bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒08‒15
forty-two papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Endocrinol Metab (Seoul). 2021 Aug 10.
      Skeletal muscle has attracted attention as endocrine organ, because exercise-dependent cytokines called myokines/exerkines are released from skeletal muscle and are involved in systemic functions. While, local mechanical loading to skeletal muscle by exercise or resistance training alters myofiber type and size and myonuclear number. Skeletal muscle-resident stem cells, known as muscle satellite cells (MuSCs), are responsible for the increased number of myonuclei. Under steady conditions, MuSCs are maintained in a mitotically quiescent state but exit from that state and start to proliferate in response to high physical activity. Alterations in MuSC behavior occur when myofibers are damaged, but the lethal damage to myofibers does not seem to evoke mechanical loading-dependent MuSC activation and proliferation. Given that MuSCs proliferate without damage, it is unclear how the different behaviors of MuSCs are controlled by different physical activities. Recent studies demonstrated that myonuclear number reflects the size of myofibers; hence, it is crucial to know the properties of MuSCs and the mechanism of myonuclear accretion by MuSCs. In addition, the elucidation of mechanical load-dependent changes in muscle resident cells, including MuSCs, will be necessary for the discovery of new myokines/exerkines and understating skeletal muscle diseases.
    Keywords:  Exercise; Hypertrophy; Muscle, skeletal; Resistance training; Skeletal muscle satellite cells
  2. Curr Mol Pharmacol. 2021 Aug 06.
      Skeletal muscles are considered the largest reservoirs of the protein pool in the body and are critical for the maintenances of body homeostasis. Skeletal muscle atrophy is supported by various physiopathological conditions that lead to loss of muscle mass and contractile capacity of the skeletal muscle. Lysosomal mediated autophagy and ubiquitin-proteasomal system (UPS) concede the major intracellular systems of muscle protein degradation that result in the loss of mass and strength. Both systems recognize ubiquitination as a signal of degradation through different mechanisms, a sign of dynamic interplay between systems. Hence, growing shreds of evidence suggest the interdependency of autophagy and UPS in the progression of skeletal muscle atrophy under various pathological conditions. Therefore, understanding the molecular dynamics as well associated factors responsible for their interdependency is a necessity for the new therapeutic insights to counteract the muscle loss. Based on current literature, the present review summarizes the factors interplay in between the autophagy and UPS in favor of enhanced proteolysis of skeletal muscle and how they affect the anabolic signaling pathways under various conditions of skeletal muscle atrophy.
    Keywords:  Skeletal muscle atrophy; autophagosome-lysosome system; mitophagy; myostatin and ubiquitination; ubiquitin-proteasomal system
  3. Front Cell Dev Biol. 2021 ;9 690577
      Skeletal muscle demonstrates a high degree of adaptability in response to changes in mechanical input. The phenotypic transformation in response to mechanical cues includes changes in muscle mass and force generating capabilities, yet the molecular pathways that govern skeletal muscle adaptation are still incompletely understood. While there is strong evidence that mechanotransduction pathways that stimulate protein synthesis play a key role in regulation of muscle mass, there are likely additional mechano-sensitive mechanisms important for controlling functional muscle adaptation. There is emerging evidence that the cell nucleus can directly respond to mechanical signals (i.e., nuclear mechanotransduction), providing a potential additional level of cellular regulation for controlling skeletal muscle mass. The importance of nuclear mechanotransduction in cellular function is evident by the various genetic diseases that arise from mutations in proteins crucial to the transmission of force between the cytoskeleton and the nucleus. Intriguingly, these diseases preferentially affect cardiac and skeletal muscle, suggesting that nuclear mechanotransduction is critically important for striated muscle homeostasis. Here we discuss our current understanding for how the nucleus acts as a mechanosensor, describe the main cytoskeletal and nuclear proteins involved in the process, and propose how similar mechanoresponsive mechanisms could occur in the unique cellular environment of a myofiber. In addition, we examine how nuclear mechanotransduction fits into our current framework for how mechanical stimuli regulates skeletal muscle mass.
    Keywords:  LINC complex; mechanotransduction; muscle adaptation; muscle mass; nesprins; nuclear lamina; nucleus
  4. Exp Cell Res. 2021 Aug 06. pii: S0014-4827(21)00319-0. [Epub ahead of print]406(2): 112766
      Duchene muscular dystrophy leads to progressive muscle structural and functional decline due to chronic degenerative-regenerative cycles. Enhancing the regenerative capacity of dystrophic muscle provides potential therapeutic options. We previously demonstrated that the circadian clock repressor Rev-erbα inhibited myogenesis and Rev-erbα ablation enhanced muscle regeneration. Here we show that Rev-erbα deficiency in the dystrophin-deficient mdx mice promotes regenerative myogenic response to ameliorate muscle damage. Loss of Rev-erbα in mdx mice improved dystrophic pathology and muscle wasting. Rev-erbα-deficient dystrophic muscle exhibit augmented myogenic response, enhanced neo-myofiber formation and attenuated inflammatory response. In mdx myoblasts devoid of Rev-erbα, myogenic differentiation was augmented together with up-regulation of Wnt signaling and proliferative pathways, suggesting that loss of Rev-erbα inhibition of these processes contributed to the improvement in regenerative myogenesis. Collectively, our findings revealed that the loss of Rev-erbα function protects dystrophic muscle from injury by promoting myogenic repair, and inhibition of its activity may have therapeutic utilities for muscular dystrophy.
    Keywords:  Circadian clock; Muscle regeneration; Muscular dystrophy; Myogenesis; Myogenic progenitor cell
  5. iScience. 2021 Aug 20. 24(8): 102838
      Skeletal muscle is composed of post-mitotic myofibers that form a syncytium containing hundreds of myonuclei. Using a progressive exercise training model in the mouse and single nucleus RNA-sequencing (snRNA-seq) for high-resolution characterization of myonuclear transcription, we show myonuclear functional specialization in muscle. After 4 weeks of exercise training, snRNA-seq reveals that resident muscle stem cells, or satellite cells, are activated with acute exercise but demonstrate limited lineage progression while contributing to muscle adaptation. In the absence of satellite cells, a portion of nuclei demonstrates divergent transcriptional dynamics associated with mixed-fate identities compared with satellite cell replete muscles. These data provide a compendium of information about how satellite cells influence myonuclear transcription in response to exercise.
