Type 2 diabetes mellitus related sarcopenia: a type of muscle loss distinct from sarcopenia and disuse muscle atrophy.

Muscle loss is a significant health concern, particularly with the increasing trend of population aging, and sarcopenia has emerged as a common pathological process of muscle loss in the elderly. Currently, there has been significant progress in the research on sarcopenia, including in-depth analysis of the mechanisms underlying sarcopenia caused by aging and the development of corresponding diagnostic criteria, forming a relatively complete system. However, as research on sarcopenia progresses, the concept of secondary sarcopenia has also been proposed. Due to the incomplete understanding of muscle loss caused by chronic diseases, there are various limitations in epidemiological, basic, and clinical research. As a result, a comprehensive concept and diagnostic system have not yet been established, which greatly hinders the prevention and treatment of the disease. This review focuses on Type 2 Diabetes Mellitus (T2DM)-related sarcopenia, comparing its similarities and differences with sarcopenia and disuse muscle atrophy. The review show significant differences between the three muscle-related issues in terms of pathological changes, epidemiology and clinical manifestations, etiology, and preventive and therapeutic strategies. Unlike sarcopenia, T2DM-related sarcopenia is characterized by a reduction in type I fibers, and it differs from disuse muscle atrophy as well. The mechanism involving insulin resistance, inflammatory status, and oxidative stress remains unclear. Therefore, future research should further explore the etiology, disease progression, and prognosis of T2DM-related sarcopenia, and develop targeted diagnostic criteria and effective preventive and therapeutic strategies to better address the muscle-related issues faced by T2DM patients and improve their quality of life and overall health.

Skeletal muscle immobilisation-induced atrophy: mechanistic insights from human studies.

Periods of skeletal muscle disuse lead to rapid declines in muscle mass (atrophy), which is fundamentally underpinned by an imbalance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB). The complex interplay of molecular mechanisms contributing to the altered regulation of muscle protein balance during disuse have been investigated but rarely synthesised in the context of humans. This narrative review discusses human models of muscle disuse and the ensuing inversely exponential rate of muscle atrophy. The molecular processes contributing to altered protein balance are explored, with a particular focus on growth and breakdown signalling pathways, mitochondrial adaptations and neuromuscular dysfunction. Finally, key research gaps within the disuse atrophy literature are highlighted providing future avenues to enhance our mechanistic understanding of human disuse atrophy.

Deciphering the Therapeutic Role of Lactate in Combating Disuse-Induced Muscle Atrophy: An NMR-Based Metabolomic Study in Mice.

Disuse muscle atrophy (DMA) is a significant healthcare challenge characterized by progressive loss of muscle mass and function resulting from prolonged inactivity. The development of effective strategies for muscle recovery is essential. In this study, we established a DMA mouse model through hindlimb suspension to evaluate the therapeutic potential of lactate in alleviating the detrimental effects on the gastrocnemius muscle. Using NMR-based metabolomic analysis, we investigated the metabolic changes in DMA-injured gastrocnemius muscles compared to controls and evaluated the beneficial effects of lactate treatment. Our results show that lactate significantly reduced muscle mass loss and improved muscle function by downregulating Murf1 expression, decreasing protein ubiquitination and hydrolysis, and increasing myosin heavy chain levels. Crucially, lactate corrected perturbations in four key metabolic pathways in the DMA gastrocnemius: the biosynthesis of phenylalanine, tyrosine, and tryptophan; phenylalanine metabolism; histidine metabolism; and arginine and proline metabolism. In addition to phenylalanine-related pathways, lactate also plays a role in regulating branched-chain amino acid metabolism and energy metabolism. Notably, lactate treatment normalized the levels of eight essential metabolites in DMA mice, underscoring its potential as a therapeutic agent against the consequences of prolonged inactivity and muscle wasting. This study not only advances our understanding of the therapeutic benefits of lactate but also provides a foundation for novel treatment approaches aimed at metabolic restoration and muscle recovery in conditions of muscle wasting.

Custom-made 3D-printed boot as a model of disuse-induced atrophy in murine skeletal muscle.

