Atrial fibrillation is associated with hypermethylation in human left atrium, and treatment with decitabine reduces atrial tachyarrhythmias in spontaneously hypertensive rats.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. As the molecular mechanisms underlying the pathology are largely unknown, this cardiac arrhythmia remains difficult to treat. To identify specific molecular actors involved in AF, we have performed a transcriptomic analysis on left atrium (LA) from patients with valvular heart disease with or without AF. We showed that 1627 genes had altered basal expression level in LA tissue of AF patients compared with the control group. The significantly enriched gene ontology biological process "anatomical structure morphogenesis" contained the highest number of genes in line with changes in structure that occur when the human heart remodels following AF development (ie, LA dilatation and interstitial fibrosis). We then focused the study on Pitx2 (paired-like homeodomain 2), being the most altered transcription factor in LA from AF patients and from which compelling evidence have indicated that its reduced expression can be considered as a marker for the disease. In addition, its expression was inversely correlated with LA size. We demonstrated that AF is associated with Pitx2 promoter hypermethylation both in humans and arrhythmic aging spontaneously hypertensive rats. Chronic administration of a DNA methylation inhibitor (ie, 5-Aza-2'-deoxycitidine) improved ECG arrhythmic profiles and superoxide dismutase activities and reduced fibrosis in the left ventricle of spontaneously hypertensive rats. Taken together, these data support the notion that AF is associated with epigenetic changes in LA and provide a proof-of-concept that hypomethylating agents have to be considered in the treatment of atrial arrhythmias.

Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion.

BACKGROUND: Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined. RESULTS AND METHODS: Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal-epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT-PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival. CONCLUSION: TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma.

Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes.

Dendritic cells (DCs), mononuclear cells that initiate immune responses, and osteoclasts (OCs), multinucleated bone-resorbing cells, are hematopoietic cells derived from monocytic precursor cells. Using in vitro generated dendritic cells, we previously showed that human and murine DCs could transdifferentiate into resorbing osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). In this study we globally compared by transcriptomic profiling this new osteoclast differentiation pathway from DCs with the canonical differentiation pathway from monocytes. DNA chip data revealed that starting from two very distinct cell types, treatment with M-CSF and RANKL generated two highly similar types of osteoclast. In particular, DC-derived osteoclasts expressed all the characteristic marker genes of monocyte-derived osteoclasts. Two major molecular events could be observed during osteoclastogenesis: downregulation of a large set of monocyte or DC specific markers, together with upregulation of characteristic osteoclast marker genes. Most interestingly, our transcriptomic data showed a closer molecular profile between DCs and OCs than between monocytes and OCs. Our data establish DCs as a new osteoclast precursor able to generate OCs more efficiently than monocytes.

Developmental plasticity of the carotid chemoafferent pathway in rats that are hypoxic during the prenatal period.

The chemoreflex pathway undergoes postnatal maturation, and the perinatal environment plays a critical role in shaping respiratory control system. We investigated the role of prenatal hypoxia on the maturation of the chemoreflex neural circuits regulating ventilation in rat. Effects of hypoxia (10% O2) from the 5th to the 20th day of gestation were studied on male offspring at birth and on postnatal days 3, 7, 21 and 68. Maturation of the respiratory control system was assessed by in vivo tyrosine hydroxylase (TH) activity measurement in peripheral chemoreceptors (carotid bodies, petrosal ganglia), and in brainstem catecholaminergic cell groups (A2C2c and A1C1 areas in the medulla, A5 and A6 areas in the pons). Resting ventilation and ventilatory response to hypoxia were evaluated as functional sequelae. In peripheral structures, prenatal hypoxia reduced TH activity within the first postnatal week and enhanced it later. In contrast, in central areas, prenatal hypoxia upregulated TH activity within the first postnatal week and downregulated it later. The in vivo TH activity impairment is therefore tissue specific, with an opposite effect on the peripheral and central neural circuits. A shift of the effect of prenatal hypoxia occurred between 1 and 3 weeks, indicating a postnatal temporal effect of prenatal hypoxia. An important period in the development of the chemoafferent pathway occurred between the first and the third postnatal week. Functionally, prenatal hypoxia impaired resting ventilation and ventilatory response to hypoxia. The alterations of the catecholaminergic components of the chemoafferent pathway resulting from prenatal hypoxia might contribute to impair postnatal respiratory behaviour.

