bims-moremu 2022-12-11 papers

bims-moremu

Biomed News

on Molecular regulators of muscle mass

Issue of 2022–12–11
eightteen papers selected by
Anna Vainshtein, Craft Science Inc.

Loss of neuromuscular junction integrity and muscle atrophy in skeletal muscle disuse.
Chemokine-like receptor 1 plays a critical role in modulating the regenerative and contractile properties of muscle tissue.
Genome-wide analysis of a cellular exercise model based on electrical pulse stimulation.
Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation.
Ultrastructural and kinetic evidence support that thick filaments slide through the Z-disc.
Meteorin-like is an injectable peptide that can enhance regeneration in aged muscle through immune-driven fibro/adipogenic progenitor signaling.
ArfGAP3 regulates vesicle transport and glucose uptake in myoblasts.
Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function.
Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control.
The bidirectional relationship between AMPK pathway activation and myokine secretion in skeletal muscle: How it affects energy metabolism.
Ginsenoside Rb1 Prevents Oxidative Stress-Induced Apoptosis and Mitochondrial Dysfunction in Muscle Stem Cells via NF-κB Pathway.
Adiponectin receptors activation performs dual effects on regulating myogenesis and adipogenesis of young and aged muscle satellite cells.
Low-frequency electrical stimulation of bilateral hind legs by belt electrodes is effective for preventing denervation-induced atrophies in multiple skeletal muscle groups in rats.
Adaptation of the transcriptional response to resistance exercise over 4 weeks of daily training.
Depressed Protein Synthesis and Anabolic Signaling Potentiate ACL Tear-Resultant Quadriceps Atrophy.
DOK7 Promotes NMJ Regeneration After Nerve Injury.
Leucine ingestion promotes mTOR translocation to the periphery and enhances total and peripheral RPS6 phosphorylation in human skeletal muscle.
Impact of 10-day bed rest on serum levels of irisin and markers of musculoskeletal metabolism.

Ageing Res Rev. 2022 Nov 26. pii: S1568-1637(22)00252-5. [Epub ahead of print]83 101810

Loss of neuromuscular junction integrity and muscle atrophy in skeletal muscle disuse.

Giuseppe Sirago, Maria A Pellegrino, Roberto Bottinelli, Martino V Franchi, Marco V Narici.

  Physical inactivity (PI) is a major risk factor of chronic diseases. A major aspect of PI is loss of muscle mass and strength. The latter phenomenon significantly impacts daily life and represent a major issue for global health. Understandably, skeletal muscle itself has been the major focus of studies aimed at understanding the mechanisms underlying loss of mass and strength. Relatively lesser attention has been given to the contribution of alterations in somatomotor control, despite the fact that these changes can start very early and can occur at multiple levels, from the cortex down to the neuromuscular junction (NMJ). It is well known that exposure to chronic inactivity or immobilization causes a disproportionate loss of force compared to muscle mass, i.e. a loss of specific or intrinsic whole muscle force. The latter phenomenon may be partially explained by the loss of specific force of individual muscle fibres, but several other players are very likely to contribute to such detrimental phenomenon. Irrespective of the length of the disuse period, the loss of force is, in fact, more than two-fold greater than that of muscle size. It is very likely that somatomotor alterations may contribute to this loss in intrinsic muscle force. Here we review evidence that alterations of one component of somatomotor control, namely the neuromuscular junction, occur in disuse. We also discuss some of the novel players in NMJ stability (e.g., homer, bassoon, pannexin) and the importance of new established and emerging molecular markers of neurodegenerative processes in humans such as agrin, neural-cell adhesion molecule and light-chain neurofilaments.

Keywords:  Ageing; Disuse; Hemichannels; NMJ; Neurofilaments; Skeletal muscle atrophy

DOI:  https://doi.org/10.1016/j.arr.2022.101810
Front Physiol. 2022 ;13 1044488

Chemokine-like receptor 1 plays a critical role in modulating the regenerative and contractile properties of muscle tissue.

Julian Boesch, Eliane Pierrel, Christian Lambert, Arno Doelemeyer, Julie Kreider, Nathalie Accart, Serge Summermatter.

