Bruno Bastide

Gap junction protein connexin40 is preferentially expressed in vascular endothelium and conductive bundles of rat myocardium and is increased under hypertensive conditions.

Gap junction channels consisting of connexin protein mediate electrical coupling between cardiac cells. Expression of two connexins, connexin40 (Cx40) and connexin43 (Cx43), has been studied in ventricular myocytes from normal and hypertensive rats. Polyclonal affinity-purified rabbit antibodies to Cx43 and Cx40 have been used for immunohistochemical analysis on frozen sections from rat heart. These studies revealed coexpression of Cx43 and Cx40 in ventricular myocytes. In addition, Cx40 is preferentially expressed in three distinct regions: first, in the endothelial layer of the heart blood vessels but not in the smooth muscle layer of the arteries; second, in the ventricular conductive myocardium, particularly in the atrioventricular bundle and bundle branches, where Cx43 is not observed; and third, in the myocyte layers close to the ventricular cavities. These results suggest that Cx40 is preferentially expressed in the fast conducting areas of myocardial tissue. Expression of both Cx40 and Cx43 was also found in immunoblots from normal and hypertensive rat myocardiocytes. Under hypertensive conditions (ie, in spontaneous hypertensive rats and in transgenic rats that exhibit hypertension due to expression of an exogenous renin gene), we found a 3.1-fold increase in Cx40 expression, compared with normal myocardium. Furthermore, we detected a 3.3-fold decrease in Cx43 protein level in transgenic hypertensive rats. The coexpression of Cx40 and Cx43 proteins in rat myocytes, their spatial distribution, and the increased amount of Cx40 protein during cardiac hypertrophy suggest that Cx40 may be involved in mediating fast conduction under normal and pathological conditions. The increased expression of Cx40 in hypertrophic heart may be a compensatory mechanism to increase conduction velocity.

Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory post-translational modification of nucleo-cytoplasmic proteins that has a complex interplay with phosphorylation. O-GlcNAc has been described as a nutritional sensor, the level of UDP-GlcNAc that serves as a donor for the uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetyl-glucosaminyltransferase being regulated by the cellular fate of glucose. Because muscular contraction is both dependent on glucose metabolism and is highly regulated by phosphorylation/dephosphorylation processes, we decided to investigate the identification of O-GlcNAc-modified proteins in skeletal muscle using a proteomic approach. Fourteen proteins were identified as being O-GlcNAc modified. These proteins can be classified in three main classes: i) proteins implicated in the signal transduction and in the translocation between the cytoplasm and the nucleus or structural proteins, ii) proteins of the glycolytic pathway and energetic metabolism, and iii) contractile proteins (myosin heavy chain). A decrease in the O-GlcNAc level was measured in the slow postural soleus muscle after 14-day hindlimb unloading, a model of functional atrophy characterized by a decrease in the force of contraction. These results strongly suggest that O-GlcNAc modification may serve as an important regulation system in skeletal muscle physiology.

Placental BDNF/TrkB signaling system is modulated by fetal growth disturbances in rat and human.

The brain-derived neurotrophic factor (BDNF) has been shown to exert an important role during implantation, placental development, and fetal growth control in mice. Its expression is closely related to the nutritional status in several tissues such as in the nervous system. In a previous study, we demonstrated that maternal undernutrition (MU), during the perinatal life, modified both the BDNF and its functional receptor, the tyrosine kinase receptor B (TrkB) gene expression in the brain of growth-restricted rat offspring during sensitive developmental windows, suggesting that these early modifications may have long-lasting consequences. In the present study, we measured BDNF/TrkB mRNA and protein levels in rat placentas from mothers submitted to a 50% food restriction during gestation, and in human placentas from pregnancies with fetal growth restriction or fetal macrosomia. In the rat, two subtypes of placental TrkB receptors have been identified: the TrkB-FL and TrkB-T1 receptors. We found that MU induced intrauterine growth restriction (IUGR) of fetuses at term and decreased the placental BDNF mRNA and protein levels. Placentae from undernourished mothers exhibited an increased mRNA expression of TrkB-FL whereas both TrkB-FL and TrkB-T1 receptors proteins levels were not modified. In human IUGR placentas, both BDNF and TrkB receptor mRNA expressions were up-regulated. Finally, although neither BDNF nor TrkB mRNA levels were altered by fetal macrosomia alone, BDNF mRNA levels were decreased when macrosomia was associated with maternal type 1 diabetes. These results show that the placental BDNF/TrkB system is modulated in rats and humans during pregnancies with fetal growth perturbations and is affected by the maternal energetic status. These data suggest that this system may exert an important role for the feto-placental unit development and that it may also be implicated in the etiology of pathologies related to placental and fetal growth disturbances.

O-Linked N-Acetylglucosaminylation Is Involved in the Ca2+ Activation Properties of Rat Skeletal Muscle

O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-Glc-NAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy. In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-d-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction.

Reversible Interruption of Gap Junctional Communication by Testosterone Propionate in Cultured Sertoli Cells and Cardiac Myocytes

Abstract. A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state.

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca2+ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.

Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK.

