bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2022–05–29
47 papers selected by
Anna Vainshtein, Craft Science Inc.
  1. Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity.
  2. Depletion of skeletal muscle satellite cells attenuates pathology in muscular dystrophy.
  3. Effects of Acute and Chronic Resistance Exercise on the Skeletal Muscle Metabolome.
  4. Functional analysis reveals that Tinagl1 is required for normal muscle development in mice through the activation of ERK signaling.
  5. MitoQ supplementation augments acute exercise-induced increases in muscle PGC1α mRNA and improves training-induced increases in peak power independent of mitochondrial content and function in untrained middle-aged men.
  6. Optimized lentiviral vector to restore full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy.
  7. Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.
  8. Taurine Administration Counteracts Aging-Associated Impingement of Skeletal Muscle Regeneration by Reducing Inflammation and Oxidative Stress.
  9. Exercise Counteracts the Deleterious Effects of Cancer Cachexia.
  10. The p97-Nploc4 ATPase complex plays a role in muscle atrophy during cancer and amyotrophic lateral sclerosis.
  11. Differential effects of calorie restriction and rapamycin on age-related molecular and functional changes in skeletal muscle.
  12. Hidden Agenda - The Involvement of Endoplasmic Reticulum Stress and Unfolded Protein Response in Inflammation-Induced Muscle Wasting.
  13. Mechanosensors control skeletal muscle mass, molecular clocks, and metabolism.
  14. Farnesol-Loaded Nanoliposomes Inhibit Inflammatory Gene Expression in Primary Human Skeletal Myoblasts.
  15. Activation and Migration of Human Skeletal Muscle Stem Cells In Vitro Differently Rely on Calcium Signals.
  16. Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes That Treat Rotator Cuff Muscle Degeneration in Mice and Are Identified in Humans by Single-Cell RNA Sequencing.
  17. Investigation of Brain Function-Related Myokine Secretion by Using Contractile 3D-Engineered Muscle.
  18. Redox Control of Signalling Responses to Contractile Activity and Ageing in Skeletal Muscle.
  19. Inflammation and Wasting of Skeletal Muscles in Kras-p53-Mutant Mice with Intraepithelial Neoplasia and Pancreatic Cancer-When Does Cachexia Start?
  20. Repeated and Interrupted Resistance Exercise Induces the Desensitization and Re-Sensitization of mTOR-Related Signaling in Human Skeletal Muscle Fibers.
  21. Thyroid Hormone Receptor Isoforms Alpha and Beta Play Convergent Roles in Muscle Physiology and Metabolic Regulation.
  22. Low Inflammatory Stimulus Increases D2 Activity and Modulates Thyroid Hormone Metabolism during Myogenesis In Vitro.
  23. Quercetin attenuates adipogenesis and fibrosis in human skeletal muscle.
  24. SS-31 does not prevent or reduce muscle atrophy 7 days after a 65 kdyne contusion spinal cord injury in young male mice.
  25. Declines in muscle protein synthesis account for short-term muscle disuse atrophy in humans in the absence of increased muscle protein breakdown.
  26. (-) - Epicatechin improves Tibialis anterior muscle repair in CD1 mice with BaCl2-induced damage.
  27. An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling.
  28. Bap1/SMN axis in Dpp4+ skeletal muscle mesenchymal cells regulates the neuromuscular system.
  29. Large1 gene transfer in older myd mice with severe muscular dystrophy restores muscle function and greatly improves survival.
  30. Pharmaceutical agents for contractile-metabolic dysfunction after volumetric muscle loss.
  31. Effect of enhanced muscle tone on the expression of atrogenes and cytoskeletal proteins during postural muscle unloading.
  32. BNIP3 is involved in muscle fiber atrophy in Late-Onset Pompe Disease patients.
  33. Interaction between mesenchymal stem cells and myoblasts in the context of facioscapulohumeral muscular dystrophy contributes to the disease phenotype.
  34. Resveratrol ameliorates muscle atrophy in chronic kidney disease via the axis of SIRT1/FoxO1.
  35. SERUM EXTRACELLULAR VESICLES FOR DELIVERY OF CRISPR-CAS9 RIBONUCLEOPROTEINS TO MODIFY THE DYSTROPHIN GENE.
  36. Full spectrum cytometry improves the resolution of highly autofluorescent biological samples: Identification of myeloid cells in regenerating skeletal muscles.
  37. Neuromuscular Aging: A Case for the Neuroprotective Effects of Dancing.
  38. Skeletal Muscle and COVID-19: The Potential Involvement of Bioactive Sphingolipids.
  39. Understanding the Human Aging Proteome Using Epidemiological Models.
  40. Size-tunable PEG-grafted copolymers as a polymeric nanoruler for passive targeting muscle tissues.
  41. The Effect of Glucagon on Protein Catabolism During Insulin Deficiency - Exchange of Amino acids Across Skeletal Muscle and The Splanchnic Bed.
  42. Aging and short-term calorie restriction differently affect the cardiac and skeletal muscle expression of genes regulating energy substrate utilization in male rats.
  43. The superior beneficial effects of exercise training versus hormone replacement therapy on skeletal muscle of ovariectomized rats.
  44. Development of Therapeutic RNA Manipulation for Muscular Dystrophy.
  45. The influence of longitudinal muscle fascicle growth on mechanical function.
  46. Resistance Training before, during, and after COVID-19 Infection: What Have We Learned So Far?
  47. Contractile force assessment methods for in vitro skeletal muscle tissues.
  1. Membranes (Basel). 2022 Apr 26. pii: 464. [Epub ahead of print]12(5):
    Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity.
    Benjamin Bydak, Taiana M Pierdoná, Samira Seif, Karim Sidhom, Patience O Obi, Hagar I Labouta, Joseph W Gordon, Ayesha Saleem.
      Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle-derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8-10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10-13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400-600, whereas myotube-Eps were largely 50-300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6-9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4-6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4-6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7-12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4-12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5-6). Myoblasts treated with control or acute stimulation-derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3-4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle-differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation-derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle-specific EVs are warranted.
    Keywords:  acute contractile activity; differential ultracentrifugation; differentiation; extracellular particles; extracellular vesicles; myoblasts; myotubes; secretome; skeletal muscle; total exosome isolation kit
    DOI:  https://doi.org/10.3390/membranes12050464
  2. Nat Commun. 2022 May 26. 13(1): 2940
    Depletion of skeletal muscle satellite cells attenuates pathology in muscular dystrophy.
    Justin G Boyer, Jiuzhou Huo, Sarah Han, Julian R Havens, Vikram Prasad, Brian L Lin, David A Kass, Taejeong Song, Sakthivel Sadayappan, Ramzi J Khairallah, Christopher W Ward, Jeffery D Molkentin.
      Skeletal muscle can repair and regenerate due to resident stem cells known as satellite cells. The muscular dystrophies are progressive muscle wasting diseases underscored by chronic muscle damage that is continually repaired by satellite cell-driven regeneration. Here we generate a genetic strategy to mediate satellite cell ablation in dystrophic mouse models to investigate how satellite cells impact disease trajectory. Unexpectedly, we observe that depletion of satellite cells reduces dystrophic disease features, with improved histopathology, enhanced sarcolemmal stability and augmented muscle performance. Mechanistically, we demonstrate that satellite cells initiate expression of the myogenic transcription factor MyoD, which then induces re-expression of fetal genes in the myofibers that destabilize the sarcolemma. Indeed, MyoD re-expression in wildtype adult skeletal muscle reduces membrane stability and promotes histopathology, while MyoD inhibition in a mouse model of muscular dystrophy improved membrane stability. Taken together these observations suggest that satellite cell activation and the fetal gene program is maladaptive in chronic dystrophic skeletal muscle.
    DOI:  https://doi.org/10.1038/s41467-022-30619-7
  3. Metabolites. 2022 May 16. pii: 445. [Epub ahead of print]12(5):
    Effects of Acute and Chronic Resistance Exercise on the Skeletal Muscle Metabolome.
    Sebastian Gehlert, Patrick Weinisch, Werner Römisch-Margl, Richard T Jaspers, Anna Artati, Jerzy Adamski, Kenneth A Dyar, Thorben Aussieker, Daniel Jacko, Wilhelm Bloch, Henning Wackerhage, Gabi Kastenmüller.
      Resistance training promotes metabolic health and stimulates muscle hypertrophy, but the precise routes by which resistance exercise (RE) conveys these health benefits are largely unknown.
    AIM: To investigate how acute RE affects human skeletal muscle metabolism.
    METHODS: We collected vastus lateralis biopsies from six healthy male untrained volunteers at rest, before the first of 13 RE training sessions, and 45 min after the first and last bouts of RE. Biopsies were analysed using untargeted mass spectrometry-based metabolomics.
    RESULTS: We measured 617 metabolites covering a broad range of metabolic pathways. In the untrained state RE altered 33 metabolites, including increased 3-methylhistidine and N-lactoylvaline, suggesting increased protein breakdown, as well as metabolites linked to ATP (xanthosine) and NAD (N1-methyl-2-pyridone-5-carboxamide) metabolism; the bile acid chenodeoxycholate also increased in response to RE in muscle opposing previous findings in blood. Resistance training led to muscle hypertrophy, with slow type I and fast/intermediate type II muscle fibre diameter increasing by 10.7% and 10.4%, respectively. Comparison of post-exercise metabolite levels between trained and untrained state revealed alterations of 46 metabolites, including decreased N-acetylated ketogenic amino acids and increased beta-citrylglutamate which might support growth. Only five of the metabolites that changed after acute exercise in the untrained state were altered after chronic training, indicating that training induces multiple metabolic changes not directly related to the acute exercise response.
    CONCLUSION: The human skeletal muscle metabolome is sensitive towards acute RE in the trained and untrained states and reflects a broad range of adaptive processes in response to repeated stimulation.
    Keywords:  beta-citrylglutamate; chenodeoxycholate; hypertrophy; metabolomics; resistance exercise; skeletal muscle; skeletal muscle adaptation
    DOI:  https://doi.org/10.3390/metabo12050445
  4. Biochim Biophys Acta Mol Cell Res. 2022 May 18. pii: S0167-4889(22)00086-6. [Epub ahead of print]1869(9): 119294
    Functional analysis reveals that Tinagl1 is required for normal muscle development in mice through the activation of ERK signaling.
    Yoriko Sato, Keisuke Kawashima, Emiko Fukui, Hiromichi Matsumoto, Fumiaki Yoshizawa, Yusuke Sato.
      Tinagl1 (tubulointerstitial nephritis antigen-like 1) is a matricellular protein involved in female infertility and breast cancer tumorigenesis. In this study, we analyzed the function of Tinagl1 in skeletal muscle using knockout mice and cell experiments. Although primary myoblasts isolated from Tinagl1-decifient (Tinagl1-/-) mice differentiated into normal myotubes, and treatment with recombinant Tinagl1 did not affect the proliferation or differentiation of C2C12 myoblasts, Tinagl1-/- mice exhibited reduced body mass and calf muscle weights compared to the control group (Tinagl1flox/flox). Furthermore, Tinagl1-/- mice showed myofibers with centrally located nuclei, which is a morphological marker of regenerating muscle or myopathy. In addition, the capillary density in the soleus muscle of Tinagl1-/- mice showed a decreasing trend compared to that of the control group. Importantly, si-RNA-mediated knockdown of TINAGL1 resulted in reduced tube formation in human umbilical vein endothelial cells (HUVECs), whereas treatment with Tinagl1 promoted tube formation. Immunoblot analysis revealed that Tinagl1 activates ERK signaling in both HUVECs and C2C12 myoblasts and myotubes, which are involved in the regulation of myogenic differentiation, proliferation, metabolism, and angiogenesis. Our results demonstrate that Tinagl1 may be required for normal muscle and capillary development through the activation of ERK signaling.
