bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2022‒01‒30
forty papers selected by
Anna Vainshtein
Craft Science Inc.

  1. FEBS J. 2022 Jan 24.
      Regeneration of the mammalian adult skeletal muscle is a well-orchestrated process regulated by multiple proteins and signaling pathways. Cytokines constitute a major class of regulators of skeletal myogenesis. It is well established that infiltrating immune cells at the site of muscle injury secrete cytokines, which play critical roles in the myofiber repair and regeneration process. In the past 10-15 years, skeletal muscle itself has emerged as a prolific producer of cytokines. Much attention in the field has been focused on the endocrine effects of muscle-secreted cytokines (myokines) on metabolic regulation. However, ample evidence suggests that muscle-derived cytokines also regulate myogenic differentiation and muscle regeneration in an autocrine manner. In this review, we survey cytokines that meet two criteria: (a) evidence of expression by muscle cells; (b) evidence demonstrating a myogenic function. Dozens of cytokines representing several major classes make up this group, and together they regulate all steps of the myogenic process. How such a large array of cytokines coordinate their signaling to form a regulatory network is a fascinating, pressing question. Functional studies that can distinguish the source of the cytokines in vivo are also much needed in order to facilitate exploration of their full therapeutic potential.
    Keywords:  Cytokine; myogenesis; myogenic differentiation; regeneration; skeletal muscle
  2. Curr Protoc. 2022 Jan;2(1): e356
      Muscular dystrophies are caused by genetic variants in genes encoding for proteins important for muscle structure or function, leading to a loss of muscle integrity and muscle wasting. To this day, no cure has been found for these diseases. Different therapeutic approaches are under intensive investigation. Cellular therapy has been extensively studied for diseases such as Duchenne Muscular Dystrophy, a debilitating disease caused by a mutation in the DMD gene, encoding for the dystrophin protein. Healthy myogenic cells transplanted into dystrophic muscles have the potential to engraft at long-term and fuse to donate their nuclei to the dystrophin-deficient myofibers, thereby restoring normal gene expression. Despite promising preclinical studies, the clinical trials had limited success so far due to many technical limitations. The recent technological advances in induced-pluripotent stem cells and genome editing opened new opportunities in this field. One of the keys to efficiently translate these new technologies into clinical benefits is to use relevant endpoints for preclinical studies. Considering that dystrophic muscles are susceptible to contraction-induced injury, the assessment of their resistance to repeated eccentric contractions is an optimal outcome to evaluate their functional recovery following cell transplantation. This protocol describes the procedure to generate induced-pluripotent stem cell-derived myoblasts, transplant these cells into skeletal muscle of immunosuppressed dystrophic mice, and assess muscle function in situ by measuring the resistance of the transplanted muscle to repeated eccentric contractions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generation of hiPSC-derived myoblasts. Basic Protocol 2: Transplantation of hiPSC-derived myoblasts in skeletal muscle of dystrophic mice. Basic Protocol 3: Assessment of muscle function in situ.
    Keywords:  contractile properties; hiPSC; muscle stem cells; muscular diseases; transplantation
  3. Front Cell Dev Biol. 2021 ;9 760260
      Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.
    Keywords:  afferents; intrafusal; mechanoreceptor; muscle spindle; myotubes; neuromuscular; proprioception; skeletal muscle
  4. J Physiol. 2022 Jan 24.
      KEY POINTS: Denervation is an experimental model of peripheral neuropathies as well as muscle disuse, and it helps us understand some aspects of the sarcopenia of aging. Muscle disuse is associated with reduced mitochondrial content and function, leading to metabolic impairments within the tissue. Although the processes that regulate mitochondrial biogenesis are understood, those that govern mitochondrial breakdown (i.e., mitophagy) are not well characterized in this context. Autophagy and mitophagy flux, measured up to the point of the lysosome (pre-lysosomal flux rates), were increased in the early stages of denervation, along with mitochondrial dysfunction, but were reduced at later time points when the degree of muscle atrophy was highest. Denervation led to progressive increases in lysosomal proteins to accommodate mitophagy flux, yet evidence for lysosomal impairment at later stages may limit the removal of dysfunctional mitochondria, stimulate reactive oxygen species signaling, and reduce muscle health as denervation time progresses.ABSTRACT: Deficits in skeletal muscle mitochondrial content and quality are observed following denervation-atrophy. This is due to alterations in the biogenesis of new mitochondria as well as their degradation via mitophagy. The regulation of autophagy and mitophagy over the course of denervation (Den) remains unknown. Further, the time-dependent changes in lysosome content, the end-stage organelle for mitophagy, remains unexplored. Here, we studied autophagic as well as mitophagic pre-lysosomal flux in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria from rat muscle subjected to Den for 1, 3, or 7 days. We also assessed flux at 1-day post-denervation in transgenic mt-keima mice. Markers of mitochondrial content were reduced at 7 days following Den, and Den further resulted in rapid decrements in mitochondrial respiration, along with increased ROS emission. Pre-lysosomal autophagy flux was upregulated at 1- and 3-days post-Den but was reduced compared to time-matched sham-operated controls at 7-days post-Den. Similarly, pre-lysosomal mitophagy flux was enhanced in SS mitochondria as early as 1- and 3-days of Den but decreased in both SS and IMF subfractions following 7 days of Den. Lysosome protein content and transcriptional regulators TFEB and TFE3 were progressively enhanced with Den, an adaptation designed to enhance autophagic capacity. However, evidence for lysosome dysfunction was apparent by 7 days, which may limit degradation capacity. This may contribute to an inability to clear dysfunctional mitochondria and increased ROS signaling, thereby accelerating muscle atrophy. Thus, therapeutic targeting of lysosome function may help to maintain autophagy and muscle health during conditions of muscle disuse or denervation. Abstract figure legend This study investigates the temporal regulation of the autophagy-lysosome system in rat skeletal muscle following neuromuscular denervation (Den) with a focus on mitochondrial decay through mitophagy. We show that mitochondrial dysfunction is time-dependant, with elevations at 3-days post-Den and further at 7 days, preceding decrements in mitochondrial protein content. Deficits in mitochondrial content may be explained by prior elevations in mitophagy as early as 1- and 3-days post-Den, but these elevations were bi-phasic, returning to lower values by 7-days post-Den. To meet the demands of increased autophagy, lysosome protein content was progressively upregulated with 3- and 7-day of Den, but evidence of lysosome dysfunction was evident, and this could impede the removal of poor-quality mitochondria. Overall, these changes in the autophagy-lysosome system following neuromuscular denervation and provide insight into the processes that contribute to Den-induced muscle atrophy. Representative graphs are Den/Sham, with the dotted line representing sham-operated control values. This article is protected by copyright. All rights reserved.
