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Journal of Cachexia, Sarcopenia and... Dec 2023Lactate, a glycolytic metabolite mainly produced in muscles, has been suggested to regulate myoblast differentiation, although the underlying mechanism remains elusive....
BACKGROUND
Lactate, a glycolytic metabolite mainly produced in muscles, has been suggested to regulate myoblast differentiation, although the underlying mechanism remains elusive. Recently, lactate-mediated histone lactylation is identified as a novel epigenetic modification that promotes gene transcription.
METHODS
We used mouse C2C12 cell line and 2-month-old male mice as in vitro and in vivo models, respectively. These models were treated with lactate to explore the biological function and latent mechanism of lactate-derived histone lactylation on myogenic differentiation by quantitative real-time PCR, western blotting, immunofluorescence staining, chromatin immunoprecipitation, cleavage under targets and tagmentation assay and RNA sequencing.
RESULTS
Using immunofluorescence staining and western blotting, we proposed that lactylation might occur in the histones. Inhibition of lactate production or intake both impaired myoblast differentiation, accompanied by diminished lactylation in the histones. Using lactylation site-specific antibodies, we demonstrated that lactate preferentially increased H3K9 lactylation (H3K9la) during myoblast differentiation (CT VS 5, 10, 15, 20, 25 mM lactate treatment, P = 0.0012, P = 0.0007, and the rest of all P < 0.0001). Notably, inhibiting H3K9la using P300 antagonist could block lactate-induced myogenesis. Through combined omics analysis using cleavage under targets and tagmentation assay and RNA sequencing, we further identified Neu2 as a potential target gene of H3K9la. IGV software analysis (P = 0.0013) and chromatin immunoprecipitation-qPCR assay (H3K9la %Input, LA group = 9.0076, control group = 2.7184, IgG = 0.3209) confirmed that H3K9la is enriched in the promoter region of Neu2. Moreover, siRNAs or inhibitors against Neu2 both abrogated myoblast differentiation despite lactate treatment, suggesting that Neu2 is required for lactate-mediated myoblast differentiation.
CONCLUSIONS
Our findings provide novel understanding of histone lysine lactylation, suggesting its role in myogenesis, and as potential therapeutic targets for muscle diseases.
Topics: Animals; Male; Mice; Cell Line; Histones; Lactic Acid; Muscle Development; Up-Regulation
PubMed: 37919243
DOI: 10.1002/jcsm.13363 -
Frontiers in Cell and Developmental... 2024Regeneration and repair are prerequisites for maintaining effective function of skeletal muscle under high energy demands, and myogenic differentiation is one of the key... (Review)
Review
Regeneration and repair are prerequisites for maintaining effective function of skeletal muscle under high energy demands, and myogenic differentiation is one of the key steps in the regeneration and repair process. A striking feature of the process of myogenic differentiation is the alteration of mitochondria in number and function. Mitochondrial dysfunction can activate a number of transcriptional, translational and post-translational programmes and pathways to maintain cellular homeostasis under different types and degrees of stress, either through its own signaling or through constant signaling interactions with the nucleus and cytoplasm, a process known as the mitochondrial stress responses (MSRs). It is now believed that mitochondrial dysfunction is closely associated with a variety of muscle diseases caused by reduced levels of myogenic differentiation, suggesting the possibility that MSRs are involved in messaging during myogenic differentiation. Also, MSRs may be involved in myogenesis by promoting bioenergetic remodeling and assisting myoblast survival during myogenic differentiation. In this review, we will take MSRs as an entry point to explore its concrete regulatory mechanisms during myogenic differentiation, with a perspective to provide a theoretical basis for the treatment and repair of related muscle diseases.
PubMed: 38681520
DOI: 10.3389/fcell.2024.1381417 -
The Journal of International Medical... Aug 2023Sclerostin, a protein encoded by the sclerostin () gene, is mostly expressed in osteocytes. First described in the pathogenesis of three disorders, sclerosteosis, van... (Review)
Review
Sclerostin, a protein encoded by the sclerostin () gene, is mostly expressed in osteocytes. First described in the pathogenesis of three disorders, sclerosteosis, van Buchem's disease, and craniodiaphyseal dysplasia, sclerostin has been identified as an important regulator of bone homeostasis, controlling bone formation by osteoblasts through inhibition of the canonical Wnt signaling pathway. Recent studies have highlighted a hypothetical role of sclerostin in myogenesis, thus modulating the interaction between bone and muscle. This narrative review provides an overview of the clinical implications of sclerostin modulation on skeletal muscle mass and function, and bone metabolism. Improving knowledge about muscle-bone crosstalk may represent a turning point in the development of therapeutic strategies for musculoskeletal disorders, particularly osteosarcopenia.