    Keywords:  Capillary; ERK signaling; Myopathy; Skeletal muscle; Tinagl1
    DOI:  https://doi.org/10.1016/j.bbamcr.2022.119294
  5. Redox Biol. 2022 May 20. pii: S2213-2317(22)00113-6. [Epub ahead of print]53 102341
    MitoQ supplementation augments acute exercise-induced increases in muscle PGC1α mRNA and improves training-induced increases in peak power independent of mitochondrial content and function in untrained middle-aged men.
    S C Broome, T Pham, A J Braakhuis, R Narang, H W Wang, A J R Hickey, C J Mitchell, T L Merry.
      The role of mitochondrial ROS in signalling muscle adaptations to exercise training has not been explored in detail. We investigated the effect of supplementation with the mitochondria-targeted antioxidant MitoQ on a) the skeletal muscle mitochondrial and antioxidant gene transcriptional response to acute high-intensity exercise and b) skeletal muscle mitochondrial content and function following exercise training. In a randomised, double-blind, placebo-controlled, parallel design study, 23 untrained men (age: 44 ± 7 years, VO2peak: 39.6 ± 7.9 ml/kg/min) were randomised to receive either MitoQ (20 mg/d) or a placebo for 10 days before completing a bout of high-intensity interval exercise (cycle ergometer, 10 × 60 s at VO2peak workload with 75 s rest). Blood samples and vastus lateralis muscle biopsies were collected before exercise and immediately and 3 h after exercise. Participants then completed high-intensity interval training (HIIT; 3 sessions per week for 3 weeks) and another blood sample and muscle biopsy were collected. There was no effect of acute exercise or MitoQ on systemic (plasma protein carbonyls and reduced glutathione) or skeletal muscle (mtDNA damage and 4-HNE) oxidative stress biomarkers. Acute exercise-induced increases in skeletal muscle peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) mRNA expression were augmented in the MitoQ group. Despite this, training-induced increases in skeletal muscle mitochondrial content were similar between groups. HIIT-induced increases in VO2peak and 20 km time trial performance were also similar between groups while training-induced increases in peak power achieved during the VO2peak test were augmented in the MitoQ group. These data suggest that training-induced increases in peak power are enhanced following MitoQ supplementation, which may be related to the augmentation of skeletal muscle PGC1α expression following acute exercise. However, these effects do not appear to be related to an effect of MitoQ supplementation on exercise-induced oxidative stress or training-induced mitochondrial biogenesis in skeletal muscle.
    Keywords:  Adaptation; Antioxidant; Exercise; Mitochondria; Performance; ROS
    DOI:  https://doi.org/10.1016/j.redox.2022.102341
  6. Mol Ther Methods Clin Dev. 2022 Jun 09. 25 491-507
    Optimized lentiviral vector to restore full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy.
    Jinhong Meng, Marc Moore, John Counsell, Francesco Muntoni, Linda Popplewell, Jennifer Morgan.
      Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally mediated delivery of such a large gene in vivo has been hindered by lack of tissue specificity, limited transduction, and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contains a muscle-specific promoter and sequence-optimized full-length dystrophin, to constrain dystrophin expression to differentiated myotubes/myofibers and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We show that these lentivirally corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58% ± 2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4% ± 2.87% of the donor-derived regenerated muscle fibers in vivo, which were able to recruit members of the dystrophin-glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle.
    Keywords:  Duchenne muscular dystrophy; lentivirus; muscle-specific promoter; myoblasts; native full-length dystrophin; sequence-optimized full-length dystrophin
    DOI:  https://doi.org/10.1016/j.omtm.2022.04.015
  7. Biomedicines. 2022 May 23. pii: 1204. [Epub ahead of print]10(5):
    Generation of hiPSC-Derived Skeletal Muscle Cells: Exploiting the Potential of Skeletal Muscle-Derived hiPSCs.
    Eric Metzler, Helena Escobar, Daniele Yumi Sunaga-Franze, Sascha Sauer, Sebastian Diecke, Simone Spuler.
      Cell therapies for muscle wasting disorders are on the verge of becoming a realistic clinical perspective. Muscle precursor cells derived from human induced pluripotent stem cells (hiPSCs) represent the key to unrestricted cell numbers indispensable for the treatment of generalized muscle wasting such as cachexia or intensive care unit (ICU)-acquired weakness. We asked how the cell of origin influences efficacy and molecular properties of hiPSC-derived muscle progenitor cells. We generated hiPSCs from primary muscle stem cells and from peripheral blood mononuclear cells (PBMCs) of the same donors (n = 4) and compared their molecular profiles, myogenic differentiation potential, and ability to generate new muscle fibers in vivo. We show that reprogramming into hiPSCs from primary muscle stem cells was faster and 35 times more efficient than from blood cells. Global transcriptome comparison revealed significant differences, but differentiation into induced myogenic cells using a directed transgene-free approach could be achieved with muscle- and PBMC-derived hiPSCs, and both cell types generated new muscle fibers in vivo. Differences in myogenic differentiation efficiency were identified with hiPSCs generated from individual donors. The generation of muscle-stem-cell-derived hiPSCs is a fast and economic method to obtain unrestricted cell numbers for cell-based therapies in muscle wasting disorders, and in this aspect are superior to blood-derived hiPSCs.
    Keywords:  epigenetic memory; hiPSCs; muscle stem cells; myogenic differentiation; reprogramming; transplantation
    DOI:  https://doi.org/10.3390/biomedicines10051204
  8. Antioxidants (Basel). 2022 May 21. pii: 1016. [Epub ahead of print]11(5):
    Taurine Administration Counteracts Aging-Associated Impingement of Skeletal Muscle Regeneration by Reducing Inflammation and Oxidative Stress.
    Alessandra Barbiera, Silvia Sorrentino, Damon Fard, Elisa Lepore, Gigliola Sica, Gabriella Dobrowolny, Luca Tamagnone, Bianca Maria Scicchitano.
      Sarcopenia, which occurs during aging, is characterized by the gradual loss of skeletal muscle mass and function, resulting in a functional decline in physical abilities. Several factors contribute to the onset of sarcopenia, including reduced regenerative capacity, chronic low-grade inflammation, mitochondrial dysfunction, and increased oxidative stress, leading to the activation of catabolic pathways. Physical activity and adequate protein intake are considered effective strategies able to reduce the incidence and severity of sarcopenia by exerting beneficial effects in improving the muscular anabolic response during aging. Taurine is a non-essential amino acid that is highly expressed in mammalian tissues and, particularly, in skeletal muscle where it is involved in the regulation of biological processes and where it acts as an antioxidant and anti-inflammatory factor. Here, we evaluated whether taurine administration in old mice counteracts the physiopathological effects of aging in skeletal muscle. We showed that, in injured muscle, taurine enhances the regenerative process by downregulating the inflammatory response and preserving muscle fiber integrity. Moreover, taurine attenuates ROS production in aged muscles by maintaining a proper cellular redox balance, acting as an antioxidant molecule. Although further studies are needed to better elucidate the molecular mechanisms responsible for the beneficial effect of taurine on skeletal muscle homeostasis, these data demonstrate that taurine administration ameliorates the microenvironment allowing an efficient regenerative process and attenuation of the catabolic pathways related to the onset of sarcopenia.
    Keywords:  PGC1α; ROS; nutrition; sarcopenia
    DOI:  https://doi.org/10.3390/antiox11051016
  9. Cancers (Basel). 2022 May 19. pii: 2512. [Epub ahead of print]14(10):
    Exercise Counteracts the Deleterious Effects of Cancer Cachexia.
    Stavroula Tsitkanou, Kevin A Murach, Tyrone A Washington, Nicholas P Greene.
      Cancer cachexia (CC) is a multifactorial syndrome characterised by unintentional loss of body weight and muscle mass in patients with cancer. The major hallmarks associated with CC development and progression include imbalanced protein turnover, inflammatory signalling, mitochondrial dysfunction and satellite cell dysregulation. So far, there is no effective treatment to counteract muscle wasting in patients with CC. Exercise training has been proposed as a potential therapeutic approach for CC. This review provides an overview of the effects of exercise training in CC-related mechanisms as well as how factors such as cancer comorbidities, exercise modality and biological sex can influence exercise effectiveness in CC. Evidence in mice and humans suggests exercise training combats all of the hallmarks of CC. Several exercise modalities induce beneficial adaptations in patients/animals with CC, but concurrent resistance and endurance training is considered the optimal type of exercise. In the case of cancer patients presenting comorbidities, exercise training should be performed only under specific guidelines and precautions to avoid adverse effects. Observational comparison of studies in CC using different biological sex shows exercise-induced adaptations are similar between male and female patients/animals with cancer, but further studies are needed to confirm this.
    Keywords:  cancer comorbidities; concurrent training; inflammation; mitochondria; physical activity; protein turnover; satellite cells
    DOI:  https://doi.org/10.3390/cancers14102512
  10. J Cachexia Sarcopenia Muscle. 2022 May 25.
    The p97-Nploc4 ATPase complex plays a role in muscle atrophy during cancer and amyotrophic lateral sclerosis.
    Andrea David Re Cecconi, Mara Barone, Simona Gaspari, Massimo Tortarolo, Caterina Bendotti, Luca Porcu, Giulia Terribile, Rosanna Piccirillo.
       BACKGROUND: The p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different protein adaptors. We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis.
    METHODS: As cancer cachexia models, we used mice bearing colon adenocarcinoma C26, human renal carcinoma RXF393, or Lewis lung carcinoma, with breast cancer 4T1-injected mice as controls. As ALS models, we employed 129/SvHsd mice carrying the mutation G93A in human SOD1. The expression of p97 and its adaptors was analysed in their muscles by quantitative real-time polymerase chain reaction (qPCR) and western blot. We electroporated plasmids into muscles or treated mice with disulfiram (DSF) to test the effects of inhibiting p97 and nuclear protein localization protein 4 (Nploc4), one of its adaptors, on atrophy.
    RESULTS: The mRNA levels of p97 were induced by 1.5-fold to 2-fold in tibialis anterior (TA) of all the cachectic models but not in the non-cachectic 4T1 tumour-bearing mice (P ≤ 0.05). Similarly, p97 was high both in mRNA and protein in TA from 17-week-old SOD1G93A mice (P ≤ 0.01). Electroporation of a shRNA for murine p97 into mouse muscle reduced the fibre atrophy caused by C26 (P = 0.0003) or ALS (P ≤ 0.01). When we interrogated a microarray, we had previously generated for the expression of p97 adaptors, we found Derl1, Herpud1, Nploc4, Rnf31, and Hsp90ab1 induced in cachectic TA from C26-mice (Fold change > 1.2, adjusted P ≤ 0.05). By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one also induced at the protein level by 1.5-fold (P ≤ 0.01). Electroporation of a CRISPR/Cas9 vector against Nploc4 into muscle reduced the fibre atrophy caused by C26 (P = 0.01) or ALS (P ≤ 0.0001). Because DSF uncouples p97 from Nploc4, we treated atrophying myotubes with DSF, and found accumulated mono and polyubiquitinated proteins and reduced degradation of long-lived proteins by 35% (P ≤ 0.0001), including actin (P ≤ 0.05). DSF halves Nploc4 in the soluble muscle fraction (P ≤ 0.001) and given to C26-bearing mice limited the body and muscle weight loss (P ≤ 0.05), with no effect on tumour growth.
    CONCLUSIONS: Overall, cancer cachexia and ALS seem to display similar mechanisms of muscle wasting at least at the catabolic level. The p97-Nploc4 complex appears to have a crucial role in muscle atrophy during these disorders and disrupting this complex might serve as a novel drug strategy.
    Keywords:  Amyotrophic lateral sclerosis; Cancer cachexia; Muscle wasting; Nploc4; Protein degradation; p97-VCP complex
    DOI:  https://doi.org/10.1002/jcsm.13011
  11. Exp Gerontol. 2022 May 24. pii: S0531-5565(22)00149-8. [Epub ahead of print] 111841
    Differential effects of calorie restriction and rapamycin on age-related molecular and functional changes in skeletal muscle.