    Keywords:  TFEB; atrophy; lysosome dysfunction; mitochondrial dysfunction; reactive oxygen species
  5. Cell Prolif. 2022 Jan 28. e13181
      OBJECTIVES: Insulin resistance in chronic kidney disease (CKD) stimulates muscle wasting, but the molecular processes behind the resistance are undetermined. However, inflammation in skeletal muscle is implicated in the pathogenesis of insulin resistance and cachexia. Toll-like receptors (TLRs) are known to regulate local innate immune responses, and microarray data have shown that Tlr13 is upregulated in the muscles of mice with CKD, but the relevance is unknown.MATERIALS AND METHODS: We performed in vitro experiments in C2C12 myotubes and constructed a CKD murine model using subtotal nephrectomy to conduct experiments in vivo.
    RESULTS: Tlr13 expression was stimulated in C2C12 myotubes treated with uremic serum. The expression of Tlr13 was also upregulated in the tibialis anterior muscles of mice with CKD. Tlr13 knockdown with siRNAs in skeletal muscle cells decreased insulin resistance despite the inclusion of uremic serum. This led to increased levels of p-AKT and suppression of protein degradation. Using immunofluorescence staining and coimmunoprecipitation assay, we found that TLR13 recruits IRF3, which activates Irf3 expression, resulting in decreased AKT activity. Moreover, insulin resistance and proteolysis are re-induced by Irf3 overexpression under Tlr13 deletion.
    CONCLUSIONS: Our results indicate that TLR13 is involved in CKD-mediated insulin resistance in muscle. In catabolic conditions where insulin signaling is impaired, targeting TLR13 may improve insulin sensitivity and prevent muscle atrophy.
    Keywords:  TLR13; chronic kidney disease; insulin resistance; muscle atrophy
  6. Am J Physiol Endocrinol Metab. 2022 Jan 24.
      microRNAs (miRs) are linked to various human diseases including Type 2 Diabetes Mellitus (T2DM) and emerging evidence suggests miRs may serve as potential therapeutic targets. Lower miR-16 content is consistent across different models of T2DM; however, the role of miR-16 in muscle metabolic health is still elusive. Therefore, the purpose of this study was to investigate how deletion of miR-16 in mice affects skeletal muscle metabolic health and contractile function in both sexes. This study was conducted using both in vitro (1) and in vivo (2) experiments. (1) We utilized C2C12 myoblasts to test if inhibition or overexpression of miR-16 affected insulin-mediated glucose handling. (2) We generated muscle-specific miR-16 knockout (KO) mice fed a high-fat diet (HFD) to assess how miR-16 content impacts metabolic and contractile properties including glucose tolerance, insulin sensitivity, muscle contractile function, protein anabolism, and mitochondrial network health. (1) Although inhibition of miR-16 induced impaired insulin signaling (p=0.002) and glucose uptake (p=0.014), overexpression of miR-16 did not attenuate lipid overload-induced insulin resistance using the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol. (2) miR-16 deletion induced both impaired muscle contractility (p=0.031-0.033), and mitochondrial network health (p=0.008-0.018) in both sexes. However, while males specifically exhibited impaired insulin sensitivity following miR-16 deletion (p=0.030), female KO mice showed pronounced glucose intolerance (p=0.046), corresponding with lower muscle weights (p=0.015), and protein hyperanabolism (p=0.023). Our findings suggest distinct sex differences in muscle adaptation in response to miR-16 deletion and miR-16 may serve as a key regulator for metabolic dysregulation in T2DM.
    Keywords:  insulin resistance; microRNA; mitochondrial quality; protein turnover; type 2 diabetes mellitus
  7. Sci Rep. 2022 Jan 26. 12(1): 1377
      Muscle wasting is a major problem leading to reduced quality of life and higher risks of mortality and various diseases. Muscle atrophy is caused by multiple conditions in which protein degradation exceeds its synthesis, including disuse, malnutrition, and microgravity. While Vitamin D receptor (VDR) is well known to regulate calcium and phosphate metabolism to maintain bone, recent studies have shown that VDR also plays roles in skeletal muscle development and homeostasis. Moreover, its expression is upregulated in muscle undergoing atrophy as well as after muscle injury. Here we show that VDR regulates simulated microgravity-induced atrophy in C2C12 myotubes in vitro. After 8 h of microgravity simulated using 3D-clinorotation, the VDR-binding motif was associated with chromatin regions closed by the simulated microgravity and enhancer regions inactivated by it, which suggests VDR mediates repression of enhancers. In addition, VDR was induced and translocated into the nuclei in response to simulated microgravity. VDR-deficient C2C12 myotubes showed resistance to simulated microgravity-induced atrophy and reduced induction of FBXO32, an atrophy-associated ubiquitin ligase. These results demonstrate that VDR contributes to the regulation of simulated microgravity-induced atrophy at least in part by controlling expression of atrophy-related genes.
  8. Elife. 2022 Jan 25. pii: e70341. [Epub ahead of print]11
      Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.
    Keywords:  developmental biology; human; human ipsc myogenesis; iMyoblasts; mouse; muscle stem cells; regenerative medicine; stem cells; xenograft
  9. Trends Genet. 2022 Jan 22. pii: S0168-9525(22)00001-4. [Epub ahead of print]
      Muscle stem cells (MuSCs) are responsible for skeletal muscle homeostasis and repair. In response to extracellular cues, MuSCs activate from quiescence, expand, differentiate into mature myofibers, and self-renew within their regenerative niche. These steps are accomplished by the dynamic action of different chromatin-modifying enzymes that, cooperating with myogenic transcription factors, coordinately regulate defined transcriptional programs. Here, we review the current knowledge on the epigenetic dynamics that allow MuSCs' fate decisions. We describe the emerging mechanisms showing how chromatin topology impacts the 3D genome architecture of MuSCs during myogenesis. Because these processes contribute to shape and maintain cell identity, we highlight how defects in proper epigenetic control of MuSCs' fate decisions underlie the pathogenesis of muscle diseases, causing the acquisition of derailed cell fates and the incapacity to properly self-renew.
    Keywords:  cell fate; chromatin; epigenetics; muscle stem cells; nuclear structure
  10. J Vis Exp. 2022 Jan 07.
      Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.
  11. PLoS One. 2022 ;17(1): e0263085
      Hibernating bears remain in their dens for 5-7 months during winter and survive without eating or drinking while staying inactive. However, they maintain their physical functions with minimal skeletal muscle atrophy and metabolic dysfunction. In bears, resistance to skeletal muscle atrophy during hibernation is likely mediated by seasonally altered systemic factors that are independent of neuromuscular activity. To determine whether there are components in bear serum that regulate protein and energy metabolism, differentiated human skeletal muscle cells were treated with bear serum (5% in DMEM/Ham's F-12, 24 h) collected during active summer (July) and hibernating winter (February) periods. The serum samples were collected from the same individual bears (Ursus thibetanus japonicus, n = 7 in each season). Total protein content in cultured skeletal muscle cells was significantly increased following a 24 h treatment with hibernating bear serum. Although the protein synthesis rate was not altered, the expression of MuRF1 protein, a muscle-specific E3 ubiquitin ligase was significantly decreased along with a concomitant activation of Akt/FOXO3a signaling. Increased levels of insulin-like growth factor-1 (IGF-1) were also observed in hibernating bear serum. These observations suggest that protein metabolism in cultured human myotubes may be altered when incubated with hibernating bear serum, with a significant increase in serum IGF-1 and diminished MuRF1 expression, a potential target of Akt/FOXO3a signaling. A protein sparing phenotype in cultured muscle cells by treatment with hibernating bear serum holds potential for the development of methods to prevent human muscle atrophy and related disorders.
