Sinus node dysfunction following targeted disruption of the murine cardiac sodium channel gene Scn5a

We have examined sino-atrial node (SAN) function in hearts from adult mice with heterozygous targeted disruption of the Scn5a gene to clarify the role of Scn5a-encoded cardiac Na⁺ channels in normal SAN function and the mechanism(s) by which reduced Na⁺ channel function might cause sinus node dysfunction. Scn5a ^+/− mice showed depressed heart rates and occasional sino-atrial (SA) block. Their isolated peripheral SAN pacemaker cells showed a reduced Na⁺ channel expression and slowed intrinsic pacemaker rates. Wild-type (WT) and Scn5a^+/− SAN preparations exhibited similar activation patterns but with significantly slower SA conduction and frequent sino-atrial conduction block in Scn5a^+/− SAN preparations. Furthermore, isolated WT and Scn5a^+/− SAN cells demonstrated differing correlations between cycle length, maximum upstroke velocity and action potential amplitude, and cell size. Small myocytes showed similar, but large myocytes reduced pacemaker rates, implicating the larger peripheral SAN cells in the reduced pacemaker rate that was observed in Scn5a^+/− myocytes. These findings were successfully reproduced in a model that implicated i _Na directly in action potential propagation through the SAN and from SAN to atria, and in modifying heart rate through a coupling of SAN and atrial cells. Functional alterations in the SAN following heterozygous-targeted disruption of Scn5a thus closely resemble those observed in clinical sinus node dysfunction. The findings accordingly provide a basis for understanding of the role of cardiac-type Na⁺ channels in normal SAN function and the pathophysiology of sinus node dysfunction and suggest new potential targets for its clinical management.     

Remodeling of cardiac fibroblasts following myocardial infarction results in increased gap junction intercellular communication

BackgroundWe have recently shown that native murine ventricular fibroblasts express both connexin43 (Cx43) and Cx45, and that the level of Cx43 expression influences intercellular coupling and cell proliferation. Relatively little is known, however, about how myocardial infarction (MI) influences expression of Cx43, or how altered Cx43 expression may affect fibroblast function post-MI. Fibroblasts are critical for infarct healing and post-infarct ventricular remodeling. They can couple electrically with cardiac myocytes and influence myocardial activation patterns. Thus, Cx43 remodeling and the level of intercellular communication in fibroblasts expressed in the infarcted heart were the subject of the present investigation.MethodsFibroblasts were isolated from both infarct scar and remote, noninfarcted regions of murine hearts 6 d after coronary ligation. Expression levels of Cx43, α-smooth muscle actin and N-cadherin were quantified by immunoblotting. Gap junctional intercellular communication was quantified by Lucifer yellow dye transfer.Results and ConclusionsFibroblasts isolated from infarcted hearts exhibited marked up-regulation of Cx43 protein expression and enhanced intercellular coupling. Exogenous administration of transforming growth factor-β (TGF-β) to fibroblast cultures from normal, non-operated hearts produced comparable up-regulation of Cx43, suggesting that increased intercellular communication between fibroblasts in infarct and peri-infarct regions may be secondary to activation of a TGF-β pathway. Unlike cardiac myocytes that down-regulate Cx43, presumably to limit intercellular transmission of biochemical mediators of ischemic injury, fibroblasts may up-regulate Cx43 to maintain electrical and metabolic coupling at a time when intercellular communication is compromised.     

Inhibition of cardiomyocyte automaticity by electrotonic application of inward rectifier current from Kir2.1 expressing cells

A biological pacemaker might be created by generation of a cellular construct consisting of cardiac cells that display spontaneous membrane depolarization, and that are electrotonically coupled to surrounding myocardial cells by means of gap junctions. Depending on the frequency of the spontaneously beating cells, frequency regulation might be required. We hypothesized that application of Kir2.1 expressing non-cardiac cells, which provide I _K1 to spontaneously active neonatal cardiomyocytes (NCMs) by electrotonic coupling in such a cellular construct, would generate an opportunity for pacemaker frequency control. Non-cardiac Kir2.1 expressing cells were co-cultured with spontaneously active rat NCMs. Electrotonic coupling between the two cell types resulted in hyperpolarization of the cardiomyocyte membrane potential and silencing of spontaneous activity. Either blocking of gap-junctional communication by halothane or inhibition of I _K1 by BaCl₂ restored the original membrane potential and spontaneous activity of the NCMs. Our results demonstrate the power of electrotonic coupling for the application of specific ion currents into an engineered cellular construct such as a biological pacemaker.     

