bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2022‒08‒14
forty-one papers selected by
Anna Vainshtein
Craft Science Inc.
  1. Collagen VI ablation in zebrafish causes neuromuscular defects during developmental and adult stages.
  2. Sex-dependent role of Pannexin 1 in regulating skeletal muscle and satellite cell function.
  3. Impact of Aging and Lifelong Exercise Training on Mitochondrial Function and Network Connectivity in Human Skeletal Muscle.
  4. SESN2 prevents the slow-to-fast myofiber shift in denervated atrophy via AMPK/PGC-1α pathway.
  5. Suppression of endoplasmic reticulum stress prevents disuse muscle atrophy in a mouse model of microgravity.
  6. Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.
  7. Myonuclear permanence in skeletal muscle memory: a systematic review and meta-analysis of human and animal studies.
  8. Nicotinamide riboside alleviates exercise intolerance in ANT1-deficient mice.
  9. Calorie Restriction Rescues Mitochondrial Dysfunction in Adck2-Deficient Skeletal Muscle.
  10. Molecular Mechanisms Underlying Intensive Care Unit-Acquired Weakness and Sarcopenia.
  11. Skeletal muscle macrophage ablation in mice.
  12. Postdevelopmental knockout of Orai1 improves muscle pathology in a mouse model of Duchenne muscular dystrophy.
  13. MiR-495-3p regulates myoblasts proliferation and differentiation through targeting cadherin 2.
  14. Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity.
  15. Postnatal Protein Intake as a Determinant of Skeletal Muscle Structure and Function in Mice-A Pilot Study.
  16. The key role of histone H3 trimethylation in skeletal muscle adaptation to exercise.
  17. Skeletal muscle deconditioning during partial weight-bearing in rodents - A systematic review and meta-analysis.
  18. OPA1 regulation of mitochondrial dynamics in skeletal and cardiac muscle.
  19. Effect of Physical Activity/Exercise on Oxidative Stress and Inflammation in Muscle and Vascular Aging.
  20. Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice.
  21. The Effect of Sleep Restriction, With or Without Exercise, on Skeletal Muscle Transcriptomic Profiles in Healthy Young Males.
  22. Exon skipping in genes encoding lineage-defining myogenic transcription factors in rhabdomyosarcoma.
  23. Identification of a novel heterozygous DYSF variant in a large family with a dominantly-inherited dysferlinopathy.
  24. Long-term voluntary wheel running effects on markers of Long Interspersed Nuclear Element-1 in skeletal muscle, liver, and brain tissue of female rats.
  25. Nuclear receptors as potential therapeutic targets in peripheral arterial disease and related myopathy.
  26. The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells.
  27. In vivo transomic analyses of glucose-responsive metabolism in skeletal muscle reveal core differences between the healthy and obese states.
  28. Computational modeling of damage in the hierarchical microstructure of skeletal muscles.
  29. Inhibition of TNF-α Restores Muscle Force, Inhibits Inflammation, and Reduces Apoptosis of Traumatized Skeletal Muscles.
  30. Monocarboxylate transporters (MCTs) in skeletal muscle and hypothalamus of less or more physically active mice exposed to aerobic training.
  31. NADPH and Mitochondrial Quality Control as Targets for a Circadian-Based Fasting and Exercise Therapy for the Treatment of Parkinson's Disease.
  32. Myonuclear addition is associated with sex-specific fiber hypertrophy and occurs in relation to fiber perimeter not cross-sectional area.
  33. Visual three-dimensional spatial distribution of motor neurons innervating superficial limb muscles in mice.
  34. Prophylactic exercise-derived circulating exosomal miR-125a-5p promotes endogenous revascularization after hindlimb ischemia by targeting endothelin converting enzyme 1.
  35. Walking or body weight squat 'activity snacks' increase dietary amino acid utilization for myofibrillar protein synthesis during prolonged sitting.
  36. A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.
  37. Nitrite lowers the oxygen cost of ATP supply in cultured skeletal muscle cells by stimulating the rate of glycolytic ATP synthesis.
  38. Matrix stiffness and architecture drive fibro-adipogenic progenitors' activation into myofibroblasts.
  39. Systemic delivery of an AAV9 exon-skipping vector significantly improves or prevents features of Duchenne muscular dystrophy in the Dup2 mouse.
  40. mRNA m5C inhibits adipogenesis and promotes myogenesis by respectively facilitating YBX2 and SMO mRNA export in ALYREF-m5C manner.
  41. Structure and Function of the Mammalian Neuromuscular Junction.
  1. Matrix Biol. 2022 Aug 09. pii: S0945-053X(22)00098-1. [Epub ahead of print]
    Collagen VI ablation in zebrafish causes neuromuscular defects during developmental and adult stages.
    Valentina Tonelotto, Chiara Consorti, Nicola Facchinello, Valeria Trapani, Patrizia Sabatelli, Chiara Giraudo, Marianna Spizzotin, Matilde Cescon, Cristiano Bertolucci, Paolo Bonaldo.
      Collagen VI (COL6) is an extracellular matrix protein exerting multiple functions in different tissues. In humans, mutations of COL6 genes cause rare inherited congenital disorders, primarily affecting skeletal muscles and collectively known as COL6-related myopathies, for which no cure is available yet. In order to get insights into the pathogenic mechanisms underlying COL6-related diseases, diverse animal models were produced. However, the roles exerted by COL6 during embryogenesis remain largely unknown. Here, we generated the first zebrafish COL6 knockout line through CRISPR/Cas9 site-specific mutagenesis of the col6a1 gene. Phenotypic characterization during embryonic and larval development revealed that lack of COL6 leads to neuromuscular defects and motor dysfunctions, together with distinctive alterations in the three-dimensional architecture of craniofacial cartilages. These phenotypic features were maintained in adult col6a1 null fish, which displayed defective muscle organization and impaired swimming capabilities. Moreover, col6a1 null fish showed autophagy defects and organelle abnormalities at both embryonic and adult stages, thus recapitulating the main features of patients affected by COL6-related myopathies. Mechanistically, lack of COL6 led to increased BMP signaling, and direct inhibition of BMP activity ameliorated the locomotor col6a1 null embryos. Finally performance of, treatment with salbutamol, a  β2-adrenergic receptor agonist, elicited a significant amelioration of the neuromuscular and motility defects of col6a1 null fish embryos. Altogether, these findings indicate that this newly generated zebrafish col6a1 null line is a valuable in vivo tool to model COL6-related myopathies and suitable for drug screenings aimed at addressing the quest for effective therapeutic strategies for these disorders.
    Keywords:  Animal model; Collagen VI; Neuromuscular system; Skeletal development; Skeletal muscle; Zebrafish
    DOI:  https://doi.org/10.1016/j.matbio.2022.08.004
  2. J Cell Physiol. 2022 Aug 08.
    Sex-dependent role of Pannexin 1 in regulating skeletal muscle and satellite cell function.
    Emily Freeman, Stéphanie Langlois, Kaylee Scott, Aymeric Ravel-Chapuis, Bernard J Jasmin, Kyle N Cowan.
      The development and regeneration of skeletal muscle are mediated by satellite cells (SCs), which ensure the efficient formation of myofibers while repopulating the niche that allows muscle repair following injuries. Pannexin 1 (Panx1) channels are expressed in SCs and their levels increase during differentiation in vitro, as well as during skeletal muscle development and regeneration in vivo. Panx1 has recently been shown to regulate muscle regeneration by promoting bleb-based myoblast migration and fusion. While skeletal muscle is largely influenced in a sex-specific way, the sex-dependent roles of Panx1 in regulating skeletal muscle and SC function remain to be investigated. Here, using global Panx1 knockout (KO) mice, we demonstrate that Panx1 loss reduces muscle fiber size and strength, decreases SC number, and alters early SC differentiation and myoblast fusion in male, but not in female mice. Interestingly, while both male and female Panx1 KO mice display an increase in the number of regenerating fibers following acute injury, the newly formed fibers in male Panx1 KO mice are smaller. Overall, our results demonstrate that Panx1 plays a significant role in regulating muscle development, regeneration, and SC number and function in male mice and reveal distinct sex-dependent functions of Panx1 in skeletal muscle.
    Keywords:  Pannexin 1; fusion; muscle development; muscle regeneration; myoblast; satellite cell; sex-dependent
    DOI:  https://doi.org/10.1002/jcp.30850
  3. J Gerontol A Biol Sci Med Sci. 2022 Aug 12. pii: glac164. [Epub ahead of print]
    Impact of Aging and Lifelong Exercise Training on Mitochondrial Function and Network Connectivity in Human Skeletal Muscle.
    Stine Ringholm, Anders Gudiksen, Jens Frey Halling, Albina Qoqaj, Philip Meizner Rasmussen, Clara Prats, Peter Plomgaard, Henriette Pilegaard.
      Aging is associated with metabolic decline and reduction in mitochondrial function in skeletal muscle which can be delayed by physical activity. Moreover, exercise training has been shown to prevent age-associated decline in mitochondrial function and fragmentation of the mitochondrial network in mouse skeletal muscle. However, whether lifelong endurance exercise training exerts the same effects in human skeletal muscle is still not clear. Therefore, the aim of the present study was to examine the effect of volume-dependent lifelong endurance exercise training on mitochondrial function and network connectivity in older human skeletal muscle. Skeletal muscle complex I+II-linked mitochondrial respiration per tissue mass was higher, but intrinsic complex I+II-linked mitochondrial respiration was lower in highly trained older than in young untrained, older untrained and older moderately trained men. Mitochondrial volume and connectivity were higher in highly trained older than in untrained and moderately trained older subjects. Furthermore, protein content of the ADP/ATP exchangers ANT1 + 2 and VDAC was higher and of the mitophagic marker Parkin lower in skeletal muscle from the highly trained older than from untrained and moderately trained older subjects. In contrast, H2O2 emission in skeletal muscle was not affected by either age or exercise training, but SOD2 protein content was higher in highly trained older than in untrained and moderately trained older subjects. This suggests that healthy aging does not induce oxidative stress or mitochondrial network fragmentation in human skeletal muscle, but high-volume exercise training increases mitochondrial volume and network connectivity, thereby increasing oxidative capacity in older human skeletal muscle.
