bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2022‒06‒26
forty-five papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Redox Biol. 2022 Jun 17. pii: S2213-2317(22)00146-X. [Epub ahead of print]54 102374
      Skeletal muscle fibers are malleable and undergo rapid remodeling in response to increased contractile activity (i.e., exercise) or prolonged periods of muscle inactivity (e.g., prolonged bedrest). Exploration of the cell signaling pathways regulating these skeletal muscle adaptations reveal that redox signaling pathways play a key role in the control of muscle remodeling during both exercise and prolonged muscle inactivity. In this regard, muscular exercise results in an acute increase in the production of reactive oxygen species (ROS) in the contracting fibers; however, this contraction-induced rise in ROS production rapidly declines when contractions cease. In contrast, prolonged muscle disuse results in a chronic elevation in ROS production within the inactive fibers. This difference in the temporal pattern of ROS production in muscle during exercise and muscle inactivity stimulates divergent cell-signaling pathways that activate both genomic and nongenomic mechanisms to promote muscle remodeling. This review examines the role that redox signaling plays in skeletal muscle adaptation in response to both prolonged muscle inactivity and endurance exercise training. We begin with a summary of the sites of ROS production in muscle fibers followed by a review of the cellular antioxidants that are responsible for regulation of ROS levels in the cell. We then discuss the specific redox-sensitive signaling pathways that promote skeletal muscle adaptation in response to both prolonged muscle inactivity and exercise. To stimulate future research, we close with a discussion of unanswered questions in this exciting field.
    Keywords:  Diaphragm; Mechanical ventilation; Muscle atrophy; Muscle wasting; Renin angiotensin system
  2. Int J Mol Sci. 2022 Jun 14. pii: 6616. [Epub ahead of print]23(12):
      Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential.
    Keywords:  Piezo1; Yoda1; muscle atrophy; myoblasts; myogenesis; myotubes; sarcopenia; satellite cells
  3. FASEB J. 2022 Jul;36(7): e22385
      Skeletal muscles can regenerate over the lifetime from resident muscle stem cells (MuSCs). Interactions between MuSCs and extracellular matrix (ECM) proteins are essential for muscle regeneration. The best-known receptors for ECM proteins are integrins, a family composed of twenty-some heterodimeric combinations of an α- and a β-subunit. β1-integrin (encoded by Itgb1) is required for quiescence, proliferation, migration, and fusion of Pax7+ MuSCs in the mouse model. β3-integrin (encoded by Itgb3) has been reported to be critical for the myogenic differentiation of C2C12 myoblasts, and Itgb3 germline mutant mice were shown to regenerate few if any myofibers after injury. To investigate the autonomous role of Itgb3 in the myogenic lineage in vivo, we conditionally inactivated a floxed Itgb3 allele (Itgb3F ) by constitutive Pax7-Cre and tamoxifen-inducible Pax7-CreERT2 drivers. Unexpectedly, we found no defects in muscle regeneration in both conditional knockout models. In vitro studies using Itgb3 mutant myoblasts or RNAi knockdown of Itgb3 in myoblasts also did not reveal a role for myogenic differentiation. As β1- and β3-integrins share ECM ligands and downstream signaling effectors, we further examined Itgb3's role in a Itgb1 haploid background. Still, we found no evidence for an autonomous role of Itgb3 in muscle regeneration in vivo. Thus, while Itgb3 is critical for the differentiation of C2C12 cells, the regenerative defects reported for the Itgb3 germline mutant are not due to its role in the MuSC. We conclude that if β3-integrin does have a role in Pax7+ MuSCs, it is compensated by β1- and/or another β-integrin(s).
    Keywords:  differentiation; mouse; muscle stem cells; regeneration; β1-integrin; β3-integrin
  4. J Cachexia Sarcopenia Muscle. 2022 Jun 20.
      BACKGROUND: Skeletal muscle exhibits remarkable plasticity under both physiological and pathological conditions. One major manifestation of this plasticity is muscle atrophy that is an adaptive response to catabolic stimuli. Because the heterogeneous transcriptome responses to catabolism in different types of muscle cells are not fully characterized, we applied single-nucleus RNA sequencing (snRNA-seq) to unveil muscle atrophy related transcriptional changes at single nucleus resolution.METHODS: Using a sciatic denervation mouse model of muscle atrophy, snRNA-seq was performed to generate single-nucleus transcriptional profiles of the gastrocnemius muscle from normal and denervated mice. Various bioinformatics analyses, including unsupervised clustering, functional enrichment analysis, trajectory analysis, regulon inference, metabolic signature characterization and cell-cell communication prediction, were applied to illustrate the transcriptome changes of the individual cell types.
    RESULTS: A total of 29 539 muscle nuclei (normal vs. denervation: 15 739 vs. 13 800) were classified into 13 nuclear types according to the known cell markers. Among these, the type IIb myonuclei were further divided into two subgroups, which we designated as type IIb1 and type IIb2 myonuclei. In response to denervation, the proportion of type IIb2 myonuclei increased sharply (78.12% vs. 38.45%, P < 0.05). Concomitantly, trajectory analysis revealed that denervated type IIb2 myonuclei clearly deviated away from the normal type IIb2 myonuclei, indicating that this subgroup underwent robust transcriptional reprogramming upon denervation. Signature genes in denervated type IIb2 myonuclei included Runx1, Gadd45a, Igfn1, Robo2, Dlg2, and Sh3d19 (P < 0.001). The gene regulatory network analysis captured a group of atrophy-related regulons (Foxo3, Runx1, Elk4, and Bhlhe40) whose activities were enhanced (P < 0.01), especially in the type IIb2 myonuclei. The metabolic landscape in the myonuclei showed that most of the metabolic pathways were down-regulated by denervation (P < 0.001), while some of the metabolic signalling, such as glutathione metabolism, was specifically activated in the denervated type IIb2 myonulei. We also investigated the transcriptomic alterations in the type I myofibres, muscle stem cells, fibro-adipogenic progenitors, macrophages, endothelial cells and pericytes and characterized their signature responses to denervation. By predicting the cell-cell interactions, we observed that the communications between myofibres and muscle resident cells were diminished by denervation.
    CONCLUSIONS: Our results define the myonuclear transition, metabolic remodelling, and gene regulation networks reprogramming associated with denervation-induced muscle atrophy and illustrate the molecular basis of the heterogeneity and plasticity of muscle cells in response to catabolism. These results provide a useful resource for exploring the molecular mechanism of muscle atrophy.
    Keywords:  Denervation; Muscle atrophy; Muscle metabolism; Skeletal muscle; snRNA-seq
  5. iScience. 2022 Jun 17. 25(6): 104444
      Skeletal muscle stem cells, or satellite cells (SCs), are essential to regenerate and maintain muscle. Quiescent SCs reside in an asymmetric niche between the basal lamina and myofiber membrane. To repair muscle, SCs activate, proliferate, and differentiate, fusing to repair myofibers or reacquiring quiescence to replenish the SC niche. Little is known about when SCs reacquire quiescence during regeneration or the cellular processes that direct SC fate decisions. We find that most SCs reacquire quiescence 5-10 days after muscle injury, following differentiation and fusion of most cells to regenerate myofibers. Single-cell sequencing of myogenic cells in regenerating muscle identifies SCs reacquiring quiescence and reveals that noncell autonomous signaling networks influence SC fate decisions during regeneration. SC transplantation experiments confirm that the regenerating environment influences SC fate. We define a window for SC repopulation of the niche, emphasizing the temporal contribution of the regenerative muscle environment on SC fate.