Our aim was to analyze the role of phosphatidylinositol 3-kinase (PI3K)-AKT and MAPK signaling pathways in the regulation of muscle mass and slow-to-fast phenotype transition during hindlimb unloading (HU). For that purpose, we studied, in rat slow soleus and fast extensor digitorum longus muscles, the time course of anabolic PI3K-AKT-mammalian target of rapamycin, catabolic PI3K-AKT-forkhead box O (FOXO), and MAPK signaling pathway activation after 7, 14, and 28 days of HU. Moreover, we performed chronic low-frequency soleus electrostimulation during HU to maintain exclusively contractile phenotype and so to determine more precisely the role of these signaling pathways in the modulation of muscle mass. HU induced a downregulation of the anabolic AKT, mammalian target of rapamycin, 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, and glycogen synthase kinase-3β targets, and an upregulation of the catabolic FOXO1 and muscle-specific RING finger protein-1 targets correlated with soleus muscle atrophy. Unexpectedly, soleus electrostimulation maintained 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, FOXO1, and muscle-specific RING finger protein-1 to control levels, but failed to reduce muscle atrophy. HU decreased ERK phosphorylation, while electrostimulation enabled the maintenance of ERK phosphorylation similar to control level. Moreover, slow-to-fast myosin heavy chain phenotype transition and upregulated glycolytic metabolism were prevented by soleus electrostimulation during HU. Taken together, our data demonstrated that the processes responsible for gradual disuse muscle plasticity in HU conditions involved both PI3-AKT and MAPK pathways. Moreover, electrostimulation during HU restored PI3K-AKT activation without counteracting soleus atrophy, suggesting the involvement of other signaling pathways. Finally, electrostimulation maintained initial contractile and metabolism properties in parallel to ERK activation, reinforcing the idea of a predominant role of ERK in the regulation of muscle slow phenotype.

Nerve influence on myosin light chain phosphorylation in slow and fast skeletal muscles

Neural stimulation controls the contractile properties of skeletal muscle fibres through transcriptional regulation of a number of proteins, including myosin isoforms. To study whether neural stimulation is also involved in the control of post‐translational modifications of myosin, we analysed the phosphorylation of alkali myosin light chains (MLC1) and regulatory myosin light chains (MLC2) in rat slow (soleus) and fast (extensor digitorum longus EDL) muscles using 2D‐gel electrophoresis and mass spectrometry. In control rats, soleus and EDL muscles differed in the proportion of the fast and slow isoforms of MLC1 and MLC2 that they contained, and also in the distribution of the variants with distinct isoelectric points identified on 2D gels. Denervation induced a slow‐to‐fast transition in myosin isoforms and increased MLC2 phosphorylation in soleus, whereas the opposite changes in myosin isoform expression and MLC2 phosphorylation were observed in EDL. Chronic low‐frequency stimulation of EDL, with a pattern mimicking that of soleus, induced a fast‐to‐slow transition in myosin isoforms, accompanied by a decreased MLC2 phosphorylation. Chronic administration (10 mg·kg−1·d−1 intraperitoneally) of cyclosporin A, a known inhibitor of calcineurin, did not change significantly the distribution of fast and slow MLC2 isoforms or the phosphorylation of MLC2. All changes in MLC2 phosphorylation were paralleled by changes in MLC kinase expression without any variation of the phosphatase subunit, PP1. No variation in MLC1 phosphorylation was detectable after denervation or cyclosporin A administration. These results suggest that the low‐frequency neural discharge, typical of soleus, determines low levels of MLC2 phosphorylation together with expression of slow myosin, and that MLC2 phosphorylation is regulated by controlling MLC kinase expression through calcineurin‐independent pathways.

Contraceptive gossypol blocks cell-to-cell communication in human and rat cells

Gossypol (a polycyclic lipophilic agent naturally present in cottonseed, known as a potent non-steroid antifertility agent and a non-specific enzyme inhibitor) irreversibly impaired the intercellular communication between homologous pairs of various cultured cells, from man or rat, involved (Sertoli or trophoblastic cells) or not involved (ventricular myocytes) in steroidogenesis, in a dose-dependent manner. In serum-free assays, a rapid junctional uncoupling occurred in non-cytotoxic conditions. At 5 microM (approximately twice the peak plasma concentration measured in human patients during chronic administration), gap junctional communication was interrupted within 4 to 10 min, without concomitant rise in the intracellular Ca2+ concentration. The latter importantly increased when gossypol treatment was prolonged (cytotoxic effect). The short term uncoupling effect of gossypol was prevented by serum proteins, but long-lasting treatments (48 h) with moderate concentrations (3 microM) elicited junctional uncoupling and impeded the in vitro differentiation of human trophoblasts.

Expression and functional implications of troponin T isoforms in soleus muscle fibers of rat after unloading

Abstract. The expression pattern of troponin T (TnT) isoforms was studied in rat soleus muscle fibers in control and after hindlimb unloading (HU) conditions. To determine the functional consequence of TnT expression, the fibers were also examined for their calcium activation characteristics. With regard to TnT expression, four populations of fibers were distinguished in control muscle. Slow fibers expressing only slow isoforms of TnT (TnT1s, 2s, 3s ) were predominant (54%). Hybrid slow fibers (16%) differed from slow fibers by the additional expression of two TnTf isoforms. Hybrid fast fibers (22%) expressed slow and fast isoforms of TnT while fast fibers (8%) expressed only fast TnT isoforms. The expression of the other regulatory protein isoforms was checked for each population. The contractile experiments revealed steeper slopes of the tension/pCa relationship from hybrid slow fibers expressing fast TnT in a completely slow molecular environment. The expression of TnTs in hybrid fast fibers did not modulate the intrinsic co-operativity. After HU, the fast population was increased and reached 55%. The slow population decreased to 41% and a very small amount of hybrid slow fibers remained (4%). These data demonstrated the implication of TnT isoforms in the calcium activation properties and, more particularly, in the modulation of co-operativity within the myofibrillar lattice. Regulation of TnT expression appeared as a very fast and complete process compared to moderate changes of TnC and TnI.