DYNAMIC GENE AND PROTEIN EXPRESSION DURING MYOBLAST DIFFERENTIATION IN VITRO

INTRODUCTION: The differentiation of myoblasts into skeletal muscle is accompanied by drastic changes of cellular morphology correlated to the appearance of contractile structures and multinucleated myotubes. Although significant progress has been made in defining biological mechanisms governing myogenesis, several biochemical and molecular events still remain to be elucidated. In this study, differentially expressed genes and proteins in the myogenesis of skeletal myoblasts were investigated in the C2C12 skeletal muscle murine cell line, a useful model to study myogenesis in vitro. MATERIALS AND METHODS: Mouse C2C12 myoblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2mM glutamine, 1% antibiotics, 0.5% anti-mycoplasma and 25mM Hepes pH7.5 at 37C° and 5%CO2. To induce myogenic differentiation, cells grown at 100% confluence were transferred in DMEM supplemented with 1%FBS, and analysed at 0, 3, 5, 7 and 9 days after differentiation induction. They have been observed with a Nikon Reverted Microscope (MR) and photographed with a digital Nikon system (1). The differential display reverse transcription (DDRT)-PCR agarose gel method (2) was used to identify genes differentially expressed during myoblast differentiation in C2C12 cell line. Northern blot assays were performed on 15 g of total RNA extracted from five stages of the cellular differentiation process. 2D-PAGE analysis (3) was carried out over a 10-day time span on differentiating C2C12 myoblasts. Both analytical (silver stained) and semi-preparative (Brilliant Blue Colloidal stained) runs were carried out. Image analysis was performed using the Melanie 3 software. Protein identification was achieved utilizing a nano ESI-Q-TOF mass spectrometer. RESULTS: Undifferentiated cells, when observed by MR, appeared flattened, non confluent, star-shaped or fusiform. Their size was 20-80 µm and one central nucleus with numerous nucleoli, was visible. Occasional rounding cells were correlated with different mitotic stages. Their organization underwent to deep changements during differentiation. They progressively indeed elongated and, over a persisting myoblast population, a number of myotubes appeared. These are multinucleated, frequently y-shaped, 100-600 µm structures showing up to 20 nuclei and the progressive assembling of contractile structures. From 16 cDNA fingerprints obtained by the DDRT-PCR approach, 20 amplicons with apparent differential expression were identified and cloned. Fifteen clones showed significant similarity to known proteins that are involved in cell division, like the MAP Kinase 3, transport across membranes, intermediary metabolism and adhesion proteins, like the alpha parvin, including about 30% of the total number of genes, with unknown functions. Northern blot analyses confirmed that few cDNAs were differentially expressed during the myoblast differentiation. In particular, the Northern assay showed a high expression level of the kinase 3 at the early stage of the cells differentiation, a higher expression level of the integral protein transcript at the final differentiation phase and a constant presence of the alpha parvin as well as the others identified genes during all myogenesis process. These data have been supported by 2D-PAGE analysis. Several analytical two dimensional electrophoresis runs on differentiating C2C12 myoblasts were performed and the electropherograms evidenced an appreciably different protein expression level related to the differentiation stage. In particular, the careful comparison of the 2-DE maps and the evaluation of different parameters, such as normalized volume and normalized optical density, revealed that the expression level of several proteins linearly increases until 7 days of the differentiation. Concurrently an increase of the number of proteins has been revealed. Moreover, the overlapping and the further matching of the electropherograms evidenced specific spots for each differentiating stage. To gain useful information concerning the mechanisms governing myoblast differentiation the MS-identification of such proteins is currently under study. Further applications of real time PCR will allow an accurate quantification analysis of their expression level at each step of the myogenesis process supporting successive study on the gene regulation.