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Abstract
Myostatin is small glycopeptide that is produced and secreted by skeletal muscle. It is a potent negative regulator of muscle growth that has been associated with conditions of frailty. In C2C12 cells, myostatin limits cell differentiation. Myostatin acts through activin receptor IIB, activin receptor-like kinase (ALK) and Smad transcription factors. microRNAs (miRNA) are short, 22 base pair nucleotides that bind to the 3′ UTR of target mRNA to repress translation or reduce mRNA stability. In the present study, expression in differentiating C2C12 cells of the myomiRs miR-1 and 133a were down-regulated following treatment with 1µg of recombinant myostatin at 1 d post-induction of differentiation while all myomiRs (miR-1, 133a/b and 206) were upregulated by SB431542, a potent ALK4/5/7 inhibitor which reduces Smad2 signaling, at 1 d and all, with the exception of miR-206, were upregulated by SB431542 at 3 d. The expression of the muscle-enriched miR-486 was greater following treatment with SB431542 but not altered by myostatin. Other highly expressed miRNAs in skeletal muscle, miR-23a/b and 145, were altered only at 1 d post-induction of differentiation. miR-27b responded differently to treatments at 1 d, where it was upregulated, as compared to 3 d, where it was downregulated. Neither myostatin nor SB431542 altered cell size or cell morphology. The data indicate that myostatin represses myomiR expression in differentiating C2C12 cells and that inhibition of Smad signaling with SB431542 can result in large changes in highly expressed miRNAs in differentiating myoblasts.
Highlights
Myostatin (GDF8) is a small, 12 kDa peptide member of the transforming growth factor-β (TGFβ) superfamily that is secreted by skeletal muscle and is a potent negative regulator of muscle growth [1]
We evaluated the effects of myostatin and/or the ALK4/5/7 inhibitor SB431542 on miRNA expression in differentiating mouse C2C12 myoblasts, which are widely employed in studies of the intracellular signals responsible for muscle atrophy and hypertrophy as well as myogenic differentiation
Examination of cells by brightfield microscopy revealed that fusion of myoblasts had not occurred under any of the conditions tested and that morphology was similar for cells treated with vehicle, myostatin or SB431543 (Fig. 1C-D)
Summary
Myostatin (GDF8) is a small, 12 kDa peptide member of the transforming growth factor-β (TGFβ) superfamily that is secreted by skeletal muscle and is a potent negative regulator of muscle growth [1]. Myostatin prevents muscle growth by suppressing hypertrophic signaling pathways and increasing transcription of genes that degrade skeletal muscle protein. [5] Myostatin signals through phosphorylation of Smad and Smad transcription factors. In the cytosol, phosphorylated Smad2/3 inhibit Akt, a major kinase that responds positively to exercise and growth factors, such as IGF-1, to increase protein synthesis and inhibit protein breakdown [7]. After phosphorylation by ALK 4 or 5, the Smad2/3 complex can bind Smad and translocate to the nucleus to increase transcription of genes, and partially activate major factors involved in muscle atrophy programs such as muscle atrophy Fbox (MAFbx) as seen with luciferase reporter studies [6]. SCI leads to rapid decreases in muscle mass below the area of injury [11,12,13] and diminished hypertrophic signaling [14].
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