Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptional analysis of genes regulating the mitochondrial dNTP pool in muscle cells.

ABSTRACT: Mutations in genes involved in dNTP metabolism can lead to tissue-specific mitochondrial depletion syndromes (MDS), likely because the expression of key enzymes is reduced to critical levels in post mitotic cells. Our goal was to establish an in vitro skeletal muscle cell model to study the muscle specificity of MDS associated with mitochondrial dNTP pool imbalance. We performed a comprehensive analysis at the mRNA level of enzymes and transporters responsible for dNTP pool imbalance in muscle cells in vitro and in vivo. Agilent Mouse Oligo Arrays 4x44K were utilized to examine expression levels in proliferating and differentiated C2C12 cells as well as in the mouse EDL (fast glycolytic) and soleus (slow oxidative) muscles. The comparison of mRNA expression profiles supports the reliability of our in vitro cell system. Proliferating mouse C2C12 myoblasts were collected at about 50 percent confluence. Myoblasts were then induced to differentiate in vitro into myotubes that were harvested after 96 hours and further purified to reduce the contribution of mononucleated cells present in the culture. Gene expression in C2C12 myoblasts and myotubes was compared with fully differentiated muscle fibers in vivo. To this aim, the extensor digitorum longus (EDL) and soleus hind limb muscles were isolated from adult CD1 mice. These muscles were selected because they have different metabolic (glycolytic vs. oxidative) and twitching (fast vs. slow) properties. Triplicate total RNA samples were submitted to gene expression profiling.

ORGANISM(S): Mus musculus

SUBMITTER: Gerolamo Lanfranchi

PROVIDER: E-GEOD-41877 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Synthesis of mitochondrial DNA precursors during myogenesis, an analysis in purified C2C12 myotubes.

Frangini Miriam M Franzolin Elisa E Chemello Francesco F Laveder Paolo P Romualdi Chiara C Bianchi Vera V Rampazzo Chiara C

The Journal of biological chemistry 20130107 8

During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has bee ...[more]

PMID: 23297407

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Project description:To provide insights into the role of Ankrd2 in the pathways controlling myogenic differentiation, the gene networks that are disturbed in Ankrd2 knockout or overexpressing primary cells were explored during differentiation. Ankrd2 plays an important role in myogenic differentiation by modulating the activity of its interacting partners (i.e p53). Since Ankrd2 binds to numerous transcription factors many signaling pathways remained to be explored to determine the functional significance of crosstalk between Ankrd2 and the activation of transcriptional programmes that regulate muscle remodeling. In contrast to previous gene expression studies in which we and others have studied the effect of Ankrd2 silencing in mouse differentiating C2C12 cells (Series GSE7542) and human differentiated CHQ5B muscle cells (Belgrano et al., 2011), we here used an in vivo experimental model in which the expression of Ankrd2 was completely abrogated. Furthermore, since Ankrd2 is known to be strongly upregulated in response to various stress stimuli, we studied the effect of Ankrd2 rescue and acute overexpression in KO and WT cells by adenoviral infection 20 hours prior to collection of cells. The pathways that change significantly are involved in distinct inflammatory response phases having NF-kB as a central node. Based on these results Ankrd2 is emerging for the first time as a repressor of immune and inflammatory responses. Gene expression profiling experiments have been performed using the SurePrint G3 Mouse 8x60K array by Agilent with 8 subarrays, each containing 39,430 60mer oligonucleotide probes for Entrez Gene RNAs, 16,251 for lincRNAs, 96 x 10 control probes, and 32 x 10 spike-in control probes. The Agilent platforms with one-color approach were selected to make possible the all-to all experiments comparison. The animal model, created and characterized in the laboratory of Dr.ML Bang, directed at knocking out Ankrd2 function was used. The adenovirus expressing Ankrd2-HA was produced using the AdEasy strategy (Agilent Technologies). Primary muscle cultures derived from wild type (WT) and Ankrd2-knockout (KO) mice infected with adenovirus expressing Ankrd2 or control GFP were collected at three different stages: proliferating, fusing and differentiated myoblasts. Three biological replicates per condition were included and a total of 36 microarray experiments were thus performed. First, the over-expression of Ankrd2 was checked by Western blot analysis. Then RNA was extracted from each sample and the relative transcriptional profiles were carried out using updated microarrays from Agilent technologies. Information from probe features was extracted from microarray scan data with the Feature Extraction Software (Agilent). Only arrays with at least 8/9 quality control metrics in range were used for the following data analyses. Intra-array normalization was performed with the Feature Extraction Software. Quantile inter-arrays normalization was performed using the Expander software (Sharan et al., 2003).

Dataset Information

Transcriptional analysis of genes regulating the mitochondrial dNTP pool in muscle cells.

Publications

Synthesis of mitochondrial DNA precursors during myogenesis, an analysis in purified C2C12 myotubes.

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