bims-moremu 2022-10-16 papers

bims-moremu

Biomed News

on Molecular regulators of muscle mass

Issue of 2022–10–16
thirty-six papers selected by
Anna Vainshtein, Craft Science Inc.

Implications of notch signaling in duchenne muscular dystrophy.
Aerobic exercise ameliorates insulin resistance in C57BL/6 J mice via activating Sestrin3.
Inhibition of High-Temperature Requirement Protein A2 Protease Activity Represses Myogenic Differentiation via UPRmt.
Inhibiting 5-lipoxygenase prevents skeletal muscle atrophy by targeting organogenesis signalling and insulin-like growth factor-1.
Atrophic skeletal muscle fibre-derived small extracellular vesicle miR-690 inhibits satellite cell differentiation during ageing.
Hypoxia-induced reprogrammed myoblasts enhance the formation of neuromuscular junctions: A pioneer study.
Generation of iMyoblasts from Human Induced Pluripotent Stem Cells.
The chemokine receptor CXCR4 regulates satellite cell activation, early expansion, and self-renewal, in response to skeletal muscle injury.
Sox6 Differentially Regulates Inherited Myogenic Abilities and Muscle Fiber Types of Satellite Cells Derived from Fast- and Slow-Type Muscles.
Aging is associated with an altered macrophage response during human skeletal muscle regeneration.
Nobiletin Prevents D-Galactose-Induced C2C12 Cell Aging by Improving Mitochondrial Function.
Glucose 6-P Dehydrogenase-An Antioxidant Enzyme with Regulatory Functions in Skeletal Muscle during Exercise.
Nanomedicine for Treating Muscle Dystrophies: Opportunities, Challenges, and Future Perspectives.
Pyroptosis and Insulin Resistance in Metabolic Organs.
Neuronal Agrin Promotes Proliferation of Primary Human Myoblasts in an Age-Dependent Manner.
Metformin attenuates an increase of calcium-dependent and ubiquitin-proteasome markers in unloaded muscle.
Reduction of senescent fibro-adipogenic progenitors in progeria-aged muscle by senolytics rescues the function of muscle stem cells.
An alternative mechanism for skeletal muscle dysfunction in long-term post-viral lung disease.
Constitutive assembly of Ca2+ entry units in soleus muscle from calsequestrin knockout mice.
Influence of 4 weeks of downhill running on calcium sensitivity of rat single muscle fibers.
The role of Sirt3 in the changes of skeletal muscle mitophagy induced by hypoxic training.
Exosomal Plasminogen Activator Inhibitor-1 Induces Ionizing Radiation-Adaptive Glioblastoma Cachexia.
Efficient co-isolation of microvascular endothelial cells and satellite cell-derived myoblasts from human skeletal muscle.
A prospective clinical study on the mechanisms underlying critical illness myopathy-A time-course approach.
Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila.
In vitro chemotherapy-associated muscle toxicity is attenuated with nutritional support, while treatment efficacy is retained.
Plants powering muscle: The effects of phytochemical PB125 on mitochondrial function and skeletal muscle health.
Low dose lithium supplementation promotes adipose tissue browning and sarco(endo)plasmic reticulum Ca2+ ATPase uncoupling in muscle.
CircCSDE1 Regulates Proliferation and Differentiation of C2C12 Myoblasts by Sponging miR-21-3p.
Mitochondrial network configuration influences sarcomere and myosin filament structure in striated muscles.
CRISPR-Based Therapeutic Gene Editing for Duchenne Muscular Dystrophy: Advances, Challenges and Perspectives.
The Impact of Spermidine on C2C12 Myoblasts Proliferation, Redox Status and Polyamines Metabolism under H2O2 Exposure.
The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts.
Measuring muscle protein synthesis in humans and the influence of nutritional state.
Profiling epigenetic age in single cells.
Evaluating Genetic Modifiers of Duchenne Muscular Dystrophy Disease Progression Using Modeling and MRI.

Front Physiol. 2022 ;13 984373

Implications of notch signaling in duchenne muscular dystrophy.

Lily Den Hartog, Atsushi Asakura.

  This review focuses upon the implications of the Notch signaling pathway in muscular dystrophies, particularly Duchenne muscular dystrophy (DMD): a pervasive and catastrophic condition concerned with skeletal muscle degeneration. Prior work has defined the pathogenesis of DMD, and several therapeutic approaches have been undertaken in order to regenerate skeletal muscle tissue and ameliorate the phenotype. There is presently no cure for DMD, but a promising avenue for novel therapies is inducing muscle regeneration via satellite cells (muscle stem cells). One specific target using this approach is the Notch signaling pathway. The canonical Notch signaling pathway has been well-characterized and it ultimately governs cell fate decision, cell proliferation, and induction of differentiation. Additionally, inhibition of the Notch signaling pathway has been directly implicated in the deficits seen with muscular dystrophies. Here, we explore the connection between the Notch signaling pathway and DMD, as well as how Notch signaling may be targeted to improve the muscle degeneration seen in muscular dystrophies.

Keywords:  muscle regeneration; muscle stem cell; muscular dystrophy; notch; satellite cell

DOI:  https://doi.org/10.3389/fphys.2022.984373
Biochim Biophys Acta Mol Basis Dis. 2022 Oct 08. pii: S0925-4439(22)00239-3. [Epub ahead of print] 166568

Aerobic exercise ameliorates insulin resistance in C57BL/6 J mice via activating Sestrin3.

Xiao Han, Yang Yang, Sujuan Liu, Yanmei Niu, Heng Shao, Li Fu.

  Skeletal muscle insulin resistance (IR) is closely linked to hyperglycemia and metabolic disorders. Regular exercise enhances insulin sensitivity in skeletal muscle, but its underlying mechanisms remain unknown. Sestrin3 (SESN3) is a stress-inducible protein that protects against obesity-induced hepatic steatosis and insulin resistance. Regular exercise training is known to increase SESN3 expression in skeletal muscle. The purpose of this study was to explore whether SESN3 mediates the metabolic effects of exercise in the mouse model of high-fat diet (HFD)-induced IR. SESN3-/- mice exhibited severer body weight gain, ectopic lipid accumulation, and dysregulation of glucose metabolism after long-term HFD feeding compared with the wild-type (WT) mice. Moreover, we found that SESN3 deficiency weakened the effects of exercise on reducing serum insulin levels and improving glucose tolerance in mice. Exercise training increased pAKT-S473 and GLUT4 expression, accompanied by enhanced pmTOR-S2481 (an indicator of mTORC2 activity) in WT quadriceps that were less pronounced in SESN3-/- mice. SESN3 overexpression in C2C12 myotubes further confirmed that SESN3 played an important role in skeletal muscle glucose metabolism. SESN3 overexpression increased the binding of Rictor to mTOR and pmTOR-S2481 in C2C12 myotubes. Moreover, SESN3 overexpression resulted in an elevation of glucose uptake and a concomitant increase of pAKT-S473 in C2C12 myotubes, whereas these effects were diminished by downregulation of mTORC2 activity. Taken together, SESN3 is a crucial protein in amplifying the beneficial effects of exercise on insulin sensitivity in skeletal muscle and systemic glucose levels. SESN3/mTORC2/AKT pathway mediated the effects of exercise on skeletal muscle insulin sensitivity.

Keywords:  Aerobic exercise; Glucose homeostasis; Insulin resistance; Metabolism; Sestrin3

DOI:  https://doi.org/10.1016/j.bbadis.2022.166568
Int J Mol Sci. 2022 Oct 04. pii: 11761. [Epub ahead of print]23(19):

Inhibition of High-Temperature Requirement Protein A2 Protease Activity Represses Myogenic Differentiation via UPRmt.

Hongyu Sun, Luyan Shen, Ping Zhang, Fu Lin, Jiaoyan Ma, Ying Wu, Huimei Yu, Liankun Sun.

  Skeletal muscles require muscle satellite cell (MuSC) differentiation to facilitate the replenishment and repair of muscle fibers. A key step in this process is called myogenic differentiation. The differentiation ability of MuSCs decreases with age and can result in sarcopenia. Although mitochondria have been reported to be involved in myogenic differentiation by promoting a bioenergetic remodeling, little is known about the interplay of mitochondrial proteostasis and myogenic differentiation. High-temperature-requirement protein A2 (HtrA2/Omi) is a protease that regulates proteostasis in the mitochondrial intermembrane space (IMS). Mice deficient in HtrA2 protease activity show a distinct phenotype of sarcopenia. To investigate the role of IMS proteostasis during myogenic differentiation, we treated C2C12 myoblasts with UCF101, a specific inhibitor of HtrA2 during differentiation process. A key step in this process is called myogenic differentiation. The differentiation ability of MuSCs decreases with age and can result in sarcopenia. Further, CHOP, p-eIF2α, and other mitochondrial unfolded protein response (UPRmt)-related proteins are upregulated. Therefore, we suggest that imbalance of mitochondrial IMS proteostasis acts via a retrograde signaling pathway to inhibit myogenic differentiation via the UPRmt pathway. These novel mechanistic insights may have implications for the development of new strategies for the treatment of sarcopenia.