[DNA microarray technology: principles and applications to the study of neurological disorders]. / Principes et intérêts pour l'étude des maladies neurologiques et technologie des puces ADN.

INTRODUCTION: DNA microarray is a powerful technology which can rapidly provide a high throughput and detailed view of the entire genome and transcriptome. In this review we discuss the basic principles behind gene expression microarrays, CGH arrays and DNA microarray genotyping, and their potential applications to neurological diseases. STATE OF THE ART: Microarray gene expression profiling is a reliable technology that has already been used with great success in the molecular classification of cancer. It is a very promising technology in the field of Neurooncology. One of the interesting characteristics of DNA microarrays is also that they can be used in a non-hypothesis-driven manner to discover new genomic characteristics that will enable to establish new pathophysiological hypotheses. Such a strategy has already yielded interesting new insights in the study of multiple sclerosis, Alzheimer disease or neuromuscular diseases. With DNA microarray genotyping it is now possible to detect mutations in many genes simultaneously. CONCLUSIONS: In Neurooncology DNA microarrays should help to establish a more accurate classification of brain tumors and recent studies have shown how gene expression profiling of brain tumors allows to uncover previously unrecognized patient subsets that differ in their survival. The applications of microarrays for the study of neurological diseases, like multiple sclerosis, Alzheimer disease or neuromuscular diseases are also promising both for generating new pathophysiological hypotheses and for enabling new molecular classifications. DNA microarray genotyping is a powerful technology that should help to discover genetic factors associated with multifactorial neurological disorders and help to diagnose complex neurogenetic diseases. This technology should also facilitate the realization of pharmacogenomic studies in neurological diseases.

Gene expression profiling in insulinomas of Men1 beta-cell mutant mice reveals early genetic and epigenetic events involved in pancreatic beta-cell tumorigenesis.

Mutations of the MEN1 gene lead to the occurrence of multiple endocrine neoplasia type 1 (MEN1). To gain insights into the mechanisms of the tumorigenesis related to MEN1 inactivation, we have used mice in which the Men1 gene was specifically disrupted in pancreatic beta-cells. In these mice, we observed full penetrance of insulinoma with defined histological characteristics of tumorigenesis. To identify the genetic factors taking part in the tumour development, we performed gene expression profiling analysis of these insulinomas at different stages. Here, we show that in late stage insulinomas, 56 genes are up-regulated and 194 are down-regulated more than fourfold compared with normal pancreatic islets. Clustering analysis reveals the deregulation of Hox gene family and the genes involved in cell proliferation and cell cycle control. The altered expression of Igf2, Igfbp3 and Igfbp6 as well as cyclin A2, B2 and D2 are confirmed by quantitative RT-PCR, with the overexpression of all the three cyclins found in early stage insulinomas. Moreover, an increased proportion of cyclin A2- and D2-expressing cells and the overexpression of insulin-like growth factor 2 (IGF2) protein are detected in mouse Men1 insulinomas by immunostaining. Interestingly, the analysis of DNA methylation patterns by quantitative serial pyrosequencing reveals that four specific CpGs in the intragenic differentially methylated region 2 (DMR2) region of the Igf2 gene known to augment transcription through methylation are significantly hypermethylated in insulinomas of Men1 beta-cell mutant mice at 6 and 10 months of age, even before IGF2 overexpression can be detected. Thus, our data indicate the involvement of both genetic and epigenetic mechanisms in early tumorigenesis of beta-cells related to MEN1 inactivation.

Cloning, sequencing, tissue distribution, and heterologous expression of rat flavin-containing monooxygenase 3.

The sequence of rat FMO3 was obtained by RT-PCR and 5'/3' terminal extension. Complete cDNA was amplified, cloned, and sequenced. The cDNA encodes a protein of 531 amino acids which contains the NADPH- and FAD-binding sites and a hydrophobic carboxyl terminus characteristic of FMOs. This sequence is 81, 81, and 91% identical to sequences of human, rabbit, and mouse FMO3, respectively, and 60% identical to rat FMO1. Rat FMO3 was expressed in Escherichia coli. The recombinant protein and the native protein purified from rat liver microsomes migrated with the same mobility (56 kDa) as determined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Recombinant rat FMO3 showed activities of methimazole S-oxidation, and NADPH oxidation associated with the N- or S-oxidation of trimethylamine and thioacetamide, in good concordance with those reported for human FMO3. When probed with rat FMO3 cDNA (bases 201 to 768), a strong signal corresponding to the 2.3-kb FMO3 transcript was detected in RNA samples from rat liver and kidney while a weak signal was observed with lung RNA samples. In contrast, the probe did not hybridize with any RNA from brain, adipose tissue, or muscle.

