bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021–08–29
forty-six papers selected by
Anna Vainshtein, Craft Science Inc.
  1. Voluntary exercise prevents abnormal muscle mitochondrial morphology in cancer cachexia mice.
  2. p21-activated kinase 4 phosphorylates peroxisome proliferator-activated receptor Υ and suppresses skeletal muscle regeneration.
  3. Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines.
  4. Reversal of deficits in aged skeletal muscle during disuse and recovery in response to treatment with a secrotome product derived from partially differentiated human pluripotent stem cells.
  5. Identification of Potentially Related Genes and Mechanisms Involved in Skeletal Muscle Atrophy Induced by Excessive Exercise in Zebrafish.
  6. Phosphorylation of Dynamin-Related Protein 1 (DRP1) Regulates Mitochondrial Dynamics and Skeletal Muscle Wasting in Cancer Cachexia.
  7. Identification of a KLF5-dependent program and drug development for skeletal muscle atrophy.
  8. Pathological Mechanism of a Constitutively Active Form of Stromal Interaction Molecule 1 in Skeletal Muscle.
  9. MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis.
  10. Interaction of Fibromodulin and Myostatin to Regulate Skeletal Muscle Aging: An Opposite Regulation in Muscle Aging, Diabetes, and Intracellular Lipid Accumulation.
  11. CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration.
  12. Effects of hyperoxia and hypoxia on the proliferation of C2C12 myoblasts.
  13. Regulation of Myogenic Differentiation by Topologically Microgrooved Surfaces for Skeletal Muscle Tissue Engineering.
  14. c-Myc overexpression increases ribosome biogenesis and protein synthesis independent of mTORC1 activation in mouse skeletal muscle.
  15. Skeletal Muscle Cell Growth Alters the Lipid Composition of Extracellular Vesicles.
  16. UBE2L3, a Partner of MuRF1/TRIM63, Is Involved in the Degradation of Myofibrillar Actin and Myosin.
  17. Polyphenols and Their Effects on Muscle Atrophy and Muscle Health.
  18. BMP2 downregulates urokinase-type plasminogen activator via p38 MAPK: Implications in C2C12 cells myogenic differentiation.
  19. SLIT3 promotes myogenic differentiation as a novel therapeutic factor against muscle loss.
  20. Curcumin and Resveratrol Improve Muscle Function and Structure through Attenuation of Proteolytic Markers in Experimental Cancer-Induced Cachexia.
  21. Insulin and 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Differentially Regulate the Skeletal Muscle Cell Secretome.
  22. Transcriptome Analysis in a Primary Human Muscle Cell Differentiation Model for Myotonic Dystrophy Type 1.
  23. Driving fibrosis in neuromuscular diseases: Role and regulation of Connective tissue growth factor (CCN2/CTGF).
  24. TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration.
  25. SESN2 protects against denervated muscle atrophy through unfolded protein response and mitophagy.
  26. Signals from the Circle: Tricarboxylic Acid Cycle Intermediates as Myometabokines.
  27. Integral Membrane Protein 2A Is a Negative Regulator of Canonical and Non-Canonical Hedgehog Signalling.
  28. Redox Signaling and Sarcopenia: Searching for the Primary Suspect.
  29. The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies.
  30. Dystrophin-negative slow-twitch soleus muscles are not susceptible to eccentric contraction induced injury over the lifespan of the mdx mouse.
  31. The Key Lnc (RNA)s in Cardiac and Skeletal Muscle Development, Regeneration, and Disease.
  32. Mesenchymal Stem Cell-Derived Exosomes Protect Muscle Loss by miR-145-5p Activity Targeting Activin A Receptors.
  33. Calpain 6 inhibits autophagy in inflammatory environments: A preliminary study on myoblasts and a chronic kidney disease rat model.
  34. Resistance training rejuvenates the mitochondrial methylome in aged human skeletal muscle.
  35. Characterization of the Skeletal Muscle Secretome Reveals a Role for Extracellular Vesicles and IL1α/IL1β in Restricting Fibro/Adipogenic Progenitor Adipogenesis.
  36. Development and progression of cancer cachexia: Perspectives from bench to bedside.
  37. The ubiquitin ligase Ozz decreases the replacement rate of embryonic myosin in myofibrils.
  38. Regular, Intense Exercise Training as a Healthy Aging Lifestyle Strategy: Preventing DNA Damage, Telomere Shortening and Adverse DNA Methylation Changes Over a Lifetime.
  39. The Leucine Catabolite and Dietary Supplement β-Hydroxy-β-Methyl Butyrate (HMB) as an Epigenetic Regulator in Muscle Progenitor Cells.
  40. Under the Hood: Skeletal Muscle Determinants of Endurance Performance.
  41. Circulating myomiRs in Muscle Denervation: From Surgical to ALS Pathological Condition.
  42. N-acetyltaurine and Acetylcarnitine Production for the Mitochondrial Acetyl-CoA Regulation in Skeletal Muscles during Endurance Exercises.
  43. Regular Physical Exercise Modulates Iron Homeostasis in the 5xFAD Mouse Model of Alzheimer's Disease.
  44. Adiponectin overexpression in C2C12 myocytes increases lipid oxidation and myofiber transition.
  45. Clock proteins and training modify exercise capacity in a daytime-dependent manner.
  46. Early Onset Physical Inactivity and Metabolic Dysfunction in Tumor-bearing Mice Is Associated with Accelerated Cachexia.
  1. Physiol Rep. 2021 Aug;9(16): e15016
    Voluntary exercise prevents abnormal muscle mitochondrial morphology in cancer cachexia mice.
    Yu Kitaoka, Mitsunori Miyazaki, Shin Kikuchi.
      This study aimed to examine the effects of voluntary wheel running on cancer cachexia-induced mitochondrial alterations in mouse skeletal muscle. Mice bearing colon 26 adenocarcinoma (C26) were used as a model of cancer cachexia. C26 mice showed a lower gastrocnemius and plantaris muscle weight, but 4 weeks of voluntary exercise rescued these changes. Further, voluntary exercise attenuated observed declines in the levels of oxidative phosphorylation proteins and activities of citrate synthase and cytochrome c oxidase in the skeletal muscle of C26 mice. Among mitochondrial morphology regulatory proteins, mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1) were decreased in the skeletal muscle of C26 mice, but exercise resulted in similar improvements as seen in markers of mitochondrial content. In isolated mitochondria, 4-hydroxynonenal and protein carbonyls were elevated in C26 mice, but exercise blunted the increases in these markers of oxidative stress. In addition, electron microscopy revealed that exercise alleviated the observed increase in the percentage of damaged mitochondria in C26 mice. These results suggest that voluntary exercise effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cachexia.
    Keywords:  cancer cachexia; mitochondria; oxidative stress; skeletal muscle; voluntary exercise
    DOI:  https://doi.org/10.14814/phy2.15016
  2. J Cachexia Sarcopenia Muscle. 2021 Aug 24.
    p21-activated kinase 4 phosphorylates peroxisome proliferator-activated receptor Υ and suppresses skeletal muscle regeneration.
    Yuancheng Mao, Chang Yeob Han, Lihua Hao, In Hyuk Bang, Eun Ju Bae, Byung-Hyun Park.
       BACKGROUND: Skeletal muscle regeneration is an adaptive response to injury that is crucial to the maintenance of muscle mass and function. A p21-activated kinase 4 (PAK4) serine/threonine kinase is critical to the regulation of cytoskeletal changes, cell proliferation, and growth. However, PAK4's role in myoblast differentiation and regenerative myogenesis remains to be determined.
    METHODS: We used a mouse model of myotoxin (notexin)-induced muscle regeneration. In vitro myogenesis was performed in the C2C12 myoblast cell line, primary myoblasts, and primary satellite cells. In vivo overexpression of PAK4 or kinase-inactive mutant PAK4S474A was conducted in skeletal muscle to examine PAK4's kinase-dependent effect on muscle regeneration. The regeneration process was evaluated by determining the number and size of multinucleated myofibres and expression patterns of myogenin and eMyHC. To explore whether PAK4 inhibition improves muscle regeneration, mice were injected intramuscularly with siRNA that targeted PAK4 or orally administered with a chemical inhibitor of PAK4.
    RESULTS: p21-activated kinase 4 was highly expressed during the myoblast stage, but expression gradually and substantially decreased as myoblasts differentiated into myotubes. PAK4 overexpression, but not kinase-inactive mutant PAK4S474A overexpression, significantly impeded myoblast fusion and MyHC-positive myotube formation in C2C12 cells, primary myoblasts, and satellite cells (P < 0.01). Conversely, PAK4 silencing led to an 8.7% and a 20.3% increase in the number of multinucleated larger myotubes in C2C12 cells and primary myoblasts. Further, in vivo overexpression of PAK4 by adenovirus injection to mice prior to and after myotoxin-induced injury led to a 52.6% decrease in the number of eMyHC-positive myofibres on Day 5 in tibialis anterior muscles as compared with those injected with control adenoviruses (P < 0.01), while Ad-PAK4S474A showed comparable muscle regeneration parameters. PAK4-induced repression of muscle regeneration coincided with an increase in phosphatase and tensin homologue (PTEN) expression and a decrease in phosphoinositide 3-kinase-Akt signalling. In contrast, PAK4 silencing reduced PTEN expression in mice. Consistent with these findings, prodrug of PAK4 inhibitor CZh-226 (30 mg/kg) orally administered to mice repressed PTEN expression and accelerated myotube formation. Subsequent mechanistic studies revealed that PAK4 directly phosphorylates PPARγ at S273 to increase its transcription activity, thereby up-regulating PTEN expression. Importantly, an analysis of the Genotype-Tissue Expression database showed a positive correlation between PAK4 and PTEN in human skeletal muscle tissues (P < 0.01).
    CONCLUSIONS: p1-activated kinase 4 is a new member of PPARγ kinase, and PAK4 inhibition may have a therapeutic role as an accelerant of muscle regeneration.
    Keywords:  Muscle regeneration; Myogenesis; PAK4; PPARγ; PTEN
    DOI:  https://doi.org/10.1002/jcsm.12774
  3. Exp Cell Res. 2021 Aug 21. pii: S0014-4827(21)00332-3. [Epub ahead of print] 112779
    Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines.
    Paige C Arneson-Wissink, Jason D Doles.
      Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in CC. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.
    Keywords:  IFNγ; NOS2; TNFα; cancer cachexia; myoblast; nitric oxide
    DOI:  https://doi.org/10.1016/j.yexcr.2021.112779
  4. Geroscience. 2021 Aug 24.
    Reversal of deficits in aged skeletal muscle during disuse and recovery in response to treatment with a secrotome product derived from partially differentiated human pluripotent stem cells.
    Dennis K Fix, Ziad S Mahmassani, Jonathan J Petrocelli, Naomi M M P de Hart, Patrick J Ferrara, Jessie S Painter, Gabriel Nistor, Thomas E Lane, Hans S Keirstead, Micah J Drummond.