    Melissa C Orenduff, Michael F Coleman, Elaine M Glenny, Kim M Huffman, Erika T Rezeli, Akshay Bareja, Carl F Pieper, Virginia B Kraus, Stephen D Hursting.
      Aging is a multifactorial process associated with progressive degradation of physiological integrity and function. One of the greatest factors contributing to the deleterious effects of aging is the decline of functional ability due to loss of muscle mass, strength, and function, a condition termed sarcopenia. Calorie restriction (CR) has consistently been shown to extend lifespan and delay the onset and progression of various age-related diseases, including sarcopenia. Additional anti-aging interventions that are receiving scientific attention are CR mimetics. Of these pharmacological compounds, rapamycin has shown similar CR-related longevity benefits without the need for diet restrictions. To investigate the potential role of rapamycin as an anti-sarcopenic alternative to CR, we conducted a study in male and female C57BL/6 J mice to assess the effects of rapamycin on age-related gene expression changes in skeletal muscle associated with loss of muscle mass, strength, and function, relative to control. We hypothesize that the effects of rapamycin will closely align with CR with respect to physical function and molecular indices associated with muscle quality. Our results indicate CR and rapamycin provide partial protection against age-related decline in muscle, while engaging uniquely different molecular pathways in skeletal muscle. Our preclinical findings of the therapeutic potential of rapamycin or a CR regimen on geroprotective benefits in muscle should be extended to translational studies towards the development of effective strategies for the prevention and management of sarcopenia.
    Keywords:  Calorie restriction; Rapamycin; Sarcopenia; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.exger.2022.111841
  12. Front Immunol. 2022 ;13 878755
    Hidden Agenda - The Involvement of Endoplasmic Reticulum Stress and Unfolded Protein Response in Inflammation-Induced Muscle Wasting.
    Melanie Kny, Jens Fielitz.
      Critically ill patients at the intensive care unit (ICU) often develop a generalized weakness, called ICU-acquired weakness (ICUAW). A major contributor to ICUAW is muscle atrophy, a loss of skeletal muscle mass and function. Skeletal muscle assures almost all of the vital functions of our body. It adapts rapidly in response to physiological as well as pathological stress, such as inactivity, immobilization, and inflammation. In response to a reduced workload or inflammation muscle atrophy develops. Recent work suggests that adaptive or maladaptive processes in the endoplasmic reticulum (ER), also known as sarcoplasmic reticulum, contributes to this process. In muscle cells, the ER is a highly specialized cellular organelle that assures calcium homeostasis and therefore muscle contraction. The ER also assures correct folding of proteins that are secreted or localized to the cell membrane. Protein folding is a highly error prone process and accumulation of misfolded or unfolded proteins can cause ER stress, which is counteracted by the activation of a signaling network known as the unfolded protein response (UPR). Three ER membrane residing molecules, protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring protein 1a (IRE1a), and activating transcription factor 6 (ATF6) initiate the UPR. The UPR aims to restore ER homeostasis by reducing overall protein synthesis and increasing gene expression of various ER chaperone proteins. If ER stress persists or cannot be resolved cell death pathways are activated. Although, ER stress-induced UPR pathways are known to be important for regulation of skeletal muscle mass and function as well as for inflammation and immune response its function in ICUAW is still elusive. Given recent advances in the development of ER stress modifying molecules for neurodegenerative diseases and cancer, it is important to know whether or not therapeutic interventions in ER stress pathways have favorable effects and these compounds can be used to prevent or treat ICUAW. In this review, we focus on the role of ER stress-induced UPR in skeletal muscle during critical illness and in response to predisposing risk factors such as immobilization, starvation and inflammation as well as ICUAW treatment to foster research for this devastating clinical problem.
    Keywords:  endoplasmic reticulum stress; inflammation; intensive care unit acquired weakness; sepsis; unfolded protein response
    DOI:  https://doi.org/10.3389/fimmu.2022.878755
  13. Cell Mol Life Sci. 2022 May 27. 79(6): 321
    Mechanosensors control skeletal muscle mass, molecular clocks, and metabolism.
    Mathias Vanmunster, Ana Victoria Rojo Garcia, Alexander Pacolet, Sebastiaan Dalle, Katrien Koppo, Ilse Jonkers, Rik Lories, Frank Suhr.
       BACKGROUND: Skeletal muscles (SkM) are mechanosensitive, with mechanical unloading resulting in muscle-devastating conditions and altered metabolic properties. However, it remains unexplored whether these atrophic conditions affect SkM mechanosensors and molecular clocks, both crucial for their homeostasis and consequent physiological metabolism.
    METHODS: We induced SkM atrophy through 14 days of hindlimb suspension (HS) in 10 male C57BL/6J mice and 10 controls (CTR). SkM histology, gene expressions and protein levels of mechanosensors, molecular clocks and metabolism-related players were examined in the m. Gastrocnemius and m. Soleus. Furthermore, we genetically reduced the expression of mechanosensors integrin-linked kinase (Ilk1) and kindlin-2 (Fermt2) in myogenic C2C12 cells and analyzed the gene expression of mechanosensors, clock components and metabolism-controlling genes.
    RESULTS: Upon hindlimb suspension, gene expression levels of both core molecular clocks and mechanosensors were moderately upregulated in m. Gastrocnemius but strongly downregulated in m. Soleus. Upon unloading, metabolism- and protein biosynthesis-related genes were moderately upregulated in m. Gastrocnemius but downregulated in m. Soleus. Furthermore, we identified very strong correlations between mechanosensors, metabolism- and circadian clock-regulating genes. Finally, genetically induced downregulations of mechanosensors Ilk1 and Fermt2 caused a downregulated mechanosensor, molecular clock and metabolism-related gene expression in the C2C12 model.
    CONCLUSIONS: Collectively, these data shed new lights on mechanisms that control muscle loss. Mechanosensors are identified to crucially control these processes, specifically through commanding molecular clock components and metabolism.
    Keywords:  Atrophy; Hindlimb suspension; Mechanosensing; Molecular clock; Skeletal muscle metabolism
    DOI:  https://doi.org/10.1007/s00018-022-04346-7
  14. Biology (Basel). 2022 May 02. pii: 701. [Epub ahead of print]11(5):
    Farnesol-Loaded Nanoliposomes Inhibit Inflammatory Gene Expression in Primary Human Skeletal Myoblasts.
    Eva Mückter, Maria Lozoya, Aline Müller, Volkmar Weissig, Mahtab Nourbakhsh.
      There is a substantial unmet need for the treatment of skeletal muscle mass loss that is associated with aging and obesity-related increases in FFA. Unsaturated FFAs stimulate the inflammatory gene expression in human skeletal myoblasts (SkMs). Farnesol is a hydrophobic acyclic sesquiterpene alcohol with potential anti-inflammatory effects. Here, we created farnesol-loaded small unilamellar (SUVs) or multilamellar lipid-based vesicles (MLVs), and investigated their effects on inflammatory gene expression in primary human skeletal myoblasts. The attachment of SUVs or MLVs to SkMs was tracked using BODIPY, a fluorescent lipid dye. The data showed that farnesol-loaded SUVs reduced FFA-induced IL6 and LIF expression by 77% and 70% in SkMs, respectively. Farnesol-loaded MLVs were less potent in inhibiting FFA-induced IL6 and LIF expression. In all experiments, equal concentrations of free farnesol did not exert significant effects on SkMs. This report suggests that farnesol, if efficiently directed into myoblasts through liposomes, may curb FFA-induced inflammation in human skeletal muscle.
    Keywords:  chemokines; cytokines; farnesol; fatty acids; human; inflammation; liposomes; nanovesicles; primary myoblasts
    DOI:  https://doi.org/10.3390/biology11050701
  15. Cells. 2022 May 19. pii: 1689. [Epub ahead of print]11(10):
    Activation and Migration of Human Skeletal Muscle Stem Cells In Vitro Differently Rely on Calcium Signals.
    Axel Tollance, Stéphane Koenig, Nicolas Liaudet, Maud Frieden.
      Muscle regeneration is essential for proper muscle homeostasis and relies primarily on muscle stem cells (MuSC). MuSC are maintained quiescent in their niche and can be activated following muscle injury. Using an in vitro model of primary human quiescent MuSC (called reserve cells, RC), we analyzed their Ca2+ response following their activation by fetal calf serum and assessed the role of Ca2+ in the processes of RC activation and migration. The results showed that RC displayed a high response heterogeneity in a cell-dependent manner following serum stimulation. Most of these responses relied on inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release associated with Ca2+ influx, partly due to store-operated calcium entry. Our study further found that blocking the IP3 production, Ca2+ influx, or both did not prevent the activation of RC. Intra- or extracellular Ca2+ chelation did not impede RC activation. However, their migration potential depended on Ca2+ responses displayed upon stimulation, and Ca2+ blockers inhibited their movement. We conclude that the two major steps of muscle regeneration, namely the activation and migration of MuSC, differently rely on Ca2+ signals.
    Keywords:  calcium signals; live cell imaging; migration; reserve cells; serum-induced activation; single-cell analysis
    DOI:  https://doi.org/10.3390/cells11101689
  16. Am J Sports Med. 2022 May 23. 3635465221095568
    Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes That Treat Rotator Cuff Muscle Degeneration in Mice and Are Identified in Humans by Single-Cell RNA Sequencing.
    Michael R Davies, Steven Garcia, Mengyao Liu, Hannah Chi, Hubert T Kim, Robert L Raffai, Xuhui Liu, Brian T Feeley.
       BACKGROUND: Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white and beige adipose tissue (BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in numerous injury models.
    PURPOSE: To characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their promyogenic effect is mediated via exosomes, and analyze human FAPs for an analogous promyogenic exosome-rich subpopulation.
    STUDY DESIGN: Controlled laboratory study.
    METHODS: FAPs from UCP1 reporter mice were isolated via fluorescence-activated cell sorting and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1-), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNA sequencing was performed on UCP1-, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ FAP-BAT exosomes (Exo-FB) as well as UCP1- non-FAP-BAT exosomes (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and mouse embryonic fibroblasts in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat wild type C57B/L6J mice that had undergone a massive rotator cuff tear. At 6 weeks mice were sacrificed, and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing was then performed on FAPs isolated from human muscle that were treated with the beta-agonist formoterol or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans.
    RESULTS: Flow cytometry analysis of sorted UCP1 reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with 3 distinct transcriptomic profiles characterized with bulk RNA sequencing. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers; UCP1- cells were marked by fibrogenic markers; and UCP1+ cells were characterized differential enrichment of white adipose tissue markers. Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, while treatment of mouse embryonic fibroblasts resulted in differentiation into myotubes. Treatment of cells with Exo-nFB resulted in poor myotube formation. Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and fatty infiltration as compared with treatment with Exo-nFB or phosphate-buffered saline. Single-cell RNA sequencing of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with one subpopulation demonstrating the presence of UCP1+ beige adipocytes with a distinct profile of BAT, mitochondrial, and extracellular vesicle-associated markers.
    CONCLUSION: FAP-BAT cells form a subpopulation of FAPs with upregulated beige gene expression and exosome production that mediate promyogenic effects in vitro and in vivo, and they are present as a transcriptomically similar subpopulation of FAPs in humans.
    CLINICAL RELEVANCE: FAP-BAT cells and their exosomes represent a potential therapeutic avenue for treating rotator cuff muscle degeneration.
    Keywords:  beige fat; fatty infiltration; fibroadipogenic progenitor; muscle stem cells
    DOI:  https://doi.org/10.1177/03635465221095568
  17. Int J Mol Sci. 2022 May 20. pii: 5723. [Epub ahead of print]23(10):
    Investigation of Brain Function-Related Myokine Secretion by Using Contractile 3D-Engineered Muscle.
