title: Role of integrin α7β1 signaling in myoblast differentiation on aligned polydioxanone scaffolds.
abstract: 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 α7 integrin subunit is upregulated during myoblast differentiation, suggesting that signaling via α7β1 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 α7β1 signaling, we measured expression and production of α7, α5, and β1 and myoblast markers in wild type cells and in cells silenced for α7 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 α7 protein. α7-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 α7β1 integrin signaling and ERK1/2 mediated gene expression.
  
  
  
  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 α7β1, where mice lacking α7 exhibit a dystrophic phenotype and impaired regeneration. Here, we report the role of α7β1 signaling in myoblast differentiation on aligned biomaterials. α7-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 α7β1 signaling plays a role in substrate-dependent tissue engineering strategies.
authors.affiliation: Physical Medicine and Rehabilitation Service, Hunter Holmes McGuire VA Medical Center, Richmond, VA, United States; Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McClure
authors.affiliation: Department of Biology, School of Integrated Life Sciences, Virginia Commonwealth University, Richmond, VA, United States.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Clark
authors.affiliation: Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, United States.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Hyzy
authors.affiliation: Physical Medicine and Rehabilitation Service, Hunter Holmes McGuire VA Medical Center, Richmond, VA, United States; Department of Biochemistry, School of Medicine, Virginia Commonwealth University, Richmond, VA, United States.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Chalfant
authors.affiliation: Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, United States.
authors.forename: Rene
authors.initials: R
authors.lastname: Olivares-Navarrete
authors.affiliation: Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, United States; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, United States. Electronic address: bboyan@vcu.edu.
authors.forename: Barbara D
authors.initials: BD
authors.lastname: Boyan
authors.affiliation: Department of Biomedical Engineering, School of Engineering, Virginia Commonwealth University, Richmond, VA, United States; Department of Periodontics, University of Texas Health Sciences Center at San Antonio, San Antonio, TX, United States.
authors.forename: Zvi
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D002454:Cell Differentiation; D002460:Cell Line; D005786:Gene Expression Regulation; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D009124:Muscle Proteins / Q000096:biosynthesis; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D016687:Polydioxanone / Q000737:chemistry*; D015398:Signal Transduction; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D000943:Antigens, Differentiation; D016023:Integrins; D009124:Muscle Proteins; D016687:Polydioxanone; C082908:integrin alpha7beta1
keywords: Biomimetic material; Muscle; Myoblast differentiation; Polydioxanone fiber alignment; Surface topography