bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒02‒28
thirty-two papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Sci Rep. 2021 Feb 25. 11(1): 4601
      Thyroid hormone signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury, via activation of thyroid nuclear receptor alpha (THRA). A mouse model of resistance to thyroid hormone carrying a frame-shift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury. The expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA-PV mice and it is known to negatively regulate myogenesis. Here, we report that in murine myoblasts COUP-TFII interacts with THRA and modulates THRA binding to thyroid response elements (TREs). Silencing of COUP-TFII expression restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts. Moreover, COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice.
  2. Nat Commun. 2021 Feb 26. 12(1): 1294
      Age-associated muscle atrophy is a debilitating condition associated with loss of muscle mass and function with age that contributes to limitation of mobility and locomotion. However, the underlying mechanisms of how intrinsic muscle changes with age are largely unknown. Here we report that, with age, Mind bomb-1 (Mib1) plays important role in skeletal muscle maintenance via proteasomal degradation-dependent regulation of α-actinin 3 (Actn3). The disruption of Mib1 in myofibers (Mib1ΔMF) results in alteration of type 2 glycolytic myofibers, muscle atrophy, impaired muscle function, and Actn3 accumulation. After chronic exercise, Mib1ΔMF mice show muscle atrophy even at young age. However, when Actn3 level is downregulated, chronic exercise-induced muscle atrophy is ameliorated. Importantly, the Mib1 and Actn3 levels show clinical relevance in human skeletal muscles accompanied by decrease in skeletal muscle function with age. Together, these findings reveal the significance of the Mib1-Actn3 axis in skeletal muscle maintenance with age and suggest the therapeutic potential for the treatment or amelioration of age-related muscle atrophy.
  3. Physiol Rep. 2021 Feb;9(4): e14751
      Spinal cord injury (SCI) leads to rapid muscle atrophy due to paralysis/paresis and subsequent disuse. SS-31 is a mitochondrial-targeting peptide that has shown efficacy in protecting skeletal muscle mass and function in non-SCI models of muscle wasting. We aimed to determine if SS-31 could prevent muscle loss after SCI. Male C57BL/6 mice aged 9 weeks underwent sham surgery or 50 kdyne contusion SCI and were administered daily injections of vehicle or 5 mg/kg SS-31 for 14 d. Both SCI groups had sustained losses in body mass compared to Sham animals and ~10% reductions in gastrocnemius, plantaris and tibialis anterior muscle mass after SCI with no clear effect of SS-31. Measurements of protein synthesis in the soleus and plantaris were similar among all groups. mRNA expression of atrophy-associated proinflammatory cytokines was also similar among all groups. There was elevation in MYH7 mRNA and a statistical reduction in MYH2 mRNA expression in the SCI+SS-31 animals compared to Sham animals. There was an SCI-induced reduction in mRNA expression of the E3 ligase FBXO32 (MAFbx), but no effect of SS-31. In summary, a 50 kdyne contusion SCI was able to reduce body mass but was not associated with substantial muscle atrophy or alterations in gene expression profiles associated with muscle health and function 14 d post-injury. SS-31 was not associated with protection against SCI-related changes in body or muscle mass, protein synthesis or gene expression in hindlimb muscles.
    Keywords:  SS-31; paralysis; skeletal muscle; spinal cord injury
  4. J Anim Sci. 2021 Feb 25. pii: skab060. [Epub ahead of print]
      Like humans, many companion animals, experience a gradual decline in skeletal muscle mass and function during later years of life. This process, analogous to sarcopenia in humans, increases risk for morbidity and mortality. Periods of reduced activity due to injury or illness, followed by an incomplete recovery, can accelerate the loss of muscle mass and function. Emerging research from human studies suggest that moderate amounts of high-quality protein may attenuate the loss of muscle, while preventing accumulation of fat during periods of disuse. Whey protein is a consumer-friendly and readily available source of high-quality protein. It supports skeletal muscle maintenance during normal aging and may also provide anabolic support during periods of illness, injury and recovery. Ongoing research efforts continue to refine our understanding of how protein quality, quantity and meal timing can be optimized to support retention of muscle mass and function during aging. Priority research areas include supplementation with high quality protein during illness/injury to stimulate anabolism by targeting molecular mechanisms that regulate skeletal muscle metabolism.
    Keywords:  aging; atrophy; disuse; humans; skeletal muscle; whey protein
  5. Aging (Albany NY). 2021 02 22. 13
      We previously determined that different vitamin D metabolites can have opposite effects on C2C12 myotubes, depending on the sites of hydroxylation or doses. Specifically, 25(OH)D3 (25VD) has an anti-atrophic activity, 1,25(OH)2D3 induces atrophy, and 24,25(OH)2D3 is anti-atrophic at low concentrations and atrophic at high concentrations. This study aimed to clarify whether cholecalciferol (VD3) too, the non-hydroxylated upstream metabolite, has a direct effect on muscle cells. Assessing the effects of VD3 treatment on mouse C2C12 skeletal muscle myotubes undergoing atrophy induced by interleukin 6 (IL6), we demonstrated that VD3 has a protective action, preserving C2C12 myotubes size, likely through promoting the differentiation and fusion of residual myoblasts and by modulating the IL6-induced autophagic flux. The lack, in C2C12 myotubes, of the hydroxylase transforming VD3 in the anti-atrophic 25VD metabolite suggests that VD3 may have a direct biological activity on the skeletal muscle. Furthermore, we found that the protective action of VD3 depended on VDR, implying that VD3 too might bind to and activate VDR. However, despite the formation of VDR-RXR heterodimers, VD3 effects do not depend on RXR activity. In conclusion, VD3, in addition to its best-known metabolites, may directly impact on skeletal muscle homeostasis.