Characterization of a Na+-Ca2+ exchanger NCX1 isoform in bovine fasciculata cells of adrenal gland.

The Na+-Ca2+ exchanger (NCX) is well known to regulate intracellular Ca2+ concentration in many excitable cells. To date, three members of the NCX exchanger gene family have been identified: NCX1, NCX2 and NCX3. In zona fasciculata of the bovine adrenal gland, a Na+-Ca2+ exchanger has been recently found to display biochemical similarities with the NCX1 exchanger of renal basolateral cells. Although a Na+-Ca2+ exchanger has already been characterized in zona medulla of adrenal gland, such an exchanger has never been studied in the adrenal cortex. Thus, we have used a reverse transcriptase-polymerase chain reaction (RT-PCR) assay with specific NCX primers on fasciculata cell cultures. The sequencing of the amplified fragment revealed, for the first time, the presence of a NACA3 isoform of the NCX1 exchanger in the adrenal cortex, similar to the one found in adrenal chromaffin cells and in renal cortex. This isoform is 92 and 94% identical in the portion compared with kidney Na+-Ca2+ exchanger NACA3 of rat and pig, respectively.

An uncoupling protein homologue putatively involved in facultative muscle thermogenesis in birds.

The cDNA of an uncoupling protein (UCP) homologue was obtained by screening a chicken skeletal-muscle library. The predicted 307-amino-acid sequence of avian UCP (avUCP) is 55, 70, 70 and 46% identical with mammalian UCP1, UCP2 and UCP3 and plant UCP respectively. avUCP mRNA expression is restricted to skeletal muscle and its abundance was increased 1.3-fold in a chicken line showing diet-induced thermogenesis, and 3.6- and 2.6-fold in cold-acclimated and glucagon-treated ducklings developing muscle non-shivering thermogenesis respectively. The present data support the implication of avUCP in avian energy expenditure.

Sympathetic control of glucagon receptor mRNA levels in brown adipose tissue of cold-exposed rats.

Brown adipose tissue (BAT) is implicated in both cold-induced thermogenesis and regulation of energy expenditure and is mainly controlled by sympathetic innervation. To clarify the permissive and/or complementary roles of glucagon in cold-induced BAT activation, glucagon receptor gene expression and its modulation by sympathetic activity were investigated in rats. One pad of interscapular BAT was surgically denervated while the other pad was sham operated, then rats were either cold-exposed (CE) for 1 week at 4 degrees C or kept near thermoneutrality (25 degrees C, TN). Using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay, it was shown that cold exposure decreased (-44%) the relative abundance of BAT glucagon receptor mRNA, an effect which was prevented by unilateral surgical sympathectomy of BAT. The present results show a negative control by sympathetic nervous activity of glucagon receptor gene expression and/or of glucagon receptor mRNA stability in BAT of cold-exposed rats. The down-regulation of glucagon receptor expression during cold exposure does not support a major role of the peptide in the thermogenic control of BAT.

Increased sodium-calcium exchange current in right ventricular cell hypertrophy induced by simulated high altitude in adult rats.

Ventricular hypertrophy is associated with an increase in action potential (AP) duration which is potentially arrhythmogenic. The implication of the Na-Ca exchange current (I(Na-Ca)) in the lengthening of the AP is controversial. The role of this current in the increased duration of the low plateau of the AP in hypertrophied adult rat ventricular myocytes by simulated chronic high-altitude exposure ( approximately 4500 m) was evaluated. Electrophysiological experiments were carried out on isolated right ventricular myocytes from exposed and control rats with the perforated patch or the conventional whole-cell technique in current or in voltage clamp condition. With the two techniques, a significant increase of the low plateau duration was observed in hypertrophied myocytes as compared to controls. The low plateau in hypertrophied myocytes was depressed when Na was replaced by Li and was no longer recorded when intracellular Ca was buffered with EGTA. Inward tail currents, evoked either on repolarization to -80 mV following a depolarizing pulse to +10 mV or by interrupted AP technique, were greater in hypertrophied than in control myocytes and were abolished when Na was replaced by Li or when intracellular Ca was buffered with EGTA, indicating an increased Na-Ca exchange activity. The Li-sensitive current-voltage curves, obtained by a voltage clamp ramp protocol with an intracellular calcium buffered solution, were not significantly different in both hypertrophied and control myocytes, suggesting no modification in the density of the Na-Ca exchange protein. This was corroborated by the lack of difference in NCX1 mRNA levels between right ventricles from control and exposed rats. We conclude that increased duration of the low plateau of rat ventricular AP in altitude cardiac hypertrophy may be attributed to an increase of the inward I(Na-Ca). This augmented I(Na-Ca)may result from a modification in the intracellular Ca homeostasis.

