bims-moremu 2023-04-02 papers

bims-moremu

Biomed News

on Molecular regulators of muscle mass

Issue of 2023‒04‒02
forty papers selected by
Anna Vainshtein
Craft Science Inc.

MicroRNA Control of the Myogenic Cell Transcriptome and Proteome: The Role of miR-16.
Going nuclear: Molecular adaptations to exercise mediated by myonuclei.
Hot topics in muscle metabolism and adaptation.
Mass spectrometry-based proteomics approaches to interrogate skeletal muscle adaptations to exercise.
Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal Myogenesis.
High Throughput Screening of Mitochondrial Bioenergetics in Myoblasts and Differentiated Myotubes.
Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.
Single-Cell Transcriptomic Analysis of Mononuclear Cell Populations in Skeletal Muscle.
Globular adiponectin ameliorates insulin resistance in skeletal muscle by enhancing the LKB1-mediated AMPK activation via SESN2.
The skeletal muscle fiber periphery: A nexus of mTOR-related anabolism.
Undercarboxylated osteocalcin and ibandronate combination ameliorates hindlimb immobilization-induced muscle wasting.
Different Resistance Exercise Loading Paradigms Similarly Affect Skeletal Muscle Gene Expression Patterns of Myostatin-Related Targets and mTORC1 Signaling Markers.
Proteome profiles of skeletal muscle connective tissue: Influence of aging and physical training.
In Vivo Investigation of Gene Function in Muscle Stem Cells by CRISPR/Cas9-Mediated Genome Editing.
Skeletal muscle mechanisms contributing to improved glycemic control following intense interval exercise and training.
Modulating fast skeletal muscle contraction protects skeletal muscle in animal models of Duchenne muscular dystrophy.
Palmitic Acid Inhibits Myogenic Activity and Expression of Myosin Heavy Chain MHC IIb in Muscle Cells through Phosphorylation-Dependent MyoD Inactivation.
Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Muscle Stem Cells Using a Seahorse Analyzer: Applicability for Aging Studies.
Skeletal Muscle Regeneration in Zebrafish.
Functional Analysis of MicroRNAs in Skeletal Muscle.
Techniques for Injury, Cell Transplantation, and Histological Analysis in Skeletal Muscle.
Murine Models of Tenotomy-Induced Mechanical Overloading and Tail-Suspension-Induced Mechanical Unloading.
Static Magnetic Fields Promote Generation of Muscle Lineage Cells from Pluripotent Stem Cells and Myoblasts.
The Role of Lonp1 on Mitochondrial Functions during Cardiovascular and Muscular Diseases.
Denervation Drives YAP/TAZ Activation in Muscular Fibro/Adipogenic Progenitors.
AMPK Phosphorylation Impacts Apoptosis in Differentiating Myoblasts Isolated from Atrophied Rat Soleus Muscle.
Multiomic characterization of disease progression in mice lacking dystrophin.
The role of Limch1 alternative splicing in skeletal muscle function.
Defective Muscle Ketone Body Oxidation Disrupts BCAA Catabolism by Altering Mitochondrial Branched-Chain Aminotransferase.
Epitranscriptome Mapping of N6-Methyladenosine Using m6A Immunoprecipitation with High Throughput Sequencing in Skeletal Muscle Stem Cells.
Uncovering the Gene Regulatory Network of Endothelial Cells in Mouse Duchenne Muscular Dystrophy: Insights from Single-Nuclei RNA Sequencing Analysis.
The role of glycogen phosphorylase in glycogen biogenesis in skeletal muscle after exercise.
Defective BVES-mediated feedback control of cAMP in muscular dystrophy.
Stretching the story of titin and muscle function.
Reduced energy metabolism contributing to aging of skeletal muscle by serum metabolomics and gut microbiota analysis.
Exercise Induces an Augmented Skeletal Muscle Mitochondrial Unfolded Protein Response in a Mouse Model of Obesity Produced by a High-Fat Diet.
USP18 is an essential regulator of muscle cell differentiation and maturation.
NAMPT-dependent NAD+ biosynthesis controls circadian metabolism in a tissue-specific manner.
Muscle cell-type diversification is driven by bHLH transcription factor expansion and extensive effector gene duplications.
Structural Biochemistry of Muscle Contraction.

Am J Physiol Cell Physiol. 2023 Mar 27.

MicroRNA Control of the Myogenic Cell Transcriptome and Proteome: The Role of miR-16.

Seongkyun Lim, David Lee, Francielly Morena Da Silva, Pieter Koopmans, Ivan J Vechetti, Ferdinand von Walden, Nicholas P Greene, Kevin A Murach.

  microRNAs (miRs) control stem cell biology and fate. Ubiquitously expressed and conserved miR-16 was the first miR implicated in tumorigenesis. miR-16 is low in muscle during developmental hypertrophy and regeneration. It is enriched in proliferating myogenic progenitor cells but is repressed during differentiation. The induction of miR-16 blocks myoblast differentiation and myotube formation while knockdown enhances it. Despite a central role for miR-16 in myogenic cell biology, how it mediates its potent effects is incompletely defined. In this investigation, global transcriptomic and proteomic analyses after miR-16 knockdown in proliferating C2C12 myoblasts revealed how miR-16 influences myogenic cell fate. Eighteen hours after miR-16 inhibition, ribosomal protein gene expression levels were higher relative to control myoblasts and p53 pathway-related gene abundance was lower. At the protein level at this same timepoint, miR-16 knockdown globally upregulated TCA cycle proteins while downregulating RNA metabolism-related proteins. miR-16 inhibition induced specific proteins associated with myogenic differentiation such as ACTA2, EEF1A2, and OPA1. We extend prior work in hypertrophic muscle tissue and show that miR-16 is lower in mechanically overloaded muscle in vivo. Our data collectively point to how miR-16 is implicated in aspects of myogenic cell differentiation. A deeper understanding of the role of miR-16 in myogenic cells has consequences for muscle developmental growth, exercise-induced hypertrophy, and regenerative repair after injury, all of which involve myogenic progenitors.

Keywords:  Proteomics; RNA-sequencing; Satellite Cells; Skeletal Muscle; lncRNA

DOI:  https://doi.org/10.1152/ajpcell.00071.2023
Sports Med Health Sci. 2023 Mar;5(1): 2-9

Going nuclear: Molecular adaptations to exercise mediated by myonuclei.

Pieter J Koopmans, Kevin A Zwetsloot, Kevin A Murach.

  Muscle fibers are multinucleated, and muscle fiber nuclei (myonuclei) are believed to be post-mitotic and are typically situated near the periphery of the myofiber. Due to the unique organization of muscle fibers and their nuclei, the cellular and molecular mechanisms regulating myofiber homeostasis in unstressed and stressed conditions (e.g., exercise) are unique. A key role myonuclei play in regulating muscle during exercise is gene transcription. Only recently have investigators had the capability to identify molecular changes at high resolution exclusively in myonuclei in response to perturbations in vivo. The purpose of this review is to describe how myonuclei modulate their transcriptome, epigenetic status, mobility and shape, and microRNA expression in response to exercise in vivo. Given the relative paucity of high-fidelity information on myonucleus-specific contributions to exercise adaptation, we identify specific gaps in knowledge and provide perspectives on future directions of research.

Keywords:  Epigenetics; Muscle memory; Skeletal muscle; Transcription; myomiR

DOI:  https://doi.org/10.1016/j.smhs.2022.11.005
Sports Med Health Sci. 2023 Mar;5(1): 1

Hot topics in muscle metabolism and adaptation.

Zhen Yan, David A Hood.

DOI: https://doi.org/10.1016/j.smhs.2023.01.004
Scand J Med Sci Sports. 2023 Mar 27.

Mass spectrometry-based proteomics approaches to interrogate skeletal muscle adaptations to exercise.

Daniel T Cervone, Roger Moreno-Justicia, Júlia Prats Quesada, Atul S Deshmukh.

