Objective: Skeletal muscle regeneration depends on the coordinated dialogue between muscle stem cells (MuSCs) and their microenvironment. While the contribution of chromatin regulators within MuSCs has been extensively documented, their function in differentiated myofibers remains largely unexplored. Here, we investigated the role of the histone methyltransferase SETDB1 in post-mitotic myonuclei.

Methods: We generated an inducible, myofiber-specific Setdb1 knockout mouse (Setdb1 ^{fl/fl; Acta1-CreERT2}) and analyzed skeletal muscle regeneration at 4, 7, and 14 days post-injury. MuSC activation was assessed in vivo and ex vivo using isolated myofiber cultures. Spatial transcriptomic analysis was initiated to map gene expression in regenerating muscle tissue with cellular resolution.

Results: In uninjured conditions, loss of Setdb1 in myofibers did not affect muscle morphology or fiber type distribution, indicating a limited impact in basal physiology. However, Setdb1-deficient muscles displayed an expansion of the PAX7 ⁺ MuSC population following injury, accompanied by an increased proportion of small regenerating fibers. Ex vivo assays on isolated myofibers further revealed that MuSCs in contact with Setdb1-deficient fibers exhibited enhanced activation and proliferation. This phenotype is similar in conditioned medium assay, suggesting a role for soluble factors secreted by the deficient fiber.To characterize this communication, we performed spatial transcriptomics of regenerating muscles. Differential analyses indicated that Setdb1-deficient myofibers activate a stress and regeneration associated transcriptional program, including enhanced expression of extracellular and signaling matrix components with potential impact on the surrounding niche. Functional enrichment supported an altered secretory and structural landscape that could directly influence MuSC behavior.

Conclusion: Together, our findings identify SETDB1 as an epigenetic gatekeeper in myofiber that restrains a latent injury-induced transcriptional program. Its loss results in the production of paracrine cues that promote MuSC activation and expansion during regeneration. This study highlights the importance of myonuclei intrinsic chromatin regulation in shaping the regenerative niche and establishes a previously unrecognized, non-cell-autonomous role for SETDB1 in muscle repair.