    Keywords:  Biological sciences; Stem cells research; Transcriptomics
  6. Pharmacol Res. 2021 Aug 10. pii: S1043-6618(21)00391-1. [Epub ahead of print] 105807
      Skeletal muscle is a crucial tissue for movement, gestural assistance, metabolic homeostasis, and thermogenesis. It makes up approximately 40% of the total body weight and 50% of total protein. However, several pathological abnormalities (e.g., chronic diseases, cancer, long-term infection, aging) can induce an imbalance in skeletal muscle protein synthesis and degradation, which triggers muscle wasting and even leads to atrophy. Skeletal muscle atrophy is characterized by weakening, shrinking, and decreasing muscle mass and fiber cross-sectional area at the histological level. It manifests as a reduction in force production, easy fatigue and decreased exercise capability, along with a lower quality of life. Mechanistically, there are several pathophysiological processes involved in skeletal muscle atrophy, including oxidative stress and inflammation, which then activate signal transduction, such as the ubiquitin proteasome system, autophagy lysosome system, and mTOR. Considering the great economic and social burden that muscle atrophy can inflict, effective prevention and treatment strategies are essential but still limited. Exercise is widely acknowledged as the most effective therapy for skeletal muscle atrophy; unfortunately, it is not applicable for all patients. Several active substances for skeletal muscle atrophy have been discovered and evaluated in clinical trials; however, they have not been marketed to date. Knowledge is being gained on the underlying mechanisms, highlighting more promising treatment strategies in the future. In this paper, the mechanisms and treatment strategies for skeletal muscle atrophy are briefly reviewed.
    Keywords:  Atrophy; Mechanism; Skeletal muscle; Treatment; Ubiquitin proteasome system; mTOR
  7. Nutrients. 2021 Jul 13. pii: 2391. [Epub ahead of print]13(7):
      There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.
    Keywords:  AMPK; ERK; IL-6; Interferon-γ; TNFα; atrophy; iNOS; myotube; protein synthesis; α-hydroxyisocaproic acid
  8. J Cachexia Sarcopenia Muscle. 2021 Aug;12(4): 1098-1116
      BACKGROUND: Spinal muscular atrophy is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Recently approved treatments aimed at increasing SMN protein levels have dramatically improved patient survival and have altered the disease landscape. While restoring SMN levels slows motor neuron loss, many patients continue to have smaller muscles and do not achieve normal motor milestones. While timing of treatment is important, it remains unclear why SMN restoration is insufficient to fully restore muscle size and function. We and others have shown that SMN-deficient muscle precursor cells fail to efficiently fuse into myotubes. However, the role of SMN in myoblast fusion is not known.METHODS: In this study, we show that SMN-deficient myoblasts readily fuse with wild-type myoblasts, demonstrating fusion competency. Conditioned media from wild type differentiating myoblasts do not rescue the fusion deficit of SMN-deficient cells, suggesting that compromised fusion may primarily be a result of altered membrane dynamics at the cell surface. Transcriptome profiling of skeletal muscle from SMN-deficient mice revealed altered expression of cell surface fusion molecules. Finally, using cell and mouse models, we investigate if myoblast fusion can be rescued in SMN-deficient myoblast and improve the muscle pathology in SMA mice.
    RESULTS: We found reduced expression of the muscle fusion proteins myomaker (P = 0.0060) and myomixer (P = 0.0051) in the muscle of SMA mice. Suppressing SMN expression in C2C12 myoblast cells reduces expression of myomaker (35% reduction; P < 0.0001) and myomixer, also known as myomerger and minion, (30% reduction; P < 0.0001) and restoring SMN levels only partially restores myomaker and myomixer expression. Ectopic expression of myomixer improves myofibre number (55% increase; P = 0.0006) and motor function (35% decrease in righting time; P = 0.0089) in SMA model mice and enhances motor function (82% decrease in righting time; P < 0.0001) and extends survival (28% increase; P < 0.01) when administered in combination with an antisense oligonucleotide that increases SMN protein levels.
    CONCLUSIONS: Here, we identified reduced expression of muscle fusion proteins as a key factor in the fusion deficits of SMN-deficient myoblasts. This discovery provides a novel target to improve SMA muscle pathology and motor function, which in combination with SMN increasing therapy could enhance clinical outcomes for SMA patients.
    Keywords:  Muscle; Myoblast; Myomaker; Myomixer; Spinal muscular atrophy
  9. Neuropharmacology. 2021 Aug 05. pii: S0028-3908(21)00299-9. [Epub ahead of print]197 108744
      Endocrine mechanisms have been largely associated with metabolic control and tissue cross talk in mammals. Classically, myokines comprise a class of signaling proteins released in the bloodstream by the skeletal muscle, which mediate physiological and metabolic responses in several tissues, including the brain. Recent exciting evidence suggests that myokines (e.g. cathepsin B, FNDC5/irisin, interleukin-6) act to control brain functions, including learning, memory, and mood, and may mediate the beneficial actions of physical exercise in the brain. However, the intricate mechanisms connecting peripherally released molecules to brain function are not fully understood. Accumulating findings further indicates that impaired skeletal muscle homeostasis impacts brain metabolism and physiology. Here we review recent findings that suggest that muscle-borne signals are essential for brain physiology and discuss perspectives on how these signals vary in response to exercise or muscle diseases. Understanding the complex interactions between skeletal muscle and brain may result in more effective therapeutic strategies to expand healthspan and to prevent brain disease.
    Keywords:  Alzheimer's disease; Cognition; Depression; Hormones; Mood; Myokines; Neurodegeneration; Physical exercise; Skeletal muscle
  10. J Nutr. 2021 Aug 12. pii: nxab251. [Epub ahead of print]
      BACKGROUND: Skeletal muscle progenitor cells (MPCs) repair damaged muscle postinjury. Pyruvate kinase M2 (PKM2) is a glycolytic enzyme (canonical activity) that can also interact with other proteins (noncanonical activity) to modify diverse cellular processes. Recent evidence links PKM2 to MPC proliferation.OBJECTIVES: This study aimed to understand cellular roles for PKM2 in MPCs and the necessity of PKM2 in MPCs for muscle regeneration postinjury.
    METHODS: Cultured, proliferating MPCs (C2C12 cells) were treated with a short hairpin RNA targeting PKM2 or small molecules that selectively affect canonical and noncanonical PKM2 activity (shikonin and TEPP-46). Cell number was measured, and RNA-sequencing and metabolic assays were used in follow-up experiments. Immunoprecipitation coupled to proteomics was used to identify binding partners of PKM2. Lastly, an MPC-specific PKM2 knockout mouse was generated and challenged with a muscle injury to determine the impact of PKM2 on regeneration.
    RESULTS: When the noncanonical activity of PKM2 was blocked or impaired, there was an increase in reactive oxygen species concentrations (1.6-2.0-fold, P < 0.01). Blocking noncanonical PKM2 activity also increased lactate excretion (1.2-1.6-fold, P < 0.05) and suppressed mitochondrial oxygen consumption (1.3-1.6-fold, P < 0.01). Glutamate dehydrogenase 1 (GLUD1) was identified as a PKM2 binding partner and blocking noncanonical PKM2 activity increased GLUD activity (1.5-1.6-fold, P < 0.05). Mice with an MPC-specific PKM2 deletion did not demonstrate impaired muscle regeneration.