Keywords:  HtrA2/Omi; UPRmt; mitonuclear imbalance; sarcopenia; skeletal muscle

DOI:  https://doi.org/10.3390/ijms231911761
J Cachexia Sarcopenia Muscle. 2022 Oct 11.

Inhibiting 5-lipoxygenase prevents skeletal muscle atrophy by targeting organogenesis signalling and insulin-like growth factor-1.

Hyun-Jun Kim, Seon-Wook Kim, Sang-Hoon Lee, Da-Woon Jung, Darren R Williams.

   BACKGROUND: Skeletal muscle atrophy can occur in response to numerous factors, such as ageing and certain medications, and produces a major socio-economic burden. At present, there are no approved drugs for treating skeletal muscle atrophy. Arachidonate 5-lipoxygenase (Alox5) is a drug target for a number of diseases. However, pharmacological targeting of Alox5, and its role in skeletal muscle atrophy, is unclear.
METHODS: The potential effects of gene knockdown and pharmacological targeting of Alox5 on skeletal muscle atrophy were investigated using cell-based models, animal models and human skeletal muscle primary cells. Malotilate, a clinically safe drug developed for enhancing liver regeneration and Alox5 inhibitor, was investigated as a repurposing candidate. Mechanism(s) of action in skeletal muscle atrophy was assessed by measuring the expression level or activation status of key regulatory pathways and validated using gene knockdown and RNA sequencing.
RESULTS: Myotubes treated with the atrophy-inducing glucocorticoid, dexamethasone, were protected from catabolic responses by treatment with malotilate (+41.29%, P < 0.01). Similar anti-atrophy effects were achieved by gene knockdown of Alox5 (+30.4%, P < 0.05). Malotilate produced anti-atrophy effects without affecting the myogenic differentiation programme. In an in vivo model of skeletal muscle atrophy, malotilate treatment preserved muscle force/strength (grip strength: +35.72%, latency to fall: +553.1%, P < 0.05), increased mass and fibre cross-sectional area (quadriceps: +23.72%, soleus: +33.3%, P < 0.01) and down-regulated atrogene expression (Atrogin-1: -61.58%, Murf-1: -66.06%, P < 0.01). Similar, beneficial effects of malotilate treatment were observed in an ageing muscle model, which also showed the preservation of fast-twitch fibres (Type 2a: +56.48%, Type 2b: +37.32%, P < 0.01). Leukotriene B4, a product of Alox5 activity with inflammatory and catabolic functions, was found to be elevated in skeletal muscle undergoing atrophy (quadriceps: +224.4%, P < 0.001). Cellular transcriptome analysis showed that targeting Alox5 up-regulated biological processes regulating organogenesis and increased the expression of insulin-like growth factor-1, a key anti-atrophy hormone (+226.5%, P < 0.05). Interestingly, these effects were restricted to the atrophy condition and not observed in normal skeletal muscle cultures with Alox5 inhibition. Human myotubes were also protected from atrophy by pharmacological targeting of Alox5 (+23.68%, P < 0.05).
CONCLUSIONS: These results shed new light on novel drug targets and mechanisms underpinning skeletal muscle atrophy. Alox5 is a regulator and drug target for muscle atrophy, and malotilate is an attractive compound for repurposing studies to treat this disease.

Keywords:  Alox5; Drug repurposing; Glucocorticoids; Malotilate; Sarcopenia; Skeletal muscle atrophy

DOI:  https://doi.org/10.1002/jcsm.13092
J Cachexia Sarcopenia Muscle. 2022 Oct 13.

Atrophic skeletal muscle fibre-derived small extracellular vesicle miR-690 inhibits satellite cell differentiation during ageing.

Xiaoyan Shao, Wang Gong, Qianjin Wang, Pu Wang, Tianshu Shi, Abdurahman Mahmut, Jianghui Qin, Yao Yao, Wenjin Yan, Dongyang Chen, Xiang Chen, Qing Jiang, Baosheng Guo.

   BACKGROUND: Sarcopenia is a common and progressive skeletal muscle disorder characterized by atrophic muscle fibres and contractile dysfunction. Accumulating evidence shows that the number and function of satellite cells (SCs) decline and become impaired during ageing, which may contribute to impaired regenerative capacity. A series of myokines/small extracellular vesicles (sEVs) released from muscle fibres regulate metabolism in muscle and extramuscular tissues in an autocrine/paracrine/endocrine manner during muscle atrophy. It is still unclear whether myokines/sEVs derived from muscle fibres can affect satellite cell function during ageing.
METHODS: Aged mice were used to investigate changes in the myogenic capacity of SCs during ageing-induced muscle atrophy. The effects of atrophic myotube-derived sEVs on satellite cell differentiation were investigated by biochemical methods and immunofluorescence staining. Small RNA sequencing was performed to identify differentially expressed sEV microRNAs (miRNAs) between the control myotubes and atrophic myotubes. The target genes of the miRNA were predicted by bioinformatics analysis and verified by luciferase activity assays. The effects of identified miRNA on the myogenic capacity of SCs in vivo were investigated by intramuscular injection of adeno-associated virus (AAV) to overexpress or silence miRNA in skeletal muscle.
RESULTS: Our study showed that the myogenic capacity of SCs was significantly decreased (50%, n = 6, P < 0.001) in the tibialis anterior muscle of aged mice. We showed that atrophic myotube-derived sEVs inhibited satellite cell differentiation in vitro (n = 3, P < 0.001) and in vivo (35%, n = 6, P < 0.05). We also found that miR-690 was the most highly enriched miRNA among all the screened sEV miRNAs in atrophic myotubes [Log2 (Fold Change) = 7, P < 0.001], which was verified in the atrophic muscle of aged mice (threefold, n = 6, P < 0.001) and aged men with mean age of 71 ± 5.27 years (2.8-fold, n = 10, P < 0.001). MiR-690 can inhibit myogenic capacity of SCs by targeting myocyte enhancer factor 2, including Mef2a, Mef2c and Mef2d, in vitro (n = 3, P < 0.05) and in vivo (n = 6, P < 0.05). Specific silencing of miR-690 in the muscle can promote satellite cell differentiation (n = 6, P < 0.001) and alleviate muscle atrophy in aged mice (n = 6, P < 0.001).
CONCLUSIONS: Our study demonstrated that atrophic muscle fibre-derived sEV miR-690 may inhibit satellite cell differentiation by targeting myocyte enhancer factor 2 during ageing.

Keywords:  Muscle fibre; Myogenic capacity; Sarcopenia; Satellite cells; Small extracellular vesicle; miR-690

DOI:  https://doi.org/10.1002/jcsm.13106
J Cell Biochem. 2022 Oct 08.

Hypoxia-induced reprogrammed myoblasts enhance the formation of neuromuscular junctions: A pioneer study.

Rachael Tolsma, Haiying Pan, Loyall Harris, John M Spitsbergen, Yong Li.

  We previously reported that muscle cells could reprogram into progenitors after traumatic injuries. These injury-induced muscle stem cells (iMuSCs) have increased migration and differentiation capacities, including neuronal differentiation. Recent studies in our laboratory suggest that the hypoxia-induced by tissue injury plays an essential role in the reprogramming process of muscle cells. We hypothesize that muscle cells reprogrammed with hypoxia have increased neuronal differentiation potentials and the neuronal differentiation extends into the formation of neuromuscular junction (NMJ)-like structures. In this study, C2C12 myoblasts were cultured under hypoxic conditions and subsequently in neural differentiation media to generate neurospheres, and then with muscle differentiation media to induce NMJ-like structure formation. Hypoxia-induced muscle cells also produced more robust NMJs compared to controls after intramuscular cell transplantation. Our results suggest hypoxia plays a role in the reprogramming of muscle stem cells, which may have the potential to form neuromuscular junctions and ultimately contribute to functional muscle healing.

Keywords:  cellular reprogramming; differentiation; hypoxia; muscle healing; muscle stem cells; neuromuscular junction

DOI:  https://doi.org/10.1002/jcb.30334
Bio Protoc. 2022 Sep 05. pii: e4500. [Epub ahead of print]12(17):

Generation of iMyoblasts from Human Induced Pluripotent Stem Cells.

Dongsheng Guo, Katelyn Daman, Danielle Fernandes Durso, Jing Yan, Charles P Emerson.

  Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment. iMyoblasts are PAX3 + /MyoD1 + myogenic stem cells with dual potential to undergo muscle differentiation and to self-renew as a regenerative cell population for muscle regeneration both ex vivo and in vivo . The simplicity and robustness of iMyoblast generation and expansion have enabled their application to model the molecular pathogenesis of Facioscapulohumeral Muscular Dystrophy and Limb-Girdle Muscular Dystrophies, to both ex vivo and in vivo muscle xenografts, and to respond efficiently to gene editing, enabling the co-development of gene correction and stem cell regenerative therapeutic technologies for the treatment of muscular dystrophies and muscle injury. Graphical abstract.

Keywords:   Differentiation ; Human induced pluripotent stem cells (hiPSCs) ; Muscular dystrophy ; Myogenesis ; Reserve cell ; iMyoblast

DOI:  https://doi.org/10.21769/BioProtoc.4500
Front Cell Dev Biol. 2022 ;10 949532

The chemokine receptor CXCR4 regulates satellite cell activation, early expansion, and self-renewal, in response to skeletal muscle injury.

Ahmed S Shams, Robert W Arpke, Micah D Gearhart, Johannes Weiblen, Ben Mai, David Oyler, Darko Bosnakovski, Omayma M Mahmoud, Gamal M Hassan, Michael Kyba.

  Acute skeletal muscle injury is followed by satellite cell activation, proliferation, and differentiation to replace damaged fibers with newly regenerated muscle fibers, processes that involve satellite cell interactions with various niche signals. Here we show that satellite cell specific deletion of the chemokine receptor CXCR4, followed by suppression of recombination escapers, leads to defects in regeneration and satellite cell pool repopulation in both the transplantation and in situ injury contexts. Mechanistically, we show that endothelial cells and FAPs express the gene for the ligand, SDF1α, and that CXCR4 is principally required for proper activation and for transit through the first cell division, and to a lesser extent the later cell divisions. In the absence of CXCR4, gene expression in quiescent satellite cells is not severely disrupted, but in activated satellite cells a subset of genes normally induced by activation fail to upregulate normally. These data demonstrate that CXCR4 signaling is essential to normal early activation, proliferation, and self-renewal of satellite cells.

Keywords:  CXCR4; activation; regeneration; satellite cells; skeletal muscle

DOI:  https://doi.org/10.3389/fcell.2022.949532
Int J Mol Sci. 2022 Sep 26. pii: 11327. [Epub ahead of print]23(19):

Sox6 Differentially Regulates Inherited Myogenic Abilities and Muscle Fiber Types of Satellite Cells Derived from Fast- and Slow-Type Muscles.

Zihao Zhang, Shudai Lin, Wen Luo, Tuanhui Ren, Xing Huang, Wangyu Li, Xiquan Zhang.

  Adult skeletal muscle is primarily divided into fast and slow-type muscles, which have distinct capacities for regeneration, metabolism and contractibility. Satellite cells plays an important role in adult skeletal muscle. However, the underlying mechanisms of satellite cell myogenesis are poorly understood. We previously found that Sox6 was highly expressed in adult fast-type muscle. Therefore, we aimed to validate the satellite cell myogenesis from different muscle fiber types and investigate the regulation of Sox6 on satellite cell myogenesis. First, we isolated satellite cells from fast- and slow-type muscles individually. We found that satellite cells derived from different muscle fiber types generated myotubes similar to their origin types. Further, we observed that cells derived from fast muscles had a higher efficiency to proliferate but lower potential to self-renew compared to the cells derived from slow muscles. Then we demonstrated that Sox6 facilitated the development of satellite cells-derived myotubes toward their inherent muscle fiber types. We revealed that higher expression of Nfix during the differentiation of fast-type muscle-derived myogenic cells inhibited the transcription of slow-type isoforms (MyH7B, Tnnc1) by binding to Sox6. On the other hand, Sox6 activated Mef2C to promote the slow fiber formation in slow-type muscle-derived myogenic cells with Nfix low expression, showing a different effect of Sox6 on the regulation of satellite cell development. Our findings demonstrated that satellite cells, the myogenic progenitor cells, tend to develop towards the fiber type similar to where they originated. The expression of Sox6 and Nfix partially explain the developmental differences of myogenic cells derived from fast- and slow-type muscles.

Keywords:  Sox6; developmental differences; muscle fiber types; muscle satellite cells

DOI:  https://doi.org/10.3390/ijms231911327
Exp Gerontol. 2022 Oct 10. pii: S0531-5565(22)00282-0. [Epub ahead of print] 111974

Aging is associated with an altered macrophage response during human skeletal muscle regeneration.

Mohadeseh Ahmadi, Anders Karlsen, Jack Mehling, Casper Soendenbroe, Abigail L Mackey, Robert D Hyldahl.

  Skeletal muscle injury in aged rodents is characterized by an asynchronous infiltration of pro- and anti-inflammatory macrophage waves, leading to improper and incomplete regeneration. It is unclear whether this aberration also occurs in aged human muscle. In this study, we quantified the macrophage responses in a human model of muscle damage and regeneration induced by electrical stimulation in 7 young and 21 older adults. At baseline, total resident macrophage (CD68+/DAPI+) content was not different between young and old subjects, but pro-inflammatory (CD206-/CD68+/DAPI+) macrophage content was lower in the old. Following damage, muscle Infiltration of CD206-/CD68+/DAPI+ macrophages was lower in old relative to young subjects. Further, only the increase in CD206-/CD68+ macrophages correlated with the change in muscle satellite cell content. Our data show that older individuals have a compromised macrophage response during muscle regeneration, pointing to an altered inflammatory response as a potential mechanism for reduced muscle regenerative efficacy in aged humans.

Keywords:  Aging; Immunosenescence; Inflammation; Muscle damage; Muscle regeneration; Satellite cell

DOI:  https://doi.org/10.1016/j.exger.2022.111974
Int J Mol Sci. 2022 Oct 08. pii: 11963. [Epub ahead of print]23(19):

Nobiletin Prevents D-Galactose-Induced C2C12 Cell Aging by Improving Mitochondrial Function.

Hui-Hui Wang, Ya-Nan Sun, Tai-Qi Qu, Xue-Qin Sang, Li-Mian Zhou, Yi-Xuan Li, Fa-Zheng Ren.

  Age-associated loss of skeletal muscle mass and function is one of the main causes of the loss of independence and physical incapacitation in the geriatric population. This study used the D-galactose-induced C2C12 myoblast aging model to explore whether nobiletin (Nob) could delay skeletal muscle aging and determine the associated mechanism. The results showed that Nob intervention improved mitochondrial function, increased ATP production, reduced reactive oxygen species (ROS) production, inhibited inflammation, and prevented apoptosis as well as aging. In addition, Nob improved autophagy function, removed misfolded proteins and damaged organelles, cleared ROS, reduced mitochondrial damage, and improved skeletal muscle atrophy. Moreover, our results illustrated that Nob can not only enhance mitochondrial function, but can also enhance autophagy function and the protein synthesis pathway to inhibit skeletal muscle atrophy. Therefore, Nob may be a potential candidate for the prevention and treatment of age-related muscle decline.

Keywords:  C2C12 myoblast; ROS; aging; mitochondrial function; nobiletin

DOI:  https://doi.org/10.3390/ijms231911963
Cells. 2022 Sep 28. pii: 3041. [Epub ahead of print]11(19):

Glucose 6-P Dehydrogenase-An Antioxidant Enzyme with Regulatory Functions in Skeletal Muscle during Exercise.

Esther García-Domínguez, Aitor Carretero, Aurora Viña-Almunia, Julio Domenech-Fernandez, Gloria Olaso-Gonzalez, Jose Viña, Mari Carmen Gomez-Cabrera.

  Hypomorphic Glucose 6-P dehydrogenase (G6PD) alleles, which cause G6PD deficiency, affect around one in twenty people worldwide. The high incidence of G6PD deficiency may reflect an evolutionary adaptation to the widespread prevalence of malaria, as G6PD-deficient red blood cells (RBCs) are hostile to the malaria parasites that infect humans. Although medical interest in this enzyme deficiency has been mainly focused on RBCs, more recent evidence suggests that there are broader implications for G6PD deficiency in health, including in skeletal muscle diseases. G6PD catalyzes the rate-limiting step in the pentose phosphate pathway (PPP), which provides the precursors of nucleotide synthesis for DNA replication as well as reduced nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is involved in the detoxification of cellular reactive oxygen species (ROS) and de novo lipid synthesis. An association between increased PPP activity and the stimulation of cell growth has been reported in different tissues including the skeletal muscle, liver, and kidney. PPP activity is increased in skeletal muscle during embryogenesis, denervation, ischemia, mechanical overload, the injection of myonecrotic agents, and physical exercise. In fact, the highest relative increase in the activity of skeletal muscle enzymes after one bout of exhaustive exercise is that of G6PD, suggesting that the activation of the PPP occurs in skeletal muscle to provide substrates for muscle repair. The age-associated loss in muscle mass and strength leads to a decrease in G6PD activity and protein content in skeletal muscle. G6PD overexpression in Drosophila Melanogaster and mice protects against metabolic stress, oxidative damage, and age-associated functional decline, and results in an extended median lifespan. This review discusses whether the well-known positive effects of exercise training in skeletal muscle are mediated through an increase in G6PD.

