bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒08‒08
forty-five papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Front Cell Dev Biol. 2021 ;9 662133
      Background: Desmin is a muscle-specific protein belonging to the intermediate filament family. Desmin mutations are linked to skeletal muscle defects, including inherited myopathies with severe clinical manifestations. The aim of this study was to examine the role of desmin in skeletal muscle remodeling and performance gain induced by muscle mechanical overloading which mimics resistance training. Methods: Plantaris muscles were overloaded by surgical ablation of gastrocnemius and soleus muscles. The functional response of plantaris muscle to mechanical overloading in desmin-deficient mice (DesKO, n = 32) was compared to that of control mice (n = 36) after 7-days or 1-month overloading. To elucidate the molecular mechanisms implicated in the observed partial adaptive response of DesKO muscle, we examined the expression levels of genes involved in muscle growth, myogenesis, inflammation and oxidative energetic metabolism. Moreover, ultrastructure and the proteolysis pathway were explored. Results: Contrary to control, absolute maximal force did not increase in DesKO muscle following 1-month mechanical overloading. Fatigue resistance was also less increased in DesKO as compared to control muscle. Despite impaired functional adaptive response of DesKO mice to mechanical overloading, muscle weight and the number of oxidative MHC2a-positive fibers per cross-section similarly increased in both genotypes after 1-month overloading. However, mechanical overloading-elicited remodeling failed to activate a normal myogenic program after 7-days overloading, resulting in proportionally reduced activation and differentiation of muscle stem cells. Ultrastructural analysis of the plantaris muscle after 1-month overloading revealed muscle fiber damage in DesKO, as indicated by the loss of sarcomere integrity and mitochondrial abnormalities. Moreover, the observed accumulation of autophagosomes and lysosomes in DesKO muscle fibers could indicate a blockage of autophagy. To address this issue, two main proteolysis pathways, the ubiquitin-proteasome system and autophagy, were explored in DesKO and control muscle. Our results suggested an alteration of proteolysis pathways in DesKO muscle in response to mechanical overloading. Conclusion: Taken together, our results show that mechanical overloading increases the negative impact of the lack of desmin on myofibril organization and mitochondria. Furthermore, our results suggest that under these conditions, the repairing activity of autophagy is disturbed. Consequently, force generation is not improved despite muscle growth, suggesting that desmin is required for a complete response to resistance training in skeletal muscle.
    Keywords:  autophagy; cytoskeleton; exercice; intermediate filament; muscle hypertrophy
  2. J Neuromuscul Dis. 2021 Jul 28.
      Adult skeletal muscle is a relatively stable tissue, as the multinucleated muscle fibres contain post-mitotic myonuclei. During early postnatal life, muscle growth occurs by the addition of skeletal muscle stem cells (satellite cells) or their progeny to growing muscle fibres. In Duchenne muscular dystrophy, which we shall use as an example of muscular dystrophies, the muscle fibres lack dystrophin and undergo necrosis. Satellite-cell mediated regeneration occurs, to repair and replace the necrotic muscle fibres, but as the regenerated muscle fibres still lack dystrophin, they undergo further cycles of degeneration and regeneration.AAV gene therapy is a promising approach for treating Duchenne muscular dystrophy. But for a single dose of, for example, AAV coding for dystrophin, to be effective, the treated myonuclei must persist, produce sufficient dystrophin and a sufficient number of nuclei must be targeted. This latter point is crucial as AAV vector remains episomal and does not replicate in dividing cells. Here, we describe and compare the growth of skeletal muscle in rodents and in humans and discuss the evidence that myofibre necrosis and regeneration leads to the loss of viral genomes within skeletal muscle. In addition, muscle growth is expected to lead to the dilution of the transduced nuclei especially in case of very early intervention, but it is not clear if growth could result in insufficient dystrophin to prevent muscle fibre breakdown. This should be the focus of future studies.
    Keywords:  Duchenne muscular dystrophy; Skeletal muscle growth; adeno-associated virus; gene therapy; myonuclei; satellite cells
  3. Elife. 2021 Aug 05. pii: e57356. [Epub ahead of print]10
      Acute skeletal muscle injury is followed by an inflammatory response, removal of damaged tissue, and the generation of new muscle fibers by resident muscle stem cells, a process well characterized in murine injury models. Inflammatory cells are needed to remove the debris at the site of injury and provide signals that are beneficial for repair. However, they also release chemokines, reactive oxygen species as well as enzymes for clearance of damaged cells and fibers, which muscle stem cells have to withstand in order to regenerate the muscle. We show here that MET and CXCR4 cooperate to protect muscle stem cells against the adverse environment encountered during muscle repair. This powerful cyto-protective role was revealed by the genetic ablation of Met and Cxcr4 in muscle stem cells of mice, which resulted in severe apoptosis during early stages of regeneration. TNFα neutralizing antibodies rescued the apoptosis, indicating that TNFα provides crucial cell-death signals during muscle repair that are counteracted by MET and CXCR4. We conclude that muscle stem cells require MET and CXCR4 to protect them against the harsh inflammatory environment encountered in an acute muscle injury.
    Keywords:  developmental biology; mouse
  4. J Cell Mol Med. 2021 Aug 04.
      Myocardin-related transcription factor-A/serum response factor (MRTF-A/SRF), a well-known transcriptional programme, has been proposed to play crucial roles in skeletal muscle development and function. However, whether MRTF-A participates in muscle regeneration and the molecular mechanisms are not completely understood. Here, we show that MRTF-A levels are highly correlated with myogenic genes using a RNA-seq assay, which reveal that MRTF-A knockdown in C2C12 cells significantly reduces PAX7 expression. Subsequent in vitro and in vivo data show that MRTF-A and PAX7 present identical expression patterns during myoblast differentiation and CTX-induced muscle injury and repair. Remarkably, MRTF-A overexpression promotes myoblast proliferation, while inhibiting cell differentiation and the expression of MyoD and MyoG. MRTF-A loss of function produces the opposite effect. Moreover, mice with lentivirus (MRTF-A) injection possesses more PAX7+ satellite cells, but less differentiating MyoD+ and MyoG+ cells, leading subsequently to diminished muscle regeneration. Our mechanistic results reveal that MRTF-A contributes to PAX7-mediated myoblast self-renewal, proliferation, and differentiation by binding to its distal CArG box. Overall, we propose that MRTF-A functions as a novel PAX7 regulator upon myoblast commitment to differentiation, which could provide pathways for dictating muscle stem cell fate and open new avenues to explore stem cell-based therapy for muscle degenerative diseases.
    Keywords:  MRTF-A; PAX7; differentiation; muscle regeneration; transcriptional regulation
  5. Int J Mol Sci. 2021 Jul 30. pii: 8179. [Epub ahead of print]22(15):
      The maintenance of mitochondrial integrity is critical for muscle health. Mitochondria, indeed, play vital roles in a wide range of cellular processes, including energy supply, Ca2+ homeostasis, retrograde signaling, cell death, and many others. All mitochondria-containing cells, including skeletal muscle cells, dispose of several pathways to maintain mitochondrial health, including mitochondrial biogenesis, mitochondrial-derived vesicles, mitochondrial dynamics (fusion and fission process shaping mitochondrial morphology), and mitophagy-the process in charge of the removal of mitochondria though autophagy. The loss of skeletal muscle mass (atrophy) is a major health problem worldwide, especially in older people. Currently, there is no treatment to counteract the progressive decline in skeletal muscle mass and strength that occurs with aging, a process termed sarcopenia. There is increasing data, including our own, suggesting that accumulation of dysfunctional mitochondria contributes to the development of sarcopenia. Impairments in mitochondrial dynamics and mitophagy were recently proposed to contribute to sarcopenia. This review summarizes the current state of knowledge on the role played by mitochondrial dynamics and mitophagy in skeletal muscle health and in the development of sarcopenia. We also highlight recent studies showing that enhancing mitophagy in skeletal muscle is a promising therapeutic target to prevent or even treat skeletal muscle dysfunction in the elderly.