Effects of intracerebroventricular injections of des-His1 (Glu9) glucagon amide on the regulatory thermogenesis in muscovy ducklings.

Recent investigations have demonstrated a modulatory action of glucagon on shivering via the central nervous system in ducklings. Such an action could be mediated by glucagon receptors that have been recently detected in several brain areas involved in the central control of the involuntary motricity in this avian species. The present study using des-His1 (Glu9) glucagon amide, was performed to investigate the central mechanisms of glucagon on shivering. This glucagon analog was found to be an antagonist of glucagon devoid of adenylate cyclase activity (GR2) by triggering the breakdown of inositol phosphate (GR1) in mammals hepatocytes. The intracerebroventricular administration of des-His1 (Glu9) glucagon amide or glucagon induced a marked inhibition of shivering in ducklings exposed to cold. It seems likely that GR1 receptors contribute to decreased shivering in ducklings exposed to cold. Central glucagon or des-His1 (Glu9) glucagon amide were devoid of thermogenic effect at thermoneutrality.

Effects of gestational hypoxia on mRNA levels of Glut3 and Glut4 transporters, hypoxia inducible factor-1 and thyroid hormone receptors in developing rat brain.

Alterations of brain development result from noxious intrauterine signals, as oxygen deprivation, which decrease glucose energetic yield. To verify the hypothesis that a defect of brain energetic adaptation is responsible for these alterations, we have studied the effects of gestational hypoxia (10% oxygen during the last 2 weeks of fetal life) on cerebral ontogenesis of glucose transporters which control the limiting step of glucose utilization by neurons. This study is realised in rats by quantification of whole brain Glut3 and Glut4 mRNA in 14- and 19-day-old embryos (E14, E19), newborn (P0) and 7 postnatal-day-old rats (P7) by using reverse transcription-polymerase chain reaction (RT-PCR) method. We have associated our study with the analysis of a transcriptional factor, the hypoxia inducible factor-1alpha (HIF-1alpha), known to control the expression of glucose transporter, and with a family of transcriptional factors, the thyroid hormone receptors (TR), regulating specific genes involved in brain development. The data show (1) for the first time the Glut4 and HIF-1alpha gene expression in fetal rat brain which are detected as soon as E14, (2) that gestational hypoxia induces an increase of mRNA transcript levels of Glut3, Glut4, TRalpha2, TRbeta1 and HIF-1alpha genes mainly or exclusively at E14, and (3) that the absence of response of Glut3 and HIF-1alpha at E19 in hypoxic vs. normoxic group could indicate an insufficient energetic adaptation at this period of development which could lead to the neural alterations observed postnatally.

Muscle-specific up-regulation of rat UCP3 mRNA expression by long-term hindlimb unloading.

The effect of long-term hindlimb unloading (2 or 5 week) on the expression of uncoupling protein-3 (UCP3) gene was investigated in rat skeletal muscles. The interaction of hindlimb unloading and thyroid status was also investigated at 2 weeks. Whatever the duration, mechanical unloading induced a similar increase in UCP3 mRNA relative abundance in the slow-twitch soleus (SOL) muscle (+80%, P < 0.05), whereas no effect was observed in the fast-twitch extensor digitorum longus (EDL) muscle. Hypothyroidism down-regulated while hyperthyroidism up-regulated UCP3 mRNA relative abundance in both SOL and EDL muscles, but thyroid status did not prevent the up-regulation of UCP3 induced by 2 weeks of suspension. These data therefore indicate for the first time that long-term hindlimb unloading up-regulates muscle UCP3 gene expression in a muscle-specific manner which is independent of thyroid status.

Biochemical and functional evidences for a GLUT-4 homologous protein in avian skeletal muscle.

