Enhancing Skeletal Muscle Fibre Type Transition through Substrate Coating Alteration in C2C12 Myoblast Cell Culture

Background: Skeletal muscle disorders—including Duchenne muscular dystrophy, sarcopenia, and volumetric muscle loss (VML)—present major clinical challenges. Duchenne and sarcopenia involve fiber-type-specific vulnerabilities and poor regeneration, while VML causes irreversible loss of region-specific fiber populations. Current treatments cannot address these deficits, highlighting the need for precision regenerative solutions. Tissue engineering, particularly through the use of extracellular matrix (ECM) scaffolds, offers promise by influencing cell behavior and differentiation. However, how different ECM substrates direct muscle fiber-type specification—toward fast-twitch or slow-twitch phenotypes—remains unclear. This study examines the effects of various ECM substrates on C2C12 myoblast differentiation, aiming to identify ECM-based strategies for targeted muscle regeneration. Methods: C2C12 myoblasts expressing Green Fluorescent Protein (C2C12GFP) were cultured and induced to differentiate on three ECM coatings: Collagen I, Fibronectin, and Geltrex™. Myogenic progression was monitored via temporal expression of key myogenic regulatory factors (MRFs), assessed using quantitative PCR. Western blotting and immunofluorescence staining were employed to detect protein expression of slow and fast myosin heavy chain (MyHC) isoforms. To capture a broader picture of gene regulation, transcriptomic profiling via RNA sequencing was conducted, followed by qPCR validation of select markers. Comprehensive bioinformatic analyses—including Gene Ontology (GO), KEGG, Gene Set Enrichment Analysis (GSEA), MetaCore, and Ingenuity Pathway Analysis (IPA)—were performed to uncover signaling pathways implicated in ECM-mediated fiber-type transitions. Results: All ECM substrates supported successful myogenic differentiation, evidenced by downregulation of Pax7 and upregulation of Myogenin by day 7. Phenotypic outcomes varied by substrate: Collagen I significantly promoted the expression of slow-twitch fiber markers at both the protein and transcriptomic levels. This effect appeared to involve the activation of key regulatory pathways, including calcineurin/NFAT, MEF2, AMPK, PI3K/AKT, ERK1, and MYOD. In contrast, Fibronectin and Geltrex™ supported fast-twitch fiber differentiation, with enhanced expression of fast-type MyHC isoforms. These effects correlated with the activation of transcription factors and pathways such as HIF1A, FOXO1, NFKB, and ERK2. Each substrate generated a distinct molecular environment conducive to specific fiber-type outcomes. Conclusion: This study demonstrates that ECM substrate composition significantly influences muscle fiber-type specification in vitro. Collagen I favored the development of oxidative, slow-twitch fibers, while Fibronectin and Geltrex™ directed differentiation toward glycolytic, fast-twitch phenotypes. These effects were mediated through unique combinations of transcriptional regulators and intracellular signaling pathways. The findings provide new insight into the role of ECM components in muscle tissue engineering and suggest that substrate-based modulation could be a viable strategy to guide therapeutic muscle regeneration. By harnessing ECM-guided fiber-type specification, future regenerative approaches may be more precisely tailored to restore the functional properties of specific muscle groups affected by injury or disease.