    Takeshi Sugimoto, Tomohiro Nakamura, Sho Yokoyama, Toshia Fujisato, Satoshi Konishi, Takeshi Hashimoto.
      Brain function-related myokines, such as lactate, irisin, and cathepsin B (CTSB), are upstream factors that control brain-derived neurotrophic factor (BDNF) expression and are secreted from skeletal muscle by exercise. However, whether irisin and CTSB are secreted by muscle contraction remains controversial. Three-dimensional (3D)-engineered muscle (3D-EM) may help determine whether skeletal muscle contraction leads to the secretion of irisin and CTSB, which has never been identified with the addition of drugs in conventional 2D muscle cell cultures. We aimed to investigate the effects of electrical pulse stimulation (EPS)-evoked muscle contraction on irisin and CTSB secretion in 3D-EM. The 3D-EM, which consisted of C2C12 myoblasts and type-1 collagen gel, was allowed to differentiate for 2 weeks and divided into the control and EPS groups. EPS was applied at 13 V, 66 Hz, and 2 msec for 3 h (on: 5 s/off: 5 s). Irisin and CTSB secretion into the culture medium was measured by Western blotting. Irisin secretion was significantly increased following EPS (p < 0.05). However, there was no significant difference in CTSB secretion between the two groups. The present study suggests that irisin may be a contractile muscle-derived myokine, but CTSB is not secreted by EPS-evoked muscle contractile stimulation in 3D-EM.
    Keywords:  C2C12; electrical pulse stimulation; muscle contraction; myokine; tissue-engineered muscle
    DOI:  https://doi.org/10.3390/ijms23105723
  18. Cells. 2022 May 20. pii: 1698. [Epub ahead of print]11(10):
    Redox Control of Signalling Responses to Contractile Activity and Ageing in Skeletal Muscle.
    Malcolm J Jackson, Natalie Pollock, Caroline Staunton, Samantha Jones, Anne McArdle.
      Research over almost 40 years has established that reactive oxygen species are generated at different sites in skeletal muscle and that the generation of these species is increased by various forms of exercise. Initially, this was thought to be potentially deleterious to skeletal muscle and other tissues, but more recent data have identified key roles of these species in muscle adaptations to exercise. The aim of this review is to summarise our current understanding of these redox signalling roles of reactive oxygen species in mediating responses of muscle to contractile activity, with a particular focus on the effects of ageing on these processes. In addition, we provide evidence that disruption of the redox status of muscle mitochondria resulting from age-associated denervation of muscle fibres may be an important factor leading to an attenuation of some muscle responses to contractile activity, and we speculate on potential mechanisms involved.
    Keywords:  contraction; muscle; sarcopenia
    DOI:  https://doi.org/10.3390/cells11101698
  19. Cells. 2022 May 11. pii: 1607. [Epub ahead of print]11(10):
    Inflammation and Wasting of Skeletal Muscles in Kras-p53-Mutant Mice with Intraepithelial Neoplasia and Pancreatic Cancer-When Does Cachexia Start?
    Wulf Hildebrandt, Jan Keck, Simon Schmich, Gabriel A Bonaterra, Beate Wilhelm, Hans Schwarzbach, Anna Eva, Mirjam Bertoune, Emily P Slater, Volker Fendrich, Ralf Kinscherf.
      Skeletal muscle wasting critically impairs the survival and quality of life in patients with pancreatic ductal adenocarcinoma (PDAC). To identify the local factors initiating muscle wasting, we studied inflammation, fiber cross-sectional area (CSA), composition, amino acid metabolism and capillarization, as well as the integrity of neuromuscular junctions (NMJ, pre-/postsynaptic co-staining) and mitochondria (electron microscopy) in the hindlimb muscle of LSL-KrasG12D/+; LSL-TrP53R172H/+; Pdx1-Cre mice with intraepithelial-neoplasia (PanIN) 1-3 and PDAC, compared to wild-type mice (WT). Significant decreases in fiber CSA occurred with PDAC but not with PanIN 1-3, compared to WT: These were found in the gastrocnemius (type 2x: -20.0%) and soleus (type 2a: -21.0%, type 1: -14.2%) muscle with accentuation in the male soleus (type 2a: -24.8%, type 1: -17.4%) and female gastrocnemius muscle (-29.6%). Significantly higher densities of endomysial CD68+ and cyclooxygenase-2+ (COX2+) cells were detected in mice with PDAC, compared to WT mice. Surprisingly, CD68+ and COX2+ cell densities were also higher in mice with PanIN 1-3 in both muscles. Significant positive correlations existed between muscular and hepatic CD68+ or COX2+ cell densities. Moreover, in the gastrocnemius muscle, suppressor-of-cytokine-3 (SOCS3) expressions was upregulated >2.7-fold with PanIN 1A-3 and PDAC. The intracellular pools of proteinogenic amino acids and glutathione significantly increased with PanIN 1A-3 compared to WT. Capillarization, NMJ, and mitochondrial ultrastructure remained unchanged with PanIN or PDAC. In conclusion, the onset of fiber atrophy coincides with the manifestation of PDAC and high-grade local (and hepatic) inflammatory infiltration without compromised microcirculation, innervation or mitochondria. Surprisingly, muscular and hepatic inflammation, SOCS3 upregulation and (proteolytic) increases in free amino acids and glutathione were already detectable in mice with precancerous PanINs. Studies of initial local triggers and defense mechanisms regarding cachexia are warranted for targeted anti-inflammatory prevention.
    Keywords:  cancer cachexia; cytokines; gastrointestinal cancer; muscle atrophy; sarcopenia; weight loss
    DOI:  https://doi.org/10.3390/cells11101607
  20. Int J Mol Sci. 2022 May 12. pii: 5431. [Epub ahead of print]23(10):
    Repeated and Interrupted Resistance Exercise Induces the Desensitization and Re-Sensitization of mTOR-Related Signaling in Human Skeletal Muscle Fibers.
    Daniel Jacko, Kirill Schaaf, Lukas Masur, Hannes Windoffer, Thorben Aussieker, Thorsten Schiffer, Jonas Zacher, Wilhelm Bloch, Sebastian Gehlert.
      The acute resistance exercise (RE)-induced phosphorylation of mTOR-related signaling proteins in skeletal muscle can be blunted after repeated RE. The time frame in which the phosphorylation (p) of mTORS2448, p70S6kT421/S424, and rpS6S235/236 will be reduced during an RE training period in humans and whether progressive (PR) loading can counteract such a decline has not been described. (1) To enclose the time frame in which pmTORS2448, prpS6S235/236, and pp70S6kT421/S424 are acutely reduced after RE occurs during repeated RE. (2) To test whether PR will prevent that reduction compared to constant loading (CO) and (3) whether 10 days without RE may re-increase blunted signaling. Fourteen healthy males (24 ± 2.8 yrs.; 1.83 ± 0.1 cm; 79.3 ± 8.5 kg) were subjected to RE with either PR (n = 8) or CO (n = 6) loading. Subjects performed RE thrice per week, conducting three sets with 10-12 repetitions on a leg press and leg extension machine. Muscle biopsies were collected at rest (T0), 45 min after the first (T1), seventh (T7), 13th (T13), and 14th (X-T14) RE session. No differences were found between PR and CO for any parameter. Thus, the groups were combined, and the results show the merged values. prpS6S235/236 and pp70s6kT421/S424 were increased at T1, but were already reduced at T7 and up to T13 compared to T1. Ten days without RE re-increased prpS6S235/236 and pp70S6kT421/S424 at X-T14 to a level comparable to that of T1. pmTORS2448 was increased from T1 to X-T14 and did not decline over the training period. Single-fiber immunohistochemistry revealed a reduction in prpS6S235/236 in type I fibers from T1 to T13 and a re-increase at X-T14, which was more augmented in type II fibers at T13 (p < 0.05). The entity of myofibers revealed a high heterogeneity in the level of prpS6S235/236, possibly reflecting individual contraction-induced stress during RE. The type I and II myofiber diameter increased from T0 and T1 to T13 and X-T14 (p < 0.05) prpS6S235/236 and pp70s6kT421/S424 reflect RE-induced states of desensitization and re-sensitization in dependency on frequent loading by RE, but also by its cessation.
    Keywords:  HSPB5; anabolic signaling; desensitization; desmin; hypertrophy; mTOR; p70S6k; resistance exercise; ribosomal protein S6; skeletal muscle; type I myofibers; type II myofibers; unloading
    DOI:  https://doi.org/10.3390/ijms23105431
  21. Metabolites. 2022 Apr 29. pii: 405. [Epub ahead of print]12(5):
    Thyroid Hormone Receptor Isoforms Alpha and Beta Play Convergent Roles in Muscle Physiology and Metabolic Regulation.
    Annarita Nappi, Melania Murolo, Annunziata Gaetana Cicatiello, Serena Sagliocchi, Emery Di Cicco, Maddalena Raia, Mariano Stornaiuolo, Monica Dentice, Caterina Miro.
      Skeletal muscle is a key energy-regulating organ, skilled in rapidly boosting the rate of energy production and substrate consumption following increased workload demand. The alteration of skeletal muscle metabolism is directly associated with numerous pathologies and disorders. Thyroid hormones (THs) and their receptors (TRs, namely, TRα and TRβ) exert pleiotropic functions in almost all cells and tissues. Skeletal muscle is a major THs-target tissue and alterations of THs levels have multiple influences on the latter. However, the biological role of THs and TRs in orchestrating metabolic pathways in skeletal muscle has only recently started to be addressed. The purpose of this paper is to investigate the muscle metabolic response to TRs abrogation, by using two different mouse models of global TRα- and TRβKO. In line with the clinical features of resistance to THs syndromes in humans, characterized by THRs gene mutations, both animal models of TRs deficiency exhibit developmental delay and mitochondrial dysfunctions. Moreover, using transcriptomic and metabolomic approaches, we found that the TRs-THs complex regulates the Fatty Acids (FAs)-binding protein GOT2, affecting FAs oxidation and transport in skeletal muscle. In conclusion, these results underline a new metabolic role of THs in governing muscle lipids distribution and metabolism.
    Keywords:  muscle metabolism; skeletal muscle; thyroid hormone receptors (TRs); thyroid hormones (THs)
    DOI:  https://doi.org/10.3390/metabo12050405
  22. Metabolites. 2022 May 06. pii: 416. [Epub ahead of print]12(5):
    Low Inflammatory Stimulus Increases D2 Activity and Modulates Thyroid Hormone Metabolism during Myogenesis In Vitro.
    Thamires Siqueira de Oliveira, Marilia Kimie Shimabukuro, Victoria Regina Siqueira Monteiro, Cherley Borba Vieira Andrade, Anita Boelen, Simone Magagnin Wajner, Ana Luiza Maia, Tania Maria Ortiga-Carvalho, Flavia Fonseca Bloise.
      Thyroid hormone (TH) signaling controls muscle progenitor cells differentiation. However, inflammation can alter muscle TH signaling by modulating the expression of TH transporters (Slc16a2), receptors (Thra1), and deiodinase enzymes (Dio2 and Dio3). Thus, a proinflammatory environment could affect myogenesis. The role of a low-grade inflammatory milieu in TH signaling during myogenesis needs further investigation. Herein, we aimed to study the impact of the bacterial lipopolysaccharide (LPS)-induced inflammatory stimulus on the TH signaling during myogenesis. C2C12 myoblasts differentiation was induced without (CTR) or with 10 ng/mL LPS presence. The myoblasts under LPS stimulus release the proinflammatory cytokines (IL-6 and IL-1β) and chemokines (CCL2 and CXCL-1). LPS decreases Myod1 expression by 28% during the initial myogenesis, thus reducing the myogenic stimulus. At the same time, LPS reduced the expression of Dio2 by 41% but doubled the D2 enzymatic activity. The late differentiation was not affected by inflammatory milieu, which only increased the Slc16a2 gene expression by 38%. LPS altered the intracellular metabolism of TH and reduced the initial myogenic stimulus. However, it did not affect late differentiation. Increased intracellular TH activation may be the compensatory pathway involved in the recovery of myogenic differentiation under a low-grade inflammatory milieu.
