bims-moremu 2024-12-08 papers

bims-moremu

Biomed News

on Molecular regulators of muscle mass

Issue of 2024–12–08
23 papers selected by
Anna Vainshtein, Craft Science Inc.

Clockwork conditioning: Aligning the skeletal muscle clock with time-of-day exercise for cardiometabolic health.
Fibro-adipogenic progenitor cells in skeletal muscle unloading: metabolic and functional impairments.
Exercise promotes skeletal muscle growth in adolescents via modulating Mettl3-mediated m6A methylation of MyoD in muscle satellite cells.
Skeletal muscle mitochondrial fragmentation predicts age-associated decline in physical capacity.
Chronic alcohol-related myopathy: a closer look at the role of lipids.
Circadian Dysfunction in the Skeletal Muscle Impairs Limb Perfusion and Muscle Regeneration in Peripheral Artery Disease.
Potential compensatory mechanisms preserving cardiac function in myotubular myopathy.
Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded noncoding RNAs.
Menstrual cycle phase does not influence muscle protein synthesis or whole-body myofibrillar proteolysis in response to resistance exercise.
Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation and metal homeostasis.
Rapamycin administration causes a decrease in muscle contractile function and systemic glucose intolerance concomitant with reduced skeletal muscle Rictor, the mTORC2 component, expression independent of energy intake in young rats.
Multiomics profiling of DNA methylation, microRNA, and mRNA in skeletal muscle from monozygotic twin pairs discordant for type 2 diabetes identifies dysregulated genes controlling metabolism.
Skeletal Muscle mRNA Splicing Variants Association With Four Different Fitness and Energetic Measures in the GESTALT Study.
SIX transcription factors are necessary for the activation of DUX4 expression in facioscapulohumeral muscular dystrophy.
An adult myogenic cell line of the Japanese fire-bellied newt Cynops pyrrhogaster.
Skeletal muscle proteome differs between young APOE3 and APOE4 targeted replacement mice in a sex-dependent manner.
Development of a Myogenin minimal promoter-based system for visualizing the degree of myogenic differentiation.
Estradiol deficiency as a consequence of aging contributes to the depletion of the satellite cell pool in female mice.
Myofibers cultured in viscoelastic hydrogels reveal the effects of integrin-binding and mechanosensing on muscle satellite cells.
Training Status Influences Regulation of Muscle and PBMC TLR4 Expression and Systemic Cytokine Responses to Vigorous Endurance Exercise.
Fibroblast diversification is an embryonic process dependent on muscle contraction.
Macrophages excite muscle spindles with glutamate to bolster locomotion.
The miR-6240 target gene Igf2bp3 promotes myoblast fusion by enhancing myomaker mRNA stability.

J Mol Cell Cardiol. 2024 Nov 29. pii: S0022-2828(24)00201-3. [Epub ahead of print]198 36-44

Clockwork conditioning: Aligning the skeletal muscle clock with time-of-day exercise for cardiometabolic health.

Spencer B Procopio, Karyn A Esser.

  Circadian rhythms have evolved to synchronize gene expression, physiology, and behavior with time-of-day changes in the external environment. In every mammalian cell exists a core clock mechanism that consists of a transcriptional-translational feedback loop that drives rhythmic gene expression. Circadian disruption, as observed in shift workers and genetic mouse models, contributes to the onset and progression of cardiometabolic disorders. The central clock, located in the hypothalamus, is uniquely sensitive to external light cues, while the peripheral clocks are responsive to non-photic stimuli such as feeding and activity in addition to signals from the central clock. Recent research has illustrated the sensitivity of the skeletal muscle circadian clock to exercise timing, offering a promising avenue for therapeutic intervention in cardiometabolic health. Here we provide an in-depth examination of the molecular mechanisms underlying skeletal muscle clock function and its impact on cardiometabolic pathways, including glucose and lipid metabolism, as well as inflammation. To highlight the role of exercise as a time-cue for the skeletal muscle clock, we discuss evidence of exercise-induced shifts in the skeletal muscle clock and the differential response to exercise performed at different times of the day. Furthermore, we present data in support of time-of-day exercise as a potential therapeutic strategy for mitigating cardiometabolic disease burden. By exploring the relationship between the skeletal muscle clock, exercise timing, and cardiometabolic health, we identify new areas for future research and offer valuable insights into novel therapeutic approaches aimed at improving cardiometabolic disease outcomes.

Keywords:  Cardiometabolic disease; Circadian rhythm; Exercise; Muscle clock; Skeletal muscle

DOI:  https://doi.org/10.1016/j.yjmcc.2024.11.011
Skelet Muscle. 2024 Dec 06. 14(1): 31

Fibro-adipogenic progenitor cells in skeletal muscle unloading: metabolic and functional impairments.

Margarita Sorokina, Danila Bobkov, Natalia Khromova, Natalia Vilchinskaya, Boris Shenkman, Anna Kostareva, Renata Dmitrieva.

   BACKGROUND: Skeletal muscle resident fibro-adipogenic progenitor cells (FAPs) control skeletal muscle regeneration providing a supportive role for muscle stem cells. Altered FAPs characteristics have been shown for a number of pathological conditions, but the influence of temporary functional unloading of healthy skeletal muscle on FAPs remains poorly studied. This work is aimed to investigate how skeletal muscle disuse affects the functionality and metabolism of FAPs.
METHODS: Hindlimb suspension (HS) rat model employed to investigate muscle response to decreased usage. FAPs were purified from m. soleus functioning muscle (Contr) and after functional unloading for 7 and 14 days (HS7 and HS14). FAPs were expanded in vitro, and tested for: immunophenotype; in vitro expansion rate, and migration activity; ability to differentiate into adipocytes in vitro; metabolic changes. Crosstalk between FAPs and muscle stem cells was estimated by influence of medium conditioned by FAP's on migration and myogenesis of C2C12 myoblasts. To reveal the molecular mechanisms behind unloading-induced alterations in FAP's functionality transcriptome analysis was performed.
RESULTS: FAPs isolated from Contr and HS muscles exhibited phenotype of MSC cells. FAPs in vitro expansion rate and migration were altered by functional unloading conditions. All samples of FAPs demonstrated the ability to adipogenic differentiation in vitro, however, HS FAPs formed fat droplets of smaller volume and transcriptome analysis showed fatty acids metabolism and PPAR signaling suppression. Skeletal muscle unloading resulted in metabolic reprogramming of FAPs: decreased spare respiratory capacity, decreased OCR/ECAR ratio detected in both HS7 and HS14 samples point to reduced oxygen consumption, decreased potential for substrate oxidation and a shift to glycolytic metabolism. Furthermore, C2C12 cultures treated with medium conditioned by FAPs showed diverse alterations: while the HS7 FAPs-derived paracrine factors supported the myoblasts fusion, the HS14-derived medium stimulated proliferation of C2C12 myoblasts; these observations were supported by increased expression of cytokines detected by transcriptome analysis.
CONCLUSION: the results obtained in this work show that the skeletal muscle functional unloading affects properties of FAPs in time-dependent manner: in atrophying skeletal muscle FAPs act as the sensors for the regulatory signals that may stimulate the metabolic and transcriptional reprogramming to preserve FAPs properties associated with maintenance of skeletal muscle homeostasis during unloading and in course of rehabilitation.

