Identification of a Beneficial Role of Proteasome-Mediated Protein Degradation in the Differentiation of Bovine Myoblasts into Myotubes

The objective of this study was to determine the role of proteasome-mediated protein degradation in the differentiation of bovine myoblasts into myotubes. This objective stemmed from an earlier, unexpected observation that the ubiquitin-proteasome pathway in bovine myoblasts was upregulated during their differentiation into myotubes. Satellite cells, the myogenic progenitor cells in adult skeletal muscle, were isolated from 5 Angus or Angus crossbred steers (experimental unit) and were expanded as myoblasts in growth medium. Myoblasts were then induced to differentiate into myotubes in differentiation medium in the presence or absence of 5 μM lactacystin, a specific inhibitor of the 20S proteasome, for 6, 12, 24, 48, and 72 h. The differentiation status of myoblasts was assessed by reverse transcription-quantitative PCR of myosin heavy chain 3 (MYH3), muscle creatine kinase (CKM), and Myomaker (TMEM8) mRNAs, markers of myotubes. Compared to control myoblasts, lactacystin-treated myoblasts expressed less MYH3, CKM, and TMEM8 mRNAs at 24, 48, and 72 h of differentiation (P < 0.01). These differences indicated that lactacystin inhibited the differentiation of bovine myoblasts into myotubes. Differentiation of myoblast into myotubes is primarily controlled by myogenin at the transcriptional level. The DNA binding and transcriptional activity of myogenin can be inhibited by the inhibitor of DNA-binding 1 (ID1) protein. Based on western blot analyses, ID1 protein expression was decreased (P < 0.05) during bovine myoblast differentiation into myotubes, and this decrease was reversed (P < 0.05) by including lactacystin in the differentiation medium. Collectively, these results suggest a beneficial role of proteasome-mediated protein degradation in bovine myoblast differentiation into myotubes, and this role may involve the degradation of the ID1 protein.