    Keywords:  C2C12; bacterial lipopolysaccharide; deiodinase; inflammation; myoblast; myogenic differentiation; triiodothyronine
    DOI:  https://doi.org/10.3390/metabo12050416
  23. Biochem Biophys Res Commun. 2022 May 14. pii: S0006-291X(22)00731-8. [Epub ahead of print]615 24-30
    Quercetin attenuates adipogenesis and fibrosis in human skeletal muscle.
    Saori Ohmae, Sota Akazawa, Toshio Takahashi, Takayuki Izumo, Tomohiro Rogi, Masaaki Nakai.
      Age-associated increase in ectopic fat degeneration and fibrosis in the skeletal muscle contribute to muscle degradation and weakness. Quercetin is a bioactive flavonoid with anti-inflammatory and anti-obesity effects. Thus, we aimed to investigate the effects of quercetin on adipogenesis and fibrosis in the human skeletal muscle, which have not yet been elucidated. Human muscle-derived PDGFRα+/CD201+ cells (mesenchymal progenitors) were incubated with various concentrations of quercetin (0, 0.3, 1, and 3 μM) under adipogenic or fibrogenic conditions. Lipid accumulation was visualized via Oil Red O staining. The expression of genes implicated in adipocyte or fibroblast differentiation and activation of signaling pathways was analyzed. The quercetin-treated PDGFRα+/CD201+ cells showed attenuated lipid accumulation and adipogenic gene expression (CEBPA and ADIPOQ) via the inhibition of CREB phosphorylation under adipocyte differentiation conditions. Additionally, quercetin treatment significantly attenuated the expression of fibrogenic genes (TIMP1, ACTA2, COL1A1 and COL3A1) by inhibiting Smad2 phosphorylation. Quercetin suppressed the differentiation of muscle-derived PDGFRα+/CD201+ cells to adipocytes and fibroblasts at concentrations achievable by dietary and dietary supplement intake, which indicated its preventive or therapeutic effect against the loss of muscle quality.
    Keywords:  Adipogenesis; Fibrosis; Muscle; Quercetin
    DOI:  https://doi.org/10.1016/j.bbrc.2022.05.033
  24. Physiol Rep. 2022 May;10(10): e15266
    SS-31 does not prevent or reduce muscle atrophy 7 days after a 65 kdyne contusion spinal cord injury in young male mice.
    Zachary A Graham, Jennifer J DeBerry, Christopher P Cardozo, Marcas M Bamman.
      Spinal cord injury (SCI) leads to major reductions in function, independent living, and quality of life. Disuse and paralysis from SCI leads to rapid muscle atrophy, with chronic muscle loss likely playing a role in the development of the secondary metabolic disorders often seen in those with SCI. Muscle disuse is associated with mitochondrial dysfunction. Previous evidence has suggested targeting the mitochondria with the tetrapeptide SS-31 is beneficial for muscle health in preclinical models that lead to mitochondrial dysfunction, such as cast immobilization or burn injury. We gave young male mice a sham (n = 8) or 65 kdyne thoracic contusion SCI with (n = 9) or without (n = 9) daily administration of 5.0 mg/kg SS-31. Hindlimb muscle mass and muscle bundle respiration were measured at 7 days post-SCI and molecular targets were investigated using immunoblotting, RT-qPCR, and metabolomics. SS-31 did not preserve body mass or hindlimb muscle mass 7 days post-SCI. SS-31 had no effect on soleus or plantaris muscle bundle respiration. SCI was associated with elevated levels of protein carbonylation, led to reduced protein expression of activated DRP1 and reductions in markers of mitochondrial fusion. SS-31 administration did result in reduced total DRP1 expression, as well as greater expression of inhibited DRP1. Gene expression of proinflammatory cytokines and their receptors were largely stable across groups, although SS-31 treatment led to greater mRNA expression of IL1B, TNF, and TNFRSF12A. In summation, SS-31 was not an efficacious treatment acutely after a moderate thoracic contusion SCI in young male mice.
    Keywords:  SS-31; metabolomics; paralysis; respirometry; spinal cord injury
    DOI:  https://doi.org/10.14814/phy2.15266
  25. J Cachexia Sarcopenia Muscle. 2022 May 23.
    Declines in muscle protein synthesis account for short-term muscle disuse atrophy in humans in the absence of increased muscle protein breakdown.
    Matthew S Brook, Tanner Stokes, Stefan H M Gorissen, Joseph J Bass, Chris McGlory, Jessica Cegielski, Daniel J Wilkinson, Bethan E Phillips, Ken Smith, Stuart M Phillips, Philip J Atherton.
       BACKGROUND: We determined the short-term (i.e. 4 days) impacts of disuse atrophy in relation to muscle protein turnover [acute fasted-fed muscle protein synthesis (MPS)/muscle protein breakdown (MPB) and integrated MPS/estimated MPB].
    METHODS: Healthy men (N = 9, 22 ± 2 years, body mass index 24 ± 3 kg m-2 ) underwent 4 day unilateral leg immobilization. Vastus lateralis (VL) muscle thickness (MT) and extensor strength and thigh lean mass (TLM) were measured. Bilateral VL muscle biopsies were collected on Day 4 at t = -120, 0, 90, and 180 min to determine integrated MPS, estimated MPB, acute fasted-fed MPS (l-[ring-13 C6 ]-phe), and acute fasted tracer decay rate representative of MPB (l-[15 N]-phe and l-[2 H8 ]-phe). Protein turnover cell signalling was measured by immunoblotting.
    RESULTS: Immobilization decreased TLM [pre: 7477 ± 1196 g, post: 7352 ± 1209 g (P < 0.01)], MT [pre: 2.67 ± 0.50 cm, post: 2.55 ± 0.51 cm (P < 0.05)], and strength [pre: 260 ± 43 N m, post: 229 ± 37 N m (P < 0.05)] with no change in control legs. Integrated MPS decreased in immob vs. control legs [control: 1.55 ± 0.21% day-1 , immob: 1.29 ± 0.17% day-1 (P < 0.01)], while tracer decay rate (i.e. MPB) (control: 0.02 ± 0.006, immob: 0.015 ± 0.015) and fractional breakdown rate (FBR) remained unchanged [control: 1.44 ± 0.51% day-1 , immob: 1.73 ± 0.35% day-1 (P = 0.21)]. Changes in MT correlated with those in MPS but not FBR. MPS increased in the control leg following feeding [fasted: 0.043 ± 0.012% h-1 , fed: 0.065 ± 0.017% h-1 (P < 0.05)] but not in immob [fasted: 0.034 ± 0.014% h-1 , fed: 0.049 ± 0.023% h-1 (P = 0.09)]. There were no changes in markers of MPB with immob (P > 0.05).
    CONCLUSIONS: Human skeletal muscle disuse atrophy is driven by declines in MPS, not increases in MPB. Pro-anabolic therapies to mitigate disuse atrophy would likely be more effective than therapies aimed at attenuating protein degradation.
    Keywords:  Atrophy; Immobilization; Muscle; Protein breakdown; Protein synthesis
    DOI:  https://doi.org/10.1002/jcsm.13005
  26. J Nutr Biochem. 2022 May 21. pii: S0955-2863(22)00140-1. [Epub ahead of print] 109069
    (-) - Epicatechin improves Tibialis anterior muscle repair in CD1 mice with BaCl2-induced damage.
    M Ramírez-Ramírez, F Fernández-Valverde, A Reséndiz-García, M G Martínez-Damas, Luis Javier Cano-Martínez, A Zentella-Dehesa, R M Coral-Vázquez.
      Several studies have shown the beneficial effect that Epicatechin (Epi) has on the skeletal muscle of murine models and patients with muscular dystrophy and in the muscles of patients with diabetes or murine sarcopenia models. This flavanol has been shown to enhance antioxidant pathways and improve muscle architecture. However, the repair process during muscle regeneration has not been analyzed. To address this, we characterize the effect of Epi in the repair process of the Tibialis anterior in a murine model with BaCl2-induced damage. CD1 mice of 10 weeks of age were randomly selected and injured with BaCl2. One hour later, they were divided into four groups (n = 6 for histology groups and n=12 for western blot groups). Epi was administered every 12h, until the time of sacrifice. Histological and morphological analysis showed that Epi significantly reduced the area of ​​damage and hypertrophy at 15 days in the damaged muscle. Furthermore, western blot assays showed that the treatment increases β-catenin (active) and myogenic proteins such as MyoD and Myogenin. These results show that Epi exerts therapeutic effects accelerating skeletal muscle repair after induced damage chemically, thus highlighting the therapeutic potential of this flavanol in different myopathies.
    Keywords:  (-)- Epicatechin; BaCl(2); chemical damage; muscular repair; skeletal muscle
    DOI:  https://doi.org/10.1016/j.jnutbio.2022.109069
  27. Cell Stem Cell. 2022 May 15. pii: S1934-5909(22)00169-2. [Epub ahead of print]
    An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling.
    Allison P Kann, Margaret Hung, Wei Wang, Jo Nguyen, Penney M Gilbert, Zhuhao Wu, Robert S Krauss.
      Many tissues harbor quiescent stem cells that are activated upon injury, subsequently proliferating and differentiating to repair tissue damage. Mechanisms by which stem cells sense injury and transition from quiescence to activation, however, remain largely unknown. Resident skeletal muscle stem cells (MuSCs) are essential orchestrators of muscle regeneration and repair. Here, with a combination of in vivo and ex vivo approaches, we show that quiescent MuSCs have elaborate, Rac GTPase-promoted cytoplasmic projections that respond to injury via the upregulation of Rho/ROCK signaling, facilitating projection retraction and driving downstream activation events. These early events involve rapid cytoskeletal rearrangements and occur independently of exogenous growth factors. This mechanism is conserved across a broad range of MuSC activation models, including injury, disease, and genetic loss of quiescence. Our results redefine MuSC activation and present a central mechanism by which quiescent stem cells initiate responses to injury.
    Keywords:  GTPase; Rac1; Rho; cytoskeleton; mechanosignaling; muscle stem cell; quiescence; satellite cell; stem cell activation; stem cell niche
    DOI:  https://doi.org/10.1016/j.stem.2022.04.016
  28. JCI Insight. 2022 May 23. pii: e158380. [Epub ahead of print]7(10):
    Bap1/SMN axis in Dpp4+ skeletal muscle mesenchymal cells regulates the neuromuscular system.
    Ji-Hoon Kim, Jong-Seol Kang, Kyusang Yoo, Jinguk Jeong, Inkuk Park, Jong Ho Park, Joonwoo Rhee, Shin Jeon, Young-Woo Jo, Sang-Hyeon Hann, Minji Seo, Seungtae Moon, Soo-Jong Um, Rho Hyun Seong, Young-Yun Kong.
      The survival of motor neuron (SMN) protein is a major component of the pre-mRNA splicing machinery and is required for RNA metabolism. Although SMN has been considered a fundamental gene for the central nervous system, due to its relationship with neuromuscular diseases, such as spinal muscular atrophy, recent studies have also revealed the requirement of SMN in non-neuronal cells in the peripheral regions. Here, we report that the fibro-adipogenic progenitor subpopulation expressing Dpp4 (Dpp4+ FAPs) is required for the neuromuscular system. Furthermore, we also reveal that BRCA1-associated protein-1 (Bap1) is crucial for the stabilization of SMN in FAPs by preventing its ubiquitination-dependent degradation. Inactivation of Bap1 in FAPs decreased SMN levels and accompanied degeneration of the neuromuscular junction, leading to loss of motor neurons and muscle atrophy. Overexpression of the ubiquitination-resistant SMN variant, SMNK186R, in Bap1-null FAPs completely prevented neuromuscular degeneration. In addition, transplantation of Dpp4+ FAPs, but not Dpp4- FAPs, completely rescued neuromuscular defects. Our data reveal the crucial role of Bap1-mediated SMN stabilization in Dpp4+ FAPs for the neuromuscular system and provide the possibility of cell-based therapeutics to treat neuromuscular diseases.
