271P Exploring the effects of the epi-drug Remodelin on murine myoblasts differentiation

Skeletal muscle differentiation is a tightly controlled and elaborated process, that consists of alignment and fusion of myoblasts to form mature myotubes. A growing interest for epigenetics involvement in guiding muscle differentiation raised the attention on epi-modulators as pivotal regulators in this process. Our in vitro study aimed to investigate the potential effects of Remodelin in muscle differentiation. Remodelin is the inhibitor of N-acetyltransferase 10, the key-player of RNA acetylation. We used the well-consolidated model of murine C2C12 cells cultivated on ultra-compliant gelatin hydrogels for long-term studies. The newly developed hydrogel scaffold promotes myotube alignment and maturation, mimicking the skeletal muscle biology observed in vivo. We differentiated C2C12 cells in low-serum conditions up to 16 days and treated them with the epi-drug Remodelin. At day 7 of differentiation, confocal images revealed that Remodelin prompted a lack of organization, proper morphology, and maturation of myotubes compared to the control. Marked twitching was neither visible upon Remodelin treatment, even in the late stage of differentiation. Remodelin downregulated the expression of protein markers of differentiation, but without any significant epi-modulation. Both transcriptomics and proteomics analyses confirmed that Remodelin effectively slowed down the process of myotube formation. RNAseq analysis revealed that the epi-drug down-regulated 749 genes, predominantly encoding proteins involved in muscle contraction, sarcomere organization, muscle structure development, and calcium ion binding. Proteomics analysis further unveiled that 37 significantly down-regulated proteins were related to extracellular matrix, cell-matrix adhesion, positive regulation of muscle cell differentiation, and calcium ion binding. Taken together, these results suggest that Remodelin exerts a broad effect on the regulatory networks of skeletal muscle differentiation.