bims-moremu 2023-06-11 papers

bims-moremu

Biomed News

on Molecular regulators of muscle mass

Issue of 2023–06–11
33 papers selected by
Anna Vainshtein, Craft Science Inc.

Single-cell transcriptomic analysis of skeletal muscle regeneration across mouse lifespan identifies altered stem cell states associated with senescence.
ATF4 regulates mitochondrial content, morphology, and function in differentiating skeletal muscle myotubes.
Effects of high-resistance wheel running on hallmarks of endurance and resistance training adaptations in mice.
MuSK-BMP signaling in adult muscle stem cells maintains quiescence and regulates myofiber size.
A concise in vitro model for evaluating interactions between macrophage and skeletal muscle cells during muscle regeneration.
Elevated phospholipid hydroperoxide glutathione peroxidase (GPX4) expression modulates oxylipin formation and inhibits age-related skeletal muscle atrophy and weakness.
Inhibition of type I PRMTs reforms muscle stem cell identity enhancing their therapeutic capacity.
Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.
Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.
Bioprinted Notch ligand to function as stem cell niche improves muscle regeneration in dystrophic muscle.
Components of Isolated Skeletal Muscle Differentiated Through Antibody Validation.
The role of myokines in cancer: crosstalk between skeletal muscle and tumor.
iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway.
AS160 expression, but not AS160 Serine-588, Threonine-642, and Serine-704 phosphorylation, is essential for elevated insulin-stimulated glucose uptake by skeletal muscle from female rats after acute exercise.
The Duration of Glucocorticoid Treatment Alters the Anabolic Response to High Force Muscle Contractions.
Myeloid cell infiltration in skeletal muscle after combined hindlimb unloading and radiation exposure in mice.
Neuromuscular junction transmission failure in aging and sarcopenia: The nexus of the neurological and muscular systems.
Round and resilient: How does exercise get myonuclei in shape?
Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.
Exercise Mediates Myokine Release and Tumor Suppression in Prostate Cancer Independent of Androgen Signaling.
Genetic deletion of skeletal muscle iPLA2γ results in mitochondrial dysfunction, muscle atrophy and alterations in whole-body energy metabolism.
Muscle Specific Promotors for Gene Therapy - A Comparative Study in Proliferating and Differentiated Cells.
Molecular mechanisms of muscle contraction: A historical perspective.
Long-term detraining reverses the improvement of lifelong exercise on skeletal muscle ferroptosis and inflammation in aging rats: fiber-type dependence of the Keap1/Nrf2 pathway.
Isometric contraction induces transient increase of REDD1 expression in non-contracted muscles partly through glucocorticoids.
Developmental, physiologic and phylogenetic perspectives on the expression and regulation of myosin heavy chains in mammalian skeletal muscles.
The D3 -creatine dilution method non-invasively measures muscle mass in mice.
Exercise therapy for sarcopenia and diabetes.
Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells.
Specific DMPK-promoter targeting by CRISPRi reverses myotonic dystrophy type 1-associated defects in patient muscle cells.
BTG2 acts as an inducer of muscle stem cell senescence.
Ablation of Sam50 is associated with fragmentation and alterations in metabolism in human myotubes.
Effects of Exercise Training on Muscle Quality in Older Individuals: A Systematic Scoping Review with Meta-Analyses.

bioRxiv. 2023 May 26. pii: 2023.05.25.542370. [Epub ahead of print]

Single-cell transcriptomic analysis of skeletal muscle regeneration across mouse lifespan identifies altered stem cell states associated with senescence.

Lauren D Walter, Jessica L Orton, Ern Hwei Hannah Fong, Viviana I Maymi, Brian D Rudd, Jennifer H Elisseeff, Benjamin D Cosgrove.

Skeletal muscle regeneration is driven by the interaction of myogenic and non-myogenic cells. In aging, regeneration is impaired due to dysfunctions of myogenic and non-myogenic cells, but this is not understood comprehensively. We collected an integrated atlas of 273,923 single-cell transcriptomes from muscles of young, old, and geriatric mice (∼5, 20, 26 months-old) at six time-points following myotoxin injury. We identified eight cell types, including T and NK cells and macrophage subtypes, that displayed accelerated or delayed response dynamics between ages. Through pseudotime analysis, we observed myogenic cell states and trajectories specific to old and geriatric ages. To explain these age differences, we assessed cellular senescence by scoring experimentally derived and curated gene-lists. This pointed to an elevation of senescent-like subsets specifically within the self-renewing muscle stem cells in aged muscles. This resource provides a holistic portrait of the altered cellular states underlying skeletal muscle regenerative decline across mouse lifespan.
EXTENDED SUMMARY: Skeletal muscle regeneration relies on the orchestrated interaction of myogenic and non-myogenic cells with spatial and temporal coordination. The regenerative capacity of skeletal muscle declines with aging due to alterations in myogenic stem/progenitor cell states and functions, non-myogenic cell contributions, and systemic changes, all of which accrue with age. A holistic network-level view of the cell-intrinsic and -extrinsic changes influencing muscle stem/progenitor cell contributions to muscle regeneration across lifespan remains poorly resolved. To provide a comprehensive atlas of regenerative muscle cell states across mouse lifespan, we collected a compendium of 273,923 single-cell transcriptomes from hindlimb muscles of young, old, and geriatric (4-7, 20, and 26 months-old, respectively) mice at six closely sampled time-points following myotoxin injury. We identified 29 muscle-resident cell types, eight of which exhibited accelerated or delayed dynamics in their abundances between age groups, including T and NK cells and multiple macrophage subtypes, suggesting that the age-related decline in muscle repair may arise from temporal miscoordination of the inflammatory response. We performed a pseudotime analysis of myogenic cells across the regeneration timespan and found age-specific myogenic stem/progenitor cell trajectories in old and geriatric muscles. Given the critical role that cellular senescence plays in limiting cell contributions in aged tissues, we built a series of tools to bioinformatically identify senescence in these single-cell data and assess their ability to identify senescence within key myogenic stages. By comparing single-cell senescence scores to co-expression of hallmark senescence genes Cdkn2a and Cdkn1a , we found that an experimentally derived gene-list derived from a muscle foreign body response (FBR) fibrosis model accurately (receiver-operator curve AUC = 0.82-0.86) identified senescent-like myogenic cells across mouse ages, injury time-points, and cell-cycle states, in a manner comparable to curated gene-lists. Further, this scoring approach pinpointed transitory senescence subsets within the myogenic stem/progenitor cell trajectory that are related to stalled MuSC self-renewal states across all ages of mice. This new resource of mouse skeletal muscle aging provides a comprehensive portrait of the changing cellular states and interaction network underlying skeletal muscle regeneration across mouse lifespan.

DOI: https://doi.org/10.1101/2023.05.25.542370
Am J Physiol Cell Physiol. 2023 Jun 05.

ATF4 regulates mitochondrial content, morphology, and function in differentiating skeletal muscle myotubes.

Jonathan M Memme, Victoria C Sanfrancesco, David A Hood.