    CONCLUSIONS: The results suggest that the noncanonical activity of PKM2 is important for MPC proliferation in vitro and demonstrate GLUD1 as a PKM2 binding partner. Because no impairments in muscle regeneration were detected in a mouse model, the endogenous environment may compensate for loss of PKM2.
    Keywords:  GLUD1; PKM2; glutamate dehydrogenase; glycolysis; muscle progenitor cell; muscle regeneration; muscle stem cell; pyruvate kinase M2
  11. Cancer Discov. 2021 Aug 13.
      Impaired BMP signaling causes denervation, neuromuscular junction dysregulation, and muscle atrophy.
  12. BMC Musculoskelet Disord. 2021 Aug 11. 22(1): 680
      BACKGROUND: Skeletal muscle atrophy and fibrosis are pathological conditions that contribute to morbidity in numerous conditions including aging, cachexia, and denervation. Muscle atrophy is characterized as reduction of muscle fiber size and loss of muscle mass while muscle fibrosis is due to fibroblasts activation and excessive production of extracellular matrix. Purinergic receptor P2Y2 has been implicated in fibrosis. This study aims to elucidate the roles of P2Y2 in sleketal muscle atrophy and fibrosis.METHODS: Primary muscle fibroblasts were isolated from wild type and P2Y2 knockout (KO) mice and their proliferating and migrating abilities were assessed by CCK-8 and Transwell migration assays respectively. Fibroblasts were activated with TGF-β1 and assessed by western blot of myofibroblast markers including α-SMA, CTGF, and collagen I. Muscle atrophy and fibrosis were induced by transection of distal sciatic nerve and assessed using Masson staining.
    RESULTS: P2Y2 KO fibroblasts proliferated and migrated significantly slower than WT fibroblasts with or without TGF-β1.The proliferation and ECM production were enhanced by P2Y2 agonist PSB-1114 and inhibited by antagonist AR-C118925. TGF-β1 induced fibrotic activation was abolished by P2Y2 ablation and inhibited by AKT, ERK, and PKC inhibitors. Ablation of P2Y2 reduced denervation induced muscle atrophy and fibrosis.
    CONCLUSIONS: P2Y2 is a promoter of skeletal muscle atrophy and activation of fibroblasts after muscle injury, which signaling through AKT, ERK and PKC. P2Y2 could be a potential intervention target after muscle injury.
    Keywords:  Extracellular matrix; Fibroblast; Muscle atrophy; Muscular fibrosis; P2Y2
  13. Mol Ther. 2021 Aug 06. pii: S1525-0016(21)00400-7. [Epub ahead of print]
      Mutations in the BIN1 (Bridging Interactor 1) gene, encoding the membrane remodeling protein amphiphysin 2, cause centronuclear myopathy associated with severe muscle weakness and myofiber disorganization and hypotrophy. There is no available therapy, and the validation of therapeutic proof-of-concepts is impaired by the lack of a faithful and easy-to-handle mammalian model. Here, we generated and characterized the Bin1mck-/- mouse through Bin1 knockout in skeletal muscle. Bin1mck-/- mice were viable, unlike the constitutive Bin1 knockout, and displayed decreased muscle force and most histological hallmarks of centronuclear myopathy including myofiber hypotrophy and intracellular disorganization. Notably, Bin1mck-/- myofibers presented strong defects in mitochondria and T-tubule networks associated with deficient calcium homeostasis and excitation-contraction coupling at the triads, potentially representing the main pathomechanisms. Systemic injection of antisense oligonucleotides targeting Dnm2 (Dynamin 2) that codes for dynamin 2, a BIN1 binding partner regulating membrane fission and mutated in other forms of centronuclear myopathy, improved muscle force and normalized the histological Bin1mck-/- phenotypes within 5 weeks. Overall, we generated a faithful mammalian model for centronuclear myopathy linked to BIN1 defects, and validated Dnm2 antisense oligonucleotides as a first translatable approach to efficiently treat BIN1-centronuclear myopathy.
    Keywords:  amphiphysin; antisense oligonucleotides; dynamin; membrane curvature; myopathy; therapy
  14. J Neuropathol Exp Neurol. 2021 Aug 07. pii: nlab062. [Epub ahead of print]
      Skeletal muscle atrophy may occur with disease, injury, decreased muscle use, starvation, and normal aging. No reliably effective treatments for atrophy are available, thus research into the mechanisms contributing to muscle loss is essential. The ERG1A K+ channel contributes to muscle loss by increasing ubiquitin proteasome proteolysis (UPP) in the skeletal muscle of both unweighted and cachectic mice. Because the mechanisms which produce atrophy vary based upon the initiating factor, here we investigate atrophy produced by denervation. Using immunohistochemistry and immunoblots, we demonstrate that ERG1A protein abundance increases significantly in the Gastrocnemius muscle of rodents 7 days after both sciatic nerve transection and hind limb unweighting. Further, we reveal that ectopic expression of a Merg1a encoded plasmid in normal mouse Gastrocnemius muscle has no effect on activity of the NFκB transcription factor family, a group of proteins which contribute to muscle atrophy by modulation of the UPP. Further, although NFκB activity increases significantly after denervation, we show that expression of a plasmid encoding a dominant negative Merg1a mutant in Gastrocnemius muscle prior to denervation, has no effect on NFκB activity. Thus, although the ERG1A K+ channel increases UPP, it does not do so through modulation of NFκB transcription factors.
    Keywords:  ERG1A potassium channel; MuRF1 E3 ligase; NFκB transcription factors; Skeletal muscle atrophy; Ubiquitin proteasome proteolysis
  15. Stem Cell Res Ther. 2021 Aug 09. 12(1): 446
      BACKGROUND: Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of COL6A1, 2, and 3 genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation.METHODS: To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs).
    RESULTS: All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (Col6a1KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in Col6a1KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (Col6a1KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not.
    CONCLUSIONS: These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice.