Keywords:  G6PD; NADPH; aging; pentose phosphate pathway; physical training; skeletal muscle

DOI:  https://doi.org/10.3390/cells11193041
Int J Mol Sci. 2022 Oct 10. pii: 12039. [Epub ahead of print]23(19):

Nanomedicine for Treating Muscle Dystrophies: Opportunities, Challenges, and Future Perspectives.

Zaheer Ahmed, Rizwan Qaisar.

  Muscular dystrophies are a group of genetic muscular diseases characterized by impaired muscle regeneration, which leads to pathological inflammation that drives muscle wasting and eventually results in weakness, functional dependency, and premature death. The most known causes of death include respiratory muscle failure due to diaphragm muscle decay. There is no definitive treatment for muscular dystrophies, and conventional therapies aim to ameliorate muscle wasting by promoting physiological muscle regeneration and growth. However, their effects on muscle function remain limited, illustrating the requirement for major advancements in novel approaches to treatments, such as nanomedicine. Nanomedicine is a rapidly evolving field that seeks to optimize drug delivery to target tissues by merging pharmaceutical and biomedical sciences. However, the therapeutic potential of nanomedicine in muscular dystrophies is poorly understood. This review highlights recent work in the application of nanomedicine in treating muscular dystrophies. First, we discuss the history and applications of nanomedicine from a broader perspective. Second, we address the use of nanoparticles for drug delivery, gene regulation, and editing to target Duchenne muscular dystrophy and myotonic dystrophy. Next, we highlight the potential hindrances and limitations of using nanomedicine in the context of cell culture and animal models. Finally, the future perspectives for using nanomedicine in clinics are summarized with relevance to muscular dystrophies.

Keywords:  dystrophies; nanoparticles; skeletal muscle; small molecules

DOI:  https://doi.org/10.3390/ijms231912039
Int J Mol Sci. 2022 Oct 01. pii: 11638. [Epub ahead of print]23(19):

Pyroptosis and Insulin Resistance in Metabolic Organs.

Huiting Wei, Di Cui.

  Skeletal muscle serves as the optimal effective organ to balance glucose homeostasis, but insulin resistance (IR) in skeletal muscle breaks this balance by impeding glucose uptake and causes metabolic disorders. IR in skeletal muscle is caused by multiple factors, and it has been reported that systemic low-grade inflammation is related to skeletal muscle IR, though its molecular mechanisms need to be ulteriorly studied. Pyroptosis is a novel inflammatory-mediated type of cell death. It has recently been reported that pyroptosis is associated with a decline in insulin sensitivity in skeletal muscle. The appropriate occurrence of pyroptosis positively eliminates pathogenic factors, whereas its excessive activation may aggravate inflammatory responses and expedite disease progression. The relationship between pyroptosis and IR in skeletal muscle and its underlined mechanism need to be further illustrated. The role of pyroptosis during the process of IR alleviation induced by non-drug interventions, such as exercise, also needs to be clarified. In this paper, we review and describe the molecular mechanisms of pyroptosis and further comb the roles of its relevant key factors in skeletal muscle IR, aiming to propose a novel theoretical basis for the relationship between pyroptosis and muscle IR and provide new research targets for the improvement of IR-related diseases.

Keywords:  GSDMs; IR; NLRP3; caspase; pyroptosis; skeletal muscle

DOI:  https://doi.org/10.3390/ijms231911638
Int J Mol Sci. 2022 Oct 04. pii: 11784. [Epub ahead of print]23(19):

Neuronal Agrin Promotes Proliferation of Primary Human Myoblasts in an Age-Dependent Manner.

Katarina Gros, Urška Matkovič, Giulia Parato, Katarina Miš, Elisa Luin, Annalisa Bernareggi, Marina Sciancalepore, Tomaž Marš, Paola Lorenzon, Sergej Pirkmajer.

  Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.

Keywords:  Lrp4; MuSK; agrin; myoblast proliferation; skeletal muscle regeneration

DOI:  https://doi.org/10.3390/ijms231911784
J Appl Physiol (1985). 2022 Oct 13.

Metformin attenuates an increase of calcium-dependent and ubiquitin-proteasome markers in unloaded muscle.

Svetlana P Belova, Ksenia Zaripova, Kristina Sharlo, Tatiana Y Kostrominova, Boris S Shenkman, Tatiana L Nemirovskaya.

  Current study tested a hypothesis that during skeletal muscle unloading, calcium-dependent signaling pathways, markers of protein synthesis and expression of E3 ubiquitin ligases can be regulated by metformin. Thirty-two male Wistar rats were randomly assigned into one of four groups: non-treated control (3C), control rats treated with metformin (3CM), 3 days of unloading/hindlimb suspension with placebo (3HS), and 3 days of unloading treated with metformin (3HSM). In soleus muscle of HS group level of phospho-AMPK (p-AMPK) was decreased by 46% while ATP content was increased by 49% when compared with the control group. There was an increase of the level of phospho-CaMK II (483%) and an up-regulation of CaN, SERCA2a, and Calpain-1 mRNA expression (87%, 41% and 62%, respectively, p<0.05) in the HS group relative to the control. HS group also had increased mRNA expression of MuRF1, MAFbx, and ubiquitin (167%, 146% and 191%, respectively, p<0.05) when compared to the control soleus muscle. Metformin treatment impeded unloading-induced changes in soleus muscle. Conclusions: metformin treatment during three days of soleus muscle unloading: 1) prevented the decrease of p-AMPK and increase of ATP content; 2) affected regulation of calcium-dependent signaling pathways via level of CaMK II phosphorylation or CaMK II, CaN, SERCA2a, and Calpain-1 mRNA expression; 3) attenuated an increase in the expression of critical markers of ubiquitin-proteasome pathways MuRF1, MAFbx, and ubiquitin while not affecting the unloading-induced increase of ULK-1 marker of autophagic/lysosomal pathway.

Keywords:  AMPK; MAFbx; MuRF1; metformin; muscle unloading

DOI:  https://doi.org/10.1152/japplphysiol.00415.2022
J Cachexia Sarcopenia Muscle. 2022 Oct 11.

Reduction of senescent fibro-adipogenic progenitors in progeria-aged muscle by senolytics rescues the function of muscle stem cells.

Lei Liu, Xianlin Yue, Zewei Sun, William S Hambright, Jianming Wei, Ying Li, Polina Matre, Yan Cui, Zhihui Wang, George Rodney, Johnny Huard, Paul D Robbins, Xiaodong Mu.

   BACKGROUND: Fibro-adipogenic progenitors (FAPs) in the muscles have been found to interact closely with muscle progenitor/stem cells (MPCs) and facilitate muscle regeneration at normal conditions. However, it is not clear how FAPs may interact with MPCs in aged muscles. Senolytics have been demonstrated to selectively eliminate senescent cells and generate therapeutic benefits on ageing and multiple age-related disease models.
METHODS: By studying the muscles and primary cells of age matched WT mice and Zmpste24-/- (Z24-/- ) mice, an accelerated ageing model for Hutchinson-Gilford progeria syndrome (HGPS), we examined the interaction between FAPs and MPCs in progeria-aged muscle, and the potential effect of senolytic drug fisetin in removing senescent FAPs and improving the function of MPCs.
RESULTS: We observed that, compared with muscles of WT mice, muscles of Z24-/- mice contained a significantly increased number of FAPs (2.4-fold; n > =6, P < 0.05) and decreased number of MPCs (2.8-fold; n > =6, P < 0.05). FAPs isolated from Z24-/- muscle contained about 44% SA-β-gal+ senescent cells, in contrast to about 3.5% senescent cells in FAPs isolated from WT muscle (n > =6, P < 0.001). The co-culture of Z24-/- FAPs with WT MPCs resulted in impaired proliferation and myogenesis potential of WT MPCs, with the number of BrdU positive proliferative cells being reduced for 3.3 times (n > =6, P < 0.001) and the number of myosin heavy chain (MHC)-positive myotubes being reduced for 4.5 times (n > =6, P < 0.001). The treatment of the in vitro co-culture system of Z24-/- FAPs and WT MPCs with the senolytic drug fisetin led to increased apoptosis of Z24-/- FAPs (14.5-fold; n > =6, P < 0.001) and rescued the impaired function of MPCs by increasing the number of MHC-positive myotubes for 3.1 times (n > =6, P < 0.001). Treatment of Z24-/- mice with fisetin in vivo was effective in reducing the number of senescent FAPs (2.2-fold, n > =6, P < 0.05) and restoring the number of muscle stem cells (2.6-fold, n > =6, P < 0.05), leading to improved muscle pathology in Z24-/- mice.
CONCLUSIONS: These results indicate that the application of senolytics in the progeria-aged muscles can be an efficient strategy to remove senescent cells, including senescent FAPs, which results in improved function of muscle progenitor/stem cells. The senescent FAPs can be a potential novel target for therapeutic treatment of progeria ageing related muscle diseases.