    Keywords:  aging; autophagy; mitochondrial dynamics; mitophagy; sarcopenia; skeletal muscle
  6. J Cachexia Sarcopenia Muscle. 2021 Aug 02.
      BACKGROUND: Type 2 diabetes and obesity are often seen concurrently with skeletal muscle wasting, leading to further derangements in function and metabolism. Muscle wasting remains an unmet need for metabolic disease, and new approaches are warranted. The neuropeptide urocortin 2 (UCN2) and its receptor corticotropin releasing factor receptor 2 (CRHR2) are highly expressed in skeletal muscle and play a role in regulating energy balance, glucose metabolism, and muscle mass. The aim of this study was to investigate the effects of modified UCN2 peptides as a pharmaceutical therapy to counteract the loss of skeletal muscle mass associated with obesity and casting immobilization.METHODS: High-fat-fed mice (C57Bl/6J; 26 weeks old) and ob/ob mice (11 weeks old) were injected daily with a PEGylated (Compound A) and non-PEGylated (Compound B) modified human UCN2 at 0.3 mg/kg subcutaneously for 14 days. A separate group of chow-fed C57Bl/6J mice (12 weeks old) was subjected to hindlimb cast immobilization and, after 1 week, received daily injections with Compound A. In vivo functional tests were performed to measure protein synthesis rates and skeletal muscle function. Ex vivo functional and molecular tests were performed to measure contractile force and signal transduction of catabolic and anabolic pathways in skeletal muscle.
    RESULTS: Skeletal muscles (extensor digitorum longus, soleus, and tibialis anterior) from high-fat-fed mice treated with Compound A were ~14% heavier than muscles from vehicle-treated mice. Chronic treatment with modified UCN2 peptides altered the expression of structural genes and transcription factors in skeletal muscle in high-fat diet-induced obesity including down-regulation of Trim63 and up-regulation of Nr4a2 and Igf1 (P < 0.05 vs. vehicle). Signal transduction via both catabolic and anabolic pathways was increased in tibialis anterior muscle, with increased phosphorylation of ribosomal protein S6 at Ser235/236 , FOXO1 at Ser256 , and ULK1 at Ser317 , suggesting that UCN2 treatment modulates protein synthesis and degradation pathways (P < 0.05 vs. vehicle). Acutely, a single injection of Compound A in drug-naïve mice had no effect on the rate of protein synthesis in skeletal muscle, as measured via the surface sensing of translation method, while the expression of Nr4a3 and Ppargc1a4 was increased (P < 0.05 vs. vehicle). Compound A treatment prevented the loss of force production from disuse due to casting. Compound B treatment increased time to fatigue during ex vivo contractions of fast-twitch extensor digitorum longus muscle. Compound A and B treatment increased lean mass and rates of skeletal muscle protein synthesis in ob/ob mice.
    CONCLUSIONS: Modified human UCN2 is a pharmacological candidate for the prevention of the loss of skeletal muscle mass associated with obesity and immobilization.
    Keywords:  Diabetes; Exercise; Insulin resistance; Muscle wasting; Obesity; Therapy
  7. Semin Cell Dev Biol. 2021 Jul 28. pii: S1084-9521(21)00200-7. [Epub ahead of print]
      While Fibro-Adipogenic Progenitors (FAPs) have been originally identified as muscle-interstitial mesenchymal cells activated in response to muscle injury and endowed with inducible fibrogenic and adipogenic potential, subsequent studies have expanded their phenotypic and functional repertoire and revealed their contribution to skeletal muscle response to a vast range of perturbations. Here we review the emerging contribution of FAPs to skeletal muscle responses to motor neuron injuries and to systemic physiological (e.g., exercise) or pathological metabolic (e.g., diabetes) perturbations. We also provide an initial blueprint of discrete sub-clusters of FAPs that are activated by specific perturbations and discuss their role in muscle adaptation to these conditions.
    Keywords:  Denervation; Diabetes; Exercise; FAPs; Neuromuscular junctions; Skeletal muscle
  8. Gene. 2021 Aug 02. pii: S0378-1119(21)00464-9. [Epub ahead of print] 145869
      Skeletal myoblasts are activated satellite cells capable of proliferation and differentiation. Studies on mammalian myoblast differentiation and myogenesis could be carried out in vitro thanks to the availability of mouse myoblast cell line C2C12. Lacking of muscle cell line hinders the studies of teleost fish myogenesis. Here, we established a continuous skeletal muscle cell line from juvenile rockfish (Sebastes schlegelii) muscle using explant method and subcultured more than 50 passages for over 150 days. Stable expression of myoblast-specific marker, MyoD (myoblast determination protein) and the potential of differentiation into multi-nucleated skeletal myotubes upon induction suggested the cell line were predominately composed of myoblasts. Transcriptome analysis revealed a total of 4,375 genes differentially expressed at four time points after the switch to differentiation medium, which were mainly involved in proliferation and differentiation of myoblasts. KIF22 (kinesin family member 22) and POLA1 (DNA polymerase alpha 1) were identified as the key genes involved in fish myoblast proliferation whereas MYL3 (myosin light chain 3) and TNNT2 (troponin T2) were determined as the crucial genes responsible for differentiation. In all, the continuous myoblasts cultured in this study provided a cell platform for future studies on marine fish myoblast differentiation and myogenesis. The molecular process of myoblast differentiation revealed in this study will open a window into the understanding of indeterminate muscle growth of large teleost.
    Keywords:  Sebastes schlegelii; muscle growth; myoblast culture, myoblast differentiation; teleost; transcriptome analysis
  9. Front Physiol. 2021 ;12 696018
      Skeletal muscle is fundamentally important for quality of life. Deterioration of skeletal muscle, such as that observed with advancing age, chronic disease, and dystrophies, is associated with metabolic and functional decline. Muscle stem/progenitor cells promote the maintenance of skeletal muscle composition (balance of muscle mass, fat, and fibrotic tissues) and are essential for the regenerative response to skeletal muscle damage. It is increasing recognized that nutrient and metabolic determinants of stem/progenitor cell function exist and are potential therapeutic targets to improve regenerative outcomes and muscle health. This review will focus on current understanding as well as key gaps in knowledge and challenges around identifying and understanding nutrient and metabolic determinants of skeletal muscle regeneration.
    Keywords:  metabolism; nutrients; regeneration; skeletal muscle; tissue recovery
  10. Curr Opin Cell Biol. 2021 Aug 02. pii: S0955-0674(21)00070-3. [Epub ahead of print]73 78-83
      Muscle stem cells (also called satellite cells or SCs) rely on their local niche for regulatory signals during homeostasis and regeneration. While a number of cell types communicate indirectly through secreted factors, here we focus on the significance of direct contact between SCs and their neighbors. During quiescence, SCs reside under a basal lamina and receive quiescence-promoting signals from their adjacent skeletal myofibers. Upon injury, the composition of the niche changes substantially, enabling the formation of new contacts that mediate proliferation, self-renewal, and differentiation. In this review, we summarize the latest work in understanding cell-cell contact within the satellite cell niche and highlight areas of open questions for future studies.
    Keywords:  Cadherin; Cell adhesion; Muscle stem cell; Notch pathway; Satellite cell; Skeletal muscle; Stem cell niche
  11. Int J Mol Sci. 2021 Jul 28. pii: 8058. [Epub ahead of print]22(15):
      The neuromuscular junction (NMJ) is a specialized synapse that bridges the motor neuron and the skeletal muscle fiber and is crucial for conversion of electrical impulses originating in the motor neuron to action potentials in the muscle fiber. The consideration of contributing factors to skeletal muscle injury, muscular dystrophy and sarcopenia cannot be restricted only to processes intrinsic to the muscle, as data show that these conditions incur denervation-like findings, such as fragmented NMJ morphology and corresponding functional changes in neuromuscular transmission. Primary defects in the NMJ also influence functional loss in motor neuron disease, congenital myasthenic syndromes and myasthenia gravis, resulting in skeletal muscle weakness and heightened fatigue. Such findings underscore the role that the NMJ plays in neuromuscular performance. Regardless of cause or effect, functional denervation is now an accepted consequence of sarcopenia and muscle disease. In this short review, we provide an overview of the pathologic etiology, symptoms, and therapeutic strategies related to the NMJ. In particular, we examine the role of the NMJ as a disease modifier and a potential therapeutic target in neuromuscular injury and disease.