    Keywords:  VDR; autophagy; cachexia; sarcopenia; vitamin D hydroxylases
  6. PLoS One. 2021 ;16(2): e0247377
      Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and β) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7-10 days. A short-term co-culture (10-11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAβ2, NKAβ3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs.
  7. Front Physiol. 2020 ;11 570170
      Cachexia is a multifactorial inflammatory syndrome with high prevalence in cancer patients. It is characterized by a metabolic chaos culminating in drastic reduction in body weight, mainly due to skeletal muscle and fat depletion. Currently, there is not a standard intervention for cachexia, but it is believed that a dynamic approach should be applied early in the course of the disease to maintain or slow the loss of physical function. The present review sought to explain the different clinical and experimental applications of different models of exercise and their contribution to a better prognosis of the disease. Here the advances in knowledge about the application of physical training in experimental models are elucidated, tests that contribute substantially to elucidate the cellular and biochemical mechanisms of exercise in different ways, as well as clinical trials that present not only the impacts of exercise in front cachexia but also the challenges of its application in clinical practice.
    Keywords:  aerobic; muscle atrophy; muscle wasting; neoplasms; resistance; systemic inflammation; therapeutic exercise; tumor
  8. Stem Cells. 2021 Feb 26.
      O-GlcNAcylation is a post-translational modification considered to be a nutrient sensor that reports nutrient scarcity or surplus. Although O-GlcNAcylation exists in a wide range of cells and/or tissues, its functional role in muscle satellite cells (SCs) remains largely unknown. Using a genetic approach, we ablated O-GlcNAc transferase (OGT), and thus O-GlcNAcylation, in SCs. We first evaluated SC function in vivo using a muscle injury model and found that OGT deficient SCs had compromised capacity to repair muscle after an acute injury compared to the wild-type SCs. By tracing SC cycling rates in vivo using the doxycycline-inducible H2B-GFP mouse model, we found that SCs lacking OGT cycled at lower rates and reduced in abundance with time. Additionally, the self-renewal ability of OGT-deficient SCs after injury was decreased compared to that of the wild-type SCs. Moreover, in vivo, in vitro, and ex vivo proliferation assays revealed that SCs lacking OGT were incapable of expanding compared to their wild-type counterparts, a phenotype that may be explained, at least in part, by an HCF1-mediated arrest in the cell cycle. Taken together, our findings suggest that O-GlcNAcylation plays a critical role in the maintenance of SC health and function in normal and injured skeletal muscle. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Satellite cells contribute to skeletal muscle health and muscle repair after injury. Satellite cell behavior is governed by nutrient availability, yet the underpinning mechanism remains poorly understood. Herein, we demonstrate that O-GlcNAcylation is critical for satellite cells to maintain their homeostasis and adjust their behaviors in normal physiological conditions and during adult regenerative myogenesis.
    Keywords:  UDP-O-GlcNAc; muscle stem cells; nutrient sensing; regenerative myogenesis
  9. J Appl Physiol (1985). 2021 Feb 25.
      There are limited and equivocal data regarding potential fiber type-specific differences in the human skeletal muscle response to sprint interval training (SIT), including how this compares to moderate-intensity continuous training (MICT). We examined mixed muscle and fiber type-specific responses to a single session (study 1) and 12 wk (study 2) of MICT and SIT using Western blotting. MICT consisted of 45 min of cycling at ~70% of maximal heart rate and SIT involved 3 x 20-sec 'all-out' sprints interspersed with 2 min of recovery. Changes in signaling proteins involved in mitochondrial biogenesis in mixed muscle and pooled fiber samples were similar after acute MICT and SIT. This included increases in the ratios of phosphorylated to total acetyl CoA carboxylase and p38 mitogen activated protein kinase protein content (main effects, p<0.05). Following training, mitochondrial content markers including the protein content of cytochrome c oxidase subunit IV and NADH:ubiquinone oxidoreductase subunit A9 were increased similarly in mixed muscle and type IIa fibers (main effects, p<0.05). In contrast, only MICT increased these markers of mitochondrial content in type I fibers (interactions, p<0.05). MICT and SIT also similarly increased the content of mitochondrial fusion proteins optic atrophy 1 (OPA1) and mitofusin 2 in mixed muscle, and OPA1 in pooled fibre samples (main effects, p<0.05). In summary, acute MICT and SIT elicited similar fiber type-specific responses of signaling proteins involved in mitochondrial biogenesis, whereas 12 wk of training revealed differential responses of mitochondrial content markers in type I but not type IIa fibers.