Keywords:  Cellular senescence; Muscle atrophy; Muscle stem cells; Progeria; Senolytics

DOI:  https://doi.org/10.1002/jcsm.13101
bioRxiv. 2022 Oct 08. pii: 2022.10.07.511313. [Epub ahead of print]

An alternative mechanism for skeletal muscle dysfunction in long-term post-viral lung disease.

Ryan A Martin, Shamus P Keeler, Kangyun Wu, William J Shearon, Devin Patel, My Hoang, Christy M Hoffmann, Michael E Hughes, Michael J Holtzman.

Chronic lung disease is often accompanied by disabling extrapulmonary symptoms, notably skeletal muscle dysfunction and atrophy. Moreover, the severity of respiratory symptoms correlates with decreased muscle mass and in turn lowered physical activity and survival rates. Previous models of muscle atrophy in chronic lung disease often modeled COPD and relied on cigarette smoke exposure and LPS-stimulation, but these conditions independently affect skeletal muscle even without accompanying lung disease. Moreover, there is an emerging and pressing need to understand the extrapulmonary manifestations of long-term post-viral lung disease (PVLD) as found in Covid-19. Here, we examine the development of skeletal muscle dysfunction in the setting of chronic pulmonary disease using a mouse model of PVLD caused by infection due to the natural pathogen Sendai virus. We identify a significant decrease in myofiber size when PVLD is maximal at 49 d after infection. We find no change in the relative types of myofibers, but the greatest decrease in fiber size is localized to fast-twitch type IIB myofibers based on myosin heavy chain immunostaining. Remarkably, all biomarkers of myocyte protein synthesis and degradation (total RNA, ribosomal abundance, and ubiquitin-proteasome expression) were stable throughout the acute infectious illness and chronic post-viral disease process. Together, the results demonstrate a distinct pattern of skeletal muscle dysfunction in a mouse model of long-term PVLD. The findings thereby provide new insight into prolonged limitations in exercise capacity in patients with chronic lung disease after viral infections and perhaps other types of lung injury.

DOI: https://doi.org/10.1101/2022.10.07.511313
J Gen Physiol. 2022 Dec 05. pii: e202213114. [Epub ahead of print]154(12):

Constitutive assembly of Ca2+ entry units in soleus muscle from calsequestrin knockout mice.

Antonio Michelucci, Laura Pietrangelo, Giorgia Rastelli, Feliciano Protasi, Robert T Dirksen, Simona Boncompagni.

Calcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter. By contrast, calsequestrin-1 (CASQ1) ablation induces a compensatory constitutive assembly of CEUs in EDL fibers, resulting in enhanced constitutive and maximum SOCE that counteracts SR Ca2+ depletion during repetitive activity. However, whether CEUs form in slow-twitch fibers, which express both the skeletal CASQ1 and the cardiac CASQ2 isoforms, is unknown. Herein, we compared the structure and function of soleus muscles from WT and knockout mice that lack either CASQ1 (CASQ1-null) or both CASQs (dCASQ-null). Ultrastructural analyses showed that SR/T-tubule junctions at the I band, virtually identical to CEUs in EDL muscle, were present and more frequent in CASQ1-null than WT mice, with dCASQ-null exhibiting the highest incidence. The greater incidence of CEUs in soleus from dCASQ-null mice correlated with increased specific force production during repetitive, high-frequency stimulation, which depended on Ca2+ entry. Consistent with this, Orai1 expression was significantly increased in soleus of CASQ1-null mice, but even more in dCASQ-null mice, compared with WT. Together, these results strengthen the concept that CEU assembly strongly depends on CASQ expression and provides an alternative source of Ca2+ needed to refill SR Ca2+ stores to maintain specific force production during sustained muscle activity.

DOI: https://doi.org/10.1085/jgp.202213114
Physiol Rep. 2022 Oct;10(19): e15450

Influence of 4 weeks of downhill running on calcium sensitivity of rat single muscle fibers.

Emma F Hubbard, Avery Hinks, Parastoo Mashouri, Geoffrey A Power.

  Improved Ca2+ sensitivity has been suggested as a mechanism behind enhancements in muscle mechanical function following eccentric training. However, little is known regarding the effects of eccentric training on single muscle fiber Ca2+ sensitivity. Adult male Sprague-Dawley rats (sacrificial age ~18 weeks; mass = 400.1 ± 34.8 g) were assigned to an eccentric training (n = 5) or sedentary control group (n = 6). Eccentric training consisted of 4 weeks of weighted downhill running 3×/week at a 15° decline and 16 m/min for 35 min per day in 5-min bouts. After sacrifice, vastus intermedius single muscle fibers were dissected, chemically permeabilized, and stored until testing. Fibers (n = 63) were isolated, and standard Ca2+ sensitivity, force, rate of force redevelopment (ktr ), and active instantaneous stiffness tests were performed using [Ca2+ ] ranging from 7.0 to 4.5. Following all mechanical testing, fiber type was determined using SDS-PAGE. There was no difference in pCa50 (i.e., [Ca2+ ] needed to elicit half of maximal force) between groups or between fiber types. However, when comparing normalized force across pCa values, fibers from the control group produced greater forces than fibers from the trained group at lower Ca2+ concentrations (p < 0.05), and this was most evident for Type I fibers (p = 0.002). Type II fibers produced faster (p < 0.001) ktr than Type I fibers, but there were no differences in absolute force, normalized force, or other measures of mechanical function between fibers from the trained and control groups. These findings indicate that eccentric training does not appear to improve single muscle fiber Ca2+ sensitivity.

Keywords:  KTR; calcium sensitivity; eccentric; rate of force development; resistance training; rodent; running

DOI:  https://doi.org/10.14814/phy2.15450
Gen Physiol Biophys. 2022 Sep;41(5): 447-455

The role of Sirt3 in the changes of skeletal muscle mitophagy induced by hypoxic training.

Chunwei Ma, Yongcai Zhao, Xiaoqing Ding, Binghong Gao.

We aimed to explore the role of Sirt3 in the regulation of skeletal muscle mitophagy with hypoxic training. C57BL/6J mice were randomly divided into four groups: C group (control), HT group (mice performed a hypoxic training of living in an environment with an oxygen concentration of 13.8% and treadmill exercise under normoxia for 6 weeks), T group (mice were subjected to an intraperitoneal (i.p.) injection of the Sirt3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) 50 mg/kg three times per week for 6 weeks) and THT group (the hypoxic training of HT group with i.p. injection of 3-TYP in T group). The results showed that 6 weeks of hypoxic training could improve ATP synthesis in skeletal muscle. After the combined intervention of 3-TYP injection and hypoxic training, Sirt3, FOXO3a, and SOD2 protein contents were still lower than those in hypoxic training group. Hypoxic training cannot improve the negative effect of Sirt3 inhibition on muscle PINK1/Parkin signal. This study demonstrated that Sirt3 plays a key role in mediating skeletal muscle mitophagy by hypoxic training. The results of our study also provided the first evidence that mitophagy caused by hypoxic training might be transduced through the Sirt3-FOXO3a signaling pathway.

DOI: https://doi.org/10.4149/gpb_2022023
Cells. 2022 Oct 01. pii: 3102. [Epub ahead of print]11(19):

Exosomal Plasminogen Activator Inhibitor-1 Induces Ionizing Radiation-Adaptive Glioblastoma Cachexia.

Eunguk Shin, Hyunkoo Kang, Haksoo Lee, Sungmin Lee, Jaewan Jeon, Kimoon Seong, Hyesook Youn, Buhyun Youn.