  Acute exercise and chronic exercise training elicit beneficial whole-body changes in physiology that ultimately depend on profound alterations to the dynamics of tissue-specific proteins. Since the work accomplished during exercise owes predominantly to skeletal muscle, it has received the majority of interest from exercise scientists that attempt to unravel adaptive mechanisms accounting for salutary metabolic effects and performance improvements that arise from training. Contemporary scientists are also beginning to use mass spectrometry-based proteomics, which is emerging as a powerful approach to interrogate the muscle protein signature in a more comprehensive manner. Collectively, these technologies facilitate the analysis of skeletal muscle protein dynamics from several viewpoints, including changes to intracellular proteins (expression proteomics), secreted proteins (secretomics), post-translational modifications as well as fiber-, cell-, and organelle-specific changes. This review aims to highlight recent literature that has leveraged new workflows and advances in mass spectrometry-based proteomics to further our understanding of training-related changes in skeletal muscle. We call attention to untapped areas in skeletal muscle proteomics research relating to exercise training and metabolism, as well as basic points of contention when applying mass spectrometry-based analyses, particularly in the study of human biology. We further encourage researchers to couple the hypothesis-generating and descriptive nature of omics data with functional analyses that propel our understanding of the complex adaptive responses in skeletal muscle that occur with acute and chronic exercise.

Keywords:  fiber-type; heterogeneity; organellar; post-translational modification; secretomics; singlecell; training

DOI:  https://doi.org/10.1111/sms.14334
Methods Mol Biol. 2023 ;2640 21-43

Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal Myogenesis.

Massimo Ganassi, Peter S Zammit, Simon M Hughes.

  Adult skeletal musculature experiences continuous physical stress, and hence requires maintenance and repair to ensure its continued efficient functioning. The population of resident muscle stem cells (MuSCs), termed satellite cells, resides beneath the basal lamina of adult myofibers, contributing to both muscle hypertrophy and regeneration. Upon exposure to activating stimuli, MuSCs proliferate to generate new myoblasts that differentiate and fuse to regenerate or grow myofibers. Moreover, many teleost fish undergo continuous growth throughout life, requiring continual nuclear recruitment from MuSCs to initiate and grow new fibers, a process that contrasts with the determinate growth observed in most amniotes. In this chapter, we describe a method for the isolation, culture, and immunolabeling of adult zebrafish myofibers that permits examination of both myofiber characteristics ex vivo and the MuSC myogenic program in vitro. Morphometric analysis of isolated myofibers is suitable to assess differences among slow and fast muscles or to investigate cellular features such as sarcomeres and neuromuscular junctions. Immunostaining for Pax7, a canonical stemness marker, identifies MuSCs on isolated myofibers for study. Furthermore, the plating of viable myofibers allows MuSC activation and expansion and downstream analysis of their proliferative and differentiative dynamics, thus providing a suitable, parallel alternative to amniote models for the study of vertebrate myogenesis.

Keywords:  Adult; MuSC; Myofiber; Myonucleus; Pax7; Skeletal muscle; Stem cell; Zebrafish

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_3
Methods Mol Biol. 2023 ;2640 89-98

High Throughput Screening of Mitochondrial Bioenergetics in Myoblasts and Differentiated Myotubes.

Kohei Takeda, Tohru Takemasa, Ryo Fujita.

  Skeletal muscles contain stem cells called satellite cells, which are essential for muscle regeneration. The population of satellite cells declines with aging and the incidence of pathological conditions such as muscular dystrophy. There is increasing evidence that metabolic switches and mitochondrial function are critical regulators of cell fate decision (quiescence, activation, differentiation, and self-renewal) during myogenesis. Thus, monitoring and identifying the metabolic profile in live cells using the Seahorse XF Bioanalyzer could provide new insights on the molecular mechanisms governing stem cell dynamics during regeneration and tissue maintenance. Here we described a method to assess mitochondrial respiration (oxygen consumption rate) and glycolysis (ECAR) in primary murine satellite cells, multinucleated myotubes, and C2C12 myoblasts.

Keywords:  Metabolism; Mitochondria; Myotube; Oxygen consumption rate; Satellite cell; Seahorse XF Analyzer; Skeletal muscle

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_7
bioRxiv. 2023 Mar 18. pii: 2023.03.17.533157. [Epub ahead of print]

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Sajedah M Hindi, Michael J Petrany, Elena Greenfeld, Leah C Focke, Alyssa A W Cramer, Michael A Whitt, Vikram Prasad, Jeffrey S Chamberlain, Benjamin Podbilewicz, Douglas P Millay.

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (μDys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

DOI: https://doi.org/10.1101/2023.03.17.533157
Methods Mol Biol. 2023 ;2640 369-395

Single-Cell Transcriptomic Analysis of Mononuclear Cell Populations in Skeletal Muscle.

Gary J He, Johanna Galvis, Tom H Cheung, Fabien Le Grand.

  Skeletal muscle possesses a remarkable regenerative capacity, mainly relying on a population of undifferentiated and unipotent muscle progenitors, called muscle stem cells (MuSCs) or satellite cells, and their interplay with various cell types within the niche. Investigating the cellular composition of skeletal muscle tissues and the heterogeneity among various cell populations is crucial to the unbiased understanding of how cellular networks work in harmony at the population level in the context of skeletal muscle homeostasis, regeneration, aging, and diseases. As opposed to probing the average profile in a cell population, single-cell RNA-seq has unlocked access to the transcriptomic landscape characterization of individual cells in a highly parallel manner. This chapter describes the workflow for single-cell transcriptomic analysis of mononuclear cells in skeletal muscle by taking advantage of the droplet-based single-cell RNA-seq platform, Chromium Single Cell 3' solution from 10x Genomics®. Using this protocol, we can reveal insights into muscle-resident cell-type identities, which can be exploited to study the muscle stem cell niche further.

Keywords:  Chromium single cell 3′ solution; Droplet-based; Mononuclear cells; Single-cell RNA-seq; Skeletal muscle

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_26
Sports Med Health Sci. 2023 Mar;5(1): 34-41

Globular adiponectin ameliorates insulin resistance in skeletal muscle by enhancing the LKB1-mediated AMPK activation via SESN2.

Xinmeng Liu, Yang Yang, Heng Shao, Sujuan Liu, Yanmei Niu, Li Fu.

  Adiponectin has been demonstrated to be a mediator of insulin sensitivity; however, the underlined mechanisms remain unclear. SESN2 is a stress-inducible protein that phosphorylates AMPK in different tissues. In this study, we aimed to validate the amelioration of insulin resistance by globular adiponectin (gAd) and to reveal the role of SESN2 in the improvement of glucose metabolism by gAd. We used a high-fat diet-induced wild-type and SESN2-/- C57BL/6J insulin resistance mice model to study the effects of six-week aerobic exercise or gAd administration on insulin resistance. In vitro study, C2C12 myotubes were used to determine the potential mechanism by overexpressing or inhibiting SESN2. Similar to exercise, six-week gAd administration decreased fasting glucose, triglyceride and insulin levels, reduced lipid deposition in skeletal muscle and reversed whole-body insulin resistance in mice fed on a high-fat diet. Moreover, gAd enhanced skeletal muscle glucose uptake by activating insulin signaling. However, these effects were diminished in SESN2-/- mice. We found that gAd administration increased the expression of SESN2 and Liver kinase B1 (LKB1) and increased AMPK-T172 phosphorylation in skeletal muscle of wild-type mice, while in SESN2-/- mice, LKB1 expression was also increased but the pAMPK-T172 was unchanged. At the cellular level, gAd increased cellular SESN2 and pAMPK-T172 expression. Immunoprecipitation experiment suggested that SESN2 promoted the formation of complexes of AMPK and LKB1 and hence phosphorylated AMPK. In conclusion, our results revealed that SESN2 played a critical role in gAd-induced AMPK phosphorylation, activation of insulin signaling and skeletal muscle insulin sensitization in mice with insulin resistance.

Keywords:  AMP-Activated protein kinase; Adiponectin; Exercise training; Glucose homeostasis; Sestrins

DOI:  https://doi.org/10.1016/j.smhs.2022.08.001
Sports Med Health Sci. 2023 Mar;5(1): 10-19

The skeletal muscle fiber periphery: A nexus of mTOR-related anabolism.

Cassidy T Tinline-Goodfellow, Matthew J Lees, Nathan Hodson.

