Yhusi Karina Riskawati, Chuang-Yu Lin, Akira Niwa, Hsi Chang
Skeletal muscle diseases often exhibit fiber-type-specific characteristics and pose substantial clinical challenges, necessitating innovative therapies. The extracellular matrix (ECM) plays a pivotal role in muscle physiology and regeneration, influencing cell differentiation. However, its specific role and mechanisms influencing muscle fiber type specification remain insufficiently understood. In this study, C2C12GFP myoblasts were differentiated into myofibers on plates coated with fibronectin, Collagen I, and Geltrex™. Differentiation occurred successfully across all ECM substrates, resulting in myofiber formation. Quantitative polymerase chain reaction (qPCR) analysis confirmed myogenic marker expression patterns, indicating decreased Pax7 and increased Myog levels by day 7. Protein analysis through Western blot and immunofluorescence assays along with transcriptomic profiling through RNA sequencing consistently indicated that Collagen I promoted slow-type fibers development, as evidenced by increased slow myofiber protein expression and the upregulation of slow fiber-associated genes, potentially mediated by pathways involving calcineurin/NFAT, MEF2, MYOD, AMPK, PI3K/AKT, and ERK1. In contrast, fibronectin and Geltrex™ led to fast-type fiber development, with elevated fast-type fiber protein levels and upregulation of fast fiber-associated genes, possibly through activation of HIF1A, FOXO1, NFKB, and ERK2. These findings elucidate ECM-mediated muscle fiber type differentiation mechanisms, informing future targeted therapies for muscle regeneration. © 2025 by the authors.
International Ph.D. Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, 110301, Taiwan; Department of Physiology, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia; Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, 807378, Taiwan; Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan; Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 110301, Taiwan; Department of Pediatrics, Taipei Medical University Hospital, Taipei, 110301, Taiwan