    Keywords:  Western blotting; aerobic exercise; mitochondria; single fiber
  10. Nat Commun. 2021 Feb 26. 12(1): 1318
      Cell-cell interactions mediated by Notch are critical for the maintenance of skeletal muscle stem cells. However, dynamics, cellular source and identity of functional Notch ligands during expansion of the stem cell pool in muscle growth and regeneration remain poorly characterized. Here we demonstrate that oscillating Delta-like 1 (Dll1) produced by myogenic cells is an indispensable Notch ligand for self-renewal of muscle stem cells in mice. Dll1 expression is controlled by the Notch target Hes1 and the muscle regulatory factor MyoD. Consistent with our mathematical model, our experimental analyses show that Hes1 acts as the oscillatory pacemaker, whereas MyoD regulates robust Dll1 expression. Interfering with Dll1 oscillations without changing its overall expression level impairs self-renewal, resulting in premature differentiation of muscle stem cells during muscle growth and regeneration. We conclude that the oscillatory Dll1 input into Notch signaling ensures the equilibrium between self-renewal and differentiation in myogenic cell communities.
  11. Stem Cell Res Ther. 2021 Feb 24. 12(1): 146
      BACKGROUND: Muscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration.METHODS: A clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration.
    RESULTS: 2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI.
    CONCLUSION: WIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.
    Keywords:  Ischemia-reperfusion injury; Muscle regeneration; Myogenic progenitor cells; Satellite cells; Stem cell; Transplantation
  12. Front Cell Dev Biol. 2021 ;9 625680
      Acute exercise increases the amount of circulating inflammatory cells and cytokines to maintain physiological homeostasis. However, it remains unclear how physical training regulates exercise-induced inflammation and performance. Here, we demonstrate that acute high intensity exercise promotes an inflammatory profile characterized by increased blood IL-6 levels, neutrophil migratory capacity, and leukocyte recruitment to skeletal muscle vessels. Moreover, we found that physical training amplified leukocyte-endothelial cell interaction induced by acute exercise in skeletal muscle vessels and diminished exercise-induced inflammation in skeletal muscle tissue. Furthermore, we verified that disruption of the gp-91 subunit of NADPH-oxidase inhibited exercise-induced leukocyte recruitment on skeletal muscle after training with enhanced exercise time until fatigue. In conclusion, the training was related to physical improvement and immune adaptations. Moreover, reactive oxygen species (ROS) could be related to mechanisms to limit aerobic performance and its absence decreases the inflammatory response elicited by exercise after training.
    Keywords:  exercise; muscular inflammation; neutrophil; oxidative stress; physical training
  13. Ageing Res Rev. 2021 Feb 21. pii: S1568-1637(21)00056-8. [Epub ahead of print] 101309
      Mitochondria are highly dynamic organelles capable of adapting their network, morphology, and function, playing a role in oxidative phosphorylation and many cellular processes in most cell types. Skeletal muscle is a very plastic tissue, subjected to many morphological changes following diverse stimuli, such as during myogenic differentiation and regenerative myogenesis. For some time now, mitochondria have been reported to be involved in myogenesis by promoting a bioenergetic remodeling and assisting myoblasts in surviving the process. However, not much is known about the interplay between mitochondrial quality control and myogenic differentiation. Sestrin2 (SESN2) is a well described regulator of autophagy and antioxidant responses and has been gaining attention due to its role in aging-associated pathologies and redox signaling promoted by reactive oxygen species (ROS) in many tissues. Current evidence involving SESN2-associated pathways suggest that it can act as a potential regulator of mitochondrial quality control following induction by ROS under stress conditions, such as during myogenesis. Yet, there are no studies directly assessing SESN2 involvement in myogenic differentiation. This review provides novel insights pertaining the involvement of SESN2 in myogenic differentiation by analyzing the interactions between ROS and mitochondrial remodeling.
    Keywords:  SESN2; differentiation; mitochondria; mitohormesis; myogenesis
  14. Clin Exp Pharmacol Physiol. 2021 Feb 21.
      Emerging evidence indicated long noncoding RNAs (lncRNAs) played important roles in diverse biological processes, including fibrosis. Here, we report that lncRNA H19 is able to promote skeletal muscle fibrosis. lnc-H19 was identified to be highly expressed in skeletal muscle fibrosis in vivo and in vitro; while lnc-H19 knockdown attenuated fibrosis in vitro. The knockdown of lnc-H19 was proved to inhibit the activation of TGFβ/Smad pathway in C2C12 myoblasts by sponging miR-20a-5p to regulate Tgfbr2 expression through the competing endogenous RNA function. Our study elucidates the roles of lnc-H19-miR-20a-5p-Tgfbr2 axis in regulating TGFβ/Smad pathway of myoblast fibrogenesis, which might provide a promising therapeutic target for skeletal muscle fibrosis.
    Keywords:  Fibrosis; Tgfbr2; lnc-H19; miR-20a-5p; myoblast; skeletal muscle
  15. Stem Cells Int. 2021 ;2021 8884283
      Stem cells have become a hot research topic in the field of regenerative medicine due to their self-renewal and differentiation capabilities. Skeletal muscle tissue is one of the most important tissues in the human body, and it is difficult to recover when severely damaged. However, conventional treatment methods can cause great pain to patients. Stem cell-based tissue engineering can repair skeletal muscle to the greatest extent with little damage. Therefore, the application of stem cells to skeletal muscle regeneration is very promising. In this review, we discuss scaffolds and stem cells for skeletal muscle regeneration and put forward our ideas for future development.