  Mitochondrial function is widely recognized as a major determinant of health, emphasizing the importance of understanding the mechanisms promoting mitochondrial quality in various tissues. Recently, the mitochondrial unfolded protein response (UPRmt) has come into focus as a modulator of mitochondrial homeostasis, particularly in stress conditions. In muscle, the necessity for ATF4 and its role in regulating mitochondrial quality control (MQC) has yet to be determined. We overexpressed (OE) and knocked down ATF4 in C2C12 myoblasts, differentiated them to myotubes for 5 days, and subjected them to acute (ACA) or chronic (CCA) contractile activity. ATF4 mediated myotube formation through the regulated expression of myogenic factors, mainly Myc and MyoD, and supressed mitochondrial biogenesis basally through PGC-1a. However, our data also show that ATF4 expression levels are directly related to mitochondrial fusion and dynamics, UPRmt activation, as well as lysosomal biogenesis and autophagy. Thus, ATF4 promoted enhanced mitochondrial networking, protein handling, and capacity for clearance of dysfunctional organelles under stress conditions, despite lower levels of mitophagy flux with OE. Indeed, we found that ATF4 promoted the formation of a smaller pool of high functioning mitochondria that are more responsive to contractile activity, have higher oxygen consumption rates and lower reactive oxygen species levels. These data provide evidence that ATF4 is both necessary and sufficient for mitochondrial quality control and adaptation during both differentiation and contractile activity, thus advancing the current understanding of ATF4 beyond its canonical functions, to include the regulation of mitochondrial morphology, lysosomal biogenesis and mitophagy in muscle cells.

Keywords:  ATF4; mitochondrial quality control; mitochondrial unfolded protein response; mitophagy and lysosomal biogenesis; skeletal muscle C2C12

DOI:  https://doi.org/10.1152/ajpcell.00080.2023
Physiol Rep. 2023 06;11(11): e15701

Effects of high-resistance wheel running on hallmarks of endurance and resistance training adaptations in mice.

Aurel B Leuchtmann, Yasmine Afifi, Danilo Ritz, Christoph Handschin.

  Exercise effectively promotes and preserves cardiorespiratory, neuromuscular, metabolic, and cognitive functions throughout life. The molecular mechanisms underlying the beneficial adaptations to exercise training are, however, still poorly understood. To improve the mechanistic study of specific exercise training adaptations, standardized, physiological, and well-characterized training interventions are required. Therefore, we performed a comprehensive interrogation of systemic changes and muscle-specific cellular and molecular adaptations to voluntary low-resistance wheel running (Run) and progressive high-resistance wheel running (RR) in young male mice. Following 10 weeks of training, both groups showed similar improvements in body composition and peak oxygen uptake (V̇O2peak ), as well as elevated mitochondrial proteins and capillarization markers in the M. plantaris. Run mice clearly outperformed RR mice in a forced treadmill running capacity test, while RR mice displayed increased grip strength as well as superior mass gains in the M. soleus, associated with distinct proteomic changes specifying the two paradigms. Thus, even though both training modalities induce overlapping adaptations, Run interventions preferably improve submaximal running performance, while progressive RR is a valid model to study training-induced gains in grip strength and plantar flexor hypertrophy.

Keywords:  endurance exercise; hypertrophy; resistance exercise; skeletal muscle; strength; training adaptation

DOI:  https://doi.org/10.14814/phy2.15701
bioRxiv. 2023 May 18. pii: 2023.05.17.541238. [Epub ahead of print]

MuSK-BMP signaling in adult muscle stem cells maintains quiescence and regulates myofiber size.

Laura A Madigan, Diego Jaime, Justin R Fallon.

A central question in the biology of adult stem cells is elucidating the signaling pathways regulating their dynamics and function in diverse physiological and age-related contexts. Adult muscle stem cells (Satellite Cells; SCs) are generally quiescent but can activate and contribute to muscle homeostasis and repair. Here we tested the role of the MuSK-BMP pathway in regulating adult SC quiescence and myofiber size. We attenuated MuSK-BMP signaling by deletion of the BMP-binding MuSK Ig3 domain ('ΔIg3-MuSK') and studied the fast TA and EDL muscles. In germ line mutants at 3 months of age SC and myonuclei numbers as well as myofiber size were comparable in ΔIg3-MuSK and WT animals. However, in 5-month-old ΔIg3-MuSK animals SC density was decreased while myofiber size, myonuclear number and grip strength were increased - indicating that SCs had activated and productively fused into the myofibers over this interval. Notably, myonuclear domain size was conserved. Following injury, the mutant muscle fully regenerated with restoration of myofiber size and SC pool to WT levels, indicating that ΔIg3-MuSK SCs maintain full stem cell function. Conditional expression of ΔIg3-MuSK in adult SCs showed that the MuSK-BMP pathway regulates quiescence and myofiber size in a cell autonomous fashion. Transcriptomic analysis revealed that SCs from uninjured ΔIg3-MuSK mice exhibit signatures of activation, including elevated Notch and epigenetic signaling. We conclude that the MuSK-BMP pathway regulates SC quiescence and myofiber size in a cell autonomous, age-dependent manner. Targeting MuSK-BMP signaling in muscle stem cells thus emerges a therapeutic strategy for promoting muscle growth and function in the settings of injury, disease, and aging.

DOI: https://doi.org/10.1101/2023.05.17.541238
Front Cell Dev Biol. 2023 ;11 1022081

A concise in vitro model for evaluating interactions between macrophage and skeletal muscle cells during muscle regeneration.

Naoya Kase, Yohko Kitagawa, Akihiro Ikenaka, Akira Niwa, Megumu K Saito.

  Skeletal muscle has a highly regenerative capacity, but the detailed process is not fully understood. Several in vitro skeletal muscle regeneration models have been developed to elucidate this, all of which rely on specialized culture conditions that limit the accessibility and their application to many general experiments. Here, we established a concise in vitro skeletal muscle regeneration model using mouse primary cells. This model allows evaluation of skeletal muscle regeneration in two-dimensional culture system similar to a typical cell culture, showing a macrophage-dependent regenerative capacity, which is an important process in skeletal muscle regeneration. Based on the concept that this model could assess the contribution of macrophages of various phenotypes to skeletal muscle regeneration, we evaluated the effect of endotoxin pre-stimulation for inducing various changes in gene expression on macrophages and found that the contribution to skeletal muscle regeneration was significantly reduced. The gene expression patterns differed from those of naive macrophages, especially immediately after skeletal muscle injury, suggesting that the difference in responsiveness contributed to the difference in regenerative efficiency. Our findings provide a concise in vitro model that enables the evaluation of the contribution of individual cell types, such as macrophages and muscle stem cells, on skeletal muscle regeneration.

Keywords:  in vitro disease modeling; innate immune memory; macrophages; regeneration efficiency; skeletal muscle regeneration

DOI:  https://doi.org/10.3389/fcell.2023.1022081
Redox Biol. 2023 Jun 01. pii: S2213-2317(23)00162-3. [Epub ahead of print]64 102761

Elevated phospholipid hydroperoxide glutathione peroxidase (GPX4) expression modulates oxylipin formation and inhibits age-related skeletal muscle atrophy and weakness.

Agnieszka Czyżowska, Jacob Brown, Hongyang Xu, Kavitha Sataranatarajan, Michael Kinter, Victoria J Tyrell, Valerie B O'Donnell, Holly Van Remmen.