  Musculoskeletal diseases are a leading contributor to mobility disability worldwide. Since the majority of patients with musculoskeletal diseases present with associated muscle weakness, treatment approaches typically comprise an element of resistance training to restore physical strength. The health-promoting effects of resistance exercise are mediated via complex, multifarious mechanisms including modulation of systemic and local inflammation. Here we investigated whether targeted inhibition of the chemerin pathway, which largely controls inflammatory processes via chemokine-like receptor 1 (CMKLR1), can improve skeletal muscle function. Using genetically modified mice, we demonstrate that blockade of CMKLR1 transiently increases maximal strength during growth, but lastingly decreases strength endurance. In-depth analyses of the underlying long-term adaptations revealed microscopic alterations in the number of Pax7-positive satellite cells, as well as molecular changes in genes governing myogenesis and calcium handling. Taken together, these data provide evidence of a critical role for CMKLR1 in regulating skeletal muscle function by modulating the regenerative and contractile properties of muscle tissue. CMKLR1 antagonists are increasingly viewed as therapeutic modalities for a variety of diseases (e.g., psoriasis, metabolic disorders, and multiple sclerosis). Our findings thus have implications for the development of novel drug substances that aim at targeting the chemerin pathway for musculoskeletal or other diseases.

Keywords:  chemerin; endurance; satellite cells; skeletal muscle; strength

DOI:  https://doi.org/10.3389/fphys.2022.1044488
Sci Rep. 2022 Dec 08. 12(1): 21251

Genome-wide analysis of a cellular exercise model based on electrical pulse stimulation.

Bora Lee, Seon Kyu Kim, Yeo Jin Shin, Young Hoon Son, Jae Won Yang, Seung-Min Lee, Yong Ryul Yang, Kwang-Pyo Lee, Ki-Sun Kwon.

Skeletal muscle communicates with other organs via myokines, which are secreted by muscle during exercise and exert various effects. Despite much investigation of the exercise, the underlying molecular mechanisms are still not fully understood. Here, we applied an in vitro exercise model in which cultured C2C12 myotubes were subjected to electrical pulse stimulation (EPS), which mimics contracting muscle. Based on the significantly up- and down-regulated genes in EPS, we constructed an in silico model to predict exercise responses at the transcriptional level. The in silico model revealed similarities in the transcriptomes of the EPS and exercised animals. Comparative analysis of the EPS data and exercised mouse muscle identified putative biomarkers in exercise signaling pathways and enabled to discover novel exercise-induced myokines. Biochemical analysis of selected exercise signature genes in muscle from exercised mice showed that EPS mimics in vivo exercise, at least in part, at the transcriptional level. Consequently, we provide a novel myokine, Amphiregulin (AREG), up-regulated both in vitro and in vivo, that would be a potential target for exercise mimetics.

DOI: https://doi.org/10.1038/s41598-022-25758-2
Metabolomics. 2022 Dec 08. 18(12): 105

Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation.

Ram B Khattri, Jason Puglise, Terence E Ryan, Glenn A Walter, Matthew E Merritt, Elisabeth R Barton.

   INTRODUCTION: Fuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates.
OBJECTIVES: The goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism.
METHODS: Isolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles.
RESULTS: Pyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation.
CONCLUSION: Isotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.

Keywords:  Glucose metabolism; Metabolic tracer; Nuclear magnetic resonance (NMR); Skeletal muscle metabolism; Substrate specificity

DOI:  https://doi.org/10.1007/s11306-022-01948-x
J R Soc Interface. 2022 Dec;19(197): 20220642

Ultrastructural and kinetic evidence support that thick filaments slide through the Z-disc.

André Tomalka, Maximilian Heim, Annika Klotz, Christian Rode, Tobias Siebert.

  How myofilaments operate at short mammalian skeletal muscle lengths is unknown. A common assumption is that thick (myosin-containing) filaments get compressed at the Z-disc. We provide ultrastructural evidence of sarcomeres contracting down to 0.44 µm-approximately a quarter of thick filament resting length-in long-lasting contractions while apparently keeping a regular, parallel thick filament arrangement. Sarcomeres produced force at such extremely short lengths. Furthermore, sarcomeres adopted a bimodal length distribution with both modes below lengths where sarcomeres are expected to generate force in classic force-length measurements. Mammalian fibres did not restore resting length but remained short after deactivation, as previously reported for amphibian fibres, and showed increased forces during passive re-elongation. These findings are incompatible with viscoelastic thick filament compression but agree with predictions of a model incorporating thick filament sliding through the Z-disc. This more coherent picture of mechanical mammalian skeletal fibre functioning opens new perspectives on muscle physiology.

