Inducible Overexpression of p21Cip1 in Myotubes Promotes Increases in Protein Synthesis and Myotube Hypertrophy

Abstract

PURPOSE: p21Cip1 is classically defined as a cyclin-dependent kinase inhibitor that promotes satellite cell differentiation within skeletal muscle. However, sparse literature has demonstrated that mechanical loading can elicit robust (i.e. ~30-50+-fold) increases in skeletal muscle p21Cip1 mRNA expression patterns up to 6 hours post-exercise; this being an event which precedes satellite cell activity. Herein we tested whether the inducible over-expression of p21Cip1 promotes alterations in muscle protein synthesis (MPS) and hypertrophy in post-differentiated myotubes. METHODS: Briefly, the p21Cip1 gene was cloned into the pINDUCER vector, which is turned on by doxycycline treatment, and a stable C2C12 p21Cip1-inducible (p21-IND) cell line was established. Empty vector C2C12 clones (EV) served as the control condition. Following 7 d of differentiation, the p21-IND and EV lines were treated for 4 days with doxycycline. RESULTS: An 86% overexpression of p21Cip1 mRNA was confirmed in p21-IND versus EV myotubes with RT-PCR (p < 0.05). p21-IND myotubes exhibited 2.5-fold greater MPS rates (p < 0.05) and a 2.2-fold greater increase in myotube size (p < 0.05) compared to EV myotubes. Select differentiation markers (i.e., MyoD mRNA and myogenin mRNA) did not differ between cell lines. Interestingly, pre-47S rRNA trended to increase in p21-IND myotubes compared to EV myotubes (1.6-fold, p = 0.09). CONCLUSIONS: These data suggest that p21Cip1 may act in post-mitotic skeletal muscle fibers to increase translational capacity and/or efficiency, thereby promoting skeletal muscle hypertrophy.