Our previous studies support a key role for mitochondrial lipid hydroperoxides as important contributors to denervation-related muscle atrophy, including muscle atrophy associated with aging. Phospholipid hydroperoxide glutathione peroxidase 4 (GPX4) is an essential antioxidant enzyme that directly reduces phospholipid hydroperoxides and we previously reported that denervation-induced muscle atrophy is blunted in a mouse model of GPX4 overexpression. Therefore, the goal of the present study was to determine whether GPX4 overexpression can reduce the age-related increase in mitochondrial hydroperoxides in skeletal muscle and ameliorate age-related muscle atrophy and weakness (sarcopenia). Male C57Bl6 WT and GPX4 transgenic (GPX4Tg) mice were studied at 3 to 5 and 23-29 months of age. Basal mitochondrial peroxide generation was reduced by 34% in muscle fibers from aged GPX4Tg compared to old WT mice. GPX4 overexpression also reduced levels of lipid peroxidation products: 4-HNE, MDA, and LOOHs by 38%, 32%, and 84% respectively in aged GPX4Tg mice compared to aged WT mice. Muscle mass was preserved in old GPX4 Tg mice by 11% and specific force generation was 21% higher in old GPX4Tg versus age matched male WT mice. Oxylipins from lipoxygenases (LOX) and cyclooxygenase (COX), as well as less abundant non-enzymatically generated isomers, were significantly reduced by GPX4 overexpression. The expression of cPLA2, 12/15-LOX and COX-2 were 1.9-, 10.5- and 3.4-fold greater in old versus young WT muscle respectively, and 12/15-LOX and COX-2 levels were reduced by 37% and 35%, respectively in muscle from old GPX4Tg mice. Our study suggests that lipid peroxidation products may play an important role in the development of sarcopenia, and their detoxification might be an effective intervention in preventing muscle atrophy.

DOI: https://doi.org/10.1016/j.redox.2023.102761
Elife. 2023 Jun 07. pii: RP84570. [Epub ahead of print]12

Inhibition of type I PRMTs reforms muscle stem cell identity enhancing their therapeutic capacity.

Claudia Dominici, Oscar D Villarreal, Junio Dort, Emilie Heckel, Yu Chang Wang, Ioannis Ragoussis, Jean-Sebastien Joyal, Nicolas Dumont, Stéphane Richard.

  In skeletal muscle, muscle stem cells (MuSC) are the main cells responsible for regeneration upon injury. In diseased skeletal muscle, it would be therapeutically advantageous to replace defective MuSCs, or rejuvenate them with drugs to enhance their self-renewal and ensure long-term regenerative potential. One limitation of the replacement approach has been the inability to efficiently expand MuSCs ex vivo, while maintaining their stemness and engraftment abilities. Herein, we show that inhibition of type I protein arginine methyltransferases (PRMTs) with MS023 increases the proliferative capacity of ex vivo cultured MuSCs. Single cell RNA sequencing (scRNAseq) of ex vivo cultured MuSCs revealed the emergence of subpopulations in MS023-treated cells which are defined by elevated Pax7 expression and markers of MuSC quiescence, both features of enhanced self-renewal. Furthermore, the scRNAseq identified MS023-specific subpopulations to be metabolically altered with upregulated glycolysis and oxidative phosphorylation (OxPhos). Transplantation of MuSCs treated with MS023 had a better ability to repopulate the MuSC niche and contributed efficiently to muscle regeneration following injury. Interestingly, the preclinical mouse model of Duchenne muscular dystrophy had increased grip strength with MS023 treatment. Our findings show that inhibition of type I PRMTs increased the proliferation capabilities of MuSCs with altered cellular metabolism, while maintaining their stem-like properties such as self-renewal and engraftment potential.

Keywords:  MS023; arginine methylation; duchenne muscular dystrophy; mouse; muscle stem cell; regenerative medicine; skeletal muscle; stem cells; type I PRMT

DOI:  https://doi.org/10.7554/eLife.84570
Development. 2023 Jun 01. pii: dev201504. [Epub ahead of print]150(11):

Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

Hasan Asfour, Estelle Hirsinger, Raquel Rouco, Faouzi Zarrouki, Shinichiro Hayashi, Sandra Swist, Thomas Braun, Ketan Patel, Frédéric Relaix, Guillaume Andrey, Sigmar Stricker, Delphine Duprez, Amalia Stantzou, Helge Amthor.

  The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

Keywords:  BMP signaling; Embryonic muscle; Fetal muscle; Hox; Limb muscle; Mouse; Muscle fiber; Myogenesis; Myogenic progenitor cell; PAX3; Patterning; SMAD6

DOI:  https://doi.org/10.1242/dev.201504
J Physiol. 2023 Jun 09.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Heather L Petrick, Rachel M Handy, Bayley Vachon, Sara M Frangos, Andrew M Holwerda, Annemarie P Gijsen, Joan M Senden, Luc J C van Loon, Graham P Holloway.

  Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species (ROS). Since dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subject to single-limb casting (3 or 7 days) and consumed drinking water with or without 1 mM sodium nitrate. Compared to the contralateral control limb, 3 days of immobilization lowered myofibrillar fractional synthesis rates (FSR, p<0.0001), resulting in muscle atrophy. While FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared to intermyofibrillar (IMF) mitochondria, immobilization for 3 days decreased FSR in both SS (p = 0.009) and IMF (p = 0.031) mitochondria. Additionally, 3 days of immobilization reduced maximal mitochondrial respiration and protein content, and increased maximal mitochondrial ROS emission without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS, IMF). While nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics following both 3 and 7 days of immobilization. However, in contrast to 3 days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR following 7 days. Therefore, while nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Since dietary nitrate can improve mitochondrial bioenergetics, we examined if nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3 day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits, and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, while dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse. Abstract figure legend In female mice consuming standard drinking water (H2 O), 3 and 7 days of single-limb immobilization decreased mitochondrial (mito) protein fractional synthesis rate (FSR), myofibrillar (myofib) protein FSR, and mitochondrial respiration, and increased mitochondrial reactive oxygen species (ROS). In contrast, sodium nitrate (NO3 ) prevented the immobilization-induced alterations in mitochondrial bioenergetics (respiration, ROS) at both timepoints (3- and 7-day). In addition, mitochondrial protein FSR was transiently (3 day) preserved in the immobilized limb of nitrate-consuming mice. Combined, while dietary nitrate was not sufficient to prevent muscle atrophy, nitrate preserved mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term muscle disuse in mice. This article is protected by copyright. All rights reserved.

Keywords:  IMF mitochondria; SS mitochondria; immobilization; mitochondrial ROS; mitochondrial respiration; nitrate; protein synthesis

DOI:  https://doi.org/10.1113/JP284701
Int J Bioprint. 2023 ;9(3): 711

Bioprinted Notch ligand to function as stem cell niche improves muscle regeneration in dystrophic muscle.

Zewei Sun, Xianlin Yue, Lei Liu, Ying Li, Jie Cui, Dong Li, Lee Weiss, Phil Campbell, Yanling Mu, Johnny Huard, Xiaodong Mu.

  299In Duchenne muscular dystrophy, dystrophic muscle phenotypes are closely associated with the exhaustion of muscle stem cells. Transplantation of muscle stem cells has been widely studied for improving muscle regeneration, but poor cell survival and self-renewal, rapid loss of stemness, and limited dispersion of grafted cells following transplantation have collectively hindered the overall success of this strategy. Optimized mechanisms for maintaining and improving stem cell function are naturally present in the microenvironment of the stem cell niche in healthy muscles. Therefore, one logical strategy toward improving stem cell function and efficiency of stem cell transplantation in diseased muscles would be the establishment of a microenvironment mimicking some key aspects of healthy native stem cell niches. Here, we applied inkjet-based bioprinting technology to engineer a mimicked artificial stem cell niche in dystrophic muscle, comprising stem cell niche regulating factors (Notch activator DLL1) bioprinted onto 3D DermaMatrix construct. The recombinant DLL1 protein, DLL1 (mouse): Fc (human) (rec), was applied here as the Notch activator. Bioprinted DermaMatrix construct was seeded with muscle stem cells in vitro, and increased stem cell maintenance and repressed myogenic differentiation process was observed. DLL1 bioprinted DermaMatrix construct was then engrafted into dystrophic muscle of mdx/scid mice, and the improved cell engraftment and progression of muscle regeneration was observed 10 days after engraftment. Our results demonstrated that bioprinting of Notch activator within 3D construct can be applied to serve as muscle stem cell niche and improve the efficacy of muscle stem cell transplantation in diseased muscle.