    Keywords:  COL6 related disease; Induced pluripotent stem cells; Mesenchymal stromal cells; Skeletal muscle regeneration; Ullrich congenital muscular dystrophy
  16. Exp Gerontol. 2021 Aug 06. pii: S0531-5565(21)00292-8. [Epub ahead of print] 111510
      Both oxidative stress and telomere transcription are up-regulated by acute endurance exercise in human skeletal muscle. Whether and how life-long exercise training influences the antioxidant system response at transcriptional level and TERRA expression is unknown, especially during aging. Response to acute endurance exercise was investigated in muscle biopsies of 3 male subjects after 45 min of cycling. MCP-1 and SOD1 mRNA levels increased up to, 15-fold and 63%, respectively, after the cycling session while the mRNA levels of SOD2 were downregulated by 25%. The effects of chronic endurance exercise and aging were tested in the blood and muscle of 34 male subjects divided into four groups: young (YU) or old (OU) untrained, young (YT) or old (OT) trained cyclists. Long-term endurance training limited the age-dependent elevation in SOD1 (OT vs OU, -26%, P = 0.03) and the decline in SOD2 mRNA levels (OU vs YU, -41%, P = 0.04). A high endurance training status alleviated the age-related increase in the aging biological marker MCP-1 in plasma (OU vs YU, +48%, P = 0.005). Similar results were observed for telomeric transcription as the age-associated increase in 16p TERRA levels (OU vs YU, +39%, P = 0.001) was counteracted by a high endurance training status (OT vs OU, -63%, P = 0.0005). In conclusion, as MCP-1, we propose that the age-related TERRA accumulation might represent a novel biological marker of aging. Those aging-related increase expression might be alleviated by a high endurance training status. Whether those biological markers of aging are linked to an elevation of oxidative stress is still an open question. Therefore, whether the positive adaptations provided by endurance training indeed reduce oxidative stress, including at telomeres, and whether TERRA plays any role in this, need to be further investigated.
    Keywords:  Exercise; Inflammation; Oxidative stress; Physical activity; Telomere
  17. Muscle Nerve. 2021 Aug 12.
      INTRODUCTION/AIM: Duchenne and Becker muscular dystrophies (DMD, BMD) are characterized by fat replacement of different skeletal muscles in a specific temporal order. Given the structural role of dystrophin in skeletal muscle mechanics, muscle architecture could be important in the progressive pathophysiology of muscle degeneration. Therefore, the aim of this study was to assess the role of muscle architecture in the progression of fat replacement in DMD and BMD.METHODS: We assessed the association between literature-based leg muscle architectural characteristics and muscle fat fraction from 22 DMD and 24 BMD patients. Dixon-based MRI estimates of fat fractions at baseline, 12 (only DMD) and 24 months were related to fiber length and physiological cross-sectional area (PCSA) using age-controlled linear mixed modelling.
    RESULTS: DMD and BMD muscles with long fibers and BMD muscles with large PCSAs were associated with increased fat fraction. The effect of fiber length was stronger in muscles with larger PCSA.
    DISCUSSION: Muscle architecture might explain the pathophysiology of muscle degeneration in dystrophinopathies, in which proximal muscles with a larger mass (fiber length x PCSA) are more susceptible, confirming the clinical observation of a temporal proximal to distal progression. These results give more insight into the mechanical role in the pathophysiology of muscular dystrophies. Ultimately, this new information can be used to help support the selection of current and the development of future therapies. This article is protected by copyright. All rights reserved.
    Keywords:  MRI; dystrophin; fat fraction; muscle degeneration; pathophysiology
  18. PLoS Genet. 2021 Aug 09. 17(8): e1009729
      Muscle precursor cells known as myoblasts are essential for muscle development and regeneration. Notch signaling is an ancient intercellular communication mechanism that plays prominent roles in controlling the myogenic program of myoblasts. Currently whether and how the myogenic cues feedback to refine Notch activities in these cells are largely unknown. Here, by mouse and human gene gain/loss-of-function studies, we report that MyoD directly turns on the expression of Notch-ligand gene Dll1 which activates Notch pathway to prevent precautious differentiation in neighboring myoblasts, while autonomously inhibits Notch to facilitate a myogenic program in Dll1 expressing cells. Mechanistically, we studied cis-regulatory DNA motifs underlying the MyoD-Dll1-Notch axis in vivo by characterizing myogenesis of a novel E-box deficient mouse model, as well as in human cells through CRISPR-mediated interference. These results uncovered the crucial transcriptional mechanism that mediates the reciprocal controls of Notch and myogenesis.
  19. Stem Cell Res Ther. 2021 Aug 09. 12(1): 448
      BACKGROUND: The skeletal muscle reconstruction occurs thanks to unipotent stem cells, i.e., satellite cells. The satellite cells remain quiescent and localized between myofiber sarcolemma and basal lamina. They are activated in response to muscle injury, proliferate, differentiate into myoblasts, and recreate myofibers. The stem and progenitor cells support skeletal muscle regeneration, which could be disturbed by extensive damage, sarcopenia, cachexia, or genetic diseases like dystrophy. Many lines of evidence showed that the level of oxygen regulates the course of cell proliferation and differentiation.METHODS: In the present study, we analyzed hypoxia impact on human and pig bone marrow-derived mesenchymal stromal cell (MSC) and mouse myoblast proliferation, differentiation, and fusion. Moreover, the influence of the transplantation of human bone marrow-derived MSCs cultured under hypoxic conditions on skeletal muscle regeneration was studied.
    RESULTS: We showed that bone marrow-derived MSCs increased VEGF expression and improved myogenesis under hypoxic conditions in vitro. Transplantation of hypoxia preconditioned bone marrow-derived MSCs into injured muscles resulted in the improved cell engraftment and formation of new vessels.
    CONCLUSIONS: We suggested that SDF-1 and VEGF secreted by hypoxia preconditioned bone marrow-derived MSCs played an essential role in cell engraftment and angiogenesis. Importantly, hypoxia preconditioned bone marrow-derived MSCs more efficiently engrafted injured muscles; however, they did not undergo myogenic differentiation.
    Keywords:  BM-MSC; Fusion; Hypoxia; Migration; Myogenic differentiation; Normoxia
  20. Dis Model Mech. 2021 Aug 11. pii: dmm.048981. [Epub ahead of print]
      Mitochondrial diseases are genetic disorders leading to an impaired mitochondrial function and resulting in exercise intolerance and muscle weakness. In patients, muscle fatigue due to defects in mitochondrial oxidative capacities commonly precedes muscle weakness. In mice, the fast-twitch skeletal muscle-specific Tfam deletion (Tfam KO) leads to deficit in the respiratory chain activity, severe muscle weakness and early death. Here, we performed a time-course study of mitochondrial and muscular dysfunctions in 11 and 14 weeks Tfam KO mice, i.e., before and when mice are about to enter the terminal stage, respectively. While force in the unfatigued state was reduced in Tfam KO mice as compared to control littermates (WT) only at 14 weeks, during repeated submaximal contractions fatigue was faster at both ages. During fatiguing stimulation, total phosphocreatine breakdown was larger in Tfam KO muscle than in WT muscle at both ages whereas phosphocreatine consumption was faster only at 14 weeks. In conclusion, the Tfam KO mouse model represents a reliable model of lethal mitochondrial myopathy where impaired mitochondrial energy production and premature fatigue occur before muscle weakness and early death.
