Stereo-cell coupled with single-cell transcriptomics identifies a transitional metabolic state in maturing skeletal muscle myofibers.

Identification of skeletal muscle fiber types and metabolic reprogramming are crucial for postnatal muscle maturation, but high-resolution metabolism and spatial heterogeneity of muscle fibers in mid-maturation remain poorly understood. Our study performed single-cell RNA sequencing (scRNA-seq) and single-cell nuclear RNA sequencing (snRNA-seq) on five hind limb muscles from 5-week-old mice, combined with Stereo-cell at single muscle fiber resolution, to elucidate myofiber subtype maturation and its metabolic changes. Integrating scRNA-seq and snRNA-seq data, a comprehensive mouse skeletal muscle cell atlas was constructed, demonstrating the complementary advantages of the two techniques in capturing interstitial cells and multinucleated muscle fibers. Our analysis resolved a continuous maturational lineage from type I to IIB myonuclei (IIB_1-3). Notably, the IIB_3 myonuclei subtype exhibited a dual hypermetabolic phenotype, with increased oxidative phosphorylation (OXPHOS) and glycolytic activity, which differs from the purely glycolytic phenotype observed in adult mice. Stereo-cell further validated this transitional metabolic state and revealed spatial heterogeneity within individual IIB myofibers, with localized high-oxidation regions. Furthermore, we observed a mixed myofiber phenotype, with subtype-specific myosin heavy chain expression enriched at the myofiber terminals, indicating directional transformation of myofiber during maturation. In summary, this study reveals a previously undescribed transitional metabolic feature of IIB-type myofiber during postnatal muscle maturation and elucidates the spatial metabolic heterogeneity of myofiber, providing new insights into the regulatory mechanisms of skeletal muscle developmental plasticity and metabolic specialization.