    Keywords:  Cell biology; Omics; Stem cells research; Transcriptomics
  6. Cells. 2022 Jun 10. pii: 1893. [Epub ahead of print]11(12):
      Immune cells can mount desirable anti-cancer immunity. However, some immune cells can support cancer disease progression. The presence of cancer can lead to production of immature myeloid cells from the bone marrow known as myeloid-derived suppressor cells (MDSCs). The immunosuppressive and pro-tumorigenic effects of MDSCs are well understood. Whether MDSCs are involved in promoting cancer cachexia is not well understood. We orthotopically injected the pancreas of mice with KPC cells or PBS. One group of tumor-bearing mice was treated with an anti-Ly6G antibody that depletes granulocytic MDSCs and neutrophils; the other received a control antibody. Anti-Ly6G treatment delayed body mass loss, reduced tibialis anterior (TA) muscle wasting, abolished TA muscle fiber atrophy, reduced diaphragm muscle fiber atrophy of type IIb and IIx fibers, and reduced atrophic gene expression in the TA muscles. Anti-ly6G treatment resulted in greater than 50% Ly6G+ cell depletion efficiency in the tumors and TA muscles. These data show that, in the orthotopic KPC model, anti-Ly6G treatment reduces the number of Ly6G+ cells in the tumor and skeletal muscle and reduces skeletal muscle atrophy. These data implicate Ly6G+ cells, including granulocytic MDSCs and neutrophils, as possible contributors to the development of pancreatic cancer-induced skeletal muscle wasting.
    Keywords:  MDSC; atrophy; cachexia; immunosuppression; skeletal muscle
  7. Exerc Sport Sci Rev. 2022 Jun 24.
      ABSTRACT: Retrotransposons are gene segments that proliferate in the genome, and the Long INterspersed Element 1 (LINE-1, or L1) retrotransposon is active in humans. Although older mammals show enhanced skeletal muscle L1 expression, exercise generally reverses this trend. We hypothesize skeletal muscle L1 expression affects muscle physiology, and additional innovative investigations are needed to confirm this hypothesis.
  8. FASEB J. 2022 Jul;36(7): e22401
      During skeletal myogenesis, the zinc-finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro-differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA-mediated gene silencing in C2C12 myoblasts and whole-transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts.
    Keywords:  E box; endoplasmic reticulum stress; gene silencing; microarray analysis; muscle regeneration; myoblast differentiation; promoter; skeletal muscle; transcription
  9. Exp Physiol. 2022 Jun 20.
      NEW FINDINGS: What is the central question of this study? The present study evaluated whether myofiber protein signaling responses to ex vivo dynamic contractions are altered by accustomization to voluntary endurance training in rats. What is the main finding and its importance? In response to ex vivo dynamic muscle contractions, canonical myofiber protein signaling pertaining to metabolic transcriptional regulation, as well as translation initiation and elongation, was not influenced by prior accustomization to voluntary endurance training in rats. Accordingly, intrinsic myofiber protein signaling responses to standardized contractile activity may be independent of prior exercise training in rat skeletal muscle.ABSTRACT: Skeletal muscle training status may influence myofiber regulatory protein signaling in response to contractile activity. The current study employed a purpose-designed ex vivo dynamic contractile protocol to evaluate the effect of exercise-accustomization on canonical myofiber protein signaling for metabolic gene expression and for translation initiation and elongation. To this end, rats completed 8 weeks of in vivo voluntary running training versus no running control intervention, whereupon an ex vivo endurance-type dynamic contraction stimulus was conducted in isolated soleus muscle preparations from both intervention groups. Protein signaling responses by phosphorylation was evaluated by immunoblotting at 0 h and 3 h following ex vivo stimulation. Phosphorylation of AMPKα and its downstream target ACC, as well as phosphorylation of eEF2 was increased immediately following the dynamic contraction protocol (at 0 h). Signaling for translation initiation and elongation was evident at 3 h after dynamic contractile activity, as evidenced by increased phosphorylation of p70S6K and 4E-BP1, as well as a decrease in phosphorylation of eEF2 back to resting control-levels. However, prior exercise training did not alter phosphorylation of the investigated signaling proteins. Accordingly, protein signaling responses to standardized endurance-type contractions may be independent of training status in rat muscle during ex vivo conditions. The present findings add to our current understanding of molecular regulatory events responsible for skeletal muscle plasticity. This article is protected by copyright. All rights reserved.
    Keywords:  ex vivo contractions; protein signaling; training status
  10. Biology (Basel). 2022 Jun 19. pii: 936. [Epub ahead of print]11(6):
      Findings from studies of muscle regeneration can significantly contribute to the treatment of age-related loss of skeletal muscle mass, which may predispose older adults to severe morbidities. We established a human experimental model using excised skeletal muscle tissues from reconstructive surgeries in eight older adults. Muscle samples from each participant were preserved immediately or maintained in agarose medium for the following 5, 9, or 11 days. Immunofluorescence analyses of the structural proteins, actin and desmin, confirmed the integrity of muscle fibers over 11 days of maintenance. Similarly, the numbers of CD80-positive M1 and CD163-positive M2 macrophages were stable over 11 days in vitro. However, the numbers of PAX7-positive satellite cells and MYOD-positive myoblasts changed in opposite ways, suggesting that satellite cells partially differentiated in vitro. Further experiments revealed that stimulation with unsaturated fatty acid C18[2]c (linoleic acid) increased resident M1 macrophages and satellite cells specifically. Thus, the use of human skeletal muscle tissue in vitro provides a direct experimental approach to study the regulation of muscle tissue regeneration by macrophages and stem cells and their responses to therapeutic compounds.
    Keywords:  actin; aging; desmin; human; immunohistology; in vitro model; myoblasts; myofibers; myofibrils; resident macrophages; satellite cells; skeletal muscle; tissue
  11. Physiol Genomics. 2022 Jun 20.
      Skeletal muscle is adaptable to a direct stimulus of exercise-induced muscle damage (EIMD). Local muscle gene networks and systemic circulatory factors respond to EIMD within days, mediating anti-inflammation and cellular proliferation. Here we show in humans that local EIMD of one muscle group is associated with a systemic response of gene networks that regulate muscle structure and cellular development in non-local homologous muscle not directly altered by EIMD. In the non-dominant knee-extensors of seven males, EIMD was induced through voluntary contractions against an electric motor that lengthened muscles. Neuromuscular assessments, vastus lateralis muscle biopsies and blood draws occurred at two days prior, and one and two days post the EIMD intervention. From the muscle and blood plasma samples, RNA-seq measured transcriptome changes of differential expression using bioinformatic analyses.Relative to the time of the EIMD intervention, local muscle that was mechanically damaged had 475 genes differentially expressed, as compared to 33 genes in the non-local homologous muscle. Gene and network analysis showed that activity of the local muscle was related to structural maintenance, repair, and energetic processes, whereas gene and network activity of the non-local muscle (that was not directly modified by the EIMD) were related to muscle cell development, stress response, and structural maintenance. Altered expression of two novel miRNAs related to the EIMD response supported that systemic factors were active. Together, these results indicate that the expression of genes and gene networks that control muscle contractile structure can be modified in response to non-local EIMD in humans.