Keywords:  actin; delta-state; muscle physiology; myosin; skeletal muscle fibre; structural biology; titin

DOI:  https://doi.org/10.1098/rsif.2022.0642
Nat Commun. 2022 Dec 09. 13(1): 7613

Meteorin-like is an injectable peptide that can enhance regeneration in aged muscle through immune-driven fibro/adipogenic progenitor signaling.

David E Lee, Lauren K McKay, Akshay Bareja, Yongwu Li, Alastair Khodabukus, Nenad Bursac, Gregory A Taylor, Gurpreet S Baht, James P White.

Pathologies associated with sarcopenia include decline in muscular strength, lean mass and regenerative capacity. Despite the substantial impact on quality of life, no pharmacological therapeutics are available to counteract the age-associated decline in functional capacity and/or, resilience. Evidence suggests immune-secreted cytokines can improve muscle regeneration, a strategy which we leverage in this study by rescuing the age-related deficiency in Meteorin-like through several in vivo add-back models. Notably, the intramuscular, peptide injection of recombinant METRNL was sufficient to improve muscle regeneration in aging. Using ex vivo media exchange and in vivo TNF inhibition, we demonstrate a mechanism of METRNL action during regeneration, showing it counteracts a pro-fibrotic gene program by triggering TNFα-induced apoptosis of fibro/adipogenic progenitor cells. These findings demonstrate therapeutic applications for METRNL to improve aged muscle, and show Fibro/Adipogenic Progenitors are viable therapeutic targets to counteract age-related loss in muscle resilience.

DOI: https://doi.org/10.1038/s41467-022-35390-3
Cell Signal. 2022 Dec 04. pii: S0898-6568(22)00313-8. [Epub ahead of print] 110551

ArfGAP3 regulates vesicle transport and glucose uptake in myoblasts.

Suting Li, Zhi Wang, Mao Chen, Ya Xiao, Jie Min, Ming Hu, Jianming Tang, Li Hong.

  Skeletal muscle injuries are common, and damaged myofibers are repaired through proliferation and differentiation of muscle satellite cells. GLUT4 is enriched in GLUT4 storage vesicles (GSVs) and plays a crucial role in the maintenance of muscle function. ArfGAP3 regulates the vesicle transport especially for COPI coat assembly, but its effects on GSVs and the repair process after skeletal muscle injury remains unclear. In this study, datasets related to skeletal muscle injury and myoblast differentiation GSE469, GSE5413, GSE45577 and GSE108040 were retrieved through the GEO database and the expression of heptameric coat protein complex I (COPI) and Golgi vesicle transport-related genes in various datasets, as well as the expression correlation between ArfGAP2, ArfGAP3 and COPI-related genes COPA, COPB1, COPB2, COPE, COPZ1, COPZ2 were analyzed. The results suggested that ArfGAP3 was expressed in the process of repair after skeletal muscle injury and myoblast differentiation and that ArfGAP3 was positively correlated with COPI-related genes. In vitro experimental results showed that ArfGAP3 was colocalized with COPA, COPB, COPG protein, and GLUT4 in C2C12 myoblasts. After the downregulation of ArfGAP3 expression, intracellular vesicle transport, and glucose uptake were blocked, the proliferation of myoblasts under low glucose culture conditions was impaired, the proportion of apoptosis increased, and myotube differentiation was impaired. In summary, ArfGAP3 regulates COPI-associated vesicle and GSVs transport and affects the proliferation and differentiation ability of myoblasts by influencing glucose uptake, thereby modulating the repair process after skeletal muscle injury.

Keywords:  ArfGAP3; GLUT4; Myoblasts; Skeletal muscle injury; Vesicle transport

DOI:  https://doi.org/10.1016/j.cellsig.2022.110551
Elife. 2022 Dec 06. pii: e82951. [Epub ahead of print]11

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function.

Jeffrey Molendijk, Ronnie Blazev, Richard J Mills, Yaan-Kit Ng, Kevin I Watt, Daryn Chau, Paul Gregorevic, Peter J Crouch, James B W Hilton, Leszek Lisowski, Peixiang Zhang, Karen Reue, Aldons J Lusis, James Hudson, David E James, Marcus M Seldin, Benjamin L Parker.

  Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

Keywords:  computational biology; human; mouse; systems biology

DOI:  https://doi.org/10.7554/eLife.82951
iScience. 2022 Dec 22. 25(12): 105654

Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control.

