bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒03‒14
38 papers selected by
Anna Vainshtein
Craft Science Inc.

  1. J Cachexia Sarcopenia Muscle. 2021 Mar 09.
      BACKGROUND: A therapeutic approach for the treatment of glucocorticoid-induced skeletal muscle atrophy should be based on the knowledge of the molecular mechanisms determining the unbalance between anabolic and catabolic processes and how to re-establish this balance. Here, we investigated whether the obestatin/GPR39 system, an autocrine signalling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against chronic glucocorticoid-induced muscle atrophy.METHODS: In this study, we used an in vivo model of muscle atrophy induced by the synthetic glucocorticoid dexamethasone to examine the liaison molecules that define the interaction between the glucocorticoid receptor and the obestatin/GPR39 systems. The findings were extended to in vitro effects on human atrophy using human KM155C25 myotubes.
    RESULTS: KLF15 and FoxO transcription factors were identified as direct targets of obestatin signalling in the control of proteostasis in skeletal muscle. The KLF15-triggered gene expression program, including atrogenes and FoxOs, was regulated via KLF15 ubiquitination by the E3 ubiquitin ligase NEDD4. Additionally, a specific pattern of FoxO post-translational modification, including FoxO4 phosphorylation by Akt pathway, was critical in the regulation of the ubiquitin-proteasome system. The functional cooperativity between Akt and NEDD4 in the regulation of FoxO and KLF15 provides integrated cues to counteract muscle proteostasis and re-establish protein synthesis.
    CONCLUSIONS: The effective control of FoxO activity in response to glucocorticoid is critical to counteract muscle-related pathologies. These results highlight the potential of the obestatin/GPR39 system to fine-tune the effects of glucocorticoids on skeletal muscle wasting.
    Keywords:  FoxO; Glucocorticoid-induced muscle atrophy; KLF15; NEDD4; Obestatin signalling; Skeletal muscle
  2. Gene. 2021 Mar 08. pii: S0378-1119(21)00156-6. [Epub ahead of print] 145562
      Skeletal muscle is the most abundant tissue in the human and animal body, loss of its function can lead to muscle aging and various myogenic diseases. The skeletal muscle development is a complex and tightly regulated process, which is driven by a variety of many factors, signaling pathways and regulatory mechanisms. Plectin (Plec), a cytolinker protein, is ubiquitously expressed in various tissues such as skin, muscle, plasma membrane, and most types of cells. Although known isoforms of Plec is well-characterized in muscle dystrophy, very little is known on the function of Plec in the skeletal muscle development. Here, we found that Plec plays a vital role in promoting C2C12 myoblasts differentiation and proliferation, but inhibits their apoptosis. Also, Plec regulates the expression of atrophy-related genes (atrogin-1 and muRF-1) to rescue muscle atrophy. Furthermore, we have demonstrated that Plec binds to Dishevelled-2 (Dvl-2) and forms a protein complex, which is then activate the canonical Wnt signaling. We also observed that Plec resists ubiquitination by stabilizing Dvl-2 and reduces the level of LC3-labeled Dvl-2 and antagonizes the autophagy system. In conclusion, our findings suggest that Plec regulates canonical Wnt signaling mediated skeletal development by stabilizing Dvl-2 and downregulating the cellular autophagic degradation system.
    Keywords:  Autophagy; Dishevelled-2; Myogenesis; Plec; Wnt Signaling
  3. Am J Respir Cell Mol Biol. 2021 Mar 09.
      Skeletal muscle dysfunction is one of the important comorbidities of COPD, however the underlying mechanisms remain largely unknown. RANKL, a key mediator in osteoclast differentiation, was also found to play a role in skeletal muscle pathogenesis. Whether RANKL is involved in COPD-related skeletal muscle dysfunction is yet unknown. We examined the expression of RANKL/RANK in skeletal muscles from mice exposed to cigarette smoke (CS) for 24 weeks. Grip strength and exercise capacity as well as muscular morphology were evaluated in CS-exposed mice with or without anti-RANKL treatment. The expressions of protein-synthesis or muscle-growth related molecules (IGF-1, myogenin, and myostatin), muscle specific ubiquitin E3 ligases (MuRF1, Atrogin1), and NF-κb inflammatory pathway were also evaluated in skeletal muscles. The effect of CS extract (CSE) on RANKL/RANK expression and that of exogenous RANKL on ubiquitin-proteasome pathway in C2C12 myotubes were investigated in vitro. Long-term CS exposure induced skeletal muscle dysfunction and atrophy, together with upregulation of RANKL/RANK expressions in a well-established mouse model of COPD. RANKL neutralization prevented skeletal muscle dysfunction and atrophy. RANKL inhibition decreased expressions of myostatin and MuRF1/Atrogin1, and suppressed NF-κb pathway in skeletal muscles from CS-exposed mice. In in vitro experiments with C2C12 myotubes, CSE induced expression of RANKL/RANK, and exogenous RANKL induced activation of ubiquitin-proteasome pathway and NF-κb pathway via RANK. Our results revealed an important role of the RANKL/RANK pathway in muscle atrophy induced by CS exposure, suggesting that RANKL may be a potential therapeutic target in COPD-related skeletal muscle dysfunction.
    Keywords:  COPD; RANKL; cigarette smoke; skeletal muscle dysfunction
  4. FASEB J. 2021 Apr;35(4): e21459
      Chronic muscle loading (overload) induces skeletal muscles to undergo hypertrophy and to increase glucose uptake. Although AMP-activated protein kinase (AMPK) reportedly serves as a negative regulator of hypertrophy and a positive regulator of glucose uptake, its role in overload-induced skeletal muscle hypertrophy and glucose uptake is unclear. This study aimed to determine whether AMPK regulates overload-induced hypertrophy and glucose uptake in skeletal muscles. To this end, skeletal muscle overload was induced through unilateral synergist ablations in wild-type (WT) and transgenic mice, expressing the dominant-negative mutation of AMPK (AMPK-DN). After 14 days, parameters, including muscle fiber cross-sectional area (CSA), glycogen level, and in vivo [3 H]-2-deoxy-D-glucose uptake, were assessed. No significant difference was observed in body weight or blood glucose level between the WT and AMPK-DN mice. However, the 14-day muscle overload activated the AMPK pathway in WT mice skeletal muscle, whereas this response was impaired in the AMPK-DN mice. Despite a normal CSA gain in each fiber type, the AMPK-DN mice demonstrated a significant impairment of overload-induced muscle glucose uptake and glycogenesis, compared to WT mice. Moreover, 14-day overload-induced changes in GLUT4 and HKII expression levels were reduced in AMPK-DN mice, compared to WT mice. This study demonstrated that AMPK activation is indispensable for overload-induced muscle glucose uptake and glycogenesis; however, it is dispensable for the induction of hypertrophy in AMPK-DN mice. Furthermore, the AMPK/GLUT4 and HKII axes may regulate overload-induced muscle glucose uptake and glycogenesis.