    Keywords:  Mitochondria; mitophagy; physical activity; respirometry; skeletal muscle
    DOI:  https://doi.org/10.1093/gerona/glac164
  4. Cell Mol Biol Lett. 2022 Aug 09. 27(1): 66
    SESN2 prevents the slow-to-fast myofiber shift in denervated atrophy via AMPK/PGC-1α pathway.
    Xiaofan Yang, Pingping Xue, Zhenyu Liu, Wenqing Li, Chuyan Li, Zhenbing Chen.
      BACKGROUND: Sestrin2 (SESN2), a stress-inducible protein, has been reported to protect against denervated muscle atrophy through unfolded protein response and mitophagy, while its role in myofiber type transition remains unknown.METHODS: A mouse sciatic nerve transection model was created to evaluate denervated muscle atrophy. Myofiber type transition was confirmed by western blot, fluorescence staining, ATP quantification, and metabolic enzyme activity analysis. Adeno-associated virus (AAV) was adopted to achieve SESN2 knockdown and overexpression in gastrocnemius. AMPK/PGC-1α signal was detected by western blot and activated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). C2C12 myotubes with rotenone treatment were adopted for in vitro experiments.
    RESULTS: SESN2 was found to be upregulated in denervated skeletal muscles and rotenone-treated C2C12 cells. Knockdown of SESN2 aggravated muscle atrophy and accelerated myofiber type transition from slow-twitch to fast-twitch. Moreover, AMPK/PGC-1α signaling was proven to be activated by SESN2 after denervation, which further induced the expression of hypoxia-inducible factor HIF2α. Exogenous activation of AMPK/PGC-1α signaling could counteract the addition of slow-to-fast myofiber shift caused by SESN2 knockdown and lead to the retainment of muscle mass after denervation.
    CONCLUSION: Collectively, the present study indicates that SESN2 prevents myofiber type transition from slow-twitch to fast-twitch and preserves muscle mass in denervated atrophy via AMPK/PGC-1α signaling. These findings contribute to a better understanding of the pathogenesis of muscle atrophy and provide novel insights into the role of SESN2 in myofiber type transition.
    Keywords:  AMPK/PGC-1α; Denervation; Myofiber type transition; SESN2; Skeletal muscle atrophy
    DOI:  https://doi.org/10.1186/s11658-022-00367-z
  5. Life Sci Space Res (Amst). 2022 Aug;pii: S2214-5524(22)00040-2. [Epub ahead of print]34 45-52
    Suppression of endoplasmic reticulum stress prevents disuse muscle atrophy in a mouse model of microgravity.
    Zeinab Ibrahim, Gopika Ramachandran, Waseem El-Huneidi, Adel Elmoselhi, Rizwan Qaisar.
      BACKGROUND: Hind-limb unloaded (HLU) mouse model exhibits skeletal muscle atrophy and weakness mimicking the conditions such as prolonged spaceflight. However, the molecular mechanisms and interventions of muscle loss during muscle unloading remain elusive. Dysfunction of protein folding by ednoplasmic reticulum (ER), a condition called ER stress, is implicated in diseases of various cell types, but its contribution to skeletal muscle detriment remains elusive. In this study, we investigated the contribution of ER stress to muscle atrophy.METHODS: Sixteen-week-old c57BL/6j male mice were grouped into ground-based controls and HLU group, which was subsequently injected with injected saline (HLU-sal.) or pan-ER stress inhibitor 4-PBA (100mg/kg/d; HLU- 4PBA) via intraperitoneal injections for three weeks.
    RESULTS: Three weeks of HLU resulted in reduction in muscle mass and strength, which were restored with 4PBA injections. We also report myofibers atrophy, myonuclear apoptosis, and aterations in the expressions of genes associated with ER stress, apoptosis, and calcium dysregulation. These findings were reversed by 4-PBA treatment.
    CONCLUSION: Altogether, our results indicate that ER stress contributes to muscle atrophy in HLU conditions. We suggest that blocking ER stress may be an effective pharmacological therapy to prevent muscle weakness and atrophy during prolonged muscle unloading.
    Keywords:  4-phenyl butyrate; Endoplasmic reticulum stress; Gastrocnemius muscle; Hindlimb unloading
    DOI:  https://doi.org/10.1016/j.lssr.2022.06.005
  6. Adv Biol Regul. 2022 Jul 31. pii: S2212-4926(22)00043-4. [Epub ahead of print]85 100903
    Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.
    Natalia Vilchinskaya, Erzhena Altaeva, Yulia Lomonosova.
      Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.
    Keywords:  E3-ubiquitin ligases; Forkhead box O 1; Myosin heavy chains; Protein synthesis; Sestrins; Skeletal muscle atrophy
    DOI:  https://doi.org/10.1016/j.jbior.2022.100903
  7. J Cachexia Sarcopenia Muscle. 2022 Aug 12.
    Myonuclear permanence in skeletal muscle memory: a systematic review and meta-analysis of human and animal studies.
    Masoud Rahmati, John J McCarthy, Fatemeh Malakoutinia.
      One aspect of skeletal muscle memory is the ability of a previously trained muscle to hypertrophy more rapidly following a period of detraining. Although the molecular basis of muscle memory remains to be fully elucidated, one potential mechanism thought to mediate muscle memory is the permanent retention of myonuclei acquired during the initial phase of hypertrophic growth. However, myonuclear permanence is debated and would benefit from a meta-analysis to clarify the current state of the field for this important aspect of skeletal muscle plasticity. The objective of this study was to perform a meta-analysis to assess the permanence of myonuclei associated with changes in physical activity and ageing. When available, the abundance of satellite cells (SCs) was also considered given their potential influence on changes in myonuclear abundance. One hundred forty-seven peer-reviewed articles were identified for inclusion across five separate meta-analyses; (1-2) human and rodent studies assessed muscle response to hypertrophy; (3-4) human and rodent studies assessed muscle response to atrophy; and (5) human studies assessed muscle response with ageing. Skeletal muscle hypertrophy was associated with higher myonuclear content that was retained in rodents, but not humans, with atrophy (SMD = -0.60, 95% CI -1.71 to 0.51, P = 0.29, and MD = 83.46, 95% CI -649.41 to 816.32, P = 0.82; respectively). Myonuclear and SC content were both lower following atrophy in humans (MD = -11, 95% CI -0.19 to -0.03, P = 0.005, and SMD = -0.49, 95% CI -0.77 to -0.22, P = 0.0005; respectively), although the response in rodents was affected by the type of muscle under consideration and the mode of atrophy. Whereas rodent myonuclei were found to be more permanent regardless of the mode of atrophy, atrophy of ≥30% was associated with a reduction in myonuclear content (SMD = -1.02, 95% CI -1.53 to -0.51, P = 0.0001). In humans, sarcopenia was accompanied by a lower myonuclear and SC content (MD = 0.47, 95% CI 0.09 to 0.85, P = 0.02, and SMD = 0.78, 95% CI 0.37-1.19, P = 0.0002; respectively). The major finding from the present meta-analysis is that myonuclei are not permanent but are lost during periods of atrophy and with ageing. These findings do not support the concept of skeletal muscle memory based on the permanence of myonuclei and suggest other mechanisms, such as epigenetics, may have a more important role in mediating this aspect of skeletal muscle plasticity.
    Keywords:  Ageing; Hypertrophy; Meta-analysis; Muscle memory; Myonuclei; Satellite cell
    DOI:  https://doi.org/10.1002/jcsm.13043
  8. Mol Metab. 2022 Aug 06. pii: S2212-8778(22)00129-6. [Epub ahead of print] 101560
    Nicotinamide riboside alleviates exercise intolerance in ANT1-deficient mice.
    Patrick M Schaefer, Jessica Huang, Arrienne Butic, Caroline Perry, Tal Yardeni, Wendy Tan, Ryan Morrow, Joseph A Baur, Douglas C Wallace.
      OBJECTIVE: Mitochondrial disorders are often characterized by muscle weakness and fatigue. Null mutations in the heart-muscle adenine nucleotide translocator isoform 1 (ANT1) of both humans and mice cause cardiomyopathy and myopathy associated with exercise intolerance and muscle weakness. Here we decipher the molecular underpinnings of ANT1-deficiency-mediated exercise intolerance.METHODS: This was achieved by correlating exercise physiology, mitochondrial function and metabolomics of mice deficient in ANT1 and comparing this to control mice.
    RESULTS: We demonstrate a peripheral limitation of skeletal muscle mitochondrial respiration and a reduced complex I respiration in ANT1-deficient mice. Upon exercise, this results in a lack of NAD+ leading to a substrate limitation and stalling of the TCA cycle and mitochondrial respiration, further limiting skeletal muscle mitochondrial respiration. Treatment of ANT1-deficient mice with nicotinamide riboside increased NAD+ levels in skeletal muscle and liver, which increased the exercise capacity and the mitochondrial respiration.
    CONCLUSION: Increasing NAD + levels with nicotinamide riboside can alleviate the exercise intolerance associated to ANT1-deficiency, indicating the therapeutic potential of NAD+-stimulating compounds in mitochondrial myopathies.
    Keywords:  Exercise; Mitochondrial disorder; NAD(+)/NADH; Nicotinamide riboside
    DOI:  https://doi.org/10.1016/j.molmet.2022.101560
  9. Front Physiol. 2022 ;13 898792
    Calorie Restriction Rescues Mitochondrial Dysfunction in Adck2-Deficient Skeletal Muscle.
    Juan Diego Hernández-Camacho, Daniel J M Fernández-Ayala, Cristina Vicente-García, Ignacio Navas-Enamorado, Guillermo López-Lluch, Clara Oliva, Rafael Artuch, Judith Garcia-Villoria, Antonia Ribes, Rafael de Cabo, Jaime J Carvajal, Plácido Navas.