    Keywords:  NMJ; exercise; muscular dystrophy; myasthenia gravis; sarcopenia
  12. Int J Mol Sci. 2021 Jul 27. pii: 8016. [Epub ahead of print]22(15):
      Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder, affecting one in 3500 to 5000 boys worldwide. The NO-sGC-cGMP pathway plays an important role in skeletal muscle function, primarily by improving blood flow and oxygen supply to the muscles during exercise. In fact, PDE5 inhibitors have previously been investigated as a potential therapy for DMD, however, a large-scale Phase III clinical trial did not meet its primary endpoint. Since the efficacy of PDE5i is dependent on sufficient endogenous NO production, which might be impaired in DMD, we investigated if NO-independent sGC stimulators, could have therapeutic benefits in a mouse model of DMD. Male mdx/mTRG2 mice aged six weeks were given food supplemented with the sGC stimulator, BAY-747 (150 mg/kg of food) or food alone (untreated) ad libitum for 16 weeks. Untreated C57BL6/J mice were used as wild type (WT) controls. Assessments of the four-limb hang, grip strength, running wheel and serum creatine kinase (CK) levels showed that mdx/mTRG2 mice had significantly reduced skeletal muscle function and severe muscle damage compared to WT mice. Treatment with BAY-747 improved grip strength and running speed, and these mice also had reduced CK levels compared to untreated mdx/mTRG2 mice. We also observed increased inflammation and fibrosis in the skeletal muscle of mdx/mTRG2 mice compared to WT. While gene expression of pro-inflammatory cytokines and some pro-fibrotic markers in the skeletal muscle was reduced following BAY-747 treatment, there was no reduction in infiltration of myeloid immune cells nor collagen deposition. In conclusion, treatment with BAY-747 significantly improves several functional and pathological parameters of the skeletal muscle in mdx/mTRG2 mice. However, the effect size was moderate and therefore, more studies are needed to fully understand the potential treatment benefit of sGC stimulators in DMD.
    Keywords:  duchenne muscular dystrophy; fibrosis; inflammation; mdx/mTRG2 mice; sGC stimulator; skeletal muscle damage; skeletal muscle function
  13. Int J Mol Sci. 2021 Jul 27. pii: 8015. [Epub ahead of print]22(15):
      Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.
    Keywords:  denervation; innervation; muscle-specific kinase (MuSK); neuromuscular junction; skeletal muscle; spinal muscular atrophy
  14. Cells. 2021 Jul 15. pii: 1786. [Epub ahead of print]10(7):
      It has been demonstrated that inhibiting Notch signaling through γ-secretase inhibitor (GSI) treatment increases myogenesis, AKT/mTOR signaling, and muscle protein synthesis (MPS) in C2C12 myotubes. The purpose of this study was to determine if GSI-mediated effects on myogenesis and MPS are dependent on AKT/mTOR signaling. C2C12 cells were assessed for indices of myotube formation, anabolic signaling, and MPS following GSI treatment in combination with rapamycin and API-1, inhibitors of mTOR and AKT, respectively. GSI treatment increased several indices of myotube fusion and MPS in C2C12 myotubes. GSI-mediated effects on myotube formation and fusion were completely negated by treatment with rapamycin and API-1. Meanwhile, GSI treatment was able to rescue MPS in C2C12 myotubes exposed to rapamycin or rapamycin combined with API-1. Examination of protein expression revealed that GSI treatment was able to rescue pGSK3β Ser9 despite AKT inhibition by API-1. These findings demonstrate that GSI treatment is able to rescue MPS independent of AKT/mTOR signaling, possibly via GSK3β modulation.
    Keywords:  AKT; GSI; mTOR; muscle protein synthesis
  15. Free Radic Biol Med. 2021 Aug 02. pii: S0891-5849(21)00423-8. [Epub ahead of print]
      High altitude exposure leads to compromised physical performance with considerable weight loss. The major stressor at high altitude is hypobaric hypoxia which leads to disturbance in redox homeostasis. Oxidative stress is a well-known trigger for many high altitude illnesses and regulates several key signaling pathways under stressful conditions. Altered redox homeostasis is considered the prime culprit of high altitude linked skeletal muscle atrophy. Hypobaric hypoxia disturbs redox homeostasis through increased RONS production and compromised antioxidant system. Increased RONS disturbs the cellular homeostasis via multiple ways such as inflammation generation, altered protein anabolic pathways, redox remodeling of RyR1 that contributed to dysregulated calcium homeostasis, enhanced protein degradation pathways via activation calcium-regulated protein, calpain, and apoptosis. Ultimately, all the cellular signaling pathways aggregately result in skeletal muscle atrophy. Dietary supplementation of phytochemicals could become a safe and effective intervention to ameliorate skeletal muscle atrophy and enhance the physical performance of the personnel who are staying at high altitude regions. The present evidence-based review explores few dietary supplementations which regulate several signaling mechanisms and ameliorate hypobaric hypoxia induced muscle atrophy and enhances physical performance. However, a clinical research trial is required to establish proof-of-concept.
    Keywords:  Diet; High altitude; Hypobaric hypoxia; Muscle; Redox homeostasis
  16. FASEB J. 2021 Sep;35(9): e21830
      Muscle disuse leads to a rapid decline in muscle mass, with reduced muscle protein synthesis (MPS) considered the primary physiological mechanism. Here, we employed a systems biology approach to uncover molecular networks and key molecular candidates that quantitatively link to the degree of muscle atrophy and/or extent of decline in MPS during short-term disuse in humans. After consuming a bolus dose of deuterium oxide (D2 O; 3 ), eight healthy males (22 ± 2 years) underwent 4 days of unilateral lower-limb immobilization. Bilateral muscle biopsies were obtained post-intervention for RNA sequencing and D2 O-derived measurement of MPS, with thigh lean mass quantified using dual-energy X-ray absorptiometry. Application of weighted gene co-expression network analysis identified 15 distinct gene clusters ("modules") with an expression profile regulated by disuse and/or quantitatively connected to disuse-induced muscle mass or MPS changes. Module scans for candidate targets established an experimentally tractable set of candidate regulatory molecules (242 hub genes, 31 transcriptional regulators) associated with disuse-induced maladaptation, many themselves potently tied to disuse-induced reductions in muscle mass and/or MPS and, therefore, strong physiologically relevant candidates. Notably, we implicate a putative role for muscle protein breakdown-related molecular networks in impairing MPS during short-term disuse, and further establish DEPTOR (a potent mTOR inhibitor) as a critical mechanistic candidate of disuse driven MPS suppression in humans. Overall, these findings offer a strong benchmark for accelerating mechanistic understanding of short-term muscle disuse atrophy that may help expedite development of therapeutic interventions.
    Keywords:  atrophy; disuse; gene network analysis; muscle protein synthesis; skeletal muscle
  17. Gen Physiol Biophys. 2021 Jul;40(4): 307-315
      Skeletal muscle secrets several bioactive molecules known as myokines. Interleukin-6 (IL-6) has been described as a myokine secreted in response to skeletal muscle injury as well as to macrophage invasion in inflammation. To our knowledge no connection between macrophages and skeletal muscle regarding IL-6 secretion has been described so far. Here we report that co-culturing of C2C12 cells with RAW macrophages enhances IL-6 secretion of the cells cultured together. However, this is not seen in cross-feeding experiments, where culture medium of RAW macrophage culture is used as the culture medium of C2C12 cells or vice versa. Pravastatin, known to induce myopathy, also stimulates IL-6 production in monocultured C2C12 cells and elevates IL-6 concentration in the culture medium of the co-cultures. These results indicate an intricate interaction between skeletal muscle and macrophages in inflammation related to IL-6 production.
