The inhibitory effects of Remodelin on murine myoblasts differentiation

Background: Myoblasts differentiation is a highly regulated and complex process leading to the formation of fused and aligned mature myotubes. Increasing interest in the role of epigenetics in muscle differentiation has highlighted epi-modulators as crucial regulators of this process. Recent findings revealed the effects of Remodelin, a selective inhibitor of the acetyltransferase Nat10, in counteracting muscle loss and muscle atrophy in in vitro and in vivo sepsis model. Remodelin was initially identified for its ability to improve nuclear architecture in cells with defective lamin A, such as those from patients with Hutchinson-Gilford Progeria Syndrome (HGPS). Our in vitro study aimed to explore the potential effects of Remodelin on myoblasts differentiation. Methods: We used a well-consolidated in vitro model of murine C2C12 myoblasts, culturing them on ultra-compliant gelatin hydrogels for long-term studies. The hydrogel scaffold promotes myotube alignment and maturation. We differentiated C2C12 cells in low-serum conditions for up to 16 days and treated them with the epi-drug Remodelin. Immunofluorescence microscopy, together with RNAseq and proteomics analyses, were used to analyse the effects of Remodelin treatment on myotube formation. Results: By day 7 of differentiation, confocal images showed that Remodelin impaired myotube organization and maturation, and proper morphology compared to untreated cells. Additionally, no significant twitching was observed upon Remodelin treatment, even in the later stage of differentiation. Intersection of transcriptomics and proteomics analyses confirmed that Remodelin effectively slowed myotube formation. RNA sequencing revealed that the epi-drug downregulated 749 genes, mainly encoding proteins involved in muscle contraction, sarcomere organization, muscle structure development, and calcium ion binding. Proteomics analysis further revealed downregulation of pathways related to myoblasts differentiation. Out of 3076 proteins quantified, 37 proteins were significantly decreased. GO analysis corroborated the sequencing results. Furthermore, Remodelin significantly downregulated the expression of protein markers associated with differentiation and it decreased histone acetylation levels. Conclusions: Collectively, these results suggest that Remodelin broadly affects the regulatory networks involved in skeletal muscle differentiation.