Microgravity has a dramatic impact on human physiology, illustrated in particular, with skeletal muscle impairment. A thorough understanding of the mechanisms leading to loss of muscle mass and structural disorders is necessary for defining efficient clinical and spaceflight countermeasures. We investigated the effects of long-term bed rest on the transcriptome of soleus (SOL) and vastus lateralis (VL) muscles in healthy women (BRC group, n = 8), and the potential beneficial impact of protein supplementation (BRN group, n = 8) and of a combined resistance and aerobic training (BRE group, n = 8). Gene expression profiles were obtained using a customized microarray containing 6,681 muscles-relevant genes. A two-class statistical analysis was applied on 2,103 genes with consolidated expression in BRC, BRN, and BRE groups. We identified 472 and 207 mRNAs whose expression was modified in SOL and VL from BRC group, respectively. Further clustering analysis, identifying relevant biological mechanisms and pathways, reported five main subclusters. Three are composed of upregulated mRNAs involved mainly in nucleic acid and protein metabolism, and two made up of downregulated transcripts encoding components involved in energy metabolism. Exercise countermeasure demonstrated drastic compensatory effects, decreasing the number of differentially expressed mRNAs by 89 and 96% in SOL and VL, respectively. In contrast, nutrition countermeasure had moderate effects and decreased the number of differentially-expressed transcripts by 40 and 25% in SOL and VL. Together, these data present a systematic, global and comprehensive view of the adaptive response of female muscle to long-term atrophy.

High liver iron content is a risk factor for developing hepatocellular carcinoma (HCC). However, HCC cells are always iron-poor. Therefore, an association between hepatocyte iron storage capacity and differentiation is suggested. To characterize biological processes involved in iron loading capacity, we used a cDNA microarray to study the differentiation of the human HepaRG cell line, from undifferentiated proliferative cells to hepatocyte differentiated cells. We were able to identify genes modulated along HepaRG differentiation, leading us to propose new genes not previously associated with HCC. Moreover, using Gene Ontology annotations, we demonstrated that HepaRG hepatocyte iron loading capacity occurred both with the repression of genes involved in cell motility, signal transduction, and biosynthesis and with the appearance of genes linked to lipid metabolism and immune response. These results provide new insights in the understanding of the relationship between iron and hepatocyte differentiation during iron-related hepatic diseases.

Gene expression profiling studies in human diseases have allowed better understanding of pathophysiological processes. In addition, they may lead to the development of new clinical tools to improve diagnosis and prognosis of patients. Most of these studies have been successfully performed for human cancers. Inspired by these results, researchers in the cardiovascular field have also started using large-scale transcriptional analysis to better understand and classify human cardiovascular disease. Here we provide an overview of the literature revealing new cardiac disease markers and encouraging results for further development of the expression profiling strategy for future clinical applications in cardiology.

AIMS:This study aimed to investigate whether a molecular profiling approach should be pursued for the classification of heart failure patients.METHODS AND RESULTS:Applying a subtraction strategy we created a cDNA library consisting of cardiac- and heart failure-relevant clones that were used to construct dedicated cDNA microarrays. We measured relative expression levels of the corresponding genes in left ventricle tissue from 17 patients (15 failing hearts and 2 nonfailing hearts). Significance analysis of microarrays was used to select 159 genes that distinguished between all patients. Two-way hierarchical clustering of the 17 patients and the 159 selected genes led to the identification of three major subgroups of patients, each with a specific molecular portrait. The two nonfailing hearts clustered closely together. Interestingly, our classification of patients based on their molecular portraits did not correspond to an identified etiological classification. Remarkably, patients with the highest medical urgency status (United Network for Organ Sharing, Status 1A) clustered together.CONCLUSION:With this pilot feasibility study we demonstrated a novel classification of end-stage heart failure patients, which encourages further development of this approach in prospective studies on heart failure patients at earlier stages of the disease.

Background-The basis for the unique effectiveness of long-term amiodarone treatment on cardiac arrhythmias is incompletely understood. The present study investigated the pharmacogenomic profile of amiodarone on genes encoding ion-channel subunits.Methods and Results-Adult male mice were treated for 6 weeks with vehicle or oral amiodarone at 30, 90, or 180 mg kg(-1).d(-1). Plasma and myocardial levels of amiodarone and N-desethylamiodarone increased dose-dependently, reaching therapeutic ranges observed in human. Plasma triiodothyronine levels decreased, whereas reverse triiodothyronine levels increased in amiodarone-treated animals. In ECG recordings, amiodarone dose-dependently prolonged the RR, PR, QRS, and corrected QT intervals. Specific microarrays containing probes for the complete ion-channel repertoire (IonChips) and real-time reverse transcription-polymerase chain reaction experiments demonstrated that amiodarone induced a dose-dependent remodeling in multiple ion-channel subunits. Genes encoding Na+ (SCN4A, SCN5A, SCN1B), connexin (GJA1), Ca2+ (CaCNA1C), and K+ channels (KCNA5, KCNB1, KCND2) were downregulated. In patch-clamp experiments, lower expression of K+ and Na+ channel genes was associated with decreased I-to,I-f, I-K,I-slow, and I-Na currents. Inversely, other K+ channel alpha- and beta-subunits, such as KCNA4, KCNK1, KCNAB1, and KCNE3, were upregulated.Conclusions-Long-term amiodarone treatment induces a dose-dependent remodeling of ion-channel expression that is correlated with the cardiac electrophysiologic effects of the drug. This profile cannot be attributed solely to the amiodarone-induced cardiac hypothyroidism syndrome. Thus, in addition to the direct effect of the drug on membrane proteins, part of the therapeutic action of long-term amiodarone treatment is likely related to its effect on ion-channel transcripts.

We propose a freely accessible web-based pipeline, which processes raw microarray scan data to obtain experimentally consolidated gene expression values. The tool MADSCAN, which stands for MicroArray Data Suites of Computed ANalysis, makes a practical choice among the numerous methods available for filtering, normalizing and scaling of raw microarray expression data in a dynamic and automatic way. Different statistical methods have been adapted to extract reliable information from replicate gene spots as well as from replicate microarrays for each biological situation under study. A carefully constructed experimental design thus allows to detect outlying expression values and to identify statistically significant expression values, together with a list of quality controls with proposed threshold values. The integrated processing procedure described here, based on multiple measurements per gene, is decisive for reliably monitoring subtle gene expression changes typical for most biological events.

Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits ("IonChips") were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I(to,f) and I(K,slow) densities, and increased I(Ks) density in hypothyroid myocytes. In addition to effects on K+ channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the alpha1C Ca2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na+, Cl-, and inwardly rectifying K+ channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel alpha- and beta-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.