  18. Pharmacol Res. 2021 Aug 02. pii: S1043-6618(21)00382-0. [Epub ahead of print] 105798
      Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus - SOL - muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.
    Keywords:  L-Alanine; L-Alanyl-L-Alanine; branched-chain amino acids; dietary supplements; hind limb unloading; skeletal muscle atrophy
  19. Cells. 2021 Jul 18. pii: 1816. [Epub ahead of print]10(7):
      IL-6 is a pleiotropic cytokine that can exert different and opposite effects. The muscle-induced and transient expression of IL-6 can act in an autocrine or paracrine manner, stimulating anabolic pathways associated with muscle growth, myogenesis, and with regulation of energy metabolism. In contrast, under pathologic conditions, including muscular dystrophy, cancer associated cachexia, aging, chronic inflammatory diseases, and other pathologies, the plasma levels of IL-6 significantly increase, promoting muscle wasting. Nevertheless, the specific physio-pathological role exerted by IL-6 in the maintenance of differentiated phenotype remains to be addressed. The purpose of this study was to define the role of increased plasma levels of IL-6 on muscle homeostasis and the mechanisms contributing to muscle loss. Here, we reported that increased plasma levels of IL-6 promote alteration in muscle growth at early stage of postnatal life and induce muscle wasting by triggering a shift of the slow-twitch fibers toward a more sensitive fast fiber phenotype. These findings unveil a role for IL-6 as a potential biomarker of stunted growth and skeletal muscle wasting.
    Keywords:  PGC-1α; interleukin-6; muscle atrophy; muscle growth; skeletal muscle
  20. Sci Rep. 2021 Aug 05. 11(1): 15865
      Muscular dystrophies are disorders characterized by progressive muscle loss and weakness that are both genotypically and phenotypically heterogenous. Progression of muscle disease arises from impaired regeneration, plasma membrane instability, defective membrane repair, and calcium mishandling. The ferlin protein family, including dysferlin and myoferlin, are calcium-binding, membrane-associated proteins that regulate membrane fusion, trafficking, and tubule formation. Mice lacking dysferlin (Dysf), myoferlin (Myof), and both dysferlin and myoferlin (Fer) on an isogenic inbred 129 background were previously demonstrated that loss of both dysferlin and myoferlin resulted in more severe muscle disease than loss of either gene alone. Furthermore, Fer mice had disordered triad organization with visibly malformed transverse tubules and sarcoplasmic reticulum, suggesting distinct roles of dysferlin and myoferlin. To assess the physiological role of disorganized triads, we now assessed excitation contraction (EC) coupling in these models. We identified differential abnormalities in EC coupling and ryanodine receptor disruption in flexor digitorum brevis myofibers isolated from ferlin mutant mice. We found that loss of dysferlin alone preserved sensitivity for EC coupling and was associated with larger ryanodine receptor clusters compared to wildtype myofibers. Loss of myoferlin alone or together with a loss of dysferlin reduced sensitivity for EC coupling, and produced disorganized and smaller ryanodine receptor cluster size compared to wildtype myofibers. These data reveal impaired EC coupling in Myof and Fer myofibers and slightly potentiated EC coupling in Dysf myofibers. Despite high homology, dysferlin and myoferlin have differential roles in regulating sarcotubular formation and maintenance resulting in unique impairments in calcium handling properties.
  21. J Biol Chem. 2021 Aug 02. pii: S0021-9258(21)00842-5. [Epub ahead of print] 101040
      Ryanodine receptor type 1 (RyR1) releases Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca2+, caffeine, and ryanodine. Cryo-electron microscopy identified binding sites for the three co-activators Ca2+, ATP, and caffeine. However, the mechanism of co-regulation and synergy between these activators remains to be determined. Here we used [3H]ryanodine ligand binding assays and molecular dynamics simulations to test the hypothesis that both the ATP and caffeine binding sites communicate with the Ca2+ binding site to sensitize RyR1 to Ca2+. We report that either AMPPCP, a nonhydrolyzable ATP analog, or caffeine can activate RyR1 in the absence or presence of Ca2+. However, enhanced RyR1 activation occurred in the presence of Ca2+, AMPPCP, and caffeine. In the absence of Ca2+, Na+ inhibited [3H]ryanodine binding without impairing RyR1 activation by AMPPCP and caffeine. Computational analysis suggested that Ca2+, ATP, and caffeine binding sites modulate RyR1 protein stability through interactions with the carboxy-terminal domain and other domains in the activation core. In the presence of ATP and caffeine but absence of Ca2+, Na+ is predicted to inhibit RyR1 by interacting with the Ca2+ binding site. Our data suggested that ATP and caffeine binding affected the conformation of the Ca2+ binding site, and conversely Ca2+ binding affected the conformation of the ATP and caffeine binding sites. We conclude that Ca2+, ATP, and caffeine regulate RyR1 through a network of allosteric interactions involving the Ca2+, ATP, and caffeine binding sites.
    Keywords:  AMPPCP; ATP; allosteric communications; caffeine; calcium; molecular dynamics simulations; ryanodine receptor; sarcoplasmic reticulum (SR); skeletal muscle
  22. Int J Mol Sci. 2021 Jul 22. pii: 7828. [Epub ahead of print]22(15):
      Glucocorticoids provide indispensable anti-inflammatory therapies. However, metabolic adverse effects including muscle wasting restrict their use. The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) modulates peripheral glucocorticoid responses through pre-receptor metabolism. This study investigates how 11β-HSD1 influences skeletal muscle responses to glucocorticoid therapy for chronic inflammation. We assessed human skeletal muscle biopsies from patients with rheumatoid arthritis and osteoarthritis for 11β-HSD1 activity ex vivo. Using the TNF-α-transgenic mouse model (TNF-tg) of chronic inflammation, we examined the effects of corticosterone treatment and 11β-HSD1 global knock-out (11βKO) on skeletal muscle, measuring anti-inflammatory gene expression, muscle weights, fiber size distribution, and catabolic pathways. Muscle 11β-HSD1 activity was elevated in patients with rheumatoid arthritis and correlated with inflammation markers. In murine skeletal muscle, glucocorticoid administration suppressed IL6 expression in TNF-tg mice but not in TNF-tg11βKO mice. TNF-tg mice exhibited reductions in muscle weight and fiber size with glucocorticoid therapy. In contrast, TNF-tg11βKO mice were protected against glucocorticoid-induced muscle atrophy. Glucocorticoid-mediated activation of catabolic mediators (FoxO1, Trim63) was also diminished in TNF-tg11βKO compared to TNF-tg mice. In summary, 11β-HSD1 knock-out prevents muscle atrophy associated with glucocorticoid therapy in a model of chronic inflammation. Targeting 11β-HSD1 may offer a strategy to refine the safety of glucocorticoids.