  Skeletal muscle anabolism is driven by numerous stimuli such as growth factors, nutrients (i.e., amino acids, glucose), and mechanical stress. These stimuli are integrated by the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signal transduction cascade. In recent years, work from our laboratory and elsewhere has sought to unravel the molecular mechanisms underpinning the mTOR-related activation of muscle protein synthesis (MPS), as well as the spatial regulation of these mechanisms within the skeletal muscle cell. These studies have suggested that the skeletal muscle fiber periphery is a region of central importance in anabolism (i.e., growth/MPS). Indeed, the fiber periphery is replete with the substrates, molecular machinery, and translational apparatus necessary to facilitate MPS. This review provides a summary of the mechanisms underpinning the mTOR-associated activation of MPS from cell, rodent, and human studies. It also presents an overview of the spatial regulation of mTORC1 in response to anabolic stimuli and outlines the factors that distinguish the periphery of the cell as a highly notable region of skeletal muscle for the induction of MPS. Future research should seek to further explore the nutrient-induced activation of mTORC1 at the periphery of skeletal muscle fibers.

Keywords:  Hypertrophy; Muscle protein synthesis; Periphery; Skeletal muscle; Translation; mTOR

DOI:  https://doi.org/10.1016/j.smhs.2022.11.004
J Physiol. 2023 Mar 31.

Undercarboxylated osteocalcin and ibandronate combination ameliorates hindlimb immobilization-induced muscle wasting.

Xuzhu Lin, Cassandra Smith, Alba Moreno-Asso, Navabeh Zarekookandeh, Tara C Brennan-Speranza, Gustavo Duque, Alan Hayes, Itamar Levinger.

  Immobilization leads to muscle wasting and insulin resistance, particularly during aging. Undercarboxylated osteocalcin (ucOC) has been suggested to improve muscle mass and glucose metabolism. Bisphosphonates, an anti-osteoporosis treatment, might protect muscle wasting independent of ucOC. We hypothesize that the combination of ucOC and ibandronate (IBN) treatments has superior protective effects against immobilization-induced muscle wasting and insulin resistance than either treatment alone. C57BL/6J mice were hindlimb-immobilized for two weeks, with injections of vehicle, ucOC (90 ng/g daily) and/or IBN (2 μg/g weekly). Insulin/oral glucose tolerance tests (ITT/OGTT) were performed. Immediately after immobilization, muscles (extensor digitorum longus [EDL], soleus, tibialis anterior [TA], gastrocnemius, and quadriceps) were isolated and measured for muscle mass. Insulin-stimulated glucose uptake (EDL and soleus) was examined. Phosphorylation/expression of proteins in anabolic/catabolic pathways were examined in quadriceps. Primary human myotubes derived from older adult muscle biopsies were treated with ucOC and/or IBN, then signaling proteins were analyzed. Combined treatment, but not individual treatments, significantly increased muscle weight/body weight ratio in immobilized soleus (31.7 %; p = 0.013) and quadriceps (20.0 %; p = 0.0008) muscles, concomitant with elevated p-Akt (S473)/Akt ratio (p = 0.0047). Combined treatment also enhanced whole-body glucose tolerance (16.6 %; p = 0.0011). In human myotubes, combined treatment stimulated greater activation of ERK1/2 (p = 0.0067 and 0.0072) and mTOR (p = 0.036), and led to a lesser expression of Fbx32 (p = 0.049) and MuRF1 (p = 0.048), than individual treatments. These findings suggest a potential therapeutic role of ucOC and bisphosphonates combination in protecting against muscle wasting induced by immobilization and aging. KEY POINTS: Undercarboxylated osteocalcin (ucOC) has been suggested to improve muscle mass and glucose metabolism. Bisphosphonates, an anti-osteoporosis treatment, might protect muscle wasting independent of ucOC. The combination treatment of ucOC and ibandronate was shown to exert greater therapeutic effect against immobilization-induced muscle wasting, and lead to greater activation of anabolic pathway and less expression of catabolic signaling proteins in myotubes derived from older adults, compared to individual treatments. The combination treatment was found to improve whole-body glucose tolerance. Our findings suggest a potential therapeutic role of ucOC and bisphosphonates combination in protecting against muscle wasting induced by immobilization and aging. Abstract figure legend Undercarboxylated osteocalcin (ucOC) and ibandronate (IBN) combination improves muscle mass and glucose disposal. Two weeks of hindlimb immobilization in mice led to leg muscle atrophy as well as muscle insulin resistance. Injections of both undercarboxylated osteocalcin (intraperitoneal [IP]) and ibandronate (subcutaneous [SC]) alleviated muscle wasting in a muscle type-specific manner. In addition, this combination treatment improved glucose disposal in mice. In both immobilized mouse muscle and human primary myotubes derived from older adults, the combination treatment with ucOC and IBN resulted in greater activation of proteins involved in anabolic signaling pathway, including Akt and mTORC1. In primary myotubes, this treatment reduced expression of proteins involved in catabolic signaling pathway, such as Fbx32 and MuRF1. These findings suggest that ucOC and bisphosphonates combination has a potential in treating muscle wasting and insulin resistance induced by immobilization and aging. This article is protected by copyright. All rights reserved.

Keywords:  bisphosphonates; glucose metabolism; hindlimb immobilization; muscle wasting; undercarboxylated osteocalcin

DOI:  https://doi.org/10.1113/JP283990
Cells. 2023 Mar 15. pii: 898. [Epub ahead of print]12(6):

Different Resistance Exercise Loading Paradigms Similarly Affect Skeletal Muscle Gene Expression Patterns of Myostatin-Related Targets and mTORC1 Signaling Markers.

Mason C McIntosh, Casey L Sexton, Joshua S Godwin, Bradley A Ruple, J Max Michel, Daniel L Plotkin, Tim N Ziegenfuss, Hector L Lopez, Ryan Smith, Varun B Dwaraka, Adam P Sharples, Vincent J Dalbo, C Brooks Mobley, Christopher G Vann, Michael D Roberts.

  Although transcriptome profiling has been used in several resistance training studies, the associated analytical approaches seldom provide in-depth information on individual genes linked to skeletal muscle hypertrophy. Therefore, a secondary analysis was performed herein on a muscle transcriptomic dataset we previously published involving trained college-aged men (n = 11) performing two resistance exercise bouts in a randomized and crossover fashion. The lower-load bout (30 Fail) consisted of 8 sets of lower body exercises to volitional fatigue using 30% one-repetition maximum (1 RM) loads, whereas the higher-load bout (80 Fail) consisted of the same exercises using 80% 1 RM loads. Vastus lateralis muscle biopsies were collected prior to (PRE), 3 h, and 6 h after each exercise bout, and 58 genes associated with skeletal muscle hypertrophy were manually interrogated from our prior microarray data. Select targets were further interrogated for associated protein expression and phosphorylation induced-signaling events. Although none of the 58 gene targets demonstrated significant bout x time interactions, ~57% (32 genes) showed a significant main effect of time from PRE to 3 h (15↑ and 17↓, p < 0.01), and ~26% (17 genes) showed a significant main effect of time from PRE to 6 h (8↑ and 9↓, p < 0.01). Notably, genes associated with the myostatin (9 genes) and mammalian target of rapamycin complex 1 (mTORC1) (9 genes) signaling pathways were most represented. Compared to mTORC1 signaling mRNAs, more MSTN signaling-related mRNAs (7 of 9) were altered post-exercise, regardless of the bout, and RHEB was the only mTORC1-associated mRNA that was upregulated following exercise. Phosphorylated (phospho-) p70S6K (Thr389) (p = 0.001; PRE to 3 h) and follistatin protein levels (p = 0.021; PRE to 6 h) increased post-exercise, regardless of the bout, whereas phospho-AKT (Thr389), phospho-mTOR (Ser2448), and myostatin protein levels remained unaltered. These data continue to suggest that performing resistance exercise to volitional fatigue, regardless of load selection, elicits similar transient mRNA and signaling responses in skeletal muscle. Moreover, these data provide further evidence that the transcriptional regulation of myostatin signaling is an involved mechanism in response to resistance exercise.

Keywords:  acute resistance exercise; gene expression; mRNA; protein

DOI:  https://doi.org/10.3390/cells12060898
J Appl Physiol (1985). 2023 Mar 30.

Proteome profiles of skeletal muscle connective tissue: Influence of aging and physical training.

Ching-Yan Chloé Yeung, Annesofie T Olesen, Richard Wilson, Shireen R Lamandé, John F Bateman, René B Svensson, S Peter Magnusson, Michael Kjaer.