      Aged individuals are at risk to experience slow and incomplete muscle recovery following periods of disuse atrophy. While several therapies have been employed to mitigate muscle mass loss during disuse and improve recovery, few have proven effective at both. Therefore, the purpose of this study was to examine the effectiveness of a uniquely developed secretome product (STEM) on aged skeletal muscle mass and function during disuse and recovery. Aged (22 months) male C57BL/6 were divided into PBS or STEM treatment (n = 30). Mice within each treatment were assigned to either ambulatory control (CON; 14 days of normal cage ambulation), 14 days of hindlimb unloading (HU), or 14 days of hindlimb unloading followed by 7 days of recovery (recovery). Mice were given an intramuscular delivery into the hindlimb muscle of either PBS or STEM every other day for the duration of their respective treatment group. We found that STEM-treated mice compared to PBS had greater soleus muscle mass, fiber cross-sectional area (CSA), and grip strength during CON and recovery experimental conditions and less muscle atrophy and weakness during HU. Muscle CD68 +, CD11b + and CD163 + macrophages were more abundant in STEM-treated CON mice compared to PBS, while only CD68 + and CD11b + macrophages were more abundant during HU and recovery conditions with STEM treatment. Moreover, STEM-treated mice had lower collagen IV and higher Pax7 + cell content compared to PBS across all experimental conditions. As a follow-up to examine the cell autonomous role of STEM on muscle, C2C12 myotubes were given STEM or horse serum media to examine myotube fusion/size and effects on muscle transcriptional networks. STEM-treated C2C12 myotubes were larger and had a higher fusion index and were related to elevated expression of transcripts associated with extracellular matrix remodeling. Our results demonstrate that STEM is a unique cocktail that possesses potent immunomodulatory and cytoskeletal remodeling properties that may have translational potential to improve skeletal muscle across a variety of conditions that adversely effect aging muscle.
    Keywords:  Atrophy; Fibrosis; Immune cells; Inflammation; Sarcopenia; Stem cells
    DOI:  https://doi.org/10.1007/s11357-021-00423-0
  5. Biology (Basel). 2021 Aug 10. pii: 761. [Epub ahead of print]10(8):
    Identification of Potentially Related Genes and Mechanisms Involved in Skeletal Muscle Atrophy Induced by Excessive Exercise in Zebrafish.
    Chen-Chen Sun, Zuo-Qiong Zhou, Zhang-Lin Chen, Run-Kang Zhu, Dong Yang, Xi-Yang Peng, Lan Zheng, Chang-Fa Tang.
      Long-term imbalance between fatigue and recovery may eventually lead to muscle weakness or even atrophy. We previously reported that excessive exercise induces pathological cardiac hypertrophy. However, the effect of excessive exercise on the skeletal muscles remains unclear. In the present study, we successfully established an excessive-exercise-induced skeletal muscle atrophy zebrafish model, with decreased muscle fiber size, critical swimming speed, and maximal oxygen consumption. High-throughput RNA-seq analysis identified differentially expressed genes in the model system compared with control zebrafish. Gene ontology and KEGG enrichment analysis revealed that the upregulated genes were enriched in autophagy, homeostasis, circadian rhythm, response to oxidative stress, apoptosis, the p53 signaling pathway, and the FoxO signaling pathway. Protein-protein interaction network analysis identified several hub genes, including keap1b, per3, ulk1b, socs2, esrp1, bcl2l1, hsp70, igf2r, mdm2, rab18a, col1a1a, fn1a, ppih, tpx2, uba5, nhlrc2, mcm4, tac1, b3gat3, and ddost, that correlate with the pathogenesis of skeletal muscle atrophy induced by excessive exercise. The underlying regulatory pathways and muscle-pressure-response-related genes identified in the present study will provide valuable insights for prescribing safe and accurate exercise programs for athletes and the supervision and clinical treatment of muscle atrophy induced by excessive exercise.
    Keywords:  FoxO signaling pathway; Wnt signaling pathway; excessive exercise; p53 signaling pathway; skeletal muscle atrophy
    DOI:  https://doi.org/10.3390/biology10080761
  6. Front Cell Dev Biol. 2021 ;9 673618
    Phosphorylation of Dynamin-Related Protein 1 (DRP1) Regulates Mitochondrial Dynamics and Skeletal Muscle Wasting in Cancer Cachexia.
    Xiangyu Mao, Yihua Gu, Xiangyu Sui, Lei Shen, Jun Han, Haiyu Wang, Qiulei Xi, Qiulin Zhuang, Qingyang Meng, Guohao Wu.
       Background: Cancer-associated cachexia (CAC) is a syndrome characterized by skeletal muscle atrophy, and the underlying mechanisms are still unclear. Recent research studies have shed light on a noteworthy link between mitochondrial dynamics and muscle physiology. In the present study, we investigate the role of dynamin-related protein 1 (DRP1), a pivotal factor of mitochondrial dynamics, in myotube atrophy during cancer-associated cachexia.
    Methods: Seventy-six surgical patients, including gastrointestinal tumor and benign disease, were enrolled in the study and divided to three groups: control, non-cachexia, and cancer-associated cachexia. Demographic data were collected. Their rectus abdominis samples were acquired intraoperatively. Muscle fiber size, markers of ubiquitin proteasome system (UPS), mitochondrial ultrastructure, and markers of mitochondrial function and dynamics were assayed. A cachexia model in vitro was established via coculturing a C2C12 myotube with media from C26 colon cancer cells. A specific DRP1 inhibitor, Mdivi-1, and a lentivirus of DRP1 knockdown/overexpression were used to regulate the expression of DRP1. Muscle diameter, mitochondrial morphology, mass, reactive oxygen species (ROS), membrane potential, and markers of UPS, mitochondrial function, and dynamics were determined.
    Results: Patients of cachexia suffered from a conspicuous worsened nutrition status and muscle loss compared to patients of other groups. Severe mitochondrial swelling and enlarged area were observed, and partial alterations in mitochondrial function were found in muscle. Analysis of mitochondrial dynamics indicated an upregulation of phosphorylated DRP1 at the ser616 site. In vitro, cancer media resulted in the atrophy of myotube. This was accompanied with a prominent unbalance of mitochondrial dynamics, as well as enhanced mitochondrial ROS and decreased mitochondrial function and membrane potential. However, certain concentrations of Mdivi-1 and DRP1 knockdown rebalanced the mitochondrial dynamics, mitigating this negative phenotype caused by cachexia. Moreover, overexpression of DRP1 aggravated these phenomena.
    Conclusion: In clinical patients, cachexia induces abnormal mitochondrial changes and possible fission activation for the atrophied muscle. Our cachexia model in vitro further demonstrates that unbalanced mitochondrial dynamics contributes to this atrophy and mitochondrial impairment, and rebuilding the balance by regulating of DRP1 could ameliorate these alterations.
    Keywords:  atrophy; cancer-associated cachexia; dynamin-related protein 1; mitochondria fission; skeletal muscle
    DOI:  https://doi.org/10.3389/fcell.2021.673618
  7. Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2102895118. [Epub ahead of print]118(35):
    Identification of a KLF5-dependent program and drug development for skeletal muscle atrophy.
    Lin Liu, Hiroyuki Koike, Takehito Ono, Shinichiro Hayashi, Fujimi Kudo, Atsushi Kaneda, Hiroyuki Kagechika, Ichiro Manabe, Tomoki Nakashima, Yumiko Oishi.
      Skeletal muscle atrophy is caused by various conditions, including aging, disuse related to a sedentary lifestyle and lack of physical activity, and cachexia. Our insufficient understanding of the molecular mechanism underlying muscle atrophy limits the targets for the development of effective pharmacologic treatments and preventions. Here, we identified Krüppel-like factor 5 (KLF5), a zinc-finger transcription factor, as a key mediator of the early muscle atrophy program. KLF5 was up-regulated in atrophying myotubes as an early response to dexamethasone or simulated microgravity in vitro. Skeletal muscle-selective deletion of Klf5 significantly attenuated muscle atrophy induced by mechanical unloading in mice. Transcriptome- and genome-wide chromatin accessibility analyses revealed that KLF5 regulates atrophy-related programs, including metabolic changes and E3-ubiquitin ligase-mediated proteolysis, in coordination with Foxo1. The synthetic retinoic acid receptor agonist Am80, a KLF5 inhibitor, suppressed both dexamethasone- and microgravity-induced muscle atrophy in vitro and oral Am80 ameliorated disuse- and dexamethasone-induced atrophy in mice. Moreover, in three independent sets of transcriptomic data from human skeletal muscle, KLF5 expression significantly increased with age and the presence of sarcopenia and correlated positively with the expression of the atrophy-related ubiquitin ligase genes FBXO32 and TRIM63 These findings demonstrate that KLF5 is a key transcriptional regulator mediating muscle atrophy and that pharmacological intervention with Am80 is a potentially preventive treatment.
    Keywords:  KLF; RAR ligand; muscle atrophy
    DOI:  https://doi.org/10.1073/pnas.2102895118
  8. Biomolecules. 2021 Jul 21. pii: 1064. [Epub ahead of print]11(8):
    Pathological Mechanism of a Constitutively Active Form of Stromal Interaction Molecule 1 in Skeletal Muscle.
    Ji Hee Park, Seung Yeon Jeong, Jun Hee Choi, Eun Hui Lee.
      Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.
    Keywords:  STIM1; cytosolic Ca2+; hyper-SOCE; skeletal muscle
    DOI:  https://doi.org/10.3390/biom11081064
  9. Elife. 2021 Aug 27. pii: e70490. [Epub ahead of print]10
    MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis.
    Alireza Ghasemizadeh, Emilie Christin, Alexandre Guiraud, Nathalie Couturier, Marie Abitbol, Valerie Risson, Emmanuelle Girard, Krzysztof Jagla, Cedric Soler, Lilia Laddada, Colline Sanchez, Francisco-Ignacio Jaque-Fernandez, Vincent Jacquemond, Jean-Luc Thomas, Marine Lanfranchi, Julien Courchet, Julien Gondin, Laurent Schaeffer, Vincent Gache.
      Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.
    Keywords:  D. melanogaster; cell biology; developmental biology; mouse
    DOI:  https://doi.org/10.7554/eLife.70490
  10. Cells. 2021 Aug 13. pii: 2083. [Epub ahead of print]10(8):
    Interaction of Fibromodulin and Myostatin to Regulate Skeletal Muscle Aging: An Opposite Regulation in Muscle Aging, Diabetes, and Intracellular Lipid Accumulation.
    Eun Ju Lee, Syed Sayeed Ahmad, Jeong Ho Lim, Khurshid Ahmad, Sibhghatulla Shaikh, Yun-Sil Lee, Sang Joon Park, Jun O Jin, Yong-Ho Lee, Inho Choi.