Skeletal muscle atrophy is characterized by a decrease in muscle mass and strength caused by an imbalance in protein synthesis and degradation. This process naturally occurs upon reduced or absent physical activity, often related to illness, forced bed rest, or unhealthy lifestyles. Currently, no treatment is available for atrophy, and it can only be prevented by overloading exercise, causing severe problems for patients who cannot exercise due to chronic diseases, disabilities, or being bedridden. The two murine models commonly used to induce muscle atrophy are hindlimb suspension and ankle joint immobilization, both of which come with criticalities. The lack of treatments and the relevance of this atrophic process require a unilateral, safe, and robust model to induce muscle atrophy. In this work, we designed and developed a 3D-printed cast to be used for the study of disuse skeletal muscle atrophy. Applying two halves of the cast is non-invasive, producing little to no swelling or skin damage. The application of the cast induces, in 2-weeks immobilized leg, the activation of atrophy-related genes, causing a muscle weight loss up to 25% in the gastrocnemius muscle, and 31% in the soleus muscle of the immobilized leg compared to the control leg. The cross-sectional area of the fibers is decreased by 31% and 34% respectively, with a peculiar effect on fiber types. In the immobilized gastrocnemius, absolute muscle force is reduced by 38%, while normalized force is reduced by 16%. The contralateral leg did not show signs of overload or hypertrophy when compared to free roaming littermates, offering a good internal control over the immobilized limb. Upon removing the cast, the mice effectively recovered mass and force in 3 weeks.

Identifying the potential therapeutic effects of miR­6516 on muscle disuse atrophy.

Muscle atrophy is a debilitating condition with various causes; while aging is one of these causes, reduced engagement in routine muscle­strengthening activities also markedly contributes to muscle loss. Although extensive research has been conducted on microRNAs (miRNAs/miRs) and their associations with muscle atrophy, the roles played by miRNA precursors remain underexplored. The present study detected the upregulation of the miR­206 precursor in cell­free (cf)RNA from the plasma of patients at risk of sarcopenia, and in cfRNAs from the muscles of mice subjected to muscle atrophy. Additionally, a decline in the levels of the miR­6516 precursor was observed in mice with muscle atrophy. The administration of mimic­miR­6516 to mice immobilized due to injury inhibited muscle atrophy by targeting and inhibiting cyclin­dependent kinase inhibitor 1b (Cdkn1b). Based on these results, the miR­206 precursor appears to be a potential biomarker of muscle atrophy, whereas miR­6516 shows promise as a therapeutic target to alleviate muscle deterioration in patients with muscle disuse and atrophy.

Transcriptome and Metabolome Profiling Provide New Insights into Disuse Muscle Atrophy in Chicken: The Potential Role of Fast-Twitch Muscle Fibers.

Disuse muscle atrophy is a disease caused by restricted activity, affecting human health and animal protein quality. While extensive research on its mechanism has been studied in mammals, comparatively little is known about this process in chickens, which are a significant source of protein for human consumption worldwide. Understanding the mechanisms underlying skeletal muscle atrophy in chickens is crucial for improving poultry health and productivity, as well as for developing strategies to mitigate muscle loss. In this study, two groups of chickens were subjected to limb immobilization for two and four weeks, respectively, in order to induce disuse muscle atrophy and uniformly sampled gastrocnemius muscle at the fourth week. A combined analysis of the transcriptome and metabolome was conducted to investigate the mechanisms of disuse-induced muscle atrophy. Through H&E staining and immunofluorescence, we found that, compared to slow-twitch muscle fibers, the fast-twitch muscle fibers showed a greater reduction in cross-sectional area in the immobilized leg, and were also the main driver of changes in cross-sectional area observed in the non-immobilized leg. Integrated analysis revealed that differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were mainly enriched in pathways related to energy metabolism, such as fatty acid metabolism, oxidative phosphorylation (OXPHOS), and glycolysis. These results provide important insights for further research on disuse muscle atrophy.

The mechanosensitive gene arrestin domain containing 2 regulates myotube diameter with direct implications for disuse atrophy with aging.

