Identification of a new population of myonuclei during skeletal muscle hypertrophy

Background Skeletal muscle represents around 40% of total human body weight and exhibits remarkable plasticity. It can hypertrophy, atrophy, or regenerate in response to changes in activity, nutrient availability, or injury. The main component of striated muscle, the myofiber, is a post-mitotic, multinucleated cell that contains the muscle’s contractile unit, the sarcomere. The myonuclei within these fibers are specialized and differ in terms of gene expression and localization. Adult muscles also contain various other cell types, including adult muscle stem cells (MuSCs), macrophages, fibro-adipogenic progenitors (FAPs), and endothelial cells. MuSCs are central to muscle plasticity, and are capable of activation, proliferation, differentiation, and fusion to form new myofibers during regeneration, or to fuse with existing myofibers during hypertrophy. Muscle hypertrophy and myofiber’s enlargement involve increased protein synthesis and reduced protein degradation, as well as myonuclear accretion following satellite cell activation. Multiple signaling pathways, such as the mTOR pathway and the RhoA/SRF mechanotransduction pathway, are involved in these processes.

We performed single-nucleus RNA sequencing (snRNA-seq) on plantaris muscles of adult mice, comparing samples 7 days after hypertrophy induction (overload, 7OV) to non-hypertrophied controls (Ctl). RNAscope experiments on isolated myofibers identified the heterogeneity of myonuclei along the myofiber.

SnRNA-seq analysis revealed a previously unknown population of myonuclei (UM). UM-Ctl, which is present only in the Ctl condition, and UM-7OV, only in the 7OV condition. These myonuclei are localised at the tips of myofibres. Furthermore, we determined that UM-7OV are not newly fused myonuclei from activated satellite cells. Trajectory analyses suggest that UM-Ctl transition into UM-7OV during hypertrophy, returning to a near-basal homeostatic state after 21 days of overload (21OV). Gene expression analysis showed that UM-Ctl and UM-7OV have distinct gene expression profiles compared to other myonuclei and respond differently to hypertrophy.

Our findings suggest the existence of a specific population of myonuclei with unique localization and gene expression profiles, which play distinct roles at baseline and during hypertrophy. These results highlight the differential properties of myonuclei in the myofiber and their potential specific functions in muscle homeostasis and adaptation. Competing Interest Statement The authors have declared no competing interest. - Abbreviations - Ctl - control - Gsn - Gelsolin - HSA - Human skeletal actin - IP - intraperitoneal - MTJ - myotendinous junction - mTmG - membrane Tomato membrane - GFP - Musc muscle stem cells - Mut - mutant - Myh - myosin heavy chain - NMJ - neuromuscular junction - OV - overload - Postn - Periostin - SnRNA-seq - single nucleus RNA sequencing - TMX - tamoxifen - Ttn - Titin - UM - unknown myonuclei - UMAP - uniform manifold approximation and projection