Topics: Humans; Hyperostosis; Knowledge; Muscles; Osteoblasts; Osteogenesis
PubMed: 37632438
DOI: 10.1177/03000605231193293 -
MedRxiv : the Preprint Server For... Jan 2024Myositis is a heterogeneous family of autoimmune muscle diseases. As myositis autoantibodies recognize intracellular proteins, their role in disease pathogenesis has...
OBJECTIVES
Myositis is a heterogeneous family of autoimmune muscle diseases. As myositis autoantibodies recognize intracellular proteins, their role in disease pathogenesis has been unclear. This study aimed to determine whether myositis autoantibodies reach their autoantigen targets within muscle cells and disrupt the normal function of these proteins.
METHODS
Confocal immunofluorescence microscopy was used to localize antibodies and other proteins of interest in myositis muscle biopsies. Bulk RNA sequencing was used to study the transcriptomic profiles of 668 samples from patients with myositis, disease controls, and healthy controls. Antibodies from myositis patients were introduced into cultured myoblasts by electroporation and the transcriptomic profiles of the treated myoblasts were studied by bulk RNA sequencing.
RESULTS
In patients with myositis autoantibodies, antibodies accumulated inside myofibers in the same subcellular compartment as the autoantigen. Each autoantibody was associated with effects consistent with dysfunction of its autoantigen, such as the derepression of genes normally repressed by Mi2/NuRD in patients with anti-Mi2 autoantibodies, the accumulation of RNAs degraded by the nuclear RNA exosome complex in patients with anti-PM/Scl autoantibodies targeting this complex, and the accumulation of lipids within myofibers of anti-HMGCR-positive patients. Internalization of patient immunoglobulin into cultured myoblasts recapitulated the transcriptomic phenotypes observed in human disease, including the derepression of Mi2/NuRD-regulated genes in anti-Mi2-positive dermatomyositis and the increased expression of genes normally degraded by the nuclear RNA exosome complex in anti-PM/Scl-positive myositis.
CONCLUSIONS
In myositis, autoantibodies are internalized into muscle fibers, disrupt the biological function of their autoantigen, and mediate the pathophysiology of the disease.
PubMed: 38313303
DOI: 10.1101/2024.01.15.24301339 -
Journal of Cachexia, Sarcopenia and... Aug 2023It has been observed that Slo1 knockout mice have reduced motor function, and people with certain Slo1 mutations have movement problems, but there is no answer whether...
BACKGROUND
It has been observed that Slo1 knockout mice have reduced motor function, and people with certain Slo1 mutations have movement problems, but there is no answer whether the movement disorder is caused by the loss of Slo1 in the nervous system, or skeletal muscle, or both. Here, to ascertain in which tissues Slo1 functions to regulate motor function and offer deeper insight in treating related movement disorder, we generated skeletal muscle-specific Slo1 knockout mice, studied the functional changes in Slo1-deficient skeletal muscle and explored the underlying mechanism.
METHODS
We used skeletal muscle-specific Slo1 knockout mice (Myf5-Cre; Slo1 mice, called CKO) as in vivo models to examine the role of Slo1 in muscle growth and muscle regeneration. The forelimb grip strength test was used to assess skeletal muscle function and treadmill exhaustion test was used to test whole-body endurance. Mouse primary myoblasts derived from CKO (myoblast/CKO) mice were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Quantitative real-time PCR, western blot and immunofluorescence approaches were used to analyse Slo1 expression during myoblast differentiation and muscle regeneration. To investigate the involvement of genes in the regulation of muscle dysfunction induced by Slo1 deletion, RNA-seq analysis was performed in primary myoblasts. Immunoprecipitation and mass spectrometry were used to identify the protein interacting with Slo1. A dual-luciferase reporter assay was used to identify whether Slo1 deletion affects NFAT activity.