  Both aging and physical activity can influence the amount of connective tissue in skeletal muscle, but the impact of these upon specific extracellular matrix (ECM) proteins in skeletal muscle is unknown. We investigated the proteome profile of connective tissue in skeletal muscle by label-free proteomic analysis of cellular protein-depleted extracts from lateral gastrocnemius muscle of old (22-23 months old) and middle-aged mice (11 months old) subjected to three different levels of regular physical activity for 10 weeks (high resistance wheel running, low resistance wheel running or sedentary controls). We hypothesized that aging is correlated with an increased amount of connective tissue proteins in skeletal muscle, and that regular physical activity can counteract these age-related changes. We found that dominating cellular proteins were diminished in the urea/thiourea extract, which was therefore used for proteomics. Proteomic analysis identified 482 proteins and showed enrichment for ECM proteins. Statistical analysis revealed that the abundances of 86 proteins were changed with age. Twenty-three of these differentially abundant proteins were identified as structural ECM proteins (e.g., collagens and laminins) and all of these were significantly more abundant with aging. No significant effect of training or interaction between training and advance in age was found for any proteins. Finally, we found a lower protein concentration in the urea/thiourea extracts from the old compared to middle-aged mice. By identifying the ECM proteome profiles of skeletal muscle connective tissue, the findings indicate that intramuscular connective tissue alters its soluble protein content with age but is unaffected by training.

Keywords:  aging; exercise; extracellular matrix; proteomics; skeletal muscle

DOI:  https://doi.org/10.1152/japplphysiol.00675.2022
Methods Mol Biol. 2023 ;2640 287-311

In Vivo Investigation of Gene Function in Muscle Stem Cells by CRISPR/Cas9-Mediated Genome Editing.

Liangqiang He, Zhiming He, Yuying Li, Hao Sun, Huating Wang.

  Skeletal muscle satellite cells (SCs) are adult stem cells responsible for muscle development and injury-induced muscle regeneration. Functional elucidation of intrinsic regulatory factors governing SC activity is constrained partially by the technological limitations in editing SCs in vivo. Although the power of CRISPR/Cas9 in genome manipulation has been widely documented, its application in endogenous SCs remains largely untested. Our recent study generates a muscle-specific genome editing system leveraging the Cre-dependent Cas9 knockin mice and AAV9-mediated sgRNAs delivery, which allows gene disruption in SCs in vivo. Here, we illustrate the step-by-step procedure for achieving efficient editing using the above system.

Keywords:  Adeno-associated virus; CRISPR/Cas9; Genome editing; Muscle satellite cell; sgRNA

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_21
Sports Med Health Sci. 2023 Mar;5(1): 20-28

Skeletal muscle mechanisms contributing to improved glycemic control following intense interval exercise and training.

Hashim Islam, Jenna B Gillen.

  High-intensity and sprint interval training (HIIT and SIT, respectively) enhance insulin sensitivity and glycemic control in both healthy adults and those with cardiometabolic diseases. The beneficial effects of intense interval training on glycemic control include both improvements seen in the hours to days following a single session of HIIT/SIT and those which accrue with chronic training. Skeletal muscle is the largest site of insulin-stimulated glucose uptake and plays an integral role in the beneficial effects of exercise on glycemic control. Here we summarize the skeletal muscle responses that contribute to improved glycemic control during and following a single session of interval exercise and evaluate the relationship between skeletal muscle remodelling and improved insulin sensitivity following HIIT/SIT training interventions. Recent evidence suggests that targeting skeletal muscle mechanisms via nutritional interventions around exercise, particularly with carbohydrate manipulation, can enhance the acute glycemic benefits of HIIT. There is also some evidence of sex-based differences in the glycemic benefits of intense interval exercise, with blunted responses observed after training in females relative to males. Differences in skeletal muscle metabolism between males and females may contribute to sex differences in insulin sensitivity following HIIT/SIT, but well-controlled studies evaluating purported muscle mechanisms alongside measurement of insulin sensitivity are needed. Given the greater representation of males in muscle physiology literature, there is also a need for more research involving female-only cohorts to enhance our basic understanding of how intense interval training influences muscle insulin sensitivity in females across the lifespan.

Keywords:  HIIT; Insulin sensitivity; Nutrition; Sex differences; Type 2 diabetes

DOI:  https://doi.org/10.1016/j.smhs.2023.01.002
J Clin Invest. 2023 Mar 30. pii: e153837. [Epub ahead of print]

Modulating fast skeletal muscle contraction protects skeletal muscle in animal models of Duchenne muscular dystrophy.

Alan J Russell, Mike DuVall, Benjamin Barthel, Ying Qian, Angela K Peter, Breanne L Newell-Stamper, Kevin Hunt, Sarah J Lehman, Molly R Madden, Stephen T Schlachter, Benjamin D Robertson, Ashleigh Van Deusen, Hector M Rodriguez, Carlos D Vera, Yu Su, Dennis R Claflin, Susan V Brooks, Peter P Nghiem, Alexis Rutledge, Twlya I Juehne, Jinsheng Yu, Elisabeth R Barton, Yangyi E Luo, Andreas Patsalos, Laszlo Nagy, H Lee Sweeney, Leslie A Leinwand, Kevin Koch.

  Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes from mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, the relationship between how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.

Keywords:  Muscle Biology; Neuromuscular disease; Skeletal muscle; Therapeutics

DOI:  https://doi.org/10.1172/JCI153837
Int J Mol Sci. 2023 Mar 19. pii: 5847. [Epub ahead of print]24(6):

Palmitic Acid Inhibits Myogenic Activity and Expression of Myosin Heavy Chain MHC IIb in Muscle Cells through Phosphorylation-Dependent MyoD Inactivation.

Izumi Matsuba, Rikako Fujita, Kaoruko Iida.

  Sarcopenia associated with aging and obesity is characterized by the atrophy of fast-twitch muscle fibers and an increase in intramuscular fat deposits. However, the mechanism of fast-twitch fiber-specific atrophy remains unclear. In this study, we aimed to assess the effect of palmitic acid (PA), the most common fatty acid component of human fat, on muscle fiber type, focusing on the expression of fiber-type-specific myosin heavy chain (MHC). Myotubes differentiated from C2C12 myoblasts were treated with PA. The PA treatment inhibited myotube formation and hypertrophy while reducing the gene expression of MHC IIb and IIx, specific isoforms of fast-twitch fibers. Consistent with this, a significant suppression of MHC IIb protein expression in PA-treated cells was observed. A reporter assay using plasmids containing the MHC IIb gene promoter revealed that the PA-induced reduction in MHC IIb gene expression was caused by the suppression of MyoD transcriptional activity through its phosphorylation. Treatment with a specific protein kinase C (PKC) inhibitor recovered the reduction in MHC IIb gene expression levels in PA-treated cells, suggesting the involvement of the PA-induced activation of PKC. Thus, PA selectively suppresses the mRNA and protein expression of fast-twitch MHC by modulating MyoD activity. This finding provides a potential pathogenic mechanism for age-related sarcopenia.

Keywords:  MyoD; myosin heavy chain; palmitic acid; protein kinase C; skeletal muscle

DOI:  https://doi.org/10.3390/ijms24065847
Methods Mol Biol. 2023 ;2640 73-88

Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Muscle Stem Cells Using a Seahorse Analyzer: Applicability for Aging Studies.

Xiaotong Hong, Pura Muñoz-Cánoves.

  In recent years, evidence showing metabolism as a fundamental regulator of stem cell functions has emerged. In skeletal muscle, its stem cells (satellite cells) sustain muscle regeneration, although they lose their regenerative potential with aging, and this has been attributed, at least in part, to changes in their metabolism. In this chapter, we describe a protocol to analyze the metabolism of satellite cells using the Seahorse technology, which can be applied to aging mice.

Keywords:  Aging; Extracellular Acidification rate; Metabolism; Oxygen consumption rate; Satellite cell; Seahorse Technology; Skeletal muscle; Stem cells; Tissue regeneration

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_6
Methods Mol Biol. 2023 ;2640 227-248

Skeletal Muscle Regeneration in Zebrafish.

Tapan G Pipalia, Sami H A Sultan, Jana Koth, Robert D Knight, Simon M Hughes.

  Muscle regeneration models have revealed mechanisms of inflammation, wound clearance, and stem cell-directed repair of damage, thereby informing therapy. Whereas studies of muscle repair are most advanced in rodents, the zebrafish is emerging as an additional model organism with genetic and optical advantages. Various muscle wounding protocols (both chemical and physical) have been published. Here we describe simple, cheap, precise, adaptable, and effective wounding protocols and analysis methods for two stages of a larval zebrafish skeletal muscle regeneration model. We show examples of how muscle damage, ingression of muscle stem cells, immune cells, and regeneration of fibers can be monitored over an extended timecourse in individual larvae. Such analyses have the potential to greatly enhance understanding, by reducing the need to average regeneration responses across individuals subjected to an unavoidably variable wound stimulus.

