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Skeletal muscle regeneration is driven by the interaction of myogenic and non-myogenic cells. In aging, regeneration is impaired due to dysfunctions of myogenic and non-myogenic cells, but this is not understood comprehensively. We collected an integrated atlas of 273,923 single-cell transcriptomes from muscles of young, old, and geriatric mice (∼5, 20, 26 months-old) at six time-points following myotoxin injury. We identified eight cell types, including T and NK cells and macrophage subtypes, that displayed accelerated or delayed response dynamics between ages. Through pseudotime analysis, we observed myogenic cell states and trajectories specific to old and geriatric ages. To explain these age differences, we assessed cellular senescence by scoring experimentally derived and curated gene-lists. This pointed to an elevation of senescent-like subsets specifically within the self-renewing muscle stem cells in aged muscles. This resource provides a holistic portrait of the altered cellular states underlying skeletal muscle regenerative decline across mouse lifespan. EXTENDED SUMMARY: Skeletal muscle regeneration relies on the orchestrated interaction of myogenic and non-myogenic cells with spatial and temporal coordination. The regenerative capacity of skeletal muscle declines with aging due to alterations in myogenic stem/progenitor cell states and functions, non-myogenic cell contributions, and systemic changes, all of which accrue with age. A holistic network-level view of the cell-intrinsic and -extrinsic changes influencing muscle stem/progenitor cell contributions to muscle regeneration across lifespan remains poorly resolved. To provide a comprehensive atlas of regenerative muscle cell states across mouse lifespan, we collected a compendium of 273,923 single-cell transcriptomes from hindlimb muscles of young, old, and geriatric (4-7, 20, and 26 months-old, respectively) mice at six closely sampled time-points following myotoxin injury. We identified 29 muscle-resident cell types, eight of which exhibited accelerated or delayed dynamics in their abundances between age groups, including T and NK cells and multiple macrophage subtypes, suggesting that the age-related decline in muscle repair may arise from temporal miscoordination of the inflammatory response. We performed a pseudotime analysis of myogenic cells across the regeneration timespan and found age-specific myogenic stem/progenitor cell trajectories in old and geriatric muscles. Given the critical role that cellular senescence plays in limiting cell contributions in aged tissues, we built a series of tools to bioinformatically identify senescence in these single-cell data and assess their ability to identify senescence within key myogenic stages. By comparing single-cell senescence scores to co-expression of hallmark senescence genes Cdkn2a and Cdkn1a , we found that an experimentally derived gene-list derived from a muscle foreign body response (FBR) fibrosis model accurately (receiver-operator curve AUC = 0.82-0.86) identified senescent-like myogenic cells across mouse ages, injury time-points, and cell-cycle states, in a manner comparable to curated gene-lists. Further, this scoring approach pinpointed transitory senescence subsets within the myogenic stem/progenitor cell trajectory that are related to stalled MuSC self-renewal states across all ages of mice. This new resource of mouse skeletal muscle aging provides a comprehensive portrait of the changing cellular states and interaction network underlying skeletal muscle regeneration across mouse lifespan.

Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

Objective Skeletal muscle regeneration relies on muscle-specific adult stem cells (MuSCs), MuSC progeny, muscle progenitor cells (MPCs), and a coordinated myogenic program that is influenced by the extracellular environment. Following injury, MPCs undergo a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Certain pathologies (e.g., metabolic diseases and muscle dystrophies) and advanced age are associated with dysregulated muscle regeneration. The availability of serine and glycine, two nutritionally non-essential amino acids, is altered in humans with these pathologies, and these amino acids have been shown to influence the proliferative state of non-muscle cells. Our objective was to determine the role of serine/glycine in MuSC/MPC function. Methods Primary human MPCs (hMPCs) were used for in vitro experiments, and young (4–6 mo) and old (>20 mo) mice were used for in vivo experiments. Serine/glycine availability was manipulated using specially formulated media in vitro or dietary restriction in vivo followed by downstream metabolic and cell proliferation analyses. Results We identified that serine/glycine are essential for hMPC proliferation. Dietary restriction of serine/glycine in a mouse model of skeletal muscle regeneration lowered the abundance of MuSCs 3 days post-injury. Stable isotope-tracing studies showed that hMPCs rely on extracellular serine/glycine for population expansion because they exhibit a limited capacity for de novo serine/glycine biosynthesis. Restriction of serine/glycine to hMPCs resulted in cell cycle arrest in G0/G1. Extracellular serine/glycine was necessary to support glutathione and global protein synthesis in hMPCs. Using an aged mouse model, we found that reduced serine/glycine availability augmented intermyocellular adipocytes 28 days post-injury. Conclusions These studies demonstrated that despite an absolute serine/glycine requirement for MuSC/MPC proliferation, de novo synthesis was inadequate to support these demands, making extracellular serine and glycine conditionally essential for efficient skeletal muscle regeneration., Highlights • Extracellular serine and glycine are necessary for muscle stem/progenitor cell population expansion. • Human muscle progenitor cells possess limited capacity for de novo serine/glycine biosynthesis. • Extracellular serine/glycine restriction increases reactive oxygen species and reduces intracellular glutathione. • Extracellular serine/glycine inhibits protein synthesis and cell cycle arrest. • Reduced extracellular serine and glycine in old mice impairs skeletal muscle regeneration.