    Keywords:  Energy metabolism; Exercise intolerance; Mitochondrial myopathy; Muscle fatigue; Muscle weakness
  21. Stem Cell Reports. 2021 Jul 29. pii: S2213-6711(21)00380-5. [Epub ahead of print]
      The health and homeostasis of skeletal muscle are preserved by a population of tissue-resident muscle stem cells (MuSCs) that maintain a state of mitotic and metabolic quiescence in adult tissues. The capacity of MuSCs to preserve the quiescent state declines with aging and metabolic insults, promoting premature activation and stem cell exhaustion. Sestrins are a class of stress-inducible proteins that act as antioxidants and inhibit the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling complex. Despite these pivotal roles, the role of Sestrins has not been explored in adult stem cells. We show that SESTRIN1,2 loss results in hyperactivation of the mTORC1 complex, increased propensity to enter the cell cycle, and shifts in metabolic flux. Aged SESTRIN1,2 knockout mice exhibited loss of MuSCs and a reduced ability to regenerate injured muscle. These findings demonstrate that Sestrins help maintain metabolic pathways in MuSCs that protect quiescence against aging.
    Keywords:  RNA sequencing; aging; mTORC1; metabolism; oxidative stress; reactive oxygen species; regeneration; satellite cells
  22. J Diabetes Res. 2021 ;2021 9979234
      Objective: To investigate if PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells. Participants. Eight lean insulin-sensitive nondiabetic participants (4 females and 4 males; age: 21.0 ± 1.0 years; BMI: 22.0 ± 0.7 kg/m2; 2-hour OGTT: 97.0 ± 6.0 mg/dl; HbA1c: 5.3 ± 0.1%; fasting plasma glucose: 87.0 ± 2.0 mg/dl; M value; 11.0 ± 1.0 mg/kgBW/min).Design: A hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects, and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies that included 8 lean insulin-sensitive participants. Cultured cells were expanded, differentiated into myotubes, and treated with 50 μM metformin for 24 hours before harvesting. PP2Ac activity was measured by a phosphatase activity assay kit (Millipore) according to the manufacturer's protocol.
    Results: The results indicated that metformin significantly increased the activity of PP2A in the myotubes for all 8 lean insulin-sensitive nondiabetic participants, and the average fold increase is 1.54 ± 0.11 (P < 0.001).
    Conclusions: These results provided the first evidence that metformin can activate PP2A in human skeletal muscle cells derived from lean healthy insulin-sensitive participants and may help to understand metformin's action in skeletal muscle in humans.
  23. Acta Naturae. 2021 Apr-Jun;13(2):13(2): 85-97
      Kozlovskaya et al. [1] and Grigoriev et al. [2] showed that enormous loss of muscle stiffness (atonia) develops in humans under true (space flight) and simulated microgravity conditions as early as after the first days of exposure. This phenomenon is attributed to the inactivation of slow motor units and called reflectory atonia. However, a lot of evidence indicating that even isolated muscle or a single fiber possesses substantial stiffness was published at the end of the 20th century. This intrinsic stiffness is determined by the active component, i.e. the ability to form actin-myosin cross-bridges during muscle stretch and contraction, as well as by cytoskeletal and extracellular matrix proteins, capable of resisting muscle stretch. The main facts on intrinsic muscle stiffness under conditions of gravitational unloading are considered in this review. The data obtained in studies of humans under dry immersion and rodent hindlimb suspension is analyzed. The results and hypotheses regarding reduced probability of cross-bridge formation in an atrophying muscle due to increased interfilament spacing are described. The evidence of cytoskeletal protein (titin, nebulin, etc.) degradation during gravitational unloading is also discussed. The possible mechanisms underlying structural changes in skeletal muscle collagen and its role in reducing intrinsic muscle stiffness are presented. The molecular mechanisms of changes in intrinsic stiffness during space flight and simulated microgravity are reviewed.
    Keywords:  atonia; collagen; cytoskeleton; dry immersion; gravitational unloading; hindlimb suspension; intrinsic stiffness; muscle stiffness; passive stiffness; sarcomeric cytoskeletal proteins; signaling; skeletal muscle; titin
  24. Nat Commun. 2021 08 12. 12(1): 4900
      Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.
  25. Mitochondrion. 2021 Aug 09. pii: S1567-7249(21)00109-4. [Epub ahead of print]
      The size and morphology of mitochondria are very heterogeneous and correlates well with their healthy functioning. In many pathological conditions, mitochondrial morphology is altered due to impaired mitochondrial dynamics (a collective term for mitochondrial fusion and fission) and dysfunction. The current study aimed at identifying the role of microRNA-128 (miR-128) in regulating mitochondrial biogenesis. Previously, peroxisome proliferator activator receptor γ coactivator 1α (PGC1α) has been shown to co-activate key intermediates of mitochondrial biogenesis, function, and dynamics; however, the upstream regulatory network remains largely unknown. We, herein using in silico analysis followed by in vitro experiments in C2C12 myoblasts, showed that miR-128 reduces mitochondrial biogenesis by directly targeting PGC1α. The expression of downstream genes, nuclear respiratory factors 1 and 2 (NRF1 and NRF2, respectively), and mitochondrial transcription factor A (TFAM) were decreased in C2C12 myoblasts upon overexpression of miR-128. Also, miR-128 is shown to promote mitochondrial dysfunction by directly targeting NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (NDUFS4). The mitochondrial dynamics and morphology were impaired post miR-128 overexpression, as revealed by downregulation of fusion proteins (mitofusin1 and 2, i.e., MFN1 and MFN2, respectively) and upregulation of fission protein (dynamin-related protein 1, i.e., DRP1). Conversely, inhibition of miR-128 expression improved mitochondrial biogenesis, function, and dynamics, as evidenced by increased mitochondrial mass and ATP production after antimiR-128 treatment. Our findings reveal that inhibition of miR-128 can be a new potential target for reversing the effects of metabolic disorders of skeletal muscle as observed during many pathophysiological conditions such as obesity and type II diabetes.