Keywords:  Muscle dystrophy; Muscle stem cell; Notch signaling; Stem cell niche

DOI:  https://doi.org/10.18063/ijb.711
bioRxiv. 2023 May 22. pii: 2023.05.20.541600. [Epub ahead of print]

Components of Isolated Skeletal Muscle Differentiated Through Antibody Validation.

Dominique C Stephens, Margaret Mungai, Amber Crabtree, Heather K Beasley, Edgar Garza-Lopez, Larry Vang, Kit Neikirk, Zer Vue, Neng Vue, Andrea G Marshall, Kyrin Turner, Jian-Qiang Shao, Bishnu Sarker, Sandra Murray, Jennifer A Gaddy, Jamaine Davis, Steven M Damo, Antentor O Hinton.

Isolation of skeletal muscles allows for the exploration of many complex diseases. Fibroblasts and myoblast play important roles in skeletal muscle morphology and function. However, skeletal muscles are complex and made up of many cellular populations and validation of these populations is highly important. Therefore, in this article, we discuss a comprehensive method to isolate mice skeletal muscle, create satellite cells for tissue culture, and use immunofluorescence to validate our approach. Novel imaging technology is increasing our ability to visualize and analyze cellular organelles and compartments.
Tweetable Abstract: Proper antibody validation of cellular populations within isolated skeletal muscle can lead to better elucidation of skeletal muscle structure and function, and their roles in complex diseases.
Method Summary: Myoblasts are isolated from mouse limb muscles. They are plated for immunofluorescence-based validation using confocal microscopy. This method demonstrates the need for reliable antibodies to correctly determine and differentiate between cellular populations within isolated skeletal muscles.

DOI: https://doi.org/10.1101/2023.05.20.541600
BMB Rep. 2023 Jun 09. pii: 5922. [Epub ahead of print]

The role of myokines in cancer: crosstalk between skeletal muscle and tumor.

Se-Young Park, Byeong-Oh Hwang, Na-Young Song.

Loss of skeletal muscle mass is a primary feature of sarcopenia and cancer cachexia. In cancer patients, tumor-derived inflammatory factors promote muscle atrophy via tumor-to-muscle effects, which is closely associated with poor prognosis. During the past decade, skeletal muscle has been considered to function as an autocrine, paracrine, and endocrine organ by releasing numerous myokines. The circulating myokines can modulate pathophysiology in the other organs, as well as in the tumor microenvironment, suggesting myokines function as muscleto-tumor signaling molecules. Here, we highlight the roles of myokines in tumorigenesis, particularly in terms of crosstalk between skeletal muscle and tumor. Better understanding of tumor-to-muscle and muscle-to-tumor effects will shed light on novel strategies for the diagnosis and treatment of cancer.
Biol Res. 2023 Jun 03. 56(1): 29

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway.

Haimei Cen, Pin Fan, Yuting Ding, Bin Luo, Hong Luo, Menglong Chen, Yu Zhang.

   BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury.
RESULTS: In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKα and ULK1 in C2C12 myotubes exposed to hypoxic damage.
CONCLUSIONS: Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

Keywords:  AMPK/ULK1 pathway; Atrogin-1; Co-culture; Duchenne muscular dystrophy; Hypoxia; LC3II/LC3I; MuRF-1

DOI:  https://doi.org/10.1186/s40659-023-00435-4
FASEB J. 2023 Jul;37(7): e23021

AS160 expression, but not AS160 Serine-588, Threonine-642, and Serine-704 phosphorylation, is essential for elevated insulin-stimulated glucose uptake by skeletal muscle from female rats after acute exercise.

Haiyan Wang, Amy Zheng, Edward B Arias, Seong Eun Kwak, Xiufang Pan, Dongsheng Duan, Gregory D Cartee.

  One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle in both sexes. We recently found that muscle expression and phosphorylation of key sites of Akt substrate of 160 kDa (AS160; also called TBC1D4) are essential for the full-exercise effect on postexercise-ISGU (PEX-ISGU) in male rats. In striking contrast, AS160's role in increased PEX-ISGU has not been rigorously tested in females. Our rationale was to address this major knowledge gap. Wild-type (WT) and AS160-knockout (KO) rats were either sedentary or acutely exercised. Adeno-associated virus (AAV) vectors were engineered to express either WT-AS160 or AS160 mutated on key serine and threonine residues (Ser588, Thr642, and Ser704) to alanine to prevent their phosphorylation. AAV vectors were delivered to the muscle of AS160-KO rats to determine if WT-AS160 or phosphorylation-inactivated AS160 would influence PEX-ISGU. AS160-KO rats have lower skeletal muscle abundance of the GLUT4 glucose transporter protein. This GLUT4 deficit was rescued using AAV delivery of GLUT4 to determine if eliminating muscle GLUT4 deficiency would normalize PEX-ISGU. The novel results were as follows: (1) AS160 expression was required for greater PEX-ISGU; (2) rescuing muscle AS160 expression in AS160-KO rats restored elevated PEX-ISGU; (3) AS160's essential role for the postexercise increase in ISGU was not attributable to reduced muscle GLUT4 content; and (4) AS160 phosphorylation on Ser588, Thr642, and Ser704 was not essential for greater PEX-ISGU. In conclusion, these novel findings revealed that three phosphosites widely proposed to influence PEX-ISGU are not required for this important outcome in female rats.

Keywords:  AMP-activated protein kinase; GLUT4 glucose transporter; acute exercise; female; glucose transport; glycogen; insulin resistance; insulin signaling; sexual dimorphism

DOI:  https://doi.org/10.1096/fj.202300282RR
J Appl Physiol (1985). 2023 Jun 08.

The Duration of Glucocorticoid Treatment Alters the Anabolic Response to High Force Muscle Contractions.

Kirsten R Dunlap, Jennifer L Steiner, Robert C Hickner, P Bryant Chase, Bradley S Gordon.

  Glucocorticoids induce a myopathy that includes loss of muscle mass and strength. Resistance exercise may reverse the muscle loss because it induces an anabolic response characterized by increases in muscle protein synthesis and potentially suppressing protein breakdown. Whether resistance exercise induces an anabolic response in glucocorticoid myopathic muscle is unknown, which is a problem because long-term glucocorticoid exposure alters the expression of genes that may prevent an anabolic response by limiting activation of pathways such as the mechanistic target of rapamycin in complex 1 (mTORC1). The purpose of this study was to assess whether high force contractions initiate an anabolic response in glucocorticoid myopathic muscle. The anabolic response was analyzed treating female mice with dexamethasone (DEX) for 7 days or 15 days. After treatment, the left tibialis anterior muscle of all mice was contracted via electrical stimulation of the sciatic nerve. Muscles were harvested 4 hr after contractions. Rates of muscle protein synthesis were estimated using the SUnSET method. After 7 days of treatment, high force contractions increased protein synthesis and mTORC1 signaling in both groups. After 15 days of treatment, high force contractions activated mTORC1 signaling equally in both groups, but protein synthesis was only increased in control mice. The failure to increase protein synthesis may be because baseline synthetic rates were elevated in DEX-treated mice. The LC3 II/I ratio marker of autophagy was decreased by contractions regardless of treatment duration. These data show duration of glucocorticoid treatment alters the anabolic response to high force contractions.