A Ca2+-inhibited non-selective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in some smooth muscles. The nature of the pacemaker conductance is unclear, but studies suggest that pacemaker activity is due to a voltage-independent, Ca²⁺-regulated, non-selective cation conductance. We investigated Ca²⁺-regulated conductances in murine intestinal ICC and found that reducing cytoplasmic Ca²⁺ activates whole-cell inward currents and single-channel currents. Both the whole-cell currents and single-channel currents reversed at 0 mV when the equilibrium potentials of all ions present were far from 0 mV. Recordings from on-cell patches revealed oscillations in unitary currents at the frequency of pacemaker currents in ICC. Voltage-clamping cells to −60 mV did not change the oscillatory activity of channels in on-cell patches. Depolarizing cells with high external K⁺ caused loss of resolvable single-channel currents, but the oscillatory single-channel currents were restored when the patches were stepped to negative potentials. Unitary currents were also resolved in excised patches. The single-channel conductance was 13 pS, and currents reversed at 0 mV. The channels responsible were strongly activated by 10⁻⁷ m Ca²⁺, and 10⁻⁶ m Ca²⁺ reduced activity. The 13 pS channels were strongly activated by the calmodulin inhibitors calmidazolium and W-7 in on-cell and excised patches. Calmidazolium and W-7 also activated a persistent inward current under whole-cell conditions. Murine ICC express Ca²⁺-inhibited, non-selective cation channels that are periodically activated at the same frequency as pacemaker currents. This conductance may contribute to the pacemaker current and generation of electrical slow waves in GI muscles.     

Jejunal afferent nerve sensitivity in wild-type and TRPV1 knockout mice

The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine intestine. Multiunit activity was recorded in vitro from mesenteric afferents supplying segments of mouse jejunum taken from wild-type (WT) and TRPV1 knockout (TRPV1^−/−) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so the pressure–response curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase in activity. Afferent response to distension was significantly lower in TRPV1^−/− than in WT mice. Single-unit analysis revealed three functional types of afferent fibres: (1) low-threshold fibres (2) wide dynamic range fibres and (3) high-threshold fibres. There was a marked downward shift of the pressure–response curve for wide dynamic range fibres in the TRPV1^−/− mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 m m ) was also attenuated in the TRPV1^−/− mice. In contrast, the response to bath application of bradykinin (1 μ m , 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μ m ) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1^−/− afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.     

Directional differentiation of human embryonic stem cells into cardiomyocytes by direct adherent culture

Abstract

Myocardial infarction is a serious and common disease in clinics, with a poor prognosis and a high mortality in the long term. The latest research progress in stem cell research, which has been at the frontier of medical research in recent years, gives hope to the patients with heart disease. Researchers have been trying to find an efficient and practical differentiation procedure to induce embryonic stem cells (ESCs) to differentiate into cardiomyocytes. This study used a direct adherent-culture method to induce the differentiation of human ESCs (hESCs) into cardiomyocytes in vitro and its differentiation efficiency was detected. The hESCs were induced into cardiomyocytes by adherence culture using the inducers activin A and BMP4. The time of appearance of beating cardiomyocytes, the percentage of beating colonies, and the beating frequency of cardiomyocytes under the microscope were observed and counted; the specific cardiomyocyte marker cTnT was started by immunofluorescence, and the electrophysiological function of cardiomyocytes was detected by patch clamp experiment. An apoptosis-Hoechst staining kit was used to detect the apoptosis ratio of beating of cardiomyocytes which had been treated by hypoxia for 24 hours. Widespread spontaneous beating cardiomyocytes was typically observed by day 13 after differentiation. The statistical result was that the average time of appearance of beating cardiomyocytes was 13·0±1·1 days, the percentage of beating colonies was 66·7%, and the beating frequency of cardiomyocytes 63·0±7·0 times/min; beating cardiomyocytes were positive to cTnT staining. Spontaneous action potentials of beating cardiomyocytes were detected, and the apoptosis ratio of beating cardiomyocytes which had been treated by hypoxia for 24 hours was 8·0±0·5%. The direct adherent-culture method was successfully used to induce the differentiation of hESCs into cardiomyocytes. The adherent method of hESC induced with activin A+BMP4 was determined to be more simple and effective than other methods. The differentiation efficiency reached 66·7%, and the differentiation time was about 13 days.     