  16. Int J Sport Nutr Exerc Metab. 2021 Feb 24. pii: ijsnem.2020-0282. [Epub ahead of print] 1-16
      This systematic review and meta-analysis examined the effects of creatine supplementation on recovery from exercise-induced muscle damage, and is reported according to the PRISMA guidelines. MEDLINE and SPORTDiscus were searched for articles from inception until April 2020. Inclusion criteria were adult participants (≥18 years); creatine provided before and/or after exercise versus a noncreatine comparator; measurement of muscle function recovery, muscle soreness, inflammation, myocellular protein efflux, oxidative stress; range of motion; randomized controlled trials in humans. Thirteen studies (totaling 278 participants; 235 males and 43 females; age range 20-60 years) were deemed eligible for analysis. Data extraction was performed independently by both authors. The Cochrane Collaboration Risk of Bias Tool was used to critically appraise the studies; forest plots were generated with random-effects model and standardized mean differences. Creatine supplementation did not alter muscle strength, muscle soreness, range of motion, or inflammation at each of the five follow-up times after exercise (<30 min, 24, 48, 72, and 96 hr; p > .05). Creatine attenuated creatine kinase activity at 48-hr postexercise (standardized mean difference: -1.06; 95% confidence interval [-1.97, -0.14]; p = .02) but at no other time points. High (I2; >75%) and significant (Chi2; p < .01) heterogeneity was identified for all outcome measures at various follow-up times. In conclusion, creatine supplementation does not accelerate recovery following exercise-induced muscle damage; however, well-controlled studies with higher sample sizes are warranted to verify these conclusions. Systematic review registration (PROSPERO CRD42020178735).
    Keywords:  antioxidants; exercise recovery; nutrition; oxidative stress; phosphocreatine
  17. Proc Natl Acad Sci U S A. 2021 Mar 02. pii: e2018342118. [Epub ahead of print]118(9):
      Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle degeneration and weakness due to mutations in the dystrophin gene. The symptoms of DMD share similarities with those of accelerated aging. Recently, hydrogen sulfide (H2S) supplementation has been suggested to modulate the effects of age-related decline in muscle function, and metabolic H2S deficiencies have been implicated in affecting muscle mass in conditions such as phenylketonuria. We therefore evaluated the use of sodium GYY4137 (NaGYY), a H2S-releasing molecule, as a possible approach for DMD treatment. Using the dys-1(eg33) Caenorhabditis elegans DMD model, we found that NaGYY treatment (100 µM) improved movement, strength, gait, and muscle mitochondrial structure, similar to the gold-standard therapeutic treatment, prednisone (370 µM). The health improvements of either treatment required the action of the kinase JNK-1, the transcription factor SKN-1, and the NAD-dependent deacetylase SIR-2.1. The transcription factor DAF-16 was required for the health benefits of NaGYY treatment, but not prednisone treatment. AP39 (100 pM), a mitochondria-targeted H2S compound, also improved movement and strength in the dys-1(eg33) model, further implying that these improvements are mitochondria-based. Additionally, we found a decline in total sulfide and H2S-producing enzymes in dystrophin/utrophin knockout mice. Overall, our results suggest that H2S deficit may contribute to DMD pathology, and rectifying/overcoming the deficit with H2S delivery compounds has potential as a therapeutic approach to DMD treatment.
    Keywords:  C. elegans; hydrogen sulfide; mitochondria; mouse; muscle
  18. Biomed Res Int. 2021 ;2021 4218086
      The capability of regeneration for skeletal muscle after injury depends on the differentiation and proliferation ability of the resident stem cells called satellite cells. It has been reported that electrical stimulation was widely used in clinical conditions to facilitate muscle regeneration after injury, but the characterization of satellite cell responses to the context of low-frequency electrical stimulation in early-phase muscle strain conditions has not been fully clarified. In this study, we aim to investigate the effects of low-frequency electrical stimulation (frequency: 20 Hz; duration: 30 minutes, twice daily) on satellite cell activities in a rat model for the early phase of muscle strain. Firstly, we adopted our previously developed rat model to mimic the early phase of muscle strain in human. After then, we examined the effects of low-frequency electrical stimulation on histopathological changes of the muscle fiber by hematoxylin and eosin (H&E) staining. Finally, we investigated the effects of low-frequency electrical stimulation on satellite cell proliferation and differentiation by quantification of the expression level of the specific proteins using western blot analyses. The muscle strain in biceps femoris muscles of rats can be induced by high-speed rotation from knee flexion 50° to full knee extension at 960°·s-1 angular velocity during its tetany by activating the sciatic nerve, as evidenced by a widening of the interstitial space between fibers, and more edema or necrosis fibers were detected in the model rats without treatment than in control rats. After treatment with low-frequency electrical stimulation (frequency: 20 Hz; duration: 30 minutes, twice daily), the acute strained biceps femoris muscles of rats showed obvious improvement of histomorphology as indicated by more mature muscle fibers with well-ordered formation with clear boundaries. Consistently, the expression levels of the MyoD and myogenin were marked higher than those in the rats in the animal model group, indicating increased satellite cell proliferating and differentiating activities by low-frequency electrical stimulation. This study shows that low-frequency electrical stimulation provides an effective stimulus to upregulate the protein expression of MyoD/myogenin and accelerate the restoration of structure during the early phase of muscle strain. This may have significance for clinical practice. Optimization of low-frequency electrical stimulation parameters may enhance the therapeutic outcome in patients.