Keywords:  Adipogenic differentiation; C2C12 myoblasts; Cellular metabolism; FAPs-myoblasts interactions; Fibro-adipogenic progenitors (FAPs); Functional unloading; Skeletal muscle regeneration; Soleus muscle; Transcriptome analysis

DOI:  https://doi.org/10.1186/s13395-024-00362-2
Cell Mol Biol Lett. 2024 Dec 04. 29(1): 150

Exercise promotes skeletal muscle growth in adolescents via modulating Mettl3-mediated m6A methylation of MyoD in muscle satellite cells.

Shujing Feng, Hao Zhou, Xingzuan Lin, Siyuan Zhu, Huifang Chen, Han Zhou, Ru Wang, Peng Wang, Xiexiang Shao, Jianhua Wang.

   BACKGROUND: Exercise exerts positive impacts on skeletal muscle health and homeostasis. Emerging evidence suggests that m6A methylation is involved in various physiological processes. However, the impact of exercise on adolescent skeletal muscle growth and the underlying epigenetic mechanisms remain poorly understood.
METHODS: The lower-limb skeletal muscles were harvested from exercise and control groups to compare the skeletal muscle growth in adolescents. mRNA sequencing was conducted to explore the mechanisms underlying enhanced skeletal muscle growth following exercise. The effects and mechanisms of Mettl3-mediated m6A methylation on adolescent skeletal muscle growth were investigated using muscle satellite cell (MuSC)-specific Mettl3 knockout (KO) mice. The potential function of MyoD for skeletal muscle growth in adolescents was explored by phenotypes after overexpression and evaluation of in vivo myogenesis. Additionally, the effects of the methyl donor betaine on adolescent skeletal muscle growth were investigated in vitro and in vivo.
RESULTS: Exercise could promote skeletal muscle growth in adolescents. Sequencing data analysis and confirmation assays uncovered that exercise significantly increased Mettl3-mediated m6A methylation and elevated the expression levels of activation marker MyoD in MuSCs. Establishment of MuSC-specific Mettl3 KO mice further demonstrated that Mettl3-mediated m6A methylation in MyoD contributed to skeletal muscle growth during adolescence. Mettl3-mediated m6A methylation regulated MyoD mRNA stability at the posttranscriptional level in MuSCs, with a functional site at 234 bp A. Increased expression of MyoD could contribute to myogenesis of adolescent MuSCs. Furthermore, the methyl donor betaine could enhance MyoD expression, contributing to MuSCs activation and skeletal muscle growth in adolescents by boosting m6A methylation levels.
CONCLUSIONS: Exercise promoted skeletal muscle growth in adolescents through facilitating MyoD mRNA stability of MuSCs in a Mettl3-mediated m6A-dependent manner. The methyl donor betaine could be a potential alternative to exercise for promoting adolescent skeletal muscle growth by directly augmenting the global levels of m6A methylation. These findings may provide a theoretical foundation for encouraging daily fitness exercise and ensuring healthy growth in adolescents.

Keywords:  Adolescent skeletal muscle growth; Betaine; Exercise; Mettl3; MuSCs; m6A methylation

DOI:  https://doi.org/10.1186/s11658-024-00670-x
Aging Cell. 2024 Dec 04. e14386

Skeletal muscle mitochondrial fragmentation predicts age-associated decline in physical capacity.

Richie P Goulding, Braeden T Charlton, Ellen A Breedveld, Matthijs van der Laan, Anne R Strating, Wendy Noort, Aryna Kolodyazhna, Brent Appelman, Michèle van Vugt, Anita E Grootemaat, Nicole N van der Wel, Jos J de Koning, Frank W Bloemers, Rob C I Wüst.

  Ageing substantially impairs skeletal muscle metabolic and physical function. Skeletal muscle mitochondrial health is also impaired with ageing, but the role of skeletal muscle mitochondrial fragmentation in age-related functional decline remains imprecisely characterized. Here, using a cross-sectional study design, we performed a detailed comparison of skeletal muscle mitochondrial characteristics in relation to in vivo markers of exercise capacity between young and middle-aged individuals. Despite similar overall oxidative phosphorylation capacity (young: 99 ± 17 vs. middle-aged: 99 ± 27 pmol O2.s-1.mg-1, p = 0.95) and intermyofibrillar mitochondrial density (young: 5.86 ± 0.57 vs. middle-aged: 5.68 ± 1.48%, p = 0.25), older participants displayed a more fragmented intermyofibrillar mitochondrial network (young: 1.15 ± 0.17 vs. middle-aged: 1.55 ± 0.15 A.U., p < 0.0001), a lower mitochondrial cristae density (young: 23.40 ± 7.12 vs. middle-aged: 13.55 ± 4.10%, p = 0.002) and a reduced subsarcolemmal mitochondrial density (young: 22.39 ± 6.50 vs. middle-aged: 13.92 ± 4.95%, p = 0.005). Linear regression analysis showed that 87% of the variance associated with maximal oxygen uptake could be explained by skeletal muscle mitochondrial fragmentation and cristae density alone, whereas subsarcolemmal mitochondrial density was positively associated with the capacity for oxygen extraction during exercise. Intramuscular lipid accumulation was positively associated with mitochondrial fragmentation and negatively associated with cristae density. Collectively, our work highlights the critical role of skeletal muscle mitochondria in age-associated declines in physical function.

Keywords:  ageing; maximal oxygen uptake; mitochondrial morphology; mitochondrial respiration; skeletal muscle

DOI:  https://doi.org/10.1111/acel.14386
Front Physiol. 2024 ;15 1492405

Chronic alcohol-related myopathy: a closer look at the role of lipids.

Muni Swamy Ganjayi, Thomas A Krauss, Craig R G Willis, Cory W Baumann.