Keywords:  Confocal live imaging; Laser injury; Multiphoton microscopy; Muscle precursor cells; Muscle regeneration; Muscle wounding; Needlestick injury; Pax7; Satellite cell; Second harmonic generation; Spinning disk imaging; Zebrafish muscle

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_17
Methods Mol Biol. 2023 ;2640 339-349

Functional Analysis of MicroRNAs in Skeletal Muscle.

Satoshi Oikawa, Takayuki Akimoto.

  MicroRNAs (miRNAs) are small non-coding RNAs that are highly conserved in vertebrates and play important roles in diverse biological processes. miRNAs function to fine-tune gene expression by accelerating the degradation of mRNA and/or by inhibiting protein translation. Identification of muscle-specific miRNAs has extended our knowledge of the molecular network in skeletal muscle. Here we describe methods that are commonly used to analyze the function of miRNAs in skeletal muscle.

Keywords:  Mature microRNAs; Precursor microRNAs; Primary microRNAs; Real-time PCR; Reporter assay; Skeletal muscle

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_24
Methods Mol Biol. 2023 ;2640 193-205

Techniques for Injury, Cell Transplantation, and Histological Analysis in Skeletal Muscle.

Norio Motohashi, Katsura Minegishi, Michihiro Imamura, Yoshitsugu Aoki.

  Skeletal muscle can adjust to changes in physiological and pathological environments by regenerating using myogenic progenitor cells or adapting muscle fiber sizes and types, metabolism, and contraction ability. To study these changes, muscle samples should be appropriately prepared. Therefore, reliable techniques to accurately analyze and evaluate skeletal muscle phenotypes are required. However, although technical approaches to genetically investigating skeletal muscle are improving, the fundamental strategies for capturing muscle pathology are the same over the decades. Hematoxylin and eosin (H&E) staining or antibodies are the simplest and standard methodologies for assessing skeletal muscle phenotypes. In this chapter, we describe fundamental techniques and protocols for inducing skeletal muscle regeneration by using chemicals and cell transplantation, in addition to methods of preparing and evaluating skeletal muscle samples.

Keywords:  Cell transplantation; Dystrophin; Muscle regeneration; Myogenic cells; mdx

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_14
Methods Mol Biol. 2023 ;2640 207-215

Murine Models of Tenotomy-Induced Mechanical Overloading and Tail-Suspension-Induced Mechanical Unloading.

Shin Fujimaki, Yusuke Ono.

  Skeletal muscle is a highly plastic tissue that can alter its mass and strength in response to mechanical stimulation, such as overloading and unloading, which lead to muscle hypertrophy and atrophy, respectively. Mechanical loading in the muscle influences muscle stem cell dynamics, including activation, proliferation, and differentiation. Although experimental models of mechanical overloading and unloading have been widely used for the investigation of the molecular mechanisms regulating muscle plasticity and stem cell function, few studies have described the methods in detail. Here, we describe the appropriate procedures for tenotomy-induced mechanical overloading and tail-suspension-induced mechanical unloading, which are the most common and simple methods to induce muscle hypertrophy and atrophy in mouse models.

Keywords:  Mechanical loading; Muscle atrophy; Muscle hypertrophy; Muscle plasticity; Tail suspension; Tenotomy

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_15
Stem Cell Rev Rep. 2023 Mar 31.

Static Magnetic Fields Promote Generation of Muscle Lineage Cells from Pluripotent Stem Cells and Myoblasts.

Zhaoyuan Guo, Jiahao Zhu, Guanyu Qin, Yumei Jia, Zheng Liu, Na Yang, Renpeng Guo.

  Static magnetic fields (SMFs) exhibit numerous biological effects and regulate the proliferation and differentiation of several adult stem cells. However, the role of SMFs in the self-renewal maintenance and developmental potential of pluripotent embryonic stem cells (ESCs) remains largely uninvestigated. Here, we show that SMFs promote the expression of the core pluripotent markers Sox2 and SSEA-1. Furthermore, SMFs facilitate the differentiation of ESCs into cardiomyocytes and skeletal muscle cells. Consistently, transcriptome analysis reveals that muscle lineage differentiation and skeletal system specification of ESCs are remarkably strengthened by SMF stimuli. Additionally, when treated with SMFs, C2C12 myoblasts exhibit an increased proliferation rate, improved expression of skeletal muscle markers and elevated myogenic differentiation capacity compared with control cells. Together, our data show that SMFs effectively promote muscle cell generation from pluripotent stem cells and myoblasts. The noninvasive and convenient physical stimuli can be used to increase the production of muscle cells in regenerative medicine and the manufacture of cultured meat in cellular agriculture.

Keywords:  C2C12 myoblasts; ESCs; Myogenic differentiation; SMFs

DOI:  https://doi.org/10.1007/s12015-023-10535-z
Antioxidants (Basel). 2023 Feb 28. pii: 598. [Epub ahead of print]12(3):

The Role of Lonp1 on Mitochondrial Functions during Cardiovascular and Muscular Diseases.

Giada Zanini, Valentina Selleri, Mara Malerba, Kateryna Solodka, Giorgia Sinigaglia, Milena Nasi, Anna Vittoria Mattioli, Marcello Pinti.

  The mitochondrial protease Lonp1 is a multifunctional enzyme that regulates crucial mitochondrial functions, including the degradation of oxidized proteins, folding of imported proteins and maintenance the correct number of copies of mitochondrial DNA. A series of recent studies has put Lonp1 at the center of the stage in the homeostasis of cardiomyocytes and muscle skeletal cells. During heart development, Lonp1 allows the metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation. Knock out of Lonp1 arrests heart development and determines cardiomyocyte apoptosis. In adults, Lonp1 acts as a cardioprotective protein, as its upregulation mitigates cardiac injury by preventing the oxidative damage of proteins and lipids, and by preserving mitochondrial redox balance. In skeletal muscle, Lonp1 is crucial for cell development, as it mediates the activation of PINK1/Parkin pathway needed for proper myoblast differentiation. Skeletal muscle-specific ablation of Lonp1 in mice causes reduced muscle fiber size and strength due to the accumulation of mitochondrial-retained protein in muscle. Lonp1 expression and activity decline with age in different tissues, including skeletal muscle, and are associated with a functional decline and structural impairment of muscle fibers. Aerobic exercise increases unfolded protein response markers including Lonp1 in the skeletal muscle of aged animals and is associated with muscle functional recovery. Finally, mutations of Lonp1 cause a syndrome named CODAS (Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies) characterized by the impaired development of multiple organs and tissues, including myocytes. CODAS patients show hypotonia and ptosis, indicative of skeletal muscle reduced performance. Overall, this body of observations points Lonp1 as a crucial regulator of mitochondrial functions in the heart and in skeletal muscle.

Keywords:  Lon protease; UPRmt; heart dysfunction; sarcopenia

DOI:  https://doi.org/10.3390/antiox12030598
Int J Mol Sci. 2023 Mar 15. pii: 5585. [Epub ahead of print]24(6):

Denervation Drives YAP/TAZ Activation in Muscular Fibro/Adipogenic Progenitors.

Felipe S Gallardo, Adriana Córdova-Casanova, Alexia Bock-Pereda, Daniela L Rebolledo, Andrea Ravasio, Juan Carlos Casar, Enrique Brandan.

  Loss of motoneuron innervation (denervation) is a hallmark of neurodegeneration and aging of the skeletal muscle. Denervation induces fibrosis, a response attributed to the activation and expansion of resident fibro/adipogenic progenitors (FAPs), i.e., multipotent stromal cells with myofibroblast potential. Using in vivo and in silico approaches, we revealed FAPs as a novel cell population that activates the transcriptional coregulators YAP/TAZ in response to skeletal muscle denervation. Here, we found that denervation induces the expression and transcriptional activity of YAP/TAZ in whole muscle lysates. Using the PdgfraH2B:EGFP/+ transgenic reporter mice to trace FAPs, we demonstrated that denervation leads to increased YAP expression that accumulates within FAPs nuclei. Consistently, re-analysis of published single-nucleus RNA sequencing (snRNA-seq) data indicates that FAPs from denervated muscles have a higher YAP/TAZ signature level than control FAPs. Thus, our work provides the foundations to address the functional role of YAP/TAZ in FAPs in a neurogenic pathological context, which could be applied to develop novel therapeutic approaches for the treatment of muscle disorders triggered by motoneuron degeneration.