    Keywords:  11beta hydroxysteroid dehydrogenase type 1; adverse effects; inflammation; myopathy; rheumatoid arthritis; sarcopenia; steroids
  23. Exp Physiol. 2021 Aug 01.
      NEW FINDINGS: What is the central question of this study? What is the impact of stress-induced premature senescence on skeletal muscle myoblast-derived extracellular vesicles and myoblast-endothelial cell crosstalk? What is the main finding and its importance? Hydrogen peroxide treatment of human myoblasts induced stress-induced premature senescence (SIPS) and increased the release of exosome-sized extracellular vesicles (30-150 nm in size) 5-fold compared to untreated controls. Treatment of SIPS myoblast-derived EVs on endothelial cells increased senescent markers and decreased proliferation. Gene expression analysis of SIPS myoblast-derived EVs revealed a 4-fold increase in senescent factor transforming growth factor-β. These results highlight potential mechanisms by which senescence imparts deleterious effects on the cellular microenvironment.ABSTRACT: Cellular senescence contributes to numerous diseases through the release of pro-inflammatory factors as part of the senescence-associated secretory phenotype (SASP). In skeletal muscle, resident muscle progenitor cells (satellite cells) express markers of senescence with advancing age and in response to various pathologies, which contributes to reduced regenerative capacities in vitro. Satellite cells regulate their microenvironment in part through the release of extracellular vesicles (EVs), but the effect of senescence on EV signaling is unknown. Primary human myoblasts were isolated following biopsies of the vastus lateralis from young healthy subjects. Hydrogen peroxide (H2 O2 ) treatment was used to achieve stress-induced premature senescence (SIPS) of myoblasts. Extracellular vesicles secreted by myoblasts with and without H2 O2 treatment were isolated, analyzed, and used to treat human umbilical vein endothelial cells (HUVECs) to assess senescent and angiogenic impact. H2 O2 treatment of primary human myoblasts in vitro increased markers of senescence (β-Galactosidase and p21Cip1 ), decreased proliferation, and increased exosome-like EV (30-150 nm) release approximately 5-fold. In HUVECs, EV treatment from H2 O2 treated myoblasts increased markers of senescence (β-Galactosidase and TGF-β), decreased proliferation, and impaired HUVEC tube formation. Analysis of H2 O2 treated myoblast-derived EV mRNA revealed a nearly 4-fold increase in TGF-β expression. Our novel results highlight the impact of SIPS on myoblast communication and identify a VasoMyo Crosstalk (VMC) by which SIPS myoblast-derived EVs impair endothelial cell function in vitro. This article is protected by copyright. All rights reserved.
    Keywords:  aging; angiogenesis; endothelial cells; extracellular vesicles; satellite cells; senescence
  24. Biomedicines. 2021 Jul 13. pii: 807. [Epub ahead of print]9(7):
      Skeletal muscle atrophy, resulting from states of hypokinesis or immobilization, leads to morphological, metabolic, and functional changes within the muscle tissue, a large variety of which are supported by the stromal cells populating the interstitium. Telocytes represent a recently discovered population of stromal cells, which has been increasingly identified in several human organs and appears to participate in sustaining cross-talk, promoting regenerative mechanisms and supporting differentiation of local stem cell niche. The aim of this morphologic study was to investigate the presence of Telocytes in the tibialis anterior muscle of healthy rats undergoing an endurance training protocol for either 4 weeks or 16 weeks compared to sedentary rats. Histomorphometric analysis of muscle fibers diameter revealed muscle atrophy in sedentary rats. Telocytes were identified by double-positive immunofluorescence staining for CD34/CD117 and CD34/vimentin. The results showed that Telocytes were significantly reduced in sedentary rats at 16 weeks, while rats subjected to regular exercise maintained a stable Telocytes population after 16 weeks. Understanding of the relationship between Telocytes and exercise offers new chances in the field of regenerative medicine, suggesting possible triggers for Telocytes in sarcopenia and other musculoskeletal disorders, promoting adapted physical activity and rehabilitation programmes in clinical practice.
    Keywords:  CD117; CD34; exercise; sedentary behavior; skeletal muscle; stem cell niche; telocytes; vimentin
  25. J Physiol. 2021 Aug 02.
    Keywords:  circadian rhythm; exercise; skeletal muscle
  26. J Clin Invest. 2021 Aug 03. pii: 146415. [Epub ahead of print]
      Decreased skeletal muscle strength and mitochondrial dysfunction are characteristic of diabetes. Action of insulin and IGF-1 through insulin receptor (IR) and IGF-1 receptor (IGF1R) maintain muscle mass via suppression of FoxOs, but whether FoxO activation coordinates atrophy in concert with mitochondrial dysfunction is unknown. We show that mitochondrial respiration and complex-I activity were decreased in streptozotocin (STZ) diabetic muscle, but these defects were reversed following muscle-specific FoxO1/3/4 triple knockout in STZ-FoxO TKO. In the absence of systemic glucose or lipid abnormalities, muscle-specific IR knockout (M-IR-/-) or combined IR/IGF1R knockout (MIGIRKO) impaired mitochondrial respiration, decreased ATP production, and increased ROS. These mitochondrial abnormalities were not present in muscle-specific IR/IGF1R and FoxO1/3/4 quintuple knockout mice (M-QKO). Acute tamoxifen-inducible deletion of IR/IGF1R also decreased muscle pyruvate respiration, complex-I activity, and supercomplex assembly. Although autophagy was increased when IR/IGF1R were deleted in muscle, mitophagy was not increased. Mechanistically, RNA-seq revealed that complex-I core subunits were decreased in STZ-diabetic and MIGIRKO muscle, and these changes were not present with FoxO knockout in STZ-FoxO TKO and M-QKO. Thus, insulin-deficient diabetes or loss of insulin/IGF-1 action in muscle decreases complex-I driven mitochondrial respiration and supercomplex assembly, in part by FoxO-mediated repression of Complex-I subunit expression.
    Keywords:  Endocrinology; Insulin; Mitochondria; Muscle
  27. Cells. 2021 Jul 15. pii: 1791. [Epub ahead of print]10(7):
      (1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10-5 M in SUR2wt/AV and 8.6 ± 0.4 × 10-6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] "knock-in" mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.
    Keywords:  ATP-sensitive potassium channel; Cantu syndrome; glibenclamide; histopathology; patch-clamp; rare disease; skeletal muscle
  28. Biology (Basel). 2021 Jul 20. pii: 686. [Epub ahead of print]10(7):
      Myocilin (MYOC) is a glycoprotein encoded by a gene associated with glaucoma pathology. In addition to the eyes, it also expresses at high transcription levels in the heart and skeletal muscle. MYOC affects the formation of the murine gastrocnemius muscle and is associated with the differentiation of mouse osteoblasts, but its role in the differentiation of C2C12 cells has not yet been reported. Here, MYOC expression was found to increase gradually during the differentiation of C2C12 cells. Overexpression of MYOC resulted in enhanced differentiation of C2C12 cells while its inhibition caused reduced differentiation. Furthermore, immunoprecipitation indicated that MYOC binds to Caveolin-1 (CAV1), a protein that influences the TGF-β pathway. Laser confocal microscopy also revealed the common sites of action of the two during the differentiation of C2C12 cells. Additionally, CAV1 was upregulated significantly as C2C12 cells differentiated, with CAV1 able to influence the differentiation of the cells. Furthermore, the Western blotting analysis demonstrated that the expression of MYOC affected the TGF-β pathway. Finally, MYOC was overexpressed while CAV1 was inhibited. The results indicate that reduced CAV1 expression blocked the promotion of C2C12 cell differentiation by MYOC. In conclusion, the results demonstrated that MYOC regulates TGF-β by influencing CAV1 to promote the differentiation of C2C12 cells.