    Keywords:  differential gene expression; eccentric; skeletal muscle
  12. FEBS J. 2022 Jun 20.
      Epigenetic regulatory mechanisms are increasingly recognized as crucial determinants of cellular specification and differentiation. During muscle cell differentiation (myogenesis), extensive remodeling of histone acetylation and methylation occurs. Several of these histone modifications aid in the expression of muscle-specific genes and the silencing of genes that block lineage commitment. Therefore, the identification of new epigenetic regulatory mechanisms is of high interest. Still, the functional relevance of numerous histone modifications during myogenesis remain completely uncertain. In this study, we focus on the function of H3K36me3 and its epigenetic writer, SET domain containing 2 (SETD2), in the context of muscle cell differentiation. We first observed that SETD2 expression increases during myogenesis. Targeted depletion of SETD2 in undifferentiated (myoblasts) and differentiated (myotubes) muscle cells reduced H3K36me3 levels and induced profound changes in gene expression and slight alterations in alternative splicing, as determined by deep RNA-sequencing analysis. Enzymes that function in metabolic pathways were upregulated in response to SETD2 depletion. Furthermore, we demonstrated that upregulation of several glycolytic enzymes was associated with an increase in intracellular pyruvate levels in SETD2 depleted cells, indicating a novel role for SETD2 in metabolic programming during myogenesis. Together, our results provide new insight into the signaling pathways controlled by chromatin modifying enzymes and their associated histone modifications during muscle cell differentiation.
    Keywords:  Alternative Splicing; Cell Differentiation; Histone Code; Metabolism; Muscle Fibers; Skeletal
  13. Genes (Basel). 2022 Jun 04. pii: 1014. [Epub ahead of print]13(6):
      Long periods of immobilization, among other etiologies, would result is muscle atrophy. Exercise is the best approach to reverse this atrophy. However, the limited or the non-ability to perform the required physical activity for such patients and the limited pharmacological options make developing novel therapeutic approaches a necessity. Within this context, secreted protein acidic and rich in cysteine (SPARC) has been characterized as an exercise-induced gene. Whereas the knock-out of this gene leads to a phenotype that mimics number of the ageing-induced and sarcopenia-related changes including muscle atrophy, overexpressing SPARC in mice or adding it to muscular cell culture produces similar effects as exercise including enhanced muscle mass, strength and metabolism. Therefore, this piece of writing aims to provide evidence supporting the potential use of SPARC/SPARC as a molecular therapy for muscle atrophy in the context of immobilization especially for elderly patients.
    Keywords:  SPARC; ageing; immobilization; muscle atrophy
  14. Mol Genet Metab. 2022 Jun 13. pii: S1096-7192(22)00340-7. [Epub ahead of print]
      Osteogenesis imperfecta (OI) is rare heritable connective tissue disorder that most often arises from mutations in the type I collagen genes, COL1A1 and COL1A2, displaying a range of symptoms including skeletal fragility, short stature, blue-gray sclera, and muscle weakness. Recent investigations into the intrinsic muscle weakness have demonstrated reduced contractile generating force in some murine models consistent with patient population studies, as well as alterations in whole body bioenergetics. Muscle weakness is found in approximately 80% of patients and has been equivocal in OI mouse models. Understanding the mechanism responsible for OI muscle weakness is crucial in building our knowledge of muscle bone cross-talk via mechanotransduction and biochemical signaling, and for potential novel therapeutic approaches. In this study we evaluated skeletal muscle mitochondrial function and whole-body bioenergetics in the heterozygous +/G610C (Amish) mouse modeling mild/moderate human type I/VI OI and minimal skeletal muscle weakness. Our analyses revealed several changes in the +/G610C mouse relative to their wildtype littermates including reduced state 3 mitochondrial respiration, increased mitochondrial citrate synthase activity, increased Parkin and p62 protein content, and an increased respiratory quotient. These changes may represent the ability of the +/G610C mouse to compensate for mitochondrial and metabolic changes that may arise due to type I collagen mutations and may also account for the lack of muscle weakness observed in the +/G610C model relative to the more severe OI models.
    Keywords:  Genetic mouse models; Mitochondrial function; Osteogenesis imperfecta; Respiratory quotient; Skeletal muscle
  15. Biomed Rep. 2022 Jul;17(1): 60
      Transient receptor potential channel 6 (TRPC6) channels constitute non-selective cation channels that are localized in the plasmalemma or sarcolemma, and have a leading permeability for the bivalent calcium ion. Animal models indicate an involvement of TRPC6 in malignant hyperthermia. The expression of TRPC6 in the sarcolemma has been demonstrated in the skeletal muscle fibers of mice. The importance of TRPC6 channels for the influx of calcium into the muscle cell has also been established. The presence of TRPC6 in tissues of human skeletal muscle is surmised. In order to confirm the presence of TRPC6 in human skeletal muscle, tissue samples of various skeletal muscles (Musculus deltoideus, pectoralis major, trizeps brachii and rectus femoris) from eight different human donors (n=8; six preserved cadavers and two non-preserved cadavers) were examined using immunohistochemistry. TRPC6 was found in all muscle fibers of all investigated bodies. Appropriate controls yielded the expected results. As demonstrated in animal studies and in studies of human cells, the presented results confirmed the presence of TRPC6 in human skeletal muscle tissue. Thus, TRPC6 is most likely important for calcium homeostasis and the proper function of human muscle fibers and may be a target for treatment in various muscular diseases.
    Keywords:  TRPC6; cation channel; human skeletal muscle tissue; immunohistochemistry; malignant hyperthermia; muscular disease
  16. J Muscle Res Cell Motil. 2022 Jun 20.
      A report on the first virtual European Muscle Conference.
    Keywords:  Cardiac muscle; Cardiomyopathy; Muscle repair; Myogenesis; Myopathy; Skeletal muscle
  17. J Pers Med. 2022 May 25. pii: 865. [Epub ahead of print]12(6):
      Facioscapulohumeral muscular dystrophy (FSHD) is arguably one of the most challenging genetic diseases to understand and treat. The disease is caused by epigenetic dysregulation of a macrosatellite repeat, either by contraction of the repeat or by mutations in silencing proteins. Both cases lead to chromatin relaxation and, in the context of a permissive allele, pathogenic misexpression of DUX4 in skeletal muscle. The complex nature of the locus and the fact that FSHD is a toxic, gain-of-function disease present unique challenges for the design of therapeutic strategies. There are three major DUX4-targeting avenues of therapy for FSHD: small molecules, oligonucleotide therapeutics, and CRISPR-based approaches. Here, we evaluate the preclinical progress of each avenue, and discuss efforts being made to overcome major hurdles to translation.
    Keywords:  AAV; CRISPR; DUX4; FSHD; antisense; facioscapulohumeral muscular dystrophy; gene therapy; muscular dystrophy; skeletal muscle; therapeutics
  18. Biomolecules. 2022 Jun 11. pii: 817. [Epub ahead of print]12(6):
      Muscle injuries are frequent in individuals with genetic myopathies and in athletes. Skeletal muscle regeneration depends on the activation and differentiation of satellite cells present in the basal lamina of muscle fibers. The skeletal muscle environment is critical for repair, metabolic and homeostatic function. Regulatory T cells (Treg) residing within skeletal muscle comprise a distinct and special cell population that modifies the inflammatory environment by secreting cytokines and amphiregulin, an epidermal growth factor receptor (EGFR) ligand that acts directly upon satellite cells, promoting tissue regeneration. This systematic review summarizes the current knowledge regarding the role of Treg in muscle repair and discusses their therapeutic potential in skeletal muscle injuries. A bibliographic search was carried out using the terms Treg and muscle regeneration and repair, covering all articles up to April 2021 indexed in the PubMed and EMBASE databases. The search included only published original research in human and experimental animal models, with further data analysis based on the PICO methodology, following PRISMA definitions and Cochrane guidelines.