Charlotte Gineste, Sonia Youhanna, Sabine U Vorrink, Sara Henriksson, Andrés Hernández, Arthur J Cheng, Thomas Chaillou, Andreas Buttgereit, Dominik Schneidereit, Oliver Friedrich, Kjell Hultenby, Joseph D Bruton, Niklas Ivarsson, Linda Sandblad, Volker M Lauschke, Håkan Westerblad.

  Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.

Keywords:  Cell biology; Cellular physiology; Developmental biology; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2022.105654
Front Physiol. 2022 ;13 1040809

The bidirectional relationship between AMPK pathway activation and myokine secretion in skeletal muscle: How it affects energy metabolism.

Mahdi Ahsan, Léa Garneau, Céline Aguer.

  Myokines are peptides and proteins secreted by skeletal muscle cells, into the interstitium, or in the blood. Their regulation may be dependent or independent of muscle contraction to induce a variety of metabolic effects. Numerous myokines have been implicated in influencing energy metabolism via AMP-activated protein kinase (AMPK) signalling. As AMPK is centrally involved in glucose and lipid metabolism, it is important to understand how myokines influence its signalling, and vice versa. Such insight will better elucidate the mechanism of metabolic regulation during exercise and at rest. This review encompasses the latest research conducted on the relationship between AMPK signalling and myokines within skeletal muscles via autocrine or paracrine signalling.

Keywords:  AMPK; cell signalling; glucose metabolism; lipid metabolism; myokines; skeletal muscle

DOI:  https://doi.org/10.3389/fphys.2022.1040809
Oxid Med Cell Longev. 2022 ;2022 9159101

Ginsenoside Rb1 Prevents Oxidative Stress-Induced Apoptosis and Mitochondrial Dysfunction in Muscle Stem Cells via NF-κB Pathway.

Wenxi Dong, Wenhao Chen, Hongbo Zou, Zile Shen, Dingye Yu, Weizhe Chen, Haojie Jiang, Xialin Yan, Zhen Yu.

Sarcopenia, featured by the progressive loss of skeletal muscle function and mass, is associated with the impaired function of muscle stem cells (MuSCs) caused by increasing oxidative stress in senescent skeletal muscle tissue during aging. Intact function of MuSCs maintains the regenerative potential as well as the homeostasis of skeletal muscle tissues during aging. Ginsenoside Rb1, a natural compound from ginseng, exhibited the effects of antioxidation and against apoptosis. However, its effects of restoring MuSC function during aging and improving age-related sarcopenia remained unknown. In this study, we investigated the role of Rb1 in improving MuSC function and inhibiting apoptosis by reducing oxidative stress levels. We found that Rb1 inhibited the accumulation of reactive oxygen species (ROS) and protected the cells from oxidative stress to attenuate the H2O2-induced cytotoxicity. Rb1 also blocked oxidative stress-induced apoptosis by inhibiting the activation of caspase-3/9, which antagonized the decrease in mitochondrial content and the increase in mitochondrial abnormalities caused by oxidative stress via promoting the protein expression of genes involved in mitochondrial biogenesis. Mechanistically, it was proven that Rb1 exerted its antioxidant effects and avoided the apoptosis of myoblasts by targeting the core regulator of the nuclear factor-kappa B (NF-κB) signal pathway. Therefore, these findings suggest that Rb1 may have a beneficial role in the prevention and treatment of MuSC exhaustion-related diseases like sarcopenia.

DOI: https://doi.org/10.1155/2022/9159101
Cell Prolif. 2022 Dec 09. e13370

Adiponectin receptors activation performs dual effects on regulating myogenesis and adipogenesis of young and aged muscle satellite cells.

Shibo Liu, Hanghang Liu, Yao Liu, Ju Zhang, Zhikai Liu, Zizhuo Zheng, En Luo.