RESULTS
We found that the body weight and size of CKO mice were not significantly different from those of Slo1 mice (called WT). Deficiency of Slo1 in muscles leads to reduced endurance (~30% reduction, P < 0.05) and strength (~30% reduction, P < 0.001). Although there was no difference in the general morphology of the muscles, electron microscopy revealed a considerable reduction in the content of mitochondria in the soleus muscle (~40% reduction, P < 0.01). We found that Slo1 was expressed mainly on the cell membrane and showed higher expression in slow-twitch fibres. Slo1 protein expression is progressively reduced during muscle postnatal development and regeneration after injury, and the expression is strongly reduced during myoblast differentiation. Slo1 deletion impaired myoblast differentiation and slow-twitch fibre formation. Mechanistically, RNA-seq analysis showed that Slo1 influences the expression of genes related to myogenic differentiation and slow-twitch fibre formation. Slo1 interacts with FAK to influence myogenic differentiation, and Slo1 deletion diminishes NFAT activity.
CONCLUSIONS
Our data reveal that Slo1 deficiency impaired skeletal muscle regeneration and slow-twitch fibre formation.
Topics: Animals; Mice; Cell Differentiation; Mice, Knockout; Movement Disorders; Muscle, Skeletal; Myoblasts
PubMed: 37212018
DOI: 10.1002/jcsm.13253 -
Journal of Cachexia, Sarcopenia and... Aug 2023Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite...
BACKGROUND
Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-β) is involved in both processes. We uncovered an increased expression of the TGF-β receptor II (TβRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TβRII signalling impairs myogenic differentiation in response to inflammation.
METHODS
We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-β/TβRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses.
RESULTS
SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1β-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TβRII, resulting in TβRII ubiquitination and destabilization. SPSB1 impaired TβRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice.
CONCLUSIONS
Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TβRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.
Topics: Animals; Mice; Cytokines; Inflammation; Interleukin-6; Muscle Development; Muscle, Skeletal; Myogenin; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha
PubMed: 37209006
DOI: 10.1002/jcsm.13252 -
Nutrients Oct 2023Age-related skeletal muscle atrophy and weakness not only reduce the quality of life of those afflicted, but also worsen the prognosis of underlying diseases. We...
Age-related skeletal muscle atrophy and weakness not only reduce the quality of life of those afflicted, but also worsen the prognosis of underlying diseases. We evaluated the effect of RGX365, a protopanaxatriol-type rare ginsenoside mixture, on improving skeletal muscle atrophy. We investigated the myogenic effect of RGX365 on mouse myoblast cells (C2C12) and dexamethasone (10 µM)-induced atrophy of differentiated C2C12. RGX365-treated myotube diameters and myosin heavy chain (MyHC) expression levels were analyzed using immunofluorescence. We evaluated the myogenic effects of RGX365 in aging sarcopenic mice. RGX365 increased myoblast differentiation and MyHC expression, and attenuated the muscle atrophy-inducing F-box (Atrogin-1) and muscle RING finger 1 (MuRF1) expression. Notably, one month of oral administration of RGX365 to 23-month-old sarcopenic mice improved muscle fiber size and the expression of skeletal muscle regeneration-associated molecules. In conclusion, rare ginsenosides, agonists of steroid receptors, can ameliorate skeletal muscle atrophy during long-term administration.
Topics: Mice; Animals; Sarcopenia; Quality of Life; Cell Line; Muscular Atrophy; Muscle, Skeletal; Muscle Fibers, Skeletal; Muscle Development
PubMed: 37836590
DOI: 10.3390/nu15194307 -
Journal of Cachexia, Sarcopenia and... Feb 2024Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse....
BACKGROUND
Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse. Age-related imbalance of protein synthesis and degradation, mainly regulated by AKT/mTOR pathway, leads to progressive loss of muscle mass. Maintenance of anabolic and regenerative capacities of skeletal muscles may be regarded as a therapeutic option for sarcopenia and other muscle wasting diseases. Our previous studies have demonstrated that BIO101, a pharmaceutical grade 20-hydroxyecdysone, increases protein synthesis through the activation of MAS receptor involved in the protective arm of renin-angiotensin-aldosterone system. The purpose of the present study was to assess the anabolic and pro-differentiating properties of BIO101 on C2C12 muscle cells in vitro and to investigate its effects on adult and old mice models in vivo.
METHODS
The effects of BIO101 on C2C12 differentiation were assessed using myogenic transcription factors and protein expression of major kinases of AKT/mTOR pathway by Western blot. The in vivo effects of BIO101 have been investigated in BIO101 orally-treated (50 mg/kg/day) adult mice (3 months) for 28 days. To demonstrate potential beneficial effect of BIO101 treatment in a sarcopenic mouse model, we use orally treated 22-month-old C57Bl6/J mice, for 14 weeks with vehicle or BIO101. Mice body and muscle weight were recorded. Physical performances were assessed using running capacity and muscle contractility tests.