    Keywords:  T regulatory cell; muscle repair; regeneration; regulatory microenvironment; skeletal muscle
  19. Cancers (Basel). 2022 Jun 11. pii: 2894. [Epub ahead of print]14(12):
      Overactivation of poly (ADPribose) polymerases (PARPs) is involved in cancer-induced cachexia. We hypothesized that the PARP inhibitor rucaparib may improve muscle mass and reduce damage in cancer cachexia mice. In mouse diaphragm and gastrocnemius (LP07 lung adenocarcinoma) treated with PARP inhibitor (rucaparib,150 mg/kg body weight/24 h for 20 days) and in non-tumor control animals, body, muscle, and tumor weights; tumor area; limb muscle strength; physical activity; muscle structural abnormalities, damage, and phenotype; PARP activity; and proteolytic and autophagy markers were quantified. In cancer cachexia mice compared to non-cachexia controls, body weight and body weight gain, muscle weight, limb strength, physical activity, and muscle fiber size significantly declined, while levels of PARP activity, plasma troponin I, muscle damage, and proteolytic and autophagy markers increased. Treatment with the PARP inhibitor rucaparib elicited a significant improvement in body weight gain, tumor size and weight, physical activity, muscle damage, troponin I, and proteolytic and autophagy levels. PARP pharmacological inhibition did not exert any significant improvements in muscle weight, fiber size, or limb muscle strength. Treatment with rucaparib, however, improved muscle damage and structural abnormalities and physical activity in cancer cachexia mice. These findings suggest that rucaparib exerts its beneficial effects on cancer cachexia performance through the restoration of muscle structure.
    Keywords:  PARP inhibitor rucaparib; lung-cancer-induced cachexia; muscle damage; muscle structural abnormalities; physical activity; proteolytic and autophagy markers; respiratory and limb muscles
  20. Skelet Muscle. 2022 Jun 23. 12(1): 14
      BACKGROUND: A common polymorphism (R577X) in the ACTN3 gene results in the complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~ 16% of the world's population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0 + 20% stretch did not cause eccentric damage. In contrast, L0 + 30% stretch produced a significant ~ 40% deficit in maximum force; here, we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this.METHODS: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA.
    RESULTS: We show that the absence of α-actinin-3 does not affect the visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically stretched. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar branching.
    CONCLUSIONS: The absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0 + 30%, but not L0 + 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3-deficient humans have been reported to have a faster decline in muscle function with increasing age, that is, as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibre breakages.
    Keywords:  Biomechatronics; Biosensors; Exercise; Myorobotics; Sarcoplasmic reticulum; Single fibre; Skeletal muscle; Skinned fibre; a-Actinin-3
  21. Front Cell Dev Biol. 2022 ;10 920569
      Muscle proteostasis is shaped by the myogenic transcription factor MyoD which regulates the expression of chaperones during muscle differentiation. Whether MyoD can also modulate chaperone expression in terminally differentiated muscle cells remains open. Here we utilized a temperature-sensitive (ts) conditional knockdown nonsense mutation in MyoD ortholog in C. elegans, HLH-1, to ask whether MyoD plays a role in maintaining muscle proteostasis post myogenesis. We showed that hlh-1 is expressed during larval development and that hlh-1 knockdown at the first, second, or third larval stages resulted in severe defects in motility and muscle organization. Motility defects and myofilament organization were rescued when the clearance of hlh-1(ts) mRNA was inhibited, and hlh-1 mRNA levels were restored. Moreover, hlh-1 knockdown modulated the expression of chaperones with putative HLH-1 binding sites in their promoters, supporting HLH-1 role in muscle maintenance during larval development. Finally, mild disruption of hlh-1 expression during development resulted in earlier dysregulation of muscle maintenance and function during adulthood. We propose that the differentiation transcription factor, HLH-1, contributes to muscle maintenance and regulates cell-specific chaperone expression post differentiation. HLH-1 may thus impact muscle proteostasis and potentially the onset and manifestation of sarcopenia.
    Keywords:  Caenorhabditis elegans (c. elegans); MyoD; chaperone; development; hlh-1; myosin; proteostasis
  22. Front Physiol. 2022 ;13 861617
      The normal decline in skeletal muscle mass that occurs with aging is exacerbated in patients with chronic obstructive pulmonary disease (COPD) and contributes to poor health outcomes, including a greater risk of death. There has been controversy about the causes of this exacerbated muscle atrophy, with considerable debate about the degree to which it reflects the very sedentary nature of COPD patients vs. being precipitated by various aspects of the COPD pathophysiology and its most frequent proximate cause, long-term smoking. Consistent with the latter view, recent evidence suggests that exacerbated aging muscle loss with COPD is likely initiated by decades of smoking-induced stress on the neuromuscular junction that predisposes patients to premature failure of muscle reinnervation capacity, accompanied by various alterations in mitochondrial function. Superimposed upon this are various aspects of COPD pathophysiology, such as hypercapnia, hypoxia, and inflammation, that can also contribute to muscle atrophy. This review will summarize the available knowledge concerning the mechanisms contributing to exacerbated aging muscle affect in COPD, consider the potential role of comorbidities using the specific example of chronic kidney disease, and identify emerging molecular mechanisms of muscle impairment, including mitochondrial permeability transition as a mechanism of muscle atrophy, and chronic activation of the aryl hydrocarbon receptor in driving COPD muscle pathophysiology.
    Keywords:  aryl hydrocarbon receptor; mitochondria; mitochondrial permeability transition; muscle atrophy; neuromuscular junction; smoking
  23. Mol Genet Metab. 2022 Jun 11. pii: S1096-7192(22)00337-7. [Epub ahead of print]
      Alglucosidase alpha is an orphan drug approved for enzyme replacement therapy (ERT) in Pompe disease (PD); however, its efficacy is limited in skeletal muscle because of a partial blockage of autophagic flux that hinders intracellular trafficking and enzyme delivery. Adjunctive therapies that enhance autophagic flux and protect mitochondrial integrity may alleviate autophagic blockage and oxidative stress and thereby improve ERT efficacy in PD. In this study, we compared the benefits of ERT combined with a ketogenic diet (ERT-KETO), daily administration of an oral ketone precursor (1,3-butanediol; ERT-BD), a multi-ingredient antioxidant diet (ERT-MITO; CoQ10, α-lipoic acid, vitamin E, beetroot extract, HMB, creatine, and citrulline), or co-therapy with the ketone precursor and multi-ingredient antioxidants (ERT-BD-MITO) on skeletal muscle pathology in GAA-KO mice. We found that two months of 1,3-BD administration raised circulatory ketone levels to ≥1.2 mM, attenuated autophagic buildup in type 2 muscle fibers, and preserved muscle strength and function in ERT-treated GAA-KO mice. Collectively, ERT-BD was more effective vs. standard ERT and ERT-KETO in terms of autophagic clearance, dampening of oxidative stress, and muscle maintenance. However, the addition of multi-ingredient antioxidants (ERT-BD-MITO) provided the most consistent benefits across all outcome measures and normalized mitochondrial protein expression in GAA-KO mice. We therefore conclude that nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants may provide an alternative to ketogenic diets for inducing ketosis and enhancing autophagic flux in PD patients.
