Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of different types of skeletal muscles from wild type and alpha tropomyosin transgenic slow mice to investigate the molecular mechanisms implicated in the mouse model of nemaline myopathy.

ABSTRACT: The aim of this study was to investigate the molecular mechanisms implicated in this mouse model of nemaline myopathy, and to further compare the molecular disease response in different skeletal muscles. For this purpose, snap frozen skeletla muscle specimens from wild type and transgenic for alpha tropomyosin slow mice were studied. Five different muscle types were used (diaphragm, plantaris, extensor digitorum longus, tibialis anterior, gastrocnemus). Mice were sacrificed between 7 and 10 months. RNA pools from 3-5 animals were created and each pool was hybridized to a U74Av2 Affymetrix GeneChip. Datasets from 36 GeneChips were included in this study. Experiment Overall Design: 36 skeletal mouse muscle RNA pools were used, from 5 different skeletal muscles, in two different conditions (wild type and transgenic)

ORGANISM(S): Mus musculus

DISEASE(S): normal

SUBMITTER: Despina Sanoudou

PROVIDER: E-GEOD-3384 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Skeletal muscle repair in a mouse model of nemaline myopathy.

Sanoudou Despina D Corbett Mark A MA Han Mei M Ghoddusi Majid M Nguyen Mai-Anh T MA Vlahovich Nicole N Hardeman Edna C EC Beggs Alan H AH

Human molecular genetics 20060728 17

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five diff ...[more]

PMID: 16877500

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Project description:In skeletal muscle, the pattern of electrical activity regulates the expression of proteins involved in synaptic transmission, contraction and metabolism. Disruptions in electrical activity, resulting from prolonged bed-rest, cast-immobilization or trauma, inevitably lead to muscle atrophy. The mechanisms that regulate muscle atrophy are poorly understood, but it seems likely that changes in gene expression play a key role in initiating and maintaining a muscle atrophy program. Previously, we found that Runx1, a transcription factor previously termed AML1, was substantially induced in muscle following denervation. More recently, we sought to determine whether this increase in Runx1 expression may be causally related to the morphological changes in skeletal muscle that accompany muscle disuse, notably muscle atrophy. We found that Runx1 is indeed required to sustain muscle and to minimize atrophy following denervation. Experiments described here are designed to identify the genes that are regulated by Runx1 in skeletal muscle with the particular goal of identifying genes that regulate muscle atrophy. We propose to use microarray analysis to identify genes, expressed in skeletal muscle, that are mis-regulated in mice lacking Runx1. We inactivated runx1 selectively in skeletal muscle and found that denervated myofibers in mutant mice atrophy far more (90% atrophy) than in wild-type mice (30% atrophy). We therefore reason that Runx1 activates and/or represses genes that are required to sustain muscle and to minimize atrophy. We generated MCK::cre; runx1f/- and runx1f/- control mice. In normal mice, an increase in runx1 expression is detected by two days after denervation and is maximal by five days after denervation. Muscle atrophy is first evident between one and two weeks after denervation. As we wish to avoid detecting global changes in gene expression that are associated with late stages of muscle atrophy, we plan to denervate muscle for three or five days and to compare gene expression in dissected innervated and denervated muscles from mutant and control mice. We will generate thirty samples for comparison-5 replicates per condition: Samples 1-3 from runx1f/- control mice. (1) innervated tibialis anterior muscles (TA); (2) 3-day-denervated TA; (3) 5-day-denervated TA. Samples 4-6 from MCK::cre; runx1f/- mice. (4) innervated TA; (5) 3-day-denervated TA; (6) 5-day-denervated TA. We obtain sufficient total RNA (10 micrograms) from each dissected muscle to avoid pooling samples. We will analyze adult mice of the same age (~six weeks after birth; most will be littermates) and sex-male. It is difficult to anticipate how many genes will be identified in this screen, as few target genes for Runx1 have been identified in any cell type and none in skeletal muscle. Moreover, although we would prefer to focus our attention on genes that are strongly dependent upon Runx1 expression (e.g. more than 5-fold difference in expression in wild-type and mutant mice), we do not know the extent to which target gene expression will depend upon Runx1. For these reasons, in these experiments, we will analyze expression from five âidenticalâ samples, so that we can be confident that even small (e.g. three-fold) differences in expression can be reliably determined. Importantly, in order to confirm results obtained from the microarray data, we will use other assays (RNase protection) to measure RNA expression of candidate genes in innervated and denervated muscles of wild-type and mutant mice.

Project description:In this study, in order to minimize the genetic variability of muscle samples we have used two approaches. First, we have analyzed the gene expression profile from a single skeletal muscle, the medial gastrocnemius (MG), and not from a pool of different muscles which could have different expression profiles. Second, we have performed the temporal gene expression profiling by extracting the MG muscles of the same individual from the both legs at two different times to minimize the inter-individual genetic variability. The MG muscle represent an excellent candidate for biopsy due to its easy accessible by surgery and also because its biopsy is well tolerated by the animals allowing us to perform another later biopsy in the other leg to obtain two MG samples of the same individual at two different times. Moreover, MG muscle is composed of approximately 20-30% type I (red) fibers and 70-80% type II (white) fibers {Ariano MA, 1973}, {Simard C, 1988}, {Zhan WZ, 1992} and therefore is more representative of skeletal muscle tissue in general than a muscle composed exclusively by red or white fibers. The transcript expression profiles in MG muscles from mdx and wild-type mice were analyzed at 3 weeks, 1.5 months and 3 months of life by using the 430 2.0 gene chips from Affymetrix (n=3 for each condition). The differentially expressed transcripts which showed differences ?1.5-fold were obtained by performing three different comparisons: 1) genes differentially expressed in mdx compared with controls at each point in time (additional file 1); 2) temporal analysis of the genes differentially expressed in mdx mice between the three points in time also compared with the variations in control mice (additional file 2); and 3) temporal analysis of the genes differentially expressed in control mice between the three points in time also compared with the variations in mdx mice (additional file 3). The first comparison that we performed, by comparing the gene expression between mdx and control mice at every point in time, was similar to that performed in previous longitudinal studies {Porter JD, 2003}, {Rouger K, 2002}, {Turk R, 2005}. However, the other two comparisons were directed to elucidate the genes that are varying throughout the period of time analyzed in every mice strain, and therefore we obtained on the one hand the genes that vary in mdx mice but not in wild-type, and on the other hand the genes that vary in control animals but remain unchanged in mdx mice between the times analyzed. To present the results in a more comprehensive form, all the genes were classified in seven different categories: Cell adhesion & extracellular matrix; Proteolysis; Muscle structure & regeneration; Inflammation & immune response; Cell signaling & cell communication; Metabolism; and Others/unknown. The resulting genes from our study were classified in their functional categories using information from Affymetrix (www.affymetrix.com) and from the Gene Ontology database accessible in the Jackson Laboratory Mouse Genome Informatics website (www.informatics.jax.org).

Dataset Information

Transcription profiling of different types of skeletal muscles from wild type and alpha tropomyosin transgenic slow mice to investigate the molecular mechanisms implicated in the mouse model of nemaline myopathy.

Publications

Skeletal muscle repair in a mouse model of nemaline myopathy.

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