  Cancer cachexia is a muscle-wasting syndrome that leads to a severely compromised quality of life and increased mortality. A strong association between cachexia and poor prognosis has been demonstrated in intractable cancers, including glioblastoma (GBM). In the present study, it was demonstrated that ionizing radiation (IR), the first-line treatment for GBM, causes cancer cachexia by increasing the exosomal release of plasminogen activator inhibitor-1 (PAI-1) from glioblastoma cells. Exosomal PAI-1 delivered to the skeletal muscle is directly penetrated in the muscles and phosphorylates STAT3 to intensify muscle atrophy by activating muscle RING-finger protein-1 (MuRF1) and muscle atrophy F-box (Atrogin1); furthermore, it hampers muscle protein synthesis by inhibiting mTOR signaling. Additionally, pharmacological inhibition of PAI-1 by TM5441 inhibited muscle atrophy and rescued muscle protein synthesis, thereby providing survival benefits in a GBM orthotopic xenograft mouse model. In summary, our data delineated the role of PAI-1 in the induction of GBM cachexia associated with radiotherapy-treated GBM. Our data also indicated that targeting PAI-1 could serve as an attractive strategy for the management of GBM following radiotherapy, which would lead to a considerable improvement in the quality of life of GBM patients undergoing radiotherapy.

Keywords:  PAI-1; cachexia; exosome; glioblastoma; radiotherapy

DOI:  https://doi.org/10.3390/cells11193102
Front Bioeng Biotechnol. 2022 ;10 964705

Efficient co-isolation of microvascular endothelial cells and satellite cell-derived myoblasts from human skeletal muscle.

Rebecca Wüst, Lisanne Terrie, Thomas Müntefering, Tobias Ruck, Lieven Thorrez.

  Vascularization of tissue-engineered constructs remains a key challenge in the field of skeletal muscle tissue engineering. One strategy for vascularizing organoids is in vitro pre-vascularization, relying on de novo assembly of undifferentiated endothelial cells into capillaries, a process termed vasculogenesis. In most endothelial cell research to date, human umbilical vein endothelial cells have been used primarily because of their availability. Nevertheless, this endothelial cell type is naturally not occurring in skeletal muscle tissue. Since endothelial cells display a tissue-specific phenotype, it is of interest to use muscle-specific microvascular endothelial cells to study pre-vascularization in skeletal muscle tissue engineering research. Thus far, tissue biopsies had to be processed in two separate protocols to obtain cells from the myogenic and the endothelial compartment. Here, we describe a novel, detailed protocol for the co-isolation of human skeletal muscle microvascular endothelial cells and satellite cell-derived myoblasts. It incorporates an automated mechanical and enzymatic tissue dissociation followed by magnetically activated cell sorting based on a combination of endothelial and skeletal muscle cell markers. Qualitative, quantitative, and functional characterization of the obtained cells is described and demonstrated by representative results. The simultaneous isolation of both cell types from the same donor is advantageous in terms of time efficiency. In addition, it may be the only possible method to isolate both cell types as the amount of tissue biopsy is often limited. The isolation of the two cell types is crucial for further studies to elucidate cell crosstalk in health and disease. Furthermore, the use of muscle-specific microvascular endothelial cells allows a shift towards engineering more physiologically relevant functional tissue, with downstream applications including drug screening and regenerative medicine.

Keywords:  cell isolation; co-isolation; human; microvascular endothelial cells; myoblasts; skeletal muscle

DOI:  https://doi.org/10.3389/fbioe.2022.964705
J Cachexia Sarcopenia Muscle. 2022 Oct 12.

A prospective clinical study on the mechanisms underlying critical illness myopathy-A time-course approach.

Nicola Cacciani, Åsa Skärlén, Ya Wen, Xiang Zhang, Alex B Addinsall, Monica Llano-Diez, Meishan Li, Lennart Gransberg, Yvette Hedström, Bo-Michael Bellander, David Nelson, Jonas Bergquist, Lars Larsson.

   BACKGROUND: Critical illness myopathy (CIM) is a consequence of modern critical care resulting in general muscle wasting and paralyses of all limb and trunk muscles, resulting in prolonged weaning from the ventilator, intensive care unit (ICU) treatment and rehabilitation. CIM is associated with severe morbidity/mortality and significant negative socioeconomic consequences, which has become increasingly evident during the current COVID-19 pandemic, but underlying mechanisms remain elusive.
METHODS: Ten neuro-ICU patients exposed to long-term controlled mechanical ventilation were followed with repeated muscle biopsies, electrophysiology and plasma collection three times per week for up to 12 days. Single muscle fibre contractile recordings were conducted on the first and final biopsy, and a multiomics approach was taken to analyse gene and protein expression in muscle and plasma at all collection time points.
RESULTS: (i) A progressive preferential myosin loss, the hallmark of CIM, was observed in all neuro-ICU patients during the observation period (myosin:actin ratio decreased from 2.0 in the first to 0.9 in the final biopsy, P < 0.001). The myosin loss was coupled to a general transcriptional downregulation of myofibrillar proteins (P < 0.05; absolute fold change >2) and activation of protein degradation pathways (false discovery rate [FDR] <0.1), resulting in significant muscle fibre atrophy and loss in force generation capacity, which declined >65% during the 12 day observation period (muscle fibre cross-sectional area [CSA] and maximum single muscle fibre force normalized to CSA [specific force] declined 30% [P < 0.007] and 50% [P < 0.0001], respectively). (ii) Membrane excitability was not affected as indicated by the maintained compound muscle action potential amplitude upon supramaximal stimulation of upper and lower extremity motor nerves. (iii) Analyses of plasma revealed early activation of inflammatory and proinflammatory pathways (FDR < 0.1), as well as a redistribution of zinc ions from plasma.
CONCLUSIONS: The mechanical ventilation-induced lung injury with release of cytokines/chemokines and the complete mechanical silencing uniquely observed in immobilized ICU patients affecting skeletal muscle gene/protein expression are forwarded as the dominant factors triggering CIM.

Keywords:  critical illness myopathy; mechanical ventilation; membrane exitability; muscle paresis; myosin loss

DOI:  https://doi.org/10.1002/jcsm.13104
Nat Aging. 2022 Jun;2(6): 494-507

Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila.

Edward T Schmid, Jung-Hoon Pyo, David W Walker.

  The effects of aging on the brain are widespread and can have dramatic implications on the overall health of an organism. Mitochondrial dysfunction is a hallmark of brain aging, but, the interplay between mitochondrial quality control, neuronal aging, and organismal health is not well understood. Here, we show that aging leads to a decline in mitochondrial autophagy (mitophagy) in the Drosophila brain with a concomitant increase in mitochondrial content. We find that induction of BCL2-interacting protein 3 (BNIP3), a mitochondrial outer membrane protein, in the adult nervous system induces mitophagy and prevents the accumulation of dysfunctional mitochondria in the aged brain. Importantly, neuronal induction of BNIP3-mediated mitophagy increases organismal longevity and healthspan. Furthermore, BNIP3-mediated mitophagy in the nervous system improves muscle and intestinal homeostasis in aged flies, indicating cell non-autonomous effects. Our findings identify BNIP3 as a therapeutic target to counteract brain aging and prolong overall organismal health with age.

Keywords:  Autophagy; Intestinal barrier dysfunction; Intestinal stem cell; Mito-QC; Mitophagy; Muscle aging; Neuronal aging

DOI:  https://doi.org/10.1038/s43587-022-00214-y
Oncotarget. 2022 Oct 08. 13 1094-1108

In vitro chemotherapy-associated muscle toxicity is attenuated with nutritional support, while treatment efficacy is retained.

Liza A Wijler, Francina J Dijk, Hanil Quirindongo, Danielle A E Raats, Bram Dorresteijn, Matthew J W Furber, Anne M May, Onno Kranenburg, Miriam van Dijk.

   PURPOSE: Muscle-wasting and treatment-related toxicities negatively impact prognosis of colorectal cancer (CRC) patients. Specific nutritional composition might support skeletal muscle and enhance treatment support. In this in vitro study we assess the effect of nutrients EPA, DHA, L-leucine and vitamin D3, as single nutrients or in combination on chemotherapy-treated C2C12-myotubes, and specific CRC-tumor cells.
MATERIALS AND METHODS: Using C2C12-myotubes, the effects of chemotherapy (oxaliplatin, 5-fluorouracil, oxaliplatin+5-fluorouracil and irinotecan) on protein synthesis, cell-viability, caspase-3/7-activity and LDH-activity were assessed. Addition of EPA, DHA, L-leucine and vitamin D3 and their combination (SNCi) were studied in presence of above chemotherapies. Tumor cell-viability was assessed in oxaliplatin-treated C26 and MC38 CRC cells, and in murine and patient-derived CRC-organoids.
RESULTS: While chemotherapy treatment of C2C12-myotubes decreased protein synthesis, cell-viability and increased caspase-3/7 and LDH-activity, SNCi showed improved protein synthesis and cell viability and lowered LDH activity. The nutrient combination SNCi showed a better overall performance compared to the single nutrients. Treatment response of tumor models was not significantly affected by addition of nutrients.
CONCLUSIONS: This in vitro study shows protective effect with specific nutrition composition of C2C12-myotubes against chemotherapy toxicity, which is superior to the single nutrients, while treatment response of tumor cells remained.