The characteristics and modulation of glucose transport were investigated in skeletal muscles of 5-wk-old Muscovy ducklings (Cairina moschata). Glucose uptake by sarcolemmal vesicles isolated from gastrocnemius muscle followed typical Michaelis-Menten kinetics with a K(m) value (17 mM) similar to that described in equivalent mammalian preparations. Western blot analysis of duckling sarcolemma using antibodies directed against rat GLUT-4 transporter revealed an immunoreactive protein of similar molecular mass (45 kDa) to that present in rats. When ducklings were killed in the postabsorptive state, GLUT-4 homologous protein was located predominantly (80%) in intracellular membranes. Insulin stimulation of a perfused leg muscle preparation in vitro led to the translocation of GLUT-4 homologous proteins from intracellular pools to the sarcolemma, with a subsequent increase in glucose uptake by sarcolemmal vesicles and perfused muscles. Glucose transport was positively controlled by the metabolic needs of skeletal muscle as reflected by the increased glucose uptake of sarcolemmal vesicles isolated from cold-acclimated ducklings. Present results, therefore, demonstrate, for the first time in an avian species, the existence in skeletal muscle of a glucose transporter showing molecular and functional homologies with the mammalian GLUT-4 transporter.

Differential regulation of uncoupling protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats.

The control of uncoupling protein-1, -2 and -3 (UCP-1, UCP-2, UCP-3) mRNA levels by sympathetic innervation in rats was investigated by specific and sensitive RT-PCR assays. In rats reared at thermoneutrality (25 degrees C), unilateral surgical sympathetic denervation of interscapular brown adipose tissue (BAT) markedly reduced the UCP-1 mRNA level (-38%) as compared with the contralateral innervated BAT pad, but was without significant effect on UCP-2 and -3 mRNA levels. Cold exposure (7 days, 4 degrees C) markedly increased UCP-1 (+180%), UCP-2 (+115%) and UCP-3 (+195%) mRNA levels in interscapular BAT. Unilateral sympathetic denervation prevented the cold-induced rise in BAT UCP-1 and UCP-2 mRNAs, but not that in BAT UCP-3 mRNA. Results were confirmed by Northern blot analysis. These data indicate a differential endocrine control of UCP-1, UCP-2 and UCP-3 gene expression in rat BAT both at thermoneutrality and during prolonged cold exposure.

Tissue-specific modulation of rat glucagon receptor mRNA by thyroid status.

The influence of thyroid status on glucagon receptor mRNA levels was investigated in rats using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Glucagon receptor mRNA was detected in liver, brown and white adipose tissues (BAT and WAT) and brain. In BAT and WAT, pharmacologically-induced moderate hypothyroidism resulted in a marked reduction in the relative abundance of glucagon receptor mRNA. Short-term treatment of hypothyroid rats with exogenous 3,3',5'-triiodo-L-thyronine (T3), resulting in a marked hyperthyroidism, reversed the phenomenon in BAT while the reversal was only partial in WAT. In the liver, there was no significant effect of mild hypothyroidism while there was a positive effect of hyperthyroidism. In brain, the relative tissue abundance of glucagon receptor mRNA was not affected by the large changes in plasma T3. The present results therefore indicate that thyroid status may modulate the relative abundance of glucagon receptor mRNA in a tissue-specific manner.

Characterization of a cDNA encoding an alpha thyroid hormone receptor in muscovy duckling.

A complementary deoxyribonucleic acid (cDNA) clone encoding an alpha thyroid hormone receptor (TR alpha) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TR alpha sequence showed a high degree of homology. Despite 45 nucleotide substitutions, the deduced peptide sequence was similar. This cDNA was used as a probe to characterize the TR alpha mRNA transcripts expressed in muscovy duckling liver and skeletal muscle.

Molecular cloning and sequencing of a cDNA encoding a beta-thyroid hormone receptor in muscovy duckling.

A cDNA clone encoding a beta-thyroid hormone receptor (TRbeta) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TRbeta sequence showed a high degree of homology. This cDNA was used as a probe to characterize the TRbeta mRNA transcripts expressed in muscovy duckling liver.

Central noradrenergic reactivity to stress in Maudsley rat strains.

Maudsley reactive (MR) and Maudsley nonreactive (MNRA) rats were submitted to a single session of acute 5-min immobilization stress and immediately sacrificed by decapitation. Subsequent neurochemical analysis revealed an elevation of 3,4-dihydroxyphenylacetic acid levels in the locus coeruleus and in the ventrolateral medulla, but not in the dorsomedial medulla, of rats of the two strains compared with nonstressed controls. This response was greater in the MR than in the MNRA group, suggesting a strain difference in the reactivity of the central noradrenergic cells to acute stress.