Novel 5'TOPmRNAs regulated by ribosomal S6 kinase are important for cardiomyocyte development: S6 kinase suppression limits cardiac differentiation and promotes pluripotent cells toward a neural lineage

Moving stem cells from bench to bedside has been a challenging task. Undermining this task is comprehending and optimizing the underlying regulatory mechanisms that drive differentiation of stem cells into desired cell and tissue types. Here we present evidence that ribosomal S6 kinase (S6K) is among the proteins upregulated as embryonic stem cells (ESCs) and human induced pluripotent stem cells differentiate into beating cardiomyocytes. We hypothesized that S6K plays a pivotal role in cardiomyogenesis, primarily because it regulates the translation of 3 cardiac-involved genes recently shown to have 5' terminal oligopyrimidine (5'TOP) sequences: connexin 43 (Cx43), desmoplakin (Dsp), and phosphatase and tensin homolog (PTEN). Along with another independent laboratory, we confirmed that S6K is indeed upregulated in beating ESC-derived cardiomyocytes compared to the surrounding nonbeating, differentiated cells. S6K short interfering RNA-transfected stem cell cultures indicate that inhibition of S6K strongly hinders development of cardiomyocyte beating and translation of Cx43, Dsp, and PTEN; these cardiac 5'TOP mRNAs were only properly translated in cells with S6K, supporting our hypothesis. An unexpected discovery took the role of S6K one step further: S6K-knockdown stem cell cultures developed significantly more neurons than seen in embryoid bodies subjected to a typical cardiac differentiation protocol. These results introduced the novel idea that in addition to its critical cardiac roles, S6K may be a significant factor that prevents stem cells from pursuing a neuronal pathway. Overall, results have indicated the necessity of S6K for normal stem cell cardiomyogenesis, as well as lowered S6K expression for stem cell neurogenesis.     

Interleukin-1 polymorphisms are associated with the inflammatory response in human muscle to acute resistance exercise

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents ( I _j) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage ( V _j) dependence. I _j records from hMSC–HeLaCx43 and hMSC–HeLaCx40 cell pairs also showed symmetrical and asymmetrical V _j dependence, while hMSC–HeLaCx45 pairs always produced asymmetrical I _j with pronounced V _j gating when the Cx45 side was negative. Symmetrical I _j suggests that the dominant functional channel is homotypic, while the asymmetrical I _j suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances ( γ _j) of 30–80pS. The macroscopic I _j obtained from hMSC–cardiac myocyte cell pairs exhibited asymmetrical V _j dependence, while single channel events revealed γ _j of the size range 40–100pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.     

Ca2+ permeability of the channel pore is not essential for the δ2 glutamate receptor to regulate synaptic plasticity and motor coordination

The δ2 glutamate receptor (GluRδ2) plays a crucial role in cerebellar functions; mice with a disrupted GluR δ 2 gene ( GluR δ 2^−/− ) display impaired synapse formation and abrogated long-term depression (LTD). However, the mechanisms by which GluRδ2 functions have remained unclear. Because a GluRδ2 mutation in lurcher mice causes channel activities characterized by Ca²⁺ permeability, GluRδ2 was previously suggested to serve as a Ca²⁺-permeable channel in Purkinje cells. To test this hypothesis, we introduced a GluR δ 2 transgene, which had a mutation (Gln618Arg) in the putative channel pore, into GluR δ 2^−/− mice. Interestingly, the mutant transgene rescued the major functional and morphological abnormalities of GluR δ 2^−/− Purkinje cells, such as enhanced paired-pulse facilitation, impaired LTD at parallel fibre synapses, and sustained innervation by multiple climbing fibres. These results indicate that the conserved glutamine residue in the channel pore, which is crucial for all Ca²⁺-permeable glutamate receptors, is not essential for the function of GluRδ2.     