  12. Stem Cell Res Ther. 2022 Jan 24. 13(1): 28
      Sarcopenia is a common age-related skeletal muscle disorder featuring the loss of muscle mass and function. In regard to tissue repair in the human body, scientists always consider the use of stem cells. In skeletal muscle, satellite cells (SCs) are adult stem cells that maintain tissue homeostasis and repair damaged regions after injury to preserve skeletal muscle integrity. Muscle-derived stem cells (MDSCs) and SCs are the two most commonly studied stem cell populations from skeletal muscle. To date, considerable progress has been achieved in understanding the complex associations between stem cells in muscle and the occurrence and treatment of sarcopenia. In this review, we first give brief introductions to sarcopenia, SCs and MDSCs. Then, we attempt to untangle the differences and connections between these two types of stem cells and further elaborate on the interactions between sarcopenia and stem cells. Finally, our perspectives on the possible application of stem cells for the treatment of sarcopenia in future are presented. Several studies emerging in recent years have shown that changes in the number and function of stem cells can trigger sarcopenia, which in turn leads to adverse influences on stem cells because of the altered internal environment in muscle. A better understanding of the role of stem cells in muscle, especially SCs and MDSCs, in sarcopenia will facilitate the realization of novel therapy approaches based on stem cells to combat sarcopenia.
    Keywords:  Muscle stem cells; Muscle-derived stem cells; Sarcopenia; Satellite cells
  13. Cell Calcium. 2022 Jan 15. pii: S0143-4160(22)00015-X. [Epub ahead of print]102 102540
      The transition of stem cells from quiescence to proliferation enables tissues to self-repair. The signaling mechanisms driving these stem-cell-status decisions are still unclear. Ca2+ and the extracellular signal-regulated kinase (Erk1/2) are two signaling pathways that have the potential to coordinate multiple signals to promote a specific cellular response. They both play important roles during nervous system development but their roles during spinal cord and muscle regeneration are not fully deciphered. Here we show in Xenopus laevis larvae that both Ca2+ and Erk1/2 signaling pathways are activated after tail amputation. In response to injury, we find that Erk1/2 signaling is activated in neural and muscle stem cells and is necessary for spinal cord and skeletal muscle regeneration. Finally, we show in vivo that Erk1/2 activity is necessary for an injury-induced increase in intracellular store-dependent Ca2+ dynamics in skeletal muscle-associated tissues but that in spinal cord, injury increases Ca2+ influx-dependent Ca2+ activity independent of Erk1/2 signaling. This study suggests that precise temporal and tissue-specific activation of Ca2+ and Erk1/2 pathways is essential for regulating spinal cord and muscle regeneration.
    Keywords:  Calcium; Extracellular signal-regulated kinase; In vivo; Regeneration; Skeletal muscle; Spinal cord
  14. iScience. 2022 Jan 21. 25(1): 103715
      Mitochondrial dysfunction causes muscle wasting in many diseases and probably also during aging. The underlying mechanism is poorly understood. We generated transgenic mice with unbalanced mitochondrial protein loading and import, by moderately overexpressing the nuclear-encoded adenine nucleotide translocase, Ant1. We found that these mice progressively lose skeletal muscle. Ant1-overloading reduces mitochondrial respiration. Interestingly, it also induces small heat shock proteins and aggresome-like structures in the cytosol, suggesting increased proteostatic burden due to accumulation of unimported mitochondrial preproteins. The transcriptome of Ant1-transgenic muscles is drastically remodeled to counteract proteostatic stress, by repressing protein synthesis and promoting proteasomal function, autophagy, and lysosomal amplification. These proteostatic adaptations collectively reduce protein content thereby reducing myofiber size and muscle mass. Thus, muscle wasting can occur as a trade-off of adaptation to mitochondria-induced proteostatic stress. This finding could have implications for understanding the mechanism of muscle wasting, especially in diseases associated with Ant1 overexpression, including facioscapulohumeral dystrophy.
    Keywords:  Biological sciences; Cell biology; Cellular physiology; Functional aspects of cell biology
  15. Int J Sports Med. 2022 Jan 27.
      Generally, skeletal muscle adaptations to exercise are perceived through a dichotomous lens where the metabolic stress imposed by aerobic training leads to increased mitochondrial adaptations while the mechanical tension from resistance training leads to myofibrillar adaptations. However, there is emerging evidence for cross over between modalities where aerobic training stimulates traditional adaptations to resistance training (e.g., hypertrophy) and resistance training stimulates traditional adaptations to aerobic training (e.g., mitochondrial biogenesis). The latter is the focus of the current review in which we propose high-volume resistance training (i.e., high time under tension) leads to aerobic adaptations such as angiogenesis, mitochondrial biogenesis, and increased oxidative capacity. As time under tension increases, skeletal muscle energy turnover, metabolic stress, and ischemia also increase, which act as signals to activate the peroxisome proliferator-activated receptor gamma coactivator 1-alpha, which is the master regulator of mitochondrial biogenesis. For practical application, the acute stress and chronic adaptations to three specific forms of high-time under tension are also discussed: Slow-tempo, low-intensity resistance training, and drop-set resistance training. These modalities of high-time under tension lead to hallmark adaptations to resistance training such as muscle endurance, hypertrophy, and strength, but little is known about their effect on traditional aerobic training adaptations.
  16. Am J Physiol Endocrinol Metab. 2022 Jan 24.
      Age-related declines in cardiorespiratory fitness and physical function are mitigated by regular endurance exercise in older adults. This may be due in part to changes in the transcriptional program of skeletal muscle following repeated bouts of exercise. However, the impact of chronic exercise training on the transcriptional response to an acute bout of endurance exercise has not been clearly determined. Here, we characterized baseline differences in muscle transcriptome and exercise-induced response in older adults who were active/endurance trained or sedentary. RNA-sequencing was performed on vastus lateralis biopsy specimens obtained before, immediately after, and 3h following a bout of endurance exercise (40-minutes of cycling at 60-70% of heart rate reserve). Using a recently developed bioinformatics approach, we found that transcript signatures related to type I myofibers, mitochondria, and endothelial cells were higher in active/endurance trained adults, and were associated with key phenotypic features including VO2peak, ATPmax, and muscle fiber proportion. Immune cell signatures were elevated in the sedentary group and linked to visceral and intermuscular adipose tissue mass. Following acute exercise, we observed distinct temporal transcriptional signatures which were largely similar among groups. Enrichment analysis revealed catabolic processes were uniquely enriched in the sedentary group at the 3h post-exercise timepoint. In summary, this study revealed key transcriptional signatures that distinguished active and sedentary adults, which were associated with difference in oxidative capacity and depot-specific adiposity. The acute response signatures were consistent with beneficial effects of endurance exercise to improve muscle health in older adults irrespective of exercise history and adiposity.