    Keywords:  Cell Biology; Mouse models; Muscle; Muscle Biology; Neurodegeneration
    DOI:  https://doi.org/10.1172/jci.insight.158380
  29. Sci Adv. 2022 May 27. 8(21): eabn0379
    Large1 gene transfer in older myd mice with severe muscular dystrophy restores muscle function and greatly improves survival.
    Takahiro Yonekawa, Adam J Rauckhorst, Sara El-Hattab, Marco A Cuellar, David Venzke, Mary E Anderson, Hidehiko Okuma, Alvin D Pewa, Eric B Taylor, Kevin P Campbell.
      Muscular dystrophy is a progressive and ultimately lethal neuromuscular disease. Although gene editing and gene transfer hold great promise as therapies when administered before the onset of severe clinical symptoms, it is unclear whether these strategies can restore muscle function and improve survival in the late stages of muscular dystrophy. Largemyd/Largemyd (myd) mice lack expression of like-acetylglucosaminyltransferase-1 (Large1) and exhibit severe muscle pathophysiology, impaired mobility, and a markedly reduced life span. Here, we show that systemic delivery of AAV2/9 CMV Large1 (AAVLarge1) in >34-week-old myd mice with advanced disease restores matriglycan expression on dystroglycan, attenuates skeletal muscle pathophysiology, improves motor and respiratory function, and normalizes systemic metabolism, which collectively and markedly extends survival. Our results in a mouse model of muscular dystrophy demonstrate that skeletal muscle function can be restored, illustrating its remarkable plasticity, and that survival can be greatly improved even after the onset of severe muscle pathophysiology.
    DOI:  https://doi.org/10.1126/sciadv.abn0379
  30. Tissue Eng Part A. 2022 May 27.
    Pharmaceutical agents for contractile-metabolic dysfunction after volumetric muscle loss.
    Jennifer McFaline-Figueroa, Albino Gian Schifino, Anna Scarlett Nichenko, Magen Lord, Edward Tatum Hunda, Elizabeth Anne Winders, Emily Noble, Sarah M Greising, Jarrod A Call.
      Volumetric muscle loss (VML) injuries represent a majority of military servicemember casualties and are common in civilian populations following blunt and/or penetrating traumas. Characterized as a skeletal muscle injury with permanent functional impairments, there are currently no standards for rehabilitation, leading to life-long disability. Toward developing rehabilitative strategies, previous research demonstrates that the remaining muscle after a VML injury lacks similar levels of plasticity or adaptability as healthy, uninjured skeletal muscle. This may be due in part to impaired innervation and vascularization of the remaining muscle as well as disrupted molecular signaling cascades commonly associated with muscle adaptation. The primary objective of this study was to assess the ability of four pharmacological agents with a strong record of modulating muscle contractile and metabolic function to improve functional deficits in a murine model of VML injury. Male C57BL/6 mice underwent a 15% multi-muscle VML injury of the posterior hindlimb and were randomized into drug treatment groups (formoterol, AICAR, pioglitazone, or sildenafil) or untreated VML group. At the end of 60 days, the injury model was first validated by comparison to age-matched injury naïve mice. Untreated VML mice had 22% less gastrocnemius muscle mass, 36% less peak-isometric torque, and 27% less maximal mitochondrial oxygen consumption rate compared to uninjured mice (p<0.01). Experimental drug groups were, then, compared to VML untreated, and there was minimal evidence of efficacy for AICAR, pioglitazone, or sildenafil in improving contractile and metabolic functional outcomes. However, formoterol-treated VML mice had 18% greater peak-isometric torque (p<0.01) and permeabilized muscle fibers had 36% greater State III mitochondrial oxygen consumption rate (p<0.01) compared to VML untreated, suggesting an overall improvement in muscle condition. There was minimal evidence that these benefits came from greater mitochondrial biogenesis and/or mitochondrial complex protein content, but could be due to greater enzyme activities levels for complex I and complex II. These findings suggest that formoterol treatment is candidate to pair with a rehabilitative approach to maximize functional improvements in VML-injured muscle.
    DOI:  https://doi.org/10.1089/ten.TEA.2022.0036
  31. Arch Biochem Biophys. 2022 May 18. pii: S0003-9861(22)00175-8. [Epub ahead of print]725 109291
    Effect of enhanced muscle tone on the expression of atrogenes and cytoskeletal proteins during postural muscle unloading.
    Svetlana P Belova, Ekaterina P Kalashnikova, Sergey A Tyganov, Tatiana Y Kostrominova, Boris S Shenkman, Tatiana L Nemirovskaya.
      Skeletal muscle unloading leads to the decreased electrical activity and decline of muscle tone.
    AIMS: Current study evaluated the effect of muscle tone preservation achieved by tetanus toxin (TeNT) treatment on signaling pathways regulating atrophic processes during unloading.
    MAIN METHODS: Four groups of rats were used: non-treated control (C), control rats with TeNT administration (CT), 7 days of unloading/hindlimb suspension with placebo (HS), and 7 days of unloading with TeNT administration (HST).
    KEY FINDINGS: Absolute and relative force of tetanic contractions was decreased by 65% in soleus muscle of HS rats when compared with C. Treatment with TeNT significantly lessened force decline in soleus muscle of HST rats when compared with HS. TeNT administration increased myosin heavy chain I beta (MyHC Iβ) expression in CT rats and prevented MyHC Iβ loss in HST group when compared with C rats. Desmin content was lower by 31.4% (p < 0.05) in HS group when compared with HST. Calpain-1 expression was increased in HS group when compared with C, CT and HST. There was a decrease in p-p70S6K content (41%, p < 0,05) and an increase in p-eEF2 content (77%, p < 0,05) in HS group when compared with C, while there were no significant differences in the content of these proteins between HST, CT and C groups.
    SIGNIFICANCE: Treatment with TeNT significantly diminished unloading-induced decline of soleus muscle mass and mechanical properties and affected the regulation of MyHC Iβ expression. These effects are mediated by signaling pathways regulating protein synthesis and degradation.
    Keywords:  Calpain-1; Desmin; MAFbx; MuRF1; Muscle tone; Muscle unloading; Myosin heavy chain
    DOI:  https://doi.org/10.1016/j.abb.2022.109291
  32. Am J Pathol. 2022 May 20. pii: S0002-9440(22)00146-8. [Epub ahead of print]
    BNIP3 is involved in muscle fiber atrophy in Late-Onset Pompe Disease patients.
    Ana Carrasco-Rozas, Esther Fernández-Simón, Xavier Suárez-Calvet, Patricia Piñol-Jurado, Jorge Alonso-Pérez, Noemí de Luna, Benedikt Schoser, Peter Meinke, Cristina Domínguez-González, Aurelio Hernández-Laín, Carmen Paradas, Eloy Rivas, Isabel Illa, Montse Olivé, Eduard Gallardo, Jordi Díaz-Manera.
      Late-onset Pompe disease (LOPD) is a rare genetic disorder produced by mutations in the GAA gene and is characterized by progressive muscle weakness. LOPD muscle biopsies show accumulation of glycogen along with the autophagic vacuoles associated with atrophic muscle fibers. The expression of molecules related to muscle fiber atrophy in muscle biopsies of LOPD patients was studied using immunofluorescence (IF) and Real-Time PCR. BNIP3, a well-known atrogene, was identified as a potential mediator of muscle fiber atrophy in LOPD muscle biopsies. We observed that vacuolated fibers in LOPD patients muscle biopsies were smaller than non-vacuolated fibers and expressed BNIP3. Our data suggested that BNIP3 expression is regulated by inhibition of the AKT-mTOR pathway, leading to phosphorylation of ULK1 at Ser317 by AMPK. We studied myoblasts and myotubes obtained from LOPD patients and age-matched controls to confirm these results using different molecular techniques. Myotubes derived from LOPD patients were likewise smaller and expressed BNIP3. Conclusively, transfection of BNIP3 into control myotubes leads to myotube atrophy. These findings suggest a cascade which starts with the inhibition of the AKT-mTOR pathway and activation of BNIP3 expression leading to progressive muscle fiber atrophy. Our results open the door to potential new treatments targeting BNIP3 to reduce its deleterious effects on muscle fiber atrophy in Pompe disease.
    DOI:  https://doi.org/10.1016/j.ajpath.2022.05.003
  33. J Cell Physiol. 2022 May 27.
    Interaction between mesenchymal stem cells and myoblasts in the context of facioscapulohumeral muscular dystrophy contributes to the disease phenotype.
    Ekaterina Kiseleva, Olesya Serbina, Anna Karpukhina, Vincent Mouly, Yegor S Vassetzky.
      Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disease associated with ectopic expression of the DUX4 gene in skeletal muscle. Muscle degeneration in FSHD is accompanied by muscle tissue replacement with fat and connective tissue. Expression of DUX4 in myoblasts stimulates mesenchymal stem cells (MSC) migration via the CXCR4-CXCL12 axis. MSCs participate in adipose and connective tissue formation and can contribute to fibrosis. Here we studied the interaction between myoblasts and MSCs and the consequences of this interaction in the FSHD context. We used cell motility assays and coculture of MSCs with myoblasts to study their mutual effects on cell migration, differentiation, proliferation, and extracellular matrix formation. The growth medium conditioned by FSHD myoblasts stimulated MSCs migration 1.6-fold (p < 0.04) compared to nonconditioned medium. Blocking the CXCL12-CXCR4 axis with the CXCR4 inhibitor (AMD3100) or neutralizing antibodies to CXCL12 abolished this effect. FSHD myoblasts stimulated MSC proliferation 1.5-2 times (p < 0.05) compared to control myoblasts, while the presence of MSCs impaired myoblast differentiation. Under inflammatory conditions, medium conditioned by FSHD myoblasts stimulated collagen secretion by MSCs 2.2-fold as compared to the nonconditioned medium, p < 0.03. FSHD myoblasts attract MSCs via the CXCL12-CXCR4 axis, stimulate MSC proliferation and collagen secretion by MSCs. Interaction between MSCs and FSHD myoblasts accounts for several important aspects of FSHD pathophysiology. The CXCL12-CXCR4 axis may serve as a potential target to improve the state of the diseased muscles.
    Keywords:  CXCL12; CXCR4; FSHD; MSCs; differentiation; fibrosis; migration; myoblasts
    DOI:  https://doi.org/10.1002/jcp.30789
  34. Phytother Res. 2022 May 23.
    Resveratrol ameliorates muscle atrophy in chronic kidney disease via the axis of SIRT1/FoxO1.
    Ruiting Wang, Weidong Yuan, Lu Li, Fei Lu, Lingling Zhang, Haifeng Gong, Xinzhong Huang.
      Chronic kidney disease (CKD) is often associated with muscle atrophy. However, the underlying molecular mechanisms are still not well understood. Here, we treated 5/6-nephrectomized (5/6Nx) rats with resveratrol and found that this treatment greatly improves renal function as evidenced by reduced proteinuria and cystatin C. Moreover, resveratrol ameliorates renal fibrosis by reducing transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF). Meanwhile, muscle atrophy in these 5/6Nx rats was largely attenuated by resveratrol. Immunoprecipitation revealed that SIRT1 physically interacts with FoxO1 in muscle, and this interaction was weakened in 5/6Nx rats. As a consequence, acetylated FoxO1 was increased in muscle of 5/6Nx rats. The application of resveratrol markedly reverses this trend. These data point out that SIRT1 is a key factor for linking renal disease and muscle atrophy. Indeed, both renal dysfunction and muscle atrophy were further aggravated by 5/6Nx in Sirt1+/- mice. Taken together, our data indicate that SIRT1 plays a pivotal role in muscle atrophy in CKD, and FoxO1 might be a substrate of SIRT1 in this process. Furthermore, resveratrol, together with other agonists of SIRT1, may hold great therapeutic potentials for treating CKD and its related muscle atrophy.