OBJECTIVES: Skeletal muscle mass and function deteriorate with ageing. Adiponectin receptors (APNrs), mainly activated by adiponectin, participate in various physiological activities and have varying signalling pathways at different ages. This study aimed to explore whether discrepant performance exists in APNr activation regulating young and aged muscle satellite cells (MUSCs) and whether age-related muscle dysfunction could be alleviated upon APNr activation.
METHODS: The gastrocnemius muscle phenotype was observed in male mice aged 2 and 18 months. An APNr agonist (AdipoRon) was used in vitro and in vivo to investigate the changes in cell biological behaviours and whether muscle dysfunction could be retarded after APNr activation.
RESULTS: Aged mice exhibited decreased muscle mass and increased fat infiltration. APNr activation inhibited C2C12 cells and young MUSCs (YMUSCs) proliferation but showed no obvious effect on aged MUSCs (AMUSCs). Moreover, APNr activation inhibited the migration of both YMUSCs and AMUSCs. Interestingly, APNr activation hampered the myogenic differentiation but advanced the adipogenic differentiation of YMUSCs, yet exact opposite results were presented in AMUSCs. It was demonstrated that Wnt and PI3K signalling pathways may mediate the phenotypic differences. Furthermore, in vivo experiments verified that APNr activation ameliorated age-related muscle atrophy and excessive fat infiltration.
CONCLUSIONS: APNr activation exerted dual effects on the regulation of myogenesis and adipogenesis of YMUSCs and AMUSCs and rescued age-related skeletal muscle dysfunction.

DOI: https://doi.org/10.1111/cpr.13370
Sci Rep. 2022 Dec 08. 12(1): 21275

Low-frequency electrical stimulation of bilateral hind legs by belt electrodes is effective for preventing denervation-induced atrophies in multiple skeletal muscle groups in rats.

Hiroyuki Uno, Shohei Kamiya, Ryuji Akimoto, Katsu Hosoki, Shunta Tadano, Karina Kouzaki, Yuki Tamura, Takaya Kotani, Mako Isemura, Koichi Nakazato.

Belt electrode skeletal muscle electrical stimulation (B-SES) can simultaneously contract multiple muscle groups. Although the beneficial effects of B-SES in clinical situations have been elucidated, its molecular mechanism remains unknown. In this study, we developed a novel rodent B-SES ankle stimulation system to test whether low-frequency stimulation prevents denervation-induced muscle atrophy. Electrical stimulations (7‒8 Hz, 30 min) with ankle belt electrodes were applied to Sprague-Dawley rats daily for one week. All animals were assigned to the control (CONT), denervation-induced atrophy (DEN), and DEN + electrical stimulation (ES) groups. The tibialis anterior (TA) and gastrocnemius (GAS) muscles were used to examine the effect of ES treatment. After seven daily sessions of continuous stimulation, muscle wet weight (n = 8-11), and muscle fiber cross-sectional area (CSA, n = 4-6) of TA and GAS muscles were lower in DEN and DEN + ES than in CON. However, it was significantly higher in DEN than DEN + ES, showing that ES partially prevented muscle atrophy. PGC-1α, COX-IV, and citrate synthase activities (n = 6) were significantly higher in DEN + ES than in DEN. The mRNA levels of muscle proteolytic molecules, Atrogin-1 and Murf1, were significantly higher in DEN than in CONT, while B-SES significantly suppressed their expression (p < 0.05). In conclusion, low-frequency electrical stimulation of the bilateral ankles using belt electrodes (but not the pad electrodes) is effective in preventing denervation-induced atrophy in multiple muscles, which has not been observed with pad electrodes. Maintaining the mitochondrial quantity and enzyme activity by low-frequency electrical stimulation is key to suppressing muscle protein degradation.

DOI: https://doi.org/10.1038/s41598-022-25359-z
FASEB J. 2023 Jan;37(1): e22686

Adaptation of the transcriptional response to resistance exercise over 4 weeks of daily training.

Mark R Viggars, Hazel Sutherland, Hermann Lanmüller, Martin Schmoll, Manfred Bijak, Jonathan C Jarvis.

We present the time course of change in the muscle transcriptome 1 h after the last exercise bout of a daily resistance training program lasting 2, 10, 20, or 30 days. Daily exercise in rat tibialis anterior muscles (5 sets of 10 repetitions over 20 min) induced progressive muscle growth that approached a new stable state after 30 days. The acute transcriptional response changed along with progressive adaptation of the muscle phenotype. For example, expression of type 2B myosin was silenced. Time courses recently synthesized from human exercise studies do not demonstrate so clearly the interplay between the acute exercise response and the longer-term consequences of repeated exercise. We highlight classes of transcripts and transcription factors whose expression increases during the growth phase and declines again as the muscle adapts to a new daily pattern of activity and reduces its rate of growth. Myc appears to play a central role.