  19. Biochem Biophys Res Commun. 2021 Feb 19. pii: S0006-291X(21)00183-2. [Epub ahead of print]547 176-182
      Skeletal muscle fibrosis and regeneration are modulated by transforming growth factor β (TGF-β) superfamily. Amongst them, TGF-β1 is a highly potent pro-fibrotic factor, while TGF-β3 has been implicated to reduce scar formation and collagen production in skin and vocal mucosa. However, little is known about the individual and combined short- and long-term effects of TGF-β1 and TGF-β3 on collagen expression in myoblasts and myotubes. Here we show that in C2C12 myoblasts TGF-β1 and/or TGF-β3 increased mRNA expression of Ctgf and Fgf-2 persistently after 3 h and of Col1A1 after 24 h, while TGF-β1+TGF-β3 mitigated these effects after 48 h incubation. Gene expression of Tgf-β1 was enhanced by TGF-β1 and/or TGF-β3 after 24 h and 48 h. However, Tgfbr1 mRNA expression was reduced at 48 h. After 48 h incubation with TGF-β1 and/or TGF-β3, Col3A1 and Col4A1 mRNA expression levels were decreased. Myoblasts produced collagen after three days incubation with TGF-β1 and/or TGF-β3 in a dose independent manner. Collagen deposition was doubled when myoblasts differentiated into myotubes and TGF-β1 and/or TGF-β3 did not stimulate collagen production any further. TGF-β type I receptor (TGFBR1) inhibitor, LY364947, suppressed TGF-βs-induced collagen production. Collagen I expression was higher in myotubes than in myoblasts. TGF-β1 and/or TGF-β3 inhibited myotube differentiation which was antagonized by LY364947. These results indicate that both C2C12 myoblasts and myotubes produce collagen. Whereas TGF-β1 and TGF-β3 individually and simultaneously stimulate collagen production in C2C12 differentiating myoblasts, in myotubes these effects are less prominent. In muscle cells, TGF-β3 is ineffective to antagonize TGF-β1-induced collagen production.
    Keywords:  Collagen; Differentiation; Fibrosis; Muscle satellite cells; TGF-β1; TGF-β3
  20. J Tissue Eng. 2021 Jan-Dec;12:12 2041731420981339
      Muscular dystrophies are a group of highly disabling disorders that share degenerative muscle weakness and wasting as common symptoms. To date, there is not an effective cure for these diseases. In the last years, bioengineered tissues have emerged as powerful tools for preclinical studies. In this review, we summarize the recent technological advances in skeletal muscle tissue engineering. We identify several ground-breaking techniques to fabricate in vitro bioartificial muscles. Accumulating evidence shows that scaffold-based tissue engineering provides topographical cues that enhance the viability and maturation of skeletal muscle. Functional bioartificial muscles have been developed using human myoblasts. These tissues accurately responded to electrical and biological stimulation. Moreover, advanced drug screening tools can be fabricated integrating these tissues in electrical stimulation platforms. However, more work introducing patient-derived cells and integrating these tissues in microdevices is needed to promote the clinical translation of bioengineered skeletal muscle as preclinical tools for muscular dystrophies.
    Keywords:  Skeletal muscle; biomaterials; drug screening platforms; muscular dystrophy; tissue engineering
  21. J Physiol Biochem. 2021 Feb 26.
      The unfolded protein response (UPR) plays a pivotal role in some exercise training-induced physiological adaptation. Our aim was to evaluate the changes in the protein kinase R-like endoplasmic reticulum kinase (PERK) arm of the UPR and hypertrophy signaling pathway following 8 weeks of resistance training and creatine (Cr) supplementation in rats. Thirty-two adult male Wistar rats (8 weeks old) were randomly divided into 4 groups of 8: untrained + placebo (UN+P), resistance training + placebo (RT+P), untrained + Cr (UN+Cr), and resistance training + Cr (RT+Cr). Trained animals were submitted to the ladder-climbing exercise training 5 days per week for a total of 8 weeks. Cr supplementation groups received creatine diluted with 1.5 ml of 5% dextrose orally. The flexor hallucis longus (FHL) muscle was extracted 48 h after the last training session and used for western blotting. After training period, the RT+Cr and RT+P groups presented a significant increase in phosphorylated and phosphorylated/total ratio hypertrophy indices, phosphorylated and phosphorylated/total ratio PERK pathway proteins, and other downstream proteins of the PERK cascade compared with their untrained counterparts (P < 0.05). The increase in hypertrophy indices were higher but PERK pathway proteins were lower in the RT-Cr group than in the RT+P group (P < 0.05). There was no significant difference between the untrained groups (P > 0.05). Our study suggests that resistance training in addition to Cr supplementation modifies PERK pathway response and improves skeletal muscle hypertrophy.
