Project description:Disuse-induced muscle atrophy commonly occurs following illness, injury, or falls and becomes increasingly frequent with ageing. Whether skeletal muscle retains a “memory” of repeated disuse remains unknown. We investigated repeated lower- limb immobilization in young adults and a refined aged rat model, integrating physiological, multi-omic, immunohistochemical, biochemical, and primary human muscle stem cell (MuSC) analyses. To enable robust age comparisons, we integrated previously published young rat data with newly generated aged rat data. In young human muscle, repeated disuse elicited attenuated transcriptional perturbations in oxidative and mitochondrial pathways, suggestive of a protective molecular memory, despite similar atrophy to initial disuse. In contrast, aged muscle exhibited a detrimental memory, characterized by greater atrophy, exaggerated suppression of aerobic metabolism genes despite recovery after initial disuse, NAD+ and mitochondrial DNA depletion, and activation of proteasomal, extracellular-matrix, and DNA-damage pathways. Whereas young rats recovered muscle mass after initial disuse, aged rats failed to do so. Across species, repeated disuse induced DNA hypermethylation and downregulation of aerobic metabolism and mitochondrial gene networks. NR4A1 and NR4A3 were among the strongest disuse- suppressed genes; NR4A1 acquired recovery-phase hypermethylation that maintained its transcriptional repression, while NR4A3 was the most downregulated gene after initial atrophy and remained persistently suppressed into recovery. Acetylcholine receptor subunit genes (CHRNA1, CHRND) were epigenetically primed, demonstrating hypomethylation and strong upregulation after disuse, and further amplification after repeated atrophy, while CHRNG was selectively induced after repeated atrophy only. NMRK2, an NAD+ biosynthesis gene, was the most downregulated gene across both atrophy periods, and supplementation with its substrate, nicotinamide riboside (NR), improved myotube size in MuSCs derived post-atrophy. Overall, repeated disuse atrophy imprints a molecular memory in skeletal muscle shaping transcriptional resilience in young adults and exaggerated susceptibility in aged muscle.

Project description:Hibernation is energy saving adaptation involving suppression of activity to survive in highly seasonal environments. Immobility and disuse generate muscle loss in most mammalian species. In contrast to other mammals, bears and ground squirrels demonstrate limited muscle atrophy over the physical inactivity of winter hibernation. This suggests that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional program underlying molecular mechanisms preventing muscle loss, we conducted a large-scale gene expression screening in hind limb muscles comparing hibernating and summer active black bears and arctic ground squirrels by the use of custom 9,600 probe cDNA microarrays. The molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during hibernation that implies induction of translation at different hibernation states. The induction of protein biosynthesis likely contributes to attenuation of disuse muscle atrophy through prolonged periods of immobility and starvation. This adaptive mechanism allows hibernating mammals to maintain full musculoskeletal function and preserve mobility during and immediately after hibernation, thus promoting survival. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction of multiply genes involved in oxidation reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation. Black bears sampled during winter hibernation were compared with the animals sampled during summer. Muscle tissue were hybridized on a custom 12,800 cDNA probe nylon membrane microarray platform . Six hibernating and six summer active bears were studied in the experiment.

Project description:Skeletal muscle atrophy is a consequence of many diseases, environmental insults, inactivity, age and injury. Atrophy is characterized by active degradation and removal of contractile proteins and a reduction in fiber size. Animal models have been extensively used to identify pathways leading to atrophic conditions. Here we have used genome-wide expression profiling analysis and quantitative PCR to identify the molecular changes that occur in two clinically relevant animal mouse models of muscle atrophy, hindlimb casting and Achilles tendon laceration (tenotomy). Gastrocnemius muscle samples were collected 2, 7 and 14 days after insult. The total amount of muscle loss as measured by wet weight and muscle fiber size was equivalent between models, although tenotomy resulted in a more rapid induction of muscle atrophy. Furthermore, tentomy resulted in the regulation of significantly more mRNA transcripts then casting. Analysis of the regulated genes and pathways suggest that the mechanism of atrophy is distinct between these models. The degradation following casting appears ubiquitin-proteasome-mediated while degradation following tenotomy appears lysosomal and matrix-metalloproteinase (MMP)-mediated. This data suggests that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat the atrophy seen under different conditions. Muscle atrophy was induced through two methods; unloading induced by tendon laceration (Tenotomy) and immobilization induced by hindlimb casting (Casting). For the tenotomy, eight week old male C57/B6 mice were anesthetized by ether inhalation and subsequent intramuscular injection of ketamine-xylazine (0.1 ml/g). The right Achilles tendon just proximal to the calcaneus was severed with a sterile scalpel. The mice were allowed to recover and move freely about their cage. An additional cohort of animals was sham operated (Sham) which involved isolation and manipulation of the Achilles tendon without laceration. For the casting, the right hindlimbs of eight week old male C57Bl/6 mice were immobilized through casting. After 2, 7 or 14 days, animals were sacrificed and the left and right gastrocnemius muscles were carefully removed, flash frozen in liquid nitrogen, and stored at -80°C for further processing. The left untreated legs served as controls. A group of naïve animals that received no treatment or manipulation was included as an additional control. Each group consisted of between 5 and 10 samples for a total of 111 individual samples.

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Mus musculus

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