    Keywords:  1,3-butanediol; Antioxidants; Autophagy; GAA; Galectin-3; Glycogen; Ketogenic diet; Ketone precursor; Ketones; Lysosome; Mitochondria; Myozyme; Oxidative Stress; Pompe; ROS; SQSTM1; Skeletal muscle; p62
  24. Int J Mol Sci. 2022 Jun 12. pii: 6566. [Epub ahead of print]23(12):
      Skeletal muscle is a tissue that has recently been recognized for its ability to produce androgens under physiological conditions. The steroidogenesis process is known to be negatively influenced by reactive oxygen species (ROS) in reproductive Leydig and ovary cells, while their effect on muscle steroidogenesis is still an unexplored field. Muscle cells are continuously exposed to ROS, resulting from both their metabolic activity and the surrounding environment. Interestingly, the regulation of signaling pathways, induced by mild ROS levels, plays an important role in muscle fiber adaptation to exercise, in a process that also elicits a significant modulation in the hormonal response. The aim of the present study was to investigate whether ROS could influence steroidogenesis in skeletal muscle cells by evaluating the release of testosterone (T) and dihydrotestosterone (DHT), as well as the evaluation of the relative expression of the key steroidogenic enzymes 5α-reductase, 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, and aromatase. C2C12 mouse myotubes were exposed to a non-cytotoxic concentration of hydrogen peroxide (H2O2), a condition intended to reproduce, in vitro, one of the main stimuli linked to the process of homeostasis and adaptation induced by exercise in skeletal muscle. Moreover, the influence of tadalafil (TAD), a phosphodiesterase 5 inhibitor (PDE5i) originally used to treat erectile dysfunction but often misused among athletes as a "performance-enhancing" drug, was evaluated in a single treatment or in combination with H2O2. Our data showed that a mild hydrogen peroxide exposure induced the release of DHT, but not T, and modulated the expression of the enzymes involved in steroidogenesis, while TAD treatment significantly reduced the H2O2-induced DHT release. This study adds a new piece of information about the adaptive skeletal muscle cell response to an oxidative environment, revealing that hydrogen peroxide plays an important role in activating muscle steroidogenesis.
    Keywords:  dihydrotestosterone; phosphodiesterase type 5; reactive oxygen species; redox status; skeletal muscles; tadalafil; testosterone
  25. Metabolism. 2022 Jun 21. pii: S0026-0495(22)00120-2. [Epub ahead of print] 155242
      INTRODUCTION AND METHODS: Skeletal muscle mitochondrial dysfunction may cause tissue oxidative stress and consequent catabolism in chronic kidney disease (CKD), contributing to patient mortality. We investigated in 5/6-nephrectomized (Nx) rats the impact of n3-polyunsaturated fatty-acids (n3-PUFA) isocaloric partial dietary replacement on gastrocnemius muscle (Gm) mitochondrial master-regulators, ATP production, ROS generation and related muscle-catabolic derangements.RESULTS: Nx had low Gm mitochondrial nuclear respiratory factor-2 and peroxisome proliferator-activated receptor gamma coactivator-1alpha, low ATP production and higher mitochondrial fission-fusion protein ratio with ROS overproduction. n3-PUFA normalized all mitochondrial derangements and pro-oxidative tissue redox state (oxydized to total glutathione ratio). n3-PUFA also normalized Nx-induced muscle-catabolic proinflammatory cytokines, insulin resistance and low muscle weight. Human uremic serum reproduced mitochondrial derangements in C2C12 myotubes, while n3-PUFA coincubation prevented all effects. n3-PUFA also enhanced muscle mitophagy in-vivo and siRNA-mediated autophagy inhibition selectively blocked n3-PUFA-induced normalization of C2C12 mitochondrial ROS production.
    CONCLUSIONS: In conclusion, dietary n3-PUFA normalize mitochondrial master-regulators, ATP production and dynamics in experimental CKD. These effects occur directly in muscle cells and they normalize ROS production through enhanced mitophagy. Dietary n3-PUFA mitochondrial effects result in normalized catabolic derangements and protection from muscle wasting, with potential positive impact on patient survival.
    Keywords:  CKD; Mitochondria; Skeletal muscle; Uremia; n3-PUFA
  26. Int J Mol Sci. 2022 Jun 16. pii: 6722. [Epub ahead of print]23(12):
      Valosin-containing protein (VCP) acts as a key regulator of cellular protein homeostasis by coordinating protein turnover and quality control. Mutations in VCP lead to (cardio-)myopathy and neurodegenerative diseases such as inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). To date, due to embryonic lethality, no constitutive VCP knockout animal model exists. Here, we generated a constitutive CRISPR/Cas9-induced vcp knockout zebrafish model. Similar to the phenotype of vcp morphant knockdown zebrafish embryos, we found that vcp-null embryos displayed significantly impaired cardiac and skeletal muscle function. By ultrastructural analysis of skeletal muscle cells and cardiomyocytes, we observed severely disrupted myofibrillar organization and accumulation of inclusion bodies as well as mitochondrial degeneration. vcp knockout was associated with a significant accumulation of ubiquitinated proteins, suggesting impaired proteasomal function. Additionally, markers of unfolded protein response (UPR)/ER-stress and autophagy-related mTOR signaling were elevated in vcp-deficient embryos, demonstrating impaired proteostasis in VCP-null zebrafish. In conclusion, our findings demonstrate the successful generation of a stable constitutive vcp knockout zebrafish line that will enable characterization of the detailed mechanistic underpinnings of vcp loss, particularly the impact of disturbed protein homeostasis on organ development and function in vivo.
    Keywords:  CRISPR/Cas9; VCP; VCPopathies; disease modeling; protein homeostasis; zebrafish
  27. Nucleic Acids Res. 2022 Jun 23. pii: gkac524. [Epub ahead of print]
      Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.
  28. Front Physiol. 2022 ;13 872719
      Despite advances over the past few decades, heart failure with reduced ejection fraction (HFrEF) remains not only a mortal but a disabling disease. Indeed, the New York Heart Association classification of HFrEF severity is based on how much exercise a patient can perform. Moreover, exercise capacity-both aerobic exercise performance and muscle power-are intimately linked with survival in patients with HFrEF. This review will highlight the pathologic changes in skeletal muscle in HFrEF that are related to impaired exercise performance. Next, it will discuss the key role that impaired nitric oxide (NO) bioavailability plays in HFrEF skeletal muscle pathology. Lastly, it will discuss intriguing new data suggesting that the inorganic nitrate 'enterosalivary pathway' may be leveraged to increase NO bioavailability via ingestion of inorganic nitrate. This ingestion of inorganic nitrate has several advantages over organic nitrate (e.g., nitroglycerin) and the endogenous nitric oxide synthase pathway. Moreover, inorganic nitrate has been shown to improve exercise performance: both muscle power and aerobic capacity, in some recent small but well-controlled, cross-over studies in patients with HFrEF. Given the critical importance of better exercise performance for the amelioration of disability as well as its links with improved outcomes in patients with HFrEF, further studies of inorganic nitrate as a potential novel treatment is critical.
    Keywords:  heart failure; nitrate; nitric oxide; nitrite; skeletal muscle
  29. Pharmaceutics. 2022 May 29. pii: 1159. [Epub ahead of print]14(6):
      Irisin is an exercise-induced hormone that regulates lipid metabolism. The present study investigates whether the anti-obesity effect of the natural flavonoid pentamethylquercetin (PMQ) is related to irisin secretion from skeletal muscle in whole animals and cultured cells. Obese mice induced by monosodium glutamate were administered oral PMQ to determine blood irisin level and in vivo parameters of lipid metabolism, and cultured mouse C2C12 myoblasts and 3T3-L1 preadipocytes were employed to investigate the related molecular identities. PMQ increased circulating irisin and decreased bodyweight, insulin, and lipid levels accompanied with increasing brown-like adipocyte formation in obese mice. The brown adipocyte marker uncoupling protein 1 (UCP-1) and other brown-like adipocyte-specific genes and/or markers were increased in mouse white fat tissue, while PMQ treatment reversed the above changes. PMQ also dose-dependently increased the reduced levels of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and fibronectin type III domain-containing 5 (FNDC5) signal molecules in obese mice. Interestingly, the irisin level was increased in the culture medium of C2C12 cells treated with PMQ, and the conditioned medium stimulated the brown-like transition of 3T3-L1 preadipocytes with the increased expression of PGC-1α, FNDC5, UCP-1, and other brown-like adipocyte-specific genes. The effects of conditioned culture medium were abolished in C2C12 cells with silenced PGC-1α. On the other hand, PMQ-induced upregulation of PGC-1α and FNDC5 expression was reduced by AMPK inhibitor Compound C in C2C12 cells. Our results demonstrate the novel information that PMQ-induced irisin secretion from skeletal muscle involves the improvement of metabolic dysfunction in obese mice via activating the AMPK/PGC-1α/FNDC5 signal pathway, suggesting that PMQ modulates skeletal muscle-adipose tissue crosstalk and may be a promising drug candidate for treating obesity and obesity-related metabolic diseases.