    Keywords:  Creatine; Ladder-climbing training; Muscle mass; PERK pathway; UPR
  22. OMICS. 2021 Feb 25.
      Regenerative medicine research and testing of new therapeutics for muscle-related human diseases call for a deeper understanding of how human myoblasts gain and maintain quiescence in vitro versus in vivo. The more closely we can experimentally simulate the in vivo environment, the more relevance in vitro research on myoblasts will have. In this context, isolation of satellite cells from muscle tissue causes activation while myoblasts remain activated in culture, thus not simulating quiescence as in their in vivo niche. Cells synchronized for cell cycle present a good starting point for experimental intervention. In the past, myoblast quiescence has been induced using suspension culture (SuCu) and, recently, by knockout serum replacement (KOSR)-supplemented culture media. We assessed the proportion of cells in G0 and molecular regulators after combining the two quiescence-inducing approaches. Quiescence was induced in primary human myoblasts (PHMs) in vitro using KOSR-treatment for 10 days or suspension in viscous media for 2 days (SuCu), or suspension combined with KOSR-treatment for 2 days (blended method, SuCu-KOSR). Quiescence and synchronization were achieved with all three protocols (G0/G1 cell cycle arrest >90% cells). Fold-change of cell cycle controller p21 mRNA for KOSR and SuCu was 3.23 ± 0.30 and 2.86 ± 0.15, respectively. Since this was already a significant change (p < 0.05), no further change was gained with the blended method. But SuCu-KOSR significantly decreased Ki67 (p = 0.0019). Myogenic regulatory factors, Myf5 and MyoD gene expression in PHMs were much more suppressed (p = 0.0004 and p = 0.0034, respectively) in SuCu-KOSR, compared to SuCu alone. In conclusion, a homogenous pool of quiescent primary myoblasts synchronized in the G0 cell cycle phase was achieved with cells from three different donors regardless of the experimental protocol. Myogenic dedifferentiation at the level of Myogenic Regulatory Factors was greater when exposed to the blend of suspension and serum-free culture. We suggest that this blended new protocol can be considered in future biomedical research if differentiation is detected too early during myoblast expansion. This shall also inform new ways to bridge the in vitro and in vivo divides in regenerative medicine research.
    Keywords:  KnockOut Serum Replacement; Myf5; muscle satellite cells; p21; quiescence; stem cells; suspension culture
  23. Dis Model Mech. 2021 Feb 22. pii: dmm047704. [Epub ahead of print]14(2):
      Besides skeletal muscle abnormalities, Duchenne muscular dystrophy (DMD) patients present with dilated cardiomyopathy development, which considerably contributes to morbidity and mortality. Because the mechanisms responsible for the cardiac complications in the context of DMD are largely unknown, evidence-based therapy approaches are still lacking. This has increased the need for basic research efforts into animal models for DMD. Here, we characterized in detail the cardiovascular abnormalities of Dmdmdx rats, with the aim of determining the suitability of this recently established dystrophin-deficient small animal as a model for DMD.Various methods were applied to compare cardiovascular properties between wild-type and Dmdmdx rats, and to characterize the Dmdmdx cardiomyopathy. These methods comprised echocardiography, invasive assessment of left ventricular hemodynamics, examination of adverse remodeling and endothelial cell inflammation, and evaluation of vascular function, employing wire myography. Finally, intracellular Ca2+ transient measurements, and recordings of currents through L-type Ca2+ channels were performed in isolated single ventricular cardiomyocytes. We found that, similar to respective observations in DMD patients, the hearts of Dmdmdx rats show significantly impaired cardiac function, fibrosis and inflammation, consistent with the development of a dilated cardiomyopathy. Moreover, in Dmdmdx rats, vascular endothelial function is impaired, which may relate to inflammation and oxidative stress, and Ca2+ handling in Dmdmdx cardiomyocytes is abnormal.These findings indicate that Dmdmdx rats represent a promising small-animal model to elucidate mechanisms of cardiomyopathy development in the dystrophic heart, and to test mechanism-based therapies aiming to combat cardiovascular complications in DMD.
    Keywords:  Cardiomyocyte; Cardiovascular dysfunction; Muscular dystrophy; Rat; Remodeling
  24. Mol Ther Nucleic Acids. 2021 Mar 05. 23 743-756
      The activation of the renin-angiotensin system (RAS) induced by increased angiotensin II (AngII) levels has been implicated in muscle atrophy, which is involved in the pathogenesis of congestive heart failure. Although peroxisome proliferator-activated receptor gamma (PPARγ) activation can suppress RAS, the exact role of PPARγ in AngII-induced muscle atrophy is unclear. Here we identified PPARγ as a negative regulator of miR-29b, a microRNA that is able to promote multiple types of muscle atrophy. Suppression of miR-29b could prevent AngII-induced muscle atrophy both in vitro and in vivo. IGF1, PI3K(p85α), and Yin Yang 1 (YY1) were identified as target genes of miR-29b, and overexpression of these targets could rescue AngII-induced muscle atrophy. Importantly, inhibition of PPARγ was sufficient to induce muscle atrophy, while PPARγ overexpression could attenuate that. These data indicate that the PPARγ/miR-29b axis mediates AngII-induced muscle atrophy, and increasing PPARγ or inhibiting miR-29b represents a promising approach to counteract AngII-induced muscle atrophy.
    Keywords:  PPARγ; angiotensin II; miR-29b; muscle atrophy
  25. Annu Rev Biophys. 2021 Feb 26.
      Two groundbreaking papers published in 1954 laid out the theory of the mechanism of muscle contraction based on force-generating interactions between myofilaments in the sarcomere that cause filaments to slide past one another during muscle contraction. The succeeding decades of research in muscle physiology have revealed a unifying interest: to understand the multiscale processes-from atom to organ-that govern muscle function. Such an understanding would have profound consequences for a vast array of applications, from developing new biomimetic technologies to treating heart disease. However, connecting structural and functional properties that are relevant at one spatiotemporal scale to those that are relevant at other scales remains a great challenge. Through a lens of multiscale dynamics, we review in this article current and historical research in muscle physiology sparked by the sliding filament theory. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 2021. Please see for revised estimates.
