Aging induces aberrant state transition kinetics in murine muscle stem cells.

Murine muscle stem cells (MuSCs) experience a transition from quiescence to activation that is required for regeneration, but it remains unknown if the trajectory and dynamics of activation change with age. Here, we use time-lapse imaging and single cell RNA-seq to measure activation trajectories and rates in young and aged MuSCs. We find that the activation trajectory is conserved in aged cells, and we develop effective machine-learning classifiers for cell age. Using cell-behavior analysis and RNA velocity, we find that activation kinetics are delayed in aged MuSCs, suggesting that changes in stem cell dynamics may contribute to impaired stem cell function with age. Intriguingly, we also find that stem cell activation appears to be a random walk-like process, with frequent reversals, rather than a continuous linear progression. These results support a view of the aged stem cell phenotype as a combination of differences in the location of stable cell states and differences in transition rates between them.

Lineage Tracing Reveals a Subset of Reserve Muscle Stem Cells Capable of Clonal Expansion under Stress.

Stem cell heterogeneity is recognized as functionally relevant for tissue homeostasis and repair. The identity, context dependence, and regulation of skeletal muscle satellite cell (SC) subsets remains poorly understood. We identify a minor subset of Pax7+ SCs that is indelibly marked by an inducible Mx1-Cre transgene in vivo, is enriched for Pax3 expression, and has reduced ROS (reactive oxygen species) levels. Mx1+ SCs possess potent stem cell activity upon transplantation but minimally contribute to endogenous muscle repair, due to their relative low abundance. In contrast, a dramatic clonal expansion of Mx1+ SCs allows extensive contribution to muscle repair and niche repopulation upon selective pressure of radiation stress, consistent with reserve stem cell (RSC) properties. Loss of Pax3 in RSCs increased ROS content and diminished survival and stress tolerance. These observations demonstrate that the Pax7+ SC pool contains a discrete population of radiotolerant RSCs that undergo clonal expansion under severe stress.

Thermal stress induces glycolytic beige fat formation via a myogenic state.

Environmental cues profoundly affect cellular plasticity in multicellular organisms. For instance, exercise promotes a glycolytic-to-oxidative fibre-type switch in skeletal muscle, and cold acclimation induces beige adipocyte biogenesis in adipose tissue. However, the molecular mechanisms by which physiological or pathological cues evoke developmental plasticity remain incompletely understood. Here we report a type of beige adipocyte that has a critical role in chronic cold adaptation in the absence of ß-adrenergic receptor signalling. This beige fat is distinct from conventional beige fat with respect to developmental origin and regulation, and displays enhanced glucose oxidation. We therefore refer to it as glycolytic beige fat. Mechanistically, we identify GA-binding protein α as a regulator of glycolytic beige adipocyte differentiation through a myogenic intermediate. Our study reveals a non-canonical adaptive mechanism by which thermal stress induces progenitor cell plasticity and recruits a distinct form of thermogenic cell that is required for energy homeostasis and survival.

Skeletal muscle satellite cells in amyotrophic lateral sclerosis.

OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving progressive muscular paralysis reflecting degeneration of motor neurons. Skeletal muscle tissue seems to play a significant role in ALS pathogenesis. Here, the role of satellite cells (SCs) in ALS muscle atrophy is investigated. METHODS: We isolated SCs from ALS human muscle biopsies and we analyzed their ability to grow and expand in vitro. Ultrastructural and immunophenotypical features were analyzed. Quantitative real-time RT-QPCR and western blot (WB) analyses were performed to evaluate MRFs and MyH1 expression. RESULTS: ALS SCs showed a high proliferative potential, but their capacity to proceed through the myogenic program and form myotubes seems altered compared to controls (Ctrls). We observed that differentiating ALS SCs showed some specific features, but they displayed an altered morphology, with a large number of vacuoles. RT-QPCR and WB showed lower Myf-4 and MyH1 compared to Ctrls. CONCLUSIONS: Our data suggest that the capacity of ALS SCs to proceed through the myogenic program seems to be altered: SCs seem to lose their ability to regenerate and restore mature myofibers.

Androgen-dependent impairment of myogenesis in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disease caused by expansion of a polyglutamine (polyQ) tract in the androgen receptor (AR). SBMA is triggered by the interaction between polyQ-AR and its natural ligands, testosterone and dihydrotestosterone (DHT). SBMA is characterized by the loss of lower motor neurons and skeletal muscle fasciculations, weakness, and atrophy. To test the hypothesis that the interaction between polyQ-AR and androgens exerts cell-autonomous toxicity in skeletal muscle, we characterized the process of myogenesis and polyQ-AR expression in DHT-treated satellite cells obtained from SBMA patients and age-matched healthy control subjects. Treatment with androgens increased the size and number of myonuclei in myotubes from control subjects, but not from SBMA patients. Myotubes from SBMA patients had a reduced number of nuclei, suggesting impaired myotube fusion and altered contractile structures. The lack of anabolic effects of androgens on myotubes from SBMA patients was not due to defects in myoblast proliferation, differentiation or apoptosis. DHT treatment of myotubes from SBMA patients increased nuclear accumulation of polyQ-AR and decreased the expression of interleukin-4 (IL-4) when compared to myotubes from control subjects. Following DHT treatment, exposure of myotubes from SBMA patients with IL-4 treatment rescued myonuclear number and size to control levels. This supports the hypothesis that androgens alter the fusion process in SBMA myogenesis. In conclusion, these results provide evidence of an androgen-dependent impairment of myogenesis in SBMA that could contribute to disease pathogenesis.

An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis.

A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development.

Satellite cell characterization from aging human muscle.

OBJECTIVES: Satellite cells (SCs) are skeletal muscle progenitor cells located between the basal lamina and the sarcolemma of muscle fibers. They are responsible for muscle growth and repair. In humans, aging results in the depletion of the SC population and in its proliferative activity, but not in its function. It has not yet been determined whether under conditions of massive muscle fiber death in vivo, the regenerative potential of SCs is totally or partially compromised in old muscle. No studies have yet tested whether advanced age is a factor that restrains the response of SCs to muscle denervation in humans; this is also due to difficulties in the isolation and in the culture of SCs from a small human surgery fragment. The aim of this study was to study in depth muscle regeneration analysing the SC ability of SCs to proliferate and differentiate in aging human patients. METHODS: In order to study in more detail the molecular mechanism, the proliferative and differentiative ability of aging SCs, we isolated SCs from aging human muscle biopsies and analysed their morphology by transmission electron microscopy and immunocytochemical analysis (antibodies against desmin, N-CAM and M-cadherin) and their capacity to grow and to expand in vitro. Moreover, in order to evaluate gene expression of myogenic regulatory factors Myf5, MyoD and myogenin (Myf4), RT-PCR was performed. RESULTS AND DISCUSSION: SCs isolated from aging human muscle biopsies and plated into favorable proliferation and differentiation conditions were able to proceed through the myogenic program and actively form myotubes, although taking longer than the young control sample. The RT-PCR analysis together with the ultrastructural SC features showed that the myogenic potential seemed to be compromised during the aging human muscle proliferation in vitro.