      The objective of this study was to investigate fibromodulin (FMOD) and myostatin (MSTN) gene expressions during skeletal muscle aging and to understand their involvements in this process. The expressions of genes related to muscle aging (Atrogin 1 and Glb1), diabetes (RAGE and CD163), and lipid accumulation (CD36 and PPARγ) and those of FMOD and MSTN were examined in CTX-injected, aged, MSTN-/-, and high-fat diet (HFD) mice and in C2C12 myoblasts treated with ceramide or grown under adipogenic conditions. Results from CTX-injected mice and gene knockdown experiments in C2C12 cells suggested the involvement of FMOD during muscle regeneration and myoblast proliferation and differentiation. Downregulation of the FMOD gene in MSTN-/- mice, and MSTN upregulation and FMOD downregulation in FMOD and MSTN knockdown C2C12 cells, respectively, during their differentiation, suggested FMOD negatively regulates MSTN gene expression, and MSTN positively regulates FMOD gene expression. The results of our in vivo and in vitro experiments indicate FMOD inhibits muscle aging by negatively regulating MSTN gene expression or by suppressing the action of MSTN protein, and that MSTN promotes muscle aging by positively regulating the expressions of Atrogin1, CD36, and PPARγ genes in muscle.
    Keywords:  fibromodulin; muscle aging; myostatin; sarcopenia; skeletal muscle
    DOI:  https://doi.org/10.3390/cells10082083
  11. Stem Cell Reports. 2021 Aug 16. pii: S2213-6711(21)00388-X. [Epub ahead of print]
    CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration.
    Krishnamoorthy Sreenivasan, Alejandra Rodríguez-delaRosa, Johnny Kim, Diana Mesquita, Jessica Segalés, Pablo Gómez-Del Arco, Isabel Espejo, Alessandro Ianni, Luciano Di Croce, Frederic Relaix, Juan Miguel Redondo, Thomas Braun, Antonio L Serrano, Eusebio Perdiguero, Pura Muñoz-Cánoves.
      Regeneration of skeletal muscle requires resident stem cells called satellite cells. Here, we report that the chromatin remodeler CHD4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. We show that conditional deletion of the Chd4 gene in satellite cells results in failure to regenerate muscle after injury. This defect is principally associated with increased stem cell plasticity and lineage infidelity during the expansion of satellite cells, caused by de-repression of non-muscle-cell lineage genes in the absence of Chd4. Thus, CHD4 ensures that a transcriptional program that safeguards satellite cell identity during muscle regeneration is maintained. Given the therapeutic potential of muscle stem cells in diverse neuromuscular pathologies, CHD4 constitutes an attractive target for satellite cell-based therapies.
    Keywords:  Chd4; NuRD; lineage maintenance; muscle stem cell; regeneration; satellite cells; skeletal muscle
    DOI:  https://doi.org/10.1016/j.stemcr.2021.07.022
  12. Am J Physiol Regul Integr Comp Physiol. 2021 08 25.
    Effects of hyperoxia and hypoxia on the proliferation of C2C12 myoblasts.
    Misa Horiike, Yoshiko Ogawa, Shigeo Kawada.
      Hyperoxic conditions are known to accelerate skeletal muscle regeneration after injuries. In the early phase of regeneration, macrophages invade the injured area and subsequently secrete various growth factors, which regulate myoblast proliferation and differentiation. Although hyperoxic conditions accelerate muscle regeneration, it is unknown whether this effect is indirectly mediated by macrophages. Here, using C2C12 cells, we show that not only hyperoxia but also hypoxia enhance myoblast proliferation directly, without accelerating differentiation into myotubes. Under hyperoxic conditions (95% O2 + 5% CO2), the cell membrane was damaged because of lipid oxidization, and a disrupted cytoskeletal structure, resulting in suppressed cell proliferation. However, a culture medium containing vitamin C (VC), an antioxidant, prevented this lipid oxidization and cytoskeletal disruption, resulting in enhanced proliferation in response to hyperoxia exposure of ≤4 h/day. In contrast, exposure to hypoxic conditions (95% N2 + 5% CO2) for ≤8 h/day enhanced cell proliferation. Hyperoxia did not promote cell differentiation into myotubes, regardless of whether the culture medium contained VC. Similarly, hypoxia did not accelerate cell differentiation. These results suggest that regardless of hyperoxia or hypoxia, changes in oxygen tension can enhance cell proliferation directly, but do not influence differentiation efficiency in C2C12 cells. Moreover, excess oxidative stress abrogated the enhancement of myoblast proliferation induced by hyperoxia. The present research will contribute to basic data for applying the effects of hyperoxia or hypoxia to muscle regeneration therapy.
    Keywords:  hyperoxia; hypoxia; myoblast; skeletal muscle regeneration
    DOI:  https://doi.org/10.1152/ajpregu.00269.2020
  13. ACS Omega. 2021 Aug 17. 6(32): 20931-20940
    Regulation of Myogenic Differentiation by Topologically Microgrooved Surfaces for Skeletal Muscle Tissue Engineering.
    Huichang Gao, Jin Xiao, Yingqi Wei, Hao Wang, Hongxia Wan, Shan Liu.
      Inspired by the natural topological structure of skeletal muscle tissue, the topological surface construction of bionic scaffolds for skeletal muscle repair has attracted great interest. Many previous studies have focused on the effects of the topological structure on myoblasts. However, these studies used only specific repeating sizes and shapes to achieve the myoblast alignment and myotube formation; moreover, the regulatory effects of the size of a topological structure on myogenic differentiation are often neglected, leading to a lack of guidance for the design of scaffolds for skeletal muscle tissue engineering. In this study, we fabricated a series of microgroove topographies with various widths and depths via a combination of soft lithography and melt-casting and studied their effects on the behaviors of skeletal muscle cells, especially myogenic differentiation, in detail. Microgrooved poly(lactic-co-glycolic acid) substrates were found to effectively regulate the proliferation, myogenic differentiation, and myotube formation of C2C12 cells, and the degree of myogenic differentiation was significantly dependent on signals in response to the size of the microgroove structure. Compared with their depth, the width of the microgroove structures can more strongly affect the myogenic differentiation of C2C12 cells, and the degree of myoblast differentiation was enhanced with increasing groove width. Microgroove structures with relatively large groove widths and small groove depths promoted the myogenic differentiation of C2C12 cells. In addition, the integrin-mediated focal adhesion kinase signaling pathway and MAPK signaling pathway were activated in cells in response to the external topological structure, and the size of the topological structure of the material surface effectively regulated the degree of the cellular response to the external topological structure. These results can guide the design of scaffolds for skeletal muscle tissue engineering and the construction of effective bionic scaffold surfaces for skeletal muscle regeneration.
    DOI:  https://doi.org/10.1021/acsomega.1c02347
  14. Am J Physiol Endocrinol Metab. 2021 08 23.
    c-Myc overexpression increases ribosome biogenesis and protein synthesis independent of mTORC1 activation in mouse skeletal muscle.
    Takahiro Mori, Satoru Ato, Jonas R Knudsen, Carlos Henriquez-Olguin, Zhencheng Li, Koki Wakabayashi, Takeshi Suginohara, Kazuhiko Higashida, Yuki Tamura, Koichi Nakazato, Thomas E Jensen, Riki Ogasawara.
      High-intensity muscle contractions (HiMC) are known to increase c-Myc expression which is known to stimulate ribosome biogenesis and protein synthesis in most cells. However, while c-Myc mRNA transcription and c-Myc mRNA translation have been shown to be upregulated following resistance exercise concomitantly with increased ribosome biogenesis, this has not been tested directly. We investigated the effect of adeno-associated virus (AAV)-mediated c-Myc overexpression, with or without fasting or percutaneous electrical stimulation-induced HiMC, on ribosome biogenesis and protein synthesis in adult mouse skeletal muscles. AAV-mediated overexpression of c-Myc in mouse skeletal muscles for 2 weeks increased the DNA polymerase subunit POL1 mRNA, 45S-pre-rRNA, total RNA, and muscle protein synthesis without altering mechanistic target of rapamycin complex 1 (mTORC1) signaling under both ad libitum and fasted conditions. RNA-seq analyses revealed that c-Myc overexpression mainly regulated ribosome biogenesis-related biological processes. The protein synthesis response to c-Myc overexpression mirrored the response with HiMC. No additional effect of combining c-Myc overexpression and HiMC was observed. Our results suggest that c-Myc overexpression is sufficient to stimulate skeletal muscle ribosome biogenesis and protein synthesis without activation of mTORC1. Therefore, the HiMC-induced increase in c-Myc may contribute to ribosome biogenesis and increased protein synthesis following HiMC.
    Keywords:  RNA-Seq; c-Myc; exercise; protein metabolism; ribosome biogenesis
    DOI:  https://doi.org/10.1152/ajpendo.00164.2021
  15. Membranes (Basel). 2021 Aug 12. pii: 619. [Epub ahead of print]11(8):
    Skeletal Muscle Cell Growth Alters the Lipid Composition of Extracellular Vesicles.
    Taylor R Valentino, Blake D Rule, C Brooks Mobley, Mariana Nikolova-Karakashian, Ivan J Vechetti.
      We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hypertrophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two- to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16- and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphingosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth.
    Keywords:  EV biogenesis; extracellular vesicles; lipids; muscle cell; muscle growth
    DOI:  https://doi.org/10.3390/membranes11080619
  16. Cells. 2021 Aug 03. pii: 1974. [Epub ahead of print]10(8):
    UBE2L3, a Partner of MuRF1/TRIM63, Is Involved in the Degradation of Myofibrillar Actin and Myosin.
    Dulce Peris-Moreno, Mélodie Malige, Agnès Claustre, Andrea Armani, Cécile Coudy-Gandilhon, Christiane Deval, Daniel Béchet, Pierre Fafournoux, Marco Sandri, Lydie Combaret, Daniel Taillandier, Cécile Polge.
      The ubiquitin proteasome system (UPS) is the main player of skeletal muscle wasting, a common characteristic of many diseases (cancer, etc.) that negatively impacts treatment and life prognosis. Within the UPS, the E3 ligase MuRF1/TRIM63 targets for degradation several myofibrillar proteins, including the main contractile proteins alpha-actin and myosin heavy chain (MHC). We previously identified five E2 ubiquitin-conjugating enzymes interacting with MuRF1, including UBE2L3/UbcH7, that exhibited a high affinity for MuRF1 (KD = 50 nM). Here, we report a main effect of UBE2L3 on alpha-actin and MHC degradation in catabolic C2C12 myotubes. Consistently UBE2L3 knockdown in Tibialis anterior induced hypertrophy in dexamethasone (Dex)-treated mice, whereas overexpression worsened the muscle atrophy of Dex-treated mice. Using combined interactomic approaches, we also characterized the interactions between MuRF1 and its substrates alpha-actin and MHC and found that MuRF1 preferentially binds to filamentous F-actin (KD = 46.7 nM) over monomeric G-actin (KD = 450 nM). By contrast with actin that did not alter MuRF1-UBE2L3 affinity, binding of MHC to MuRF1 (KD = 8 nM) impeded UBE2L3 binding, suggesting that differential interactions prevail with MuRF1 depending on both the substrate and the E2. Our data suggest that UBE2L3 regulates contractile proteins levels and skeletal muscle atrophy.