Arrestin domain containing 2 and 3 (Arrdc2/3) are genes whose mRNA contents are decreased in young skeletal muscle following mechanical overload. Arrdc3 is linked to the regulation of signaling pathways in nonmuscle cells that could influence skeletal muscle size. Despite a similar amino acid sequence, Arrdc2 function remains undefined. The purpose of this study was to further explore the relationship of Arrdc2/Arrdc3 expression with changes in mechanical load in young and aged muscle and define the effect of Arrdc2/3 expression on C2C12 myotube diameter. In young and aged mice, mechanical load was decreased using hindlimb suspension whereas mechanical load was increased by reloading previously unloaded muscle or inducing high-force contractions. Arrdc2 and Arrdc3 mRNAs were overexpressed in C2C12 myotubes using adenoviruses. Myotube diameter was determined 48-h posttransfection, and RNA sequencing was performed on those samples. Arrdc2 and Arrdc3 mRNA content was higher in the unloaded muscle within 1 day of disuse and remained higher up through 10 days. The induction of Arrdc2 mRNA was more pronounced in aged muscle than young muscle in response to unloading. Reloading previously unloaded muscle of young and aged mice restored Arrdc2 and Arrdc3 levels to ambulatory levels. Increasing mechanical load beyond normal ambulatory levels lowered Arrdc2 mRNA, but not Arrdc3 mRNA, in young and aged muscle. Arrdc2 overexpression only was sufficient to lower myotube diameter in C2C12 cells in part by altering the transcriptome favoring muscle atrophy. These data are consistent with Arrdc2 contributing to disuse atrophy, particularly in aged muscle.NEW & NOTEWORTHY We establish Arrdc2 as a novel mechanosensitive gene highly induced in response to mechanical unloading, particularly in aged muscle. Arrdc2 induction in C2C12 myotubes is sufficient to produce thinner myotubes and a transcriptional landscape consistent with muscle atrophy and disuse.

Skeletal muscle-specific inducible AMPKα1/α2 knockout mice develop muscle weakness, glycogen depletion, and fibrosis that persists during disuse atrophy.

The 5' adenosine monophosphate-activated protein kinase (AMPK) is an important skeletal muscle regulator implicated as a possible therapeutic target to ameliorate the local undesired deconditioning of disuse atrophy. However, the muscle-specific role of AMPK in regulating muscle function, fibrosis, and transcriptional reprogramming during physical disuse is unknown. The purpose of this study was to determine how the absence of both catalytic subunits of AMPK in skeletal muscle influences muscle force production, collagen deposition, and the transcriptional landscape. We generated skeletal muscle-specific tamoxifen-inducible AMPKα1/α2 knockout (AMPKα-/-) mice that underwent 14 days of hindlimb unloading (HU) or remained ambulatory for 14 days (AMB). We found that AMPKα-/- during ambulatory conditions altered body weight and myofiber size, decreased muscle function, depleted glycogen stores and TBC1 domain family member 1 (TBC1D1) phosphorylation, increased collagen deposition, and altered transcriptional pathways. Primarily, pathways related to cellular senescence and mitochondrial biogenesis and function were influenced by the absence of AMPKα. The effects of AMPKα-/- persisted, but were not worsened, following hindlimb unloading. Together, we report that AMPKα is necessary to maintain skeletal muscle quality.NEW & NOTEWORTHY We determined that skeletal muscle-specific AMPKα knockout (KO) mice display functional, fibrotic, and transcriptional alterations before and during muscle disuse atrophy. We also observed that AMPKα KO drives muscle fibrosis and pathways related to cellular senescence that continues during the hindlimb unloading period.

Proteomic Identification of Seasonally Expressed Proteins Contributing to Heart Function and the Avoidance of Skeletal Muscle Disuse Atrophy in a Hibernating Mammal.