Keywords:  FAPs; YAP/TAZ; denervation; fibrosis; skeletal muscle

DOI:  https://doi.org/10.3390/ijms24065585
Cells. 2023 Mar 16. pii: 920. [Epub ahead of print]12(6):

AMPK Phosphorylation Impacts Apoptosis in Differentiating Myoblasts Isolated from Atrophied Rat Soleus Muscle.

Natalia A Vilchinskaya, Sergey V Rozhkov, Olga V Turtikova, Timur M Mirzoev, Boris S Shenkman.

  Regrowth of atrophied myofibers depends on muscle satellite cells (SCs) that exist outside the plasma membrane. Muscle atrophy appears to result in reduced number of SCs due to apoptosis. Given reduced AMP-activated protein kinase (AMPK) activity during differentiation of primary myoblasts derived from atrophic muscle, we hypothesized that there may be a potential link between AMPK and susceptibility of differentiating myoblasts to apoptosis. The aim of this study was to estimate the effect of AMPK activation (via AICAR treatment) on apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle. Thirty rats were randomly assigned to the following two groups: control (C, n = 10) and 7-day hindlimb suspension (HS, n = 20). Myoblasts derived from the soleus muscles of HS rats were divided into two parts: AICAR-treated cells and non-treated cells. Apoptotic processes were evaluated by using TUNEL assay, RT-PCR and WB. In differentiating myoblasts derived from the atrophied soleus, there was a significant decrease (p < 0.05) in AMPK and ACC phosphorylation in parallel with increased number of apoptotic nuclei and a significant upregulation of pro-apoptotic markers (caspase-3, -9, BAX, p53) compared to the cells derived from control muscles. AICAR treatment of atrophic muscle-derived myoblasts during differentiation prevented reductions in AMPK and ACC phosphorylation as well as maintained the number of apoptotic nuclei and the expression of pro-apoptotic markers at the control levels. Thus, the maintenance of AMPK activity can suppress enhanced apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle.

Keywords:  AICAR; AMPK; TUNEL; apoptosis; caspase-3; hindlimb suspension; primary myoblasts

DOI:  https://doi.org/10.3390/cells12060920
PLoS One. 2023 ;18(3): e0283869

Multiomic characterization of disease progression in mice lacking dystrophin.

Mirko Signorelli, Roula Tsonaka, Annemieke Aartsma-Rus, Pietro Spitali.

Duchenne muscular dystrophy (DMD) is caused by genetic mutations leading to lack of dystrophin in skeletal muscle. A better understanding of how objective biomarkers for DMD vary across subjects and over time is needed to model disease progression and response to therapy more effectively, both in pre-clinical and clinical research. We present an in-depth characterization of disease progression in 3 murine models of DMD by multiomic analysis of longitudinal trajectories between 6 and 30 weeks of age. Integration of RNA-seq, mass spectrometry-based metabolomic and lipidomic data obtained in muscle and blood samples by Multi-Omics Factor Analysis (MOFA) led to the identification of 8 latent factors that explained 78.8% of the variance in the multiomic dataset. Latent factors could discriminate dystrophic and healthy mice, as well as different time-points. MOFA enabled to connect the gene expression signature in dystrophic muscles, characterized by pro-fibrotic and energy metabolism alterations, to inflammation and lipid signatures in blood. Our results show that omic observations in blood can be directly related to skeletal muscle pathology in dystrophic muscle.

DOI: https://doi.org/10.1371/journal.pone.0283869
Life Sci Alliance. 2023 Jun;pii: e202201868. [Epub ahead of print]6(6):

The role of Limch1 alternative splicing in skeletal muscle function.

Matthew S Penna, Rong-Chi Hu, George G Rodney, Thomas A Cooper.

Postnatal skeletal muscle development is a highly dynamic period associated with widespread alternative splicing changes required to adapt tissues to adult function. These splicing events have significant implications because the reversion of adult mRNA isoforms to fetal isoforms is observed in forms of muscular dystrophy. LIMCH1 is a stress fiber-associated protein that is alternatively spliced to generate uLIMCH1, a ubiquitously expressed isoform, and mLIMCH1, a skeletal muscle-specific isoform containing six additional exons simultaneously included after birth in the mouse. CRISPR/Cas9 was used to delete the six alternatively spliced exons of LIMCH1 in mice, thereby forcing the constitutive expression of the predominantly fetal isoform, uLIMCH1. mLIMCH1 knockout mice had significant grip strength weakness in vivo, and maximum force generated was decreased ex vivo. Calcium-handling deficits were observed during myofiber stimulation that could explain the mechanism by which mLIMCH1 knockout leads to muscle weakness. In addition, LIMCH1 is mis-spliced in myotonic dystrophy type 1, with the muscleblind-like (MBNL) family of proteins acting as the likely major regulator of Limch1 alternative splicing in skeletal muscle.

DOI: https://doi.org/10.26508/lsa.202201868
Am J Physiol Endocrinol Metab. 2023 Mar 29.

Defective Muscle Ketone Body Oxidation Disrupts BCAA Catabolism by Altering Mitochondrial Branched-Chain Aminotransferase.

Hamza Mechchate, Abdualrahman Mohammed Abdualkader, James Bradshaw Bernacchi, Keshav Gopal, S Amirhossein Tabatabaei Dakhili, Kunyan Yang, Amanda A Greenwell, Xingxing Kong, Peter A Crawford, Rami Al Batran.

  Ketone bodies are an endogenous fuel source generated primarily by the liver to provide alternative energy for extrahepatic tissues during prolonged fasting and exercise. Skeletal muscle is an important site of ketone body oxidation which occurs through a series of reactions requiring the enzyme succinyl-CoA:3-ketoacid-CoA transferase (SCOT/Oxct1). We have previously shown that deleting SCOT in the skeletal muscle protects against obesity-induced insulin resistance by increasing pyruvate dehydrogenase (PDH) activity, the rate-limiting enzyme of glucose oxidation. However, it remains unclear whether inhibiting muscle ketone body oxidation causes hypoglycemia and affects fuel metabolism in the absence of obesity. Here, we show that lean mice lacking skeletal muscle SCOT (SCOTSkM-/-) exhibited no overt phenotypic differences in glucose and fat metabolism from their human α-skeletal actin-Cre (HSACre) littermates. Of interest, we found that plasma and muscle branched-chain amino acid (BCAA) levels are elevated in SCOTSkM-/- lean mice compared to their HSACre littermates. Interestingly, this alteration in BCAA catabolism was only seen in SCOTSkM-/- mice under low-fat feeding and associated with decreased expression of mitochondrial branched-chain aminotransferases (BCATm/Bcat2), the first enzyme in BCAA catabolic pathway. Loss- and gain-of-function studies in C2C12 myotubes demonstrated that suppressing SCOT markedly diminished BCATm expression, whereas overexpressing SCOT resulted in an opposite effect without influencing BCAA oxidation enzymes. Further, SCOT overexpression in C2C12 myotubes significantly increased luciferase activity driven by a Bcat2 promoter construct. Together, our findings indicate that SCOT regulates the expression of the Bcat2 gene, which, through the abundance of its product BCATm, may influence circulating BCAA concentrations.

Keywords:  BCAAs; BCATm; Ketone body; SCOT; Skeletal Muscle

DOI:  https://doi.org/10.1152/ajpendo.00206.2022
Methods Mol Biol. 2023 ;2640 431-443

Epitranscriptome Mapping of N6-Methyladenosine Using m6A Immunoprecipitation with High Throughput Sequencing in Skeletal Muscle Stem Cells.

Justin Law, Stefan Günther, Shuichi Watanabe.

  N6-Methyladenosine (m6A), one of the most abundant chemical modifications in mRNA (epitranscriptome), contributes to the regulation of biological processes by iterating gene expression post-transcriptionally. A number of publications on m6A modification have escalated in the recent past, due to the advancements in profiling m6A along the transcriptome using different approaches. The vast majority of studies primarily focused on m6A modification on cell lines but not primary cells. We present in this chapter a protocol for m6A immunoprecipitation with high throughput sequencing (MeRIP-Seq) that profiles m6A on mRNA with merely 100 μg total RNA worth of muscle stem cells as starting material. With this MeRIP-Seq, we observed epitranscriptome landscape in muscle stem cells.