Connexin 43 mimetic peptide Gap26 confers protection to intact heart against myocardial ischemia injury

Unapposed connexin 43 hemichannels (Cx43Hc) are present on sarcolemma of cardiomyocytes. Whereas Cx43Hc remain closed during physiological conditions, their opening under ischemic stress contributes to irreversible tissue injury and cell death. To date, conventional blockers of connexin channels act unselectively on both gap junction channels and unapposed hemichannels. Here, we test the hypothesis that Gap26, a synthetic structural mimetic peptide deriving from the first extracellular loop of Cx43 and a presumed selective blocker of Cx43Hc, confers resistance to intact rat heart against ischemia injury. Langendorff-perfused intact rat hearts were utilized. Regional ischemia was induced by 40-min occlusion of the left anterior descendent coronary and followed by 180 min of reperfusion. Gap26 was applied either 10 min before or 30 min after the initiation of ischemia. Interestingly, myocardial infarct size was reduced by 48% and 55% in hearts treated with Gap26 before or during ischemia, respectively, compared to untreated hearts. Additionally, myocardial perfusate flow was increased in both groups during reperfusion by 37% and 32%, respectively. Application of Gap26 increased survival of isolated cardiomyocytes after simulated ischemia–reperfusion by nearly twofold compared to untreated cells. On the other hand, superfusion of tsA201 cells transiently expressing Cx43 with Gap26 caused 61% inhibition of Cx43Hc-mediated currents recorded using the patch clamp technique. In summary, we demonstrate for the first time that Cx43 mimetic peptide Gap26 confers protection to intact heart against ischemia–reperfusion injury whether administered before or after the occurrence of ischemia. In addition, we provide unequivocal evidence for the inhibitory effect of Gap26 on genuine Cx43Hc.     

Tumor necrosis factor alpha is reparative via TNFR2 [corrected] in the hippocampus and via TNFR1 [corrected] in the striatum after virus-induced encephalitis

Differentiating between injurious and reparative factors facilitates appropriate therapeutic intervention. We evaluated the role of tumor necrosis factor α (TNFα) in parenchymal brain pathology resolution following virus-induced encephalitis from a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). We infected the following animals with TMEV for 7 to 270 days: B6/129 TNF^−/− mice (without TNFα expression), B6/129 TNFR1^−/− mice (without TNFα receptor 1 expression), and B6/129 TNFR2^−/− mice (without TNFα receptor 2 expression). Normal TNFα-expressing controls were TMEV-infected B6, 129/J, B6/129F1 and B6/129F2 mice. Whereas all strains developed inflammation and neuronal injury in the hippocampus and striatum 7 to 21 days postinfection (dpi), the control mice resolved the pathology by 45 to 90 dpi. However, parenchymal hippocampal and striatal injury persisted in B6/129 TNF^−/− mice following infection. Treating virus-infected mice with active recombinant mouse TNFα resulted in less hippocampal and striatal pathology, whereas TNFα-neutralizing treatment worsened pathology. T1 "black holes" appeared on MRI during early infection in the hippocampus and striatum in all mice but persisted only in TNF^−/− mice. TNFR1 mediated hippocampal pathology resolution whereas TNFR2 mediated striatal healing. These findings indicate the role of TNFα in resolution of sublethal hippocampal and striatal injury.     

Regulatory T cells in spontaneous autoimmune encephalomyelitis

Summary: Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4⁺αβ T-cell repertoire. Monoclonality of the αβ T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG^−/− mice or TCR α^−/−/TCR β^−/− double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4⁺ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25⁺ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR α and β chain composition. T-reg cells expand poorly in vivo , and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.     

Connexin expression and intercellular communication in two- and three-dimensional in vitro cultures of human bladder carcinoma.