    Keywords:  FoxO1; SIRT1; chronic kidney diseases; muscle atrophy; resveratrol
    DOI:  https://doi.org/10.1002/ptr.7499
  35. Mol Ther. 2022 May 25. pii: S1525-0016(22)00321-5. [Epub ahead of print]
    SERUM EXTRACELLULAR VESICLES FOR DELIVERY OF CRISPR-CAS9 RIBONUCLEOPROTEINS TO MODIFY THE DYSTROPHIN GENE.
    Nathalie Majeau, Annabelle Fortin-Archambault, Catherine Gérard, Joël Rousseau, Pouiré Yamégo, Jacques P Tremblay.
      Extracellular vesicles (EVs) mediate intercellular biomolecule exchanges in the body, making them promising delivery vehicles for therapeutic cargo. Genetic engineering by CRISPR system is an interesting therapeutic avenue for genetic diseases such as Duchenne Muscular Dystrophy (DMD). We developed a simple method for loading EVs with CRISPR ribonucleoproteins (RNPs) consisting of SpCas9 proteins and guide RNAs (gRNAs). EVs were first purified from human or mouse serum using ultrafiltration and size-exclusion chromatography. Using protein transfectant to load RNPs into serum EVs, we showed that EVs are good carriers of RNPs in vitro and restored the expression of the tdTomato fluorescent protein in muscle fibers of Ai9 mice. EVs carrying RNPs targeting introns 22 and 24 of the DMD gene were also injected into muscles of mdx mice having a non-sense mutation in exon 23. Up to 19% of the cDNA extracted from treated mdx mice had the intended deletion of exons 23 and 24, allowing dystrophin expression in muscle fibers. RNPs alone, without EVs, were inefficient in generating detectable deletions in mouse muscles. This method opens new opportunities for rapid and safe delivery of CRISPR components to treat DMD.
    DOI:  https://doi.org/10.1016/j.ymthe.2022.05.023
  36. Cytometry A. 2022 May 24.
    Full spectrum cytometry improves the resolution of highly autofluorescent biological samples: Identification of myeloid cells in regenerating skeletal muscles.
    Yacine Kharraz, Vera Lukesova, Antonio L Serrano, Adam Davison, Pura Muñoz-Cánoves.
      Autofluorescence (AF) is an intrinsic characteristic of cells caused by the presence of fluorescent biological compounds within the cell; these can include structural proteins (e.g., collagen and elastin), cellular organelles, and metabolites (e.g., aromatic amino acids). In flow cytometric studies, the presence of AF can lead to reduced antigen and population resolution, as well as the presence of artifacts due to false positive events. Here, we describe a methodology that uses the inherent ability of full spectrum cytometry to treat AF as a fluorochrome and to thereby separate it from the other fluorochromes of the assay. This method can be applied to complex inflamed tissues; for instance, in regenerating skeletal muscle we have developed a 16-color panel targeting highly autofluorescent myeloid cells. This represents a first step toward overcoming technological limitations in flow cytometry due to AF.
    Keywords:  Aurora; antigen resolution; full spectrum; macrophages; skeletal muscle; tissue regeneration
    DOI:  https://doi.org/10.1002/cyto.a.24568
  37. Gerontology. 2022 May 23. 1-9
    Neuromuscular Aging: A Case for the Neuroprotective Effects of Dancing.
    Martino V Franchi, Francesca Badiali, Fabio Sarto, Patrick Müller, Notger G Müller, Kathrin Rehfeld, Elena Monti, Debbie Rankin, Stefano Longo, Jon Lund, Anita Hökelmann, Marco Narici.
       AIM: We planned a cross-sectional investigation (study 1) and a longitudinal training intervention (study 2) to investigate whether recreational dancing affords greater neuroprotective effects against age-related neuromuscular junction (NMJ) degeneration compared to general fitness exercise training.
    METHODS: In study 1, we recruited 19 older volunteers regularly practising dancing (older dancers [OD]) and 15 recreationally physically active older individuals (OA) and physical performance, muscle morphology, muscle function, and NMJ stability (from serum C-terminal agrin fragment [CAF] concentration) were assessed. In study 2, employing a longitudinal study design in a different cohort (composed of 37 older adults), we aimed to study whether a 6-month dancing intervention decreased CAF concentration compared to general fitness exercise training in older adults.
    RESULTS: Our findings show that OD had a lower CAF concentration (suggesting an increased NMJ stability) compared to OA. This result was accompanied by superior functional performance despite no differences in muscle size. In study 2, we observed a reduction in CAF concentration only in the dancing group.
    CONCLUSION: Overall, these findings suggest that dancing is an effective training modality to promote neuroprotection and increase muscle function in healthy older individuals.
    Keywords:  C-terminal agrin fragment; Dance; Muscle power; Neuromuscular junction; Physical activity
    DOI:  https://doi.org/10.1159/000524843
  38. Biomedicines. 2022 May 04. pii: 1068. [Epub ahead of print]10(5):
    Skeletal Muscle and COVID-19: The Potential Involvement of Bioactive Sphingolipids.
    Elisabetta Meacci, Federica Pierucci, Mercedes Garcia-Gil.
      SARS-CoV-2 virus infection is the cause of the coronavirus disease 2019 (COVID-19), which is still spreading over the world. The manifestation of this disease can range from mild to severe and can be limited in time (weeks) or persist for months in about 30-50% of patients. COVID-19 is considered a multiple organ dysfunction syndrome and the musculoskeletal system manifestations are beginning to be considered of absolute importance in both COVID-19 patients and in patients recovering from the SARS-CoV-2 infection. Musculoskeletal manifestations of COVID-19 and other coronavirus infections include loss of muscle mass, muscle weakness, fatigue or myalgia, and muscle injury. The molecular mechanisms by which SARS-CoV-2 can cause damage to skeletal muscle (SkM) cells are not yet well understood. Sphingolipids (SLs) represent an important class of eukaryotic lipids with structural functions as well as bioactive molecules able to modulate crucial processes, including inflammation and viral infection. In the last two decades, several reports have highlighted the role of SLs in modulating SkM cell differentiation, regeneration, aging, response to insulin, and contraction. This review summarizes the consequences of SARS-CoV-2 infection on SkM and the potential involvement of SLs in the tissue responses to virus infection. In particular, we highlight the role of sphingosine 1-phosphate signaling in order to aid the prediction of novel targets for preventing and/or treating acute and long-term musculoskeletal manifestations of virus infection in COVID-19.
    Keywords:  COVID-19; SARS-CoV-2; amyotrophic lateral sclerosis; ceramide; multiple sclerosis; myasthenia gravis; skeletal muscle; sphingolipids; sphingosine 1-phosphate; sphingosine 1-phosphate receptors
    DOI:  https://doi.org/10.3390/biomedicines10051068
  39. Methods Mol Biol. 2022 ;2399 173-192
    Understanding the Human Aging Proteome Using Epidemiological Models.
    Ceereena Ubaida-Mohien, Ruin Moaddel, Zenobia Moore, Pei-Lun Kuo, Ravi Tharakan, Toshiko Tanaka, Luigi Ferrucci.
      Human aging is a complex multifactorial process associated with a decline of physical and cognitive function and high susceptibility to chronic diseases, influenced by genetic, epigenetic, environmental, and demographic factors. This chapter will provide an overview on the use of epidemiological models with proteomics data as a method that can be used to identify factors that modulate the aging process in humans. This is demonstrated with proteomics data from human plasma and skeletal muscle, where the combination with epidemiological models identified a set of mitochondrial, spliceosome, and senescence proteins as well as the role of energetic pathways such as glycolysis, and electron transport pathways that regulate the aging process.
    Keywords:  Aging; BLSA; Data model; Epidemiology; GESTALT; Plasma; Proteomics; SOMAscan; Skeletal muscle; TMT
    DOI:  https://doi.org/10.1007/978-1-0716-1831-8_8
  40. J Control Release. 2022 May 22. pii: S0168-3659(22)00301-7. [Epub ahead of print]
    Size-tunable PEG-grafted copolymers as a polymeric nanoruler for passive targeting muscle tissues.
    Mitsuru Naito, Yusuke Watanuki, Kazuko Toh, Jongmin Yum, Beob Soo Kim, Kaori Taniwaki, Satomi Ogura, Hiroki Ishida, Masaru Cho, Hiroyuki Chaya, Ken Miyajima, Yuichi Yamasaki, Kensuke Osada, Katsura Minegishi, Yoshitsugu Aoki, Kanjiro Miyata.
      Muscle-targeted drug delivery is a major challenge in nanomedicine. The extravasation of nanomedicines (or nanoparticles) from the bloodstream into muscle tissues is hindered by the continuous endothelium, the so-called blood-muscle barrier. This study aimed to evaluate the optimal size of macromolecular drugs for extravasation (or passive targeting) into muscle tissues. We constructed a size-tunable polymeric delivery platform as a polymeric nanoruler by grafting poly(ethylene glycol)s (PEGs) onto the poly(aspartic acid) (PAsp) backbone. A series of PEG-grafted copolymers (gPEGs) with a narrow size distribution between 11 and 32 nm in hydrodynamic diameter (DH) were prepared by changing the molecular weight of the PEGs. Biodistribution analyses revealed that accumulation amounts of gPEGs in the muscle tissues of normal mice tended to decrease above their size of ~15 nm (or ~ 11 nm for the heart). The gPEGs accumulated in the skeletal muscles of Duchenne muscular dystrophy model mice (mdx mice) at a 2-3-fold higher level than in the skeletal muscles of normal mice. At the same time, there was a reduced accumulation of gPEGs in the spleen and liver. Intravital confocal laser scanning microscopy and immunohistochemical analysis showed extravasation and locally enhanced accumulation of gPEGs in the skeletal muscle of mdx mice. This study outlined the pivotal role of macromolecular drug size in muscle-targeted drug delivery and demonstrated the enhanced permeability of 11-32 nm-sized macromolecular drugs in mdx mice.
    Keywords:  Graft copolymer; Muscle targeting; Muscular dystrophy; PEG; Size effect
    DOI:  https://doi.org/10.1016/j.jconrel.2022.05.030
  41. Diabetes. 2022 May 27. pii: db220079. [Epub ahead of print]
    The Effect of Glucagon on Protein Catabolism During Insulin Deficiency - Exchange of Amino acids Across Skeletal Muscle and The Splanchnic Bed.
    Haleigh James, Wilson I Gonsalves, Shankarappa Manjunatha, Surendra Dasari, Ian R Lanza, Katherine A Klaus, Adrian Vella, James C Andrews, K Sreekumaran Nair.
      Transient insulin deprivation with concurrent hyperglucagonemia is a catabolic state that can occur in type 1 diabetes. To evaluate glucagon's catabolic effect in the setting of its glucogenic effect, we measured the regional exchanges of amino-metabolites across muscle and splanchnic beds in 16 healthy humans during either somatostatin followed by glucagon or a saline infusion alone. Despite a ≥2-fold increase in the regional exchange of amino-metabolites by glucagon, whole body kinetics and concentrations of amino acids (AAs) remained stable. Glucagon increased the splanchnic uptake of not only gluconeogenic but also essential amino acids (EAAs) while increasing their release from the muscle bed. Regional tracer-based kinetics and 3-methyl-histidine release indicate that EAA release from muscle is likely caused by reduced protein synthesis rather than increased protein degradation. Furthermore, many metabolites known to affect insulin action and metabolism were altered by hyperglucagonemia including increase in branched amino acids and keto acids of leucine and isoleucine in arterial plasma. Further, an increase in arterial concentrations of α-aminoadipic acid arising from increased conversion from lysine in the splanchnic bed was noted. These results demonstrate that hyperglucagonemia during hypoinsulinemia increases net muscle protein catabolism and substantially increases the exchange of amino metabolites across splanchnic and muscle beds.