    Keywords:  AMP-activated protein kinase; functional overload; glucose uptake; hypertrophy; skeletal muscle
  5. Sci Adv. 2021 Mar;pii: eabe4501. [Epub ahead of print]7(11):
      The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.
  6. J Steroid Biochem Mol Biol. 2021 Mar 03. pii: S0960-0760(21)00054-6. [Epub ahead of print] 105861
      BACKGROUND: Evidence is growing for a role of vitamin D in regulating skeletal muscle mass, strength and functional capacity. Given the role the kidneys play in activating total vitamin D, and the high prevalence of vitamin D deficiency in Chronic Kidney Disease (CKD), it is possible that deficiency contributes to the low levels of physical function and muscle mass in these patients.METHODS: This is a secondary cross-sectional analysis of previously published interventional study, within vitro follow up work. 34 CKD patients at stages G3b-5 (eGFR 25.5 ± 8.3 ml/min/1.73m2; age 61 ± 12 years) were recruited, with a sub-group (n = 20) also donating a muscle biopsy. Vitamin D and associated metabolites were analysed in plasma by liquid chromatography tandem-mass spectroscopy and correlated to a range of physiological tests of muscle size, function, exercise capacity and body composition. The effects of 1α,25(OH)2D3 supplementation on myogenesis and myotube size was investigated in primary skeletal muscle cells from vitamin D deficient donors.
    RESULTS: In vivo, there was no association between total or active vitamin D and muscle size or strength, but a significant correlation with V̇O2Peak was seen with total vitamin D (25OHD). In vitro, 1α,25(OH)2D3 supplementation reduced IL-6 mRNA expression, but had no effect upon proliferation, differentiation or myotube diameter.
    CONCLUSIONS: Vitamin D deficiency is not a prominent factor driving the loss of muscle mass in CKD, but may play a role in reduced exercise capacity.
    Keywords:  Exercise Capacity; Physical function; Skeletal muscle; Vitamin D
  7. Vitam Horm. 2021 ;pii: S0083-6729(20)30069-8. [Epub ahead of print]115 535-570
      Aging involves numerous changes in body composition that include a decrease in skeletal muscle mass. The gradual reduction in muscle mass is associated with a simultaneous decrease in muscle strength, which leads to reduced mobility, fragility and loss of independence. This process called sarcopenia is secondary to several factors such as sedentary lifestyle, inadequate nutrition, chronic inflammatory state and neurological alterations. However, the endocrine changes associated with aging seem to be of special importance in the development of sarcopenia. On one hand, advancing age is associated with a decreased secretion of the main hormones that stimulate skeletal muscle mass and function (growth hormone, insulin-like growth factor 1 (IGFI), testosterone and estradiol). On the other hand, the alteration of the IGF-I signaling along with decreased insulin sensitivity also have an important impact on myogenesis. Other hormones that decline with aging such as the adrenal-derived dehydroepiandrosterone, thyroid hormones and vitamin D seem to also be involved in sarcopenia. Adipokines released by adipose tissue show important changes during aging and can affect muscle physiology and metabolism. In addition, catabolic hormones such as cortisol and angiotensin II can accelerate aged-induced muscle atrophy, as they are involved in muscle wasting and their levels increase with age. The role played by all of these hormones and the possible use of some of them as therapeutic tools for treating sarcopenia will be discussed.
    Keywords:  Adiponectin; Angiotensin II; Cortisol; Estrogens; IGF-I; Insulin; Leptin; Sarcopenia; Skeletal muscle atrophy; Testosterone
  8. Life Sci. 2021 Mar 03. pii: S0024-3205(21)00281-2. [Epub ahead of print]273 119296
      AIMS: Skeletal muscle mass and strength are reduced in asthma and contribute to compromised functional capacity in asthmatic patients. However, an effective pharmacological intervention remains elusive, partly because molecular mechanisms dictating muscle decline in asthma are not known.MATERIALS: We investigated the potential contribution(s) of skeletal muscle sarcoplasmic reticulum Ca2+ ATPase (SERCA) to muscle atrophy and weakness in asthmatic patients. Quadriceps muscle biopsies were taken from 58 to 72 years old male patients with mild and advanced asthma and the SERCA activity was analyzed in association with cellular redox environment and myonuclear domain (MND) size.
    KEY FINDINGS: Maximal SERCA activity was reduced in skeletal muscles of mild and advanced asthmatics and was associated with reduced expression of SERCA2 protein and upregulation of sarcolipin, a SERCA inhibitory lipoprotein. We also found downregulation of Ca2+ release protein calstabin and upregulation of Ca2+ buffer, calsequestrin in skeletal muscles of asthmatic patients. The atrophic single muscle fibers had smaller cytoplasmic domains per myonucleus possibly indicating the reduced transcriptional reserves of individual myonuclei. Plasma periostin and CAF22 levels were significantly elevated in asthmatic patients and showed a strong correlation with hand-grip strength. These changes were accompanied by substantially elevated markers of global oxidative stress including lipid peroxidation and mitochondrial ROS production.
    CONCLUSION: Taken together, our data suggest that muscle weakness and atrophy in asthma is in part driven by SERCA dysfunction and oxidative stress. The data propose SERCA dysfunction as a therapeutic intervention to address muscle decline in asthma.
    Keywords:  Asthma; Biomarkers; Oxidative stress; SERCA dysfunction; Skeletal muscle
  9. Anim Biotechnol. 2021 Mar 11. 1-9
      Leucine can promote slow-twitch muscle fibers formation, and this effect may be mediated by AMPK signaling pathway. In addition, adiponectin (AdipoQ) plays an important role in regulation of muscle fiber type transformation. AdipoQ is located in the upstream of AMPK and its secretion can be regulated by leucine. Therefore, the aim of this study was to explore whether leucine affects muscle fiber type transformation through AdipoQ signaling pathway. Our data showed that 4 mM leucine significantly increased protein expression levels of slow MyHC, Myoglobin, Troponin I-SS, AdipoQ, AdipoR1, phospho-AMPK (p-AMPK) and PGC-1α and mRNA expression levels of AMPKα2, PGC-1α, AdipoQ and AdipoR1, and significantly decreased fast MyHC protein expression. In addition, 4 mM leucine significantly increased the SDH activity while significantly decreased the LDH activity. However, knockdown of AdipoR1 expression by AdipoR1-siRNA abolished leucine-induced upregulation of protein expressions of slow MyHC, AdipoR1, p-AMPK, PGC-1α and NRF1, mRNA expressions of MyHC I, MyHC IIa, AdipoR1, AMPKα2 and PGC-1α, ATP5G, TFAM and NRF1, and mtDNA level, as well as downregulation of protein expression of fast MyHC and mRNA expression of MyHC IIb. Together, our data revealed that leucine promotes muscle fiber type transformation from fast-twitch to slow-twitch through AdipoQ signaling pathway.