DOI: https://doi.org/10.1096/fj.202201418R
Am J Sports Med. 2022 Dec 07. 3635465221135769

Depressed Protein Synthesis and Anabolic Signaling Potentiate ACL Tear-Resultant Quadriceps Atrophy.

Alexander R Keeble, Camille R Brightwell, Christine M Latham, Nicholas T Thomas, C Brooks Mobley, Kevin A Murach, Darren L Johnson, Brian Noehren, Christopher S Fry.

   BACKGROUND: Anterior cruciate ligament (ACL) tear (ACLT) leads to protracted quadriceps muscle atrophy. Protein turnover largely dictates muscle size and is highly responsive to injury and loading. Regulation of quadriceps molecular protein synthetic machinery after ACLT has largely been unexplored, limiting development of targeted therapies.
PURPOSE: To define the effect of ACLT on (1) the activation of protein synthetic and catabolic signaling within quadriceps biopsy specimens from human participants and (2) the time course of alterations to protein synthesis and its molecular regulation in a mouse ACL injury model.
STUDY DESIGN: Descriptive laboratory study.
METHODS: Muscle biopsy specimens were obtained from the ACL-injured and noninjured vastus lateralis of young adult humans after an overnight fast (N = 21; mean ± SD, 19 ± 5 years). Mice had their limbs assigned to ACLT or control, and whole quadriceps were collected 6 hours or 1, 3, or 7 days after injury with puromycin injected before tissue collection for assessment of relative protein synthesis. Muscle fiber size and expression and phosphorylation of protein anabolic and catabolic signaling proteins were assessed at the protein and transcript levels (RNA sequencing).
RESULTS: Human quadriceps showed reduced phosphorylation of ribosomal protein S6 (-41%) in the ACL-injured limb (P = .008), in addition to elevated phosphorylation of eukaryotic initiation factor 2α (+98%; P = .006), indicative of depressed protein anabolic signaling in the injured limb. No differences in E3 ubiquitin ligase expression were noted. Protein synthesis was lower at 1 day (P = .01 vs control limb) and 3 days (P = .002 vs control limb) after ACLT in mice. Pathway analyses revealed shared molecular alterations between human and mouse quadriceps after ACLT.
CONCLUSION: (1) Global protein synthesis and anabolic signaling deficits occur in the quadriceps in response to ACL injury, without notable changes in measured markers of muscle protein catabolism. (2) Importantly, these deficits occur before the onset of significant atrophy, underscoring the need for early intervention.
CLINICAL RELEVANCE: These findings suggest that blunted protein anabolism as opposed to increased catabolism likely mediates quadriceps atrophy after ACL injury. Thus, future interventions should aim to restore muscle protein anabolism rapidly after ACLT.

Keywords:  E3 ubiquitin ligase; anterior cruciate ligament; protein metabolism; skeletal muscle

DOI:  https://doi.org/10.1177/03635465221135769
Mol Neurobiol. 2022 Dec 05.

DOK7 Promotes NMJ Regeneration After Nerve Injury.

Ethan D Kosco, Hongyang Jing, Peng Chen, Wen-Cheng Xiong, Ivy S Samuels, Lin Mei.

  Motor function recovery from injury requires the regeneration of not only muscle fibers, but also the neuromuscular junction-the synapse between motor nerve terminals and muscle fibers. However, unlike muscle regeneration which has been extensively studied, little is known about the molecular mechanisms of NMJ regeneration. Recognizing the critical role of agrin-LRP4-MuSK signaling in NMJ formation and maintenance, we investigated whether increasing MuSK activity promotes NMJ regeneration. To this end, we evaluated the effect of DOK7, a protein that stimulates MuSK, on NMJ regeneration. Reinnervation, AChR cluster density, and endplate area were improved, and fragmentation was reduced in the AAV9-DOK7-GFP-injected muscles compared with muscles injected with AAV9-GFP. These results demonstrated expedited NMJ regeneration associated with increased DOK7 expression and support the hypothesis that increasing agrin signaling benefits motor function recovery after injury. Our findings propose a potentially new therapeutic strategy for functional recovery after muscle and nerve injury, i.e., promoting NMJ regeneration by increasing agrin signaling.

Keywords:  Adeno-associated virus serotype 9; DOK7; Nerve injury; Neuromuscular junction; Regeneration

DOI:  https://doi.org/10.1007/s12035-022-03143-4
Amino Acids. 2022 Dec 06.