The identification of gap-junctional proteins (connexins) and the preparation of related antibodies provides new tools to study patterns of intercellular communication in tumors. Focusing on the biology of human bladder carcinoma, we compared the expression of gap-junctional proteins (connexins Cx26, Cx32, and Cx43) with a dye-coupling assay for gap-junctional intercellular communication in three cell lines with different urothelial differentiation. The cell lines HCV-29, RT4, and J82 were initially grown as monolayers of different ages. Connexin expression was found mostly positive over the time of culture and found constantly negative only in J82 cells for Cx26 and HCV-29 cells for Cx32. In HCV-29 cells, Cx26 increased in positivity over the time of culture. Western blotting with the antibodies confirmed the findings. Comparisons of dye transfer using Lucifer Yellow showed an increase of coupling in the normal urothelial cell line HCV-29 in contrast to a decrease of coupling in the tumor cell lines. Data were extended by multicellular spheroid (MCS) co-cultures with the stromal fibroblast line N1. In three-dimensional cultures as MCSs, Cx26 was increased in proximity of RT4 tumor cells to fibroblasts, and positivity was maintained in J82 cells. E-cadherin expression in cell lines showed no change in dependence of growth state. The data suggest that Cx26 plays a role in negative growth control or differentiation of urothelial cells. Preliminary comparative data on normal and neoplastic urothelium show all three connexins in normal urothelium, in contrast to varying amounts of Cx43 and low amounts of Cx32 in tumors and evident loss of Cx26 in low-grade tumors. Discrepancies between monolayer and MCS cultures are most likely due to higher differentiation in MCSs, and the continuation of systematic work with heterologous MCSs is indicated for more information on the role of gap-junctional proteins in human tumors.     

Fibroblast Growth-Stimulatory Activity Released from Human Monocytes

Human monoeytcs release fibroblast growth-stimulatory activity. In this study tumour necrosis factor (TNF) has been identified as a major contributor to the monocyte-derived fibroblast growth-stimulaiory activity. A neutralizing monoclonal antibody (MoAb) against recombinant TNF (rTNF) inhibited growth of FS-4 fibroblasts and skin biopsy fibroblasts induced by monocyte supernatants, indicating that TNF was involved in the stimulation. Optimal growth of FS-4 fibroblasts was induced by monocyte supernatants at dilutions which cointained TNF at a concentration between 1.6 × 10⁻⁶ and 4.0 × 10⁻⁵μg/ml, less growth being induced at higher TNF concentrations. Contrary to this, no optimal concentration interval was found lor rTNF, since increasing rTNF concentrations always resulted in increased growth stimulation. It also appeared that natural TNF in the monocyte supernatants induced growth at a much lower concentration than rTNF tested in the absence of monocyte supernatant.     

Differentiation of mouse-induced pluripotent stem cells into cardiomyocytes in vitro

In order to investigate the effective method to induce mice-induced pluripotent stem (miPS) cells into cardiomyocytes in vitro and to investigate the effect of vitamin C on cardiomyocyte differentiation from miPS cells to find a highly efficient and clinically safe method. MiPS cells were isolated and expanded to form embryoid bodies (EBs) using the hanging drop way. EBs were induced using differentiation medium containing vitamin C (10^?4 mmol/ml). The control group did not receive any form of inducer. The time and frequency at which beating cardiomyocytes appeared and the percentage of beating colonies were determined to investigate the function of vitamin C on cardiac myocytes differentiation from miPS cells. Beating cell areas were found in (62.5 ± 1.7%) of EBs when using differentiation medium containing vitamin C, which was at a significantly greater frequency than in the control group (7.6 ± 2.6%). Beating cardiomyocytes within the two groups were positive for troponin (cTnT) staining. Vitamin C markedly increased the productivity of miPS cell differentiation into cardiomyocytes, as supported by expression of the unique cardiac protein cTnT. The vitamin C is suitable candidate for the induction of miPS cell differentiation into cardiomyocytes in vitro.     

Glycinergic input of widefield, displaced amacrine cells of the mouse retina

Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRα1 ( Glra1^spd-ot , 'oscillator' mouse), the GlyRα2 ( Glra2 ^−/−) and the GlyRα3 subunit ( Glra3 ^−/−). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, τ∼25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1^spd-ot and Glra3 ^−/− mice (τ∼25 ms); however, glycinergic currents were absent in group II cells of Glra2 ^−/− mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (τ∼50–70 ms). We propose that GlyRs of group II cells are dominated by the α2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the α4 subunit.     