Keywords:  Anabolic Resistance; Protein Synthesis; Resistance Exercise; mTORC1

DOI:  https://doi.org/10.1152/japplphysiol.00113.2023
NPJ Microgravity. 2023 Jun 07. 9(1): 40

Myeloid cell infiltration in skeletal muscle after combined hindlimb unloading and radiation exposure in mice.

Eric B Emanuelsson, Bjorn Baselet, Mieke Neefs, Sarah Baatout, Brit Proesmans, Lisa Daenen, Carl Johan Sundberg, Helene Rundqvist, Rodrigo Fernandez-Gonzalo.

The skeletal muscle and the immune system are heavily affected by the space environment. The crosstalk between these organs, although established, is not fully understood. This study determined the nature of immune cell changes in the murine skeletal muscle following (hindlimb) unloading combined with an acute session of irradiation (HLUR). Our findings show that 14 days of HLUR induces a significant increase of myeloid immune cell infiltration in skeletal muscle.

DOI: https://doi.org/10.1038/s41526-023-00289-w
Ageing Res Rev. 2023 Jun 01. pii: S1568-1637(23)00125-3. [Epub ahead of print]89 101966

Neuromuscular junction transmission failure in aging and sarcopenia: The nexus of the neurological and muscular systems.

W David Arnold, Brian C Clark.

  Sarcopenia, or age-related decline in muscle form and function, exerts high personal, societal, and economic burdens when untreated. Integrity and function of the neuromuscular junction (NMJ), as the nexus between the nervous and muscular systems, is critical for input and dependable neural control of muscle force generation. As such, the NMJ has long been a site of keen interest in the context of skeletal muscle function deficits during aging and in the context of sarcopenia. Historically, changes of NMJ morphology during aging have been investigated extensively but primarily in aged rodent models. Aged rodents have consistently shown features of NMJ endplate fragmentation and denervation. Yet, the presence of NMJ changes in older humans remains controversial, and conflicting findings have been reported. This review article describes the physiological processes involved in NMJ transmission, discusses the evidence that supports NMJ transmission failure as a possible contributor to sarcopenia, and speculates on the potential of targeting these defects for therapeutic development. The technical approaches that are available for assessment of NMJ transmission, whether each approach has been applied in the context of aging and sarcopenia, and the associated findings are summarized. Like morphological studies, age-related NMJ transmission deficits have primarily been studied in rodents. In preclinical studies, isolated synaptic electrophysiology recordings of endplate currents or potentials have been mostly used, and paradoxically, have shown enhancement, rather than failure, with aging. Yet, in vivo assessment of single muscle fiber action potential generation using single fiber electromyography and nerve-stimulated muscle force measurements show evidence of NMJ failure in aged mice and rats. Together these findings suggest that endplate response enhancement may be a compensatory response to post-synaptic mechanisms of NMJ transmission failure in aged rodents. Possible, but underexplored, mechanisms of this failure are discussed including the simplification of post-synaptic folding and altered voltage-gated sodium channel clustering or function. In humans, there is limited clinical data that has selectively investigated single synaptic function in the context of aging. If sarcopenic older adults turn out to exhibit notable impairments in NMJ transmission (this has yet to be examined but based on available evidence appears to be plausible) then these NMJ transmission defects present a well-defined biological mechanism and offer a well-defined pathway for clinical implementation. Investigation of small molecules that are currently available clinically or being testing clinically in other disorders may provide a rapid route for development of interventions for older adults impacted by sarcopenia.

Keywords:  Dynapenia; Mobility; Muscle weakness; Older adults; Physical function; Skeletal muscle function deficits

DOI:  https://doi.org/10.1016/j.arr.2023.101966
J Physiol. 2023 Jun 07.

Round and resilient: How does exercise get myonuclei in shape?

Ines Kortebi, Paul Babits, Celine Bailleul.



Keywords:  aging; exercise adaptations; lamin A; muscle protein synthesis; myonuclei

DOI:  https://doi.org/10.1113/JP285046
Sci Rep. 2023 Jun 08. 13(1): 9333

Primary mouse myoblast metabotropic purinoceptor profiles and calcium signalling differ with their muscle origin and are altered in mdx dystrophinopathy.

Justyna Róg, Aleksandra Oksiejuk, Dariusz C Górecki, Krzysztof Zabłocki.

Mortality of Duchenne Muscular Dystrophy (DMD) is a consequence of progressive wasting of skeletal and cardiac muscle, where dystrophinopathy affects not only muscle fibres but also myogenic cells. Elevated activity of P2X7 receptors and increased store-operated calcium entry have been identified in myoblasts from the mdx mouse model of DMD. Moreover, in immortalized mdx myoblasts, increased metabotropic purinergic receptor response was found. Here, to exclude any potential effects of cell immortalization, we investigated the metabotropic response in primary mdx and wild-type myoblasts. Overall, analyses of receptor transcript and protein levels, antagonist sensitivity, and cellular localization in these primary myoblasts confirmed the previous data from immortalised cells. However, we identified significant differences in the pattern of expression and activity of P2Y receptors and the levels of the "calcium signalling toolkit" proteins between mdx and wild-type myoblasts isolated from different muscles. These results not only extend the earlier findings on the phenotypic effects of dystrophinopathy in undifferentiated muscle but, importantly, also reveal that these changes are muscle type-dependent and endure in isolated cells. This muscle-specific cellular impact of DMD may not be limited to the purinergic abnormality in mice and needs to be taken into consideration in human studies.

DOI: https://doi.org/10.1038/s41598-023-36545-y
Exerc Sport Sci Rev. 2023 Jun 08.

Exercise Mediates Myokine Release and Tumor Suppression in Prostate Cancer Independent of Androgen Signaling.

Jin-Soo Kim, Dennis R Taaffe, Daniel A Galvão, Fred Saad, Robert U Newton.

ABSTRACT: A prominent toxicity of androgen suppression in prostate cancer patients is loss of skeletal muscle. Exercise may induce tumor-suppression through the endocrinal function of skeletal muscle, however, this is currently unknown. In this review we summarise our work demonstrating the acute and chronic myokine response to exercise and the tumor-suppressive effect of circulatory milieu alteration in prostate cancer patients.

DOI: https://doi.org/10.1249/JES.0000000000000323
iScience. 2023 Jun 16. 26(6): 106895

Genetic deletion of skeletal muscle iPLA2γ results in mitochondrial dysfunction, muscle atrophy and alterations in whole-body energy metabolism.

Sung Ho Moon, Beverly Gibson Dilthey, Shaoping Guan, Harold F Sims, Sara K Pittman, Amy L Keith, Christopher M Jenkins, Conrad C Weihl, Richard W Gross.

  Skeletal muscle is the major site of glucose utilization in mammals integrating serum glucose clearance with mitochondrial respiration. To mechanistically elucidate the roles of iPLA2γ in skeletal muscle mitochondria, we generated a skeletal muscle-specific calcium-independent phospholipase A2γ knockout (SKMiPLA2γKO) mouse. Genetic ablation of skeletal muscle iPLA2γ resulted in pronounced muscle weakness, muscle atrophy, and increased blood lactate resulting from defects in mitochondrial function impairing metabolic processing of pyruvate and resultant bioenergetic inefficiency. Mitochondria from SKMiPLA2γKO mice were dysmorphic displaying marked changes in size, shape, and interfibrillar juxtaposition. Mitochondrial respirometry demonstrated a marked impairment in respiratory efficiency with decreases in the mass and function of oxidative phosphorylation complexes and cytochrome c. Further, a pronounced decrease in mitochondrial membrane potential and remodeling of cardiolipin molecular species were prominent. Collectively, these alterations prevented body weight gain during high-fat feeding through enhanced glucose disposal without efficient capture of chemical energy thereby altering whole-body bioenergetics.

