Role of integrin α7β1 signaling in myoblast differentiation on aligned polydioxanone scaffolds

The aligned structural environment in skeletal muscle is believed to be a crucial component in functional muscle regeneration. Myotube formation is increased on aligned biomaterials, but we do not fully understand the mechanisms that direct this enhanced fusion. Previous studies indicate that the alpha 7 integrin subunit is upregulated during myoblast differentiation, suggesting that signaling via alpha 7 beta 1 mediates the effect of alignment. To test this hypothesis, we took advantage of an in vitro model using random and aligned polydioxanone (PDO) matrices and C2C12 myoblasts. We measured expression and production of myoblast markers: paired box-7 (Pax7), myogenic differentiation factor-1 (MyoD), myogenin (MyoG), myogenic factor-6 (Myf6), and myosin heavy chain (MyHC). To examine the role of alpha 7 beta 1 signaling, we measured expression and production of alpha 7, alpha 5, and beta 1 and myoblast markers in wild type cells and in cells silenced for alpha 7 and assessed effects of silencing on myogenic differentiation. Downstream signaling via ERK1/2 mitogen activated protein kinase (MAPK) was examined using a specific MEK1/2 inhibitor. Alignment increased mRNAs and protein for early (MyoD) and late (MyoG, MyHC) myoblast markers in comparison to non-aligned matrices, and these levels corresponded with increased alpha 7 protein. alpha 7-silencing reduced MyoG and MyHC protein in cells cultured on tissue culture polystyrene and aligned PDO matrices compared to wild type cells. Inhibition of ERK1/2 blocked effects of alignment. These data suggest that alignment regulates myogenic differentiation via alpha 7 beta 1 integrin signaling and ERK1/2 mediated gene expression. Statement of Significance Muscle regeneration in severe muscle injuries is complex, requiring a sequence of events to promote healing and not fibrosis. Aligned biomaterials that recapitulate muscle environments hold potential to facilitate regeneration, but it is important to understand cell-substrate signaling to form functional muscle. A critical component of muscle signaling is integrin alpha 7 beta 1, where mice lacking alpha 7 exhibit a dystrophic phenotype and impaired regeneration. Here, we report the role of alpha 7 beta 1 signaling in myoblast differentiation on aligned biomaterials. alpha 7-silenced myoblasts were found to regulate myogenic differentiation and demonstrate defective fusion. Our data shows reduced levels of myogenin and myosin heavy chain protein, while MyoD remains unchanged. These results support the hypothesis that alpha 7 beta 1 signaling plays a role in substrate-dependent tissue engineering strategies. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.