    Keywords:  RNA-seq; cardiorespiratory fitness; endothelial cell; mitochondria
  17. Exp Cell Res. 2022 Jan 23. pii: S0014-4827(21)00545-0. [Epub ahead of print] 112989
      Circadian rhythms generate 24 h-long oscillations, which are key regulators of many aspects of behavior and physiology. Recent circadian transcriptome studies have discovered rhythmicity at the transcriptional level of hundreds of skeletal muscle genes, with roles in skeletal muscle growth, maintenance, and metabolic functions. These rhythms allow this tissue to perform molecular functions at the appropriate time of the day in order to anticipate environmental changes. However, while the last decade of research has characterized several aspects of the skeletal muscle molecular clock, many still are unexplored, including its functions, regulatory mechanisms, and interactions with other tissues. The central clock is believed to be located in the suprachiasmatic nucleus (SCN) of the brain hypothalamus, providing entrainment to peripheral organs through humoral and neuronal signals. However, these mechanisms of action are still unknown. Conversely, muscle tissue can be entrained through extrinsic, SCN-independent factors, such as feeding and physical activity. In this review, we provide an overview of the recent research about the extrinsic and intrinsic factors required for skeletal muscle clock regulation. Furthermore, we discuss the need for future studies to elucidate the mechanisms behind this regulation, which will in turn help dissect the role of circadian disruption at the onset of aging and diseases.
    Keywords:  Circadian rhythm; Clock regulation; Satellite cell; Skeletal muscle
  18. Front Cell Dev Biol. 2021 ;9 793088
      Aging promotes most degenerative pathologies in mammals, which are characterized by progressive decline of function at molecular, cellular, tissue, and organismal levels and account for a host of health care expenditures in both developing and developed nations. Sarcopenia is a prominent age-related disorder in musculoskeletal system. Defined as gradual and generalized chronic skeletal muscle disorder, sarcopenia involves accelerated loss of muscle mass, strength and function, which is associated with increased adverse functional outcomes and evolutionally refers to muscle wasting accompanied by other geriatric syndromes. More efforts have been made to clarify mechanisms underlying sarcopenia and new findings suggest that it may be feasible to delay age-related sarcopenia by modulating fundamental mechanisms such as cellular senescence. Cellular senescence refers to the essentially irreversible growth arrest mainly regulated by p53/p21CIP1 and p16INK4a/pRB pathways as organism ages, possibly detrimentally contributing to sarcopenia via muscle stem cells (MuSCs) dysfunction and the senescence-associated secretory phenotype (SASP) while cellular senescence may have beneficial functions in counteracting cancer progression, tissue regeneration and wound healing. By now diverse studies in mice and humans have established that targeting cellular senescence is a powerful strategy to alleviating sarcopenia. However, the mechanisms through which senescent cells contribute to sarcopenia progression need to be further researched. We review the possible mechanisms involved in muscle stem cells (MuSCs) dysfunction and the SASP resulting from cellular senescence, their associations with sarcopenia, current emerging therapeutic opportunities based on targeting cellular senescence relevant to sarcopenia, and potential paths to developing clinical interventions genetically or pharmacologically.
    Keywords:  aging; cellular senescence; muscle stem cells (MuSCs) dysfunction; sarcopenia; senescence-associated secretory phenotype (SASP)
  19. Skelet Muscle. 2022 Jan 22. 12(1): 2
      BACKGROUND: The sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The SC coordinates changes in phosphorylation and Ca++-flux during mechanical deformation, and these processes are disrupted with loss-of-function mutations in gamma-sarcoglycan (Sgcg) that cause Limb girdle muscular dystrophy 2C/R5.METHODS: To gain insight into how the SC mediates mechano-signaling in muscle, we utilized LC-MS/MS proteomics of SC-associated proteins in immunoprecipitates from enriched sarcolemmal fractions. Criteria for inclusion were co-immunoprecipitation with anti-Sgcg from C57BL/6 control muscle and under-representation in parallel experiments with Sgcg-null muscle and with non-specific IgG. Validation of interaction was performed in co-expression experiments in human RH30 rhabdomyosarcoma cells.
    RESULTS: We identified 19 candidates as direct or indirect interactors for Sgcg, including the other 3 SC proteins. Novel potential interactors included protein-phosphatase-1-catalytic-subunit-beta (Ppp1cb, PP1b) and Na+-K+-Cl--co-transporter NKCC1 (SLC12A2). NKCC1 co-localized with Sgcg after co-expression in human RH30 rhabdomyosarcoma cells, and its cytosolic domains depleted Sgcg from cell lysates upon immunoprecipitation and co-localized with Sgcg after detergent permeabilization. NKCC1 localized in proximity to the dystrophin complex at costameres in vivo. Bumetanide inhibition of NKCC1 cotransporter activity in isolated muscles reduced SC-dependent, strain-induced increases in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In silico analysis suggests that candidate SC interactors may cross-talk with survival signaling pathways, including p53, estrogen receptor, and TRIM25.
    CONCLUSIONS: Results support that NKCC1 is a new SC-associated signaling protein. Moreover, the identities of other candidate SC interactors suggest ways by which the SC and NKCC1, along with other Sgcg interactors such as the membrane-cytoskeleton linker archvillin, may regulate kinase- and Ca++-mediated survival signaling in skeletal muscle.
    Keywords:  Archvillin; Limb girdle muscular dystrophy; NKCC1; PP1β/δ; Sarcoglycans; Sarcolemma; Skeletal muscle; Svil
  20. Front Physiol. 2021 ;12 756626
      Introduction: The increasingly popular microbiopsy is an appealing alternative to the more invasive Bergström biopsy given the challenges associated with harvesting skeletal muscle in older populations. Parameters of muscle fiber morphology and composition derived from the microbiopsy have not been compared between young and older adults. Purpose: The purpose of this study was to examine muscle fiber morphology and composition in young (YM) and older (OM) males using the microbiopsy sampling technique. A secondary aim was to determine if specific strength is associated with serum levels of C-terminal agrin fragment [CAF; an indicator of neuromuscular junction (NMJ) degradation]. Methods: Thirty healthy, YM (n = 15, age = 20.7 ± 2.2 years) and OM (n = 15, age = 71.6 ± 3.9 years) underwent ultrasound imaging to determine whole-muscle cross-sectional area (CSA) of the vastus lateralis and rectus femoris as well as isometric and isokinetic (60°⋅s-1 and 180°⋅s-1) peak torque testing of the knee extensors. Microbiopsy samples of the vastus lateralis were collected from 13 YM and 11 OM, and immunofluorescence was used to calculate CSA and proportion of type I and type II fibers. Results: Peak torque was lower in OM at all velocities (p ≤ 0.001; d = 1.39-1.86) but only lower at 180°⋅s-1 (p = 0.003; d = 1.23) when normalized to whole-muscle CSA. Whole-muscle CSA was smaller in OM (p = 0.001; d = 1.34), but atrophy was not present at the single fiber level (p > 0.05). Per individual, ∼900 fibers were analyzed, and type I fiber CSA was larger (p = 0.05; d = 0.94) in OM which resulted in a smaller type II/I fiber CSA ratio (p = 0.015; d = 0.95). CAF levels were not sensitive to age (p = 0.159; d = 0.53) nor associated with specific strength or whole-muscle CSA in OM. Conclusion: The microbiopsy appears to be a viable alternative to the Bergström biopsy for histological analyses of skeletal muscle in older adults. NMJ integrity was not influential for age-related differences in specific strength in our healthy, non-sarcopenic older sample.