Hibernation in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) takes place over 4-6 months and is characterized by multiday bouts of hypothermic torpor (5-7 °C core body temperature) that are regularly interrupted every 1-2 weeks by brief (12-24 h) normothermic active periods called interbout arousals. Our goal was to gain insight into the molecular mechanisms that underlie the hibernator's ability to preserve heart function and avoid the deleterious effects of skeletal muscle disuse atrophy over prolonged periods of inactivity, starvation, and near-freezing body temperatures. To achieve this goal, we performed organelle enrichment of heart and skeletal muscle at five seasonal time points followed by LC-MS-based label-free quantitative proteomics. In both organs, we saw an increase in the levels of many proteins as ground squirrels transition from an active state to a prehibernation state in the fall. Interestingly, seasonal abundance patterns identified DHRS7C, SRL, TRIM72, RTN2, and MPZ as potential protein candidates for mitigating disuse atrophy in skeletal muscle, and ex vivo contractile mechanics analysis revealed no deleterious effects in the ground squirrel's muscles despite prolonged sedentary activity. Overall, an increased understanding of protein abundance in hibernators may enable novel therapeutic strategies to treat muscle disuse atrophy and heart disease in humans.

A non-invasive mouse model that recapitulates disuse-induced muscle atrophy in immobilized patients.

Disuse muscle atrophy occurs consequent to prolonged limb immobility or bed rest, which represents an unmet medical need. As existing animal models of limb immobilization often cause skin erosion, edema, and other untoward effects, we here report an alternative method via thermoplastic immobilization of hindlimbs in mice. While significant decreases in the weight and fiber size were noted after 7 days of immobilization, no apparent skin erosion or edema was found. To shed light onto the molecular mechanism underlying this muscle wasting, we performed the next-generation sequencing analysis of gastrocnemius muscles from immobilized versus non-mobilized legs. Among a total of 55,487 genes analyzed, 787 genes were differentially expressed (> fourfold; 454 and 333 genes up- and down-regulated, respectively), which included genes associated with muscle tissue development, muscle system process, protein digestion and absorption, and inflammation-related signaling. From a clinical perspective, this model may help understand the molecular/cellular mechanism that drives muscle disuse and identify therapeutic strategies for this debilitating disease.

The Structural Adaptations That Mediate Disuse-Induced Atrophy of Skeletal Muscle.

The maintenance of skeletal muscle mass plays a fundamental role in health and issues associated with quality of life. Mechanical signals are one of the most potent regulators of muscle mass, with a decrease in mechanical loading leading to a decrease in muscle mass. This concept has been supported by a plethora of human- and animal-based studies over the past 100 years and has resulted in the commonly used term of 'disuse atrophy'. These same studies have also provided a great deal of insight into the structural adaptations that mediate disuse-induced atrophy. For instance, disuse results in radial atrophy of fascicles, and this is driven, at least in part, by radial atrophy of the muscle fibers. However, the ultrastructural adaptations that mediate these changes remain far from defined. Indeed, even the most basic questions, such as whether the radial atrophy of muscle fibers is driven by the radial atrophy of myofibrils and/or myofibril hypoplasia, have yet to be answered. In this review, we thoroughly summarize what is known about the macroscopic, microscopic, and ultrastructural adaptations that mediated disuse-induced atrophy and highlight some of the major gaps in knowledge that need to be filled.

Plasticity changes in neuromuscular junction morphology and related regulatory proteins in the hibernating ground squirrel.

Skeletal muscle disuse atrophy can cause degenerative changes in neuromuscular junction morphology. Although Daurian ground squirrels (Spermophilus dauricus) are a natural anti-disuse animal model for studying muscle atrophy during hibernation, little is known about the morphological and regulatory mechanisms of their neuromuscular junctions. Here, we found that morphological indices of the soleus muscle were significantly lower during hibernation (torpor and interbout arousal) compared with pre-hibernation but recovered during post-hibernation. In the extensor digitorum longus muscle, neuromuscular junction morphology did not change significantly during hibernation. Agrin-Lrp4-MuSK is a key pathway for the formation and maintenance of the neuromuscular junction. Our results showed that low-density lipoprotein receptor-associated protein 4 (Lrp4) expression in the soleus (slow muscle) decreased by 46.2% in the interbout arousal group compared with the pre-hibernation group (P = 0.019), with recovery in the post-hibernation group. Compared with the pre-hibernation group, agrin expression in the extensor digitorum longus (fast muscle) increased by 67.0% in the interbout arousal group (P = 0.016). In conclusion, periodic up-regulation in agrin expression during interbout arousal may be involved in the maintenance of neuromuscular junction morphology in the extensor digitorum longus muscle during hibernation. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus during hibernation, these changes recovered to the pre-hibernation levels in the post-hibernation group, exhibiting significant plasticity. This plasticity may be one of the important mechanisms for resisting disuse atrophy in hibernating animals.NEW & NOTEWORTHY This study is the first to explore the neuromuscular junction morphology of slow- and fast-twitch muscles in Daurian ground squirrels during different periods of hibernation. Results showed that the neuromuscular junction maintained stable morphology in the extensor digitorum longus muscle. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus muscle during hibernation recovered in post-hibernation, exhibiting significant plasticity.