Keywords:  Cell biology; Cellular physiology; Physiology

DOI:  https://doi.org/10.1016/j.isci.2023.106895
J Neuromuscul Dis. 2023 Jun 02.

Muscle Specific Promotors for Gene Therapy - A Comparative Study in Proliferating and Differentiated Cells.

Julienne Dietz, Frank Jacobsen, Heidi Zhuge, Nassam Daya, Anne Bigot, Wenli Zhang, Anja Ehrhardt, Matthias Vorgerd, Eric Ehrke-Schulz.

   BACKGROUND: Depending on the therapy approach and disease background, the heterogeneity of muscular tissues complicates the development of targeted gene therapy, where either expression in all muscle types or restriction to only one muscle type is warranted. Muscle specificity can be achieved using promotors mediating tissue specific and sustained physiological expression in the desired muscle types but limited activity in non-targeted tissue. Several muscle specific promotors have been described, but direct comparisons between them are lacking.
OBJECTIVE: Here we present a direct comparison of muscle specific Desmin-, MHCK7, microRNA206- and Calpain3 promotor.
METHODS: To directly compare these muscle specific promotors we utilized transfection of reporter plasmids using an in vitro model based on electrical pulse stimulation (EPS) to provoke sarcomere formation in 2D cell culture for quantification of promotor activities in far differentiated mouse and human myotubes.
RESULTS: We found that Desmin- and MHCK7 promotors showed stronger reporter gene expression levels in proliferating and differentiated myogenic cell lines than miR206 and CAPN3 promotor. However, Desmin and MHCK7 promotor promoted gene expression also cardiac cells whereas miR206 and CAPN3 promotor expression was restricted to skeletal muscle.
CONCLUSIONS: Our results provides direct comparison of muscle specific promotors with regard to expression strengths and specificity as this is important feature to avoid undesired transgene expression in non-target muscle cells for a desired therapy approach.

Keywords:  Muscle; gene therapy; promotor; targeted expression

DOI:  https://doi.org/10.3233/JND-221574
J Biomech. 2023 May 25. pii: S0021-9290(23)00228-2. [Epub ahead of print]155 111659

Molecular mechanisms of muscle contraction: A historical perspective.

Walter Herzog, Gudrun Schappacher-Tilp.

  Studies of muscle structure and function can be traced to at least 2,000 years ago. However, the modern era of muscle contraction mechanisms started in the 1950s with the classic works by AF Huxley and HE Huxley, both born in the United Kingdom, but not related and working independently. HE Huxley was the first to suggest that muscle contraction occurred through the sliding of two sets of filamentous structures (actin or thin filaments and myosin or thick filaments). AF Huxley then developed a biologically inspired mathematical model suggesting a possible molecular mechanism of how this sliding of actin and myosin might take place. This model then evolved from a two-state to a multi-state model of myosin-actin interactions, and from one that suggested a linear motor causing the sliding to a rotating motor. This model, the cross-bridge model of muscle contraction, is still widely used in biomechanics, and even the more sophisticated cross-bridge models of today still contain many of the features originally proposed by AF Huxley. In 2002, we discovered a hitherto unknown property of muscle contraction that suggested the involvement of passive structures in active force production, the so-called passive force enhancement. It was quickly revealed that this passive force enhancement was caused by the filamentous protein titin, and the three-filament (actin, myosin, and titin) sarcomere model of muscle contraction evolved. There are many suggestions of how these three proteins interact to cause contraction and produce active force, and one such suggestion is described here, but the molecular details of this proposed mechanism still need careful evaluation.

Keywords:  Actin; Mathematical models of contraction; Myosin; Passive force enhancement; Residual force depression; Residual force enhancement; Skeletal muscle; Theories of contraction; Titin

DOI:  https://doi.org/10.1016/j.jbiomech.2023.111659
Biogerontology. 2023 Jun 08.

Long-term detraining reverses the improvement of lifelong exercise on skeletal muscle ferroptosis and inflammation in aging rats: fiber-type dependence of the Keap1/Nrf2 pathway.

Zhuang-Zhi Wang, Hai-Chen Xu, Huan-Xia Zhou, Chen-Kai Zhang, Bo-Ming Li, Jia-Han He, Pin-Shi Ni, Xiao-Ming Yu, Yun-Qing Liu, Fang-Hui Li.

  We investigated the effects of lifelong aerobic exercise and 8 months of detraining after 10 months of aerobic training on circulation, skeletal muscle oxidative stress, and inflammation in aging rats. Sprague-Dawley rats were randomly assigned to the control (CON), detraining (DET), and lifelong aerobic training (LAT) groups. The DET and LAT groups began aerobic treadmill exercise at the age of 8 months and stopped training at the 18th and 26th month, respectively; all rats were sacrificed when aged 26 months. Compared with CON, LAT remarkably decreased serum and aged skeletal muscle 4-hydroxynonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels. Superoxide dismutase 2(SOD2) levels were higher in the LAT group than in the CON group in skeletal muscle. However, DET remarkably decreased SOD2 protein expression and content in the skeletal muscle and increased malondialdehyde (MDA) level compared with LAT. Compared with LAT, DET remarkably downregulated adiponectin and upregulated tumor necrosis factor alpha (TNF-α) expression, while phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and 70-kDa ribosomal protein S6 kinase (P70S6K) protein expression decreased, and that of FoxO1 and muscle atrophy F-box (MAFbX) proteins increased in the quadriceps femoris. Adiponectin and TNF-α expression in the soleus muscle did not change between groups, whereas that of AKT, mammalian target of rapamycin (mTOR), and P70S6K was lower in the soleus in the DET group than in that in the LAT group. Compared with that in the LAT group, sestrin1 (SES1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression in the DET group was lower, whereas Keap1 mRNA expression was remarkably upregulated in the quadriceps femoris. Interestingly, the protein and mRNA levels of SES1, Nrf2, and Keap1 in soleus muscle did not differ between groups. LAT remarkably upregulated ferritin heavy polypeptide 1(FTH), glutathione peroxidase 4(GPX4), and solute carrier family 7member 11 (SLC7A11) protein expression in the quadriceps femoris and soleus muscles, compared with CON. However, compared with LAT, DET downregulated FTH, GPX4, and SLC7A11 protein expression in the quadriceps femoris and soleus muscles. Long-term detraining during the aging phase reverses the improvement effect of lifelong exercise on oxidative stress, inflammation, ferroptosis, and muscle atrophy in aging skeletal muscle. The quadriceps femoris is more evident than the soleus, which may be related to the different changes in the Keap1/Nrf2 pathway in different skeletal muscles.

Keywords:  Aged skeletal muscle; Detraining; Inflammatory; Nrf2 pathway; Oxidation stress

DOI:  https://doi.org/10.1007/s10522-023-10042-1
Physiol Rep. 2023 06;11(11): e15745

Isometric contraction induces transient increase of REDD1 expression in non-contracted muscles partly through glucocorticoids.

Taro Murakami.