Keywords:  Epitranscriptome; Immunoprecipitation; Mettl3/14; RNA metabolism; YTH RNA binding proteins; m6A

DOI:  https://doi.org/10.1007/978-1-0716-3036-5_29
Biology (Basel). 2023 Mar 10. pii: 422. [Epub ahead of print]12(3):

Uncovering the Gene Regulatory Network of Endothelial Cells in Mouse Duchenne Muscular Dystrophy: Insights from Single-Nuclei RNA Sequencing Analysis.

Yan Shen, Il-Man Kim, Mark Hamrick, Yaoliang Tang.

  INTRODUCTION: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disorder caused by mutations in the dystrophin gene, which leads to heart and respiratory failure. Despite the critical impact of DMD on endothelial cells (ECs), there is limited understanding of its effect on the endothelial gene network. The aim of this study was to investigate the impact of DMD on the gene regulatory network of ECs.METHODS AND RESULTS: To gain insights into the role of the dystrophin muscular dystrophy gene (DMD) in ECs from Duchenne muscular dystrophy; the study utilized single-nuclei RNA sequencing (snRNA-seq) to evaluate the transcriptomic profile of ECs from skeletal muscles in DMD mutant mice (DMDmut) and wild-type control mice. The analysis showed that the DMD mutation resulted in the suppression of several genes, including SPTBN1 and the upregulation of multiple long noncoding RNAs (lncRNAs). GM48099, GM19951, and GM15564 were consistently upregulated in ECs and skeletal muscle cells from DMDmut, indicating that these dysregulated lncRNAs are conserved across different cell types. Gene ontology (GO) enrichment analysis revealed that the DMD mutation activated the following four pathways in ECs: fibrillary collagen trimer, banded collagen fibril, complex of collagen trimers, and purine nucleotide metabolism. The study also found that the metabolic pathway activity of ECs was altered. Oxidative phosphorylation (OXPHOS), fatty acid degradation, glycolysis, and pyruvate metabolism were decreased while purine metabolism, pyrimidine metabolism, and one carbon pool by folate were increased. Moreover, the study investigated the impact of the DMD mutation on ECs from skeletal muscles and found a significant decrease in their overall number, but no change in their proliferation.
CONCLUSIONS: Overall, this study provides new insights into the gene regulatory program in ECs in DMD and highlights the importance of further research in this area.

Keywords:  Duchenne muscular dystrophy; cell metabolism; dystrophin; single-nuclear RNA sequencing (snRNA-seq); skeletal muscle-derived endothelial cells

DOI:  https://doi.org/10.3390/biology12030422
Sports Med Health Sci. 2023 Mar;5(1): 29-33

The role of glycogen phosphorylase in glycogen biogenesis in skeletal muscle after exercise.

Abram Katz.

  Initially it was believed that phosphorylase was responsible for both glycogen breakdown and synthesis in the living cell. The discovery of glycogen synthase and McArdle's disease (lack of phosphorylase activity), together with the high Pi/glucose 1-P ratio in skeletal muscle, demonstrated that glycogen synthesis could not be attributed to reversal of the phosphorylase reaction. Rather, glycogen synthesis was attributable solely to the activity of glycogen synthase, subsequent to the transport of glucose into the cell. However, the well-established observation that phosphorylase was inactivated (i.e., dephosphorylated) during the initial recovery period after prior exercise, when the rate of glycogen accumulation is highest and independent of insulin, suggested that phosphorylase could play an active role in glycogen accumulation. But the quantitative contribution of phosphorylase inactivation was not established until recently, when studying isolated murine muscle preparations during recovery from repeated contractions at temperatures ranging from 25 to 35 °C. Thus, in both slow-twitch, oxidative and fast-twitch, glycolytic muscles, inactivation of phosphorylase accounted for 45%-75% of glycogen accumulation during the initial hours of recovery following repeated contractions. Such data indicate that phosphorylase inactivation may be the most important mechanism for glycogen accumulation under defined conditions. These results support the initial belief that phosphorylase plays a quantitative role in glycogen formation in the living cell. However, the mechanism is not via activation of phosphorylase, but rather via inactivation of the enzyme.

Keywords:  Exercise; Glycogen; Glycogen synthase; Muscle; Phosphorylase

DOI:  https://doi.org/10.1016/j.smhs.2022.11.001
Nat Commun. 2023 Mar 30. 14(1): 1785

Defective BVES-mediated feedback control of cAMP in muscular dystrophy.

Haiwen Li, Peipei Wang, Chen Zhang, Yuanbojiao Zuo, Yuan Zhou, Renzhi Han.

Biological processes incorporate feedback mechanisms to enable positive and/or negative regulation. cAMP is an important second messenger involved in many aspects of muscle biology. However, the feedback mechanisms for the cAMP signaling control in skeletal muscle are largely unknown. Here we show that blood vessel epicardial substance (BVES) is a negative regulator of adenylyl cyclase 9 (ADCY9)-mediated cAMP signaling involved in maintaining muscle mass and function. BVES deletion in mice reduces muscle mass and impairs muscle performance, whereas virally delivered BVES expressed in Bves-deficient skeletal muscle reverses these defects. BVES interacts with and negatively regulates ADCY9's activity. Disruption of BVES-mediated control of cAMP signaling leads to an increased protein kinase A (PKA) signaling cascade, thereby promoting FoxO-mediated ubiquitin proteasome degradation and autophagy initiation. Our study reveals that BVES functions as a negative feedback regulator of ADCY9-cAMP signaling in skeletal muscle, playing an important role in maintaining muscle homeostasis.

DOI: https://doi.org/10.1038/s41467-023-37496-8
J Biomech. 2023 Mar 23. pii: S0021-9290(23)00122-7. [Epub ahead of print]152 111553

Stretching the story of titin and muscle function.

Wolfgang A Linke.

  The discovery of the giant protein titin, also known as connectin, dates almost half a century back. In this review, I recapitulate major advances in the discovery of the titin filaments and the recognition of their properties and function until today. I briefly discuss how our understanding of the layout and interactions of titin in muscle sarcomeres has evolved and review key facts about the titin sequence at the gene (TTN) and protein levels. I also touch upon properties of titin important for the stability of the contractile units and the assembly and maintenance of sarcomeric proteins. The greater part of my discussion centers around the mechanical function of titin in skeletal muscle. I cover milestones of research on titin's role in stretch-dependent passive tension development, recollect the reasons behind the enormous elastic diversity of titin, and provide an update on the molecular mechanisms of titin elasticity, details of which are emerging even now. I reflect on current knowledge of how muscle fibers behave mechanically if titin stiffness is removed and how titin stiffness can be dynamically regulated, such as by posttranslational modifications or calcium binding. Finally, I highlight novel and exciting, but still controversially discussed, insight into the role titin plays in active tension development, such as length-dependent activation and contraction from longer muscle lengths.

Keywords:  Elasticity; Muscle mechanics; Passive tension; Sarcomere; Skeletal muscle

DOI:  https://doi.org/10.1016/j.jbiomech.2023.111553
Life Sci. 2023 Mar 23. pii: S0024-3205(23)00253-9. [Epub ahead of print] 121619

Reduced energy metabolism contributing to aging of skeletal muscle by serum metabolomics and gut microbiota analysis.

Pan He, Guanhua Du, Xuemei Qin, Zhenyu Li.

  AIMS: Sarcopenia is an age-related syndrome characterized by a gradual loss of the muscle mass, strength, and function. It is associated with a high risk of adverse consequences such as poorer quality of life, falls, disability and mortality among the elderly. The aim in this study is to investigate the pathological mechanism of sarcopenia.MAIN METHODS: The aging of skeletal muscle was investigated by the D-galactose induced accelerated aging model combining with constrained motion. After 10 weeks, muscle function and gastrocnemius muscle index, and morphology of muscle fibers were evaluated, and myostatin, IGF-1 and ATP in skeletal muscle were also determined. Then the mechanism of aging-related skeletal muscle dysfunctions was investigated based on untargeted serum metabolomics and 16S rRNA gene sequencing. Four key metabolites were validated by the D-galactose-induced C2C12 senescent cell model in vitro.
KEY FINDINGS: Results showed that gastrocnemius muscle mass was decreased significantly, morphology of muscle fibers was altered, and muscle function was damaged in the aged group. Furthermore, increased MSTN, and decreased IGF-1 and ATP were also observed in the aging skeletal muscle. Importantly, alteration of the key pathways including riboflavin biosynthesis and energy metabolism contributed to the aging of skeletal muscle. Four key metabolites, including riboflavin, α-ketoglutaric acid and two dicarboxylic acids, which were involved in these metabolic pathways, could promote the proliferation of C2C12 cells.
SIGNIFICANCE: These findings provide novel insights into pathological mechanism of sarcopenia, and will facilitate the development of therapeutic and preventive strategies for sarcopenia.