Skeletal muscle-specific DJ-1 ablation-induced atrogenes expression and mitochondrial dysfunction contributing to muscular atrophy.

BACKGROUND: DJ-1 is a causative gene for Parkinson's disease. DJ-1-deficient mice develop gait-associated progressive behavioural abnormalities and hypoactive forearm grip strength. However, underlying activity mechanisms are not fully explored. METHODS: Western blotting and quantitative real-time polymerase chain reaction approaches were adopted to analyse DJ-1 expression in skeletal muscle from aged humans or mice and compared with young subjects. Skeletal muscle-specific-DJ-1 knockout (MDKO) mice were generated, followed by an assessment of the physical activity phenotypes (grip strength, maximal load capacity, and hanging, rotarod, and exercise capacity tests) of the MDKO and control mice on the chow diet. Muscular atrophy phenotypes (cross-sectional area and fibre types) were determined by imaging and quantitative real-time polymerase chain reaction. Mitochondrial function and skeletal muscle morphology were evaluated by oxygen consumption rate and electron microscopy, respectively. Tail suspension was applied to address disuse atrophy. RNA-seq analysis was performed to indicate molecular changes in muscles with DJ-1 ablation. Dual-luciferase reporter assays were employed to identify the promoter region of Trim63 and Fbxo32 genes, which were indirectly regulated by DJ-1 via the FoxO1 pathway. Cytoplasmic and nuclear fractions of DJ-1-deleted muscle cells were analysed by western blotting. Compound 23 was administered into the gastrocnemius muscle to mimic the of DJ-1 deletion effects. RESULTS: DJ-1 expression decreased in atrophied muscles of aged human (young men, n = 2; old with aged men, n = 2; young women, n = 2; old with aged women, n = 2) and immobilization mice (n = 6, P < 0.01). MDKO mice exhibited no body weight difference compared with control mice on the chow diet (Flox, n = 8; MDKO, n = 9). DJ-1-deficient muscles were slightly dystrophic (Flox, n = 7; MDKO, n = 8; P < 0.05), with impaired physical activities and oxidative capacity (n = 8, P < 0.01). In disuse-atrophic conditions, MDKO mice showed smaller cross-sectional area (n = 5, P < 0.01) and more central nuclei than control mice (Flox, n = 7; MDKO, n = 6; P < 0.05), without alteration in muscle fibre types (Flox, n = 6; MDKO, n = 7). Biochemical analysis indicated that reduced mitochondrial function and upregulated of atrogenes induced these changes. Furthermore, RNA-seq analysis revealed enhanced activity of the FoxO1 signalling pathway in DJ-1-ablated muscles, which was responsible for the induction of atrogenes. Finally, compound 23 (an inhibitor of DJ-1) could mimic the effects of DJ-1 ablation in vivo. CONCLUSIONS: Our results illuminate the crucial of skeletal muscle DJ-1 in the regulation of catabolic signals from mechanical stimulation, providing a therapeutic target for muscle wasting diseases.

Impaired proteostatic mechanisms other than decreased protein synthesis limit old skeletal muscle recovery after disuse atrophy.