    Keywords:  MiR-128; NDUFS4; PGC1α; mitochondrial biogenesis; mitochondrial dysfunction
  26. J Aging Sci. 2020 ;pii: 005. [Epub ahead of print]8(Suppl 3):
      Sarcopenia is a debilitating muscle-wasting disease that is the major cause of frailty and disability in aging. Ghrelin (aka acylated ghrelin, AG) is a circulating peptide hormone with an unique octanoylation on Ser3. AG induces growth hormone (GH) secretion, increases food intake, and promotes adiposity and insulin resistance via its receptor, Growth Hormone Secretagogue Receptor (GHS-R). Unlike AG, unacylated ghrelin (UAG) is a peptide generated from the same ghrelin gene with amino acid sequence identical to AG but without the octanoylation modification, so UAG does not activate GHS-R. Intriguingly, both AG and UAG have been shown to promote differentiation and fusion of muscle C2C12 cells, regulate metabolic and mitochondrial signaling pathways in myotubes, and attenuate fasting- or denervation-induced muscle atrophy. Furthermore, it has also been shown that ghrelin gene deficiency increases vulnerability to fasting-induced muscle loss in aging mice, and AG and UAG effectively protects against muscle atrophy of aging mice. Because UAG doesn't bind to GHS-R, it doesn't have the undesired side-effects of elevated GH-release and increased obesity as AG. In summary, UAG has an impressive anti-atrophic effect in muscle protecting against muscle atrophy in aging, it has potential to be a unique and superior therapeutic candidate for muscle-wasting diseases such as sarcopenia.
    Keywords:  Aging; GHS-R; Ghrelin; Growth hormone; Sarcopenia
  27. Pflugers Arch. 2021 Aug 13.
      Functional hyperemia is fundamental to provide enhanced oxygen delivery during exercise in skeletal muscle. Different mechanisms are suggested to contribute, mediators from skeletal muscle, transmitter spillover from the neuromuscular synapse as well as endothelium-related dilators. We hypothesized that redundant mechanisms that invoke adenosine, endothelial autacoids, and KATP channels mediate the dilation of intramuscular arterioles in mice. Arterioles (maximal diameter: 20-42 µm, n = 65) were studied in the cremaster by intravital microscopy during electrical stimulation of the motor nerve to induce twitch or tetanic skeletal muscle contractions (10 or 100 Hz). Stimulation for 1-60 s dilated arterioles rapidly up to 65% of dilator capacity. Blockade of nicotinergic receptors blocked muscle contraction and arteriolar dilation. Exclusive blockade of adenosine receptors (1,3-dipropyl-8-(p-sulfophenyl)xanthine) or of NO and prostaglandins (nitro-L-arginine and indomethacin, LN + Indo) exerted only a minor attenuation. Combination of these blockers, however, reduced the dilation by roughly one-third during longer stimulation periods (> 1 s at 100 Hz). Blockade of KATP channels (glibenclamide) which strongly reduced adenosine-induced dilation reduced responses upon electrical stimulation only moderately. The attenuation was strongly enhanced if glibenclamide was combined with LN + Indo and even observed during brief stimulation. LN was more efficient than indomethacin to abrogate dilations if combined with glibenclamide. Arteriolar dilations induced by electrical stimulation of motor nerves require muscular contractions and are not elicited by acetylcholine spillover from neuromuscular synapses. The dilations are mediated by redundant mechanisms, mainly activation of KATP channels and release of NO. The contribution of K+ channels and hyperpolarization sets the stage for ascending dilations that are crucial for a coordinated response in the network.
    Keywords:  Active hyperemia; Adenosine; Endothelial autacoids; Glibenclamide; KATP channels
  28. Biophys J. 2021 Aug 10. pii: S0006-3495(21)00609-3. [Epub ahead of print]
      Muscles sense internally generated and externally applied forces, responding to these in a coordinated hierarchical manner at different time scales. The center of the basic unit of the muscle, the sarcomeric M-band, is perfectly placed to sense the different types of load to which the muscle is subjected. In particular, the kinase domain (TK) of titin located at the M-band is a known candidate for mechanical signaling. Here, we develop the quantitative mathematical model that describes the kinetics of TK-based mechanosensitive signaling, and predicts trophic changes in response to exercise and rehabilitation regimes. First, we build the kinetic model for TK conformational changes under force: opening, phosphorylation, signaling and autoinhibition. We find that TK opens as a metastable mechanosensitive switch, which naturally produces a much greater signal after high-load resistance exercise than an equally energetically costly endurance effort. Next, in order for the model to be stable, give coherent predictions, in particular the lag following the onset of an exercise regime, we have to account for the associated kinetics of phosphate (carried by ATP), and for the non-linear dependence of protein synthesis rates on muscle fibre size. We suggest that the latter effect may occur via the steric inhibition of ribosome diffusion through the sieve-like myofilament lattice. The full model yields a steady-state solution (homeostasis) for muscle cross-sectional area and tension, and a quantitatively plausible hypertrophic response to training as well as atrophy following an extended reduction in tension.
  29. Skelet Muscle. 2021 Aug 13. 11(1): 20
      BACKGROUND: Caenorhabditis elegans has been widely used as a model to study muscle structure and function. Its body wall muscle is functionally and structurally similar to vertebrate skeletal muscle with conserved molecular pathways contributing to sarcomere structure, and muscle function. However, a systematic investigation of the relationship between muscle force and sarcomere organization is lacking. Here, we investigate the contribution of various sarcomere proteins and membrane attachment components to muscle structure and function to introduce C. elegans as a model organism to study the genetic basis of muscle strength.METHODS: We employ two recently developed assays that involve exertion of muscle forces to investigate the correlation of muscle function to sarcomere organization. We utilized a microfluidic pillar-based platform called NemaFlex that quantifies the maximum exertable force and a burrowing assay that challenges the animals to move in three dimensions under a chemical stimulus. We selected 20 mutants with known defects in various substructures of sarcomeres and compared the physiological function of muscle proteins required for force generation and transmission. We also characterized the degree of sarcomere disorganization using immunostaining approaches.
    RESULTS: We find that mutants with genetic defects in thin filaments, thick filaments, and M-lines are generally weaker, and our assays are successful in detecting the functional changes in response to each sarcomere location tested. We find that the NemaFlex and burrowing assays are functionally distinct informing on different aspects of muscle physiology. Specifically, the burrowing assay has a larger bandwidth in phenotyping muscle mutants, because it could pick ten additional mutants impaired while exerting normal muscle force in NemaFlex. This enabled us to combine their readouts to develop an integrated muscle function score that was found to correlate with the score for muscle structure disorganization.
    CONCLUSIONS: Our results highlight the suitability of NemaFlex and burrowing assays for evaluating muscle physiology of C. elegans. Using these approaches, we discuss the importance of the studied sarcomere proteins for muscle function and structure. The scoring methodology we have developed enhances the utility of  C. elegans as a genetic model to study muscle function.
    Keywords:  Burrowing assay; Microfluidics; Muscle genetics; Muscle physiology; Sarcomere structure
  30. Hum Mol Genet. 2021 Aug 09. pii: ddab220. [Epub ahead of print]
      Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~ 17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with a SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~ 20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.