    Keywords:  AdipoQ signaling pathway; Leucine; muscle fiber type transformation; porcine skeletal muscle satellite cells
  10. Front Physiol. 2021 ;12 619447
      The methylome and transcriptome signatures following exercise that are physiologically and metabolically relevant to sporting contexts such as team sports or health prescription scenarios (e.g., high intensity interval training/HIIT) has not been investigated. To explore this, we performed two different sport/exercise relevant high-intensity running protocols in five male sport team members using a repeated measures design of: (1) change of direction (COD) versus; (2) straight line (ST) running exercise with a wash-out period of at least 2 weeks between trials. Skeletal muscle biopsies collected from the vastus lateralis 30 min and 24 h post exercise, were assayed using 850K methylation arrays and a comparative analysis with recent (subject-unmatched) sprint and acute aerobic exercise meta-analysis transcriptomes was performed. Despite COD and ST exercise being matched for classically defined intensity measures (speed × distance and number of accelerations/decelerations), COD exercise elicited greater movement (GPS-Playerload), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) exertion measures compared with ST exercise, suggesting COD exercise evoked a higher exercise intensity. The exercise response alone across both conditions evoked extensive alterations in the methylome 30 min and 24 h post exercise, particularly in MAPK, AMPK and axon guidance pathways. COD evoked a considerably greater hypomethylated signature across the genome compared with ST exercise, particularly at 30 min post exercise, enriched in: Protein binding, MAPK, AMPK, insulin, and axon guidance pathways. Comparative methylome analysis with sprint running transcriptomes identified considerable overlap, with 49% of genes that were altered at the expression level also differentially methylated after COD exercise. After differential methylated region analysis, we observed that VEGFA and its downstream nuclear transcription factor, NR4A1 had enriched hypomethylation within their promoter regions. VEGFA and NR4A1 were also significantly upregulated in the sprint transcriptome and meta-analysis of exercise transcriptomes. We also confirmed increased gene expression of VEGFA, and considerably larger increases in the expression of canonical metabolic genes PPARGC1A (that encodes PGC1-α) and NR4A3 in COD vs. ST exercise. Overall, we demonstrate that increased physiological/metabolic load via COD exercise in human skeletal muscle evokes considerable epigenetic modifications that are associated with changes in expression of genes responsible for adaptation to exercise.
    Keywords:  AMPK; DNA methylation; MAPK; NR4A1 (Nur77); NR4A3; PGC1 alpha; VEGF; change of direction
  11. J Clin Endocrinol Metab. 2021 Feb 26. pii: dgab106. [Epub ahead of print]
      CONTEXT: The early events regulating the remodelling programme following skeletal muscle damage are poorly understood.OBJECTIVE: The objective of this study was to determine the association between myofibrillar protein synthesis (myoPS) and nuclear factor-kappa B (NF-κB) signalling by nutritionally accelerating recovery of muscle function following damage.
    DESIGN, SETTING, PARTICIPANTS, AND INTERVENTIONS: Healthy males and females consumed daily post-exercise and pre-bed protein-polyphenol (PP; n=9; 4 females) or isocaloric maltodextrin placebo (PLA; n=9; 3 females) drinks (parallel design), 6 days before and 3 days after 300 unilateral eccentric quadriceps contractions (EC) during complete dietary control.
    MAIN OUTCOME MEASURES: Muscle function was assessed daily, and skeletal muscle biopsies were taken after 24, 27 and 36 h for measurements of myoPS rates using deuterated water, and gene ontology and NF-κB signalling analysis using an RT-qPCR gene array.
    RESULTS: EC impaired muscle function for 48 h in PLA, but for just 24 h in PP (P=0.047). EC increased myoPS compared to the control leg during post-exercise (24-27 h; 0.14±0.01 vs 0.11±0.01%·h -1, respectively; P=0.075) and overnight periods (27-36 h; 0.10±0.01 vs 0.07±0.01%·h -1, respectively; P=0.020), but was not further increased by PP (P>0.05). PP decreased post-exercise and overnight muscle IL1R1 (PLA=2.8±0.4, PP=1.1±0.4 and PLA=1.9±0.4, PP=0.3±0.4 log2 fold change, respectively) and IL1RL1 (PLA=4.9±0.7, PP=1.6±0.8 and PLA=3.7±0.6, PP=0.7±0.7 log2 fold change, respectively) mRNA expression (P<0.05) and downstream NF-κB signalling compared to PLA.
    CONCLUSION: PP ingestion likely accelerates recovery of muscle function by attenuating inflammatory NF-κB transcriptional signalling, possibly to reduce aberrant tissue degradation rather than increase myoPS rates.
    Keywords:  Inflammation; deuterated water; muscle damage; protein-polyphenol
  12. Muscle Nerve. 2021 Mar 08.
      INTRODUCTION/AIMS: One of the hallmarks of injured skeletal muscle is the appearance of elevated skeletal muscle proteins in circulation. Human skeletal muscle generally consists of a mosaic of slow (type I) and fast (type IIa, IIx/d) fibers, defined by their myosin isoform expression. Recently, measurement of circulating fiber-type specific isoforms of troponin I has been used as a biomarker to suggest that muscle injury in healthy volunteers (HV) results in the appearance of muscle proteins from fast but not slow fibers. We sought to understand if this is also the case in severe myopathy patients with Becker and Duchenne muscular dystrophy (BMD, DMD).METHODS: An ELISA that selectively measures fast and slow skeletal troponin I (TNNI2 and TNNI1) was used to measure a cross-section of patient plasma samples from HV (N=50), BMD (N=49) and DMD (N=132) patients. Creatine kinase (CK) activity was also measured from the same samples for comparison.
    RESULTS: TNNI2 was elevated in BMD and DMD and correlated with the injury biomarker, CK. In contrast, TNNI1 levels were indistinguishable from levels in HV. There was an inverse relationship between CK and TNNI2 levels and age but no relationship for TNNI1.