RESULTS
Anabolic properties of BIO101 were confirmed by the rapid activation of AKT/mTOR, leading to an increase of C2C12 myotubes diameters (+26%, P < 0.001). Pro-differentiating effects of BIO101 on C2C12 myoblasts were revealed by increased expression of muscle-specific differentiation transcription factors (MyoD, myogenin), resulting in increased fusion index and number of nuclei per myotube (+39% and +53%, respectively, at day 6). These effects of BIO101 were like those of angiotensin (1-7) and were abolished with the use of A779, a MAS receptor specific antagonist. Chronic BIO101 oral treatment induced AKT/mTOR activation and anabolic effects accompanied with improved physical performances in adult and old animals (maximal running distance and maximal running velocity).
CONCLUSIONS
Our data suggest beneficial anabolic and pro-differentiating effects of BIO101 rendering BIO101 a potent drug candidate for treating sarcopenia and possibly other muscle wasting disorders.
Topics: Mice; Animals; Sarcopenia; Proto-Oncogene Proteins c-akt; Muscle, Skeletal; Muscular Diseases; Muscular Atrophy; TOR Serine-Threonine Kinases; Myoblasts; Transcription Factors
PubMed: 38064183
DOI: 10.1002/jcsm.13326 -
Journal of Clinical Medicine Sep 2023Dysferlinopathy is a disease caused by a dysferlin deficiency due to mutations in the gene. Dysferlin is a membrane protein in the sarcolemma and is involved in... (Review)
Review
Dysferlinopathy is a disease caused by a dysferlin deficiency due to mutations in the gene. Dysferlin is a membrane protein in the sarcolemma and is involved in different functions, such as membrane repair and vesicle fusion, T-tubule development and maintenance, Ca signalling, and the regulation of various molecules. Miyoshi Myopathy type 1 (MMD1) and Limb-Girdle Muscular Dystrophy 2B/R2 (LGMD2B/LGMDR2) are two possible clinical presentations, yet the same mutations can cause both presentations in the same family. They are therefore grouped under the name dysferlinopathy. Onset is typically during the teenage years or young adulthood and is characterized by a loss of Achilles tendon reflexes and difficulty in standing on tiptoes or climbing stairs, followed by a slow progressive loss of strength in limb muscles. The MRI pattern of patient muscles and their biopsies show various fibre sizes, necrotic and regenerative fibres, and fat and connective tissue accumulation. Recent tools were developed for diagnosis and research, especially to evaluate the evolution of the patient condition and to prevent misdiagnosis caused by similarities with polymyositis and Charcot-Marie-Tooth disease. The specific characteristic of dysferlinopathy is dysferlin deficiency. Recently, mouse models with patient mutations were developed to study genetic approaches to treat dysferlinopathy. The research fields for dysferlinopathy therapy include symptomatic treatments, as well as antisense-mediated exon skipping, myoblast transplantation, and gene editing.
PubMed: 37762951
DOI: 10.3390/jcm12186011 -
Nature Communications Jul 2023Muscle stem cells, the engine of muscle repair, are affected in myotonic dystrophy type 1 (DM1); however, the underlying molecular mechanism and the impact on the...
Muscle stem cells, the engine of muscle repair, are affected in myotonic dystrophy type 1 (DM1); however, the underlying molecular mechanism and the impact on the disease severity are still elusive. Here, we show using patients' samples that muscle stem cells/myoblasts exhibit signs of cellular senescence in vitro and in situ. Single cell RNAseq uncovers a subset of senescent myoblasts expressing high levels of genes related to the senescence-associated secretory phenotype (SASP). We show that the levels of interleukin-6, a prominent SASP cytokine, in the serum of DM1 patients correlate with muscle weakness and functional capacity limitations. Drug screening revealed that the senolytic BCL-XL inhibitor (A1155463) can specifically remove senescent DM1 myoblasts by inducing their apoptosis. Clearance of senescent cells reduced the expression of SASP, which rescued the proliferation and differentiation capacity of DM1 myoblasts in vitro and enhanced their engraftment following transplantation in vivo. Altogether, this study identifies the pathogenic mechanism associated with muscle stem cell defects in DM1 and opens a therapeutic avenue that targets these defective cells to restore myogenesis.
Topics: Humans; Myotonic Dystrophy; Senotherapeutics; Muscle Fibers, Skeletal; Satellite Cells, Skeletal Muscle; Muscle Development
PubMed: 37468473
DOI: 10.1038/s41467-023-39663-3