Leucine ingestion promotes mTOR translocation to the periphery and enhances total and peripheral RPS6 phosphorylation in human skeletal muscle.

Maksym N H Holowaty, Matthew J Lees, Sidney Abou Sawan, Kevin J M Paulussen, Ralf Jäger, Martin Purpura, Scott A Paluska, Nicholas A Burd, Nathan Hodson, Daniel R Moore.

  The activation of the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, by anabolic stimuli (such as muscle contraction or essential amino acids) involves its translocation to the cell periphery. Leucine is generally considered the most anabolic of amino acids for its ability to independently modulate muscle protein synthesis. However, it is currently unknown if free leucine impacts region-specific mTORC1-mediated phosphorylation events and protein-protein interactions. In this clinical trial (NCT03952884; registered May 16, 2019), we used immunofluorescence methods to investigate the role of dietary leucine on the postprandial regulation of mTORC1 and ribosomal protein S6 (RPS6), an important downstream readout of mTORC1 activity. Eight young, healthy, recreationally active males (n = 8; 23 ± 3 yrs) ingested 2 g of leucine with vastus lateralis biopsies collected at baseline, 30, 60, and 180 min postprandial. Leucine promoted mTOR translocation to the periphery (~ 18-29%; p ≤ 0.012) and enhanced mTOR localization with the lysosome (~ 16%; both p = 0.049) at 30 and 60 min post-feeding. p-RPS6Ser240/244 staining intensity, a readout of mTORC1 activity, was significantly elevated at all postprandial timepoints in both the total fiber (~ 14-30%; p ≤ 0.032) and peripheral regions (~ 16-33%; p ≤ 0.014). Additionally, total and peripheral p-RPS6Ser240/244 staining intensity at 60 min was positively correlated (r = 0.74, p = 0.036; r = 0.80, p = 0.016, respectively) with rates of myofibrillar protein synthesis over 180 min. The ability of leucine to activate mTORC1 in peripheral regions favors an enhanced rate of MPS, as this is the intracellular space thought to be replete with the cellular machinery that facilitates this anabolic process.

Keywords:  Amino acids; Anabolism; Immunofluorescence; Muscle protein synthesis; Protein trafficking; mRNA translation

DOI:  https://doi.org/10.1007/s00726-022-03221-w
FASEB J. 2023 Jan;37(1): e22668

Impact of 10-day bed rest on serum levels of irisin and markers of musculoskeletal metabolism.

Angela Oranger, Giuseppina Storlino, Manuela Dicarlo, Roberta Zerlotin, Patrizia Pignataro, Lorenzo Sanesi, Marco Narici, Rado Pišot, Bostjan Simunič, Graziana Colaianni, Maria Grano, Silvia Colucci.

  The bed rest (BR) is a ground-based model to simulate microgravity mimicking skeletal-muscle alterations as in spaceflight. Molecular coupling between bone and muscle might be involved in physiological and pathological conditions. Thus, the new myokine irisin and bone-muscle turnover markers have been studied during and after 10 days of BR. Ten young male individuals were subjected to 10 days of horizontal BR. Serum concentrations of irisin, myostatin, sclerostin, and haptoglobin were assessed, and muscle tissue gene expression on vastus lateralis biopsies was determined. During 10-days BR, we observed no significant fluctuation levels of irisin, myostatin, and sclerostin. Two days after BR (R+2), irisin serum levels significantly decreased while myostatin, sclerostin, and haptoglobin were significantly increased compared with BR0. Gene expression of myokines, inflammatory molecules, transcription factors, and markers of muscle atrophy and senescence on muscle biopsies were not altered, suggesting that muscle metabolism of young, healthy subjects is able to adapt to the hypomobility condition during 10-day BR. However, when subjects were divided according to irisin serum levels at BR9, muscle ring finger-1 mRNA expression was significantly lower in subjects with higher irisin serum levels, suggesting that this myokine may prevent the triggering of muscle atrophy. Moreover, the negative correlation between p21 mRNA and irisin at BR9 indicated a possible inhibitory effect of the myokine on the senescence marker. In conclusion, irisin could be a prognostic marker of hypomobility-induced muscle atrophy, and its serum levels could protect against muscle deterioration by preventing and/or delaying the expression of atrophy and senescence cellular markers.

Keywords:  bed rest; cell senescence; haptoglobin; irisin; muscle atrophy; myostatin; sclerostin

DOI:  https://doi.org/10.1096/fj.202201005RR