maintained in RPMI 1640 medium supplemented with fetal calf serum. Nodules were forming in all human pulp primary cultures (HPPc) and human pulp subcultures observed until their fifth passage (HPSc<5). Mineralization of the nodules was confirmed by the presence of calcium and phosphate that were quantified by X-ray microanalysis. Specific immunolabeling revealed alpha-smooth muscle actin and vimentin in both HPPc and HPSc<5 cells. Cells positive for alpha-smooth muscle actin were either isolated or gathered together in the nodules. Under transmission electron microscopy, some cells in primary pulp cultures exhibited features typical of myofibroblasts or pericytes, such as stress fibers, fibronexus, indented nuclei and gap-junctions. These cells were frequently in close contact with mineral deposits. This work demonstrates for the first time the presence of pericytes or myofibroblasts in mineralized human pulp cultures, but further investigation is required to determine their origin, role and degree of differentiation.

The intracellular domain of cadherin-11 is not required for the induction of cell aggregation, adhesion or gap-junction formation.

The cadherin family of cell adhesion molecules demonstrates calcium-dependent homophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type I cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind beta-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.