  31. J Exp Biol. 2021 Aug 01. pii: jeb242968. [Epub ahead of print]224(15):
      Naked mole-rats reduce their metabolic requirements to tolerate severe hypoxia. However, the regulatory mechanisms that underpin this metabolic suppression have yet to be elucidated. 5'-AMP-activated protein kinase (AMPK) is the cellular 'master' energy effector and we hypothesized that alterations in the AMPK pathway contribute to metabolic reorganization in hypoxic naked mole-rat skeletal muscle. To test this hypothesis, we exposed naked mole-rats to 4 h of normoxia (21% O2) or severe hypoxia (3% O2), while indirectly measuring whole-animal metabolic rate and fuel preference. We then isolated skeletal muscle and assessed protein expression and post-translational modification of AMPK, and downstream changes in key glucose and fatty acid metabolic proteins mediated by AMPK, including acetyl-CoA carboxylase (ACC1), glycogen synthase (GS) and glucose transporters (GLUTs) 1 and 4. We found that in hypoxic naked mole-rats (1) metabolic rate decreased ∼80% and fuel use switched to carbohydrates, and that (2) levels of activated phosphorylated AMPK and GS, and GLUT4 expression were downregulated in skeletal muscle, while ACC1 was unchanged. To explore the regulatory mechanism underlying this hypometabolic state, we used RT-qPCR to examine 55 AMPK-associated microRNAs (miRNAs), which are short non-coding RNA post-transcriptional silencers. We identified changes in 10 miRNAs (three upregulated and seven downregulated) implicated in AMPK downregulation. Our results suggest that miRNAs and post-translational mechanisms coordinately reduce AMPK activity and downregulate metabolism in naked mole-rat skeletal muscle during severe hypoxia. This novel mechanism may support tissue-specific prioritization of energy for more essential organs in hypoxia.
    Keywords:   Heterocephalus glaber ; GLUT4; Glycogen synthase; Glycolysis; Hypometabolism; Hypoxic metabolic response; miRNA
  32. Anal Chem. 2021 Aug 12.
      Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality.1 Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles. Oxidative peak currents (Ip) from DNA immobilized layer by layer (LbL) were monitored using square-wave voltammetry (SWV) via Ru(bpy)32+ electrocatalysis. Ip significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in the guanine content. nDNA Ip significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical-level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.
  33. FASEB Bioadv. 2021 Aug;3(8): 639-651
      Duchenne muscular dystrophy (DMD) is a genetic disorder that results in the absence of dystrophin, a cytoskeletal protein. Individuals with this disease experience progressive muscle destruction, which leads to muscle weakness. Studies have been conducted to find solutions for the relief of individuals with this disease, several of which have shown that utrophin, a protein closely related to dystrophin, when overexpressed in mdx neonatal mice (the murine model of DMD), is able to prevent the progressive muscle destruction observed in the absence of dystrophin. Furthermore, recent studies have shown that L-arginine induces utrophin upregulation in adult mdx mice. We hypothesized that L-arginine treatment also induces utrophin upregulation to prevent the development of muscle weakness in neonatal mdx mice. Hence, L-arginine should also prevent progressive muscle destruction via utrophin upregulation in mdx neonatal mice. Mdx neonatal mice were injected intraperitoneally daily with 800 mg/kg of L-arginine for 6 weeks, whereas control mice were injected with a physiological saline. The following experiments were performed on the tibialis anterior (TA) muscle: muscle contractility and resistance to mechanical stress; central nucleation and peripheral nucleation, utrophin, and creatine kinase quantification as well as a nitric oxide (NO) assay. Our findings show that early administration of L-arginine in mdx neonatal mice prevents the destruction of the tibialis anterior (TA) muscle. However, this improvement was related to nitric oxide (NO) production rather than the expected utrophin upregulation.
    Keywords:  L‐arginine; mdx; neonates; nitric oxide; utrophin
  34. Eur J Clin Invest. 2021 Sep;51(9): e13574
      BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies.PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity.
    RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes.
    CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.
    Keywords:  acetyl-CoA-driven respiration; electron transport chain; frozen skeletal muscle biopsy; high-resolution respirometry; mitochondrial diseases; oxygen consumption rate
  35. Aging Cell. 2021 Aug 08. e13448
      Aged skeletal muscle is characterized by poor muscle recovery following disuse coinciding with an impaired muscle pro-inflammatory macrophage response. Macrophage inflammatory status is regulated by its metabolic state, but little is understood of macrophage metabolism and its relation to macrophage inflammation in the context of muscle recovery and aging. Therefore, the purpose of this study was to thoroughly characterize macrophage metabolism and inflammation in aged muscle during early recovery following disuse atrophy using single cell transcriptomics and functional assays. Young (4-5 months) and old (20-22 months) male C57BL/6 mice underwent 14 days of hindlimb unloading followed by 4 days of ambulatory recovery. CD45+ cells were isolated from solei muscles and analyzed using 10x Genomics single cell RNA sequencing. We found that aged pro-inflammatory macrophage clusters were characterized with an impaired inflammatory and glycolytic transcriptome, and this dysregulation was accompanied by a suppression of HIF-1α and its immediate downstream target, Glut1. As a follow-up, bone marrow-derived macrophages were isolated from a separate cohort of young and old mice at 4-d recovery and were polarized to a pro-inflammatory phenotype and used for glycolysis stress test, phagocytosis activity assay, and targeted GC-MS metabolomics. Aged bone marrow-derived pro-inflammatory macrophages were characterized with impaired glycolysis and phagocytosis function, decreased succinate and an accumulation of glycolytic metabolic intermediates overall supporting reduced glycolytic flux and macrophage function. Our results indicate that the metabolic reprograming and function of aged skeletal muscle pro-inflammatory macrophages are dysfunctional during early recovery from disuse atrophy possibly attributing to attenuated regrowth.
    Keywords:  glycolysis; inflammation; metabolomics; scRNASeq; single cell transcriptomics
  36. Sci Rep. 2021 Aug 12. 11(1): 16423
      This study explores if unhealthy lipoprotein distribution (LPD) impairs the anabolic and amino acid sensing responses to whey-protein feeding. Thus, if impairment of such anabolic response to protein consumption is seen by the LPD this may negatively affect the skeletal muscle mass. Muscle protein synthesis (MPS) was measured by puromycin labeling in Apolipoprotein E knockout (Apoe KO), characterized by an unhealthy LPD, and wild type mice post-absorptive at 10 and 20 weeks, and post-prandial after whey-protein feeding at 20 weeks. Hypertrophy signaling and amino acid sensing mechanisms were studied and gut microbiome diversity explored. Surprisingly, whey-protein feeding did not affect MPS. p-mTOR and p-4E-BP1 was increased 2 h after whey-protein feeding in both genotypes, but with general lower levels in Apoe KO compared to wild type. At 20 weeks of age, Apoe KO had a greater mRNA-expression for SNAT2, CD98, ATF4 and GCN2 compared to wild type. These responses were not associated with gut microbiota compositional differences. Regardless of LPD status, MPS was similar in Apoe KO and wild type. Surprisingly, whey-protein did not stimulate MPS. However, Apoe KO had lower levels of hypertrophy signaling, was amino acid deprived, and had impaired amino acid sensing mechanisms.