    DISCUSSION: We define a surprising discrepancy between TNNI1 and TNNI2 in patient plasma that may have implications for the interpretation of elevated muscle protein levels in dystrophinopathies.
    Keywords:  Muscular dystrophy; biomarker; creatine kinase; muscle injury; troponin
  13. J Biol Chem. 2021 Mar 04. pii: S0021-9258(21)00292-1. [Epub ahead of print] 100516
      Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.
    Keywords:  DAGK); PDZ domain; Ras homolog gene family; Ras‐related C3 botulinum toxin substrate 1 (Rac1); bleb; cell biology; cell migration; cell signaling; diacylglycerol; diacylglycerol kinase (DGK; fibroblast; lamellipodium; member A (RhoA); membrane; phosphatidic acid; phosphorylation; protein kinase C (PKC); scaffold protein; skeletal muscle; syntrophin
  14. Hum Mol Genet. 2021 Mar 09. pii: ddab063. [Epub ahead of print]
      Facioscapulohumeral muscular dystrophy (FSHD) is an inherited muscle disease caused by misexpression of the DUX4 gene in skeletal muscle. DUX4 is a transcription factor which is normally expressed in the cleavage-stage embryo and regulates gene expression involved in early embryonic development. Recent studies revealed that DUX4 also activates the transcription of repetitive elements such as endogenous retroviruses (ERVs), mammalian apparent LTR-retrotransposons (MaLRs), and pericentromeric satellite repeats (HSATII). DUX4-bound ERV sequences also create alternative promoters for genes or long non-coding RNAs (lncRNAs), producing fusion transcripts. To further understand transcriptional regulation by DUX4, we performed nanopore long-read direct RNA sequencing (dRNA-seq) of human muscle cells induced by DUX4, because long reads show whole isoforms with greater confidence. We successfully detected differential expression of known DUX4-induced genes, and discovered 61 differentially-expressed repeat loci, which are near DUX4-ChIP peaks. We also identified 247 gene-ERV fusion transcripts, of which 216 were not reported previously. In addition, long-read dRNA-seq clearly shows that RNA splicing is a common event in DUX4-activated ERV transcripts. Long-read analysis showed non-LTR transposons including Alu elements are also transcribed from LTRs. Our findings revealed further complexity of DUX4-induced ERV transcripts. This catalogue of DUX4-activated repetitive elements may provide useful information to elucidate the pathology of FSHD. Also, our results indicate that nanopore dRNA-seq has complementary strengths to conventional short read cDNA sequencing.
  15. J Cell Commun Signal. 2021 Mar 10.
      Muscular dystrophies (MDs) are a diverse group of severe disorders characterized by increased skeletal muscle feebleness. In many cases, respiratory and cardiac muscles are also compromised. Skeletal muscle inflammation and fibrosis are hallmarks of several skeletal muscle diseases, including MDs. Until now, several keys signaling pathways and factors that regulate inflammation and fibrosis have been identified. However, no curative treatments are available. Therefore, it is necessary to find new therapeutic targets to fight these diseases and improve muscle performance. Lysophosphatidic acid (LPA) is an active glycerophospholipid mainly synthesized by the secreted enzyme autotaxin (ATX), which activates six different G protein-coupled receptors named LPA1 to LPA6 (LPARs). In conjunction, they are part of the ATX/LPA/LPARs axis, involved in the inflammatory and fibrotic response in several organs-tissues. This review recapitulates the most relevant aspects of inflammation and fibrosis in MDs. It analyzes experimental evidence of the effects of the ATX/LPA/LPARs axis on inflammatory and fibrotic responses. Finally, we speculate about its potential role as a new therapeutic pharmacological target to treat these diseases.
    Keywords:  Autotaxin; Fibrosis; Inflammation; Lysophosphatidic acid; Muscular dystrophies
  16. J Gerontol A Biol Sci Med Sci. 2021 Mar 11. pii: glab077. [Epub ahead of print]
      Periods of inactivity experienced by older adults induce nutrient anabolic resistance creating a cascade of skeletal muscle transcriptional and translational aberrations contributing to muscle dysfunction. The purpose of this study was to identify how inactivity alters leucine-stimulated translation of molecules and pathways within the skeletal muscle of older adults. We performed ribosomal profiling alongside RNA sequencing from skeletal muscle biopsies taken from older adults (n=8; ~72y; 6F/2M) in response to a leucine bolus before (Active) and after (Reduced Activity) 2-weeks of reduced physical activity. At both visits, muscle biopsies were taken at baseline, 60min (early response), and 180min (late response) after leucine ingestion. Previously identified inactivity-related gene transcription changes (PFKFB3, GADD45A, NMRK2) were heightened by leucine with corresponding changes in translation. In contrast, leucine also stimulated translational efficiency (T.E.) of several transcripts in a manner not explained by corresponding changes in mRNA abundance ("uncoupled translation"). Inactivity eliminated this uncoupled translational response for several transcripts, and reduced the translation of most mRNAs encoding for ribosomal proteins. Ingenuity Pathway Analysis identified discordant circadian translation and transcription as a result of inactivity such as translation changes to PER2 and PER3 despite unchanged transcription. We demonstrate inactivity alters leucine-stimulated "uncoupled translation" of ribosomal proteins and circadian regulators otherwise not detectable by traditional RNA-sequencing. Innovative techniques such as ribosomal profiling continues to further our understanding of how physical activity mediates translational regulation, and will set a path towards therapies that can restore optimal protein synthesis on the transcript specific level to combat negative consequences of inactivity on aging muscle.