      ADCK2 haploinsufficiency-mediated mitochondrial coenzyme Q deficiency in skeletal muscle causes mitochondrial myopathy associated with defects in beta-oxidation of fatty acids, aged-matched metabolic reprogramming, and defective physical performance. Calorie restriction has proven to increase lifespan and delay the onset of chronic diseases associated to aging. To study the possible treatment by food deprivation, heterozygous Adck2 knockout mice were fed under 40% calorie restriction (CR) and the phenotype was followed for 7 months. The overall glucose and fatty acids metabolism in muscle was restored in mutant mice to WT levels after CR. CR modulated the skeletal muscle metabolic profile of mutant mice, partially rescuing the profile of WT animals. The analysis of mitochondria isolated from skeletal muscle demonstrated that CR increased both CoQ levels and oxygen consumption rate (OCR) based on both glucose and fatty acids substrates, along with mitochondrial mass. The elevated aerobic metabolism fits with an increase of type IIa fibers, and a reduction of type IIx in mutant muscles, reaching WT levels. To further explore the effect of CR over muscle stem cells, satellite cells were isolated and induced to differentiate in culture media containing serum from animals in either ad libitum or CR diets for 72 h. Mutant cells showed slower differentiation alongside with decreased oxygen consumption. In vitro differentiation of mutant cells was increased under CR serum reaching levels of WT isolated cells, recovering respiration measured by OCR and partially beta-oxidation of fatty acids. The overall increase of skeletal muscle bioenergetics following CR intervention is paralleled with a physical activity improvement, with some increases in two and four limbs strength tests, and weights strength test. Running wheel activity was also partially improved in mutant mice under CR. These results demonstrate that CR intervention, which has been shown to improve age-associated physical and metabolic decline in WT mice, also recovers the defective aerobic metabolism and differentiation of skeletal muscle in mice caused by ADCK2 haploinsufficiency.
    Keywords:  coenzyme Q; fatty acids; food deprivation; metabolism; mitochondria
    DOI:  https://doi.org/10.3389/fphys.2022.898792
  10. Int J Mol Sci. 2022 Jul 29. pii: 8396. [Epub ahead of print]23(15):
    Molecular Mechanisms Underlying Intensive Care Unit-Acquired Weakness and Sarcopenia.
    Marcela Kanova, Pavel Kohout.
      Skeletal muscle is a highly adaptable organ, and its amount declines under catabolic conditions such as critical illness. Aging is accompanied by a gradual loss of muscle, especially when physical activity decreases. Intensive care unit-acquired weakness is a common and highly serious neuromuscular complication in critically ill patients. It is a consequence of critical illness and is characterized by a systemic inflammatory response, leading to metabolic stress, that causes the development of multiple organ dysfunction. Muscle dysfunction is an important component of this syndrome, and the degree of catabolism corresponds to the severity of the condition. The population of critically ill is aging; thus, we face another negative effect-sarcopenia-the age-related decline of skeletal muscle mass and function. Low-grade inflammation gradually accumulates over time, inhibits proteosynthesis, worsens anabolic resistance, and increases insulin resistance. The cumulative consequence is a gradual decline in muscle recovery and muscle mass. The clinical manifestation for both of the above conditions is skeletal muscle weakness, with macromolecular damage, and a common mechanism-mitochondrial dysfunction. In this review, we compare the molecular mechanisms underlying the two types of muscle atrophy, and address questions regarding possible shared molecular mechanisms, and whether critical illness accelerates the aging process.
    Keywords:  intensive care unit-acquired weakness; muscle atrophy; proteostasis; rapamycin system; sarcopenia; ubiquitin–proteasome system
    DOI:  https://doi.org/10.3390/ijms23158396
  11. J Immunol Methods. 2022 Aug 03. pii: S0022-1759(22)00116-8. [Epub ahead of print] 113329
    Skeletal muscle macrophage ablation in mice.
    Jennifer T W Krall, Kevin W Gibbs, Lanazha Belfield, Chun Liu, Lina Purcell, Joseph J Bivona, Matthew E Poynter, Renee D Stapleton, Michael J Toth, D Clark Files.
      Macrophages are important mediators of skeletal muscle function in both healthy and diseased states. In vivo specific depletion of macrophages provides an experimental method to understand physiological and pathophysiological effects of macrophages. Systemic depletion of macrophages can deplete skeletal muscle macrophages but also alters systemic inflammatory responses and metabolism, which confounds the muscle specific effects of macrophage depletion. The primary aim of this manuscript is to evaluate two methods of murine intramuscular macrophage depletion in an acute lung injury-associated indirect skeletal muscle wasting mouse model. Adult C57BL/6 (WT) and Macrophage Fas-Induced Apoptosis (MaFIA, C57BL/6-Tg) mice received clodronate liposomes or the dimerization drug AP20187 through intramuscular injection of the tibialis anterior muscle compartment, respectively. Vehicle control was injected in the contralateral muscle. We demonstrate intramuscular AP20187 in the MaFIA mouse depletes macrophages but causes an infiltration of CD45 intermediate neutrophils. In contrast, intramuscular clodronate liposomes successfully depletes macrophages without an associated increase in CD45 intermediate cells. In conclusion, intramuscular clodronate is effective for selective depletion of muscle macrophages without eliciting acute inflammation seen with AP20187 in MaFIA mice. This technique is an important tool to study the functional roles of macrophages in skeletal muscle.
    Keywords:  Clodronate liposomes; Conditional macrophage depletion; Inflammation; Skeletal muscle; Transgenic mouse model
    DOI:  https://doi.org/10.1016/j.jim.2022.113329
  12. J Gen Physiol. 2022 Sep 05. pii: e202213081. [Epub ahead of print]154(9):
    Postdevelopmental knockout of Orai1 improves muscle pathology in a mouse model of Duchenne muscular dystrophy.
    Maricela García-Castañeda, Antonio Michelucci, Nan Zhao, Sundeep Malik, Robert T Dirksen.
      Duchenne muscular dystrophy (DMD), an X-linked disorder caused by loss-of-function mutations in the dystrophin gene, is characterized by progressive muscle degeneration and weakness. Enhanced store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism coordinated by STIM1 sensors of luminal Ca2+ within the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the sarcolemma, is proposed to contribute to Ca2+-mediated muscle damage in DMD. To directly determine the impact of Orai1-dependent SOCE on the dystrophic phenotype, we crossed mdx mice with tamoxifen-inducible, muscle-specific Orai1 knockout mice (mdx-Orai1 KO mice). Both constitutive and SOCE were significantly increased in flexor digitorum brevis fibers from mdx mice, while SOCE was absent in fibers from both Orai1 KO and mdx-Orai1 KO mice. Compared with WT mice, fibers from mdx mice exhibited (1) increased resting myoplasmic Ca2+ levels, (2) reduced total releasable Ca2+ store content, and (3) a prolonged rate of electrically evoked Ca2+ transient decay. These effects were partially normalized in fibers from mdx-Orai1 KO mice. Intact extensor digitorum longus muscles from mdx mice exhibited a significant reduction of maximal specific force, which was rescued in muscles from mdx-Orai1 KO mice. Finally, during exposure to consecutive eccentric contractions, muscles from mdx mice displayed a more pronounced decline in specific force compared with that of WT mice, which was also significantly attenuated by Orai1 ablation. Together, these results indicate that enhanced Orai1-dependent SOCE exacerbates the dystrophic phenotype and that Orai1 deficiency improves muscle pathology by both normalizing Ca2+ homeostasis and promoting sarcolemmal integrity/stability.
    DOI:  https://doi.org/10.1085/jgp.202213081
  13. Anim Biotechnol. 2022 Aug 11. 1-9
    MiR-495-3p regulates myoblasts proliferation and differentiation through targeting cadherin 2.
    Chengchuang Song, Qi Wang, Qi Qi, Xi Chen, Yanhong Wang, Chunlei Zhang, Fang Xintang.
      MircoRNAs (miRNAs) play an important role in skeletal muscle development. Previous study had found that miR-495-3p was differentially expressed in fetal and adult goat skeletal muscle, but its function in myogenic proliferation and differentiation are unclear. Herein, we found the expression of miR-495-3p in C2C12 was downregulated during proliferation stage and upregulated during differentiation stage. Functionally, overexpression of miR-495-3p in C2C12 inhibited proliferation, and promoted myogenic differentiation. Mechanistically, the luciferase reporter assay demonstrated that cadherin 2 (CDH2) was a potential target gene of miR-495-3p. Importantly, overexpression of miR-495-3p inhibited CDH2 expression. Furthermore, knockdown of CDH2 in C2C12 inhibited proliferation and promoted myogenic differentiation. Together, the results showed that miR-495-3p inhibits C2C12 proliferation and promotes myogenic differentiation through targeting CDH2.
    Keywords:  cadherin 2; differentiation; miR-495-3p; myoblast; proliferation
    DOI:  https://doi.org/10.1080/10495398.2022.2109042
  14. J Cachexia Sarcopenia Muscle. 2022 Aug 12.
    Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity.
    Lai Wang, Zuguo Zheng, Lijun Zhu, Lingchang Meng, Hanling Liu, Keke Wang, Jun Chen, Ping Li, Hua Yang.
      BACKGROUND: Statins are widely prescribed cholesterol-lowering drugs but have been shown to increase the risk of type 2 diabetes mellitus. However, the molecular mechanisms underlying the diabetogenic effect of statins are still not fully understood.METHODS: The effects of geranylgeranyl transferase I and II (GGTase I and II) inhibition on insulin-stimulated glucose uptake and GLUT4 translocation, and the dependence of these effects on insulin signalling were investigated in skeletal muscle cells. The protective effects of geranylgeranyl pyrophosphate (GGPP) and its precursor geranylgeraniol (GGOH) on simvastatin-induced insulin resistance were evaluated in vitro and in vivo. The effect of GGTase II inhibition in skeletal muscle on insulin sensitivity in vivo was confirmed by adeno-associated virus serotype 9 (AAV9)-mediated knockdown of the specific subunit of GGTase II, RABGGTA. The regulatory mechanisms of GGTase I on insulin signalling and GGTase II on insulin-stimulated GLUT4 translocation were investigated by knockdown of RhoA, TAZ, IRS1, geranylgeranylation site mutation of RhoA, RAB8A, and RAB13.