    Keywords:  AMP-activated protein kinase; browning of white adipose tissue; fibronectin type III domain-containing 5; irisin; pentamethylquercetin; peroxisome proliferator-activated receptor-γ coactivator-1α; skeletal muscle
  30. J Brachial Plex Peripher Nerve Inj. 2022 Jan;17(1): e12-e21
      Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective  The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods  In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results  Follistatin treatment resulted in smaller muscles after 3 months denervation ( p  = 0.019) and reduced force after 3 months sham denervation ( p  < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p  = 0.066). Follistatin increased satellite cell counts after denervation ( p  < 0.001) but reduced satellite cell counts after sham denervation ( p  = 0.037). Conclusion  Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.
    Keywords:  anabolic; denervation atrophy; follistatin; hypertrophy; muscle force; nerve regeneration; nerve repair; peripheral nerve; progenitor cells; rodent
  31. Cell Death Dis. 2022 Jun 22. 13(6): 561
      Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.
  32. Pflugers Arch. 2022 Jun 21.
      Transmembrane potassium (K) gradients are key determinants of membrane potential that can modulate action potentials, control muscle contractility, and influence ion channel and transporter activity. Daily K intake is normally equal to the amount of K in the entire extracellular fluid (ECF) creating a critical challenge - how to maintain ECF [K] and membrane potential in a narrow range during feast and famine. Adaptations to maintain ECF [K] include sensing the K intake, sensing ECF [K] vs. desired set-point and activating mediators that regulate K distribution between ECF and ICF, and regulate renal K excretion. In this focused review, we discuss the basis of these adaptions, including (1) potential mechanisms for rapid feedforward signaling to kidney and muscle after a meal (before a rise in ECF [K]), (2) how skeletal muscles sense and respond to changes in ECF [K], (3) effects of K on aldosterone biosynthesis, and (4) how the kidney responds to changes in ECF [K] to modify K excretion. The concepts of sexual dimorphisms in renal K handling adaptation are introduced, and the molecular mechanisms that can account for the benefits of a K-rich diet to maintain cardiovascular health are discussed. Although the big picture of K homeostasis is becoming more clear, we also highlight significant pieces of the puzzle that remain to be solved, including knowledge gaps in our understanding of initiating signals, sensors and their connection to homeostatic adjustments of ECF [K].
    Keywords:  Aldosterone; Feed-forward signaling; Feedback signaling; Na,K-ATPase alpha2; Plasma potassium; Potassium channels; Skeletal muscle; Sodium transporters
  33. Biomedicines. 2022 Jun 16. pii: 1428. [Epub ahead of print]10(6):
      Limb girdle muscular dystrophies (LGMD), caused by mutations in 29 different genes, are the fourth most prevalent group of genetic muscle diseases. Although the link between LGMD and its genetic origins has been determined, LGMD still represent an unmet medical need. Here, we describe a platform for modeling LGMD based on the use of human induced pluripotent stem cells (hiPSC). Thanks to the self-renewing and pluripotency properties of hiPSC, this platform provides a renewable and an alternative source of skeletal muscle cells (skMC) to primary, immortalized, or overexpressing cells. We report that skMC derived from hiPSC express the majority of the genes and proteins that cause LGMD. As a proof of concept, we demonstrate the importance of this cellular model for studying LGMDR9 by evaluating disease-specific phenotypes in skMC derived from hiPSC obtained from four patients.
    Keywords:  induced pluripotent stem cells; limb girdle muscular dystrophies; pathological modeling; skeletal muscle cells
  34. J Cachexia Sarcopenia Muscle. 2022 Jun 19.
      BACKGROUND: Cachexia is a life-threatening condition observed in several pathologies, such as cancer or chronic diseases. Interleukin 10 (Il10) gene transfer is known to improve cachexia by downregulating Il6. Here, we used an IL10-knockout mouse model to simulate cachexia and investigate the effects of eggshell membrane (ESM), a resistant protein, on general pre-cachexia symptoms, which is particularly important for the development of cachexia therapeutics.METHODS: Five-week-old male C57BL6/J mice were fed an AIN-93G powdered diet (WT), and 5-week-old male B6.129P2-Il10 < tm1Cgn>/J (IL10-/- ) mice were fed either the AIN-93G diet (KO) or an 8% ESM-containing diet (KOE) for 28 weeks. The tissue weight and levels of anaemia-, blood glucose-, lipid metabolism-, and muscular and colonic inflammation-related biochemical markers were measured. Transcriptomic analysis on liver and colon mucus and proteomic analysis on skeletal muscle were performed. Ingenuity Pathway Analysis was used to identify molecular pathways and networks. Caecal short-chain fatty acids (SCFAs) were identified using HPLC, and caecal bacteria DNA were subjected to metagenomic analysis. Flow cytometry analysis was performed to measure the CD4+ IL17+ T cells in mesenteric lymph nodes.
    RESULTS: The body weight, weight of gastrocnemius muscle and fat tissues, colon weight/length ratio, plasma HDL and NEFA, muscular PECAM-1 levels (P < 0.01), plasma glucose and colonic mucosal myeloperoxidase activity (P < 0.05) and T helper (Th) 17 cell abundance (P = 0.071) were improved in KOE mice over KO mice. Proteomic analysis indicated the protective role of ESM in muscle weakness and maintenance of muscle formation (>1.5-fold). Transcriptomic analysis revealed that ESM supplementation suppressed the LPS/IL1-mediated inhibition of RXR function pathway in the liver and downregulated the colonic mucosal expression of chemokines and Th cell differentiation-related markers (P < 0.01) by suppressing the upstream BATF pathway. Analysis of the intestinal microenvironment revealed that ESM supplementation ameliorated the microbial alpha diversity and the abundance of microbiota associated with the degree of inflammation (P < 0.05) and increased the level of total organic acids, particularly of SCFAs such as butyrate (2.3-fold), which could inhibit Th1 and Th17 production.
    CONCLUSIONS: ESM supplementation ameliorated the chief symptoms of cachexia, including anorexia, lean fat tissue mass, skeletal muscle wasting and reduced physical function. ESM also improved colon and skeletal muscle inflammation, lipid metabolism and microbial dysbiosis. These results along with the suppressed differentiation of Th cells could be associated with the beneficial intestinal microenvironment and, subsequently, attenuation of pre-cachexia. Our findings provide insights into the potential of ESM in complementary interventions for pre-cachexia prevention.