  Chronic alcohol-related myopathy (CAM), characterized by muscle atrophy and weakness, arises from prolonged excessive ethanol (EtOH) intake. The precise mechanisms by which EtOH induces skeletal muscle atrophy are not fully understood. This article posits that the pathophysiology of CAM may be significantly influenced by how EtOH modifies lipid profiles and alters lipid composition and content in skeletal muscle. We review existing literature on lipid alterations in CAM-afflicted individuals and analogous animal models, discuss EtOH's direct and indirect effects on skeletal muscle lipids, and present specific instances where lipids contribute to muscle atrophy. This article advocates for a novel viewpoint, suggesting that lipid dysregulation may be the principal factor in EtOH-induced muscle wasting, offering a different angle to approach CAM research and treatment strategies.

Keywords:  adipose tissue; atrophy; metabolism; skeletal muscle; weakness

DOI:  https://doi.org/10.3389/fphys.2024.1492405
Arterioscler Thromb Vasc Biol. 2024 Dec 05.

Circadian Dysfunction in the Skeletal Muscle Impairs Limb Perfusion and Muscle Regeneration in Peripheral Artery Disease.

Pei Zhu, Calvin L Chao, Adam W T Steffeck, Caitlyn Dang, Noah X Hamlish, Eric M Pfrender, Bin Jiang, Clara B Peek.

   BACKGROUND: Peripheral artery disease (PAD), caused by atherosclerosis, leads to limb ischemia, muscle damage, and impaired mobility in the lower extremities. Recent studies suggest that circadian rhythm disruptions can hinder vascular repair during ischemia, but the specific tissues involved and the impact on muscle health remain unclear. This study investigates the role of the skeletal muscle circadian clock in muscle adaptation to ischemic stress using a surgical mouse model of hindlimb ischemia.
METHODS: We performed secondary analysis of publicly available RNA-sequencing data sets derived from patients with PAD to identify the differential expression of circadian-related genes in endothelial cells and ischemic limb skeletal muscles. We used mice with specific genetic loss of the circadian clock activator, BMAL1 (brain and muscle ARNT-like 1), in adult skeletal muscle tissues (Bmal1muscle). Bmal1muscle mice and controls underwent femoral artery ligation surgery to induce hindlimb ischemia. Laser Doppler imaging was used to assess limb perfusion at various time points after the surgery. Muscle tissues were analyzed with RNA sequencing and histological examination to investigate PAD-related muscle pathologies. Additionally, we studied the role of BMAL1 in muscle fiber adaptation to hypoxia using RNA and assay for transposase-accessible chromatin with sequencing analyses in primary myotube culture model.
RESULTS: Disrupted expression of circadian rhythm-related genes was observed in existing RNA-sequencing data sets from endothelial cells and ischemic limb skeletal muscles derived from patients with PAD. Genetic loss of Bmal1 specifically in adult mouse skeletal muscle tissues delayed reperfusion recovery following induction of hindlimb ischemia. Histological examination of muscle tissues showed reduced regenerated myofiber number and a decreased proportion of type IIB fast-twitch myofibers in Bmal1muscle mouse muscles in the ischemic limbs but not in their contralateral nonischemic limbs. Transcriptomic analysis revealed abrogated metabolic, angiogenic, and myogenic pathways relevant to hypoxia adaptation in Bmal1muscle mouse muscles. These changes were corroborated in Bmal1-deficient cultured primary myotubes cultured under hypoxic conditions.
CONCLUSIONS: Circadian clock in skeletal muscle is crucial for the muscle's response to hypoxia during hindlimb ischemia. Targeting the muscle circadian clock may have therapeutic potential for enhancing muscle response to reduced blood flow and promoting recovery in conditions such as PAD.

Keywords:  atherosclerosis; femoral artery; ischemia; perfusion; reperfusion

DOI:  https://doi.org/10.1161/ATVBAHA.124.321772
Cell Mol Life Sci. 2024 Dec 03. 81(1): 476

Potential compensatory mechanisms preserving cardiac function in myotubular myopathy.

Alix Simon, Nadège Diedhiou, David Reiss, Marie Goret, Erwan Grandgirard, Jocelyn Laporte.

  X-Linked myotubular myopathy (XLMTM) is characterized by severe skeletal muscle weakness and reduced life expectancy. The pathomechanism and the impact of non-muscular defects affecting survival, such as liver dysfunction, are poorly understood. Here, we investigated organ-specific effects of XLMTM using the Mtm1-/y mouse model. We performed RNA-sequencing to identify a common mechanism in different skeletal muscles, and to explore potential phenotypes and compensatory mechanisms in the heart and the liver. The cardiac and hepatic function and structural integrity were assessed both in vivo and in vitro. Our findings revealed no defects in liver function or morphology. A disease signature common to several skeletal muscles highlighted dysregulation of muscle development, inflammation, cell adhesion and oxidative phosphorylation as key pathomechanisms. The heart displayed only mild functional alterations without obvious structural defects. Transcriptomic analyses revealed an opposite dysregulation of mitochondrial function, cell adhesion and beta integrin trafficking pathways in cardiac muscle compared to skeletal muscles. Despite this dysregulation, biochemical and cellular experiments demonstrated that these pathways were strongly affected in skeletal muscle and normal in cardiac muscle. Moreover, biomarkers reflecting the molecular activity of MTM1, such as PtdIns3P and dynamin 2 levels, were increased in the skeletal muscles but not in cardiac muscle. Overall, these data suggest a compensatory mechanism preserving cardiac function, pointing to potential therapeutic targets to cure the severe skeletal muscle defects in XLMTM.

Keywords:  Centronuclear myopathy; Dynamin; Integrin; Myotubularin; Omics; Phosphoinositides

DOI:  https://doi.org/10.1007/s00018-024-05512-9
FASEB J. 2024 Dec 15. 38(23): e70223

Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded noncoding RNAs.

Jessica Silver, Adam J Trewin, Stella Loke, Larry Croft, Mark Ziemann, Megan Soria, Hayley Dillon, Søren Nielsen, Séverine Lamon, Glenn D Wadley.

Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localize within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localize within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimized to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small RNA sequencing libraries were successfully constructed from as little as 1.8 ng mitochondrial RNA input. Small RNA sequencing of mitochondria from rat myoblasts revealed the enrichment of over 200 miRNAs. Whole-transcriptome RNA sequencing of enzymatically purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localized within skeletal muscle mitochondria.

DOI: https://doi.org/10.1096/fj.202401618R
J Physiol. 2024 Dec 04.

Menstrual cycle phase does not influence muscle protein synthesis or whole-body myofibrillar proteolysis in response to resistance exercise.

Lauren M Colenso-Semple, James McKendry, Changhyun Lim, Philip J Atherton, Daniel J Wilkinson, K Smith, Stuart M Phillips.