    RESULTS: Both inhibition of GGTase I and II mimicked simvastatin-induced insulin resistance in skeletal muscle cells. GGPP and GGOH were able to prevent simvastatin-induced skeletal muscle insulin resistance in vitro and in vivo. GGTase I inhibition suppressed the phosphorylation of AKT (Ser473) (-51.3%, P < 0.01), while GGTase II inhibition had no effect on it. AAV9-mediated knockdown of RABGGTA in skeletal muscle impaired glucose disposal without disrupting insulin signalling in vivo (-46.2% for gastrocnemius glucose uptake, P < 0.001; -52.5% for tibialis anterior glucose uptake, P < 0.001; -17.8% for soleus glucose uptake, P < 0.05; -31.4% for extensor digitorum longus glucose uptake, P < 0.01). Inhibition of RhoA, TAZ, IRS1, or geranylgeranylation deficiency of RhoA attenuated the beneficial effect of GGPP on insulin signalling in skeletal muscle cells. Geranylgeranylation deficiency of RAB8A inhibited insulin-stimulated GLUT4 translocation and concomitant glucose uptake in skeletal muscle cells (-42.8% for GLUT4 translocation, P < 0.01; -50.6% for glucose uptake, P < 0.001).
    CONCLUSIONS: Geranylgeranyl pyrophosphate regulates glucose uptake via GGTase I-mediated insulin signalling-dependent way and GGTase II-mediated insulin signalling-independent way in skeletal muscle. Supplementation of GGPP/GGOH could be a potential therapeutic strategy for statin-induced insulin resistance.
    Keywords:  GGPP; Insulin resistance; RAB8A; RhoA; Skeletal muscle; Statin
    DOI:  https://doi.org/10.1002/jcsm.13061
  15. Int J Mol Sci. 2022 Aug 08. pii: 8815. [Epub ahead of print]23(15):
    Postnatal Protein Intake as a Determinant of Skeletal Muscle Structure and Function in Mice-A Pilot Study.
    Ifigeneia Giakoumaki, Natalie Pollock, Turki Aljuaid, Anthony J Sannicandro, Moussira Alameddine, Euan Owen, Ioanna Myrtziou, Susan E Ozanne, Ioannis Kanakis, Katarzyna Goljanek-Whysall, Aphrodite Vasilaki.
      Sarcopenia is characterised by an age-related decrease in the number of muscle fibres and additional weakening of the remaining fibres, resulting in a reduction in muscle mass and function. Many studies associate poor maternal nutrition during gestation and/or lactation with altered skeletal muscle homeostasis in the offspring and the development of sarcopenia. The aim of this study was to determine whether the musculoskeletal physiology in offspring born to mouse dams fed a low-protein diet during pregnancy was altered and whether any physiological changes could be modulated by the nutritional protein content in early postnatal stages. Thy1-YFP female mice were fed ad libitum on either a normal (20%) or a low-protein (5%) diet. Newborn pups were cross-fostered to different lactating dams (maintained on a 20% or 5% diet) to generate three groups analysed at weaning (21 days): Normal-to-Normal (NN), Normal-to-Low (NL) and Low-to-Normal (LN). Further offspring were maintained ad libitum on the same diet as during lactation until 12 weeks of age, creating another three groups (NNN, NLL, LNN). Mice on a low protein diet postnatally (NL, NLL) exhibited a significant reduction in body and muscle weight persisting up to 12 weeks, unlike mice on a low protein diet only prenatally (LN, LNN). Muscle fibre size was reduced in mice from the NL but not LN group, showing recovery at 12 weeks of age. Muscle force was reduced in NLL mice, concomitant with changes in the NMJ site and changes in atrophy-related and myosin genes. In addition, μCT scans of mouse tibiae at 12 weeks of age revealed changes in bone mass and morphology, resulting in a higher bone mass in the NLL group than the control NNN group. Finally, changes in the expression of miR-133 in the muscle of NLL mice suggest a regulatory role for this microRNA in muscle development in response to postnatal diet changes. Overall, this data shows that a low maternal protein diet and early postnatal life low-protein intake in mice can impact skeletal muscle physiology and function in early life while postnatal low protein diet favours bone integrity in adulthood.
    Keywords:  maternal protein restriction; microRNAs; neuromuscular junction; offspring; skeletal muscle
    DOI:  https://doi.org/10.3390/ijms23158815
  16. J Physiol. 2022 Aug 09.
    The key role of histone H3 trimethylation in skeletal muscle adaptation to exercise.
    Tom Normand-Gravier, Robert Solsona, Anthony M J Sanchez.
      
    Keywords:  DNA methylation; endurance exercise; histone
    DOI:  https://doi.org/10.1113/JP283661
  17. Life Sci Space Res (Amst). 2022 Aug;pii: S2214-5524(22)00042-6. [Epub ahead of print]34 68-86
    Skeletal muscle deconditioning during partial weight-bearing in rodents - A systematic review and meta-analysis.
    Patrick Swain, Marie Mortreux, Jonathan M Laws, Harry Kyriacou, Enrico De Martino, Andrew Winnard, Nick Caplan.
      Space agencies are planning to send humans back to the Lunar surface, in preparation for crewed exploration of Mars. However, the effect of hypogravity on human skeletal muscle is largely unknown. A recently established rodent partial weight-bearing model has been employed to mimic various levels of hypogravity loading and may provide valuable insights to better understanding how human muscle might respond to this environment. The aim of this study was to perform a systematic review regarding the effects of partial weight-bearing on the morphology and function of rodent skeletal muscle. Five online databases were searched with the following inclusion criteria: population (rodents), intervention (partial weight-bearing for ≥1 week), control (full weight-bearing), outcome(s) (skeletal muscle morphology/function), and study design (animal intervention). Of the 2,993 studies identified, eight were included. Partial weight-bearing at 20%, 40%, and 70% of full loading caused rapid deconditioning of skeletal muscle morphology and function within the first one to two weeks of exposure. Calf circumference, hindlimb wet muscle mass, myofiber cross-sectional area, front/rear paw grip force, and nerve-stimulated plantarflexion force were reduced typically by medium to very large effects. Higher levels of partial weight-bearing often attenuated deconditioning but failed to entirely prevent it. Species and sex mediated the deconditioning response. Risk of bias was low/unclear for most studies. These findings suggest that there is insufficient stimulus to mitigate muscular deconditioning in hypogravity settings highlighting the need to develop countermeasures for maintaining astronaut/cosmonaut muscular health on the Moon and Mars.
    Keywords:  Atrophy; Hypogravity; Mars; Moon; Musculoskeletal
    DOI:  https://doi.org/10.1016/j.lssr.2022.06.007
  18. Trends Endocrinol Metab. 2022 Aug 06. pii: S1043-2760(22)00134-5. [Epub ahead of print]
    OPA1 regulation of mitochondrial dynamics in skeletal and cardiac muscle.
    John Noone, Donal J O'Gorman, Helena C Kenny.
      The mitochondria are double-membrane organelles integral for energy metabolism. Mitochondrial dynamics is regulated by inner and outer mitochondrial membrane (IMM and OMM) proteins, which promote fission and fusion. Optic atrophy 1 (OPA1) regulates IMM fusion, prevents apoptosis, and is a key regulator of morphological change in skeletal and cardiac muscle physiology and pathophysiology. OPA1 fuses the inner membranes of adjacent mitochondria, allowing for an increase in oxidative phosphorylation (OXPHOS). Considering the importance of energy metabolism in whole-body physiology, OPA1 and its regulators have been proposed as novel targets for the treatment of skeletal muscle atrophy and heart failure. Here, we review the role and regulation of OPA1 in skeletal muscle and cardiac pathophysiology, epitomizing its critical role in the cell.
    Keywords:  exercise; heart; metabolism; mitochondria; skeletal muscle
    DOI:  https://doi.org/10.1016/j.tem.2022.07.003
  19. Int J Mol Sci. 2022 Aug 05. pii: 8713. [Epub ahead of print]23(15):
    Effect of Physical Activity/Exercise on Oxidative Stress and Inflammation in Muscle and Vascular Aging.
    Mariam El Assar, Alejandro Álvarez-Bustos, Patricia Sosa, Javier Angulo, Leocadio Rodríguez-Mañas.
      Functional status is considered the main determinant of healthy aging. Impairment in skeletal muscle and the cardiovascular system, two interrelated systems, results in compromised functional status in aging. Increased oxidative stress and inflammation in older subjects constitute the background for skeletal muscle and cardiovascular system alterations. Aged skeletal muscle mass and strength impairment is related to anabolic resistance, mitochondrial dysfunction, increased oxidative stress and inflammation as well as a reduced antioxidant response and myokine profile. Arterial stiffness and endothelial function stand out as the main cardiovascular alterations related to aging, where increased systemic and vascular oxidative stress and inflammation play a key role. Physical activity and exercise training arise as modifiable determinants of functional outcomes in older persons. Exercise enhances antioxidant response, decreases age-related oxidative stress and pro-inflammatory signals, and promotes the activation of anabolic and mitochondrial biogenesis pathways in skeletal muscle. Additionally, exercise improves endothelial function and arterial stiffness by reducing inflammatory and oxidative damage signaling in vascular tissue together with an increase in antioxidant enzymes and nitric oxide availability, globally promoting functional performance and healthy aging. This review focuses on the role of oxidative stress and inflammation in aged musculoskeletal and vascular systems and how physical activity/exercise influences functional status in the elderly.
    Keywords:  aging; cardiovascular system; exercise; inflammation; muscle; oxidative stress; physical activity
    DOI:  https://doi.org/10.3390/ijms23158713
  20. Nucleic Acids Res. 2022 Aug 10. pii: gkac641. [Epub ahead of print]
    Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice.
