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We demonstrate that early life disuse resulted in less grip strength in adulthood. Analysis of muscle composition demonstrated no loss of whole muscle or myofiber size indicating lower muscle quality akin to premature aging. This poor muscle quality was characterized by altered muscle macrophages and extracellular matrix area. We demonstrate intriguing correlations between this loss of grip strength and muscle macrophages and also area of noncontractile tissue in the muscle.

We provide evidence that the whole body loss of CCL2 in mice has adverse impacts on whole body function and skeletal muscle-specific contractile characteristics and collagen content. These defects in muscle function worsened during the recovery from disuse atrophy and corresponded with decreased recovery of muscle mass. We conclude that the absence of CCL2 decreased recruitment of proinflammatory macrophages to the muscle during the regrowth phase following disuse atrophy resulting in impaired collagen remodeling events and full resolution of muscle morphology and function.

SummaryReactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacologic neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediate age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacologic suppression.

Background Skeletal muscle (SkM) is a large, secretory organ that produces and releases myokines that can have autocrine, paracrine, and endocrine effects. Whether extracellular vesicles (EVs) also play a role in the SkM adaptive response and ability to communicate with other tissues is not well understood. The purpose of this study was to investigate EV biogenesis factors, marker expression, and localization across cell types in the skeletal muscle. We also aimed to investigate whether EV concentrations are altered by disuse atrophy. Methods To identify the potential markers of SkM-derived EVs, EVs were isolated from rat serum using density gradient ultracentrifugation, followed by fluorescence correlation spectroscopy measurements or qPCR. Single-cell RNA sequencing (scRNA-seq) data from rat SkM were analyzed to assess the EV biogenesis factor expression, and cellular localization of tetraspanins was investigated by immunohistochemistry. Finally, to assess the effects of mechanical unloading on EV expression in vivo, EV concentrations were measured in the serum by nanoparticle tracking analysis in both a rat and human model of disuse. Results In this study, we show that the widely used markers of SkM-derived EVs, α-sarcoglycan and miR-1, are undetectable in serum EVs. We also found that EV biogenesis factors, including the tetraspanins CD63, CD9, and CD81, are expressed by a variety of cell types in SkM. SkM sections showed very low detection of CD63, CD9, and CD81 in myofibers and instead accumulation within the interstitial space. Furthermore, although there were no differences in serum EV concentrations following hindlimb suspension in rats, serum EV concentrations were elevated in human subjects after bed rest. Conclusions Our findings provide insight into the distribution and localization of EVs in SkM and demonstrate the importance of methodological guidelines in SkM EV research.

Periods of inactivity experienced by older adults induce nutrient anabolic resistance creating a cascade of skeletal muscle transcriptional and translational aberrations contributing to muscle dysfunction. The purpose of this study was to identify how inactivity alters leucine-stimulated translation of molecules and pathways within the skeletal muscle of older adults. We performed ribosomal profiling alongside RNA sequencing from skeletal muscle biopsies taken from older adults (n = 8; ~72 years; 6 F/2 M) in response to a leucine bolus before (Active) and after (Reduced Activity) 2 weeks of reduced physical activity. At both visits, muscle biopsies were taken at baseline, 60 minutes (early response), and 180 minutes (late response) after leucine ingestion. Previously identified inactivity-related gene transcription changes (PFKFB3, GADD45A, NMRK2) were heightened by leucine with corresponding changes in translation. In contrast, leucine also stimulated translational efficiency of several transcripts in a manner not explained by corresponding changes in mRNA abundance (“uncoupled translation”). Inactivity eliminated this uncoupled translational response for several transcripts, and reduced the translation of most mRNAs encoding for ribosomal proteins. Ingenuity Pathway Analysis identified discordant circadian translation and transcription as a result of inactivity such as translation changes to PER2 and PER3 despite unchanged transcription. We demonstrate inactivity alters leucine-stimulated “uncoupled translation” of ribosomal proteins and circadian regulators otherwise not detectable by traditional RNA sequencing. Innovative techniques such as ribosomal profiling continues to further our understanding of how physical activity mediates translational regulation, and will set a path toward therapies that can restore optimal protein synthesis on the transcript-specific level to combat negative consequences of inactivity on aging muscle.