    Keywords:  C2C12; CAV1; MYOC; TGF-β; differentiation
  29. J Biol Chem. 2021 Jul 31. pii: S0021-9258(21)00825-5. [Epub ahead of print] 101023
      Ammonia is a cytotoxic molecule generated during normal cellular functions. Dysregulated ammonia metabolism, which is evident in many chronic diseases such as liver cirrhosis, heart failure and chronic obstructive pulmonary disease, initiates a hyperammonemic stress response (HASR) in tissues including skeletal muscle and in myotubes. Perturbations in levels of specific regulatory molecules have been reported, but the global responses to hyperammonemia are unclear. In this study we used a multiomics approach to vertically integrate unbiased data generated using transposase-accessible chromatin sequencing (ATACseq), RNA sequencing (RNAseq), and proteomics. We then horizontally integrated these data across different models of hyperammonemia, including myotubes and mouse and human muscle tissues. Changes in chromatin accessibility and/or expression of genes resulted in distinct clusters of temporal molecular changes including transient, persistent, and delayed responses during hyperammonemia in myotubes. Known responses to hyperammonemia, including mitochondrial and oxidative dysfunction, protein homeostasis disruption, and oxidative stress pathway activation were enriched in our datasets. During hyperammonemia, pathways that impact skeletal muscle structure and function that were consistently enriched were those that contribute to mitochondrial dysfunction, oxidative stress, and senescence. We made several novel observations, including an enrichment in antiapoptotic Bcl2 family protein expression, increased calcium flux, and increased protein glycosylation in myotubes and muscle tissue upon hyperammonemia. Critical molecules in these pathways were validated experimentally. Human skeletal muscle from patients with cirrhosis displayed similar responses, establishing translational relevance. These data can be used to identify complex molecular interactions during adaptive and maladaptive responses during the cellular stress response to hyperammonemia.
    Keywords:  bioinformatics; glycosylation; hypoxia-inducible factor (HIF); senescence; skeletal muscle metabolism
  30. Int J Mol Sci. 2021 Jul 26. pii: 7958. [Epub ahead of print]22(15):
      Biological aging research is expected to reveal modifiable molecular mechanisms that can be harnessed to slow or possibly reverse unhealthy trajectories. However, there is first an urgent need to define consensus molecular markers of healthy and unhealthy aging. Established aging hallmarks are all linked to metabolism, and a 'rewired' metabolic circuitry has been shown to accelerate or delay biological aging. To identify metabolic signatures distinguishing healthy from unhealthy aging trajectories, we performed nontargeted metabolomics on skeletal muscles from 2-month-old and 21-month-old mice, and after dietary and lifestyle interventions known to impact biological aging. We hypothesized that common metabolic signatures would highlight specific pathways and processes promoting healthy aging, while revealing the molecular underpinnings of unhealthy aging. Here, we report 50 metabolites that commonly distinguished aging trajectories in all cohorts, including 18 commonly reduced under unhealthy aging and 32 increased. We stratified these metabolites according to known relationships with various aging hallmarks and found the greatest associations with oxidative stress and nutrient sensing. Collectively, our data suggest interventions aimed at maintaining skeletal muscle arginine and lysine may be useful therapeutic strategies to minimize biological aging and maintain skeletal muscle health, function, and regenerative capacity in old age.
    Keywords:  aging; diet; exercise; lifestyle; metabolic signatures; metabolomics; skeletal muscle
  31. J Biosci Bioeng. 2021 Aug 01. pii: S1389-1723(21)00179-1. [Epub ahead of print]
      Electric pulse-stimulated C2C12 myotubes are gaining interest in the field of muscle physiology and biotechnology because electric pulse stimulation (EPS) enhances sarcomere structure development and active tension generation capability. Recently, we found that termination of EPS results in the rapid loss of active tension generation accompanied by disassembly of the sarcomere structure, which may represent an in vitro muscle atrophy model. To elucidate the molecular mechanism underlying this rapid loss of active tension generation and sarcomere structure disassembly after termination of EPS, we performed transcriptomic analysis using microarray. After termination of EPS, 74 genes were upregulated and 120 genes were downregulated after 30 min; however, atrophy-related genes were not found among these genes. To further assess the effect of EPS on gene expression, we re-applied EPS after its termination for 8 h and searched for genes whose expression was reversed. Four genes were upregulated by termination of EPS and downregulated by the re-application of EPS, whereas two genes were downregulated by termination of EPS and upregulated by the re-application of EPS. Although none of these genes were atrophy- or hypertrophy-related, the results presented in this study will contribute to the understanding of gene expression changes that mediate rapid loss of active tension generation and sarcomere structure disassembly following termination of EPS in C2C12 myotubes.
    Keywords:  Atrophy; Differentiation; Microarray; Myogenesis; Skeletal muscle
  32. Sci Transl Med. 2021 Aug 04. pii: eaay9592. [Epub ahead of print]13(605):
      Most patients with advanced solid cancers exhibit features of cachexia, a debilitating syndrome characterized by progressive loss of skeletal muscle mass and strength. Because the underlying mechanisms of this multifactorial syndrome are incompletely defined, effective therapeutics have yet to be developed. Here, we show that diminished bone morphogenetic protein (BMP) signaling is observed early in the onset of skeletal muscle wasting associated with cancer cachexia in mouse models and in patients with cancer. Cancer-mediated factors including Activin A and IL-6 trigger the expression of the BMP inhibitor Noggin in muscle, which blocks the actions of BMPs on muscle fibers and motor nerves, subsequently causing disruption of the neuromuscular junction (NMJ), denervation, and muscle wasting. Increasing BMP signaling in the muscles of tumor-bearing mice by gene delivery or pharmacological means can prevent muscle wasting and preserve measures of NMJ function. The data identify perturbed BMP signaling and denervation of muscle fibers as important pathogenic mechanisms of muscle wasting associated with tumor growth. Collectively, these findings present interventions that promote BMP-mediated signaling as an attractive strategy to counteract the loss of functional musculature in patients with cancer.
  33. J Cachexia Sarcopenia Muscle. 2021 Aug 01.
      BACKGROUND: Hyperphosphatemia has been related to the development of sarcopenia in aging mice. We describe the intracellular mechanisms involved in the impairment of the myogenic differentiation promoted by hyperphosphatemia and analyse these mechanisms in the muscle from older mice.METHODS: C2 C12 cells were grown in 2% horse serum in order to promote myogenic differentiation, in the presence or absence of 10 mM beta-glycerophosphate (BGP) for 7 days. Troponin T, paired box 7 (Pax-7), myogenic factor 5 (Myf5), myogenic differentiation 1 (MyoD), myogenin (MyoG), myocyte enhancer factor 2 (MEF2C), P300/CBP-associated factor (PCAF), histone deacetylase 1 (HDAC1), fibronectin, vimentin, and collagen I were analysed at 48, 72, and 168 h, by western blotting or by immunofluorescence staining visualized by confocal microscopy. Studies in mice were performed in 5- and 24-month-old C57BL6 mice. Three months before sacrifice, 21-month-old mice were fed with a standard diet or a low phosphate diet, containing 0.6% or 0.2% phosphate, respectively. Serum phosphate concentration was assessed by a colorimetric method and forelimb strength by a grip test. Fibrosis was observed in the tibialis anterior muscle by Sirius Red staining. In gastrocnemius muscle, MyoG, MEF2C, and fibronectin expressions were analysed by western blotting.
    RESULTS: Cells differentiated in the presence of BGP showed near five times less expression of troponin T and kept higher levels of Pax-7 than control cells indicating a reduced myogenic differentiation. BGP reduced Myf5 about 50% and diminished MyoD transcriptional activity by increasing the expression of HDAC1 and reducing the expression of PCAF. Consequently, BGP reduced to 50% the expression of MyoG and MEF2C. A significant increase in the expression of fibrosis markers as collagen I, vimentin, and fibronectin was found in cells treated with BGP. In mice, serum phosphate (17.24 ± 0.77 mg/dL young; 23.23 ± 0.81 mg/dL old; 19.09 ± 0.75 mg/dL old with low phosphate diet) correlates negatively (r = -0.515, P = 0.001) with the muscular strength (3.13 ± 0.07 gf/g young; 1.70 ± 0.12 gf/g old; 2.10 ± 0.09 gf/g old with low phosphate diet) and with the expression of MyoG (r = -0.535, P = 0.007) and positively with the expression of fibronectin (r = 0.503, P = 0.001) in gastrocnemius muscle. The tibialis anterior muscle from old mice showed muscular fibrosis. Older mice fed with a low phosphate diet showed improved muscular parameters relative to control mice of similar age.