    Cody A Desjardins, Monica Yao, John Hall, Emma O'Donnell, Reshmii Venkatesan, Sean Spring, Aiyun Wen, Nelson Hsia, Peiyi Shen, Ryan Russo, Bo Lan, Tyler Picariello, Kim Tang, Timothy Weeden, Stefano Zanotti, Romesh Subramanian, Oxana Ibraghimov-Beskrovnaya.
      Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.
    DOI:  https://doi.org/10.1093/nar/gkac641
  21. Front Endocrinol (Lausanne). 2022 ;13 863224
    The Effect of Sleep Restriction, With or Without Exercise, on Skeletal Muscle Transcriptomic Profiles in Healthy Young Males.
    Wentao Lin, Nicholas J Saner, Xiquan Weng, Nikeisha J Caruana, Javier Botella, Jujiao Kuang, Matthew J-C Lee, Nicholas A Jamnick, Nathan W Pitchford, Andrew Garnham, Jonathan D Bartlett, Hao Chen, David J Bishop.
      Background: Inadequate sleep is associated with many detrimental health effects, including increased risk of developing insulin resistance and type 2 diabetes. These effects have been associated with changes to the skeletal muscle transcriptome, although this has not been characterised in response to a period of sleep restriction. Exercise induces a beneficial transcriptional response within skeletal muscle that may counteract some of the negative effects associated with sleep restriction. We hypothesised that sleep restriction would down-regulate transcriptional pathways associated with glucose metabolism, but that performing exercise would mitigate these effects.Methods: 20 healthy young males were allocated to one of three experimental groups: a Normal Sleep (NS) group (8 h time in bed per night (TIB), for five nights (11 pm - 7 am)), a Sleep Restriction (SR) group (4 h TIB, for five nights (3 am - 7 am)), and a Sleep Restriction and Exercise group (SR+EX) (4 h TIB, for five nights (3 am - 7 am) and three high-intensity interval exercise (HIIE) sessions (performed at 10 am)). RNA sequencing was performed on muscle samples collected pre- and post-intervention. Our data was then compared to skeletal muscle transcriptomic data previously reported following sleep deprivation (24 h without sleep).
    Results: Gene set enrichment analysis (GSEA) indicated there was an increased enrichment of inflammatory and immune response related pathways in the SR group post-intervention. However, in the SR+EX group the direction of enrichment in these same pathways occurred in the opposite directions. Despite this, there were no significant changes at the individual gene level from pre- to post-intervention. A set of genes previously shown to be decreased with sleep deprivation was also decreased in the SR group, but increased in the SR+EX group.
    Conclusion: The alterations to inflammatory and immune related pathways in skeletal muscle, following five nights of sleep restriction, provide insight regarding the transcriptional changes that underpin the detrimental effects associated with sleep loss. Performing three sessions of HIIE during sleep restriction attenuated some of these transcriptional changes. Overall, the transcriptional alterations observed with a moderate period of sleep restriction were less evident than previously reported changes following a period of sleep deprivation.
    Keywords:  circadian rhythm; high-intensity interval exercise (HIE); inflammation; mitochondria; skeletal muscle; sleep loss; sleep restriction; transcriptomics
    DOI:  https://doi.org/10.3389/fendo.2022.863224
  22. Cold Spring Harb Mol Case Stud. 2022 Aug 06. pii: mcs.a006190. [Epub ahead of print]
    Exon skipping in genes encoding lineage-defining myogenic transcription factors in rhabdomyosarcoma.
    Erin Butler, Lin Xu, Dinesh Rakheja, Blake Schwettmann, Shireen Toubbeh, Lei Guo, Jiwoon Kim, Stephen X Skapek, Yanbin Zheng.
      Rhabdomyosarcoma (RMS) is a childhood sarcoma composed of myoblast-like cells, which suggests a defect in terminal skeletal muscle differentiation. To explore potential defects in the differentiation program, we searched for mRNA splicing variants in genes encoding transcription factors driving skeletal muscle lineage commitment and differentiation. We studied two RMS cases and identified altered splicing resulting in "skipping" the second of three exons in MYOD1. RNA-Seq data from 42 tumors and additional RMS cell lines revealed exon 2 skipping in both MYOD1 and MYF5 but not in MYF6 or MYOG. Complementary molecular analysis of MYOD1 mRNA found evidence for exon skipping in 5 additional RMS cases. Functional studies showed that so-called MYODΔEx2 protein failed to robustly induce muscle-specific genes, and its ectopic expression conferred a selective advantage in cultured fibroblasts and an RMS xenograft. In summary, we present previously unrecognized exon skipping within MYOD1 and MYF5 in RMS, and we propose that alternative splicing can represent a mechanism to alter the function of these two transcription factors in RMS.
    Keywords:  Alveolar rhabdomyosarcoma; Embryonal rhabdomyosarcoma
    DOI:  https://doi.org/10.1101/mcs.a006190
  23. Neuropathol Appl Neurobiol. 2022 Aug 12. e12846
    Identification of a novel heterozygous DYSF variant in a large family with a dominantly-inherited dysferlinopathy.
    Chiara Folland, Russell Johnsen, Adriana Botero Gomez, Daniel Trajanoski, Mark R Davis, Ursula Moore, Volker Straub, Rita Barresi, Michela Guglieri, Hannah Hayhurst, Andrew M Schaefer, Nigel G Laing, Philipa J Lamont, Gianina Ravenscroft.
      AIMS: Dysferlinopathy is an autosomal recessive muscular dystrophy, caused by bi-allelic variants in the gene encoding dysferlin (DYSF). Onset typically occurs in the second to third decade and is characterised by slowly progressive skeletal muscle weakness and atrophy of the proximal and/or distal muscles of the four limbs. There are rare cases of symptomatic DYSF variant carriers. Here, we report a large family with a dominantly inherited hyperCKaemia and late-onset muscular dystrophy.METHODS AND RESULTS: Genetic analysis identified a co-segregating novel DYSF variant [NM_003494.4:c.6207del p.(Tyr2070Metfs*4)]. No secondary variants in DYSF or other dystrophy-related genes were identified on whole genome sequencing and analysis of the proband's DNA. Skeletal muscle involvement was milder and later onset than typical dysferlinopathy presentations; these clinical signs manifested in four individuals, all between the fourth and sixth decades of life. All individuals heterozygous for the c.6207del variant had hyperCKaemia. Histological analysis of skeletal muscle biopsies across three generations showed clear dystrophic signs, including inflammatory infiltrates, regenerating myofibres, increased variability in myofibre size, and internal nuclei. Muscle magnetic resonance imaging revealed fatty replacement of muscle in two individuals. Western blot and immunohistochemical analysis of muscle biopsy demonstrated consistent reduction of dysferlin staining. Allele-specific quantitative PCR analysis of DYSF mRNA from patient muscle found that the variant, localised to the extreme C-terminus of dysferlin, does not activate post-transcriptional mRNA decay.
    CONCLUSIONS: We propose that this inheritance pattern may be underappreciated and that other late-onset muscular dystrophy cases with mono-allelic DYSF variants, particularly C-terminal premature truncation variants, may represent dominant forms of disease.
    Keywords:  NGS; dominant; dysferlin; dysferlinopathy; hyperCKaemia; myopathy
    DOI:  https://doi.org/10.1111/nan.12846
  24. Am J Physiol Cell Physiol. 2022 Aug 08.
    Long-term voluntary wheel running effects on markers of Long Interspersed Nuclear Element-1 in skeletal muscle, liver, and brain tissue of female rats.
    Shelby C Osburn, Paulo Mesquita, Frances K Neal, Melissa Rumbley, Matthew T Holmes, Bradley A Ruple, C Brooks Mobley, Michael D Brown, Danielle J McCullough, Andreas N Kavazis, Michael D Roberts.
      We sought to determine the effects of long-term voluntary wheel running on markers of Long Interspersed Nuclear Element-1 (L1) in skeletal muscle, liver, and the hippocampus of female rats. Additionally, markers of the cGAS-STING DNA sensing pathway that results in inflammation were interrogated. Female Lewis rats (n=34) were separated into one of three groups including a 6-month-old group to serve as a young comparator group (CTL, n=10), a group that had access to a running wheel for voluntary wheel running (EX, n=12), and an age-matched group that did not (SED, n=12). Both SED and EX groups were carried out from 6 months to 15 months of age. There were no significant differences in L1 mRNA expression for any of the tissues between groups. Methylation of the L1 promoter in the soleus and hippocampus was significantly higher in SED and EX compared to CTL (p<0.05). ORF1p expression was higher in older SED and EX rats compared to CTL for every tissue (p<0.05). There were no differences between groups for L1 mRNA or cGAS-STING pathway markers. Our results suggest there is an increased ORF1 protein expression across tissues with aging that is not mitigated by voluntary wheel running. Additionally, while previous data imply that L1 methylation changes may play a role in acute exercise for L1 RNA expression, this does not seem to occur during extended periods of voluntary wheel running.
    Keywords:  L1; cGAS-STING; exercise; methylation
    DOI:  https://doi.org/10.1152/ajpcell.00234.2022
  25. FEBS J. 2022 Aug 09.
    Nuclear receptors as potential therapeutic targets in peripheral arterial disease and related myopathy.
    Ashok Kumar, Vihang A Narkar.
      Peripheral arterial disease (PAD) is a prevalent cardiovascular complication of limb vascular insufficiency, causing ischemic injury, mitochondrial metabolic damage, and functional impairment in the skeletal muscle, and ultimately leading to immobility and mortality. While potential therapies have been mostly focused on revascularization, none of the currently available pharmacological treatments are fully effective in PAD, often leading to amputations, particularly in chronic metabolic diseases. One major limitation of focused angiogenesis and revascularization as a therapeutic strategy is limited effect on metabolic restoration and muscle regeneration in the affected limb. Therefore, additional pre-clinical investigations are needed to discover novel treatment options for PAD preferably targeting multiple aspects of muscle recovery. In this review, we propose nuclear receptors expressed in the skeletal muscle as potential candidates for ischemic muscle repair in PAD. We review classic steroid and orphan receptors that have been reported to be involved in the regulation of paracrine muscle angiogenesis, oxidative metabolism, mitochondrial biogenesis and muscle regeneration, and discuss how these receptors could be critical for recovery from ischemic muscle damage. Furthermore, we identify existing gaps in our understanding of nuclear receptor signaling in the skeletal muscle, and propose future areas of research that could be instrumental in exploring nuclear receptors as therapeutic candidates for treating PAD.