Keywords:  C2C12 myotubes; chemotherapy; chemotherapy-associated toxicity; colorectal cancer; nutrients; tumor (organoid) cells

DOI:  https://doi.org/10.18632/oncotarget.28279
J Physiol. 2022 Oct 11.

Plants powering muscle: The effects of phytochemical PB125 on mitochondrial function and skeletal muscle health.

Fasih Ahmad Rahman, Dylan James Hian-Cheong, James Patrick Thoms.



Keywords:  NRF2 activator; mitochondrial quality control; proteostasis; sarcopenia

DOI:  https://doi.org/10.1113/JP283779
J Biol Chem. 2022 Oct 06. pii: S0021-9258(22)01012-2. [Epub ahead of print] 102568

Low dose lithium supplementation promotes adipose tissue browning and sarco(endo)plasmic reticulum Ca2+ ATPase uncoupling in muscle.

Mia S Geromella, Chantal R Ryan, Jessica L Braun, Michael S Finch, Lucas A Maddalena, Olivia Bagshaw, Briana Hockey, Fereshteh Moradi, Rachel K Fenech, Jisook Ryoo, Daniel M Marko, Roopan Dhaliwal, Jake Sweezey-Munroe, Sophie I Hamstra, Georgina Gardner, Sebastian Silvera, Rene Vandenboom, Brian D Roy, Jeffrey A Stuart, Rebecca E K MacPherson, Val A Fajardo.

  Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) uncoupling in skeletal muscle, and mitochondrial uncoupling via uncoupling protein 1 (UCP1) in brown/beige adipose tissue are two primary mechanisms implicated in energy expenditure. Here, we investigated the effects of glycogen synthase kinase 3 (GSK3) inhibition via lithium chloride (LiCl) treatment on SERCA uncoupling in skeletal muscle and UCP1 expression in adipose. C2C12 and 3T3-L1 cells treated with LiCl had increased SERCA uncoupling and UCP1 protein levels, respectively, ultimately raising cellular respiration; however, this was only observed when LiCl treatment occurred throughout differentiation. In vivo, LiCl treatment (10 mg/kg/day) increased food intake in chow-fed and high-fat diet (HFD, 60% kcal) fed male mice without increasing body mass - a result attributed to elevated daily energy expenditure. In soleus muscle, we determined LiCl treatment promoted SERCA uncoupling via increased expression of SERCA uncouplers, sarcolipin and/or neuronatin, under chow and HFD-fed conditions. We attribute these effects to the GSK3 inhibition observed with LiCl treatment as partial muscle-specific GSK3 knockdown produced similar effects. In adipose, LiCl treatment inhibited GSK3 in inguinal WAT (iWAT) but not in brown adipose tissue under chow-fed conditions, which in turn led to an increase in UCP1 in iWAT and a beiging-like effect with a multilocular phenotype. We did not observe this beiging-like effect and increase in UCP1 when mice were fed a HFD, as LiCl could not overcome the ensuing overactivation of GSK3. Nonetheless, our study establishes novel regulatory links between GSK3 and SERCA uncoupling in muscle and GSK3 and UCP1 and beiging in iWAT.

Keywords:  SERCA; UCP1; beige; efficiency; neuronatin; sarcolipin; uncoupling

DOI:  https://doi.org/10.1016/j.jbc.2022.102568
Int J Mol Sci. 2022 Oct 10. pii: 12038. [Epub ahead of print]23(19):

CircCSDE1 Regulates Proliferation and Differentiation of C2C12 Myoblasts by Sponging miR-21-3p.

Di Sun, Jiaqi An, Zixu Cui, Jiao Li, Ziwei You, Chang Lu, Yang Yang, Pengfei Gao, Xiaohong Guo, Bugao Li, Chunbo Cai, Guoqing Cao.

  The growth and development of skeletal muscle is regulated by many factors, and recent studies have shown that circular RNAs (circRNAs) can participate in this process. The model of porcine skeletal muscle injury was constructed to search for circRNAs that can regulate the growth and development of skeletal muscle in pigs. Using whole-transcriptome sequencing and bioinformatics analysis, a novel circRNA (circCSDE1) was screened out, which is highly expressed in skeletal muscle. Functional studies in C2C12 cells demonstrated that circCSDE1 could promote proliferation and inhibit myoblast differentiation, while opposing changes were observed by circCSDE1 knockdown. A dual-luciferase reporter assay revealed that circCSDE1 directly targeted miR-21-3p to regulate the expression of the downstream target gene (Cyclin-dependent kinase 16, CDK16). Moreover, miR-21-3p could inhibit proliferation and promote myoblast differentiation in C2C12 cells, opposite with the effects of circCSDE1. Additionally, the rescue experiments offered further evidence that circCSDE1 and its target, miR-21-3p, work together to regulate myoblast proliferation and differentiation. This study provides a theoretical basis for further understanding the regulatory mechanisms of circRNAs.

Keywords:  C2C12; circCSDE1; differentiation; pig; proliferation; skeletal muscle injury

DOI:  https://doi.org/10.3390/ijms231912038
Nat Commun. 2022 Oct 13. 13(1): 6058

Mitochondrial network configuration influences sarcomere and myosin filament structure in striated muscles.

Prasanna Katti, Alexander S Hall, Hailey A Parry, Peter T Ajayi, Yuho Kim, T Bradley Willingham, Christopher K E Bleck, Han Wen, Brian Glancy.

Sustained muscle contraction occurs through interactions between actin and myosin filaments within sarcomeres and requires a constant supply of adenosine triphosphate (ATP) from nearby mitochondria. However, it remains unclear how different physical configurations between sarcomeres and mitochondria alter the energetic support for contractile function. Here, we show that sarcomere cross-sectional area (CSA) varies along its length in a cell type-dependent manner where the reduction in Z-disk CSA relative to the sarcomere center is closely coordinated with mitochondrial network configuration in flies, mice, and humans. Further, we find myosin filaments near the sarcomere periphery are curved relative to interior filaments with greater curvature for filaments near mitochondria compared to sarcoplasmic reticulum. Finally, we demonstrate variable myosin filament lattice spacing between filament ends and filament centers in a cell type-dependent manner. These data suggest both sarcomere structure and myofilament interactions are influenced by the location and orientation of mitochondria within muscle cells.

DOI: https://doi.org/10.1038/s41467-022-33678-y
Cells. 2022 Sep 22. pii: 2964. [Epub ahead of print]11(19):

CRISPR-Based Therapeutic Gene Editing for Duchenne Muscular Dystrophy: Advances, Challenges and Perspectives.

Guofang Chen, Tingyi Wei, Hui Yang, Guoling Li, Haisen Li.

  Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the dystrophin gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common types of fatal genetic diseases, there is no curative treatment for this devastating disorder. In recent years, gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system has paved a new path toward correcting pathological mutations at the genetic source, thus enabling the permanent restoration of dystrophin expression and function throughout the musculature. To date, the therapeutic benefits of CRISPR genome-editing systems have been successfully demonstrated in human cells, rodents, canines, and piglets with diverse DMD mutations. Nevertheless, there remain some nonignorable challenges to be solved before the clinical application of CRISPR-based gene therapy. Herein, we provide an overview of therapeutic CRISPR genome-editing systems, summarize recent advancements in their applications in DMD contexts, and discuss several potential obstacles lying ahead of clinical translation.

Keywords:  CRISPR; DMD; base editing; double cut; dystrophin; gene therapy; prime editing; single cut

DOI:  https://doi.org/10.3390/cells11192964
Int J Mol Sci. 2022 Sep 20. pii: 10986. [Epub ahead of print]23(19):

The Impact of Spermidine on C2C12 Myoblasts Proliferation, Redox Status and Polyamines Metabolism under H2O2 Exposure.

Roberta Ceci, Guglielmo Duranti, Stefano Giuliani, Marianna Nicoletta Rossi, Ivan Dimauro, Stefania Sabatini, Paolo Mariottini, Manuela Cervelli.

