Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of rat extraocular muscle and extensor digitorum longus samples from animals aged 6, 18 and 30 months

ABSTRACT: The fast and constant activity of the extraocular muscles (EOMs) impose mechanical and metabolic stresses not typically seen in limb skeletal muscles. These functional properties may explain why EOMs seem to age at a faster rate than other skeletal muscles. Using high-density cDNA microarrays, this study investigated the gene expression profile of EOMs and extensor digitorum longus muscles (EDL) of Fischer 344/Brown Norway F1 hybrid rats at 6-, 18- and 30-months of age. At 6-mo, 705 genes and expressed sequence tags (ESTs) were differentially expressed in EOMs (436 up, 269 down). Overall, the EOM profile at this age was mostly consistent with the increased expression of fetal, developmental and EOM-specific myosin isoforms, enzymes involved in glycolysis and TCA cycle, and ion transporters and pumps, confirming the notion that EOM may represent a distinct muscle group (PNAS 98:12062, 2001). Interestingly, at 18-mo only 36 probes were significantly different in EOM (15 up, 21 down), most of them ESTs. However, at 30-mo EOMs had 655 differentially expressed genes and ESTs (480 up, 175 down). In this age group, the EOM expression profile reverted to a pattern similar to that found at 6-mo, with evidence of ongoing tissue remodeling and increased expression of antioxidant enzymes. These results indicate that the gene expression profile of EOM and EDL evolve differently throughout the lifespan. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 4 animals were combined into each RNA sample to decrease inter-subject variability. Biotinylated cRNA samples were hybridized to Affymetrix Rat Genome U34 gene chips (n=24 chips) described previously [McMullen et al. 2004]. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Comparisons the one-sided Wilcoxonâs signed rank test to estimate âincrease/no change/ decreaseâ difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 2.00 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Colleen McMullen

PROVIDER: E-GEOD-3309 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: To examine and characterize the expression profile of genes expressed at the neuromuscular junctions (NMJs) of extraocular muscles (EOMs) in comparison to the NMJs of tibialis anterior muscle (TA). Methods: Adult rat rectus EOMs and TAs were dissected, flash-frozen, serially sectioned and stained for acetylcholinesterase to identify NMJs. Approximately 6000 NMJs for EOM (EOMsyn) and 6000 NMJs for TA (TAsyn) and equal amounts of NMJ-free fiber regions (EOMfib, TAfib) and underlying myonuclei were captured using laser capture microdissection (LCM). RNA was isolated, processed and used for microarray-based expression profiling. Profiles were generated for genes differentially expressed at synaptic and non-synaptic regions of TA (TAsyn vs TAfib) and EOM (EOMsyn vs EOMfib) using a false discovery rate (FDR) of 5% as well as an 'interaction list' revealing the most significantly differentially expressed genes at an FDR of 1%. We validated the profiles by real-time quantitative reverse transcription-polymerase chain reaction (qPCR). Results: The regional transcriptomes associated with NMJ of EOMs and TAs were identified. We found 275 genes that were preferentially expressed in EOMsyn and 230 known transcripts that were preferentially expressed in TAsyn; 288 of the transcripts were common to both synapses; these included well-known, evolutionarily conserved, synaptic markers (e.g. nicotinic Acetylcholine receptor (ACHR) alpha and epsilon subunits, nestin) as well as a large number of novel genes. Conclusion: Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. Our definition of the synaptic transcriptome provides insight into the mechanism of formation and functioning of the unique synapses of EOM and their differential involvement in diseases noted in the EOM allotype. Tissue preparation: A total of 4 rats were killed by CO2 inhalation. The bony orbit was removed from the skull and opened at the lamina cribrosa. The globe with the four recti EOMs still attached was carefully dissected from the bony orbit. The eyeball with muscles was placed on cryomolds, covered with OCT tissue embedding medium (Tissue-Tek: Sakura Finetek, Tokyo, Japan) and flash-frozen in isopentane, cooled in liquid nitrogen and stored at -80 degreeC. The tibialis anterior (TA) muscles of all rats were dissected and frozen in the same way. The EOM and TA were then cut transversely into 10 um sections using a Microm HM 500 cryostat (Zeiss, Oberkochen, Germany), mounted on PEN (poly-ethylene-naphthalene) Membrane Slides (Arcturus) and refrozen immediately. Unfixed sections were stored at -80 degreeC until needed. Section staining: Sections for LCM were stained for acetylcholinesterase based on the method of Karnowsky and Roots to visualize NMJ. Palm microdissection: The PALM MicroBeam System was used for microdissection and for catapulting isolated tissue into a microfuge cap containing 80 ul RLT-Lysis Buffer (Quiagen). Approximately 1000 NMJ and equal amount of non-synaptic regions were collected for each muscle.