  This study investigated whether muscle contraction induces expression of regulated in development and DNA damage 1 (REDD1), a potent inhibitor of mTORC1, in mice muscle. Gastrocnemius muscle was unilaterally and isometrically contracted with electrical stimulation, and changes in muscle protein synthesis, mTORC1 signaling phosphorylation, and REDD1 protein, and mRNA were measured at time points of 0, 3, 6, 12, and 24 h after the contraction. At time point 0 and 3 h, muscle protein synthesis was blunted by the contraction, accompanied by a decrease in phosphorylation of 4E-BP1 at time point 0 h, suggesting suppression of mTORC1 was involved in blunting of muscle protein synthesis during and shortly after the contraction. REDD1 protein was not increased in the contracted muscle at these time points, but at time point 3 h, both REDD1 protein and mRNA were increased in the contralateral non-contracted muscle. The induction of REDD1 expression in the non-contracted muscle was attenuated by RU-486, an antagonist of the glucocorticoid receptor, suggesting that glucocorticoids are involved in this process. These findings suggest that muscle contraction induces temporal anabolic resistance in non-contracted muscle, potentially increasing the availability of amino acids for contracted muscle, allowing for the synthesis of muscle protein.

Keywords:  REDD1; RU-486; isometric contraction; mTORC1; muscle protein synthesis

DOI:  https://doi.org/10.14814/phy2.15745
J Comp Physiol B. 2023 Jun 05.

Developmental, physiologic and phylogenetic perspectives on the expression and regulation of myosin heavy chains in mammalian skeletal muscles.

Joseph Foon Yoong Hoh.

  The kinetics of myosin controls the speed and power of muscle contraction. Mammalian skeletal muscles express twelve kinetically different myosin heavy chain (MyHC) genes which provides a wide range of muscle speeds to meet different functional demands. Myogenic progenitors from diverse craniofacial and somitic mesoderm specify muscle allotypes with different repertoires for MyHC expression. This review provides a brief synopsis on the historical and current views on how cell lineage, neural impulse patterns, and thyroid hormone influence MyHC gene expression in muscles of the limb allotype during development and in adult life and the molecular mechanisms thereof. During somitic myogenesis, embryonic and foetal myoblast lineages form slow and fast primary and secondary myotube ontotypes which respond differently to postnatal neural and thyroidal influences to generate fully differentiated fibre phenotypes. Fibres of a given phenotype may arise from myotubes of different ontotypes which retain their capacity to respond differently to neural and thyroidal influences during postnatal life. This gives muscles physiological plasticity to adapt to fluctuations in thyroid hormone levels and patterns of use. The kinetics of MyHC isoforms vary inversely with animal body mass. Fast 2b fibres are specifically absent in muscles involved in elastic energy saving in hopping marsupials and generally absent in large eutherian mammals. Changes in MyHC expression are viewed in the context of the physiology of the whole animal. The roles of myoblast lineage and thyroid hormone in regulating MyHC gene expression are phylogenetically the most ancient while that of neural impulse patterns the most recent.

Keywords:  Gene expression; Myogenesis; Myosin heavy chain; Phylogeny; Plasticity; Skeletal muscle

DOI:  https://doi.org/10.1007/s00360-023-01499-0
Aging Cell. 2023 Jun 05. e13897

The D3 -creatine dilution method non-invasively measures muscle mass in mice.

Lauren Wimer, Elena Goncharova, Sofiya Galkina, Edna Nyangau, Mahalakshmi Shankaran, Asia Davis, Leandro Prado, Maria Castro Munoz, Sharon Epstein, Cavan Patterson, Nicholas Shaum, Mark Hellerstein, William Evans, Simon Melov.

  Developing accurate methods to quantify age-related muscle loss (sarcopenia) could greatly accelerate development of therapies to treat muscle loss in the elderly, as current methods are inaccurate or expensive. The current gold standard method for quantifying sarcopenia is dual-energy X-ray absorptiometry (DXA) but does not measure muscle directly-it is a composite measure quantifying "lean mass" (muscle) excluding fat and bone. In humans, DXA overestimates muscle mass, which has led to erroneous conclusions about the importance of skeletal muscle in human health and disease. In animal models, DXA is a popular method for measuring lean mass. However, instrumentation is expensive and is potentially limited by anesthesia concerns. Recently, the D3 -creatine (D3 Cr) dilution method for quantifying muscle mass was developed in humans and rats. This method is faster, cheaper, and more accurate than DXA. Here, we demonstrate that the D3 Cr method is a specific assay for muscle mass in mice, and we test associations with DXA and body weight. We evaluated the D3 Cr method compared to DXA-determined lean body mass (LBM) in aged mice and reported that DXA consistently overestimates muscle mass with age. Overall, we provide evidence that the D3 Cr dilution method directly measures muscle mass in mice. Combined with its ease of use, accessibility, and non-invasive nature, the method may prove to more quickly advance development of preclinical therapies targeting sarcopenia.

Keywords:  aging; mice; sarcopenia; skeletal muscle

DOI:  https://doi.org/10.1111/acel.13897
World J Diabetes. 2023 May 15. 14(5): 565-572

Exercise therapy for sarcopenia and diabetes.

Seung-Taek Lim, Sunghwun Kang.

  Aging is characterized by the gradual deterioration of function at the molecular, cellular, tissue, and organism levels in humans. The typical diseases caused by changes in body composition, as well as functional decline in the human body's organs due to aging include sarcopenia and metabolic disorders. The accumulation of dysfunctional aging β cells with age can cause decreased glucose tolerance and diabetes. Muscle decline has a multifactorial origin, involving lifestyle habits, disease triggers, and age-dependent biological changes. The reduced function of β cells in elderly people lowers insulin sensitivity, which affects protein synthesis and interferes with muscle synthesis. The functional decrease and aggravation of disease in elderly people with less regular exercise or physical activity causes imbalances in food intake and a continuous, vicious cycle. In contrast, resistance exercise increases the function of β cells and protein synthesis in elderly people. In this review, we discuss regular physical activities or exercises to prevent and improve health, which is sarcopenia as decreased muscle mass and metabolic disorders as diabetes in the elderly.

Keywords:  Aging; Diabetes; Elderly; Muscle; Resistance exercise; Sarcopenia

DOI:  https://doi.org/10.4239/wjd.v14.i5.565
bioRxiv. 2023 May 22. pii: 2023.05.20.541599. [Epub ahead of print]

Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells.

Dominique C Stephens, Amber Crabtree, Heather K Beasley, Edgar Garza-Lopez, Kit Neikirk, Margaret Mungai, Larry Vang, Zer Vue, Neng Vue, Andrea G Marshall, Kyrin Turner, Jianqiang Shao, Sandra Murray, Jennifer A Gaddy, Celestine Wanjalla, Jamaine Davis, Steven Damo, Antentor O Hinton.

Proximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs).
Tweetable Abstract: Proximity Ligation Assays can be used in skeletal muscle tissue and myoblasts to explore the protein-protein interactions involved in MERC sites.
Highlights: Skeletal muscle tissue and cells are plated on glass coverslips for evaluation by proximity ligation assay (PLA).Following fixation, cells are probed and stained for Mfn1, Mfn2, mitochondria, and ER and imaged using fluorescence confocal microscopy.This method shows that PLA can be used in mouse SkM and is adaptable to other models.Protocol for detection of protein-protein interactions using PLA.
Key Resources Table:

DOI: https://doi.org/10.1101/2023.05.20.541599
Mol Ther Nucleic Acids. 2023 Jun 13. 32 857-871

Specific DMPK-promoter targeting by CRISPRi reverses myotonic dystrophy type 1-associated defects in patient muscle cells.

Florent Porquet, Lin Weidong, Kévin Jehasse, Hélène Gazon, Maria Kondili, Silvia Blacher, Laurent Massotte, Emmannuel Di Valentin, Denis Furling, Nicolas Albert Gillet, Arnaud François Klein, Vincent Seutin, Luc Willems.

  Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3' untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats (CUGexp). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK-promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp-RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK-promoter silencing by CRISPRi as a promising therapeutic approach for DM1.