    Keywords:  MicroScale-Thermophoresis; MuRF1/TRIM63; UBE2L3/UbcH7; alpha-actin; contractile proteins; glucocorticoids; myosin; skeletal muscle atrophy; ubiquitinating enzymes
    DOI:  https://doi.org/10.3390/cells10081974
  17. Molecules. 2021 Aug 12. pii: 4887. [Epub ahead of print]26(16):
    Polyphenols and Their Effects on Muscle Atrophy and Muscle Health.
    Takeshi Nikawa, Anayt Ulla, Iori Sakakibara.
      Skeletal muscle atrophy is the decrease in muscle mass and strength caused by reduced protein synthesis/accelerated protein degradation. Various conditions, such as denervation, disuse, aging, chronic diseases, heart disease, obstructive lung disease, diabetes, renal failure, AIDS, sepsis, cancer, and steroidal medications, can cause muscle atrophy. Mechanistically, inflammation, oxidative stress, and mitochondrial dysfunction are among the major contributors to muscle atrophy, by modulating signaling pathways that regulate muscle homeostasis. To prevent muscle catabolism and enhance muscle anabolism, several natural and synthetic compounds have been investigated. Recently, polyphenols (i.e., natural phytochemicals) have received extensive attention regarding their effect on muscle atrophy because of their potent antioxidant and anti-inflammatory properties. Numerous in vitro and in vivo studies have reported polyphenols as strongly effective bioactive molecules that attenuate muscle atrophy and enhance muscle health. This review describes polyphenols as promising bioactive molecules that impede muscle atrophy induced by various proatrophic factors. The effects of each class/subclass of polyphenolic compounds regarding protection against the muscle disorders induced by various pathological/physiological factors are summarized in tabular form and discussed. Although considerable variations in antiatrophic potencies and mechanisms were observed among structurally diverse polyphenolic compounds, they are vital factors to be considered in muscle atrophy prevention strategies.
    Keywords:  antioxidants; flavonoid; mitochondrial biogenesis; mitochondrial dysfunction; muscle atrophy; myogenesis; oxidative stress; polyphenols; proteolysis
    DOI:  https://doi.org/10.3390/molecules26164887
  18. Acta Histochem. 2021 Aug 23. pii: S0065-1281(21)00096-9. [Epub ahead of print]123(6): 151774
    BMP2 downregulates urokinase-type plasminogen activator via p38 MAPK: Implications in C2C12 cells myogenic differentiation.
    Juan F Santibanez, Hristina Obradović, Jelena Krstić.
      Bone morphogenetic protein (BMP)2 strongly affects the differentiation program of myoblast cells by inhibiting myogenesis and inducing osteogenic differentiation. In turn, extracellular matrix (ECM) proteinases, such as urokinase-type plasminogen activator (uPA), can influence the fate of muscle stem cells by participating in ECM reorganization. Although both BMP2 and uPA have antagonistic roles in muscles cells differentiation, no connection between them has been elucidated so far. This study aims to determine whether BMP2 regulates uPA expression in the myogenic C2C12 cell line and its impact on muscle cell fate differentiation. Our results showed that BMP2 did not modify C2C12 cell proliferation in a growth medium or myogenic differentiation medium. Although BMP2 inhibited myogenesis and induced osteogenesis, these effects were achieved with different doses of BMP2. Low concentrations of BMP2 blocked myogenesis, while a higher concentration was needed to induce osteogenesis. Reduced uPA expression was noticed alongside myogenic inhibition at low concentrations of BMP2. BMP2 activated p38 MAPK signaling to inhibit uPA activity. Furthermore, ectopic human uPA expression reduced BMP2's ability to inhibit the myogenic differentiation of C2C12 cells. In conclusion, BMP2 inhibits uPA expression through p38 MAPK and in vitro myogenesis at non-osteogenic concentrations, while uPA ectopic expression prevents BMP2 from inhibiting myogenesis in C2C12 cells.
    Keywords:  BMP2; Myoblast; Myogenesis; Myotubes; p38; uPA
    DOI:  https://doi.org/10.1016/j.acthis.2021.151774
  19. J Cachexia Sarcopenia Muscle. 2021 Aug 22.
    SLIT3 promotes myogenic differentiation as a novel therapeutic factor against muscle loss.
    Han Jin Cho, Hyeonmok Kim, Young-Sun Lee, Sung Ah Moon, Jin-Man Kim, Hanjun Kim, Min Ji Kim, Jiyoung Yu, Kyunggon Kim, In-Jeoung Baek, Seung Hun Lee, Kyong Hoon Ahn, Sungsub Kim, Jong-Sun Kang, Jung-Min Koh.
       BACKGROUND: Sarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently discovered as a novel therapeutic factor against osteoporosis, and a SLIT3 fragment containing the second leucine-rich repeat domain (LRRD2) had a therapeutic efficacy against osteoporosis. However, a role of SLIT3 in the skeletal muscle is unknown.
    METHODS: Skeletal muscle mass, strength, and/or physical activity were evaluated in Slit3-/- , ovariectomized, and aged mice, based on the measurements of muscle weight and grip strength, Kondziella's inverted hanging test, and/or wheel-running test. Skeletal muscles were also histologically evaluated by haematoxylin and eosin staining and/or immunofluorescence. The ovariectomized and aged mice were intravenously injected with recombinant SLIT3 LRRD2 for 4 weeks. C2C12 cells were used to know cellular effects of SLIT3, such as in vitro myogenesis, fusion, cell viability, and proliferation, and also used to evaluate its molecular mechanisms by immunocytochemistry, immunoprecipitation, western blotting, real-time PCR, siRNA transfection, and receptor-ligand binding ELISA.
    RESULTS: Slit3-deficient mice exhibited decreased skeletal muscle mass, muscle strength, and physical activity. The relative masses of gastrocnemius and soleus were lower in the Slit3-/- mice (0.580 ± 0.039% and 0.033 ± 0.003%, respectively) than those in the WT littermates (0.622 ± 0.043% and 0.038 ± 0.003%, respectively) (all, P < 0.05). Gastrocnemius of Slit3-/- mice showed the reduced number of Type I and Type IIa fibres (all, P < 0.05), but not of Type IIb and Type IIx fibres. SLIT3 activated β-catenin signalling by promoting its release from M-cadherin, thereby increasing myogenin expression to stimulate myoblast differentiation. In vitro experiments involving ROBO2 expression, knockdown, and interaction with SLIT3 indicated that ROBO2 functions as a SLIT3 receptor to aid myoblast differentiation. SLIT3 LRRD2 dissociated M-cadherin-bound β-catenin and up-regulated myogenin expression to increase myoblast differentiation, in a manner similar to full-length SLIT3. Systemic treatment with SLIT3 LRRD2 increased skeletal muscle mass in both ovariectomized and aged mice (all, P < 0.05). The relative masses of gastrocnemius and soleus were higher in the treated aged mice (0.548 ± 0.045% and 0.033 ± 0.005%, respectively) than in the untreated aged mice (0.508 ± 0.016% and 0.028 ± 0.003%, respectively) (all, P < 0.05). SLIT3 LRRD2 treatment increased the hanging duration of the aged mice by approximately 1.7-fold (P < 0.05).
    CONCLUSIONS: SLIT3 plays a sarcoprotective role by activating β-catenin signalling. SLIT3 LRRD2 can potentially be used as a therapeutic agent against muscle loss.
    Keywords:  LRRD2; Muscle loss; Robo2; Slit3
    DOI:  https://doi.org/10.1002/jcsm.12769
  20. Molecules. 2021 Aug 13. pii: 4904. [Epub ahead of print]26(16):
    Curcumin and Resveratrol Improve Muscle Function and Structure through Attenuation of Proteolytic Markers in Experimental Cancer-Induced Cachexia.
    Antonio Penedo-Vázquez, Xavier Duran, Javier Mateu, Adrián López-Postigo, Esther Barreiro.
      Muscle wasting and cachexia are prominent comorbidities in cancer. Treatment with polyphenolic compounds may partly revert muscle wasting. We hypothesized that treatment with curcumin or resveratrol in cancer cachectic mice may improve muscle phenotype and total body weight through attenuation of several proteolytic and signaling mechanisms in limb muscles. In gastrocnemius and soleus muscles of cancer cachectic mice (LP07 adenocarcinoma cells, N = 10/group): (1) LC-induced cachexia, (2) LC-cachexia+curcumin, and (3) LC-cachexia + resveratrol, muscle structure and damage (including blood troponin I), sirtuin-1, proteolytic markers, and signaling pathways (NF-κB and FoxO3) were explored (immunohistochemistry and immunoblotting). Compared to nontreated cachectic mice, in LC-cachexia + curcumin and LC-cachexia + resveratrol groups, body and muscle weights (gastrocnemius), limb muscle strength, muscle damage, and myofiber cross-sectional area improved, and in both muscles, sirtuin-1 increased, while proteolysis (troponin I), proteolytic markers, and signaling pathways were attenuated. Curcumin and resveratrol elicited beneficial effects on fast- and slow-twitch limb muscle phenotypes in cachectic mice through sirtuin-1 activation, attenuation of atrophy signaling pathways, and proteolysis in cancer cachectic mice. These findings have future therapeutic implications as these natural compounds, separately or in combination, may be used in clinical settings of muscle mass loss and dysfunction including cancer cachexia.
    Keywords:  atrophy signaling pathways; cancer-induced cachexia mouse model; curcumin; muscle function and structure; muscle proteolysis; resveratrol; sirtuin-1; troponin I
    DOI:  https://doi.org/10.3390/molecules26164904
  21. Proteomes. 2021 Aug 03. pii: 37. [Epub ahead of print]9(3):
    Insulin and 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Differentially Regulate the Skeletal Muscle Cell Secretome.
    Alba Gonzalez-Franquesa, Lone Peijs, Daniel T Cervone, Ceren Koçana, Juleen R Zierath, Atul S Deshmukh.
      Skeletal muscle is a major contributor to whole-body glucose homeostasis and is an important endocrine organ. To date, few studies have undertaken the large-scale identification of skeletal muscle-derived secreted proteins (myokines), particularly in response to stimuli that activate pathways governing energy metabolism in health and disease. Whereas the AMP-activated protein kinase (AMPK) and insulin-signaling pathways have received notable attention for their ability to independently regulate skeletal muscle substrate metabolism, little work has examined their ability to re-pattern the secretome. The present study coupled the use of high-resolution MS-based proteomics and bioinformatics analysis of conditioned media derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR-an AMPK activator)- and insulin-treated differentiated C2C12 myotubes. We quantified 858 secreted proteins, including cytokines and growth factors such as fibroblast growth factor-21 (Fgf21). We identified 377 and 118 proteins that were significantly altered by insulin and AICAR treatment, respectively. Notably, the family of insulin growth factor binding-proteins (Igfbp) was differentially regulated by each treatment. Insulin- but not AICAR-induced conditioned media increased the mitochondrial respiratory capacity of myotubes, potentially via secreted factors. These findings may serve as an important resource to elucidate secondary metabolic effects of insulin and AICAR stimulation in skeletal muscle.