  It has been hypothesised that skeletal muscle protein turnover is affected by menstrual cycle phase with a more anabolic environment during the follicular vs. the luteal phase. We assessed the influence of menstrual cycle phase on muscle protein synthesis and myofibrillar protein breakdown in response to 6 days of controlled resistance exercise in young females during peak oestrogen and peak progesterone, using stable isotopes, unbiased metabolomics and muscle biopsies. We used comprehensive menstrual cycle phase-detection methods, including cycle tracking, blood samples and urinary test kits, to classify menstrual phases. Participants (n = 12) completed two 6 day study phases in a randomised order: late follicular phase and mid-luteal phase. Participants performed unilateral resistance exercise in each menstrual cycle phase, exercising the contralateral leg in each phase in a counterbalanced manner. Follicular phase myofibrillar protein synthesis (MPS) rates were 1.33 ± 0.27% h-1 in the control leg and 1.52 ± 0.27% h-1 in the exercise leg. Luteal phase MPS was 1.28 ± 0.27% h-1 in the control leg and 1.46 ± 0.25% h-1 in the exercise leg. We observed a significant effect of exercise (P < 0.001) but no effect of cycle phase or interaction. There was no significant effect of menstrual cycle phase on whole-body myofibrillar protein breakdown (P = 0.24). Using unbiased metabolomics, we observed no notable phase-specific changes in circulating blood metabolites associated with any particular menstrual cycle phase. Fluctuations in endogenous ovarian hormones influenced neither MPS, nor MPB in response to resistance exercise. Skeletal muscle is not more anabolically responsive to resistance exercise in a particular menstrual cycle phase. KEY POINTS: It has been hypothesised that the follicular (peak oestrogen) vs. the luteal (peak progesterone) phase of the menstrual cycle is more advantageous for skeletal muscle anabolism in response to resistance exercise. Using best practice methods to assess menstrual cycle status, we measured integrated (over 6 days) muscle protein synthesis (MPS) and myofibrillar protein breakdown (MPB) following resistance exercise in females (n = 12) in their follicular and luteal phases. We observed the expected differences in oestrogen and progesterone concentrations that confirmed our participants' menstrual cycle phase; however, there were no notable metabolic pathway differences, as measured using metabolomics, between cycle phases. We observed that resistance exercise stimulated MPS, but there was no effect of menstrual cycle phase on either resting or exercise-stimulated MPS or MPB. Our data show no greater anabolic effect of resistance exercise in the follicular vs. the luteal phase of the menstrual cycle.

Keywords:  exercise; human muscle; menstrual cycle; protein metabolism

DOI:  https://doi.org/10.1113/JP287342
PLoS Genet. 2024 Dec 05. 20(12): e1011495

Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation and metal homeostasis.

Odette Verdejo-Torres, David C Klein, Lorena Novoa-Aponte, Jaime Carrazco-Carrillo, Denzel Bonilla-Pinto, Antonio Rivera, Arpie Bakhshian, Fa'alataitaua M Fitisemanu, Martha L Jiménez-González, Lyra Flinn, Aidan T Pezacki, Antonio Lanzirotti, Luis Antonio Ortiz Frade, Christopher J Chang, Juan G Navea, Crysten E Blaby-Haas, Sarah J Hainer, Teresita Padilla-Benavides.

Copper (Cu) is essential for respiration, neurotransmitter synthesis, oxidative stress response, and transcription regulation, with imbalances leading to neurological, cognitive, and muscular disorders. Here we show the role of a novel Cu-binding protein (Cu-BP) in mammalian transcriptional regulation, specifically on skeletal muscle differentiation using murine primary myoblasts. Utilizing synchrotron X-ray fluorescence-mass spectrometry, we identified murine cysteine-rich intestinal protein 2 (mCrip2) as a key Cu-BP abundant in both nuclear and cytosolic fractions. mCrip2 binds two to four Cu+ ions with high affinity and presents limited redox potential. CRISPR/Cas9-mediated deletion of mCrip2 impaired myogenesis, likely due to Cu accumulation in cells. CUT&RUN and transcriptome analyses revealed its association with gene promoters, including MyoD1 and metallothioneins, suggesting a novel Cu-responsive regulatory role for mCrip2. Our work describes the significance of mCrip2 in skeletal muscle differentiation and metal homeostasis, expanding understanding of the Cu-network in myoblasts. Copper (Cu) is essential for various cellular processes, including respiration and stress response, but imbalances can cause serious health issues. This study reveals a new Cu-binding protein (Cu-BP) involved in muscle development in primary myoblasts. Using unbiased metalloproteomic techniques and high throughput sequencing, we identified mCrip2 as a key Cu-BP found in cell nuclei and cytoplasm. mCrip2 binds up to four Cu+ ions and has a limited redox potential. Deleting mCrip2 using CRISPR/Cas9 disrupted muscle formation due to Cu accumulation. Further analyses showed that mCrip2 regulates the expression of genes like MyoD1, essential for muscle differentiation, and metallothioneins in response to copper supplementation. This research highlights the importance of mCrip2 in muscle development and metal homeostasis, providing new insights into the Cu-network in cells.

DOI: https://doi.org/10.1371/journal.pgen.1011495
PLoS One. 2024 ;19(12): e0312859

Rapamycin administration causes a decrease in muscle contractile function and systemic glucose intolerance concomitant with reduced skeletal muscle Rictor, the mTORC2 component, expression independent of energy intake in young rats.

Satoru Ato, Chieri Oya, Riki Ogasawara.

Emerging evidence suggests the potential of rapamycin, an antibiotic from Streptomyces hygroscopicus that functions as a mechanistic target of rapamycin (mTOR) inhibitor, as a mimetic of caloric restriction (CR) for maintaining skeletal muscle health. Several studies showed that rapamycin administration (RAP) reduced appetite and energy intake. However, the physiological and molecular differences between RAP and CR in skeletal muscle are not fully understood. Here we observed the effects of 4 weeks of RAP administration and CR corresponding to the reduction in energy intake produced by RAP administration (PF, paired feeding) on fast glycolytic and slow oxidative muscle in young adult rats. We found that 4 weeks of RAP demonstrated low fast-glycolytic muscle mass with smaller type I and IIb/x myofiber size independent of the energy intake. In addition, PF improved the contractile function of the plantar flexor muscle, whereas RAP did not improve its function. The suppressing response of mTORC1 signaling to RAP is greater in slow-oxidative muscles than in fast-glycolytic muscles. In addition, systemic glucose tolerance was exacerbated by RAP, with reduced expression of Rictor and hexokinase in skeletal muscle. These observations imply that RAP may have a slight but significant negative impact and it obviously different to CR in young adult skeletal muscle.