    Keywords:  Cachexia; Egg shell membrane; Gut microbiota; IL10-knockout mice; SCFA; T helper cells differentiation
  35. Metabolites. 2022 Jun 17. pii: 556. [Epub ahead of print]12(6):
      The transcription factor Krüppel-like factor 10 (Klf10), also known as Tieg1 for TGFβ (Inducible Early Gene-1) is known to control numerous genes in many cell types that are involved in various key biological processes (differentiation, proliferation, apoptosis, inflammation), including cell metabolism and human disease. In skeletal muscle, particularly in the soleus, deletion of the Klf10 gene (Klf10 KO) resulted in ultrastructure fiber disorganization and mitochondrial metabolism deficiencies, characterized by muscular hypertrophy. To determine the metabolic profile related to loss of Klf10 expression, we analyzed blood and soleus tissue using UHPLC-Mass Spectrometry. Metabolomics analyses on both serum and soleus revealed profound differences between wild-type (WT) and KO animals. Klf10 deficient mice exhibited alterations in metabolites associated with energetic metabolism. Additionally, chemical classes of aromatic and amino-acid compounds were disrupted, together with Krebs cycle intermediates, lipids and phospholipids. From variable importance in projection (VIP) analyses, the Warburg effect, citric acid cycle, gluconeogenesis and transfer of acetyl groups into mitochondria appeared to be possible pathways involved in the metabolic alterations observed in Klf10 KO mice. These studies have revealed essential roles for Klf10 in regulating multiple metabolic pathways whose alterations may underlie the observed skeletal muscle defects as well as other diseases.
    Keywords:  Klf10; UHPLC-MS; Warburg effect; metabolic pathways; metabolomics; mice; serum; soleus
  36. Eur J Appl Physiol. 2022 Jun 25.
      Resistance exercise training (RET) is a key modality to enhance sports performance, injury prevention and rehabilitation, and improving overall health via increases in muscular strength. Yet, the contribution of neural mechanisms to increases in muscular strength are highly debated. This is particularly true for the involvement of the motor unit, which is the link between neural (activation) and mechanical (muscle fiber twitch forces) mechanisms. A plethora of literature that examines the effects of RET on skeletal muscle speculate the role of motor units, such as increases in firing rates partially explains muscular strength gains. Results, however, are mixed regarding changes in firing rates in studies that utilize single motor unit recordings. The lack of clarity could be related to vast or subtle differences in RET programs, methods to record motor units, muscles tested, types of contractions and intensities used to record motor units, etc. Yet to be discussed, mixed findings could be the result of non-uniform MU behavior that is not typically accounted for in RET research. The purpose of this narration is to discuss the effects of acute resistance exercise training studies on MU behavior and to provide guidance for future research.
    Keywords:  Action potential waveforms; Firing rates; Motor unit; Neural adaptations; Resistance exercise training
  37. PLoS Genet. 2022 Jun 23. 18(6): e1010287
      Myofibrils of the skeletal muscle are comprised of sarcomeres that generate force by contraction when myosin-rich thick filaments slide past actin-based thin filaments. Surprisingly little is known about the molecular processes that guide sarcomere assembly in vivo, despite deficits within this process being a major cause of human disease. To overcome this knowledge gap, we undertook a forward genetic screen coupled with reverse genetics to identify genes required for vertebrate sarcomere assembly. In this screen, we identified a zebrafish mutant with a nonsense mutation in mob4. In Drosophila, mob4 has been reported to play a role in spindle focusing as well as neurite branching and in planarians mob4 was implemented in body size regulation. In contrast, zebrafish mob4geh mutants are characterised by an impaired actin biogenesis resulting in sarcomere defects. Whereas loss of mob4 leads to a reduction in the amount of myofibril, transgenic expression of mob4 triggers an increase. Further genetic analysis revealed the interaction of Mob4 with the actin-folding chaperonin TRiC, suggesting that Mob4 impacts on TRiC to control actin biogenesis and thus myofibril growth. Additionally, mob4geh features a defective microtubule network, which is in-line with tubulin being the second main folding substrate of TRiC. We also detected similar characteristics for strn3-deficient mutants, which confirmed Mob4 as a core component of STRIPAK and surprisingly implicates a role of the STRIPAK complex in sarcomerogenesis.
  38. Front Sports Act Living. 2022 ;4 903992
      As the fields of kinesiology, exercise science, and human movement developed, the majority of the research focused on male physiology and extrapolated findings to females. In the medical sphere, basing practice on data developed in only males resulted in the removal of drugs from the market in the late 1990s due to severe side effects (some life-threatening) in females that were not observed in males. In response to substantial evidence demonstrating exercise-induced health benefits, exercise is often promoted as a key modality in disease prevention, management, and rehabilitation. However, much like the early days of drug development, a historical literature knowledge base of predominantly male studies may leave the exercise field vulnerable to overlooking potentially key biological differences in males and females that may be important to consider in prescribing exercise (e.g., how exercise responses may differ between sexes and whether there are optimal approaches to consider for females that differ from conventional approaches that are based on male physiology). Thus, this review will discuss anatomical, physiological, and skeletal muscle molecular differences that may contribute to sex differences in exercise responses, as well as clinical considerations based on this knowledge in athletic and general populations over the continuum of age. Finally, this review summarizes the current gaps in knowledge, highlights the areas ripe for future research, and considerations for sex-cognizant research in exercise fields.
    Keywords:  exercise medicine; molecular biology; muscle; physiology; sex differences
  39. Cell Mol Life Sci. 2022 Jun 21. 79(7): 374
      Myostatin is a negative regulator of skeletal muscle growth secreted by skeletal myocytes. In the past years, myostatin inhibition sparked interest among the scientific community for its potential to enhance muscle growth and to reduce, or even prevent, muscle atrophy. These characteristics make it a promising target for the treatment of muscle atrophy in motor neuron diseases, namely, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), which are rare neurological diseases, whereby the degeneration of motor neurons leads to progressive muscle loss and paralysis. These diseases carry a huge burden of morbidity and mortality but, despite this unfavorable scenario, several therapeutic advancements have been made in the past years. Indeed, a number of different curative therapies for SMA have been approved, leading to a revolution in the life expectancy and outcomes of SMA patients. Similarly, tofersen, an antisense oligonucleotide, is now undergoing clinical trial phase for use in ALS patients carrying the SOD1 mutation. However, these therapies are not able to completely halt or reverse progression of muscle damage. Recently, a trial evaluating apitegromab, a myostatin inhibitor, in SMA patients was started, following positive results from preclinical studies. In this context, myostatin inhibition could represent a useful strategy to tackle motor symptoms in these patients. The aim of this review is to describe the myostatin pathway and its role in motor neuron diseases, and to summarize and critically discuss preclinical and clinical studies of myostatin inhibitors in SMA and ALS. Then, we will highlight promises and pitfalls related to the use of myostatin inhibitors in the human setting, to aid the scientific community in the development of future clinical trials.
    Keywords:  Activin receptors, type II; Monoclonal antibodies; Motor neuron diseases; Muscle atrophy; Myostatin
  40. Mol Metab. 2022 Jun 16. pii: S2212-8778(22)00097-7. [Epub ahead of print] 101528
      Mitochondrial capacity is critical to adapt the high energy demand of the heart to circadian oscillations and diseased states. Glucocorticoids regulate the circadian cycle of energy metabolism, but little is known about how circadian timing of exogenous glucocorticoid dosing directly regulates heart metabolism through cardiomyocyte-autonomous mechanisms. While chronic once-daily intake of glucocorticoids promotes metabolic stress and heart failure, we recently discovered that intermittent once-weekly dosing of exogenous glucocorticoids promoted muscle metabolism in normal and obese skeletal muscle. However, the effects of glucocorticoid intermittence on heart metabolism and heart failure remain unknown. Here we investigated the extent to which circadian time of dosing regulates the effects of the glucocorticoid prednisone in heart metabolism and function in conditions of single pulse or chronic intermittent dosing. In WT mice, we found that prednisone improved cardiac content of NAD+ and ATP with light-phase dosing (ZT0), while the effects were blocked by dark-phase dosing (ZT12). The drug effects on mitochondrial function were cardiomyocyte-autonomous, as shown by inducible cardiomyocyte-restricted glucocorticoid receptor (GR) ablation, and depended on an intact cardiomyocyte clock, as shown by inducible cardiomyocyte-restricted ablation of Brain and Muscle ARNT-like 1 (BMAL1). Conjugating time-of-dosing with chronic intermittence, we found that once-weekly prednisone improved metabolism and function in heart after myocardial injury dependent on circadian time of intake, i.e. with light-phase but not dark-phase dosing. Our study identifies cardiac-autonomous mechanisms through which circadian-specific intermittent dosing reconverts glucocorticoid drugs to metabolic boosters for the heart.