    CONCLUSIONS: Hyperphosphatemia impairs myogenic differentiation, by inhibiting the transcriptional activity of MyoD, and enhances the expression of fibrotic genes in cultured myoblasts. Experiments carried out in older mice demonstrate a close relationship between age-related hyperphosphatemia and the decrease in the expression of myogenic factors and the increase in factors related to muscle fibrosis.
    Keywords:  Hyperphosphatemia; Muscular fibrosis; Myogenic differentiation; Skeletal muscle
  34. Cells. 2021 Jul 08. pii: 1730. [Epub ahead of print]10(7):
      Store-operated Ca2+ entry (SOCE) is a ubiquitous mechanism regulating extracellular Ca2+ entry to control a multitude of Ca2+-dependent signaling pathways and cellular processes. SOCE relies on the concerted activity of the reticular Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel ORAI1, and dysfunctions of these key factors result in human pathologies. STIM1 and ORAI1 gain-of-function (GoF) mutations induce excessive Ca2+ influx through SOCE over-activation, and cause tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK), two overlapping disorders characterized by muscle weakness and additional multi-systemic signs affecting growth, platelets, spleen, skin, and intellectual abilities. In order to investigate the pathophysiological effect of overactive SOCE on muscle function and structure, we combined transcriptomics with morphological and functional studies on a TAM/STRMK mouse model. Muscles from Stim1R304W/+ mice displayed aberrant expression profiles of genes implicated in Ca2+ handling and excitation-contraction coupling (ECC), and in vivo investigations evidenced delayed muscle contraction and relaxation kinetics. We also identified signs of reticular stress and abnormal mitochondrial activity, and histological and respirometric analyses on muscle samples revealed enhanced myofiber degeneration associated with reduced mitochondrial respiration. Taken together, we uncovered a molecular disease signature and deciphered the pathomechanism underlying the functional and structural muscle anomalies characterizing TAM/STRMK.
    Keywords:  STIM2; York platelet syndrome; calcium; congenital myopathy; muscle weakness; neuromuscular disorder
  35. Nat Commun. 2021 Aug 06. 12(1): 4773
      The relationship between the age-associated decline in mitochondrial function and its effect on skeletal muscle physiology and function remain unclear. In the current study, we examined to what extent physical activity contributes to the decline in mitochondrial function and muscle health during aging and compared mitochondrial function in young and older adults, with similar habitual physical activity levels. We also studied exercise-trained older adults and physically impaired older adults. Aging was associated with a decline in mitochondrial capacity, exercise capacity and efficiency, gait stability, muscle function, and insulin sensitivity, even when maintaining an adequate daily physical activity level. Our data also suggest that a further increase in physical activity level, achieved through regular exercise training, can largely negate the effects of aging. Finally, mitochondrial capacity correlated with exercise efficiency and insulin sensitivity. Together, our data support a link between mitochondrial function and age-associated deterioration of skeletal muscle.
  36. J Pers Med. 2021 Jul 16. pii: 671. [Epub ahead of print]11(7):
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that leads to progressive degeneration of motor neurons (MNs) and severe muscle atrophy without effective treatment. Most research on ALS has been focused on the study of MNs and supporting cells of the central nervous system. Strikingly, the recent observations of pathological changes in muscle occurring before disease onset and independent from MN degeneration have bolstered the interest for the study of muscle tissue as a potential target for delivery of therapies for ALS. Skeletal muscle has just been described as a tissue with an important secretory function that is toxic to MNs in the context of ALS. Moreover, a fine-tuning balance between biosynthetic and atrophic pathways is necessary to induce myogenesis for muscle tissue repair. Compromising this response due to primary metabolic abnormalities in the muscle could trigger defective muscle regeneration and neuromuscular junction restoration, with deleterious consequences for MNs and thereby hastening the development of ALS. However, it remains puzzling how backward signaling from the muscle could impinge on MN death. This review provides a comprehensive analysis on the current state-of-the-art of the role of the skeletal muscle in ALS, highlighting its contribution to the neurodegeneration in ALS through backward-signaling processes as a newly uncovered mechanism for a peripheral etiopathogenesis of the disease.
    Keywords:  ALS; distal axonopathy; metabolism; neuromuscular disorder; neuromuscular junction; skeletal muscle; vesicles
  37. J Appl Physiol (1985). 2021 Aug 05.
      Mitochondrial derived peptides (MDPs) humanin (HN) and mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) are involved in cell survival, suppression of apoptosis and metabolism. Circulating levels of MDPs are altered in chronic diseases such as diabetes type 2 and chronic kidney disease. Whether acute resistance (RE) or endurance (EE) exercise modulates circulating levels of HN and MOTS-c in humans is unknown. Following familiarization, subjects were randomized to EE (n=10, 45 min cycling at 70% of estimated VO2max), RE (n=10, 4 sets x 7RM, leg press and knee extension), or control (CON, n=10). Skeletal muscle biopsies and blood samples were collected before and at 30 minutes and 3 hours following exercise. Plasma concentration of HN and MOTS-c, skeletal muscle MOTS-c as well as gene expression of exercise related genes were analyzed. Acute EE and RE promoted changes in skeletal muscle gene expression typically seen in response to each exercise modality (c-Myc, 45S pre-rRNA, PGC-1α-total and PGC-1α-ex1b). At rest, circulating levels of HN were positively correlated to MOTS-c levels and age. Plasma levels of MDPs were not correlated to fitness outcomes (VO2max, leg strength or muscle mitochondrial (mt) DNA copy number). Circulating levels of HN were significantly elevated by acute EE but not RE. MOTS-C levels showed a trend to increase after EE. These results indicate that plasma MDP levels are not related to fitness status but that acute EE increases circulating levels of MDPs, in particular HN.
    Keywords:  Skeletal muscle; exercise; humanin; mitochondria; mots-c
  38. Life (Basel). 2021 Jul 04. pii: 648. [Epub ahead of print]11(7):
      Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a pathogenic disruption of the DYSTROPHIN gene that results in non-functional dystrophin protein. DMD patients experience loss of ambulation, cardiac arrhythmia, metabolic syndrome, and respiratory failure. At the molecular level, the lack of dystrophin in the muscle results in myofiber death, fibrotic infiltration, and mitochondrial dysfunction. There is no cure for DMD, although dystrophin-replacement gene therapies and exon-skipping approaches are being pursued in clinical trials. Mitochondrial dysfunction is one of the first cellular changes seen in DMD myofibers, occurring prior to muscle disease onset and progresses with disease severity. This is seen by reduced mitochondrial function, abnormal mitochondrial morphology and impaired mitophagy (degradation of damaged mitochondria). Dysfunctional mitochondria release high levels of reactive oxygen species (ROS), which can activate pro-inflammatory pathways such as IL-1β and IL-6. Impaired mitophagy in DMD results in increased inflammation and further aggravates disease pathology, evidenced by increased muscle damage and increased fibrosis. This review will focus on the critical interplay between mitophagy and inflammation in Duchenne muscular dystrophy as a pathological mechanism, as well as describe both candidate and established therapeutic targets that regulate these pathways.
    Keywords:  DMD; dystrophin; dystrophy; inflammation; mitophagy
  39. EMBO Rep. 2021 Aug 06. e52247
      Our knowledge of the coordination of fuel usage in skeletal muscle is incomplete. Whether and how microRNAs are involved in the substrate selection for oxidation is largely unknown. Here we show that mice lacking miR-183 and miR-96 have enhanced muscle oxidative phenotype and altered glucose/lipid homeostasis. Moreover, loss of miR-183 and miR-96 results in a shift in substrate utilization toward fat relative to carbohydrates in mice. Mechanistically, loss of miR-183 and miR-96 suppresses glucose utilization in skeletal muscle by increasing PDHA1 phosphorylation via targeting FoxO1 and PDK4. On the other hand, loss of miR-183 and miR-96 promotes fat usage in skeletal muscle by enhancing intramuscular lipolysis via targeting FoxO1 and ATGL. Thus, our study establishes miR-183 and miR-96 as master coordinators of fuel selection and metabolic homeostasis owing to their capability of modulating both glucose utilization and fat catabolism. Lastly, we show that loss of miR-183 and miR-96 can alleviate obesity and improve glucose metabolism in high-fat diet-induced mice, suggesting that miR-183 and miR-96 may serve as therapeutic targets for metabolic diseases.
