Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse denervated muscle from runx1f mutants vs. controls

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Elizabeth Salomon

PROVIDER: E-GEOD-4411 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 “identical” 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. Keywords: other

Project description:Background: Skeletal muscle crucially depends on motor innervation, and, when damaged, on the resident muscle stem cells (MuSCs). However, the role and function of MuSCs in the context of denervation remains poorly understood. Methods: Alterations of MuSCs and their myofiber niche after denervation were investigated in a surgery-based mouse model of unilateral sciatic nerve transection. FACS-isolated MuSCs were subjected to RNA-sequencing and mass spectrometry for the analysis of intrinsic changes after denervation and in vivo assays, such as Cardiotoxin-induced muscle injury or MuSC transplantation, were performed to assess MuSC functions after denervation. Bioinformatic and histological analyses were conducted to further examine MuSCs and their myofiber niche after denervation. Results: Muscle cross section analysis revealed a significant increase in Pax7 (p-value= 0.0441), Pax7/Ki67 (p-value= 0.0023), MyoD (p-value= 0.0016) and Myog (p-value= 0.0057) positive cells after denervation, illustrating a break of quiescence and commitment to the myogenic lineage. An Omics approach showed profound intrinsic alterations on the mRNA (2613 differentially expressed genes, p-value <0.05) and protein (1096 differentially abundant proteins, q-value <0.05) level of MuSCs 21 days after denervation. Skeletal muscle injury together with denervation surgery caused deregulated regeneration, indicated by the reduced number of proliferating MuSCs and sustained high levels of developmental myosin heavy chain (Sham: 1 % vs DEN: 40 % of all myofibers), at 21 days post-surgery. In a transplantation assay, MuSCs from a denervated host were still able to engraft and fuse to form new myofibers, irrespective of the innervation status of the recipient muscle. Analysis of myofibers revealed not only massive changes in the expression profile (10492 differentially expressed genes, p-value <0.05) after denervation, but it was also shown that secretion of Opn and Tgfb1 from denervated myofibers was increased 30-fold and 6000-fold, respectively. Bioinformatic analyses indicated strong upregulation of gene expression of the transcription factor Junb in MuSCs from denervated muscles (log2 fold change = 3.27). Of interest, Tgfb1 recombinant protein was able to induce Junb gene expression in vitro, demonstrating that myofiber-secreted ligands can induce gene expression changes in MuSCs, which might result in the phenotypes observed after denervation. Conclusion: Skeletal muscle denervation is altering myofiber secretion, causing MuSC activation and profound intrinsic changes, leading to reduced regenerative capacity. As MuSCs possess a remarkable regenerative potential, they might represent a promising target for novel treatment options for neuromuscular disorders and peripheral nerve injuries.

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Transcription profiling of mouse denervated muscle from runx1f mutants vs. controls

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