    Keywords:  C-terminal agrin fragment (CAF); aging; atrophy; immunofluorescence; microbiopsy; muscle quality; myofiber; vastus lateralis
  21. Front Physiol. 2021 ;12 686119
      Skeletal muscle is one of the most important tissues of the human body. It comprises up to 40% of the body mass and is crucial to survival. Hence, the maintenance of skeletal muscle mass and strength is pivotal. It is well-established that resistance exercise provides a potent anabolic stimulus to increase muscle mass and strength in men and women of all ages. Resistance exercise consists of mechano-biological descriptors, such as load, muscle action, number of repetitions, repetition duration, number of sets, rest interval between sets, frequency, volitional muscular failure, and range of motion, which can be manipulated. Herein, we discuss the evidence-based contribution of these mechano-biological descriptors to muscle mass and strength.
    Keywords:  contribution; descriptors; evidence-based; mechano-biological; resistance exercise
  22. Epigenomes. 2021 Dec 22. pii: 1. [Epub ahead of print]6(1):
      Striated muscle has especially large energy demands. We identified 97 genes preferentially expressed in skeletal muscle and heart, but not in aorta, and found significant enrichment for mitochondrial associations among them. We compared the epigenomic and transcriptomic profiles of the 27 genes associated with striated muscle and mitochondria. Many showed strong correlations between their tissue-specific transcription levels, and their tissue-specific promoter, enhancer, or open chromatin as well as their DNA hypomethylation. Their striated muscle-specific enhancer chromatin was inside, upstream, or downstream of the gene, throughout much of the gene as a super-enhancer (CKMT2, SLC25A4, and ACO2), or even overlapping a neighboring gene (COX6A2, COX7A1, and COQ10A). Surprisingly, the 3' end of the 1.38 Mb PRKN (PARK2) gene (involved in mitophagy and linked to juvenile Parkinson's disease) displayed skeletal muscle/myoblast-specific enhancer chromatin, a myoblast-specific antisense RNA, as well as brain-specific enhancer chromatin. We also found novel tissue-specific RNAs in brain and embryonic stem cells within PPARGC1A (PGC-1α), which encodes a master transcriptional coregulator for mitochondrial formation and metabolism. The tissue specificity of this gene's four alternative promoters, including a muscle-associated promoter, correlated with nearby enhancer chromatin and open chromatin. Our in-depth epigenetic examination of these genes revealed previously undescribed tissue-specific enhancer chromatin, intragenic promoters, regions of DNA hypomethylation, and intragenic noncoding RNAs that give new insights into transcription control for this medically important set of genes.
    Keywords:  DNA methylation; PGC-1α/PPARGC1A; PRKN/PARK2; Parkinson’s disease; enhancer; epigenetics; heart; mitochondria; mitophagy; skeletal muscle
  23. F1000Res. 2021 ;10 1259
      The energy sensor AMP kinase (AMPK) and the master scaffolding protein, AXIN, are two major regulators of biological processes in metazoans. AXIN-dependent regulation of AMPK activation plays a crucial role in maintaining metabolic homeostasis during glucose-deprived and energy-stressed conditions. The two proteins are also required for muscle function. While studies have refined our knowledge of various cellular events that promote the formation of AXIN-AMPK complexes and the involvement of effector proteins, more work is needed to understand precisely how the pathway is regulated in response to various forms of stress. In this review, we discuss recent data on AXIN and AMPK interaction and its role in physiological changes leading to improved muscle health and an extension of lifespan. We argue that AXIN-AMPK signaling plays an essential role in maintaining muscle function and manipulating the pathway in a tissue-specific manner could delay muscle aging. Therefore, research on understanding the factors that regulate AXIN-AMPK signaling holds the potential for developing novel therapeutics to slow down or revert the age-associated decline in muscle function, thereby extending the healthspan of animals.
    Keywords:  AAK-2; AMPK; Axin; C. elegans; LKB1; PRY-1; aging; muscle
  24. Cell Biochem Biophys. 2022 Jan 24.
      During myofiber regeneration, myoblasts are continuously subjected to shear stress. It is currently not known whether shear stress affects the regenerative capacity of myoblasts when extracellular matrix (ECM) stiffness increases (e.g. upon aging). Therefore, we aimed to assess (1) whether matrix stiffness and pulsating fluid shear stress affect myoblast proliferation and/or expression of differentiation-associated genes in myoblasts, and (2) whether matrix stiffness modulates the mechanoresponse of myoblasts to pulsating fluid shear stress. Myoblasts were seeded on matrigel-coated polyacrylamide gel matrices of varying stiffness, mimicking young ("soft", 0.5 kPa) and old ECM ("stiff", 20 kPa), as well as on matrigel-coated glass matrices with very high stiffness (40 ϺPa), and subjected to 1 h pulsating fluid shear stress (3 Pa/s or 4 Pa/s, 1 Hz). We found enhanced proliferation of myoblasts on stiff matrices, but reduced differentiation compared to myoblasts on soft matrices. Pulsating fluid shear stress significantly upregulated gene expression of proliferation-associated genes C-fos and Il-6, as well as expression of cytoskeletal α-actin in myoblasts seeded on glass. In contrast, pulsating fluid shear stress significantly downregulated gene expression of α-actin and Myogenin in myoblasts seeded on soft matrices. In conclusion, these results suggest that age and disease-associated increased ECM stiffness may contribute to declined regenerative capacity of myoblasts, by reducing their capacity to differentiate into new muscular tissue, at least in the absence of mechanical stimulation.
    Keywords:  Aging; Myoblasts; Myogenesis; Shear stress; Substrate stiffness
  25. Med Sci Sports Exerc. 2022 Jan 25.
      PURPOSE: Resistance training induces skeletal muscle hypertrophy via the summated effects of post-exercise elevations in myofibrillar protein synthesis (MyoPS) that persist for up to 48 h, although research in females is currently lacking. MyoPS is regulated by mTOR translocation and colocalization; however, the effects of resistance training on these intracellular processes is unknown. We hypothesized that MyoPS would correlate with hypertrophy only after training in both sexes and would be associated with intracellular redistribution of mTOR.METHODS: Recreationally active males and females (n = 10 each) underwent 8 weeks of whole-body resistance exercise 3x/week. Fasted muscle biopsies were obtained immediately before (REST) and 24 and 48 h after acute resistance exercise in the untrained (UT) and trained (T) state to determine integrated MyoPS over 48 h (D2O ingestion) and intracellular mTOR colocalization (immunofluorescence microscopy).
