Enhancing Skeletal Muscle Fiber Types Transition through Substrate Coating Alteration in Myoblast Cell Culture

Skeletal muscle diseases often exhibit fiber type-specific characteristics and posing significant clinical challenges, such as Duchenne muscular dystrophy (DMD), sarcopenia, and volumetric muscle loss injuries. Current treatments are primarily palliative, highlighting the need for innovative therapies. The extracellular matrix (ECM) plays a pivotal role in muscle physiology and regeneration by influencing cell differentiation and repair. Understanding the mechanism of specific ECM coatings affect muscle fiber type differentiation in vitro could elicit the targeted therapies for muscle regeneration. In this study, C2C12GFP myoblasts were differentiated into myotubes on the plates coated with Fibronectin, Collagen I, and Geltrex™. The myoblasts differentiated successfully on all ECM substrates, forming myofiber. qPCR confirmed myogenic marker expression patterns, with decreased Pax7 and increased Myog by day 7. Protein analysis (Western blot and immunofluorescence) and transcriptome profiling (RNA-Sequencing) consistently demonstrated that Collagen I enhanced the development of slow-type fibers, evidenced by increased slow myotube protein expression and the upregulation of slow fiber-associated genes (e.g., Myh7, Myl2), potentially via Calcineurin/NFAT, MEF2, MYOD, AMPK, PI3K/AKT and ERK1 pathways. In contrast, Fibronectin and Geltrex™ favored fast-type fiber development, as shown by elevated fast-type fiber protein levels and upregulation of fast fiber-associated genes (e.g., Fyn, Hk2), possibly through HIF1A, FOXO1, NFKB and ERK2 activation.