BACKGROUND AND AIMS. Metformin is the most commonly prescribed medication to treat diabetes. Emerging evidence suggests that metformin could have off target effects that might help promote healthy muscle aging, but these effects have not been thoroughly studied in glucose tolerant older individuals. The purpose of this study was to investigate the short-term effects of metformin consumption on skeletal muscle mitochondrial bioenergetics in healthy older adults. METHODS. We obtained muscle biopsy samples from 16 healthy older adults previously naïve to metformin and treated with metformin (METF; 3F, 5M), or placebo (CON; 3F, 5M), for two weeks using a randomized and blinded study design. Samples were analyzed using high-resolution respirometry, immunofluorescence, and immunoblotting to assess muscle mitochondrial bioenergetics, satellite cell (SC) content, and associated protein markers. RESULTS. We found that metformin treatment did not alter maximal mitochondrial respiration rates in muscle compared to CON. In contrast, mitochondrial H(2)O(2) emission and production were elevated in muscle samples from METF versus CON (METF emission: 2.59 ± 0.72 SE Fold, P=0.04; METF production: 2.29 ± 0.53 SE Fold, P=0.02). Furthermore, the change in H(2)O(2) emission was positively correlated with the change in type 1 myofiber SC content and this was biased in METF participants (Pooled: R(2)=0.5816, P=0.0006; METF: R(2)=0.674, P=0.0125). CONCLUSIONS. These findings suggest that acute exposure to metformin does not impact mitochondrial respiration in aged, glucose-tolerant muscle, but rather, influences mitochondrial-free radical and SC dynamics. CLINICAL TRIAL REGISTRATION. NCT03107884, clinicaltrials.gov

With this cohort, we previously demonstrated preservation of thigh lean tissue with neuromuscular electrical stimulation combined with protein supplementation (NMES+PRO) treatment during bed rest in healthy older adults. Because macrophage polarization plays a significant role in the repair and maintenance of muscle size and insulin sensitivity, we hypothesized that muscle macrophages would be induced by NMES+PRO and would correspond to an increase in lean mass and an attenuated insulin resistance response altered by bed rest. Older adults (60–80 years old; body mass index < 30 kg/m2) underwent 5 days of bed rest and were randomized to either thrice daily treatment of NMES+PRO (n = 8) or CON (n = 8). Lean mass, insulin sensitivity, and markers of muscle macrophages, inflammation, and connective tissue were determined before and after bed rest. Glucose intolerance and insulin resistance occurred after bed rest but there was not a treatment effect (p > 0.10). Proinflammatory-like macrophages (CD11b+, CD206−) increased (p < 0.05) with NMES+PRO treatment and was different than CON. Minor changes in noncontractile tissue were observed. However, changes in muscle macrophages or extracellular matrix were not related to the preservation of thigh lean mass or insulin resistance. Daily NMES+PRO treatment during bed rest induced a muscle proinflammatory-like macrophage response and was unrelated to muscle size or metabolic function. This study is listed as clinical trial NCT02566590. Novelty Neuromuscular electrical stimulation combined with protein supplementation (NMES+PRO) increased proinflammatory-like macrophages and extracellular matrix content in older adults after bed rest. NMES+PRO changes in macrophages and noncontractile tissue macrophages were not related to muscle size preservation or insulin sensitivity.

Objective Inactivity and inflammation are linked to obesity and insulin resistance. It was hypothesized that MyD88 (mediates inflammation) knockout from muscle (MusMyD88-/- ) would prevent, whereas miR146a-/- (MyD88 inhibitor) would exacerbate, inactivity-induced metabolic disturbances. Methods Cre-control, MusMyD88-/- , and miR146a-/- mice were given running wheels for 5 weeks to model an active phenotype. Afterward, half were placed into a small mouse cage (SMC) to restrict movement for 8 days. Body composition, muscle (3 H)2-deoxyglucose uptake, visceral fat histology, and tissue weight (hind limb muscles, visceral fat, and liver) were assessed. In skeletal muscle and visceral fat, RNA sequencing and mitochondrial function were performed on female MusMyD88-/- and Cre-control SMC mice. Results The SMC induced adiposity, hyperinsulinemia, and muscle insulin-stimulated glucose uptake, which was worsened in miR146a-/- mice. In females, MusMyD88-/- mice were protected. Female MusMyD88-/- mice during the SMC period (vs. Cre-control) exhibited higher Igf1 and decreased Ip6k3 and Trim63 muscle expression. Visceral fat transcript changes corresponded to improved lipid metabolism, decreased adipose expansion (Gulp1↑, Anxa2↓, Ehd1↓) and meta-inflammation (Hmox1↓), and increased beiging (Fgf10↑). Ralgapa2, negative regulator of GLUT4 translocation, and inflammation-related gene 993011J21Rik2 were decreased in both muscle and fat. Conclusions Whole-body miR146a-/- exacerbated inactivity-induced fat gain and muscle insulin resistance, whereas MusMyD88-/- prevented insulin resistance in female mice.