    Keywords:  Aging; Circadian Rhythm; Disuse; Translation; ribosome profiling
  17. J Gen Physiol. 2021 Apr 05. pii: e202012637. [Epub ahead of print]153(4):
      Huntington's disease (HD) is a fatal and progressive condition with severe debilitating motor defects and muscle weakness. Although classically recognized as a neurodegenerative disorder, there is increasing evidence of cell autonomous toxicity in skeletal muscle. We recently demonstrated that skeletal muscle fibers from the R6/2 model mouse of HD have a decrease in specific membrane capacitance, suggesting a loss of transverse tubule (t-tubule) membrane in R6/2 muscle. A previous report also indicated that Cav1.1 current was reduced in R6/2 skeletal muscle, suggesting defects in excitation-contraction (EC) coupling. Thus, we hypothesized that a loss and/or disruption of the skeletal muscle t-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle. We used live-cell imaging with multiphoton confocal microscopy and transmission electron microscopy to assess the t-tubule architecture in late-stage R6/2 muscle and found no significant differences in the t-tubule system density, regularity, or integrity. However, electron microscopy images revealed that the cross-sectional area of t-tubules at the triad were 25% smaller in R6/2 compared with age-matched control skeletal muscle. Computer simulation revealed that the resulting decrease in the R6/2 t-tubule luminal conductance contributed to, but did not fully explain, the reduced R6/2 membrane capacitance. Analyses of bridging integrator-1 (Bin1), which plays a primary role in t-tubule formation, revealed decreased Bin1 protein levels and aberrant splicing of Bin1 mRNA in R6/2 muscle. Additionally, the distance between the t-tubule and sarcoplasmic reticulum was wider in R6/2 compared with control muscle, which was associated with a decrease in junctophilin 1 and 2 mRNA levels. Altogether, these findings can help explain dysregulated EC coupling and motor impairment in Huntington's disease.
  18. Neurobiol Aging. 2021 Feb 05. pii: S0197-4580(21)00019-1. [Epub ahead of print]
      Sarcopenia, or pathological loss of muscle mass and strength during aging, is an important contributor to loss of physical function in older adults. Sarcopenia is a multifactorial syndrome associated with intrinsic muscle and upstream neurological dysfunction. Exercise is well-established as an effective intervention for sarcopenia, but less is known about the long-term neurobiological impact of exercise. The goals of this study were to investigate the effects of exercise, alone or in combination with follistatin (FST) overexpression (antagonist of myostatin), on neuromuscular junction transmission and motor unit numbers in mice between the age of 22 and 27 months, ages at which prior studies have demonstrated that some motor unit loss is already evident. C57BL/6J mice underwent baseline assessment and were randomized to housing with or without voluntary running wheels and injection with adeno-associated virus to overexpress FST or vehicle. Groups for comparison included sedentary and running with and without FST. Longitudinal assessments showed significantly increased muscle mass and contractility in the 'running plus FST' group, but running, with and without FST, showed no effect on motor unit degeneration. In contrast, running, with and without FST, demonstrated marked improvement of neuromuscular junction transmission stability.
    Keywords:  Aging; Exercise; Follistatin; Motor Unit; Neuromuscular Junction
  19. Am J Physiol Endocrinol Metab. 2021 Mar 08.
      Exercise improves obesity-induced insulin resistance and metabolic disorders via mechanisms that remain unclear. Here, we show that the levels of the hepatokine transthyretin (TTR) in circulation are elevated in insulin-resistant individuals including high-fat diet (HFD)-induced obese mice, db/db mice, and patients with metabolic syndrome. Liver Ttr mRNA and circulating TTR levels were reduced in mice by treadmill training, as was the TTR levels in quadriceps femoris muscle; however, AMPK signalling activity was enhanced. Transgenic overexpression of TTR or injection of purified TTR triggered insulin resistance in mice fed on regular chow (RC). Furthermore, TTR overexpression reduced the beneficial effects of exercise on insulin sensitivity in HFD-fed mice. TTR was internalized by muscle cells via the membrane receptor Grp78 and the internalization into the quadriceps femoris was reduced by treadmill training. The TTR/Grp78 combination in C2C12 cells was increased, whereas the AMPK activity of C2C12 cells was decreased as the TTR concentration rose. Additionally, Grp78 silencing prevented the TTR internalization and reversed its inhibitory effect on AMPK activity in C2C12 cells. Our study suggests that elevated circulating TTR may contribute to insulin resistance and counteract the exercise-induced insulin sensitivity improvement; the TTR suppression might be an adaptive response to exercise through enhancing AMPK activity in skeletal muscles.
    Keywords:  AMPK; Grp78; Transthyretin; exercise; insulin resistance
  20. J Vis Exp. 2021 Feb 18.
      Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as 'MyoTACTIC', and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.
  21. Front Genet. 2021 ;12 607910
      Maternally expressed gene 3 (MEG3) is a long non-coding RNA that is a crucial regulator of skeletal muscle development. Some single-nucleotide polymorphism (SNP) mutants in MEG3 had strong associations with meat quality traits. Nevertheless, the function and mechanism of MEG3 mutants on porcine skeletal muscle development have not yet been well-demonstrated. In this study, eight SNPs were identified in MEG3 of fat- and lean-type pig breeds. Four of these SNPs (g.3087C > T, g.3108C > T, g.3398C > T, and g.3971A > C) were significantly associated with meat quality and consisted of the CCCA haplotype for fat-type pigs and the TTCC haplotype for lean-type pigs. Quantitative real-time PCR results showed that the expression of MEG3-TTCC was higher than that of MEG3-CCCA in transcription level (P < 0.01). The stability assay showed that the lncRNA stability of MEG3-TTCC was lower than that of MEG3-CCCA (P < 0.05). Furthermore, the results of qRT-PCR, Western blot, and Cell Counting Kit-8 assays demonstrated that the overexpression of MEG3-TTCC more significantly inhibited the proliferation of porcine skeletal muscle satellite cells (SCs) than that of MEG3-CCCA (P < 0.05). Moreover, the overexpression of MEG3-TTCC more significantly promoted the differentiation of SCs than that of MEG3-CCCA (P < 0.05). The Western blot assay suggested that the overexpression of MEG3-TTCC and MEG3-CCCA inhibited the proliferation of SCs by inhibiting PI3K/AKT and MAPK/ERK1/2 signaling pathways. The overexpression of the two haplotypes also promoted the differentiation of SCs by activating the JAK2/STAT3 signaling pathway in different degrees. These data are valuable for further studies on understanding the crucial role of lncRNAs in skeletal muscle development.
    Keywords:  MEG3; SNPs; long non-coding RNA; pig; skeletal muscle
  22. J Med Chem. 2021 Mar 11.
      Troponin regulates the calcium-mediated activation of skeletal muscle. Muscle weakness in diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy occurs from diminished neuromuscular output. The first direct fast skeletal troponin activator, tirasemtiv, amplifies the response of muscle to neuromuscular input. Tirasemtiv binds selectively and strongly to fast skeletal troponin, slowing the rate of calcium release and sensitizing muscle to calcium. We report the solution NMR structure of tirasemtiv bound to a fast skeletal troponin C-troponin I chimera. The structure reveals that tirasemtiv binds in a hydrophobic pocket between the regulatory domain of troponin C and the switch region of troponin I, which overlaps with that of Anapoe in the X-ray structure of skeletal troponin. Multiple interactions stabilize the troponin C-troponin I interface, increase the affinity of troponin C for the switch region of fast skeletal troponin I, and drive the equilibrium toward the active state.