    RESULTS: Training increased (P < 0.01) muscle strength (~20-126%), muscle thickness (MT;~8-11%), and average fibre cross sectional area (fCSA;~15-20%). MyoPS increased above REST in UT (P = 0.032) and T (P < 0.01), but to a greater extent in males (~23%;P = 0.023), and was positively (P < 0.01) associated with MT and fCSA at T only in both males and females. mTOR colocalization with the cell periphery increased (P < 0.01) in T, irrespective of sex or acute exercise. Training increased (P ≤ 0.043) total mTOR, LAMP2 (lysosomal marker), and their colocalization (P < 0.01), although their colocalization was greater in males at 24 and 48 h independent of training status (P < 0.01).
    CONCLUSIONS: MyoPS during prolonged recovery from exercise is greater in males but related to muscle hypertrophy regardless of sex only in the trained state, which may be underpinned by altered mTOR localization.
  26. Am J Aging Sci Res. 2021 ;2(1): 19-23
      In prior work, we analyzed gene expression profiles of mouse, rat and human gastrocnemius muscles to identify conserved regulators of muscle aging processes. By further comparing data obtained from different muscles we found stronger conservation of aging-related factors at the level of molecular pathways than at the level of individual genes. Here we compared the predictive power of models based on gene expression levels and those based on transcription factor motif activities for an individual's age. Although somewhat less accurate than models based on gene expression, models based on motif activities achieve good prediction of muscle age, further providing insights into aging-related molecular pathways.
    Keywords:  Gene expression; Inflammation; Motif activity; Muscle aging; Muscle homeostasis; Regression
  27. J Physiol. 2022 Jan 26.
      KEY POINTS: IUGR is associated with large-scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the FGF21 co-receptor, KLB, are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma KLB mediates a reduction in muscle development through inhibition of mTOR signaling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals.ABSTRACT: Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified β-Klotho (KLB), a Fibroblast Growth Factor 21 (FGF21) co-receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally-occurring disease model, we performed transcriptome-wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal-weight (NW) littermates. We found that, alongside large-scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs) we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mTOR activity. Moreover, transfection of MPCs with KLB siRNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data sheds new light into the pathogenesis of IUGR, a significant cause of lifelong ill-health in humans and animals. Abstract figure legend In IUGR fetuses, reduced placental supply induces an adaptive response characterized by preferential shunting of blood and therefore oxygen and nutrients to vital tissues such as brain and heart, at the expense of other tissues including skeletal muscle. Using a pig model, we found skeletal muscle from IUGR fetuses to display large-scale gene expression dysregulation including developmental, tissue injury and metabolic genes. Among upregulated genes in IUGR muscle was the FGF21 co-receptor, KLB, whereas FGF21 levels were distinctly elevated in the circulation of IUGR fetuses. Subsequent studies with muscle progenitor cells showed that signaling through FGF21 and KLB inhibits mTOR activation and reduces differentiation and myotube formation by both pig and human cells. These results identify FGF21/KLB signaling as a novel mediator of reduced muscle growth in IUGR fetuses. This article is protected by copyright. All rights reserved.
    Keywords:  FGF21; Fetal; IUGR; KLB; Skeletal Muscle; Transcriptome
  28. Hum Gene Ther. 2022 Jan 26.
      Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy that progresses despite currently available therapy in some patients. The development of gene therapy with adeno-associated virus (AAV) vectors revealed a sex-dependent decrease in efficacy in female mice with Pompe disease. This study evaluated the effect of testosterone on gene therapy with an AAV2/8 vector containing a liver-specific promoter to drive expression of GAA (AAV2/8-LSPhGAA) in female GAA-knockout (KO) mice that were implanted with pellets containing testosterone propionate prior to vector administration. Six weeks following treatment, neuromuscular function and muscle strength were improved as demonstrated by increased Rotarod and wirehang latency for female mice treated with testosterone and vector, in comparison with vector alone. Biochemical correction improved following the addition of testosterone as demonstrated by increased GAA activity and decreased glycogen content in the skeletal muscles of female mice treated with testosterone and vector, in comparison with vector alone. An alternative androgen, oxandrolone, was evaluated similarly to reveal increased GAA in the diaphragm and EDL of female GAA-KO mice following oxandrolone administration; however, glycogen content was unchanged by oxandrolone treatment. The efficacy of androgen hormone treatment in females correlated with increased mannose-6-phosphate receptor in skeletal muscle. These data confirmed the benefits of brief treatment with an androgen hormone in mice with Pompe disease during gene therapy.
  29. J Biomech. 2022 Jan 10. pii: S0021-9290(22)00013-6. [Epub ahead of print]132 110954
      Skeletal muscle design studies, based on anatomical structure, extend back several hundred years. Accurate anatomical drawings show that many muscle fibers are oriented at an angle relative to a muscle's axis of force generation. This pennation angle has been reported in the skeletal muscle biomechanics literature, primarily in the context of trying to understand muscle force generation. In this perspective, I will describe several discoveries that changed my understanding of pennation and I will suggest that muscle pennation has little if any functional significance. I believe that the correct view of pennation is that it represents a packing strategy whereby short fibers can be packed into a limited volume. While surface pennation angle is easily measured, is very descriptive, and changes during force generation, I believe it has no functional significance.
    Keywords:  Fiber length; Fiber type; Mulitpennate; Muscle architecture; Physiological; Ross-sectional area; Unipennate
  30. Cell Rep. 2022 Jan 25. pii: S2211-1247(22)00001-8. [Epub ahead of print]38(4): 110295
      Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. Here, we provide evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM.
    Keywords:  FGFR; Twist; Yorkie; lineage plasticity; muscle; reprogramming
  31. Hum Gene Ther. 2022 Jan 25.
      Facioscapulohumeral muscular dystrophy (FSHD) is a rare muscle dystrophy causing muscle weakness initially in the face, shoulders and upper arms, and extended to lower body muscles as the disease progresses. Respiratory restriction in FSHD is increasingly reported to be more common and severe than previously thought, with the involvement of diaphragm weakness in pulmonary insufficiency being under debate. As aberrant expression of the double homeobox 4 (DUX4) gene is the prime cause of FSHD, we and others have developed numerous strategies and reported promising results on downregulating DUX4 expression in both cellular and animal models of FSHD. However, the effect of DUX4 and anti-DUX4 approaches on diaphragm muscle has not been elucidated. Here we show that toxic DUX4 expression causes pathology that affects the diaphragm of ACTA1-MCM/FLExDUX4 mouse model of FSHD at both molecular and histological levels. Of importance, a systemic antisense treatment that suppresses DUX4 and target genes expression by 50% significantly improves muscle regeneration and muscle fibrosis, and prevents modification in myofiber type composition, supporting its development as a treatment for FSHD.
  32. J Cancer Res Clin Oncol. 2022 Jan 27.
      PURPOSE: Cancer-induced muscle wasting (i.e., cancer cachexia, CC) is a common and devastating syndrome that results in the death of more than 1 in 5 patients. Although primarily a result of elevated inflammation, there are multiple mechanisms that complement and amplify one another. Research on the use of exercise to manage CC is still limited, while exercise for CC management has been recently discouraged. Moreover, there is a lack of understanding that exercise is not a single medicine, but mode, type, dosage, and timing (exercise prescription) have distinct health outcomes. The purpose of this review was to examine the effects of these modes and subtypes to identify the most optimal form and dosage of exercise therapy specific to each underlying mechanism of CC.METHODS: The relevant literatures from MEDLINE and Scopus databases were examined.