Obesity alters skeletal muscle lipidome and promotes myopathy, but it is unknown whether aberrant muscle lipidome contributes to the reduction in skeletal muscle contractile force-generating capacity. Comprehensive lipidomic analyses of mouse skeletal muscle revealed a very strong positive correlation between the abundance of lysophosphatidylcholine (lyso-PC), a class of lipids that is known to be downregulated with obesity, with maximal tetanic force production. The level of lyso-PC is regulated primarily by lyso-PC acyltransferase 3 (LPCAT3), which acylates lyso-PC to form phosphatidylcholine. Tamoxifen-inducible skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) was sufficient to reduce muscle lyso-PC content in both standard chow diet- and high-fat diet (HFD)-fed conditions. Strikingly, the assessment of skeletal muscle force-generating capacity ex vivo revealed that muscles from LPCAT3-MKI mice were weaker regardless of diet. Defects in force production were more apparent in HFD-fed condition, where tetanic force production was 40% lower in muscles from LPCAT3-MKI compared to that of control mice. These observations were partly explained by reductions in the cross-sectional area in type IIa and IIx fibers, and signs of muscle edema in the absence of fibrosis. Future studies will pursue the mechanism by which LPCAT3 may alter protein turnover to promote myopathy.

Loss of muscle mass and strength after disuse followed by impaired muscle recovery commonly occurs with aging. Metformin and leucine individually have shown positive effects in skeletal muscle during atrophy conditions but have not been evaluated in combination nor tested as a remedy to enhance muscle recovery following disuse atrophy in aging. The purpose of this study was to determine if a dual treatment of metformin and leucine (MET+LEU) would prevent disuse-induced atrophy and/or promote muscle recovery in aged mice and if these muscle responses correspond to changes in satellite cells and collagen remodeling. Aged mice (22–24 mo) underwent 14 days of hindlimb unloading (HU) followed by 7 or 14 days of reloading (7 or 14d RL). Metformin (MET), leucine (LEU), or MET+LEU was administered via drinking water and were compared to Vehicle (standard drinking water) and Ambulatory baseline (AMB). We observed that during HU, MET+LEU resolved whole body grip strength and soleus muscle specific force decrements caused by HU. Gastrocnemius satellite cell abundance was increased with MET+LEU treatment but did not alter muscle size during disuse or recovery conditions. Moreover, MET+LEU treatment alleviated gastrocnemius collagen accumulation caused by HU and increased collagen turnover during 7 and 14d RL driven by a decrease in collagen IV content. Transcriptional pathway analysis revealed that MET+LEU altered muscle hallmark pathways related to inflammation and myogenesis during HU. Together, the dual treatment of metformin and leucine was able to increase muscle function, satellite cell content, and reduce collagen accumulation, thus improving muscle quality during disuse and recovery in aging.

Aged skeletal muscle is characterized by poor muscle recovery following disuse coinciding with an impaired muscle pro‐inflammatory macrophage response. Macrophage inflammatory status is regulated by its metabolic state, but little is understood of macrophage metabolism and its relation to macrophage inflammation in the context of muscle recovery and aging. Therefore, the purpose of this study was to thoroughly characterize macrophage metabolism and inflammation in aged muscle during early recovery following disuse atrophy using single cell transcriptomics and functional assays. Young (4–5 months) and old (20–22 months) male C57BL/6 mice underwent 14 days of hindlimb unloading followed by 4 days of ambulatory recovery. CD45+ cells were isolated from solei muscles and analyzed using 10x Genomics single cell RNA sequencing. We found that aged pro‐inflammatory macrophage clusters were characterized with an impaired inflammatory and glycolytic transcriptome, and this dysregulation was accompanied by a suppression of HIF‐1α and its immediate downstream target, Glut1. As a follow‐up, bone marrow‐derived macrophages were isolated from a separate cohort of young and old mice at 4‐d recovery and were polarized to a pro‐inflammatory phenotype and used for glycolysis stress test, phagocytosis activity assay, and targeted GC‐MS metabolomics. Aged bone marrow‐derived pro‐inflammatory macrophages were characterized with impaired glycolysis and phagocytosis function, decreased succinate and an accumulation of glycolytic metabolic intermediates overall supporting reduced glycolytic flux and macrophage function. Our results indicate that the metabolic reprograming and function of aged skeletal muscle pro‐inflammatory macrophages are dysfunctional during early recovery from disuse atrophy possibly attributing to attenuated regrowth., Summary of metabolic dysfunction in aged pro‐inflammatory macrophages after 4 days of recovery from disuse atrophy. Figure depicts aged skeletal muscle macrophages during the early recovery phase following disuse atrophy (4 days of reloading). Aged pro‐inflammatory macrophages during recovery are present with impaired inflammation and glycolytic metabolism and accompanied by lower HIF‐1α. The lack of HIF‐1α transcription in the single cell sequencing may be regulated by the presence of succinate which typical increases in normal (young) pro‐inflammatory macrophages.