  23. J Neuromuscul Dis. 2021 Mar 01.
      Myotonic dystrophy type 1 (DM1) is the most common monogenetic muscular disorder of adulthood. This multisystemic disease is caused by CTG repeat expansion in the 3'-untranslated region of the DM1 protein kinase gene called DMPK. DMPK encodes a myosin kinase expressed in skeletal muscle cells and other cellular populations such as smooth muscle cells, neurons and fibroblasts. The resultant expanded (CUG)n RNA transcripts sequester RNA binding factors leading to ubiquitous and persistent splicing deregulation. The accumulation of mutant CUG repeats is linked to increased activity of glycogen synthase kinase 3 beta (GSK3β), a highly conserved and ubiquitous serine/threonine kinase with functions in pathways regulating inflammation, metabolism, oncogenesis, neurogenesis and myogenesis. As GSK3β-inhibition ameliorates defects in myogenesis, muscle strength and myotonia in a DM1 mouse model, this kinase represents a key player of DM1 pathobiochemistry and constitutes a promising therapeutic target. To better characterise DM1 patients, and monitor treatment responses, we aimed to define a set of robust disease and severity markers linked to GSK3βby unbiased proteomic profiling utilizing fibroblasts derived from DM1 patients with low (80- 150) and high (2600- 3600) CTG-repeats. Apart from GSK3β increase, we identified dysregulation of nine proteins (CAPN1, CTNNB1, CTPS1, DNMT1, HDAC2, HNRNPH3, MAP2K2, NR3C1, VDAC2) modulated by GSK3β. In silico-based expression studies confirmed expression in neuronal and skeletal muscle cells and revealed a relatively elevated abundance in fibroblasts. The potential impact of each marker in the myopathology of DM1 is discussed based on respective function to inform potential uses as severity markers or for monitoring GSK3β inhibitor treatment responses.
    Keywords:  CAPN1; CTNNB1; CTPS1; GSK3β ; HDAC2; fibroblast proteomics
  24. Sci Adv. 2021 Mar;pii: eabe9446. [Epub ahead of print]7(11):
      Here, we present an approach to model and adapt the mechanical regulation of morphogenesis that uses contractile cells as sculptors of engineered tissue anisotropy in vitro. Our method uses heterobifunctional cross-linkers to create mechanical boundary constraints that guide surface-directed sculpting of cell-laden extracellular matrix hydrogel constructs. Using this approach, we engineered linearly aligned tissues with structural and mechanical anisotropy. A multiscale in silico model of the sculpting process was developed to reveal that cell contractility increases as a function of principal stress polarization in anisotropic tissues. We also show that the anisotropic biophysical microenvironment of linearly aligned tissues potentiates soluble factor-mediated tenogenic and myogenic differentiation of mesenchymal stem cells. The application of our method is demonstrated by (i) skeletal muscle arrays to screen therapeutic modulators of acute oxidative injury and (ii) a 3D microphysiological model of lung cancer cachexia to study inflammatory and oxidative muscle injury induced by tumor-derived signals.
  25. J Diabetes Res. 2021 ;2021 5123241
      Insulin resistance is a state of impaired responsiveness to insulin action. This condition not only results in deficient glucose uptake but increases the risk for cardiovascular diseases (CVD), stroke, and obesity. The present work investigates the molecular mechanisms of high carbohydrate and fat diet in inducing prediabetic hyperinsulinemia and effect of exercise on InsR signaling events, muscular AChE, and lactate dehydrogenase activity. Adult male Wistar rats were divided into the control (C) diet group, high-carbohydrate diet (HCD) group, high-fat diet (HFD) group, and HCD and HFD groups with exercise (HCD Ex and HFD Ex, respectively). Acetyl choline esterase activity, lactate dehydrogenase activity, total lactate levels, IRS1 phosphorylations, and Glut4 expression patterns were studied in the muscle tissue among these groups. High carbohydrate and fat feeding led to hyperinsulinemic status with reduced acetylcholine esterase (AChE) activity and impaired phosphorylation of IRS1 along with increased lactate concentrations in the muscle. Exercise significantly upregulated phosphoinositide 3 kinase (PI3K) docking site phosphorylation and downregulated the negative IRS1 phosphorylations thereby increasing the glucose transporter (GLUT) expressions and reducing the lactate accumulation. Also, the levels of second messengers like IP3 and cAMP were increased with exercise. Increased second messenger levels induce calcium release thereby activating the downstream pathway promoting the translocation of GLUT4 to the plasma membrane. Our results showed that the metabolic and signaling pathway dysregulations seen during diet-induced hyperinsulinemia, a metabolic condition seen during the early stages in the development of prediabetes, were improved with vigorous physical exercise. Thus, exercise can be considered as an excellent management approach over drug therapy for diabetes and its complications.
  26. Front Physiol. 2021 ;12 635094
      Background: Power-oriented resistance training (PRT) is one of the most effective exercise programs to counteract neuromuscular and physical function age-related declines. However, the optimal load that maximizes these outcomes or the load-specific adaptations induced on muscle power determinants remain to be better understood. Furthermore, to investigate whether these adaptations are potentially transferred to an untrained limb (i.e., cross-education phenomenon) could be especially relevant during limb-immobilization frequently observed in older people (e.g., after hip fracture).Methods: At least 30 well-functioning older participants (>65 years) will participate in a within-person randomized controlled trial. After an 8-week control period, the effects of two 12-week PRT programs using light vs. heavy loads will be compared using an unilateral exercise model through three study arms (light-load PRT vs. non-exercise; heavy-load PRT vs. non-exercise; and light- vs. heavy- load PRT). Muscle-tendon function, muscle excitation and morphology and physical function will be evaluated to analyze the load-specific effects of PRT in older people. Additionally, the effects of PRT will be examined on a non-exercised contralateral limb.
    Discussion: Tailored exercise programs are largely demanded given their potentially greater efficiency preventing age-related negative consequences, especially during limb-immobilization. This trial will provide evidence supporting the use of light- or heavy-load PRT on older adults depending on individual needs, improving decision making and exercise program efficacy.
    Clinical Trial Registration: NCT03724461 registration data: October 30, 2018.