Project description:The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other skeletal muscles. Previously, we and others have shown that EOMs have a unique transcriptome and proteome. Here, we investigated the expression pattern of microRNAs (miRNAs) in EOM, as they may play a role in generating the unique EOM allotype. We screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. 74 miRNAs were found to be differentially regulated (p-value < 0.05) and 31 miRNAs (14 up-regulated and 17 down-regulated) were found to be differentially regulated at a signal strength > 500 including the muscle-specific miR-206, miR-1, miR-133a, miR-133b and miR-499. qPCR analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using co-transfection of precursor miRNAs (pre-miRNAs) along with reporter constructs containing the 3â-untranslated region (3âUTR) of their predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular make-up of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne's muscular dystrophy (DMD) and may assist in development of therapeutic strategies. Total RNA from four EOM and four TA tissue samples dissected from four adult male C57/Bl10 mice were used (TA served as control) to screen four LC Sciences microRNA Microarray chips. The chips contained microRNA sequences based on miRBase content 10.0 totalling 568 different miRNAs. Samples were labelled with Cy3 and Cy5 using dye-swap. Relative differences of miRNA expression was expressed as fold-changes EOM/TA, which were calculated after normalization across all four arrays.

Project description:Sarcopenia is the decreased muscle mass and weakness associated with aging and a major cause of morbidity and mortality in the elderly. To what extent non-locomotive muscles are susceptible to this condition is unclear. For example, age affects laryngeal function (ventilation, airway protective reflexes, swallowing and phonation). Age-related laryngeal dysfunction may be due to effects on its intrinsic muscles that have a unique phenotype: very small, mostly fast oxidative muscle fibers. For this study, we examined how age alters the functional characteristics and gene expression profile of posterior cricoarytenoid (PCA), an intrinsic laryngeal muscle. PCA muscles from Fischer 344-Brown Norway F1 hybrid rats (6 and 30 months of age) were used for cDNA microarrays, light and electron (EM) microscopy, and in vitro contractile function. Histological analyses demonstrated a ~40% increase in mean PCA fiber size and in the number of fibers with low myosin ATPase activity. There was also evidence of ragged-red fibers, a hallmark of mitochondrial dysfunction. In turn, mitochondrial volume density, determined by EM, was significantly higher in PCA muscles at 30 months (43% vs. 21% at 6 months). In vitro function showed a decrease in velocity of unloaded shortening at 30 months. Finally, cDNA microarrays demonstrated a transcriptome shift in PCA muscle with age. Gene classes with the largest changes were: signal transduction, transcription factors, and metabolic enzymes. These data demonstrate that PCA muscles are significantly altered by age. Moreover, the observed changes in muscle fiber size, mitochondrial content and gene expression profile suggest that the PCA response to age diverges from that seen in more typical skeletal muscles. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 4 animals were combined into each RNA sample to decrease inter-subject variability. Biotinylated cRNA samples were hybridized to Affymetrix Rat Genome U34 gene chips (n=9 chips) described previously [McMullen et al. 2004]. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all three independent replicates per group were considered for further analysis. Comparisons used the 6-mo transcriptome as the baseline and the one-sided Wilcoxonâs signed rank test to estimate âincrease/no change/ decreaseâ difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.70 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

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Transcription profiling of rat extraocular muscle and extensor digitorum longus samples from animals aged 6, 18 and 30 months

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