Akt1 and Akt2 Regulate Human Skeletal Myoblast Differentiation

Abstract

While considerable research to date has examined the roles of Akt isoforms in skeletal muscle development and regeneration in mice, there is surprisingly little data regarding Akt isoforms in human skeletal muscle development. To determine the extent to which observations in mice extend to those in human skeletal muscle tissue, we performed studies using differentiating primary human skeletal myoblasts. Primary human myoblasts were allowed to differentiate for 72 hours following seeding, and RNA was analyzed for transcript abundances of AKT1, AKT2, and AKT3. We found that AKT2 transcripts were ~2.5‐ to 3‐fold more abundant than AKT1 and AKT3 transcripts throughout the period of differentiation (P<0.001). There were no differences between expression of AKT1 or AKT3 across time or relative to each other. Myoblasts were next transduced with vectors encoding cDNA for wild‐type Akt1, wild type Akt2, kinase‐dead Akt1 (K179M), kinase‐dead Akt2 (K181M), or vector control. Expression of KD‐Akt2, but not expression of KD‐Akt1, reduced myogenin protein by ~41% after 24 hours differentiation (P<0.05) as determined by Western blot. Following seventy‐two hours of differentiation, myosin heavy chain (MyHC) immunoreactivity was reduced by ~48% in cells expressing kinase‐dead Akt1 (P<0.05), and by ~63% in cells expressing kinase‐dead Akt2 (P<0.01). Cells simultaneously expressing KD‐Akt1 and KD‐Akt2 showed an ~90% reduction in MyHC abundance as compared to control cells, suggesting that contributions of individual Akt isoforms may be additive. Together, these data indicate that both Akt1 and Akt2 regulate differentiation of primary human skeletal myoblasts.