    Keywords:  AMPK; insulin; metabolism; secretomics; skeletal muscle
    DOI:  https://doi.org/10.3390/proteomes9030037
  22. Int J Mol Sci. 2021 Aug 10. pii: 8607. [Epub ahead of print]22(16):
    Transcriptome Analysis in a Primary Human Muscle Cell Differentiation Model for Myotonic Dystrophy Type 1.
    Vanessa Todorow, Stefan Hintze, Alastair R W Kerr, Andreas Hehr, Benedikt Schoser, Peter Meinke.
      Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.
    Keywords:  human primary muscle cell culture; myotonic dystrophy type 1; splicing; transcriptomics
    DOI:  https://doi.org/10.3390/ijms22168607
  23. Matrix Biol Plus. 2021 Aug;11 100059
    Driving fibrosis in neuromuscular diseases: Role and regulation of Connective tissue growth factor (CCN2/CTGF).
    Daniela L Rebolledo, Kenneth E Lipson, Enrique Brandan.
      Connective tissue growth factor or cellular communication network 2 (CCN2/CTGF) is a matricellular protein member of the CCN family involved in several crucial biological processes. In skeletal muscle, CCN2/CTGF abundance is elevated in human muscle biopsies and/or animal models for diverse neuromuscular pathologies, including muscular dystrophies, neurodegenerative disorders, muscle denervation, and muscle overuse. In this context, CCN2/CTGF is deeply involved in extracellular matrix (ECM) modulation, acting as a strong pro-fibrotic factor that promotes excessive ECM accumulation. Reducing CCN2/CTGF levels or biological activity in pathological conditions can decrease fibrosis, improve muscle architecture and function. In this work, we summarize information about the role of CCN2/CTGF in fibrosis associated with neuromuscular pathologies and the mechanisms and signaling pathways that regulate their expression in skeletal muscle.
    Keywords:  ALS, Amyotrophic Lateral Sclerosis; CCN2/CTGF; CCN2/CTGF, connective tissue growth factor; DMD, Duchenne muscular dystrophy; ECM, extracellular matrix; FG-3019; Fibrosis; LPA, lysophosphatidic acid; Neuromuscular diseases; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.mbplus.2021.100059
  24. J Cachexia Sarcopenia Muscle. 2021 Aug 23.
    TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration.
    Wen Luo, Zetong Lin, Jiahui Chen, Genghua Chen, Siyu Zhang, Manqing Liu, Hongmei Li, Danlin He, Shaodong Liang, Qingbin Luo, Dexiang Zhang, Qinghua Nie, Xiquan Zhang.
       BACKGROUND: Transmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms of action in myogenesis and muscle disorders remain largely unexplored. It has been found that transmembrane protein 182 (TMEM182) is dramatically up-regulated during myogenesis, but its detailed functions remain unclear. This study aimed to analyse the function of TMEM182 during myogenesis and muscle regeneration.
    METHODS: RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence approaches were used to analyse TMEM182 expression during myoblast differentiation. A dual-luciferase reporter assay was used to identify the promoter region of the TMEM182 gene, and a chromatin immunoprecipitation assay was used to investigate the regulation TMEM182 transcription by MyoD. We used chickens and TMEM182-knockout mice as in vivo models to examine the function of TMEM182 in muscle growth and muscle regeneration. Chickens and mouse primary myoblasts were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Co-immunoprecipitation and mass spectrometry were used to identify the interaction between TMEM182 and integrin beta 1 (ITGB1). The molecular mechanism by which TMEM182 regulates myogenesis and muscle regeneration was examined by Transwell migration, cell wound healing, adhesion, glutathione-S-transferse pull down, protein purification, and RNA immunoprecipitation assays.
    RESULTS: TMEM182 was specifically expressed in skeletal muscle and adipose tissue and was regulated at the transcriptional level by the myogenic regulatory factor MyoD1. Functionally, TMEM182 inhibited myoblast differentiation and fusion. The in vivo studies indicated that TMEM182 induced muscle fibre atrophy and delayed muscle regeneration. TMEM182 knockout in mice led to significant increases in body weight, muscle mass, muscle fibre number, and muscle fibre diameter. Skeletal muscle regeneration was accelerated in TMEM182-knockout mice. Furthermore, we revealed that the inhibitory roles of TMEM182 in skeletal muscle depend on ITGB1, an essential membrane receptor involved in cell adhesion and muscle formation. TMEM182 directly interacted with ITGB1, and this interaction required an extracellular hybrid domain of ITGB1 (aa 387-470) and a conserved region (aa 52-62) within the large extracellular loop of TMEM182. Mechanistically, TMEM182 modulated ITGB1 activation by coordinating the association between ITGB1 and laminin and regulating the intracellular signalling of ITGB1. Myogenic deletion of TMEM182 increased the binding activity of ITGB1 to laminin and induced the activation of the FAK-ERK and FAK-Akt signalling axes during myogenesis.
    CONCLUSIONS: Our data reveal that TMEM182 is a novel negative regulator of myogenic differentiation and muscle regeneration.
    Keywords:  FAK signalling; Integrin beta 1; Muscle regeneration; Myogenesis; TMEM182
    DOI:  https://doi.org/10.1002/jcsm.12767
  25. Cell Death Dis. 2021 Aug 24. 12(9): 805
    SESN2 protects against denervated muscle atrophy through unfolded protein response and mitophagy.
    Xiaofan Yang, Pingping Xue, Meng Yuan, Xiang Xu, Cheng Wang, Wenqing Li, Hans-Günther Machens, Zhenbing Chen.
      Denervation of skeletal muscles results in a rapid and programmed loss of muscle size and performance, termed muscle atrophy, which leads to a poor prognosis of clinical nerve repair. Previous researches considered this process a result of multiple factors, such as protein homeostasis disorder, mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and apoptosis, while their intrinsic association remains to be explored. In this study, Sestrin2 (SESN2), a stress-inducible protein, was shown to act as a key protective signal involved in the crosstalk therein. SESN2 expression was induced in the gastrocnemius two weeks post denervation, which was accompanied by ERS, mitochondrial dysfunction, and apoptosis. Knockdown of SESN2 aggravated this situation and resulted in severer atrophy. Similar results were also found in rotenone-treated C2C12 cells. Furthermore, SESN2 was demonstrated to be induced by an ERS-activated transcription factor CCAAT-enhancer-binding protein beta (C/EBPβ). Once induced, SESN2 halted protein synthesis by inhibiting the mammalian target of rapamycin complex 1 (mTORC1), thereby attenuating ERS. Moreover, increased SESN2 activated the specific autophagic machinery and facilitated the aggregation of sequestosome 1 (SQSTM1, p62) on the mitochondrial surface, which promoted the clearance of damaged mitochondria through mitophagy. Collectively, the SESN2-mediated unfolded protein response (UPR) and mitophagy play a critical role in protecting against denervated muscle atrophy, which may provide novel insights into the mechanism of skeletal muscle atrophy following denervation.
    DOI:  https://doi.org/10.1038/s41419-021-04094-9
  26. Metabolites. 2021 Jul 23. pii: 474. [Epub ahead of print]11(8):
    Signals from the Circle: Tricarboxylic Acid Cycle Intermediates as Myometabokines.
    Jennifer Maurer, Miriam Hoene, Cora Weigert.
      Regular physical activity is an effective strategy to prevent and ameliorate aging-associated diseases. In particular, training increases muscle performance and improves whole-body metabolism. Since exercise affects the whole organism, it has countless health benefits. The systemic effects of exercise can, in part, be explained by communication between the contracting skeletal muscle and other organs and cell types. While small proteins and peptides known as myokines are the most prominent candidates to mediate this tissue cross-talk, recent investigations have paid increasing attention to metabolites. The purpose of this review is to highlight the potential role of tricarboxylic acid (TCA) metabolites as humoral mediators of exercise adaptation processes. We focus on TCA metabolites that are released from human skeletal muscle in response to exercise and provide an overview of their potential auto-, para- or endocrine health-promoting effects.
    Keywords:  TCA cycle; arterio-venous difference; citrate; exercise; exercise adaptation; liver; myometabokine; succinate
    DOI:  https://doi.org/10.3390/metabo11080474
  27. Cells. 2021 Aug 06. pii: 2003. [Epub ahead of print]10(8):
    Integral Membrane Protein 2A Is a Negative Regulator of Canonical and Non-Canonical Hedgehog Signalling.
    Cintli C Morales-Alcala, Ioanna Ch Georgiou, Alex J Timmis, Natalia A Riobo-Del Galdo.
      The Hedgehog (Hh) receptor PTCH1 and the integral membrane protein 2A (ITM2A) inhibit autophagy by reducing autolysosome formation. In this study, we demonstrate that ITM2A physically interacts with PTCH1; however, the two proteins inhibit autophagic flux independently, since silencing of ITM2A did not prevent the accumulation of LC3BII and p62 in PTCH1-overexpressing cells, suggesting that they provide alternative modes to limit autophagy. Knockdown of ITM2A potentiated PTCH1-induced autophagic flux blockade and increased PTCH1 expression, while ITM2A overexpression reduced PTCH1 protein levels, indicating that it is a negative regulator of PTCH1 non-canonical signalling. Our study also revealed that endogenous ITM2A is necessary for timely induction of myogenic differentiation markers in C2C12 cells since partial knockdown delays the timing of differentiation. We also found that basal autophagic flux decreases during myogenic differentiation at the same time that ITM2A expression increases. Given that canonical Hh signalling prevents myogenic differentiation, we investigated the effect of ITM2A on canonical Hh signalling using GLI-luciferase assays. Our findings demonstrate that ITM2A is a strong negative regulator of GLI transcriptional activity and of GLI1 stability. In summary, ITM2A negatively regulates canonical and non-canonical Hh signalling.
    Keywords:  GLI; ITM2A; autophagy; hedgehog; patched1; skeletal muscle
    DOI:  https://doi.org/10.3390/cells10082003
  28. Int J Mol Sci. 2021 Aug 22. pii: 9045. [Epub ahead of print]22(16):
    Redox Signaling and Sarcopenia: Searching for the Primary Suspect.
    Nicholas A Foreman, Anton S Hesse, Li Li Ji.