    RESULTS: Exercise can counteract the most prominent mechanisms and signs of CC including muscle wasting, increased protein turnover, systemic inflammation, reduced appetite and anorexia, increased energy expenditure and fat wasting, insulin resistance, metabolic dysregulation, gut dysbiosis, hypogonadism, impaired oxidative capacity, mitochondrial dysfunction, and cancer treatments side-effects. There are different modes of exercise, and each mode has different sub-types that induce vastly diverse changes when performed over multiple sessions. Choosing suboptimal exercise modes, types, or dosages can be counterproductive and could further contribute to the mechanisms of CC without impacting muscle growth.
    CONCLUSION: Available evidence shows that patients with CC can safely undertake higher-intensity resistance exercise programs, and benefit from increases in body mass and muscle mass.
    Keywords:  Cancer cachexia; Exercise; Inflammation; Muscle atrophy; Muscle wasting; Tumor
  33. J Neuropathol Exp Neurol. 2022 Jan 23. pii: nlab136. [Epub ahead of print]
      Phenotyping intramuscular immune cells is essential for the characterization of dysimmune/inflammatory myopathies (DIM). Flow cytometry (FC) is the most reliable technique for analyzing leukocyte subpopulations and evaluating their activation levels. We developed a purely mechanical protocol for extracting cells from muscle tissue allowing us to preserve cell surface epitopes and determined its applicability to experimental pathology in mice and myopathological diagnosis in human. Skeletal muscle regeneration in mice was associated with a transient enrichment of macrophages (CD11bhighGr-1+), myeloid dendritic cells (CD3-C8+CD11bhigh), CD8+ T cells (CD3+C8+), and NK cells (CD3- CD11bhighNKp46+). In murine models of inherited muscle dystrophies, leukocytes represented 23%-84% of intramuscular mononuclear cells, with a percentage of CD8+ T cells (4%-17%) mirroring that of all CD45+ cells, while MDCs remained a minority. In human 16 samples (DIM: n = 9; nonimmune conditions: n = 7), DIM was associated with intramuscular recruitment of CD8+ T cells, but not CD4+ T cells and NK cells. FC allowed concomitant quantification of HLA-DR, CD25, CD38, and CD57 activation/differentiation biomarkers and showed increased activation levels of CD4+ and CD8+ T cells in DIM. In conclusion, FC is an appropriate method for quantifying intramuscular leukocyte subpopulations and analyzing their activation states.
    Keywords:  Flow cytometry; Inflammatory myopathy; Lymphocytes; Muscular dystrophy; Myofiber necrosis; Myopathy
  34. EBioMedicine. 2022 Jan 23. pii: S2352-3964(22)00026-3. [Epub ahead of print]76 103842
      BACKGROUND: Sarcolipin and uncoupling protein 3 (UCP3) mediate muscle-based non-shivering thermogenesis (NST) to improve metabolic homeostasis. The impacts of maternal obesity (MO) and maternal exercise (ME) on NST in offspring muscle remain unexamined.METHODS: Female mice were fed with a control diet or high fat diet to induce obesity. Then, obese mice were further separated into two groups: obesity only (OB) and OB plus daily exercise (OB/Ex). Fetal muscle was collected at embryonic day 18.5 and offspring mice at 3-month-old. Apelin administration during pregnancy and apelin receptor (APJ) knockout mouse were further used for investigating the mediatory role of APJ on muscle-based thermogenesis. To explore the direct effects of exercise on AMP-activated protein kinase (AMPK) downstream targets, AMPK knockout mouse was used.
    FINDINGS: MO inhibited while ME activated AMPK and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in fetal muscle. AMPK activation increased sarcolipin expression, which inhibited the uptake of calcium ions into sarcoplasmic reticulum, thereby activating CaMKK2. Consistently, the expression of UCP3 and sarcolipin was suppressed due to MO but activated in ME fetal muscle. Importantly, changes of UCP3 and sarcolipin maintained in offspring muscle, showing the transgenerational effects. Furthermore, apelin administration during pregnancy mimicked the effects of ME on AMPK and CaMKK2 activation, and UCP3 and sarcolipin expression, underscoring the mediatory roles of apelin-AMPK signaling in improving fetal muscle development.
    INTERPRETATION: ME, via activation of apelin signaling-AMPK axis, enhances NST gene expression in fetal and offspring muscle impaired due to MO, which intergenerationally protects offspring from diet-induced obesity and metabolic disorders.
    FUNDING: This work was supported by National Institutes of Health Grant R01-HD067449.
    Keywords:  Calcium activity; Maternal obesity; PGC-1α; Sarcolipin; UCP3
  35. Tissue Cell. 2022 Jan 19. pii: S0040-8166(22)00013-1. [Epub ahead of print]75 101741
      To investigate the effects of the previous administration of testosterone propionate (TP) on the morphology of the gastrocnemius muscle of Wistar rats submitted to ladder-based resistance training (LRT). Twenty-eight rats were divided equally into groups: initial control (CI), 4-week TP (CT4), 4-week TP + LRT (TRT), and placebo + LRT (RT). The rats from the CT4 and TRT groups were treated with TP for four weeks (10 mg/kg/week). TRT and RT trained for ten weeks. The rodents were euthanized at the end of the experiment, and gastrocnemius muscle, prostate, and left and right testicles were collected. There was no statistical difference between the RT and TRT for final volume load. The prostate mass of the TRT and RT groups was statistically heavier than the CT4 group (P < 0.01). The TRT group's prostate/body mass ratio was statistically different from the CT4 group (P < 0.05). The TRT group was shown to have larger type I, type II, and mean fCSA fibers than all other groups (P < 0.001). Regarding the nuclei/fiber ratio (N/f), the CT4, RT, and TRT groups had higher values than CI (P < 0.01). In addition, the RT group showed a higher N/f ratio than CT4 (P < 0.001) but lower than TRT (P < 0.001). In conclusion, short-term TP administration before resistance training can elicit a greater N/f ratio and size of the mean fCSA of the Gastrocnemius muscle of young adult Wistar rats than resistance training alone.