BACKGROUND: Skeletal muscle mass and strength diminish during periods of disuse but recover upon return to weight bearing in healthy adults but are incomplete in old muscle. Efforts to improve muscle recovery in older individuals commonly aim at increasing myofibrillar protein synthesis via mammalian target of rapamycin (mTOR) stimulation despite evidence demonstrating that old muscle has chronically elevated levels of mammalian target of rapamycin complex 1 (mTORC1) activity. We hypothesized that protein synthesis is higher in old muscle than adult muscle, which contributes to a proteostatic stress that impairs recovery. METHODS: We unloaded hindlimbs of adult (10-month) and old (28-month) F344BN rats for 14 days to induce atrophy, followed by reloading up to 60 days with deuterium oxide (D2 O) labelling to study muscle regrowth and proteostasis. RESULTS: We found that old muscle has limited recovery of muscle mass during reloading despite having higher translational capacity and myofibrillar protein synthesis (0.029 k/day ± 0.002 vs. 0.039 k/day ± 0.002, P < 0.0001) than adult muscle. We showed that collagen protein synthesis was not different (0.005 k (1/day) ± 0.0005 vs. 0.004 k (1/day) ± 0.0005, P = 0.15) in old compared to adult, but old muscle had higher collagen concentration (4.5 µg/mg ± 1.2 vs. 9.8 µg/mg ± 0.96, P < 0.01), implying that collagen breakdown was slower in old muscle than adult muscle. This finding was supported by old muscle having more insoluble collagen (4.0 ± 1.1 vs. 9.2 ± 0.9, P < 0.01) and an accumulation of advanced glycation end products (1.0 ± 0.06 vs. 1.5 ± 0.08, P < 0.001) than adult muscle during reloading. Limited recovery of muscle mass during reloading is in part due to higher protein degradation (0.017 1/t ± 0.002 vs. 0.028 1/t ± 0.004, P < 0.05) and/or compromised proteostasis as evidenced by accumulation of ubiquitinated insoluble proteins (1.02 ± 0.06 vs. 1.22 ± 0.06, P < 0.05). Last, we showed that synthesis of individual proteins related to protein folding/refolding, protein degradation and neural-related biological processes was higher in old muscle during reloading than adult muscle. CONCLUSIONS: Our data suggest that the failure of old muscle to recover after disuse is not due to limitations in the ability to synthesize myofibrillar proteins but because of other impaired proteostatic mechanisms (e.g., protein folding and degradation). These data provide novel information on individual proteins that accumulate in protein aggregates after disuse and certain biological processes such as protein folding and degradation that likely play a role in impaired recovery. Therefore, interventions to enhance regrowth of old muscle after disuse should be directed towards the identified impaired proteostatic mechanisms and not aimed at increasing protein synthesis.

An electrical stimulation intervention protocol to prevent disuse atrophy and muscle strength decline: an experimental study in rat.

BACKGROUND: Skeletal muscle is negatively impacted by conditions such as spaceflight or prolonged bed rest, resulting in a dramatic decline in muscle mass, maximum contractile force, and muscular endurance. Electrical stimulation (ES) is an essential tool in neurophysiotherapy and an effective means of preventing skeletal muscle atrophy and dysfunction. Historically, ES treatment protocols have used either low or high frequency electrical stimulation (LFES/HFES). However, our study tests the use of a combination of different frequencies in a single electrical stimulation intervention in order to determine a more effective protocol for improving both skeletal muscle strength and endurance. METHODS: An adult male SD rat model of muscle atrophy was established through 4 weeks of tail suspension (TS). To investigate the effects of different frequency combinations, the experimental animals were treated with low (20 Hz) or high (100 Hz) frequency before TS for 6 weeks, and during TS for 4weeks. The maximum contraction force and fatigue resistance of skeletal muscle were then assessed before the animals were sacrificed. The muscle mass, fiber cross-sectional area (CSA), fiber type and related protein expression were examined and analyzed to gain insights into the mechanisms by which the ES intervention protocol used in this study regulates muscle strength and endurance. RESULTS: After 4 weeks of unloading, the soleus muscle mass and fiber CSA decreased by 39% and 58% respectively, while the number of glycolytic muscle fibers increased by 21%. The gastrocnemius muscle fibers showed a 51% decrease in CSA, with a 44% decrease in single contractility and a 39% decrease in fatigue resistance. The number of glycolytic muscle fibers in the gastrocnemius also increased by 29%. However, the application of HFES either prior to or during unloading showed an improvement in muscle mass, fiber CSA, and oxidative muscle fibers. In the pre-unloading group, the soleus muscle mass increased by 62%, while the number of oxidative muscle fibers increased by 18%. In the during unloading group, the soleus muscle mass increased by 29% and the number of oxidative muscle fibers increased by 15%. In the gastrocnemius, the pre-unloading group showed a 38% increase in single contractile force and a 19% increase in fatigue resistance, while in the during unloading group, a 21% increase in single contractile force and a 29% increase in fatigue resistance was observed, along with a 37% and 26% increase in the number of oxidative muscle fibers, respectively. The combination of HFES before unloading and LFES during unloading resulted in a significant elevation of the soleus mass by 49% and CSA by 90%, with a 40% increase in the number of oxidative muscle fibers in the gastrocnemius. This combination also resulted in a 66% increase in single contractility and a 38% increase in fatigue resistance. CONCLUSION: Our results indicated that using HFES before unloading can reduce the harmful effects of muscle unloading on the soleus and gastrocnemius muscles. Furthermore, we found that combining HFES before unloading with LFES during unloading was more effective in preventing muscle atrophy in the soleus and preserving the contractile function of the gastrocnemius muscle.