      Sarcopenia, the age-related decline in muscle mass and function, derives from multiple etiological mechanisms. Accumulative research suggests that reactive oxygen species (ROS) generation plays a critical role in the development of this pathophysiological disorder. In this communication, we review the various signaling pathways that control muscle metabolic and functional integrity such as protein turnover, cell death and regeneration, inflammation, organismic damage, and metabolic functions. Although no single pathway can be identified as the most crucial factor that causes sarcopenia, age-associated dysregulation of redox signaling appears to underlie many deteriorations at physiological, subcellular, and molecular levels. Furthermore, discord of mitochondrial homeostasis with aging affects most observed problems and requires our attention. The search for the primary suspect of the fundamental mechanism for sarcopenia will likely take more intense research for the secret of this health hazard to the elderly to be unlocked.
    Keywords:  aging; mitochondria; peroxiredoxin; redox signaling; sarcopenia; skeletal muscle
    DOI:  https://doi.org/10.3390/ijms22169045
  29. Cell Rep. 2021 Aug 24. pii: S2211-1247(21)01039-1. [Epub ahead of print]36(8): 109601
    The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies.
    Nicolas Vignier, Maria Chatzifrangkeskou, Luca Pinton, Hugo Wioland, Thibaut Marais, Mégane Lemaitre, Caroline Le Dour, Cécile Peccate, Déborah Cardoso, Alain Schmitt, Wei Wu, Maria-Grazia Biferi, Naïra Naouar, Coline Macquart, Maud Beuvin, Valérie Decostre, Gisèle Bonne, Guillaume Romet-Lemonne, Howard J Worman, Francesco Saverio Tedesco, Antoine Jégou, Antoine Muchir.
      Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations.
    Keywords:  ERK1/2 signaling; cofilin-1; muscular dystrophy; sarcomeric organization; skeletal muscle
    DOI:  https://doi.org/10.1016/j.celrep.2021.109601
  30. Am J Physiol Cell Physiol. 2021 08 25.
    Dystrophin-negative slow-twitch soleus muscles are not susceptible to eccentric contraction induced injury over the lifespan of the mdx mouse.
    Leonit Kiriaev, Sindy Kueh, John W Morley, Peter J Houweling, Stephen Chan, Kathryn N North, Stewart I Head.
      Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease in humans and is characterized by the absence of a functional copy of the protein dystrophin from skeletal muscle. In dystrophin-negative humans and rodents, regenerated skeletal muscle fibers show abnormal branching. The number of fibers with branches and the complexity of branching increases with each cycle of degeneration/regeneration. Previously, using the mdx mouse model of DMD, we have proposed that once the number and complexity of branched fibers present in dystrophic fast-twitch EDL muscle surpasses a stable level, we term "tipping point" the branches, in and of themselves, mechanically weaken the muscle by rupturing when subjected to high forces during eccentric contractions. Here we use the slow-twitch soleus muscle from the dystrophic mdx mouse to study pre-diseased "peri-ambulatory" dystrophic at 2-3 weeks, the peak regenerative "adult" phase at 6-9 weeks and "old" at 58-112 weeks. Using isolated mdx soleus muscles we examined contractile function and response to eccentric contraction correlated with amount and complexity of regenerated branched fibers. The intact muscle was enzymatically dispersed into individual fibers in order to count fiber branching and some muscles were optically cleared to allow laser scanning confocal microscopy. We demonstrate throughout the lifespan of the mdx mouse dystrophic slow-twitch soleus muscle is no more susceptible to eccentric contraction induced injury than age matched littermate controls and that this is correlated with a reduction in the number and complexity of branched fibers compared to fast-twitch dystrophic EDL muscles.
    Keywords:  Duchenne Muscular Dystrophy; Slow-twitch; fiber branching; soleus
    DOI:  https://doi.org/10.1152/ajpcell.00122.2021
  31. J Cardiovasc Dev Dis. 2021 Jul 25. pii: 84. [Epub ahead of print]8(8):
    The Key Lnc (RNA)s in Cardiac and Skeletal Muscle Development, Regeneration, and Disease.
    Amanda Pinheiro, Francisco J Naya.
      Non-coding RNAs (ncRNAs) play a key role in the regulation of transcriptional and epigenetic activity in mammalian cells. Comprehensive analysis of these ncRNAs has revealed sophisticated gene regulatory mechanisms which finely tune the proper gene output required for cellular homeostasis, proliferation, and differentiation. However, this elaborate circuitry has also made it vulnerable to perturbations that often result in disease. Among the many types of ncRNAs, long non-coding RNAs (lncRNAs) appear to have the most diverse mechanisms of action including competitive binding to miRNA targets, direct binding to mRNA, interactions with transcription factors, and facilitation of epigenetic modifications. Moreover, many lncRNAs display tissue-specific expression patterns suggesting an important regulatory role in organogenesis, yet the molecular mechanisms through which these molecules regulate cardiac and skeletal muscle development remains surprisingly limited. Given the structural and metabolic similarities of cardiac and skeletal muscle, it is likely that several lncRNAs expressed in both of these tissues have conserved functions in establishing the striated muscle phenotype. As many aspects of regeneration recapitulate development, understanding the role lncRNAs play in these processes may provide novel insights to improve regenerative therapeutic interventions in cardiac and skeletal muscle diseases. This review highlights key lncRNAs that function as regulators of development, regeneration, and disease in cardiac and skeletal muscle. Finally, we highlight lncRNAs encoded by imprinted genes in striated muscle and the contributions of these loci on the regulation of gene expression.
    Keywords:  cardiac; development; disease; epigenetics; genomic imprinting; long noncoding RNA; regeneration; skeletal
    DOI:  https://doi.org/10.3390/jcdd8080084
  32. Cells. 2021 Aug 23. pii: 2169. [Epub ahead of print]10(8):
    Mesenchymal Stem Cell-Derived Exosomes Protect Muscle Loss by miR-145-5p Activity Targeting Activin A Receptors.
    Kyung-Ah Cho, Da-Won Choi, Yu-Hee Kim, Jungwoo Kim, Kyung-Ha Ryu, So-Youn Woo.
      Skeletal muscle mass is decreased under a wide range of pathologic conditions. In particular, chemotherapy is well known for inducing muscle loss and atrophy. Previous studies using tonsil-derived mesenchymal stem cells (T-MSCs) or a T-MSC-conditioned medium showed effective recovery of total body weight in the chemotherapy-preconditioned bone marrow transplantation mouse model. This study investigated whether extracellular vesicles of T-MSCs, such as exosomes, are a key player in the recovery of body weight and skeletal muscle mass in chemotherapy-treated mice. T-MSC exosomes transplantation significantly decreased loss of total body weight and muscle mass in the busulfan-cyclophosphamide conditioning regimen in BALB/c recipient mice containing elevated serum activin A. Additionally, T-MSC exosomes rescued impaired C2C12 cell differentiation in the presence of activin A in vitro. We found that T-MSC exosomes possess abundant miR-145-5p, which targets activin A receptors, ACVR2A, and ACVR1B. Indeed, T-MSC exosomes rescue muscle atrophy both in vivo and in vitro via miR-145-5p dependent manner. These results suggest that T-MSC exosomes have therapeutic potential to maintain or improve skeletal muscle mass in various activin A elevated pathologic conditions.
    Keywords:  activin A; exosomes; mesenchymal stem cell; skeletal muscle
    DOI:  https://doi.org/10.3390/cells10082169
  33. Int J Mol Med. 2021 Oct;pii: 194. [Epub ahead of print]48(4):
    Calpain 6 inhibits autophagy in inflammatory environments: A preliminary study on myoblasts and a chronic kidney disease rat model.
    Yue Yue Zhang, Li Jie Gu, Nan Zhu, Ling Wang, Min Chao Cai, Jie Shuang Jia, Shu Rong, Wei Jie Yuan.
      A non‑classical calpain, calpain 6 (CAPN6), can inhibit skeletal muscle differentiation and regeneration. In the present study, the role of CAPN6 in the regulation of the autophagy of myoblasts in vitro was investigated. The underlying molecular events and the CAPN6 level in atrophic skeletal muscle in a rat model of chronic kidney disease (CKD) were also investigated. In vitro, CAPN6 was overexpressed, or knocked down, in rat L6 myoblasts to assess autophagy and related gene expression and co‑localization. Subsequently, myoblasts were treated with a mixture of cytokines, and relative gene expression and autophagy were assessed. A rat model of CKD for muscle atrophy was established, and blood chemical level and the expression of CAPN6 in muscle were assessed. The data revealed that the knockdown of CAPN6 in rat myoblasts resulted in increased microtubule‑associated protein 1 light chain 3 (LC3) levels, while its overexpression decreased LC3 levels and impaired autophagy. Additionally, it was observed that the co‑localization of mammalian target of rapamycin (mTOR) and lysosomal‑associated membrane protein 1 (LAMP1), a lysosomal marker, proteins was increased. In addition, mTOR, Raptor and α‑tubulin (a marker of microtubules) increased in the CAPN6 overexpression group. However, inflammatory cytokines, such as interleukin (IL)‑6, tumor necrosis factor (TNF)‑α, interferon (INF)‑γ and lipopolysaccharides upregulated CAPN6 expression, inhibited L6 myoblast autophagy and stabilized mTOR activity. Furthermore, the animal model successfully mimicked human disease as regards an increase in body weight, and a reduction in muscle mass, cross‑sectional area and blood biomarker concentrations; a slight increase in CAPN6 mRNA and protein levels in muscles was observed. Finally, the data of the present study suggested that CAPN6 reduced autophagy via the maintenance of mTOR signaling, which may play a role in CKD‑related muscle atrophy. However, future studies are required to determine whether CAPN6 may be used as an intervention target for CKD‑related skeletal muscle atrophy.
    Keywords:  autophagy; calpain 6; chronic kidney disease; inflammation; mTOR; muscle atrophy
    DOI:  https://doi.org/10.3892/ijmm.2021.5027
  34. FASEB J. 2021 09;35(9): e21864
    Resistance training rejuvenates the mitochondrial methylome in aged human skeletal muscle.
    Bradley A Ruple, Joshua S Godwin, Paulo H C Mesquita, Shelby C Osburn, Christopher G Vann, Donald A Lamb, Casey L Sexton, Darren G Candow, Scott C Forbes, Andrew D Frugé, Andreas N Kavazis, Kaelin C Young, Robert A Seaborne, Adam P Sharples, Michael D Roberts.
      Resistance training (RT) dynamically alters the skeletal muscle nuclear DNA methylome. However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older, Caucasian untrained males (65 ± 7 y.o.) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 h following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Biochemical assays were also performed, and older male data were compared to younger trained males (22 ± 2 y.o., n = 7, n = 6 Caucasian & n = 1 African American). RT increased whole-body lean tissue mass (p = .017), VL thickness (p = .012), and knee extensor torque (p = .029) in older males. RT also affected the mtDNA methylome, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p < .05). Several mtDNA sites presented a more "youthful" signature in older males after RT in comparison to younger males. The 1.12 kilobase mtDNA D-loop/control region, which regulates replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and complex III/IV protein levels were also observed (p < .05). While limited to a shorter-term intervention, this is the first evidence showing that RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT evokes mitochondrial methylome profiles to mimic younger men. The significance of these findings relative to broader RT-induced epigenetic changes needs to be elucidated.