    Keywords:  Exercise; Morphology; Skeletal muscle; Steroids; Testosterone
  36. Am J Physiol Endocrinol Metab. 2022 Jan 24.
      Neuromedin B (NB), a bombesin-like peptide, exerts its specific actions by binding to the neuromedin B receptor (NBR), a G protein-coupled receptor. Female NBR-knockout (NBR-KO) mice exhibit resistance to diet-induced obesity, without hyperphagia, suggesting possible increase in energy expenditure. Skeletal muscle (SM) is crucial for whole-body energy homeostasis, however the presence of NB-NBR signaling and effects in SM are unknown. Here we show that male and female wild type express Nmbr and Nmb mRNA in SM, with higher levels in females. Female NBR-KO gastrocnemius showed increased Myh7 mRNA level, which characterizes type I fibers (oxidative profile). Their permeabilized gastrocnemius fibers, studied by high-resolution respirometry, exhibited higher consumption of O2 coupled to ATP synthesis and unaltered uncoupled respiration. NBR-KO gastrocnemius had higher protein levels of ATP-synthase, and of Nduf9 mRNA, corresponding to mitochondrial complex I subunit. NBR-KO gastrocnemius exhibited slight increase in mitochondria number, increased thickness of Z line at electron microscopy, and unaltered mitochondrial dynamics markers. Therefore, in the females´ gastrocnemius, a predominantly glycolytic SM, the NBR absence promotes changes that favor mitochondrial oxidative phosphorylation capacity. Additionally, in L6 myocytes, NB treatment (5 μg/mL/16 h) promoted lower O2 consumption coupled to ATP synthesis, suggesting direct action at SM cells. Altogether, the study reinforces the hypothesis that inhibition of NB-NBR signaling enhances the capacity for oxidative phosphorylation of white SM, encouraging future studies to elucidate their contribution on other types of SM and to whole body energy expenditure, which may lead to a new target to drug development for obesity treatment.
    Keywords:  G protein-coupled receptor; energy metabolism; mitochondrial energetics; neuromedin B receptor; skeletal muscle
  37. RNA. 2022 Jan 26. pii: rna.078993.121. [Epub ahead of print]
      Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the polypyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.
    Keywords:  Alternative splicing; Myogenesis; RNA-binding proteins
  38. J Cachexia Sarcopenia Muscle. 2022 Jan 26.
      BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by DMD mutations leading to dystrophin loss. Full-length Dp427 is the primary dystrophin isoform expressed in muscle and is also expressed in the central nervous system (CNS). Two shorter isoforms, Dp140 and Dp71, are highly expressed in the CNS. While a role for Dp140 and Dp71 on DMD CNS comorbidities is well known, relationships between mutations expected to disrupt Dp140 and Dp71 and motor outcomes are not.METHODS: Functional outcome data from 387 DMD boys aged 4-15 years were subdivided by DMD mutation expected effects on dystrophin isoform expression; Group 1 (Dp427 absent, Dp140/Dp71 present, n = 201); Group 2 (Dp427/Dp140 absent, Dp71 present, n = 152); and Group 3 (Dp427/Dp140/Dp71 absent, n = 34). Relationships between isoform group and North Star ambulatory assessment (NSAA) scores, 10 m walk/run velocities and rise time velocities were explored using regression analysis. Western blot analysis was used to study Dp427, Dp140 and Dp71 production in myogenic cells (control and DMD human), control skeletal muscle, DMD skeletal muscle from the three isoform groups and cerebral cortex from mice (wild-type and DMD models). Grip strength and rotarod running test were studied in wild-type mice and DMD mouse models. DMD mouse models were mdx (Dp427 absent, Dp140/Dp71 present), mdx52 (Dp427/Dp140 absent, Dp71 present) and DMD-null (lacking all isoforms).
    RESULTS: In DMD boys, mean NSAA scores at 5 years of age were 6.1 points lower in Group 3 than Group 1 (P < 0.01) and 4.9 points lower in Group 3 than Group 2 (P = 0.05). Mean peak NSAA scores were 4.0 points lower in Group 3 than Group 1 (P < 0.01) and 1.6 points lower in Group 2 than Group 1 (P = 0.04). Mean four-limb grip strength was 1.5 g/g lower in mdx52 than mdx mice (P = 0.003) and 1.5 g/g lower in DMD-null than mdx mice (P = 0.002). Dp71 was produced in myogenic cells (control and DMD human) and skeletal muscle from humans in Groups 1 and 2 and mdx mice, but not skeletal muscle from human controls, myogenic cells and skeletal muscle from humans in Group 3 or skeletal muscle from wild-type, mdx52 or DMD-null mice.
    CONCLUSIONS: Our results highlight the importance of considering expected effects of DMD mutations on dystrophin isoform production when considering patterns of DMD motor impairment and the implications for clinical practice and clinical trials. Our results suggest a complex relationship between dystrophin isoforms expressed in the brain and DMD motor function.
    Keywords:  Duchenne muscular dystrophy; Isoform; Motor function
  39. PLoS One. 2022 ;17(1): e0262488
      Cellular senescence is accompanied by metabolic and epigenomic remodeling, but the transcriptional mechanism of this process is unclear. Our previous RNA interference-based screen of chromatin factors found that lysine methyltransferases including SETD8 and NSD2 inhibited the senescence program in cultured fibroblasts. Here, we report that loss of the zinc finger and homeobox protein 3 (ZHX3), a ubiquitously expressed transcription repressor, induced senescence-associated gene expression and mitochondrial-nucleolar activation. Chromatin immunoprecipitation-sequencing analyses of growing cells revealed that ZHX3 was enriched at the transcription start sites of senescence-associated genes such as the cyclin-dependent kinase inhibitor (ARF-p16INK4a) gene and ribosomal RNA (rRNA) coding genes. ZHX3 expression was consistently downregulated in cells with replicative or oncogene-induced senescence. Mass spectrometry-based proteomics identified 28 proteins that interacted with ZHX3, including ATP citrate lyase and RNA metabolism proteins. Loss of ZHX3 or ZHX3-interaction partners by knockdown similarly induced the expression of p16INK4a and rRNA genes. Zhx3-knockout mice showed upregulation of p16INK4a in the testes, thymus and skeletal muscle tissues, together with relatively short survival periods in males. These data suggested that ZHX3 plays an essential role in transcriptional control to prevent cellular senescence.
  40. Eur J Sport Sci. 2022 Jan 22. 1-20
      AbstractThe aim of this review was to perform a meta-analysis examining the effects of CWI coupled with resistance training on gains in muscular strength. Four databases were searched to find relevant studies. Their methodological quality and risk of bias were evaluated using the PEDro checklist. The effects of CWI vs. control on muscular strength were examined in a random-effects meta-analysis. Ten studies (n = 170; 92% males), with 11 comparisons across 22 groups, were included in the analysis. Studies were classified as of good or fair methodological quality. The main meta-analysis found that CWI attenuated muscular strength gains (effect size [ES]: -0.23; 95% confidence interval [CI]: -0.45, -0.01; p = 0.041). In the analysis of data from studies applying CWI only to the trained limbs, CWI attenuated muscular strength gains (ES: -0.31; 95% CI: -0.61, -0.01; p = 0.041). In the analysis of data from studies using whole-body CWI, there was no significant difference in muscular strength gains between CWI and control (ES: -0.08; 95% CI: -0.53, 0.38; p = 0.743). In summary, this meta-analysis found that the use of CWI following resistance exercise sessions attenuates muscular strength gains in males. However, when CWI was applied to the whole body, there was no significant difference between CWI and control for muscular strength. Due to the attenuated gains in muscular strength found with single limb CWI, the use and/or timing of CWI in resistance training should be carefully considered and individualized.
    Keywords:  1RM; exercise; isokinetic strength; isometric strength