    Keywords:  Nuclear receptors; angiogenesis; ischemia; mitochondria; peripheral arterial disease; regeneration; skeletal muscle
    DOI:  https://doi.org/10.1111/febs.16593
  26. Cells. 2022 Aug 06. pii: 2440. [Epub ahead of print]11(15):
    The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells.
    Nicole K Nakamura, Darcy S Tokunaga, Herena Y Ha, Noemi Polgar.
      In obesity, chronic membrane-localization of CD36 free fatty acid (FFA) translocase, but not other FFA transporters, enhances FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation promotes insulin resistance by inhibiting insulin-induced GLUT4 glucose transporter trafficking and glucose uptake. GLUT4 and CD36 cell surface delivery is triggered by insulin- and contraction-induced signaling, which share conserved downstream effectors. While we have gathered detailed knowledge on GLUT4 trafficking, the mechanisms regulating CD36 membrane delivery and subsequent FFA uptake in skeletal muscle are not fully understood. The exocyst trafficking complex facilitates the docking of membrane-bound vesicles, a process underlying the controlled surface delivery of fuel transporters. The exocyst regulates insulin-induced glucose uptake via GLUT4 membrane trafficking in adipocytes and skeletal muscle cells and plays a role in lipid uptake in adipocytes. Based on the high degree of conservation of the GLUT4 and CD36 trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst also contributes to lipid uptake in skeletal muscle and acts through the targeted plasma membrane delivery of CD36 in response to insulin and contraction. Here, we show that the exocyst complex is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA uptake in muscle cells.
    Keywords:  CD36 fatty acid translocase; exocyst; skeletal muscle
    DOI:  https://doi.org/10.3390/cells11152440
  27. Sci Rep. 2022 Aug 12. 12(1): 13719
    In vivo transomic analyses of glucose-responsive metabolism in skeletal muscle reveal core differences between the healthy and obese states.
    Toshiya Kokaji, Miki Eto, Atsushi Hatano, Katsuyuki Yugi, Keigo Morita, Satoshi Ohno, Masashi Fujii, Ken-Ichi Hironaka, Yuki Ito, Riku Egami, Saori Uematsu, Akira Terakawa, Yifei Pan, Hideki Maehara, Dongzi Li, Yunfan Bai, Takaho Tsuchiya, Haruka Ozaki, Hiroshi Inoue, Hiroyuki Kubota, Yutaka Suzuki, Akiyoshi Hirayama, Tomoyoshi Soga, Shinya Kuroda.
      Metabolic regulation in skeletal muscle is essential for blood glucose homeostasis. Obesity causes insulin resistance in skeletal muscle, leading to hyperglycemia and type 2 diabetes. In this study, we performed multiomic analysis of the skeletal muscle of wild-type (WT) and leptin-deficient obese (ob/ob) mice, and constructed regulatory transomic networks for metabolism after oral glucose administration. Our network revealed that metabolic regulation by glucose-responsive metabolites had a major effect on WT mice, especially carbohydrate metabolic pathways. By contrast, in ob/ob mice, much of the metabolic regulation by glucose-responsive metabolites was lost and metabolic regulation by glucose-responsive genes was largely increased, especially in carbohydrate and lipid metabolic pathways. We present some characteristic metabolic regulatory pathways found in central carbon, branched amino acids, and ketone body metabolism. Our transomic analysis will provide insights into how skeletal muscle responds to changes in blood glucose and how it fails to respond in obesity.
    DOI:  https://doi.org/10.1038/s41598-022-17964-9
  28. J Mech Behav Biomed Mater. 2022 Jul 27. pii: S1751-6161(22)00294-6. [Epub ahead of print]134 105386
    Computational modeling of damage in the hierarchical microstructure of skeletal muscles.
    Jens Lamsfuss, Swantje Bargmann.
      One of the skeletal muscle's exceptional properties is its high damage tolerance in terms of its high toughness, which allows the muscle to withstand cracks of millimeter length while maintaining most of its strength (Taylor et al., 2012). In skeletal muscles, damage occurs on different hierarchical levels of the microstructure. We analyze the damage behavior on hierarchy levels 3 (muscle fiber) and 4 (fascicle) on which the most common serious muscle injuries occur. Our model captures damage initiation and rupture of activated muscle fibers resulting from eccentric contractions. We consider the interaction between muscle fibers and endomysium and investigate the influence of the components titin and endomysium on the mechanical behavior in pre-damaged fascicles. Endomysium generally transmits contractile forces. Our results show that high strains in pre-damaged fiber regions are not transferred by the endomysium and, thus, adjacent undamaged fibers are well protected. Moreover, the results show titin's extraordinary stabilization properties of pre-damaged muscle fibers, so that macroscopic strains of fascicles are hardly reduced in case of strongly pre-damaged fibers and intact titin.
    Keywords:  Damage modeling; Eccentric contraction; Finite element method; Muscle injury; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.jmbbm.2022.105386
  29. Cells. 2022 Aug 03. pii: 2397. [Epub ahead of print]11(15):
    Inhibition of TNF-α Restores Muscle Force, Inhibits Inflammation, and Reduces Apoptosis of Traumatized Skeletal Muscles.
    Ioannis Stratos, Ann-Kathrin Behrendt, Christian Anselm, Aldebarani Gonzalez, Thomas Mittlmeier, Brigitte Vollmar.
      BACKGROUND: Muscle injuries are common in humans and are often associated with irrecoverable damage and disability. Upon muscle injury, TNF-α signaling pathways modulate the healing process and are predominantly associated with tissue degradation. In this study we assumed that TNF-α inhibition could reduce the TNF-α-associated tissue degradation after muscle injury.MATERIALS AND METHODS: Therefore, the left soleus muscle of 42 male Wistar rats was injured using a standardized open muscle injury model. All rats were treated immediately after injury either with infliximab (single i.p. injection; 10 mg/kg b.w.) or saline solution i.p. Final measurements were conducted at day one, four, and 14 post injury. The muscle force, the muscle cell proliferation, the muscle cell coverage as well as the myofiber diameter served as read out parameters of our experiment.
    RESULTS: Systemic application of infliximab could significantly reduce the TNF-α levels in the injured muscle at day four upon trauma compared to saline treated animals. The ratio of muscle weight to body weight was increased and the twitch muscle force showed a significant rise 14 days after trauma and TNF-α inhibition. Quantification of myofiber diameter in the penumbra zone showed a significant difference between both groups at day one and four after injury, indicated by muscle hypertrophy in the infliximab group. Planimetric analysis of the injured muscle at day 14 revealed increased muscle tissue fraction in the infliximab group compared to the control animals. Muscle cell proliferation did not differ between both groups.
    CONCLUSIONS: These data provide evidence that the TNF-α blockade positively regulates the restauration of skeletal muscles upon injury.
    Keywords:  infliximab; muscle injury; regeneration; tumor necrosis factor alpha
    DOI:  https://doi.org/10.3390/cells11152397
  30. Life Sci. 2022 Aug 07. pii: S0024-3205(22)00572-0. [Epub ahead of print] 120872
    Monocarboxylate transporters (MCTs) in skeletal muscle and hypothalamus of less or more physically active mice exposed to aerobic training.
    P P M Scariot, F B Manchado-Gobatto, W R Beck, M Papoti, Van Ginkel, C A Gobatto.
      AIMS: It is known that the synthesis of monocarboxylate transporters (MCTs) can be stimulated by aerobic training, but few about this effect associated or not with non-voluntary daily activities. We examined the effect of eight weeks of aerobic training in MCTs on the skeletal muscle and hypothalamus of less or more physically active mice, which can be achieved by keeping them in two different housing models, a small cage (SC) and a large cage (LC).MAIN METHODS: Forty male C57BL/6 J mice were divided into four groups. In each housing condition, mice were divided into untrained (N) and trained (T). For 8 weeks, the trained animals ran on a treadmill with an intensity equivalent to 80 % of the individual critical velocity (CV), considered aerobic capacity, 40 min/day, 5 times/week. Protein expression of MCTs was determined with fluorescence Western Blot.
    KEY FINDINGS: T groups had higher hypothalamic MCT2 than N groups (ANOVA, P = 0.032). Significant correlations were detected between hypothalamic MCT2 and CV. There was a difference between the SC and LC groups in relation to MCT4 in the hypothalamus (LC > SC, P = 0.044). Trained mice housed in LC (but not SC-T) exhibited a reduction in MCT4 muscle (P < 0.001).
    SIGNIFICANCE: Our findings indicate that aerobically trained mice increased the expression of MCT2 protein in the hypothalamus, which has been related to the uptake of lactate in neurons. Changes in energy metabolism in physically active mice (kept in LC) may be related to upregulation of hypothalamic MCT4, probably participating in the regulation of satiety.
    Keywords:  Active lifestyle; Aerobic training; C57BL/6 J mice; Critical velocity; Hypothalamus; Monocarboxylate transporters (MCTs); Skeletal muscle
    DOI:  https://doi.org/10.1016/j.lfs.2022.120872
  31. Cells. 2022 Aug 04. pii: 2416. [Epub ahead of print]11(15):
    NADPH and Mitochondrial Quality Control as Targets for a Circadian-Based Fasting and Exercise Therapy for the Treatment of Parkinson's Disease.
    William M Curtis, William A Seeds, Mark P Mattson, Patrick C Bradshaw.