Keywords:  D-galactose; Gut microbiota; Mechanism; Metabolomics; Sarcopenia

DOI:  https://doi.org/10.1016/j.lfs.2023.121619
Int J Mol Sci. 2023 Mar 16. pii: 5654. [Epub ahead of print]24(6):

Exercise Induces an Augmented Skeletal Muscle Mitochondrial Unfolded Protein Response in a Mouse Model of Obesity Produced by a High-Fat Diet.

Pía Apablaza, Juan Carlos Bórquez, Rodrigo Mendoza, Mónica Silva, Gladys Tapia, Alejandra Espinosa, Rodrigo Troncoso, Luis A Videla, Nevenka Juretić, Andrea Del Campo.

  Increase in body fat contributes to loss of function and changes in skeletal muscle, accelerating sarcopenia, a phenomenon known as sarco-obesity or sarcopenic obesity. Studies suggest that obesity decreases the skeletal muscle (SM)'s ability to oxidize glucose, increases fatty acid oxidation and reactive oxygen species production, due to mitochondrial dysfunction. Exercise improves mitochondrial dysfunction in obesity; however, it is not known if exercise regulates the mitochondrial unfolded protein response (UPRmt) in the SM. Our study aimed to determine the mito-nuclear UPRmt in response to exercise in a model of obesity, and how this response is associated with the improvement in SM functioning after exercise training. C57BL/6 mice were fed a normal diet and high-fat diet (HFD) for 12 weeks. After 8 weeks, animals were subdivided into sedentary and exercised for the remaining 4 weeks. Grip strength and maximal velocity of mice submitted to HFD improved after training. Our results show an increase in the activation of UPRmt after exercise while in obese mice, proteostasis is basally decreased but shows a more pronounced increase with exercise. These results correlate with improvement in the circulating triglycerides, suggesting mitochondrial proteostasis could be protective and could be related to mitochondrial fuel utilization in SM.

Keywords:  UPRmt; exercise; obesity; skeletal muscle

DOI:  https://doi.org/10.3390/ijms24065654
Cell Death Dis. 2023 Mar 31. 14(3): 231

USP18 is an essential regulator of muscle cell differentiation and maturation.

Cyriel Sebastiaan Olie, Adán Pinto-Fernández, Andreas Damianou, Iolanda Vendrell, Hailiang Mei, Bianca den Hamer, Erik van der Wal, Jessica C de Greef, Vered Raz, Benedikt M Kessler.

The ubiquitin proteasomal system is a critical regulator of muscle physiology, and impaired UPS is key in many muscle pathologies. Yet, little is known about the function of deubiquitinating enzymes (DUBs) in the muscle cell context. We performed a genetic screen to identify DUBs as potential regulators of muscle cell differentiation. Surprisingly, we observed that the depletion of ubiquitin-specific protease 18 (USP18) affected the differentiation of muscle cells. USP18 depletion first stimulated differentiation initiation. Later, during differentiation, the absence of USP18 expression abrogated myotube maintenance. USP18 enzymatic function typically attenuates the immune response by removing interferon-stimulated gene 15 (ISG15) from protein substrates. However, in muscle cells, we found that USP18, predominantly nuclear, regulates differentiation independent of ISG15 and the ISG response. Exploring the pattern of RNA expression profiles and protein networks whose levels depend on USP18 expression, we found that differentiation initiation was concomitant with reduced expression of the cell-cycle gene network and altered expression of myogenic transcription (co) factors. We show that USP18 depletion altered the calcium channel gene network, resulting in reduced calcium flux in myotubes. Additionally, we show that reduced expression of sarcomeric proteins in the USP18 proteome was consistent with reduced contractile force in an engineered muscle model. Our results revealed nuclear USP18 as a critical regulator of differentiation initiation and maintenance, independent of ISG15 and its role in the ISG response.

DOI: https://doi.org/10.1038/s41419-023-05725-z
Proc Natl Acad Sci U S A. 2023 Apr 04. 120(14): e2220102120

NAMPT-dependent NAD+ biosynthesis controls circadian metabolism in a tissue-specific manner.

Astrid L Basse, Karen N Nielsen, Iuliia Karavaeva, Lars R Ingerslev, Tao Ma, Jesper F Havelund, Thomas S Nielsen, Mikkel Frost, Julia Peics, Emilie Dalbram, Morten Dall, Juleen R Zierath, Romain Barrès, Nils J Færgeman, Jonas T Treebak, Zachary Gerhart-Hines.

  Molecular clocks in the periphery coordinate tissue-specific daily biorhythms by integrating input from the hypothalamic master clock and intracellular metabolic signals. One such key metabolic signal is the cellular concentration of NAD+, which oscillates along with its biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT). NAD+ levels feed back into the clock to influence rhythmicity of biological functions, yet whether this metabolic fine-tuning occurs ubiquitously across cell types and is a core clock feature is unknown. Here, we show that NAMPT-dependent control over the molecular clock varies substantially between tissues. Brown adipose tissue (BAT) requires NAMPT to sustain the amplitude of the core clock, whereas rhythmicity in white adipose tissue (WAT) is only moderately dependent on NAD+ biosynthesis, and the skeletal muscle clock is completely refractory to loss of NAMPT. In BAT and WAT, NAMPT differentially orchestrates oscillation of clock-controlled gene networks and the diurnality of metabolite levels. NAMPT coordinates the rhythmicity of TCA cycle intermediates in BAT, but not in WAT, and loss of NAD+ abolishes these oscillations similarly to high-fat diet-induced circadian disruption. Moreover, adipose NAMPT depletion improved the ability of animals to defend body temperature during cold stress but in a time-of-day-independent manner. Thus, our findings reveal that peripheral molecular clocks and metabolic biorhythms are shaped in a highly tissue-specific manner by NAMPT-dependent NAD+ synthesis.

Keywords:  Brown adipose tissue; Circadian metabolism; Clock rhythm; NAD; Skeletal muscle

DOI:  https://doi.org/10.1073/pnas.2220102120
Nat Commun. 2023 Mar 29. 14(1): 1747

Muscle cell-type diversification is driven by bHLH transcription factor expansion and extensive effector gene duplications.

Alison G Cole, Stefan M Jahnel, Sabrina Kaul, Julia Steger, Julia Hagauer, Andreas Denner, Patricio Ferrer Murguia, Elisabeth Taudes, Bob Zimmermann, Robert Reischl, Patrick R H Steinmetz, Ulrich Technau.

Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the emergence of new cell types remain unclear. Here we address the origin and diversification of muscle cells in the non-bilaterian, diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations, which differ by extensive sets of paralogous structural protein genes. We find that the regulatory gene set of the slow cnidarian muscles is remarkably similar to the bilaterian cardiac muscle, while the two fast muscles differ substantially from each other in terms of transcription factor profiles, though driving the same set of structural protein genes and having similar physiological characteristics. We show that anthozoan-specific paralogs of Paraxis/Twist/Hand-related bHLH transcription factors are involved in the formation of fast and slow muscles. Our data suggest that the subsequent recruitment of an entire effector gene set from the inner cell layer into the neural ectoderm contributes to the evolution of a novel muscle cell type. Thus, we conclude that extensive transcription factor gene duplications and co-option of effector modules act as an evolutionary mechanism underlying cell type diversification during metazoan evolution.

DOI: https://doi.org/10.1038/s41467-023-37220-6
Annu Rev Biochem. 2023 Mar 31.

Structural Biochemistry of Muscle Contraction.

Zhexin Wang, Stefan Raunser.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles relies on the sliding of two types of filaments-the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin-myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo-electron microscopy and cryo-electron tomography in leading muscle research into a new era. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-biochem-052521-042909