Nicotinamide Phosphoribosyltransferase-elevated NAD+ biosynthesis prevents muscle disuse atrophy by reversing mitochondrial dysfunction.

BACKGROUND: It is well known that muscle disuse atrophy is associated with mitochondrial dysfunction, which is implicated in reduced nicotinamide adenine dinucleotide (NAD+ ) levels. Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in NAD+ biosynthesis, may serve as a novel strategy to treat muscle disuse atrophy by reversing mitochondrial dysfunction. METHODS: To investigate the effects of NAMPT on the prevention of disuse atrophy of skeletal muscles predominantly composed of slow-twitch (type I) or fast-twitch (type II) fibres, rabbit models of rotator cuff tear-induced supraspinatus muscle atrophy and anterior cruciate ligament (ACL) transection-induced extensor digitorum longus (EDL) atrophy were established and then administered NAMPT therapy. Muscle mass, fibre cross-sectional area (CSA), fibre type, fatty infiltration, western blot, and mitochondrial function were assayed to analyse the effects and molecular mechanisms of NAMPT in preventing muscle disuse atrophy. RESULTS: Acute disuse of the supraspinatus muscle exhibited significant loss of mass (8.86 ± 0.25 to 5.10 ± 0.79 g; P < 0.001) and decreased fibre CSA (3939.6 ± 136.1 to 2773.4 ± 217.6 µm2 , P < 0.001), which were reversed by NAMPT (muscle mass 6.17 ± 0.54 g, P = 0.0033; fibre CSA, 3219.8 ± 289.4 µm2 , P = 0.0018). Disuse-induced impairment of mitochondrial function were significantly improved by NAMPT, including citrate synthase activity (40.8 ± 6.3 to 50.5 ± 5.6 nmol/min/mg, P = 0.0043), and NAD+ biosynthesis (279.9 ± 48.7 to 392.2 ± 43.2 pmol/mg, P = 0.0023). Western blot revealed that NAMPT increases NAD+ levels by activating NAMPT-dependent NAD+ salvage synthesis pathway. In supraspinatus muscle atrophy due to chronic disuse, a combination of NAMPT injection and repair surgery was more effective than repair in reversing muscle atrophy. Although the predominant composition of EDL muscle is fast-twitch (type II) fibre type that differ from supraspinatus muscle, its mitochondrial function and NAD+ levels are also susceptible to disuse. Similar to the supraspinatus muscle, NAMPT-elevated NAD+ biosynthesis was also efficient in preventing EDL disuse atrophy by reversing mitochondrial dysfunction. CONCLUSIONS: NAMPT-elevated NAD+ biosynthesis can prevent disuse atrophy of skeletal muscles that predominantly composed with either slow-twitch (type I) or fast-twitch (type II) fibres by reversing mitochondrial dysfunction.