      Dysfunctional mitochondrial quality control (MQC) is implicated in the pathogenesis of Parkinson's disease (PD). The improper selection of mitochondria for mitophagy increases reactive oxygen species (ROS) levels and lowers ATP levels. The downstream effects include oxidative damage, failure to maintain proteostasis and ion gradients, and decreased NAD+ and NADPH levels, resulting in insufficient energy metabolism and neurotransmitter synthesis. A ketosis-based metabolic therapy that increases the levels of (R)-3-hydroxybutyrate (BHB) may reverse the dysfunctional MQC by partially replacing glucose as an energy source, by stimulating mitophagy, and by decreasing inflammation. Fasting can potentially raise cytoplasmic NADPH levels by increasing the mitochondrial export and cytoplasmic metabolism of ketone body-derived citrate that increases flux through isocitrate dehydrogenase 1 (IDH1). NADPH is an essential cofactor for nitric oxide synthase, and the nitric oxide synthesized can diffuse into the mitochondrial matrix and react with electron transport chain-synthesized superoxide to form peroxynitrite. Excessive superoxide and peroxynitrite production can cause the opening of the mitochondrial permeability transition pore (mPTP) to depolarize the mitochondria and activate PINK1-dependent mitophagy. Both fasting and exercise increase ketogenesis and increase the cellular NAD+/NADH ratio, both of which are beneficial for neuronal metabolism. In addition, both fasting and exercise engage the adaptive cellular stress response signaling pathways that protect neurons against the oxidative and proteotoxic stress implicated in PD. Here, we discuss how intermittent fasting from the evening meal through to the next-day lunch together with morning exercise, when circadian NAD+/NADH is most oxidized, circadian NADP+/NADPH is most reduced, and circadian mitophagy gene expression is high, may slow the progression of PD.
    Keywords:  DJ-1; IDH1; NAD; NADPH; PINK1; Parkin; Parkinson’s disease; circadian; coffee; exercise; fasting; metabolic therapy; mitochondrial biogenesis; mitochondrial quality control; mitophagy; nicotinamide adenine dinucleotide; nicotinamide adenine dinucleotide phosphate
    DOI:  https://doi.org/10.3390/cells11152416
  32. J Appl Physiol (1985). 2022 Aug 11.
    Myonuclear addition is associated with sex-specific fiber hypertrophy and occurs in relation to fiber perimeter not cross-sectional area.
    Lukas Moesgaard, Søren Jessen, Abigail L Mackey, Morten Hostrup.
      It is unclear whether resistance training-induced myofiber hypertrophy is affected by sex, and whether myonuclear addition occurs in relation to the myonuclear domain and can contribute to explaining a potential sex-specific hypertrophic response. This study investigated the effect of 8 weeks of resistance training on myofiber hypertrophy and myonuclear addition in 12 males (28±7 years; mean±SD) and 12 females (27±7 years). Muscle biopsies were collected from m. vastus lateralis before and after the training intervention and analyzed by immunohistochemistry for fiber type and size, satellite cells, and myonuclei. Hypertrophy of type I fibers was greater in males than females (P<0.05), whereas hypertrophy of type II fibers was similar between sexes (P=0.158‒0.419). Expansion of the satellite cell pool (P=0.132‒0.667) and myonuclear addition (P=0.064‒0.228) did not differ significantly between sexes, irrespective of myofiber type. However, when individual responses to resistance training were assessed, myonuclear addition was strongly correlated with fiber hypertrophy (r=0.68‒0.85, P<0.001). While myofiber hypertrophy was accompanied by an increase in myonuclear domain (P<0.05), fiber perimeter per myonucleus remained constant throughout the study (P=0.096‒0.666). These findings indicate that myonuclear addition occurs in relation to the fiber perimeter per myonucleus, not the myonuclear domain, and has a substantial role in muscle hypertrophy, but does not fully explain greater hypertrophy of type I fibers in males than females.
    Keywords:  Fiber Hypertrophy; Myonuclear addition; Myonuclear domain; Resistance training; Sex
    DOI:  https://doi.org/10.1152/japplphysiol.00235.2022
  33. Front Cell Neurosci. 2022 ;16 904172
    Visual three-dimensional spatial distribution of motor neurons innervating superficial limb muscles in mice.
    Zhidan Qi, Shuai Han, Shen Wang, Xinyi Gu, Jin Deng, Chen Huang, Xiaofeng Yin.
      The coordination of motor function in the spinal cord depends on selective connections between distinct classes of motor neurons and their target muscles. However, knowledge regarding the anatomical connections between the superficial limb skeletal muscles and the motor neurons that innervate them is limited. In this study, with a combination of the multiple retrograde tracing method with 3DISCO clearing, we explored the spatial distribution of different motor neuron pools targeting specific superficial muscles of the forelimbs or hindlimbs in mouse spinal cords, which were dominated by the radial, median, ulnar, or sciatic nerve. This study reveals the precise interrelationship among different motor neuron pools innervating limb muscles under the same space and time. The data will help to further understand the neural loop and muscular motor coordination.
    Keywords:  3D imaging; motor neuron; optical clearing; retrograde tracing; skeletal muscle; spinal cord
    DOI:  https://doi.org/10.3389/fncel.2022.904172
  34. Front Cardiovasc Med. 2022 ;9 881526
    Prophylactic exercise-derived circulating exosomal miR-125a-5p promotes endogenous revascularization after hindlimb ischemia by targeting endothelin converting enzyme 1.
    Xueting Qiu, Jipeng Zhou, Yanying Xu, Longsheng Liao, Huijun Yang, Yuan Xiang, Zhengshi Zhou, Quan Sun, Minghong Chen, Jiaxiong Zhang, Wanzhou Wu, Lingping Zhu, Baiyang You, Lingfang He, Ying Luo, Zhenyu Li, Chuanchang Li, Yongping Bai.
      Background: Prophylactic exercise improves clinical outcomes in patients experiencing severe ischemic diseases. Previous studies have shown that exercise could alter the amount or content of circulating exosomes. However, little is known about the role of precursory exercise-derived circulating exosomes (Exe-Exo) in ischemic diseases. We therefore aimed to explore the function and mechanism of Exe-Exo in endogenous revascularization and perfusion recovery in peripheral arterial disease.Methods and Results: We first determined that 4 weeks of precursory treadmill exercise improved perfusion recovery on days 7, 14 and 21 after unilateral femoral artery ligation (FAL) but had no effect immediately after ligation. Then, local muscle delivery of Exe-Exo promotes arteriogenesis, angiogenesis and perfusion recovery, which could be abolished by GW4869, a well-recognized pharmacological agent inhibiting exosome release. This suggests that Exe-Exo mediated exercise-induced revascularization. In vitro, Exe-Exo enhanced endothelial cell proliferation, migration and tube formation. In addition, we identified miR-125a-5p as a novel exerkine through exosomal miRNA sequencing and RT-qPCR validation. Inhibition of miR-125a-5p abrogated the beneficial effects of Exe-Exo both in vivo and in vitro. Mechanistically, these exercise-afforded benefits were attributed to the exosomal miR-125a-5p downregulation of ECE1 expression and the subsequent activation of the AKT/eNOS downstream signaling pathway. Specifically, skeletal muscle may be a major tissue source of exercise-induced exosomal miR-125a-5p via fluorescence in situ hybridization.
    Conclusions: Endogenous circulating exosomal miR-125a-5p promotes exercise-induced revascularization via targeting ECE1 and activating AKT/eNOS downstream signaling pathway. Identify exosomal miR-125a-5p as a novel exerkine, and highlight its potential therapeutic role in the prevention and treatment of peripheral arterial disease.
    Keywords:  circulating; exercise; exosome; miR-125a-5p; revascularization
    DOI:  https://doi.org/10.3389/fcvm.2022.881526
  35. J Appl Physiol (1985). 2022 Aug 11.
    Walking or body weight squat 'activity snacks' increase dietary amino acid utilization for myofibrillar protein synthesis during prolonged sitting.
    Daniel R Moore, Eric P Williamson, Nathan Hodson, Stephanie Estafanos, Michael Mazzulla, Dinesh Kumbhare, Jenna B Gillen.
      Interrupting prolonged sitting with intermittent exercise enhances postprandial glycemic control but has unknown effects on sensitizing skeletal muscle to dietary amino acids. We hypothesized that brief walking or body weight squats would enhance the utilization of dietary phenylalanine for myofibrillar protein synthesis (MyoPS) during prolonged sitting. Participants (7 males and 5 females; ~23y; ~25.1kg/m2; ~7300 steps/d) completed three 7.5h trials consisting of prolonged sitting (SIT) or sitting with intermittent (every 30 minutes) walking (WALK) or body weight squatting (SQUAT). Two mixed-macronutrient meals (~55:30:15% carbohydrate:fat:protein), enriched with L-[ring-2H5]phenylalanine or L-[ring-13C6]phenylalanine, were provided to mimic breakfast and lunch. Tracer incorporation into myofibrillar protein was determined from the vastus lateralis with MyoPS estimated using plasma enrichment as precursor surrogate. Phosphorylation of candidate anabolic signaling proteins were determined by immunoblotting. There was no difference between conditions (p≥0.78) in the time course or area under the curve for plasma phenylalanine enrichment. MyoPS was greater (p<0.05, weighted planned comparison) in SQUAT (0.103±0.030%/h) and WALK (0.118±0.037%/h) compared to SIT (0.080±0.032%/h). Compared to SIT, there were moderate-to-large effect sizes, respectively, for SQUAT (ES=0.75; 95% CI -0.10-1.55) and WALK (ES=1.10; 95% CI 0.20-1.91). Fold change in rpS6Ser240/244 phosphorylation was greater in SQUAT compared to SIT (7.6±2.7 vs. 1.6±0.45 fold, p<0.05) with no difference (p≥0.21) in any other targets measured (4E-BP1Thr37/46, eEF2Thr56, mTORSer2448, ERK1/2Thr202/Tyr204). Interrupting prolonged sitting with short 'activity snacks' improves the utilization of dietary amino acids for MyoPS. The long term impact of this practical lifestyle modification for muscle mass or quality should be investigated.
    Keywords:  dietary protein; muscle mass; muscle protein synthesis; physical activity; sedentary
    DOI:  https://doi.org/10.1152/japplphysiol.00106.2022
  36. Elife. 2022 Aug 08. pii: e78923. [Epub ahead of print]11
    A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag.