    Keywords:  aging; force-velocity; intensity; physical function; power training; strength training
  27. BMC Musculoskelet Disord. 2021 Mar 10. 22(1): 262
      BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a patchy and slowly progressive disease of skeletal muscle. For MRI to be a useful biomarker in an FSHD clinical trial, it should reliably detect changes over relatively short time-intervals (~ 1 year). We hypothesized that fatty change over the study course would be most likely in muscles already demonstrating disease progression, and that the degree of MRI burden would be correlated with function.METHODS: We studied 36 patients with FSHD and lower-extremity weakness at baseline. Thirty-two patients returned in our 12-month longitudinal observational study. We analyzed DIXON MRI images of 16 lower-extremity muscles in each patient and compared them to quantitative strength measurement and ambulatory functional outcome measures.
    RESULTS: There was a small shift to higher fat fractions in the summed muscle data for each patient, however individual muscles demonstrated much larger magnitudes of change. The greatest increase in fat fraction was observed in muscles having an intermediate fat replacement at baseline, with minimally (baseline fat fraction < 0.10) or severely (> 0.70) affected muscles less likely to progress. Functional outcome measures did not demonstrate marked change over the interval; however, overall MRI disease burden was correlated with functional outcome measures. Direct comparison of the tibialis anterior (TA) fat fraction and quantitative strength measurement showed a sigmoidal relationship, with steepest drop being when the muscle gets more than ~ 20% fatty replaced.
    CONCLUSIONS: Assessing MRI changes in 16 lower-extremity muscles across 1 year demonstrated that those muscles having an intermediate baseline fat fraction were more likely to progress. Ambulatory functional outcome measures are generally related to overall muscle MRI burden but remain unchanged in the short term. Quantitative strength measurement of the TA showed a steep loss of strength when more fatty infiltration is present suggesting that MRI may be preferable for following incremental change or modulation with drug therapy.
    Keywords:  All neuromuscular disease; Facioscapulohumeral muscular dystrophy (FSHD); MRI; Muscle disease; Outcome measures
  28. Diabetologia. 2021 Mar 12.
      AIMS/HYPOTHESIS: Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells.METHODS: C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT.
    RESULTS: HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1β and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55 pmol ATP/μg RNA) compared with controls (32 ± 10 pmol ATP/μg RNA). ATP release in obese mice fibres was reduced by application of 100 μmol/l oleamide (OLE) and 5 μmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2 U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10 pmol ATP/μg RNA vs 252 ± 37 pmol ATP/μg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300 μmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10 mg/kg each day) for 14 days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10 pmol ATP/μg RNA vs 222 ± 28 pmol ATP/μg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management.
    CONCLUSIONS/INTERPRETATION: In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.
    Keywords:  Carbenoxolone; Extracellular nucleotides; High-fat diet; PANX1; Purinergic signalling; Skeletal muscle
  29. Sports Med. 2021 Mar 09.
      BACKGROUND: Flavonoid polyphenols are bioactive phytochemicals found in fruits and teas among other sources. It has been postulated that foods and supplements containing flavonoid polyphenols may enhance recovery from exercise-induced muscle damage (EIMD) through upregulation of cell signalling stress response pathways, particularly the nuclear factor erythroid 2-related factor 2 (NRF2) pathway.OBJECTIVES: This study aims to investigate the ability of polyphenol treatments containing flavonoids to enhance recovery of skeletal muscle strength, soreness and creatine kinase post EIMD.
    METHODS: Medline (Pubmed), Embase and SPORTdiscus were searched from inception to August 2020 for randomised placebo-controlled trials which assessed the impact of 6 or more days of flavonoid containing polyphenol ingestion on skeletal muscle recovery in the 96-h period following a single bout of EIMD. A total of 2983 studies were screened in duplicate resulting in 26 studies included for analysis. All meta-analyses were undertaken using a random-effects model.
    RESULTS: The pooled results of these meta-analyses show flavonoid polyphenol treatments can enhance recovery of muscle strength by 7.14% (95% CI [5.50-8.78], P < 0.00001) and reduce muscle soreness by 4.12% (95% CI [- 5.82 to - 2.41] P = 0.00001), no change in the recovery of creatine kinase concentrations was observed.
    CONCLUSION: These results indicate that ingestion of polyphenol treatments which contain flavonoids has significant potential to improve recovery of muscular strength and reduce muscle soreness in the 4-day period post EIMD. However, the characterisation of polyphenol dosage and composition of study treatments should be prioritised in future research to facilitate the development of specific guidelines for the inclusion of flavonoid-rich foods in the diet of athletes and active individuals.
  30. Drug Des Devel Ther. 2021 ;15 927-936
      Purpose: To assess the cellular and molecular effects of lidocaine on muscles/myoblasts.Methods: Cultured myogenic precursor (C2C12) cells were treated with varying concentrations of lidocaine.
    Results: Cell viability of C2C12 cells was inhibited by lidocaine in a concentration-dependent manner, with concentrations ≥0.08%, producing a dramatic reduction in cell viability. These ≥0.08% concentrations of lidocaine arrested cell cycles of C2C12 cells in the G0/G1 phase. Moreover, lidocaine inhibited cell migration and myogenic processes in C2C12 cells at low concentrations. Results from QRT-PCR assays revealed that following treatment with lidocaine, Notch1, Notch2, Hes1, Csl and Dll4 all showed higher levels of expression, while no changes were observed in Mmal1, Hey1, Dll1 and Jag1.
    Conclusion: This work provides the first description of the effects of lidocaine upon the regeneration of muscles and maintenance of satellite cells at the cellular and molecular levels. In specific, we found that the Dll4-Notch-Csl-Hes1 axis was up-regulated suggesting that the Notch signaling pathway was involved in producing these effects of lidocaine. These findings provide a new and important foundation for future investigations into the effects of drug therapies in muscle diseases.
    Keywords:  C2C12 cells; Notch signaling pathway; local anesthetics; myogenic differentiation; strabismus
  31. Biosci Biotechnol Biochem. 2021 Feb 01. pii: zbab018. [Epub ahead of print]
      Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs is significantly higher than the PCs in a time-dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.