    Keywords:  aging; methylation; mitochondrial DNA; resistance training
    DOI:  https://doi.org/10.1096/fj.202100873RR
  35. Biomolecules. 2021 Aug 08. pii: 1171. [Epub ahead of print]11(8):
    Characterization of the Skeletal Muscle Secretome Reveals a Role for Extracellular Vesicles and IL1α/IL1β in Restricting Fibro/Adipogenic Progenitor Adipogenesis.
    Simone Vumbaca, Giulio Giuliani, Valeria Fiorentini, Flavia Tortolici, Andrea Cerquone Perpetuini, Federica Riccio, Simona Sennato, Cesare Gargioli, Claudia Fuoco, Luisa Castagnoli, Gianni Cesareni.
      Repeated mechanical stress causes injuries in the adult skeletal muscle that need to be repaired. Although muscle regeneration is a highly efficient process, it fails in some pathological conditions, compromising tissue functionality. This may be caused by aberrant cell-cell communication, resulting in the deposition of fibrotic and adipose infiltrates. Here, we investigate in vivo changes in the profile of skeletal muscle secretome during the regeneration process to suggest new targetable regulatory circuits whose failure may lead to tissue degeneration in pathological conditions. We describe the kinetic variation of expression levels of 76 secreted proteins during the regeneration process. In addition, we profile the gene expression of immune cells, endothelial cells, satellite cells, and fibro-adipogenic progenitors. This analysis allowed us to annotate each cell-type with the cytokines and receptors they have the potential to synthetize, thus making it possible to draw a cell-cell interaction map. We next selected 12 cytokines whose receptors are expressed in FAPs and tested their ability to modulate FAP adipogenesis and proliferation. We observed that IL1α and IL1β potently inhibit FAP adipogenesis, while EGF and BTC notably promote FAP proliferation. In addition, we characterized the cross-talk mediated by extracellular vesicles (EVs). We first monitored the modulation of muscle EV cargo during tissue regeneration. Using a single-vesicle flow cytometry approach, we observed that EVs differentially affect the uptake of RNA and proteins into their lumen. We also investigated the EV capability to interact with SCs and FAPs and to modulate their proliferation and differentiation. We conclude that both cytokines and EVs secreted during muscle regeneration have the potential to modulate adipogenic differentiation of FAPs. The results of our approach provide a system-wide picture of mechanisms that control cell fate during the regeneration process in the muscle niche.
    Keywords:  adipogenesis; cytokine; extracellular vesicles; faps; muscle regeneration; secretome
    DOI:  https://doi.org/10.3390/biom11081171
  36. Sports Med Health Sci. 2020 Dec;2(4): 177-185
    Development and progression of cancer cachexia: Perspectives from bench to bedside.
    Seongkyun Lim, Jacob L Brown, Tyrone A Washington, Nicholas P Greene.
      Cancer cachexia (CC) is a devastating syndrome characterized by weight loss, reduced fat mass and muscle mass that affects approximately 80% of cancer patients and is responsible for 22%-30% of cancer-associated deaths. Understanding underlying mechanisms for the development of CC are crucial to advance therapies to treat CC and improve cancer outcomes. CC is a multi-organ syndrome that results in extensive skeletal muscle and adipose tissue wasting; however, CC can impair other organs such as the liver, heart, brain, and bone as well. A considerable amount of CC research focuses on changes that occur within the muscle, but cancer-related impairments in other organ systems are understudied. Furthermore, metabolic changes in organ systems other than muscle may contribute to CC. Therefore, the purpose of this review is to address degenerative mechanisms which occur during CC from a whole-body perspective. Outlining the information known about metabolic changes that occur in response to cancer is necessary to develop and enhance therapies to treat CC. As much of the current evidences in CC are from pre-clinical models we should note the majority of the data reviewed here are from preclinical models.
    Keywords:  Lewis lung carcinoma; Mitochondrial dysfunction; Muscle atrophy; Protein turnover; Tumor-bearing mouse
    DOI:  https://doi.org/10.1016/j.smhs.2020.10.003
  37. Physiol Rep. 2021 Sep;9(17): e15003
    The ubiquitin ligase Ozz decreases the replacement rate of embryonic myosin in myofibrils.
    Emi Ichimura, Koichi Ojima, Susumu Muroya, Takahiro Suzuki, Ken Kobayashi, Takanori Nishimura.
      Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.
    Keywords:  myofibril; myosin; skeletal muscle; thick filament; ubiquitin ligase; ubiquitin-proteasome system
    DOI:  https://doi.org/10.14814/phy2.15003
  38. Front Genet. 2021 ;12 652497
    Regular, Intense Exercise Training as a Healthy Aging Lifestyle Strategy: Preventing DNA Damage, Telomere Shortening and Adverse DNA Methylation Changes Over a Lifetime.
    Maha Sellami, Nicola Bragazzi, Mohammad Shoaib Prince, Joshua Denham, Mohamed Elrayess.
      Exercise training is one of the few therapeutic interventions that improves health span by delaying the onset of age-related diseases and preventing early death. The length of telomeres, the 5'-TTAGGG n -3' tandem repeats at the ends of mammalian chromosomes, is one of the main indicators of biological age. Telomeres undergo shortening with each cellular division. This subsequently leads to alterations in the expression of several genes that encode vital proteins with critical functions in many tissues throughout the body, and ultimately impacts cardiovascular, immune and muscle physiology. The sub-telomeric DNA is comprised of heavily methylated, heterochromatin. Methylation and histone acetylation are two of the most well-studied examples of the epigenetic modifications that occur on histone proteins. DNA methylation is the type of epigenetic modification that alters gene expression without modifying gene sequence. Although diet, genetic predisposition and a healthy lifestyle seem to alter DNA methylation and telomere length (TL), recent evidence suggests that training status or physical fitness are some of the major factors that control DNA structural modifications. In fact, TL is positively associated with cardiorespiratory fitness, physical activity level (sedentary, active, moderately trained, or elite) and training intensity, but is shorter in over-trained athletes. Similarly, somatic cells are vulnerable to exercise-induced epigenetic modification, including DNA methylation. Exercise-training load, however, depends on intensity and volume (duration and frequency). Training load-dependent responses in genomic profiles could underpin the discordant physiological and physical responses to exercise. In the current review, we will discuss the role of various forms of exercise training in the regulation of DNA damage, TL and DNA methylation status in humans, to provide an update on the influence exercise training has on biological aging.
    Keywords:  epigenetic clock; epigenetics; gene; oxidative stress; physical activity; skeletal muscle; telomerase
    DOI:  https://doi.org/10.3389/fgene.2021.652497
  39. Metabolites. 2021 Aug 04. pii: 512. [Epub ahead of print]11(8):
    The Leucine Catabolite and Dietary Supplement β-Hydroxy-β-Methyl Butyrate (HMB) as an Epigenetic Regulator in Muscle Progenitor Cells.
    Virve Cavallucci, Giovambattista Pani.
      β-Hydroxy-β-Methyl Butyrate (HMB) is a natural catabolite of leucine deemed to play a role in amino acid signaling and the maintenance of lean muscle mass. Accordingly, HMB is used as a dietary supplement by sportsmen and has shown some clinical effectiveness in preventing muscle wasting in cancer and chronic lung disease, as well as in age-dependent sarcopenia. However, the molecular cascades underlying these beneficial effects are largely unknown. HMB bears a significant structural similarity with Butyrate and β-Hydroxybutyrate (βHB), two compounds recognized for important epigenetic and histone-marking activities in multiple cell types including muscle cells. We asked whether similar chromatin-modifying actions could be assigned to HMB as well. Exposure of murine C2C12 myoblasts to millimolar concentrations of HMB led to an increase in global histone acetylation, as monitored by anti-acetylated lysine immunoblotting, while preventing myotube differentiation. In these effects, HMB resembled, although with less potency, the histone deacetylase (HDAC) inhibitor Sodium Butyrate. However, initial studies did not confirm a direct inhibitory effect of HMB on HDACs in vitro. β-Hydroxybutyrate, a ketone body produced by the liver during starvation or intense exercise, has a modest effect on histone acetylation of C2C12 cells or in vitro HDAC inhibitor activities, and, unlike Butyrate and HMB, did not interfere with myotube formation in a myoblast differentiation assay. Instead, βHB dramatically increased lysine β-hydroxybutyrylation (Kbhb) of histone tails, an epigenetic mark associated with fasting responses and muscle catabolic states. However, when C2C12 cells were exposed to βHB in the presence of equimolar HMB this chromatin modification was drastically reduced, pointing to a role for HMB in attenuating ketosis-associated muscle wasting. In conclusion, while their mechanistic underpinnings remain to be clarified, these preliminary observations highlight novel and potentially important activities of HMB as an epigenetic regulator and βHB antagonist in muscle precursor cells, to be further explored in their biomedical implications.
    Keywords:  HDACs; butyrate; dietary supplements; histone acetylation; ketone bodies; lysine β-hydroxybutyrylation (Kbhb); myoblasts; myotubes; sarcopenia; β-Hydroxy-β-Methyl Butyrate (HMB)
    DOI:  https://doi.org/10.3390/metabo11080512
  40. Front Sports Act Living. 2021 ;3 719434
    Under the Hood: Skeletal Muscle Determinants of Endurance Performance.
    Stephan van der Zwaard, Franck Brocherie, Richard T Jaspers.
      In the past decades, researchers have extensively studied (elite) athletes' physiological responses to understand how to maximize their endurance performance. In endurance sports, whole-body measurements such as the maximal oxygen consumption, lactate threshold, and efficiency/economy play a key role in performance. Although these determinants are known to interact, it has also been demonstrated that athletes rarely excel in all three. The leading question is how athletes reach exceptional values in one or all of these determinants to optimize their endurance performance, and how such performance can be explained by (combinations of) underlying physiological determinants. In this review, we advance on Joyner and Coyle's conceptual framework of endurance performance, by integrating a meta-analysis of the interrelationships, and corresponding effect sizes between endurance performance and its key physiological determinants at the macroscopic (whole-body) and the microscopic level (muscle tissue, i.e., muscle fiber oxidative capacity, oxygen supply, muscle fiber size, and fiber type). Moreover, we discuss how these physiological determinants can be improved by training and what potential physiological challenges endurance athletes may face when trying to maximize their performance. This review highlights that integrative assessment of skeletal muscle determinants points toward efficient type-I fibers with a high mitochondrial oxidative capacity and strongly encourages well-adjusted capillarization and myoglobin concentrations to accommodate the required oxygen flux during endurance performance, especially in large muscle fibers. Optimisation of endurance performance requires careful design of training interventions that fine tune modulation of exercise intensity, frequency and duration, and particularly periodisation with respect to the skeletal muscle determinants.