Global deletion of CCL2 has adverse impacts on recovery of skeletal muscle fiber size and function and is muscle specific.

Timely and complete recovery of muscle mass and function following a bout of physical disuse are critical components of returning to normal activities of daily living and lifestyle. Proper cross talk between the muscle tissue and myeloid cells (e.g., macrophages) throughout the recovery period from disuse atrophy plays a significant role in the complete resolution of muscle size and function. Chemokine C-C motif ligand 2 (CCL2) has a critical function of recruiting macrophages during the early phase of muscle damage. However, the importance of CCL2 has not been defined in the context of disuse and recovery. Here, we utilized a mouse model of whole body CCL2 deletion (CCL2KO) and subjected them to a period of hindlimb unloading followed by reloading to investigate the importance of CCL2 on the regrowth of muscle following disuse atrophy using ex vivo muscle tests, immunohistochemistry, and fluorescence-activated cell sorting approaches. We show mice that lack CCL2 display an incomplete recovery of gastrocnemius muscle mass, myofiber cross-sectional area, and EDL muscle contractile characteristics during the recovery from disuse atrophy. The soleus and plantaris had limited impact as a result of CCL2 deficiency suggesting a muscle-specific effect. Mice that lack CCL2 have decreased skeletal muscle collagen turnover, which may be related to defects in muscle function and stiffness. In addition, we show that the recruitment of macrophages to gastrocnemius muscle was dramatically reduced in CCL2KO mice during the recovery from disuse atrophy, which likely precipitated poor recovery of muscle size and function and aberrant collagen remodeling.NEW & NOTEWORTHY 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.

Extracellular vesicle distribution and localization in skeletal muscle at rest and following disuse atrophy.

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.

LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice.

Spinobulbar muscular atrophy (SBMA) is caused by CAG expansions in the androgen receptor gene. Androgen binding to polyQ-expanded androgen receptor triggers SBMA through a combination of toxic gain-of-function and loss-of-function mechanisms. Leveraging cell lines, mice, and patient-derived specimens, we show that androgen receptor co-regulators lysine-specific demethylase 1 (LSD1) and protein arginine methyltransferase 6 (PRMT6) are overexpressed in an androgen-dependent manner specifically in the skeletal muscle of SBMA patients and mice. LSD1 and PRMT6 cooperatively and synergistically transactivate androgen receptor, and their effect is enhanced by expanded polyQ. Pharmacological and genetic silencing of LSD1 and PRMT6 attenuates polyQ-expanded androgen receptor transactivation in SBMA cells and suppresses toxicity in SBMA flies, and a preclinical approach based on miRNA-mediated silencing of LSD1 and PRMT6 attenuates disease manifestations in SBMA mice. These observations suggest that targeting overexpressed co-regulators can attenuate androgen receptor toxic gain-of-function without exacerbating loss-of-function, highlighting a potential therapeutic strategy for patients with SBMA.

Treadmill Exercise Suppresses Histological Progression of Disuse Atrophy in Articular Cartilage in Rat Knee Joints during Hindlimb Suspension.

OBJECTIVE: The purpose of this study was to determine the preventive effects of treadmill exercise or physiological loading on disuse atrophy in the rat knee joint cartilage and bone during hindlimb suspension. DESIGN: Twenty male rats were divided into 4 experimental groups, including the control, hindlimb suspension, physiological loading, and treadmill walking groups. Histological changes in the articular cartilage and bone of the tibia were histomorphometrically and immunohistochemically evaluated 4 weeks after the intervention. RESULTS: Compared with the control group, the hindlimb suspension group showed thinning of cartilage thickness, decreased matrix staining, and decreased proportion of noncalcified layers. Cartilage thinning, decreased matrix staining, and decreased noncalcified layers were suppressed in the treadmill walking group. The physiological loading group exhibited no significant suppression of cartilage thinning or decreased noncalcified layers, but the decreased matrix staining was significantly suppressed. No significant prevention of bone mass loss or changes in subchondral bone thickness were detected after physiological loading or treadmill walking. CONCLUSION: Disuse atrophy of the articular cartilage caused by unloading conditions could be prevented by treadmill walking in rat knee joints.