    Keywords:  fuel metabolism; lipolysis; metabolic flexibility; miR-183/96; skeletal muscle
  40. Curr Mol Med. 2021 May 21.
      BACKGROUND: The antibacterial mechanism of doxycycline is known, but on the nerve-muscle apparatus is yet unclear.OBJECTIVE: To combine molecular targets of the neuromuscular machinery using the neuronal blocker effect doxycycline, a semisynthetic second-generation tetracycline derivative, on mice neuromuscular preparations, in situ.
    METHODS: Doxycycline was assessed at the neurotransmission; presynaptic; synaptic cleft; and postsynaptic, including the muscle fiber, using the traditional myographic technique. Preliminarily, doxycycline showed an "all or nothing" effect, being "all" obtained with 4 µM and "nothing", with 1-3 µM. The rationale of this study was to apply known pharmacological tools against the blocker effect of 4 µM doxycycline such as F55-6 (Casearia sylvestris), CaCl2 (or Ca2+), atropine, neostigmine, polyethylene glycol (PEG 400), and d-Tubocurarine. The evaluation of cholinesterase enzyme activity, the diaphragm muscle histology, and protocols on the neuromuscular preparation submitted to indirect or direct stimuli were complementary.
    RESULTS: Doxycycline does not affect cholinesterase activity nor cause damage to skeletal muscle diaphragm; acts on ryanodine receptor, sarcolemmal membrane, and on neuronal sodium channel with a postjunctional consequence due to the decreased availability of muscle nicotinic acetylcholine receptors.
    CONCLUSIONS: In conclusion, using the blocker effect we showed that doxycycline acts on multiple targets, among them, is antagonized by F55-6, a neuronal Na+-channel agonist and Ca2+, but not by neostigmine.
    Keywords:  Antibiotics; Doxycycline; Molecular targets; Neuromuscular Junction; Phrenic-nerve diaphragm preparation; Tetracycline
  41. Front Physiol. 2021 ;12 702826
      Branched-chain amino acids (BCAAs) are critical for skeletal muscle and whole-body anabolism and energy homeostasis. They also serve as signaling molecules, for example, being able to activate mammalian/mechanistic target of rapamycin complex 1 (mTORC1). This has implication for macronutrient metabolism. However, elevated circulating levels of BCAAs and of their ketoacids as well as impaired catabolism of these amino acids (AAs) are implicated in the development of insulin resistance and its sequelae, including type 2 diabetes, cardiovascular disease, and of some cancers, although other studies indicate supplements of these AAs may help in the management of some chronic diseases. Here, we first reviewed the catabolism of these AAs especially in skeletal muscle as this tissue contributes the most to whole body disposal of the BCAA. We then reviewed emerging mechanisms of control of enzymes involved in regulating BCAA catabolism. Such mechanisms include regulation of their abundance by microRNA and by post translational modifications such as phosphorylation, acetylation, and ubiquitination. We also reviewed implications of impaired metabolism of BCAA for muscle and whole-body metabolism. We comment on outstanding questions in the regulation of catabolism of these AAs, including regulation of the abundance and post-transcriptional/post-translational modification of enzymes that regulate BCAA catabolism, as well the impact of circadian rhythm, age and mTORC1 on these enzymes. Answers to such questions may facilitate emergence of treatment/management options that can help patients suffering from chronic diseases linked to impaired metabolism of the BCAAs.
    Keywords:  branched-chain amino acids; catabolism; mTORC1; protein synthesis; skeletal muscle
  42. Anal Biochem. 2021 Jul 29. pii: S0003-2697(21)00220-7. [Epub ahead of print] 114319
      Evidence suggests acetylation of human adenine nucleotide translocase 1 (ANT1) at lysine 23 (Lys23) reduces binding of ADP. Lys23 contributes to the positive charge that facilitates this interaction. This study was undertaken to characterize ANT1 abundance and acetylation by a novel method using small amounts of human skeletal muscle biopsies. Lysates of whole muscle or mitochondria from the same tissue were prepared from needle biopsies of vastus lateralis muscle of healthy volunteers. Lysed proteins were resolved on gels, the section containing ANT1 (surrounding 30 Kd) was excised, digested with trypsin, spiked with labeled unacetylated and acetylated synthetic standard peptides and analyzed by mass spectrometry. Natural logarithm transformation of data linearized ion intensities over a 10-fold range of peptide mass. Coefficients of variation ranged from 7 to 30% for ANT1 abundance and Lys23 acetylation. In three volunteers, ANT1 content was 8.36 ± 0.33 nmoles/gram wet weight muscle and 0.64 ± 0.05 nmoles/mg mitochondria, so mitochondrial content was 13.3 ± 2.4 mg mitochondria per gram muscle. Acetylation of Lys23 averaged 14.3 ± 4.2% and 4.87 ± 1.84% in whole muscle and mitochondria, respectively. This assay makes it possible to assess effects of acetylation on the function of ANT1 in human muscle.
    Keywords:  Adenine nucleotide translocase; Lysine acetylation; Mitochondria; Proteomics; Skeletal muscle
  43. Front Immunol. 2021 ;12 666879
      Muscular dystrophies and inflammatory myopathies are heterogeneous muscular disorders characterized by progressive muscle weakness and mass loss. Despite the high variability of etiology, inflammation and involvement of both innate and adaptive immune response are shared features. The best understood immune mechanisms involved in these pathologies include complement cascade activation, auto-antibodies directed against muscular proteins or de-novo expressed antigens in myofibers, MHC-I overexpression in myofibers, and lymphocytes-mediated cytotoxicity. Intravenous immunoglobulins (IVIGs) administration could represent a suitable immunomodulator with this respect. Here we focus on mechanisms of action of immunoglobulins in muscular dystrophies and inflammatory myopathies highlighting results of IVIGs from pre-clinical and case reports evidences.
    Keywords:  autoantibodies; autoimmunity; immunoglobulins; inflammatory myopathies; muscle inflammation; muscular dystrophies
  44. Cells. 2021 Jul 03. pii: 1680. [Epub ahead of print]10(7):
      Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn-/- cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn-/- cells and could serve as basis for further research.
    Keywords:  GDF8; hypoxia; ischemia; muscle; myostatin; reoxygenation; reperfusion; skeletal
  45. Cell Metab. 2021 Jul 30. pii: S1550-4131(21)00331-4. [Epub ahead of print]
      Exercise is a powerful driver of physiological angiogenesis during adulthood, but the mechanisms of exercise-induced vascular expansion are poorly understood. We explored endothelial heterogeneity in skeletal muscle and identified two capillary muscle endothelial cell (mEC) populations that are characterized by differential expression of ATF3/4. Spatial mapping showed that ATF3/4+ mECs are enriched in red oxidative muscle areas while ATF3/4low ECs lie adjacent to white glycolytic fibers. In vitro and in vivo experiments revealed that red ATF3/4+ mECs are more angiogenic when compared with white ATF3/4low mECs. Mechanistically, ATF3/4 in mECs control genes involved in amino acid uptake and metabolism and metabolically prime red (ATF3/4+) mECs for angiogenesis. As a consequence, supplementation of non-essential amino acids and overexpression of ATF4 increased proliferation of white mECs. Finally, deleting Atf4 in ECs impaired exercise-induced angiogenesis. Our findings illustrate that spatial metabolic angiodiversity determines the angiogenic potential of muscle ECs.
    Keywords:  amino acid metabolism; endothelial heterogeneity; endothelial metabolism; exercise; muscle angiogenesis; single-cell RNA-seq