  26. Dev Cell. 2021 Feb 15. pii: S1534-5807(21)00074-5. [Epub ahead of print]
      Cell-cell fusion is a frequent and essential event during development, and its dysregulation causes diseases ranging from infertility to muscle weakness. Fusing cells need to repeatedly remodel their plasma membrane through orchestrated formation and disassembly of actin filaments, but how the dynamic reorganization of the cortical actin cytoskeleton is controlled is still poorly understood. Here, we identified a ubiquitin-dependent toggle switch that establishes reversible actin bundling during mammalian cell fusion. We found that EPS8-IRSp53 complexes stabilize cortical actin bundles at sites of cell contact to promote close membrane alignment. EPS8 monoubiquitylation by CUL3KCTD10 displaces EPS8-IRSp53 from membranes and counteracts actin bundling, a dual activity that restricts actin bundling to allow paired cells to progress with fusion. We conclude that cytoskeletal rearrangements during development are precisely controlled by ubiquitylation, raising the possibility of modulating the efficiency of cell-cell fusion for therapeutic benefit.
    Keywords:  CUL3; EPS8; actin; actin bundle; cell fusion; monoubiquitylation; ubiquitin
  27. Aging (Albany NY). 2021 Feb 11. 13(3): 3313-3341
      By combining transcriptomic data with other data sources, inferences can be made about functional changes during ageing. Thus, we conducted a meta-analysis on 127 publicly available microarray and RNA-Seq datasets from mice, rats and humans, identifying a transcriptomic signature of ageing across species and tissues. Analyses on subsets of these datasets produced transcriptomic signatures of ageing for brain, heart and muscle. We then applied enrichment analysis and machine learning to functionally describe these signatures, revealing overexpression of immune and stress response genes and underexpression of metabolic and developmental genes. Further analyses revealed little overlap between genes differentially expressed with age in different tissues, despite ageing differentially expressed genes typically being widely expressed across tissues. Additionally we show that the ageing gene expression signatures (particularly the overexpressed signatures) of the whole meta-analysis, brain and muscle tend to include genes that are central in protein-protein interaction networks. We also show that genes underexpressed with age in the brain are highly central in a co-expression network, suggesting that underexpression of these genes may have broad phenotypic consequences. In sum, we show numerous functional similarities between the ageing transcriptomes of these important tissues, along with unique network properties of genes differentially expressed with age in both a protein-protein interaction and co-expression networks.
    Keywords:  Artificial Intelligence; functional genomics; machine learning; microarray; mitochondria
  28. Front Cell Dev Biol. 2021 ;9 634853
      Extracellular vesicles (EVs) have emerged as key players of intercellular communication and mediate crosstalk between tissues. Metastatic tumors release tumorigenic EVs, capable of pre-conditioning distal sites for organotropic metastasis. Growing evidence identifies muscle cell-derived EVs and myokines as potent mediators of cellular differentiation, proliferation, and metabolism. Muscle-derived EVs cargo myokines and other biological modulators like microRNAs, cytokines, chemokines, and prostaglandins hence, are likely to modulate the remodeling of niches in vital sites, such as liver and adipose tissues. Despite the scarcity of evidence to support a direct relationship between muscle-EVs and cancer metastasis, their indirect attribution to the regulation of niche remodeling and the establishment of pre-metastatic homing niches can be put forward. This hypothesis is supported by the role of muscle-derived EVs in findings gathered from other pathologies like inflammation and metabolic disorders. In this review, we present and discuss studies that evidently support the potential roles of muscle-derived EVs in the events of niche pre-conditioning and remodeling of metastatic tumor microenvironment. We highlight the potential contributions of the integrin-mediated interactions with an emerging myokine, irisin, to the regulation of EV-driven microenvironment remodeling in tumor metastasis. Further research into muscle-derived EVs and myokines in cancer progression is imperative and may hold promising contributions to advance our knowledge in the pathophysiology, progression and therapeutic management of metastatic cancers.
    Keywords:  extracellular vesicles; homing niche; integrins; irisin; muscle; myokines; tissue microenvironment; tumor metastasis
  29. Food Nutr Res. 2021 ;65
      Background: Saturated fatty acids (SFAs) generally have been thought to worsen insulin-resistance and increase the risk of developing type 2 diabetes mellitus (T2DM). Recently, accumulating evidence has revealed that SFAs are not a single homogeneous group, instead different SFAs are associated with T2DM in opposing directions. Pentadecanoic acid (C15:0, PA) is directly correlated with dairy products, and a negative association between circulating PA and metabolic disease risk was observed in epidemiological studies. Therefore, the role of PA in human health needs to be reinforced. Whether PA has a direct benefit on glucose metabolism and insulin sensitivity needs further investigation.Objective: The present study aimed to investigate the effect and potential mechanism of action of PA on basal and insulin stimulated glucose uptake in C2C12 myotubes.
    Methods: Glucose uptake was determined using a 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. The phosphorylation levels of proteins involved in the insulin and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined by western blotting.