DOI: https://doi.org/10.1371/journal.pone.0312859
BMC Med. 2024 12 02. 22(1): 572

Multiomics profiling of DNA methylation, microRNA, and mRNA in skeletal muscle from monozygotic twin pairs discordant for type 2 diabetes identifies dysregulated genes controlling metabolism.

Charlotte Ling, Magdalena Vavakova, Bilal Ahmad Mir, Johanna Säll, Alexander Perfilyev, Melina Martin, Per-Anders Jansson, Cajsa Davegårdh, Olof Asplund, Ola Hansson, Allan Vaag, Emma Nilsson.

   BACKGROUND: A large proportion of skeletal muscle insulin resistance in type 2 diabetes (T2D) is caused by environmental factors.
METHODS: By applying multiomics mRNA, microRNA (miRNA), and DNA methylation platforms in biopsies from 20 monozygotic twin pairs discordant for T2D, we aimed to delineate the epigenetic and transcriptional machinery underlying non-genetic muscle insulin resistance in T2D.
RESULTS: Using gene set enrichment analysis (GSEA), we found decreased mRNA expression of genes involved in extracellular matrix organization, branched-chain amino acid catabolism, metabolism of vitamins and cofactors, lipid metabolism, muscle contraction, signaling by receptor tyrosine kinases pathways, and translocation of glucose transporter 4 (GLUT4) to the plasma membrane in muscle from twins with T2D. Differential expression levels of one or more predicted target relevant miRNA(s) were identified for approximately 35% of the dysregulated GSEA pathways. These include miRNAs with a significant overrepresentation of targets involved in GLUT4 translocation (miR-4643 and miR-548z), signaling by receptor tyrosine kinases pathways (miR-607), and muscle contraction (miR-4658). Acquired DNA methylation changes in skeletal muscle were quantitatively small in twins with T2D compared with the co-twins without T2D. Key methylation and expression results were validated in muscle, myotubes, and/or myoblasts from unrelated subjects with T2D and controls. Finally, mimicking T2D-associated changes by overexpressing miR-548 and miR-607 in cultured myotubes decreased expression of target genes, GLUT4 and FGFR4, respectively, and impaired insulin-stimulated phosphorylation of Akt (Ser473) and TBC1D4.
CONCLUSIONS: Together, we show that T2D is associated with non- and epigenetically determined differential transcriptional regulation of pathways regulating skeletal muscle metabolism and contraction.

Keywords:  DNA methylation; Discordant monozygotic twins; Epigenetics; Gene expression; MicroRNA (miRNA); Skeletal muscle; Type 2 diabetes (T2D)

DOI:  https://doi.org/10.1186/s12916-024-03789-y
J Cachexia Sarcopenia Muscle. 2024 Dec 02.

Skeletal Muscle mRNA Splicing Variants Association With Four Different Fitness and Energetic Measures in the GESTALT Study.

Stefano Donega, Nirad Banskota, Esha Gupta, Marta Gonzalez-Freire, Ann Zenobia Moore, Ceereena Ubaida-Mohien, Rachel Munk, Linda Zukley, Yulan Piao, Chris Bergeron, Jan Bergeron, Arsun Bektas, Marta Zampino, Carole Stagg, Fred Indig, Lisa M Hartnell, Mary Kaileh, Kenneth Fishbein, Richard G Spencer, Myriam Gorospe, Supriyo De, Josephine M Egan, Ranjan Sen, Luigi Ferrucci.

   BACKGROUND: Physical activity is essential for maintaining muscle mitochondrial function and aerobic capacity. The molecular mechanisms underlying such protective effects are incompletely understood, in part because it is difficult to separate the effects of disease status and physical activity. We explored the association of human skeletal muscle transcriptomic with four measures of energetics and mitochondria oxidative capacity in healthy individuals.
METHODS: Using RNA sequencing of vastus lateralis muscle biopsies from 82 GESTALT participants (52 males, aged 22-89 years), we explored gene and splicing variant expression profiles associated with self-reported physical activity, peak oxygen consumption (VO2 peak), muscle oxidative capacity (kPCr) and mitochondrial respiration (Mit-O2 flux). The effect of aging on gene expression was examined in participants with low and high VO2 peak.
RESULTS: The four measures of energetics were negative correlated with age and generally intercorrelated. We identified protein-coding genes associated with four energetic measures adjusting for age, muscle fiber-ratio, sex and batch effect. Mitochondrial pathways were overrepresented across all energetic variables, albeit with little overlap at the gene level. Alternative spliced transcript isoforms associated with energetics were primarily enriched for cytoplasmic ribonucleoprotein granules. The splicing pathway was up-regulated with aging in low but not in high fitness participants, and transcript isoforms detected in the low fitness group pertain to processes such as cell cycle regulation, RNA/protein localization, nuclear transport and catabolism.
CONCLUSIONS: A consistent mitochondrial signature emerged across all energetic measures. Alternative splicing was enhanced in older, low fitness participants supporting the energy-splicing axis hypothesis. The identified splicing variants were enriched in pathways involving the accumulation of ribonucleoproteins in cytoplasmic granules, whose function remains unclear. Further research is needed to understand the function of these proteoforms in promoting adaptation to low energy availability.

Keywords:  VO2; aging; alternative splicing; energy; exercise; kPCr; mitochondria respirometry; muscle; physical activity

DOI:  https://doi.org/10.1002/jcsm.13603
Skelet Muscle. 2024 Dec 03. 14(1): 30

SIX transcription factors are necessary for the activation of DUX4 expression in facioscapulohumeral muscular dystrophy.

Amelia Fox, Jonathan Oliva, Rajanikanth Vangipurapu, Francis M Sverdrup.

   BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a common and progressive muscle wasting disease that is characterized by muscle weakness often first noticed in the face, the shoulder girdle and upper arms before progressing to the lower limb muscles. FSHD is caused by the misexpression of the Double Homeobox 4 (DUX4) transcription factor in skeletal muscle. While epigenetic derepression of D4Z4 macrosatellite repeats underlies DUX4 misexpression, our understanding of the complex transcriptional activation of DUX4 is incomplete.
METHODS: To identify potential DUX4-regulatory factors, we used small interfering RNAs (siRNAs) to knockdown SIX family transcription factors (SIX1, 2, 4, 5) in patient-derived FSHD1 and FSHD2 myoblasts that were differentiated to form multinucleated myotubes. Quantitative real-time polymerase chain reaction was used to measure changes in DUX4 mRNA, DUX4 target gene expression and myogenic markers. Staining for SIX1 and SIX2 with specific antibodies was performed in FSHD myoblasts and myotubes. To assess reciprocal effects of DUX4 on SIX1, 2, and 4 expression, we utilized a doxycycline-inducible DUX4 myoblast cell line.
RESULT: We show that SIX1, 2 and 4 transcription factors, regulators of embryonic development, muscle differentiation, regeneration and homeostasis, are necessary for myogenic differentiation-dependent DUX4 expression in FSHD muscle cells. Using siRNA, we demonstrate SIX1, SIX2, and SIX4 to be critical factors involved in the induction of DUX4 transcription in differentiating FSHD myotubes in vitro. siRNA dual knockdown of SIX1 and SIX2 resulted in a ~ 98% decrease of DUX4 and DUX4 target genes, suggesting that SIX1 and SIX2 are the most critical in promoting DUX4 expression. Importantly, we show that DUX4 downregulates SIX RNA levels, suggesting negative feedback regulation.
CONCLUSIONS: In this study, we identified a family of developmental regulators that promote aberrant DUX4 expression in FSHD1 and FSHD2 differentiating muscle cells. Our findings highlight the critical involvement of SIX transcription factors (SIX1, 2, 4) in the pathogenesis of FSHD by serving as necessary factors that function in the promotion of DUX4 expression following epigenetic derepression of the D4Z4 repeats.

Keywords:  D4Z4 macrosatellite repeats; DUX4 Double homeobox 4; FSHD Facioscapulohumeral muscular dystrophy; SIX transcription factors; siRNA Small interfering RNA

DOI:  https://doi.org/10.1186/s13395-024-00361-3
Sci Rep. 2024 12 03. 14(1): 30041

An adult myogenic cell line of the Japanese fire-bellied newt Cynops pyrrhogaster.

Shota Shiga, Yuri Murakami, Zixiao Wang, Ryo Ando, Martin Miguel Casco-Robles, Fumiaki Maruo, Fubito Toyama, Chikafumi Chiba.

  Adult myogenic cell lines are useful to study muscle development, repair and regeneration. In newts, which are known for their high regenerative capacity, myogenic cell lines have not been established in species other than the Eastern newt Notophthalmus viridescens. In this study, we established another myogenic cell line, named CpM01, from the skeletal muscle of the forearm of the adult Japanese fire-bellied newt Cynops pyrrhogaster. CpM01 maintained high proliferative ability even after numerous passages, and could be induced to differentiate into myotubes by changing the culture medium. CpM01 expressed myogenic regulatory factors (MRFs) such as Myf5, MRF4 and myogenin. Changes in the immunorectivities of MRFs during differentiation of CpM01 into myotubes were consistent with those during new muscle generation in limb regeneration. In newts, myogenic cells have two origins, muscle fibers or satellite cells. CpM01 expressed Pax7, suggesting the origin might be satellite cells. scRNA-seq analysis deeply characterized CpM01 and demonstrated that the expression patterns of myogenic genes (Pax3, Pax7, myocyte-specific enhancer factor 2 A, and genes encoding MRFs) in CpM01 are related to progress of the cell cycle. CpM01 can be a useful tool for future studies of limb muscle regeneration in adult newts.

Keywords:  Adult myogenic cell line; Limb regeneration; Myogenic regulatory factors; Newts; Satellite cells

DOI:  https://doi.org/10.1038/s41598-024-81899-6
Front Aging Neurosci. 2024 ;16 1486762

Skeletal muscle proteome differs between young APOE3 and APOE4 targeted replacement mice in a sex-dependent manner.

Chelsea N Johnson, Colton R Lysaker, Colin S McCoin, Mara R Evans, John P Thyfault, Heather M Wilkins, Jill K Morris, Paige C Geiger.

   Introduction: Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), yet it's unclear how this allele mediates risk. APOE4 carriers experience reduced mobility and faster decline in muscle strength, suggesting skeletal muscle involvement. Mitochondria are critical for muscle function and although we have reported defects in muscle mitochondrial respiration during early cognitive decline, APOE4-mediated effects on muscle mitochondria are unknown.
Methods: Here, we sought to determine the impact of APOE4 on skeletal muscle bioenergetics using young, male and female APOE3 (control) and APOE4 targeted replacement mice (n = 8 per genotype/sex combination). We examined the proteome, mitochondrial respiration, fiber size, and fiber-type distribution in skeletal muscle.
Results: We found that APOE4 alters mitochondrial pathway expression in young mouse muscle in a sex-dependent manner without affecting respiration and fiber size or composition relative to APOE3. In both sexes, the expression of mitochondrial pathways involved in electron transport, ATP synthesis, and heat production by uncoupling proteins and mitochondrial dysfunction significantly differed between APOE4 and APOE3 muscle. For pathways with predicted direction of activation, electron transport and oxidative phosphorylation were upregulated while mitochondrial dysfunction and sirtuin signaling were downregulated in female APOE4 vs. APOE3 muscle. In males, sulfur amino acid metabolism was upregulated in APOE4 vs. APOE3 muscle.
Discussion: This work highlights early involvement of skeletal muscle in a mouse model of APOE4-linked AD, which may contribute to AD pathogenesis or serve as a biomarker for brain health.

Keywords:  APOE4; Alzheimer's disease; mice; mitochondria; proteomics; skeletal muscle

DOI:  https://doi.org/10.3389/fnagi.2024.1486762
Biochem Biophys Res Commun. 2024 Nov 28. pii: S0006-291X(24)01627-9. [Epub ahead of print]741 151091

Development of a Myogenin minimal promoter-based system for visualizing the degree of myogenic differentiation.

Yoshizuki Fumoto, Shingo Takada, Yasuhito Onodera, Shigetsugu Hatakeyama, Tsukasa Oikawa.

Myogenic differentiation plays a fundamental role in myogenesis during development and in muscle regeneration. Sequential expression of myogenic regulatory factors (MRFs) including myogenin in the progenitor cells triggers the expression of effector proteins such as myosin heavy chain (MHC), leading to the terminal muscle differentiation. Although we have a snapshot-like understanding of molecules at each stage of the differentiation, how these molecules are interrelated in the continuum of myogenic differentiation remains poorly understood. In this study, we analyzed the dynamics of the minimal Myogenin promoter activity in live myoblasts. With the development of a new co-expression analysis method, we were able to reveal in detail the relationship between this Myogenin promoter activity and the expression of endogenous myogenin or MHC, as differentiation markers. Consequently, we found that our visualization system of myogenic differentiation is suitable for monitoring the transition from myoblasts to myotubes, in which the Myogenin promoter activity quantitatively represents the degree of myogenic differentiation. Thus, this system allows simultaneous observation of the degree of myoblast differentiation in relation to other molecules, which would contribute to deepening our understanding of myogenic differentiation as a continuous process.