Keywords:  CRISPRi; MT: RNA/DNA Editing; gene silencing; gene therapy; myotonic dystrophy type 1; neuromuscular disease

DOI:  https://doi.org/10.1016/j.omtn.2023.05.007
Biochem Biophys Res Commun. 2023 Aug 20. pii: S0006-291X(23)00684-8. [Epub ahead of print]669 113-119

BTG2 acts as an inducer of muscle stem cell senescence.

Baozhou Peng, Yihan Chen, Yaning Wang, Yixi Fu, Xinrui Zeng, Hanmeng Zhou, Zibaidan Abulaiti, Shuaiyu Wang, Hongbo Zhang.

   BACKGROUND: Muscle aging is associated with muscle stem cell (MuSC) senescence, a process of whose DNA damage accumulation is considered as one of the leading causes. BTG2 had been identified as a mediator of genotoxic and cellular stress signaling pathways, however, its role in senescence of stem cells, including MuSC, remains unknown.
METHOD: We first compared MuSCs isolated from young and old mice to evaluate our in vitro model of natural senescence. CCK8 and EdU assays were utilized to assess the proliferation capacity of the MuSCs. Cellular senescence was further assessed at biochemical levels by SA-β-Gal and γHA2.X staining, and at molecular levels by quantifying the expression of senescence-associated genes. Next, by performing genetic analysis, we identified Btg2 as a potential regulator of MuSC senescence, which was experimentally validated by Btg2 overexpression and knockdown in primary MuSCs. Lastly, we extended our research to humans by analyzing the potential links between BTG2 and muscle function decline in aging.
RESULTS: BTG2 is highly expressed in MuSCs from elder mice showing senescent phenotypes. Overexpression and knockdown of Btg2 stimulates and prevents MuSCs senescence, respectively. In humans, high level of BTG2 is associated with low muscle mass in aging, and is a risk factor of aging-related diseases, such as diabetic retinopathy and HDL cholesterol.
CONCLUSION: Our work demonstrates BTG2 as a regulator of MuSC senescence and may serve as an intervention target for muscle aging.

Keywords:  Aging; Btg2; Muscle stem cell; Senescence

DOI:  https://doi.org/10.1016/j.bbrc.2023.05.098
bioRxiv. 2023 May 22. pii: 2023.05.20.541602. [Epub ahead of print]

Ablation of Sam50 is associated with fragmentation and alterations in metabolism in human myotubes.

Zer Vue, Chia Vang, Kit Neikirk, Edgar Garza-Lopez, Jian-Qiang Shao, Margaret Mungai, Larry Vang, Heather Beasley, Andrea G Marshall, Amber Crabtree, Dominique Stephens, Melanie McReynolds, Sandra A Murray, Prasanna Katti, Steven Damo, Jennifer A Gaddy, Antentor Hinton.

The Sorting and Assembly Machinery (SAM) Complex functions in the assembly of β-barrel in the mitochondrial membrane. The SAM complex is made up of three subunits, Sam35, Sam37, and Sam50. While both Sam35 and Sam37 are peripheral membrane proteins that are not required for survival, Sam50 interacts with the MICOS complex to connect the inner and outer mitochondrial membranes and forms the mitochondrial intermembrane space bridging (MIB) complex. Specifically, Sam50 stabilizes the MIB complex for protein transport, respiratory chain complex assembly, and cristae integrity regulation. To structurally form and sustain the cristae, the MICOS complex assembles at the cristae junction and binds directly to Sam50. However, the role of Sam50 in overall mitochondrial structure and metabolism in skeletal muscle remains unclear. Here, we use SBF-SEM and Amira software perform 3D renderings of mitochondria and autophagosomes in human myotubes. Beyond this, Gas Chromatography-Mass Spectrometry-based metabolomics was utilized to interrogate differential changes of the metabolites in wild-type (WT) and Sam50 deficient myotubes. Ablation of Sam50 , revealed increases in ß-Alanine, propanoate, and phenylalanine, and tyrosine metabolism. Additionally, we observed that mitochondrial fragmentation and autophagosome formation was increased in Sam50 -deficient myotubes compared to control myotubes. Beyond this, the metabolomic analysis revealed an increase in amino acid metabolism and fatty acid metabolism. XF24 Seahorse Analyzer shows that oxidative capacity is further impaired upon ablation of Sam50 in both murine and human myotubes. Together, these data suggest Sam50 is critical for establishing and maintaining mitochondria, mitochondrial cristae structure, and mitochondrial metabolism.

DOI: https://doi.org/10.1101/2023.05.20.541602
Sports Med Open. 2023 Jun 06. 9(1): 41

Effects of Exercise Training on Muscle Quality in Older Individuals: A Systematic Scoping Review with Meta-Analyses.

Tibor Hortobágyi, Tomas Vetrovsky, Jennifer S Brach, Martijn van Haren, Krystof Volesky, Regis Radaelli, Pedro Lopez, Urs Granacher.

   BACKGROUND: The quantity and quality of skeletal muscle are important determinants of daily function and metabolic health. Various forms of physical exercise can improve muscle function, but this effect can be inconsistent and has not been systematically examined across the health-neurological disease continuum. The purpose of this systematic scoping review with meta-analyses was to determine the effects and potential moderators of exercise training on morphological and neuromuscular muscle quality (MMQ, NMQ) in healthy older individuals. In addition and in the form of a scoping review, we examined the effects of exercise training on NMQ and MMQ in individuals with neurological conditions.
METHODS: A systematic literature search was performed in the electronic databases Medline, Embase, and Web of Science. Randomized controlled trials were included that examined the effects of exercise training on muscle quality (MQ) in older individuals with and without neurological conditions. Risk of bias and study quality were assessed (Cochrane Risk of Bias Tool 2.0). We performed random-effects models using robust variance estimation and tested moderators using the approximate Hotelling-Zhang test.
RESULTS: Thirty studies (n = 1494, 34% females) in healthy older individuals and no studies in individuals with neurological conditions were eligible for inclusion. Exercise training had small effects on MMQ (g = 0.21, 95% confidence interval [CI]: 0.03-0.40, p = 0.029). Heterogeneity was low (median I2 = 16%). Training and demographic variables did not moderate the effects of exercise on MMQ. There was no association between changes in MMQ and changes in functional outcomes. Exercise training improved NMQ (g = 0.68, 95% CI 0.35-1.01, p < 0.000) across all studies, in particular in higher-functioning older individuals (g = 0.72, 95% CI 0.38-1.06, p < 0.001), in lower extremity muscles (g = 0.74, 95% CI 0.35-1.13, p = 0.001), and after resistance training (g = 0.91; 95% CI 0.42-1.41, p = 0.001). Heterogeneity was very high (median I2 = 79%). Of the training and demographic variables, only resistance training moderated the exercise-effects on NMQ. High- versus low-intensity exercise moderated the exercise-effects on NMQ, but these effects were considered unreliable due to a low number of studies at high intensity. There was no association between changes in NMQ and changes in functional outcomes.
CONCLUSION: Exercise training has small effects on MMQ and medium-large effects on NMQ in healthy older individuals. There was no association between improvements in MQ and increases in muscle strength, mobility, and balance. Information on dose-response relations following training is currently lacking. There is a critical gap in muscle quality data for older individuals with lower function and neurological conditions after exercise training. Health practitioners should use resistance training to improve muscle function in older individuals. Well-designed studies are needed to examine the relevance of exercise training-induced changes in MQ in daily function in older individuals, especially to those with lower function and neurological conditions.

Keywords:  Aging; Intramuscular fat; Muscle mass; Neurological disease; Resistance training

DOI:  https://doi.org/10.1186/s40798-023-00585-5