Muscle progenitor cells (MPCs) in aged muscle exhibit impaired activation into proliferating myoblasts, thereby impairing fusion and changes in secreted factors. The antihyperglycemic drug metformin, currently studied as a candidate antiaging therapy, may have potential to promote function of aged MPCs. We evaluated the impact of 2 wk of metformin ingestion on primary myoblast function measured in vitro after being extracted from muscle biopsies of older adult participants. MPCs were isolated from muscle biopsies of community-dwelling older (4 male/4 female, ∼69 yr) adult participants before (pre) and after (post) the metformin ingestion period and studied in vitro. Cells were extracted from Young participants (4 male/4 female, ∼27 yr) to serve as a “youthful” comparator. MPCs from Old subjects had lower fusion index and myoblast-endothelial cell homing compared with Young, while Old MPCs, extracted after short-term metformin ingestion, performed better at both tasks. Transcriptomic analyses of Old MPCs (vs. Young) revealed decreased histone expression and increased myogenic pathway activity, yet this phenotype was partially restored by metformin. However, metformin ingestion exacerbated pathways related to inflammation signaling. Together, this study demonstrated that 2 wk of metformin ingestion induced persistent effects on Old MPCs that improved function in vitro and altered their transcriptional signature including histone and chromatin remodeling.

Toll-like receptor 4 (TLR4) is a proposed mediator of ceramide accumulation, muscle atrophy, and insulin resistance in skeletal muscle. It is currently unknown whether pharmacological inhibition of TLR4, using the TLR4-specific inhibitor TAK-242 during muscle disuse, is able to prevent changes in intracellular ceramide species and consequently preserve muscle size and insulin sensitivity in physically active mice. To address this question, we subjected running wheel-conditioned C57BL/6 male mice (13 wk old; ∼10/group) to 7 days of hindlimb suspension (HS), 7 days of continued wheel running (WR), or daily injections of TAK-242 during HS (HS + TAK242) for 7 days. We measured hindlimb muscle morphology, intramuscular and liver ceramide content, HOMA-IR, mRNA proxies of ceramide turnover and lipid trafficking, and muscle fatty acid and glycerolipid content. As a result, soleus and liver ceramide abundance was greater ( P < 0.05) in HS vs. WR but was reduced with TLR4 inhibition (HS + TAK-242 vs. HS). Muscle mass declined ( P < 0.01) with HS (vs. WR), but TLR4 inhibition did not prevent this loss (soleus: P = 0.08; HS vs. HS + TAK-242). HOMA-IR was impaired ( P < 0.01) in HS versus WR mice, but only fasting blood glucose was reduced with TLR4 inhibition (HS + TAK-242 vs HS, P < 0.05). Robust decreases in muscle Spt2 and Cd36 mRNA and muscle lipidomic trafficking may partially explain reductions in ceramides with TLR4 inhibition. In conclusion, pharmacological TLR4 inhibition in wheel-conditioned mice prevented ceramide accumulation during the early phase of hindlimb suspension (7 days) but had little effect on muscle size and insulin sensitivity.

Intramuscular lipid accumulation has been associated with insulin resistance (IR), aging, diabetes, dyslipidemia, and obesity. A substantial body of evidence has implicated ceramides, a sphingolipid intermediate, as potent antagonists of insulin action that drive insulin resistance. Indeed, genetic mouse studies that lower ceramides are potently insulin sensitizing. Surprisingly less is known about how physical activity (skeletal muscle contraction) regulates ceramides, especially in light that muscle contraction regulates insulin sensitivity. The purpose of this review is to critically evaluate studies (rodent and human) concerning the relationship between skeletal muscle ceramides and IR in response to increased physical activity. Our review of the literature indicates that chronic exercise reduces ceramide levels in individuals with obesity, diabetes, or hyperlipidemia. However, metabolically healthy individuals engaged in increased physical activity can improve insulin sensitivity independent of changes in skeletal muscle ceramide content. Herein we discuss these studies and provide context regarding the technical limitations (e.g., difficulty assessing the myriad ceramide species, the challenge of obtaining information on subcellular compartmentalization, and the paucity of flux measurements) and a lack of mechanistic studies that prevent a more sophisticated assessment of the ceramide pathway during increased contractile activity that lead to divergences in skeletal muscle insulin sensitivity.