DOI: https://doi.org/10.1016/j.bbrc.2024.151091
Aging Cell. 2024 Dec 06. e14441

Estradiol deficiency as a consequence of aging contributes to the depletion of the satellite cell pool in female mice.

Brian P Sullivan, Alexie A Larson, Ahmed S Shams, Shawna L McMillin, Mara C Ebeling, Sydney Peng, Michael Kyba, Dawn A Lowe.

  The effects of aging on the satellite cell pool have primarily been studied in male mice, where the role of cell-intrinsic versus environmental changes on satellite cell function remains contentious. Estradiol is necessary for maintenance of satellite cell pool size in adult female mice-here we investigate the hypothesis that in females, estradiol is a major environmental driver of age-associated effects on satellite cells. In 24-26 month-old ovarian senescent mice, we find the satellite cell pool size is severely diminished in certain muscles (TA and EDL) but only marginally affected in others (soleus and gastrocnemius). Supplementation with 17-beta estradiol significantly increases satellite cell pool size in the TA and EDL. To assess cell-intrinsic versus environmental regulation, we perform two transplantation experiments, Adult or Aged satellite cells transplanted into Adult recipients, and Adult satellite cells transplanted into Adult or Aged mice. These results demonstrate that the aged environment dominates over cell-autonomous age in terms of the specification of satellite cell pool size. Transcriptional profiling on satellite cells from Adult, Aged and ovariectomized mice revealed commonalities across the two estradiol-deficient conditions, Aged and ovariectomized, in GO terms from differentially expressed genes. Our findings support the hypothesis that the lack of estradiol contributes to reductions in satellite cell number in Aged female muscle, yet cells that remain are functional in terms of proliferative potential and self-renewal capacity. These findings have implications for sex hormone treatment of menopausal women and highlight the vital role of estradiol in the maintenance of the satellite cell pool.

Keywords:  aging; estradiol; satellite stem cell; sex hormones; skeletal muscle

DOI:  https://doi.org/10.1111/acel.14441
Acta Biomater. 2024 Nov 28. pii: S1742-7061(24)00710-4. [Epub ahead of print]

Myofibers cultured in viscoelastic hydrogels reveal the effects of integrin-binding and mechanosensing on muscle satellite cells.

Tze-Ling Chang, Alexandra N Borelli, Alicia A Cutler, Bradley B Olwin, Kristi S Anseth.

  Quiescent skeletal muscle satellite cells (SCs) located on myofibers activate in response to muscle injury to regenerate muscle; however, identifying the role of specific matrix signals on SC behavior in vivo is difficult. Therefore, we developed a viscoelastic hydrogel with tunable properties to encapsulate myofibers while maintaining stem cell niche polarity and SC-myofiber interactions to investigate how matrix signals, including viscoelasticity and the integrin-binding ligand arginyl-glycyl-aspartic acid (RGD), influence SC behavior during muscle regeneration. Viscoelastic hydrogels support myofiber culture while preserving SC stemness for up to 72 hours post-encapsulation, minimizing myofiber hypercontraction and SC hyperproliferation compared to Matrigel. Pax7 is continuously expressed in SCs on myofibers embedded in hydrogels with higher stress relaxation while SCs differentiate when embedded in elastic hydrogels. Increasing RGD concentrations activates SCs and translocates YAP/TAZ to the nucleus as revealed by photo-expansion microscopy. Deleting YAP/TAZ abrogates RGD-mediated activation of SCs, and thus, YAP/TAZ mediates RGD ligand-induced SC activation and subsequent proliferation. STATEMENT OF SIGNIFICANCE: Satellite cells (SCs) are responsible for muscle maintenance and regeneration, but how the extracellular matrix regulates SC function is less understood and would benefit from new biomaterial models that can recapitulate the complexity of SC niche in vitro. Upon isolation of myofibers, SCs exit quiescence, becoming activated. To circumvent this issue, we developed a viscoelastic hydrogel for encapsulating myofibers, which maintains SC quiescence and limits differentiation, allowing the study of RGD effects. We showed that increasing RGD concentration promotes activation and suppresses differentiation. Finally, to allow high resolution imaging for resolving the subcellular localization of YAP/TAZ transcriptional co-activators, we applied photo-expansion microscopy and gel-to-gel transfer techniques to quantify YAP/TAZ nuclear-cytoplasmic ratio, revealing that RGD-mediated activation relies on YAP/TAZ nuclear translocation.

Keywords:  expansion microscopy; mechanosensing; muscle satellite cell; myofiber; viscoelastic hydrogel

DOI:  https://doi.org/10.1016/j.actbio.2024.11.044
Med Sci Sports Exerc. 2024 Nov 29.

Training Status Influences Regulation of Muscle and PBMC TLR4 Expression and Systemic Cytokine Responses to Vigorous Endurance Exercise.

Jeremy B Ducharme, Jonathan W Specht, Alyssa R Bailly, Zachary J Fennel, Roberto C Nava, Christine M Mermier, Orlando Laitano, Michael R Deyhle.

INTRODUCTION: A bout of vigorous endurance exercise transiently activates Toll-like receptor 4 (TLR4) and reduces TLR4 protein expressed on peripheral blood mononuclear cells (PBMCs). Endurance training, on the other hand, reduces TLR4-mediated signaling and minimizes the physiological stress imposed by exercise. Less is known about what occurs in skeletal muscle regarding TLR4 regulation and signaling. Therefore, this study aimed to investigate the regulation of TLR4 expressed in different tissue types (PBMCs and skeletal muscle samples) between endurance-trained and untrained men following vigorous endurance exercise and determine the effect of training status on cytokine responses associated with TLR4 activation.
METHODS: Endurance-trained (n = 7) and untrained (n = 5) men cycled for 1-hr at their respiratory compensation point, with blood and skeletal muscle samples collected pre- and 3-hours post-exercise.
RESULTS: In response to vigorous exercise, untrained men experienced a decrease in inhibitor of κBα (IκBα) protein (suggesting IκB degradation and the activation of TLR4-associated transcription factor NF-κB) and TLR4 protein levels, along with a simultaneous increase in TLR4 mRNA expression in both skeletal muscle and PBMCs. Moreover, this exercise session led to elevated levels of circulating interleukin-6, tumor necrosis factor-α, and interleukin-1β. Collectively, these results suggest a heightened TLR4-mediated signaling pathway in untrained men. However, no changes in these targets were observed in endurance-trained men, possibly indicating a potential mechanism by which regular endurance training blunts systemic inflammation.
CONCLUSIONS: These findings highlight the potential of endurance training to mitigate TLR4-mediated signaling, such as systemic inflammation, and shed light on the effects of exercise on TLR4 expression in PBMCs and skeletal muscle.