    Keywords:  Cardiac bioenergetics; Circadian rhythm; Glucocorticoid steroids; Inducible cardiomyocyte-specific GR knockout; Molecular clock
  41. BMC Bioinformatics. 2022 Jun 18. 23(1): 241
      BACKGROUND: The biological relevance and accuracy of gene expression data depend on the adequacy of data normalization. This is both due to its role in resolving and accounting for technical variation and errors, and its defining role in shaping the viewpoint of biological interpretations. Still, the choice of the normalization method is often not explicitly motivated although this choice may be particularly decisive for conclusions in studies involving pronounced cellular plasticity. In this study, we highlight the consequences of using three fundamentally different modes of normalization for interpreting RNA-seq data from human skeletal muscle undergoing exercise-training-induced growth. Briefly, 25 participants conducted 12 weeks of high-load resistance training. Muscle biopsy specimens were sampled from m. vastus lateralis before, after two weeks of training (week 2) and after the intervention (week 12), and were subsequently analyzed using RNA-seq. Transcript counts were modeled as (1) per-library-size, (2) per-total-RNA, and (3) per-sample-size (per-mg-tissue).RESULT: Initially, the three modes of transcript modeling led to the identification of three unique sets of stable genes, which displayed differential expression profiles. Specifically, genes showing stable expression across samples in the per-library-size dataset displayed training-associated increases in per-total-RNA and per-sample-size datasets. These gene sets were then used for normalization of the entire dataset, providing transcript abundance estimates corresponding to each of the three biological viewpoints (i.e., per-library-size, per-total-RNA, and per-sample-size). The different normalization modes led to different conclusions, measured as training-associated changes in transcript expression. Briefly, for 27% and 20% of the transcripts, training was associated with changes in expression in per-total-RNA and per-sample-size scenarios, but not in the per-library-size scenario. At week 2, this led to opposite conclusions for 4% of the transcripts between per-library-size and per-sample-size datasets (↑ vs. ↓, respectively).
    CONCLUSION: Scientists should be explicit with their choice of normalization strategies and should interpret the results of gene expression analyses with caution. This is particularly important for data sets involving a limited number of genes or involving growing or differentiating cellular models, where the risk of biased conclusions is pronounced.
    Keywords:  Normalization; RNA-seq; Resistance training; Skeletal muscle
  42. Cytokine. 2022 Jun 20. pii: S1043-4666(22)00145-4. [Epub ahead of print]157 155936
      Brown/beige adipocytes, which are derived from skeletal muscle/smooth muscle-lineage cells, consume excess energy as heat through the expression of mitochondrial uncoupling protein 1 (UCP1). Previous studies have shown that forced expression of PR/SET domain (PRDM)-16 or early B-cell factor (EBF)-2 induced UCP1-positive adipocytes in C2C12 myogenic cells. Here, we explored the culture conditions to induce Ucp1 expression in C2C12 cells without introducing exogenous genes. Treatment with rosiglitazone (a peroxisome proliferator-activated receptor (PPAR)-γ agonist), GW501516 (a PPARδ agonist), and bone morphogenetic protein (BMP)-7 for 8 days efficiently increased Ucp1 expression in response to treatment with forskolin, an activator of the protein kinase A pathway. BMP7 dose-dependently increased forskolin-induced Ucp1 expression in the presence of rosiglitazone and GW501516; however, GW501516 was not required for Ucp1 induction. Additionally, the structurally related proteins, BMP6 and BMP9, efficiently increased forskolin-induced Ucp1 expression in rosiglitazone-treated cells. UCP1 protein was localized in cells with lipid droplets, but adipocytes were not always positive for UCP1. Continuous treatment with BMP7 was needed for the efficient induction of Ucp1 by forskolin treatment. Significant expression of Prdm16 was not detected, irrespective of the treatment, and treatment with rosiglitazone, GW501516, and BMP7 did not affect the expression levels of Ebf2. Fibroblast growth factor receptor (Fgfr)-3 expression levels were increased by BMP9 in rosiglitazone-treated cells, and molecules that upregulate Fgfr3 transcription partly overlapped with those that stimulate Ucp1 transcription. The present results provide basic information on the practical differentiation of myogenic cells to brown adipocytes.
    Keywords:  Adipocyte; BMP; Myogenic cell; UCP1
  43. Genes (Basel). 2022 May 26. pii: 950. [Epub ahead of print]13(6):
      Ageing is the effect of time on biological entities. It represents a risk factor for a variety of diseases and health disorders; thus, therapeutic options are required to tackle ageing issues. Modern geriatric medicine prescribes exercise to counteract ageing effects. This work presents secreted protein acidic and rich in cysteine (SPARC) as a potential antiageing therapy. Indeed, SPARC declines with ageing, exercise induces SPARC, and SPARC overexpression in mice mimics exercise. Thus, we hypothesize that SPARC is an exercise-induced factor that is beyond-at least part of-the antiageing effects induced by exercise. This could become a potential antiageing therapy for the elderly that counteracts ageing by mimicking the effects of exercise without needing to perform exercise. This is of particular importance because ageing usually reduces mobility and age-related diseases can reduce the ability to perform the required physical activity. On the other hand, the possibilities of mimicking exercise benefits via SPARC are not limited to ageing, and can be applied in various contexts in which exercise cannot be performed because of physical disabilities, health disorders, or limited mobility.
    Keywords:  ageing; antiageing; exercise; secreted protein acidic and rich in cysteine
  44. Am J Physiol Endocrinol Metab. 2022 Jun 22.
      Rapid oscillations in cytosolic calcium (Ca2+) coordinate muscle contraction, relaxation, and physical movement. Intriguingly, dietary nitrate decreases ATP cost of contraction, increases force production, and increases cytosolic Ca2+; which would seemingly necessitate a greater demand for sarcoplasmic reticulum Ca2+ ATPase (SERCA) to sequester Ca2+ within the SR during relaxation. As SERCA is highly regulated, we aimed to determine the effect of 7-day nitrate supplementation (1 mM via drinking water) on SERCA enzymatic properties and the functional interaction between SERCA and mitochondrial oxidative phosphorylation. In soleus, we report that dietary nitrate increased force production across all stimulation frequencies tested, and throughout a 25 min fatigue protocol. Mice supplemented with nitrate also displayed an ~25% increase in submaximal SERCA activity and SERCA efficiency (p=0.053) in soleus. To examine a possible link between ATP consumption and production, we established a methodology coupling SERCA and mitochondria in permeabilized muscle fibers. The premise of this experiment is that the addition of Ca2+ in the presence of ATP generates ADP from SERCA to support mitochondrial respiration. Similar to submaximal SERCA activity, mitochondrial respiration supported by SERCA-derived ADP was increased ~20% following nitrate in red gastrocnemius. This effect was fully attenuated by the SERCA inhibitor cyclopiazonic acid and was not attributed to differences in mitochondrial oxidative capacity, ADP sensitivity, protein content, or reactive oxygen species emission. Overall, these findings suggest improvements in submaximal SERCA kinetics may contribute to the effects of nitrate on force production during fatigue.
    Keywords:  Calcium homeostasis; Contractile function; Mitochondria; Nitrate; SERCA