    Results: We found that PA significantly promoted glucose uptake and GLUT4 translocation to the plasma membrane. PA had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (Tyr632) and protein kinase B (PKB/Akt), but increased phosphorylation of AMPK and Akt substrate of 160 kDa (AS160). Compound C (an AMPK inhibitor) blocked PA-induced AMPK activation and reversed PA-induced GLUT4 translocation, indicating that PA promotes glucose uptake via the AMPK pathway in vitro. Moreover, PA significantly promoted insulin-stimulated glucose uptake in myotubes. Under insulin stimulation, PA did not affect the insulin-dependent pathway, but still activated AMPK.
    Conclusion: PA, an odd-chain SFA, significantly stimulates glucose uptake via the AMPK-AS160 pathway and exhibits an insulin-sensitizing effect in myotubes.
    Keywords:  AMP-activated protein kinase; C2C12 myotubes; glucose uptake; insulin sensitivity; pentadecanoic acid
  30. Tissue Eng Regen Med. 2021 Feb 24.
      BACKGROUND: Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. Adult stem cells isolated from various sources (i.e., cord blood, bone marrow, adipose tissue) are being considered as a realistic option due to their well-documented therapeutic potentials. Our previous studies standardized a method to isolate circulating multipotent cells (CMCs) that are able to sustain long term in vitro culture and differentiate towards mesodermal lineages.METHODS: In this work, long-term cultures of CMCs were stimulated to study in vitro neuronal and myogenic differentiation. After induction, cells were analysed at different time points. Morphological studies were performed by scanning electron microscopy and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. For myogenic plasticity study, CMCs were transplanted into in vivo model of chemically-induced muscle damage.
    RESULTS: After neurogenic induction, CMCs showed characteristic dendrite-like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of CMCs under stimulation with potassium chloride and the secretion of noradrenalin confirmed their ability to acquire a functional phenotype. In parallel, the myogenic potential of CMCs was confirmed by their ability to form syncytium-like structures in vitro and express myogenic markers both at early and late phases of differentiation. Interestingly, in a rat model of bupivacaine-induced muscle damage, CMCs integrated within the host tissue taking part in tissue repair.
    CONCLUSION: Overall, collected data demonstrated long-term cultured CMCs retain proliferative and differentiative potentials suggesting to be a good candidate for cell therapy.
    Keywords:  Circulating stem cells; Degenerative diseases; Myogenesis; Neurogenesis; Regenerative medicine
  31. Cell Stem Cell. 2021 Feb 18. pii: S1934-5909(21)00017-5. [Epub ahead of print]
      Tissue damage dramatically alters how cells interact with their microenvironment. These changes in turn dictate cellular responses, such as stem cell activation, yet early cellular responses in vivo remain ill defined. We generated single-cell and nucleus atlases from intact, dissociated, and injured muscle and liver and identified a common stress response signature shared by multiple cell types across these organs. This prevalent stress response was detected in published datasets across a range of tissues, demonstrating high conservation but also a significant degree of data distortion in single-cell reference atlases. Using quiescent muscle stem cells as a paradigm of cell activation following injury, we captured early cell activation following muscle injury and found that an essential ERK1/2 primary proliferation signal precedes initiation of the Notch-regulated myogenic program. This study defines initial events in response to tissue perturbation and identifies a broadly conserved transcriptional stress response that acts in parallel with cell-specific adaptive alterations.
    Keywords:  ERK signaling; Notch signaling; muscle stem cells; polyamine synthesis; quiescence/activation; single-cell/single-nucleus atlases; stress response
  32. Neurotherapeutics. 2021 Feb 23.
      This phase 2, double-blind, placebo-controlled, hypothesis-generating study evaluated the effects of oral reldesemtiv, a fast skeletal muscle troponin activator, in patients with spinal muscular atrophy (SMA). Patients ≥ 12 years of age with type II, III, or IV SMA were randomized into 2 sequential, ascending reldesemtiv dosing cohorts (cohort 1: 150 mg bid or placebo [2:1]; cohort 2: 450 mg bid or placebo [2:1]). The primary objective was to determine potential pharmacodynamic effects of reldesemtiv on 8 outcome measures in SMA, including 6-minute walk distance (6MWD) and maximum expiratory pressure (MEP). Changes from baseline to weeks 4 and 8 were determined. Pharmacokinetics and safety were also evaluated. Patients were randomized to reldesemtiv 150 mg, 450 mg, or placebo (24, 20, and 26, respectively). The change from baseline in 6MWD was greater for reldesemtiv 450 mg than for placebo at weeks 4 and 8 (least squares [LS] mean difference, 35.6 m [p = 0.0037] and 24.9 m [p = 0.058], respectively). Changes from baseline in MEP at week 8 on reldesemtiv 150 and 450 mg were significantly greater than those on placebo (LS mean differences, 11.7 [p = 0.038] and 13.2 cm H2O [p = 0.03], respectively). For 6MWD and MEP, significant changes from placebo were seen in the highest reldesemtiv peak plasma concentration quartile (Cmax > 3.29 μg/mL; LS mean differences, 43.3 m [p = 0.010] and 28.8 cm H2O [p = 0.0002], respectively). Both dose levels of reldesemtiv were well tolerated. Results suggest reldesemtiv may offer clinical benefit and support evaluation in larger SMA patient populations.
    Keywords:  Reldesemtiv; pharmacodynamics; pharmacokinetics; six-minute walk test; spinal muscular atrophy clinical trial