    Keywords:  V̇O2max; aerobic performance; hypertrophy; metabolism; mitochondria; muscle fiber type; oxygen transport; training
    DOI:  https://doi.org/10.3389/fspor.2021.719434
  41. Cells. 2021 Aug 10. pii: 2043. [Epub ahead of print]10(8):
    Circulating myomiRs in Muscle Denervation: From Surgical to ALS Pathological Condition.
    Irene Casola, Bianca Maria Scicchitano, Elisa Lepore, Silvia Mandillo, Elisabetta Golini, Carmine Nicoletti, Laura Barberi, Gabriella Dobrowolny, Antonio Musarò.
      ALS is a fatal neurodegenerative disease that is associated with muscle atrophy, motoneuron degeneration and denervation. Different mechanisms have been proposed to explain the pathogenesis of the disease; in this context, microRNAs have been described as biomarkers and potential pathogenetic factors for ALS. MyomiRs are microRNAs produced by skeletal muscle, and they play an important role in tissue homeostasis; moreover, they can be released in blood circulation in pathological conditions, including ALS. However, the functional role of myomiRs in muscle denervation has not yet been fully clarified. In this study, we analyze the levels of two myomiRs, namely miR-206 and miR-133a, in skeletal muscle and blood samples of denervated mice, and we demonstrate that surgical denervation reduces the expression of both miR-206 and miR-133a, while miR-206 but not miR-133a is upregulated during the re-innervation process. Furthermore, we quantify the levels of miR-206 and miR-133a in serum samples of two ALS mouse models, characterized by different disease velocities, and we demonstrate a different modulation of circulating myomiRs during ALS disease, according to the velocity of disease progression. Moreover, taking into account surgical and pathological denervation, we describe a different response to increasing amounts of circulating miR-206, suggesting a hormetic effect of miR-206 in relation to changes in neuromuscular communication.
    Keywords:  ALS; circulating miRNA; muscle denervation
    DOI:  https://doi.org/10.3390/cells10082043
  42. Metabolites. 2021 Aug 06. pii: 522. [Epub ahead of print]11(8):
    N-acetyltaurine and Acetylcarnitine Production for the Mitochondrial Acetyl-CoA Regulation in Skeletal Muscles during Endurance Exercises.
    Teruo Miyazaki, Yuho Nakamura-Shinya, Kei Ebina, Shoichi Komine, Song-Gyu Ra, Keisuke Ishikura, Hajime Ohmori, Akira Honda.
      During endurance exercises, a large amount of mitochondrial acetyl-CoA is produced in skeletal muscles from lipids, and the excess acetyl-CoA suppresses the metabolic flux from glycolysis to the TCA cycle. This study evaluated the hypothesis that taurine and carnitine act as a buffer of the acetyl moiety of mitochondrial acetyl-CoA derived from the short- and long-chain fatty acids of skeletal muscles during endurance exercises. In human subjects, the serum concentrations of acetylated forms of taurine (NAT) and carnitine (ACT), which are the metabolites of acetyl-CoA buffering, significantly increased after a full marathon. In the culture medium of primary human skeletal muscle cells, NAT and ACT concentrations significantly increased when they were cultured with taurine and acetate or with carnitine and palmitic acid, respectively. The increase in the mitochondrial acetyl-CoA/free CoA ratio induced by acetate and palmitic acid was suppressed by taurine and carnitine, respectively. Elevations of NAT and ACT in the blood of humans during endurance exercises might serve the buffering of the acetyl-moiety in mitochondria by taurine and carnitine, respectively. The results suggest that blood levels of NAT and ACT indicate energy production status from fatty acids in the skeletal muscles of humans undergoing endurance exercise.
    Keywords:  ACT; NAT; acetyl-CoA; carnitine; endurance exercise; mitochondria; skeletal muscle; taurine
    DOI:  https://doi.org/10.3390/metabo11080522
  43. Int J Mol Sci. 2021 Aug 13. pii: 8715. [Epub ahead of print]22(16):
    Regular Physical Exercise Modulates Iron Homeostasis in the 5xFAD Mouse Model of Alzheimer's Disease.
    Irina Belaya, Nina Kucháriková, Veronika Górová, Kai Kysenius, Dominic J Hare, Peter J Crouch, Tarja Malm, Mustafa Atalay, Anthony R White, Jeffrey R Liddell, Katja M Kanninen.
      Dysregulation of brain iron metabolism is one of the pathological features of aging and Alzheimer's disease (AD), a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. While physical inactivity is one of the risk factors for AD and regular exercise improves cognitive function and reduces pathology associated with AD, the underlying mechanisms remain unclear. The purpose of the study is to explore the effect of regular physical exercise on modulation of iron homeostasis in the brain and periphery of the 5xFAD mouse model of AD. By using inductively coupled plasma mass spectrometry and a variety of biochemical techniques, we measured total iron content and level of proteins essential in iron homeostasis in the brain and skeletal muscles of sedentary and exercised mice. Long-term voluntary running induced redistribution of iron resulted in altered iron metabolism and trafficking in the brain and increased iron content in skeletal muscle. Exercise reduced levels of cortical hepcidin, a key regulator of iron homeostasis, coupled with interleukin-6 (IL-6) decrease in cortex and plasma. We propose that regular exercise induces a reduction of hepcidin in the brain, possibly via the IL-6/STAT3/JAK1 pathway. These findings indicate that regular exercise modulates iron homeostasis in both wild-type and AD mice.
    Keywords:  5xFAD mouse; Alzheimer’s disease; cortex; hepcidin; il-6; iron; regular voluntary exercise; skeletal muscle
    DOI:  https://doi.org/10.3390/ijms22168715
  44. J Physiol Biochem. 2021 Aug 23.
    Adiponectin overexpression in C2C12 myocytes increases lipid oxidation and myofiber transition.
    Marta Lopez-Yus, Rebeca Lopez-Perez, Maria Pilar Garcia-Sobreviela, Raquel Del Moral-Bergos, Silvia Lorente-Cebrian, Jose M Arbones-Mainar.
      Metabolic syndrome and obesity have detrimental effects on the metabolic function of the skeletal muscle. Mounting evidence indicates that patients with those conditions may present an increased ratio of glycolytic to oxidative fibers associated with a decrease in oxidative capacity. In this regard, adiponectin, a hormone mainly secreted by adipocytes that regulates glucose and lipid metabolism, has emerged as a myokine that could play an important role in this process. We aimed to investigate whether adiponectin overexpression in skeletal muscle might be a local protective mechanism, favoring fatty acid utilization. To that end, we generated an in vitro model of myocytes with upregulated endogenous adiponectin using a lentiviral carrier. We demonstrated that the adiponectin-transduced myocytes were able to produce and secrete fully functional adiponectin complexes. Adiponectin overexpression remarkably upregulated the mRNA level of myogenic regulatory factors as well as genes implicated in lipolysis (HSL, ATGL) and cellular and mitochondrial fatty acid transport (LPL, CD36, CPT1B). This was accompanied by increased isoproterenol-induced lipolysis and β-oxidation and reduced lipogenesis, whereas insulin-stimulated glucose uptake was unaltered in transduced myocytes. Lastly, the relative expression of the more glycolytic myofibers (MyHC IIb) compared to the more oxidative ones (MyHC I) was notably reduced. Our results showed that the released adiponectin acted in an autocrine/paracrine manner, increasing lipid oxidation in myocytes and leading to a transition of myofibers from the glycolytic to the oxidative type. In conclusion, muscle adiponectin overexpression might be a way to relieve muscle diseases caused by oxidative muscle fiber deficiency.
    Keywords:  Adipokine; Lentivirus; Muscle; Myopathy
    DOI:  https://doi.org/10.1007/s13105-021-00836-7
  45. Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2101115118. [Epub ahead of print]118(35):
    Clock proteins and training modify exercise capacity in a daytime-dependent manner.
    Yaarit Adamovich, Vaishnavi Dandavate, Saar Ezagouri, Gal Manella, Ziv Zwighaft, Jonathan Sobel, Yael Kuperman, Marina Golik, Asher Auerbach, Maxim Itkin, Sergey Malitsky, Gad Asher.
      Exercise and circadian biology are closely intertwined with physiology and metabolism, yet the functional interaction between circadian clocks and exercise capacity is only partially characterized. Here, we tested different clock mutant mouse models to examine the effect of the circadian clock and clock proteins, namely PERIODs and BMAL1, on exercise capacity. We found that daytime variance in endurance exercise capacity is circadian clock controlled. Unlike wild-type mice, which outperform in the late compared with the early part of their active phase, PERIODs- and BMAL1-null mice do not show daytime variance in exercise capacity. It appears that BMAL1 impairs and PERIODs enhance exercise capacity in a daytime-dependent manner. An analysis of liver and muscle glycogen stores as well as muscle lipid utilization suggested that these daytime effects mostly relate to liver glycogen levels and correspond to the animals' feeding behavior. Furthermore, given that exercise capacity responds to training, we tested the effect of training at different times of the day and found that training in the late compared with the early part of the active phase improves exercise performance. Overall, our findings suggest that clock proteins shape exercise capacity in a daytime-dependent manner through changes in liver glycogen levels, likely due to their effect on animals' feeding behavior.
    Keywords:  circadian clocks; exercise; glycogen; metabolism; training
    DOI:  https://doi.org/10.1073/pnas.2101115118
  46. Med Sci Sports Exerc. 2021 Aug 24.
    Early Onset Physical Inactivity and Metabolic Dysfunction in Tumor-bearing Mice Is Associated with Accelerated Cachexia.
    Brittany R Counts, Jessica L Halle, James A Carson.
      Cancer-induced skeletal muscle mass loss is a critical characteristic of cachexia. While physical inactivity and systemic metabolic dysfunction can precede cachexia development, how these early-onset disruptions are related to cachexia's eventual severity is not well understood. The well-established Lewis Lung Carcinoma (LLC) pre-clinical cachexia model exhibits a varying degree of cachexia. Therefore, we examined if the early onset of physical inactivity and metabolic dysfunction were associated with accelerated cachexia development in LLC tumor-bearing mice.
    METHODS: Male C57BL/6 J (12 wks. age) were injected with 1 x 106 LLC cells or PBS subcutaneously in the right flank, and tissue was collected 26-28 days post cell injection. Tumor volume was measured every 5 days throughout the study to calculate the tumor growth rate. Fifteen days post tumor inoculation, a subset of PBS (n = 11) and LLC (n = 16) mice were individually housed in metabolic CLAMs cages for 5 days.
    RESULTS: LLC mice exhibited greater body weight loss (-5.1%), decreased muscle mass (-7%), decreased fat mass (-22%), and increased plasma IL-6 (212%) compared to PBS mice. Before the onset of cachexia, total cage activity was decreased in tumor-bearing mice. Cage activity was negatively associated to tumor mass and positively associated to hindlimb muscle mass. Additionally, LLC mice had greater lipid oxidation than PBS mice.
    CONCLUSION: LLC mice exhibit early-onset physical inactivity and altered systemic lipid oxidation, which are associated with the eventual development of cachexia.
    DOI:  https://doi.org/10.1249/MSS.0000000000002772
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