  A central feature of the skeletal muscle is its ability to regenerate through the activation, by environmental signals, of satellite cells. Once activated, these cells proliferate as myoblasts, and defects in this process profoundly affect the subsequent process of regeneration. High levels of reactive oxygen species such as hydrogen peroxide (H2O2) with the consequent formation of oxidized macromolecules increase myoblasts' cell death and strongly contribute to the loss of myoblast function. Recently, particular interest has turned towards the beneficial effects on muscle of the naturally occurring polyamine spermidine (Spd). In this work, we tested the hypothesis that Spd, upon oxidative challenge, would restore the compromised myoblasts' viability and redox status. The effects of Spd in combination with aminoguanidine (Spd-AG), an inhibitor of bovine serum amine oxidase, on murine C2C12 myoblasts treated with a mild dose of H2O2 were evaluated by analyzing: (i) myoblast viability and recovery from wound scratch; (ii) redox status and (iii) polyamine (PAs) metabolism. The treatment of C2C12 myoblasts with Spd-AG increased cell number and accelerated scratch wound closure, while H2O2 exposure caused redox status imbalance and cell death. The combined treatment with Spd-AG showed an antioxidant effect on C2C12 myoblasts, partially restoring cellular total antioxidant capacity, reducing the oxidized glutathione (GSH/GSSG) ratio and increasing cell viability through a reduction in cell death. Moreover, Spd-AG administration counteracted the induction of polyamine catabolic genes and PA content decreased due to H2O2 challenges. In conclusion, our data suggest that Spd treatment has a protective role in skeletal muscle cells by restoring redox balance and promoting recovery from wound scratches, thus making myoblasts able to better cope with an oxidative insult.

Keywords:  glutathione; myoblasts proliferation; polyamines homeostasis; redox homeostasis; spermidine

DOI:  https://doi.org/10.3390/ijms231910986
Int J Mol Sci. 2022 Sep 21. pii: 11103. [Epub ahead of print]23(19):

The TRPV1 Receptor Is Up-Regulated by Sphingosine 1-Phosphate and Is Implicated in the Anandamide-Dependent Regulation of Mitochondrial Activity in C2C12 Myoblasts.

Sara Standoli, Sara Pecchioli, Daniel Tortolani, Camilla Di Meo, Federico Fanti, Manuel Sergi, Marina Bacci, Isabelle Seidita, Caterina Bernacchioni, Chiara Donati, Paola Bruni, Mauro Maccarrone, Cinzia Rapino, Francesca Cencetti.

  The sphingosine 1-phosphate (S1P) and endocannabinoid (ECS) systems comprehend bioactive lipids widely involved in the regulation of similar biological processes. Interactions between S1P and ECS have not been so far investigated in skeletal muscle, where both systems are active. Here, we used murine C2C12 myoblasts to investigate the effects of S1P on ECS elements by qRT-PCR, Western blotting and UHPLC-MS. In addition, the modulation of the mitochondrial membrane potential (ΔΨm), by JC-1 and Mitotracker Red CMX-Ros fluorescent dyes, as well as levels of protein controlling mitochondrial function, along with the oxygen consumption were assessed, by Western blotting and respirometry, respectively, after cell treatment with methanandamide (mAEA) and in the presence of S1P or antagonists to endocannabinoid-binding receptors. S1P induced a significant increase in TRPV1 expression both at mRNA and protein level, while it reduced the protein content of CB2. A dose-dependent effect of mAEA on ΔΨm, mediated by TRPV1, was evidenced; in particular, low doses were responsible for increased ΔΨm, whereas a high dose negatively modulated ΔΨm and cell survival. Moreover, mAEA-induced hyperpolarization was counteracted by S1P. These findings open new dimension to S1P and endocannabinoids cross-talk in skeletal muscle, identifying TRPV1 as a pivotal target.

Keywords:  C2C12 myoblasts; methanandamide; mitochondrial membrane potential; sphingosine 1-phosphate; transient receptor potential vanilloid type 1

DOI:  https://doi.org/10.3390/ijms231911103
Clin Sci (Lond). 2022 Oct 14. 136(19): 1425-1431

Measuring muscle protein synthesis in humans and the influence of nutritional state.

Philip C Calder, Nicolaas E P Deutz.

  In 1982 and 2011, Clinical Science published papers that used infusion of stable isotope-labeled amino acids to assess skeletal muscle protein synthesis in the fasted and fed state and before and after a period of increased intake of omega-3 fatty acids, respectively; both of these papers have been highly cited. An overview of the study designs, key findings and novel features, and a consideration of the lasting impact of these two papers is presented. The earlier paper introduced stable isotope tracer approaches in humans that showed consuming a meal will increase whole body oxidation, synthesis, and breakdown of protein, but that protein synthesis is greater than breakdown resulting in net accumulation of protein. The paper also demonstrated that consuming a meal promotes net protein synthesis in skeletal muscle. The later paper introduced the concept that omega-3 polyunsaturated fatty acids are able to improve anabolism by reporting that 8 weeks consumption of high-dose omega-3 fatty acids by healthy young and middle-aged adults increased skeletal muscle protein synthesis during a hyperaminoacidemic-hyperinsulinemic clamp compared with what was seen during the clamp at study entry. Omega-3 fatty acids also increased the phosphorylation of important signaling proteins in muscle, including mammalian target of rapamycin, p70s6k, and Akt, during the clamp. These two papers remain relevant because they offer experimental approaches to study human (patho)physiology in different contexts, they present novel insights into the impact of nutritional state (feeding) and specific nutrients (omega-3 fatty acids) on muscle protein synthesis, and they suggest ways to explore the potential of interventions to help prevent and reverse the age-, disease-, and disuse-associated decline in muscle mass.

Keywords:  amino acid metabolism; protein synthesis; stable isotopes

DOI:  https://doi.org/10.1042/CS20211171
Nat Aging. 2021 Dec;1(12): 1189-1201

Profiling epigenetic age in single cells.

Alexandre Trapp, Csaba Kerepesi, Vadim N Gladyshev.

DNA methylation dynamics emerged as a promising biomarker of mammalian aging, with multivariate machine learning models ('epigenetic clocks') enabling measurement of biological age in bulk tissue samples. However, intrinsically sparse and binarized methylation profiles of individual cells have so far precluded the assessment of aging in single-cell data. Here, we introduce scAge, a statistical framework for epigenetic age profiling at single-cell resolution, and validate our approach in mice. Our method recapitulates the chronological age of tissues, while uncovering heterogeneity among cells. We show accurate tracking of the aging process in hepatocytes, demonstrate attenuated epigenetic aging in muscle stem cells, and track age dynamics in embryonic stem cells. We also use scAge to reveal, at the single-cell level, a natural and stratified rejuvenation event occurring during early embryogenesis. We provide our framework as a resource to enable exploration of epigenetic aging trajectories at single-cell resolution.

DOI: https://doi.org/10.1038/s43587-021-00134-3
Neurology. 2022 Sep 02. pii: 10.1212/WNL.0000000000201163. [Epub ahead of print]

Evaluating Genetic Modifiers of Duchenne Muscular Dystrophy Disease Progression Using Modeling and MRI.

Alison M Barnard, David W Hammers, William T Triplett, Sarah Kim, Sean C Forbes, Rebecca J Willcocks, Michael J Daniels, Claudia R Senesac, Donovan J Lott, Ishu Arpan, William D Rooney, Richard T Wang, Stanley F Nelson, H Lee Sweeney, Krista Vandenborne, Glenn A Walter.

BACKGROUND AND OBJECTIVES: Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder with a well-characterized disease phenotype but considerable interindividual heterogeneity that is not well understood. The aim of the study was to evaluate the effects of dystrophin mutations and genetic modifiers of DMD on rate and age of muscle replacement by fat.
METHODS: 175 corticosteroid treated participants from the ImagingDMD natural history study underwent repeated magnetic resonance spectroscopy (MRS) of the vastus lateralis (VL) and soleus (SOL) to determine muscle fat fraction. MRS was performed annually in the majority of instances; however, some individuals had additional visits at 3 or 6 month intervals. Fat fraction changes over time were modeled using nonlinear mixed effects to estimate disease trajectories based on the age that the VL or SOL reached half-maximum change in fat fraction (mu) and the time required for fat fraction change (sigma). Computed mu and sigma values were evaluated for dystrophin mutations that have demonstrated the ability to lead to a mild phenotype as well as compared between different genetic polymorphism groups.
RESULTS: Participants with dystrophin gene deletions amenable to exon 8 skipping (n=4) had minimal increases in SOL fat fraction and had an increase in VL mu value by 4.4 years compared to a reference cohort (p=0.039). Participants with nonsense mutations within exons that may produce milder phenotypes (n=11) also had minimal increases in SOL and VL fat fractions. No differences in estimated fat fraction trajectories were seen for individuals amenable to exon 44 skipping (n=10). Modeling of the SPP1, LTBP4, and THBS1 genetic modifiers did not result in significant differences in muscle fat fraction trajectories between genotype groups (p>0.05); however, trends were noted for the polymorphisms associated with long-range regulation of LTBP4 and THBS1 that deserve further follow-up.
DISCUSSION: The results of this study link the historically mild phenotypes seen in individuals amenable to exon 8 skipping and with certain nonsense mutations with alterations in trajectories of lower extremity muscle replacement by fat.

DOI: https://doi.org/10.1212/WNL.0000000000201163