    Keywords:  2D and 3D culture; C2C12; cell expansion; myogenesis; spheroids
  32. Eur J Transl Myol. 2021 Mar 02.
      Mobility-impaired persons, either very old or younger but suffering with systemic neuromuscular disorders or chronic organ failures, spend small amounts of time for daily physical activity, contributing to aggravate their poor mobility by resting muscle atrophy. Sooner or later the limitations to their mobility enforce them to bed and to more frequent hospitalizations. We include among these patients at risk those who are negative for the SARS-COV-2 infection, but suffering with COVID-19 pandemic syndrome. Beside managements of psychological symptoms, it is mandatory to offer to the last group physical rehabilitation approaches easy to learn and self-managed at home. Inspired by the proven capability to recover skeletal muscle contractility and strength by home-based volitional exercises and functional electrical stimulation, we suggest also for chronic COVID-19 pandemic syndrome a 10-20 min long daily routine of easy and safe physical exercises that can activate, and recover from weakness, the main 400 skeletal muscles used for every-day mobility activities. Persons can do many of them in bed (Full-Body in-Bed Gym), and hospitalized patients can learn this light training before leaving the hospital. It is, indeed, an extension of well-established cardiovascular-respiratory rehabilitation training performed after heavy surgical interventions. Blood pressure readings, monitored before and after daily routine, demonstrate a transient decrease in peripheral resistance due to increased blood flow of many muscles. Continued regularly, Full-Body in-Bed Gym may help maintaining independence of frail people, including those suffering with the COVID-19 pandemic syndrome.
  33. G3 (Bethesda). 2021 Mar 13. pii: jkab047. [Epub ahead of print]
      At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 caused levamisole hypersensitivity and had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. We also examined other genes that resulted in a levamisole hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.
    Keywords:   C. elegans ; epn-1 ; gas-1 ; ATP; GABA; acetylcholine; endocytosis; levamisole
  34. Biochem Biophys Res Commun. 2021 Mar 08. pii: S0006-291X(21)00363-6. [Epub ahead of print]550 184-190
      Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. Here, we investigated the role of linear ubiquitin E3 ligase (LUBEL), a DrosophilaHOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, we propose a new role of linear ubiquitination in proteostasis in the muscle aging.
    Keywords:  Aging; Drosophila; LUBEL; Linear ubiquitination; Muscle; Proteostasis
  35. BMJ Open Sport Exerc Med. 2021 ;7(1): e000876
      Objectives: In this study, we investigated daily fluctuations in molecular (gene expression) and physiological (biomechanical muscle properties) features in human peripheral cells and their correlation with exercise performance.Methods: 21 healthy participants (13 men and 8 women) took part in three test series: for the molecular analysis, 15 participants provided hair, blood or saliva time-course sampling for the rhythmicity analysis of core-clock gene expression via RT-PCR. For the exercise tests, 16 participants conducted strength and endurance exercises at different times of the day (9h, 12h, 15h and 18h). Myotonometry was carried out using a digital palpation device (MyotonPRO), five muscles were measured in 11 participants. A computational analysis was performed to relate core-clock gene expression, resting muscle tone and exercise performance.
    Results: Core-clock genes show daily fluctuations in expression in all biological samples tested for all participants. Exercise performance peaks in the late afternoon (15-18 hours for both men and women) and shows variations in performance, depending on the type of exercise (eg, strength vs endurance). Muscle tone varies across the day and higher muscle tone correlates with better performance. Molecular daily profiles correlate with daily variation in exercise performance.
    Conclusion: Training programmes can profit from these findings to increase efficiency and fine-tune timing of training sessions based on the individual molecular data. Our results can benefit both professional athletes, where a fraction of seconds may allow for a gold medal, and rehabilitation in clinical settings to increase therapy efficacy and reduce recovery times.
    Keywords:  circadian rhythms; exercise performance times; molecular rhythmicity analysis; muscle tone
  36. Hum Mol Genet. 2021 Mar 09. pii: ddab065. [Epub ahead of print]
      Spinal Muscular Atrophy (SMA) is an autosomal recessive degenerative motor neuron disease characterized by symmetrical muscle weakness and atrophy of limb and trunk muscles being the most severe genetic disease in children. In SMA mouse models, motor nerve terminals display neurotransmitter release reduction, endocytosis decrease, and mitochondria alterations. The relationship between these changes is, however, not well understood. In the present study, we investigated whether the endocytosis impairment could be related to the functional alteration of the presynaptic mitochondria during action potential (AP) firing. To this aim, we generated a Synaptophysin-pHluorin (SypHy) transgenic mouse, crossed it with Taiwanese SMA mice, and recorded exo- and endocytosis and mitochondria Ca2+ signaling in real-time at ex vivo motor nerve terminals of Taiwanese-SypHy mice. The experiments were performed at the beginning of the motor symptoms to get an integrated view of the nerve terminal's functional state before degeneration. Our electrophysiological and live imaging results demonstrated that the mitochondria's capacity to increase matrix-free Ca2+ in SMA mice was significantly limited during nerve AP firing, except when the rate of Ca2+ entry to the cytosol was considerably reduced. These results indicate that both the mitochondrial Ca2+ signaling alterations and the secretion machinery defects are significant players in the dysfunction of the presynaptic terminal in SMA.
  37. Nano Lett. 2021 Mar 12.
      Extracellular vesicles (EVs) have emerged as a promising strategy to promote tissue regeneration. However, overcoming the low EV production yield remains a big challenge in translating EV-based therapies to the clinical practice. Current EV production relies heavily on 2D cell culture, which is not only less physiologically relevant to cells but also requires substantial medium and space. In this study, we engineered tissues seeded with stem cells from dental pulp or adipose tissues, or skeletal muscle cells, and significantly enhanced the EV production yield by applying mechanical stimuli, including flow and stretching, in bioreactors. Further mechanistic investigation revealed that this process was mediated by yes-associated protein (YAP) mechanosensitivity. EVs from mechanically stimulated dental pulp stem cells on 3D scaffolds displayed superior capability in inducing axonal sprouting than the 2D counterparts. Our results demonstrate the promise of this strategy to boost EV production and optimize their functional performance toward clinical translation.
    Keywords:  Extracellular vesicles; YAP; biomechanics; mechanosensitivity; tissue engineering
  38. Annu Rev Biophys. 2021 Mar 12.
      The ability of cells to generate mechanical forces, but also to sense, adapt to, and respond to mechanical signals, is crucial for many developmental, postnatal homeostatic, and pathophysiological processes. However, the molecular mechanisms underlying cellular mechanotransduction have remained elusive for many decades, as techniques to visualize and quantify molecular forces across individual proteins in cells were missing. The development of genetically encoded molecular tension sensors now allows the quantification of piconewton-scale forces that act upon distinct molecules in living cells and even whole organisms. In this review, we discuss the physical principles, advantages, and limitations of this increasingly popular method. By highlighting current examples from the literature, we demonstrate how molecular tension sensors can be utilized to obtain access to previously unappreciated biophysical parameters that define the propagation of mechanical forces on molecular scales. We discuss how the methodology can be further developed and provide a perspective on how the technique could be applied to uncover entirely novel aspects of mechanobiology in the future. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 2021. Please see for revised estimates.