DOI: https://doi.org/10.1249/MSS.0000000000003618
Cell Rep. 2024 Dec 04. pii: S2211-1247(24)01385-8. [Epub ahead of print]43(12): 115034

Fibroblast diversification is an embryonic process dependent on muscle contraction.

Lavi Coren, Shelly Zaffryar-Eilot, Anas Odeh, Anna Kaganovsky, Peleg Hasson.

  Fibroblasts, the most common cell type found in connective tissues, play major roles in development, homeostasis, regeneration, and disease. Although specific fibroblast subpopulations have been associated with different biological processes, the mechanisms and unique activities underlying their diversity have not been thoroughly examined. Here, we set out to dissect the variation in skeletal-muscle-resident fibroblasts (mrFibroblasts) during development. Our results demonstrate that mrFibroblasts diversify following the transition from embryonic to fetal myogenesis prior to birth. We find that mrFibroblasts segregate into two major subpopulations occupying distinct niches, with interstitial fibroblasts residing between the muscle fibers and delineating fibroblasts sheathing the muscle. We further show that these subpopulations entail distinct cellular dynamics and transcriptomes. Notably, we find that mrFibroblast subpopulations exert distinct regulatory roles on myoblast proliferation and differentiation. Finally, we demonstrate that this diversification depends on muscle contraction. Altogether, these findings establish that mrFibroblasts diversify in a spatiotemporal embryonic process into distinct cell types, entailing different characteristics and roles.

Keywords:  CP: Developmental biology; cell diversification; embryonic development; fibroblast subpopulations; fibroblasts; msenchymal cells; muscle; muscle connective tissue; myogenesis

DOI:  https://doi.org/10.1016/j.celrep.2024.115034
Nature. 2024 Dec 04.

Macrophages excite muscle spindles with glutamate to bolster locomotion.

Yuyang Yan, Nuria Antolin, Luming Zhou, Luyang Xu, Irene Lisa Vargas, Carlos Daniel Gomez, Guiping Kong, Ilaria Palmisano, Yi Yang, Jessica Chadwick, Franziska Müller, Anthony M J Bull, Cristina Lo Celso, Guido Primiano, Serenella Servidei, Jean François Perrier, Carmelo Bellardita, Simone Di Giovanni.

The stretch reflex is a fundamental component of the motor system that orchestrates the coordinated muscle contractions underlying movement. At the heart of this process lie the muscle spindles (MS), specialized receptors finely attuned to fluctuations in tension within intrafusal muscle fibres. The tension variation in the MS triggers a series of neuronal events including an initial depolarization of sensory type Ia afferents that subsequently causes the activation of motoneurons within the spinal cord1,2. This neuronal cascade culminates in the execution of muscle contraction, underscoring a presumed closed-loop mechanism between the musculoskeletal and nervous systems. By contrast, here we report the discovery of a new population of macrophages with exclusive molecular and functional signatures within the MS that express the machinery for synthesizing and releasing glutamate. Using mouse intersectional genetics with optogenetics and electrophysiology, we show that activation of MS macrophages (MSMP) drives proprioceptive sensory neuron firing on a millisecond timescale. MSMP activate spinal circuits, motor neurons and muscles by means of a glutamate-dependent mechanism that excites the MS. Furthermore, MSMP respond to neural and muscle activation by increasing the expression of glutaminase, enabling them to convert the uptaken glutamine released by myocytes during muscle contraction into glutamate. Selective silencing or depletion of MSMP in hindlimb muscles disrupted the modulation of the stretch reflex for force generation and sensory feedback correction, impairing locomotor strategies in mice. Our results have identified a new cellular component, the MSMP, that directly regulates neural activity and muscle contraction. The glutamate-mediated signalling of MSMP and their dynamic response to sensory cues introduce a new dimension to our understanding of sensation and motor action, potentially offering innovative therapeutic approaches in conditions that affect sensorimotor function.

DOI: https://doi.org/10.1038/s41586-024-08272-5
Cell Mol Biol Lett. 2024 Dec 05. 29(1): 152

The miR-6240 target gene Igf2bp3 promotes myoblast fusion by enhancing myomaker mRNA stability.

Yuxin Huang, Wei Wang, Xinhao Fan, Xiaoqin Liu, Weiwei Liu, Zishuai Wang, Yixing Li, Yalan Yang, Zhonglin Tang.

   BACKGROUND: Myoblast fusion plays a crucial role in myogenesis. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) functions as an RNA N6-methyladenosine reader and exerts important roles in various biological processes. While our prior study suggested Igf2bp3 contributes to myogenesis, its molecular regulatory mechanism is largely unclear.
METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were used for gene expression analysis. siRNA and CRISPRi technologies were conducted to knockdown the expression of Igf2bp3. CRISPR/Cas9 technology was performed to knockout Igf2bp3. The Igf2bp3 overexpression vector was designed using the pcDNA3.1(+) vector. Immunofluorescence detection was employed for subcellular localization and cell differentiation analysis. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were conducted for cell proliferation and fusion detection. The dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized for regulatory mechanism analysis of Igf2bp3.
RESULTS: The overexpression of Igf2bp3 enhances myoblast fusion while knockdown of Igf2bp3 blocks the formation of myotubes. miR-6240 promotes myoblast proliferation while preventing myoblast differentiation and fusion by targeting the 3' untranslated rgion (UTR) of Igf2bp3. Notably, the impacts of miR-6240 mimics on myoblast proliferation, differentiation, and fusion can be effectively counteracted by the overexpression of Igf2bp3. Moreover, our findings elucidate a direct interaction between Igf2bp3 and the myoblast fusion factor myomaker (Mymk). Igf2bp3 binds to Mymk to enhance its mRNA stability. This interaction results in increased expression of Mymk and heightened myoblast fusion.
CONCLUSIONS: Our study unveils Igf2bp3 as a novel post-transcriptional regulator of myoblast fusion through the miR-6240/Mymk axis, significantly contributing to our understanding of skeletal muscle development.

Keywords:   Igf2bp3 ; Mymk ; Myobalst fusion; Myogenesis; mRNA stability; miR-6240

DOI:  https://doi.org/10.1186/s11658-024-00650-1