  37. Front Immunol. 2021 ;12 628822
      Background: Skeletal muscle ischemia/reperfusion (I/R) injury is an important clinical issue that can cause remote organ injury. Although its pathogenesis has not been fully elucidated, recent studies have suggested that damage-associated molecular patterns (DAMPs) are mediators of remote organ injury in sterile inflammation. The purpose of this study was to investigate the possible involvement of DAMPs, including the nuclear proteins high-mobility group box 1 (HMGB1) and histone H3, in the pathogenesis of skeletal muscle I/R injury in mice.Methods: Hindlimb ischemia was induced in mice through bilateral ligation of inguinal regions using rubber grommets. Reperfusion was induced by cutting the rubber grommets after 2-12 h of ischemic period. Survival rates, localization of HMGB1 and histone H3 in the gastrocnemius muscle, and circulating HMGB1 and histone H3 levels were analyzed. The effect of anti-HMGB1 and anti-histone H3 antibodies on survival was analyzed in mice with I/R injury.
    Results: All mice with hindlimb ischemia survived for at least 36 h, while all mice died within 24 h if the hindlimbs were reperfused after ischemia for 4-12 h. Immunohistochemical analysis revealed that HMGB1 translocated from the nucleus to the cytoplasm in the ischemic gastrocnemius muscle, while histone H3 was confined to the nucleus. Accordingly, serum HMGB1 levels were significantly elevated in mice with hindlimb I/R compared with normal mice or mice with hindlimb ischemia (P < 0.05). Serum histone H3 levels were not elevated after I/R. Treatment with anti-HMGB1 antibodies significantly improved survival of mice with hindlimb I/R injury compared with control antibodies (P < 0.05).
    Conclusions: HMGB1, but not histone H3, translocated to the cytoplasm during skeletal muscle ischemia, and was released into the systemic circulation after reperfusion in mice with I/R injury. Treatment with anti-HMGB1 antibodies partially improved survival.
    Keywords:  damage-associated molecular patterns; extracellular histones; high mobility group box 1; ischemia reperfusion injury; skeletal muscle
  38. Circ Res. 2021 Aug 10.
      Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of DMD patients, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional and transcriptional abnormalities in DMD-associated cardiac disease.Methods and Results: We generated induced pluripotent stem cells (iPSCs) from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD (cDMD) iPSC lines, wherein the DMD open reading frame was restored via reframing (RF) or exon skipping (ES). While DMD cardiomyocytes (CMs) demonstrated morphologic, structural and functional deficits compared to control CMs, CMs from both cDMD lines were similar to control CMs. Bulk RNA-sequencing of DMD CMs showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in cDMD CMs. We then corrected dysfunctional DMD CMs by adenoviral delivery of Cas9/gRNA and showed that correction of DMD CMs post-differentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in CMs and fibroblasts, which in corrected mice was reduced to similar levels as wildtype mice.Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in iPSC-CMs and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.
  39. J Orthop Res. 2021 Aug 11.
      Prolonged mechanical ventilation for critically-ill patients with respiratory distress can result in severe muscle wasting with preferential loss of myosin. Systemic inflammation triggered by lung mechanical injury likely contributes to this myopathy, although the exact mechanisms are unknown. In this study, we hypothesized that muscle wasting following mechanical ventilation is accompanied by bone loss. The objective was to determine the rate, nature, and extent of bone loss in the femora of rats ventilated up to 10 days and to relate the bone changes to muscle deterioration. We have developed a rat model of ventilator-induced muscle wasting and established its feasibility and clinical validity. This model involves pharmacologic paralysis, parenteral nutrition, and continuous mechanical ventilation. We assessed the hindlimb muscle and bone of rats ventilated for 0, 2, 5, 8, and 10 days. Routine histology, microCT, and biomechanical evaluations were performed. Hindlimb muscles developed changes consistent with myopathy, whereas the femurs demonstrated progressive decline in trabecular bone volume, mineral density, and microarchitecture beginning day 8 of mechanical ventilation. Biomechanical testing showed reduction in flexural strength and stiffness on day 10. The bone changes correlated with the loss of muscle mass and myosin. These results demonstrate that mechanical ventilation leads to progressive trabecular bone loss parallel to muscle deterioration. Statement of Clinical Significance: The results of our study suggest that mechanically ventilated patients may be at risk of compromised bone integrity and muscle weakness, predisposing to post-ventilator falls and fractures, thereby warranting interventions to prevent progressive bone and muscle decline. This article is protected by copyright. All rights reserved.
    Keywords:  bone fragility.; critical illness myopathy; mechanical ventilation; trabecular bone loss
  40. Nutrients. 2021 Jul 20. pii: 2473. [Epub ahead of print]13(7):
      With energy intake restriction and exercise remaining the key diet and lifestyle approaches to weight loss, this is not without potential negative implications for body composition, metabolic health, and quality and quantity of life. Ideally, weight loss should be derived almost exclusively from the fat mass compartment as this is the main driver of metabolic disease, however, several studies have shown that there is an accompanying loss of tissue from the fat-free compartment, especially skeletal muscle. Population groups including post-menopausal women, the elderly, those with metabolic disease and athletes may be particularly at risk of skeletal muscle loss when following a weight management programme. Research studies that have addressed this issue across a range of population groups are reviewed with a focus upon the contribution of resistance and endurance forms of exercise and a higher intake dietary protein above the current guideline of 0.8 g/kg body weight/day. While findings can be contradictory, overall, the consensus appears that fat-free and skeletal muscle masses can be preserved, albeit to varying degrees by including both forms of exercise (but especially resistance forms) in the weight management intervention. Equally, higher intakes of protein can protect loss of these body compartments, acting either separately or synergistically with exercise. Elderly individuals in particular may benefit most from this approach. Thus, the evidence supports the recommendations for intakes of protein above the current guidelines of 0.8 g/kg body weight/d for the healthy elderly population to also be incorporated into the dietary prescription for weight management in this age group.
    Keywords:  body composition; exercise; fat-free mass; interventions; obesity; protein intake; sarcopenia; skeletal muscle mass; weight loss
  41. STAR Protoc. 2021 Sep 17. 2(3): 100694
      Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021).
    Keywords:  Cell Differentiation; Cell isolation; Flow Cytometry/Mass Cytometry; RNAseq; Single Cell; Stem Cells
  42. Life Sci Alliance. 2021 Oct;pii: e202101014. [Epub ahead of print]4(10):
      Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.