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

Skeletal muscle fibers are among the largest cells in the body and rely on the spatial distribution of numerous nuclei to maintain intracellular function across vast cytoplasmic volumes. How nuclear organization adapts when nuclear number is reduced remains unclear. Here, we analyzed three-dimensional myonuclear positioning during postnatal development in a mouse model with impaired myonuclear accretion. Across more than 1,000 fibers and nearly 15,000 nuclei, reduced myonuclear number led to increased spatial regularity, including more even longitudinal spacing, lower variability in inter-nuclear distances, and the emergence of a preferred inter-nuclear spacing. These changes persist after accounting for fiber size and nuclear density, indicating an active reorganization rather than a passive geometric consequence. Mapping myonuclei onto the fiber surface further revealed more uniform and locally ordered two-dimensional organization, although this surface pattern was largely explained by the improved longitudinal spacing. Together, these results show that muscle fibers do not simply tolerate fewer nuclei, but actively adapt myonuclear positioning in a manner that promotes efficient cytoplasmic coverage across large cellular dimensions.