Characterization of connexin 30.3 and 43 in thymocytes

During maturation, thymocytes interact directly and indirectly with different cell types of the thymic microenvironment. Such a cellular communication has been basically ascribed to soluble factors and surface receptors. However, little attention has been given to cellular communication mediated by gap junctions. The existence of these intercellular channels in the immune system remained a controversial issue since the 1970s until recently, when a growing body of evidence has indicated their presence and physiological roles in the immune system. In this work, we investigated whether thymocytes express gap junction-forming proteins (connexins, Cx) and are capable of forming functional intercellular channels. Using RT-PCR, we demonstrated that thymocytes express the mRNA for two Cx isoforms: Cx30.3 and Cx43, but not for Cx26, Cx30, Cx31, Cx31.1, Cx32, Cx33, Cx36, Cx37, Cx40, Cx45, Cx46, and Cx50. In addition, the presence of Cx30.3 and Cx43 was confirmed using different techniques (RNase protection assay, western blot and immunofluorescence). However, despite the expression of these two Cxs, we did not detect functional homocellular coupling between thymocytes or between EL-4 cells (a Cx43 expressing thymic lymphoma-derived cell line) or heterocellular coupling between thymocytes and thymic epithelial cells (TEC) or between EL-4 and TEC in unstimulated conditions. Concluding, in this study, we described for the first time the expression of connexins in thymocytes, which may constitute a new molecule having a functional role in thymocytes maturation.     

On the different roles of AT1 and AT2 receptors in stretch-induced changes of connexin43 expression and localisation

Cyclic mechanical stretch (CMS) and angiotensin II (ATII) play an important role in cardiac remodelling. Thus, we aimed to examine how ATII affects CMS-induced changes in localisation and expression of the gap junction protein connexin43 (Cx43). Neonatal rat cardiomyocytes cultured on gelatin-coated Flexcell cell culture plates were kept static or were exposed to CMS (110?% of resting length, 1?Hz) for 24?h with or without additional ATII (0.1?μmol/L). Moreover, inhibitors of ATII receptors (AT-R) were used (for AT₁-R: losartan 0.1?μmol/L, for AT₂-R: PD123177 0.1?μmol/L). Thereafter, the cardiomyocytes were investigated by immunohistology, PCR and Western blot. After 24?h of CMS, cardiomyocytes were significantly elongated and orientated 75?±?1.6° nearly perpendicular to the stretch axis. Furthermore, CMS significantly accentuated Cx43 at the cell poles (ratio Cx43 polar/lateral static: 2.32?±?0.17; CMS: 10.08?±?3.2). Additional ATII application significantly reduced Cx43 polarisation (ratio Cx43 polar/lateral ATII: 4.61?±?0.42). The combined administration of ATII and losartan to CMS further reduced Cx43 polarisation to control levels, whilst the AT₂-R blocker PD123177 restored polarisation. Moreover, CMS and ATII application resulted in a significant Cx43 protein and Cx43 mRNA up-regulation which could be blocked by losartan but not by PD123177. Thus, CMS results in a self-organisation of the cardiomyocytes leading to elongated cells orientated transversely towards the stretch axis with enhanced Cx43 expression and Cx43 accentuation at the cell poles. ATII enhances total Cx43 mRNA and protein expression probably via AT₁-R (=inhibitory effect of losartan) and reduces Cx43 polarisation presumably via AT₂-R, since PD123177 (but not losartan) inhibited the negative effects of ATII on polarisation.     

α5 subunit-containing GABAA receptors affect the dynamic range of mouse hippocampal kainate-induced gamma frequency oscillations in vitro

Though all in vitro models of gamma frequency network oscillations are critically dependent on GABA_A receptor-mediated synaptic transmission little is known about the specific role played by different subtypes of GABA_A receptor. Strong expression of the α5 subunit of the GABA_A receptor is restricted to few brain regions, amongst them the hippocampal dendritic layers. Receptors containing this subunit may be expressed on the extrasynaptic membrane of principal cells and can mediate a tonic GABA_A conductance. Using hippocampal slices of wild-type (WT) and α5−/− mice we investigated the role of α5 subunits in the generation of kainate-induced gamma frequency oscillations (20–80 Hz). The change in power of the oscillations evoked in CA3 by increasing network drive (kainate, 50–400 n m ) was significantly greater in α5−/− than in WT slices. However, the change in frequency of gamma oscillations with increasing network drive seen in WT slices was absent in α5−/− slices. Raising the concentration of extracellular GABA by bathing slices in the GABA transaminase inhibitor vigabatrin and blocking uptake with tiagabine reduced the power of gamma oscillations more in WT slices than α5−/− slices (43% versus 15%). The data suggest that loss of this GABA_A receptor subunit alters the dynamic profile of gamma oscillations to changes in network drive, possibly via actions of GABA at extrasynaptic receptors.