    Willa Wen-You Yim, Hayashi Yamamoto, Noboru Mizushima.
      Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the GFP-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g., GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant RFP accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g., Halo-LC3 and Halo-GFP) are proteolytically processed to generate Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.
    Keywords:  cell biology; human; mouse
    DOI:  https://doi.org/10.7554/eLife.78923
  37. PLoS One. 2022 ;17(8): e0266905
    Nitrite lowers the oxygen cost of ATP supply in cultured skeletal muscle cells by stimulating the rate of glycolytic ATP synthesis.
    Anthony G Wynne, Charles Affourtit.
      Dietary nitrate lowers the oxygen cost of human exercise. This effect has been suggested to result from stimulation of coupling efficiency of skeletal muscle oxidative phosphorylation by reduced nitrate derivatives. In this paper, we report the acute effects of sodium nitrite on the bioenergetic behaviour of cultured rat (L6) myocytes. At odds with improved efficiency of mitochondrial ATP synthesis, extracellular flux analysis reveals that a ½-hour exposure to NaNO2 (0.1-5 μM) does not affect mitochondrial coupling efficiency in static myoblasts or in spontaneously contracting myotubes. Unexpectedly, NaNO2 stimulates the rate of glycolytic ATP production in both myoblasts and myotubes. Increased ATP supply through glycolysis does not emerge at the expense of oxidative phosphorylation, which means that NaNO2 acutely increases the rate of overall myocellular ATP synthesis, significantly so in myoblasts and tending towards significance in contractile myotubes. Notably, NaNO2 exposure shifts myocytes to a more glycolytic bioenergetic phenotype. Mitochondrial oxygen consumption does not decrease after NaNO2 exposure, and non-mitochondrial respiration tends to drop. When total ATP synthesis rates are expressed in relation to total cellular oxygen consumption rates, it thus transpires that NaNO2 lowers the oxygen cost of ATP supply in cultured L6 myocytes.
    DOI:  https://doi.org/10.1371/journal.pone.0266905
  38. Sci Rep. 2022 Aug 09. 12(1): 13582
    Matrix stiffness and architecture drive fibro-adipogenic progenitors' activation into myofibroblasts.
    Taryn Loomis, Lin-Ya Hu, Ross P Wohlgemuth, Rosemary R Chellakudam, Pooja D Muralidharan, Lucas R Smith.
      Fibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle, but in muscle pathologies FAPs the are main source of excess extracellular matrix (ECM) resulting in fibrosis. Fibrotic ECM has altered mechanical and architectural properties, but the feedback onto FAPs of stiffness or ECM properties is largely unknown. In this study, FAPs' sensitivity to their ECM substrate was assessed using collagen coated polyacrylamide to control substrate stiffness and collagen hydrogels to engineer concentration, crosslinking, fibril size, and alignment. FAPs on substrates of fibrotic stiffnesses had increased myofibroblast activation, depicted by αSMA expression, compared to substrates mimicking healthy muscle, which correlated strongly YAP nuclear localization. Surprisingly, fibrosis associated collagen crosslinking and larger fibril size inhibited myofibroblast activation, which was independent of YAP localization. Additionally, collagen crosslinking and larger fibril diameters were associated with decreased remodeling of the collagenous substrate as measured by second harmonic generation imaging. Inhibition of YAP activity through verteporfin reduced myofibroblast activation on stiff substrates but not substrates with altered architecture. This study is the first to demonstrate that fibrotic muscle stiffness can elicit FAP activation to myofibroblasts through YAP signaling. However, fibrotic collagen architecture actually inhibits myofibroblast activation through a YAP independent mechanism. These data expand knowledge of FAPs sensitivity to ECM and illuminate targets to block FAP's from driving progression of muscle fibrosis.
    DOI:  https://doi.org/10.1038/s41598-022-17852-2
  39. Mol Ther Methods Clin Dev. 2022 Sep 08. 26 279-293
    Systemic delivery of an AAV9 exon-skipping vector significantly improves or prevents features of Duchenne muscular dystrophy in the Dup2 mouse.
    Nicolas Wein, Tatyana A Vetter, Adeline Vulin, Tabatha R Simmons, Emma C Frair, Adrienne J Bradley, Liubov V Gushchina, Camila F Almeida, Nianyuan Huang, Daniel Lesman, Dhanarajan Rajakumar, Robert B Weiss, Kevin M Flanigan.
      Duchenne muscular dystrophy (DMD) is typically caused by mutations that disrupt the DMD reading frame, but nonsense mutations in the 5' part of the gene induce utilization of an internal ribosomal entry site (IRES) in exon 5, driving expression of a highly functional N-truncated dystrophin. We have developed an AAV9 vector expressing U7 small nuclear RNAs targeting DMD exon 2 and have tested it in a mouse containing a duplication of exon 2, in which skipping of both exon 2 copies induces IRES-driven expression, and skipping of one copy leads to wild-type dystrophin expression. One-time intravascular injection either at postnatal days 0-1 or at 2 months results in efficient exon skipping and dystrophin expression, and significant protection from functional and pathologic deficits. Immunofluorescence quantification showed 33%-53% average dystrophin intensity and 55%-79% average dystrophin-positive fibers in mice treated in adulthood, with partial amelioration of DMD pathology and correction of DMD-associated alterations in gene expression. In mice treated neonatally, dystrophin immunofluorescence reached 49%-85% of normal intensity and 76%-99% dystrophin-positive fibers, with near-complete correction of dystrophic pathology, and these beneficial effects persisted for at least 6 months. Our results demonstrate the robustness, durability, and safety of exon 2 skipping using scAAV9.U7snRNA.ACCA, supporting its clinical use.
    Keywords:  Becker muscular dystrophy; Duchenne muscular dystrophy; U7snRNA; dystrophin; exon skipping; gene therapy
    DOI:  https://doi.org/10.1016/j.omtm.2022.07.005
  40. Cell Mol Life Sci. 2022 Aug 12. 79(9): 481
    mRNA m5C inhibits adipogenesis and promotes myogenesis by respectively facilitating YBX2 and SMO mRNA export in ALYREF-m5C manner.
    Youhua Liu, Ying Yang, Ruifan Wu, Chun-Chun Gao, Xing Liao, Xiao Han, Botao Zeng, Chaoqun Huang, Yaojun Luo, Yuxi Liu, Yushi Chen, Wei Chen, Jiaqi Liu, Qin Jiang, Yuanling Zhao, Zhen Bi, Guanqun Guo, Yongxi Yao, Yun Xiang, Xiaojun Zhang, Teresa G Valencak, Yizhen Wang, Xinxia Wang.
      Although 5-methylcytosine (m5C) has been identified as a novel and abundant mRNA modification and associated with energy metabolism, its regulation function in adipose tissue and skeletal muscle is still limited. This study aimed at investigating the effect of mRNA m5C on adipogenesis and myogenesis using Jinhua pigs (J), Yorkshire pigs (Y) and their hybrids Yorkshire-Jinhua pigs (YJ). We found that Y grow faster than J and YJ, while fatness-related characteristics observed in Y were lower than those of J and YJ. Besides, total mRNA m5C levels and expression rates of NSUN2 were higher both in backfat layer (BL) and longissimus dorsi muscle (LDM) of Y compared to J and YJ, suggesting that higher mRNA m5C levels positively correlate with lower fat and higher muscle mass. RNA bisulfite sequencing profiling of m5C revealed tissue-specific and dynamic features in pigs. Functionally, hyper-methylated m5C-containing genes were enriched in pathways linked to impaired adipogenesis and enhanced myogenesis. In in vitro, m5C inhibited lipid accumulation and promoted myogenic differentiation. Furthermore, YBX2 and SMO were identified as m5C targets. Mechanistically, YBX2 and SMO mRNAs with m5C modification were recognized and exported into the cytoplasm from the nucleus by ALYREF, thus leading to increased YBX2 and SMO protein expression and thereby inhibiting adipogenesis and promoting myogenesis, respectively. Our work uncovered the critical role of mRNA m5C in regulating adipogenesis and myogenesis via ALYREF-m5C-YBX2 and ALYREF-m5C-SMO manners, providing a potential therapeutic target in the prevention and treatment of obesity, skeletal muscle dysfunction and metabolic disorder diseases.
    Keywords:  ALYREF; Adipogenesis; Myogenesis; SMO; YBX2; m5C modification
    DOI:  https://doi.org/10.1007/s00018-022-04474-0
  41. Compr Physiol. 2022 Aug 11. 12(4): 1-36
    Structure and Function of the Mammalian Neuromuscular Junction.
    Leah A Davis, Matthew J Fogarty, Alyssa Brown, Gary C Sieck.
      The mammalian neuromuscular junction (NMJ) comprises a presynaptic terminal, a postsynaptic receptor region on the muscle fiber (endplate), and the perisynaptic (terminal) Schwann cell. As with any synapse, the purpose of the NMJ is to transmit signals from the nervous system to muscle fibers. This neural control of muscle fibers is organized as motor units, which display distinct structural and functional phenotypes including differences in pre- and postsynaptic elements of NMJs. Motor units vary considerably in the frequency of their activation (both motor neuron discharge rate and duration/duty cycle), force generation, and susceptibility to fatigue. For earlier and more frequently recruited motor units, the structure and function of the activated NMJs must have high fidelity to ensure consistent activation and continued contractile response to sustain vital motor behaviors (e.g., breathing and postural balance). Similarly, for higher force less frequent behaviors (e.g., coughing and jumping), the structure and function of recruited NMJs must ensure short-term reliable activation but not activation sustained for a prolonged period in which fatigue may occur. The NMJ is highly plastic, changing structurally and functionally throughout the life span from embryonic development to old age. The NMJ also changes under pathological conditions including acute and chronic disease. Such neuroplasticity often varies across motor unit types. © 2022 American Physiological Society. Compr Physiol 12:1-36, 2022.
    DOI:  https://doi.org/10.1002/cphy.c210022
This page is being maintained by Thomas Krichel. It was last updated on 2022‒08‒14 at 03:30:36.