Introduction Muscle functionality and adaptability depend on the precise specification of muscle fiber types, a process that directly influences contraction dynamics and overall performance. While the critical role of extracellular matrix (ECM) components in muscle development is recognized, the specific mechanisms through which ECM influences the differentiation of myoblasts into distinct muscle fiber types remain inadequately explored. This gap in knowledge, particularly regarding how different ECM cues guide the activation of myogenic regulatory factors (MRFs) and mediate fiber type-specific transitions, limits our understanding of muscle physiology and the development of targeted therapeutic interventions. Addressing this gap, our study delves into the modulation of C2C12 myoblast differentiation by ECM components present in Matrigel™, aiming to elucidate the intricate processes of muscle contraction regulation and fiber type specification. Through this investigation, we seek to contribute to the broader comprehension of muscle tissue engineering and the potential for ECM-based strategies in regenerative medicine.

Method C2C12-GFP myoblasts were differentiated on a Matrigel™ substrate, enabling the assessment of myotube formation. The expression patterns of key MRFs were evaluated via quantitative PCR (qPCR), while the expression of myosin heavy chain (MyHC) genes and proteins, indicative of fast and slow skeletal muscle fibers, was analyzed using Western blotting and immunocytochemistry. RNA sequencing was employed to dissect the signaling pathways and gene expression profiles underlying muscle fiber type differentiation in response to ECM cues.

Results Observations from Day 7 post-differentiation highlighted a pronounced shift towards fast-type myotube predominance, with decreased Pax7 and increased Myogenin expressions reflecting advanced differentiation stages. This trend was validated through Western blot and immunofluorescence analyses, revealing an altered expression ratio favoring fast-type over slow-type myotube proteins. qPCR analysis corroborated these findings, demonstrating upregulation of the MYH4 gene and downregulation of MYH7, alongside modulations in HIF-1α and PGC-1α levels. RNA sequencing analysis utilizing Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to analyze differentially expressed genes (DEGs), revealed a gene expression profile distinct from controls, with pathway analyses emphasizing activity within specific pathways such as C5 isoprenoid biosynthesis and inositol phosphate metabolism. Central genes to these pathways, including Cenpf, Brca1, and Nek2, were pinpointed as potential targets for modulating muscle fiber type differentiation.

Conclusions The ECM components in Matrigel™ play a significant role in steering C2C12 myoblast differentiation towards specific muscle fiber types, with significant implications for muscle contraction and overall muscle health. Our study enhances the understanding of the molecular landscape governing muscle fiber specificity and opens avenues for targeted strategies in muscle repair and regeneration. By elucidating the mechanisms through which ECM influences muscle cell fate, we contribute to the broader knowledge base in muscle physiology, offering insights into optimizing muscle function and combating muscle-related diseases.