    DOI:  https://doi.org/10.2337/db22-0079
  42. Biogerontology. 2022 May 23.
    Aging and short-term calorie restriction differently affect the cardiac and skeletal muscle expression of genes regulating energy substrate utilization in male rats.
    Aleksandra Ławniczak, Agata Wrońska, Piotr Wierzbicki, Zbigniew Kmieć.
      Aging affects the energy metabolism differently in the cardiac and skeletal muscles. The study aim was to assess the effects of short-term calorie restriction (SCR) and refeeding on the expression of genes involved in the control of cardiac and skeletal muscle energy metabolism in old vs. young male rats. Young (4 mo) and old (24 mo) rats were subjected to 60% SCR for 30 days, and refed ad libitum for 2 or 4 days. In the cardiac (CM) and skeletal muscles (SM) we compared the gene expression (qPCR) of carnitine palmitoyltransferase-I (Cpt-I), peroxisome proliferator-activated receptor beta/delta (Ppar-β/δ), glucose transporter 4 (Glut4), peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc-1α), and sirtuin 3 (Sirt3). In CM, aging increased Cpt-I expression but did not affect the other genes. In SM, Cpt-I, Glut4, Pgc-1α, and Sirt3 mRNA levels were lower in old than young rats. In CM of only young rats SCR increased Cpt-I expression which remained elevated after refeeding. Upon SCR, the expression of Ppar-β/δ, Glut4, Pgc-1α, and Sirt3 in CM increased in young but not old rats, and refeeding re-established control levels. In SM of young rats SCR increased Ppar-β/δ and Pgc-1α, and decreased Sirt3 expression, whereas refeeding generally decreased these mRNA levels. In SM of old rats SCR decreased only Pgc-1α expression. The adaptive response to SCR and subsequent refeeding is muscle tissue-specific and differs in young and old male rats. SCR appears to increase the efficiency of glucose and fatty acid utilization in the cardiac muscle of young, but not old male rats.
    Keywords:  Aging; Heart muscle; Refeeding; Short-term calorie restriction; Skeletal muscle
    DOI:  https://doi.org/10.1007/s10522-022-09965-y
  43. Sci Rep. 2022 May 24. 12(1): 8764
    The superior beneficial effects of exercise training versus hormone replacement therapy on skeletal muscle of ovariectomized rats.
    Sara Barros Silva, Kinulpe Honorato-Sampaio, Sabrina Paula Costa, Talita Emanuela Domingues, Timilly Mayra Martins da Cruz, Cíntia Maria Rodrigues, Karine Beatriz Costa, Jousielle Márcia Dos Santos, Vanessa Kelly da Silva Lage, Thais Peixoto Gaiad, Ana Paula Santos, Marco Fabrício Dias-Peixoto, Cândido Celso Coimbra, Adelina Martha Dos Reis, Raphael Escorsim Szawka, Pedro Henrique Scheidt Figueiredo, Henrique Silveira Costa, Murilo Xavier Oliveira, Vanessa Amaral Mendonça, Ana Cristina Rodrigues Lacerda.
      Previous studies have highlighted the positive effects of Estradiol (E2) replacement therapy and physical exercise on skeletal muscle during menopause. However, the comparison effects of exercise training (ET) and estradiol replacement therapy during menopause on skeletal muscle have not been investigated to date. This study aimed to compare the effects of endurance exercise training versus E2 replacement therapy on mitochondrial density, redox status, and inflammatory biomarkers in the skeletal muscle of ovariectomized rats. Thirty female Wistar rats (12-week-old) were randomly assigned into three groups: Untrained ovariectomized rats (UN-OVX, n = 10); untrained ovariectomized rats treated with estradiol replacement therapy (E2-OVX); and, trained ovariectomized rats (TR-OVX). After ovariectomy, the E2-OVX rats were treated subcutaneously with E2 (implanted Silastic® capsule containing 360 μg of 17β-estradiol/mL) while the TR-OVX group performed an exercise training protocol (50-70% of maximal running speed on a treadmill, 60 min/day, 5 days/week for 8 weeks). After euthanasia, the soleus muscle was processed for histological and biochemical evaluations. Only exercise prevented the reduction of maximal oxygen consumption and increased mechanical efficiency (ME). While mitochondrial muscle density, total antioxidant capacity (FRAP), catalase (CAT) activity, and interleukin 10 levels were higher in TR-OVX, only OVX-E2 presented higher CAT activity and lower interleukin 6 levels. Endurance exercise training compared with E2 replacement therapy maintains the aerobic capacity improving the ME of OVX rats. In addition, only endurance exercise training raises the skeletal muscle mitochondrial content and tends to balance the redox and inflammatory status in the skeletal muscle of OVX rats.
    DOI:  https://doi.org/10.1038/s41598-022-12739-8
  44. Front Genome Ed. 2022 ;4 863651
    Development of Therapeutic RNA Manipulation for Muscular Dystrophy.
    Saifullah, Norio Motohashi, Toshifumi Tsukahara, Yoshitsugu Aoki.
      Approval of therapeutic RNA molecules, including RNA vaccines, has paved the way for next-generation treatment strategies for various diseases. Oligonucleotide-based therapeutics hold particular promise for treating incurable muscular dystrophies, including Duchenne muscular dystrophy (DMD). DMD is a severe monogenic disease triggered by deletions, duplications, or point mutations in the DMD gene, which encodes a membrane-linked cytoskeletal protein to protect muscle fibers from contraction-induced injury. Patients with DMD inevitably succumb to muscle degeneration and atrophy early in life, leading to premature death from cardiac and respiratory failure. Thus far, the disease has thwarted all curative strategies. Transcriptomic manipulation, employing exon skipping using antisense oligonucleotides (ASO), has made significant progress in the search for DMD therapeutics. Several exon-skipping drugs employing RNA manipulation technology have been approved by regulatory agencies and have shown promise in clinical trials. This review summarizes recent scientific and clinical progress of ASO and other novel RNA manipulations, including RNA-based editing using MS2 coat protein-conjugated adenosine deaminase acting on the RNA (MCP-ADAR) system illustrating the efficacy and limitations of therapies to restore dystrophin. Perhaps lessons from this review will encourage the application of RNA-editing therapy to other neuromuscular disorders.
    Keywords:  DMD; RNA editing therapy; RNA engineering; antisense oligonucleotides; exon skipping; molecular therapy
    DOI:  https://doi.org/10.3389/fgeed.2022.863651
  45. J Appl Physiol (1985). 2022 May 19.
    The influence of longitudinal muscle fascicle growth on mechanical function.
    Avery Hinks, Martino V Franchi, Geoffrey A Power.
      Skeletal muscle has the remarkable ability to remodel and adapt, such as the increase in serial sarcomere number (SSN) or fascicle length (FL) observed after overstretching a muscle. This type of remodelling is termed longitudinal muscle fascicle growth, and its impact on biomechanical function has been of interest since the 1960s due to its clinical applications in muscle strain injury, muscle spasticity, and sarcopenia. Despite simplified hypotheses on how longitudinal muscle fascicle growth might influence mechanical function, existing literature presents conflicting results partly due to a breadth of methodologies. The purpose of this review is to outline what is currently known about the influence of longitudinal muscle fascicle growth on mechanical function and suggest future directions to address current knowledge gaps and methodological limitations. Various interventions indicate longitudinal muscle fascicle growth can increase the optimal muscle length for active force, but whether the whole force-length relationship widens has been less investigated. Future research should also explore the ability for longitudinal fascicle growth to broaden the torque-angle relationship's plateau region, and the relation to increased force during shortening. Without a concurrent increase in intramuscular collagen, longitudinal muscle fascicle growth also reduces passive tension at long muscle lengths; further research is required to understand whether this translates to increased joint range of motion. Lastly, some evidence suggests longitudinal fascicle growth can increase maximum shortening velocity and peak isotonic power, however, there has yet to be direct assessment of these measures in a neurologically intact model of longitudinal muscle fascicle growth.
    Keywords:  Fascicle length; Force-velocity relationship; Sarcomerogenesis; force-length relationship; serial sarcomere number
    DOI:  https://doi.org/10.1152/japplphysiol.00114.2022
  46. Int J Environ Res Public Health. 2022 May 23. pii: 6323. [Epub ahead of print]19(10):
    Resistance Training before, during, and after COVID-19 Infection: What Have We Learned So Far?
    Paulo Gentil, Claudio Andre Barbosa de Lira, Carlos Alexandre Vieira, Rodrigo Ramirez-Campillo, Amir Hossein Haghighi, Filipe Manuel Clemente, Daniel Souza.
      At the end of 2019, a severe acute respiratory syndrome caused by SARS-CoV-2 started a pandemic, leading to millions of deaths and many important political and social changes. Even in the absence of contamination, the mobility reduction, social distancing and closing of exercise facilities negatively affected physical activity and conditioning, which is associated with muscle atrophy, loss of muscle strength, and reductions in functional capacity. In cases of infection, it has been shown that increased physical capacity is associated with decreased hospitalization and mortality risk. Although millions of people have died from COVID-19, most contaminated individuals survived the infection, but carried different sequelae, such as the severe loss of physical function and a reduced quality of life. Among different physical exercise models that might help to prevent and treat COVID-19-related conditions, resistance training (RT) might be particularly relevant. Among its benefits, RT can be adapted to be performed in many different situations, even with limited space and equipment, and is easily adapted to an individual's characteristics and health status. The current narrative review aims to provide insights into how RT can be used in different scenarios to counteract the negative effects of COVID-19. By doing this, the authors expect to provide insights to help deal with the current pandemic and similar events the world may face in the future.
    Keywords:  coronavirus; human physical conditioning; muscle strength; musculoskeletal and neural physiological phenomena; resistance training
    DOI:  https://doi.org/10.3390/ijerph19106323
  47. Elife. 2022 May 23. pii: e77204. [Epub ahead of print]11
    Contractile force assessment methods for in vitro skeletal muscle tissues.
    Camila Vesga-Castro, Javier Aldazabal, Ainara Vallejo-Illarramendi, Jacobo Paredes.
      Over the last few years, there has been growing interest in measuring the contractile force (CF) of engineered muscle tissues to evaluate their functionality. However, there are still no standards available for selecting the most suitable experimental platform, measuring system, culture protocol, or stimulation patterns. Consequently, the high variability of published data hinders any comparison between different studies. We have identified that cantilever deflection, post deflection, and force transducers are the most commonly used configurations for CF assessment in 2D and 3D models. Additionally, we have discussed the most relevant emerging technologies that would greatly complement CF evaluation with intracellular and localized analysis. This review provides a comprehensive analysis of the most significant advances in CF evaluation and its critical parameters. In order to compare contractile performance across experimental platforms, we have used the specific force (sF, kN/m2), CF normalized to the calculated cross-sectional area (CSA). However, this parameter presents a high variability throughout the different studies, which indicates the need to identify additional parameters and complementary analysis suitable for proper comparison. We propose that future contractility studies in skeletal muscle constructs report detailed information about construct size, contractile area, maturity level, sarcomere length, and, ideally, the tetanus-to-twitch ratio. These studies will hopefully shed light on the relative impact of these variables on muscle force performance of engineered muscle constructs. Prospective advances in muscle tissue engineering, particularly in muscle disease models, will require a joint effort to develop standardized methodologies for assessing CF of engineered muscle tissues.
    Keywords:  contractile force; neuroscience; skeletal muscle; stimulation; tissue engineering
    DOI:  https://doi.org/10.7554/eLife.77204
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