Reduced Myonuclear Number Drives Spatial Optimization of Nuclear Positioning in Multinucleated Muscle Fibers

Abstract

Skeletal muscle fibers are large syncytial cells containing hundreds of spatially distributed myonuclei that collectively provide transcriptional output across a shared cytoplasm. Whether the spatial organization of myonuclei adjusts when myonuclear accretion does not keep pace with cytoplasmic growth remains unresolved. We therefore asked whether a smaller myonuclear population is distributed more uniformly within a growing fiber, potentially limiting cytoplasmic regions disproportionately remote from a source of nucleus-derived products. To test this hypothesis, we analyzed three-dimensional myonuclear organization throughout postnatal growth in a mouse model in which inducible Myomaker deletion in muscle stem cells restricts their fusion with growing fibers and thereby limits the addition of new myonuclei. Across 1,029 EDL fibers containing nearly 15,000 myonuclei, sampled from postnatal day 13 (P13) to approximately 5 months of age (P150), the resulting reduction in myonuclear number left fewer nuclear sites of transcriptional output within the growing fiber and increased the characteristic distance between neighboring myonuclei. Despite this greater separation, the myonuclei present were distributed more regularly along the fiber axis, reducing the prevalence of cytoplasmic regions disproportionately remote from a myonucleus. This spatial regularization may limit local disparities in access to myonuclear gene products as the cytoplasmic volume associated with each nucleus increases. Importantly, this organization was expressed primarily along the fiber's long axis. When evaluated across the full fiber surface, the more regular longitudinal arrangement accounted for nearly all of the increase in spatial uniformity, whereas circumferential positioning made only a minimal additional contribution. Thus, the elongated geometry of a myofiber makes longitudinal organization particularly important for limiting nucleus-distant cytoplasmic regions when fewer myonuclei are available relative to fiber size.