Skeletal Muscle最新文献

Background: Skeletal muscle comprises 30-40% of a mammal's body mass, maintaining its integrity through efficient muscle fiber regeneration, which involves myoblast differentiation into myotubes. Previously, we reported that N-acetylglucosamine (GlcNAc) promotes myogenesis in C2C12 cells, although the underlying processes remained unclear. GlcNAc's activated form, UDP-GlcNAc, is critical for the biosynthesis of highly branched (N-acetyllactosamine-rich) N-linked oligosaccharides, which are recognized by galectin-3 (Gal-3), a protein that facilitates dynamic cell-cell and cell-matrix interactions and modulating the motility dynamics of membrane-associated proteins.

Methods: In this study, we used primary myoblasts from both wild-type and Gal-3 null (Gal-3KO) mice, observing myotube formation through long-term live-cell imaging and single-cell tracking to reveal the dynamic process that occurred during the myotube formation.

Results: We found that GlcNAc enhances myoblast fusion in a dose-dependent manner, and that the addition of Gal-3 with GlcNAc leads to the formation of larger myotubes. Gal-3KO myoblasts exhibited a reduced capacity for myotube formation-a deficiency that was rectified by supplementing with GlcNAc and Gal-3. Our results highlight the role of Gal-3 interaction with oligosaccharides, whose synthesis is promoted by GlcNAc in facilitating myotube formation. Single-cell tracking revealed that GlcNAc and Gal-3 increase myoblast motility, leading to a faster, coordinated, flow-like movement-a collective behavior, along which myotubes form through cell fusion. Interestingly, myoblasts contributing to myotube formation were pre-positioned along the eventual shape of the myotubes before this flow-like movement was fully established. These myoblasts moved along the flow, paused, and even moved against it, suggesting that both coordinated flow and initial spatial positioning contribute to myoblast alignment along the axis of future myotubes.

Conclusion: Our findings suggest that GlcNAc, in conjunction with Gal-3, enhances myotube formation by fostering an environment conducive to myoblast positioning, establishing optimal coordinated flow-like movement, and facilitating fusion. This suggests potential therapeutic applications of GlcNAc in muscle repair and muscle disorders.

Limb-girdle muscular dystrophy 2D (or LGMDR3) is a rare autosomal recessive disorder caused by mutations in the SGCA gene, which encode α-sarcoglycan (α-SG). α-SG is a critical component of the dystrophin-associated protein complex, whose role in differentiated muscle is to distribute contraction force and protect the sarcolemma from mechanical damage. Most SGCA mutations are missense, leading to a folding-defective α-SG that is degraded by the ubiquitin-proteasome system, destabilizing the sarcolemma and causing progressive muscle weakness. Notably, pharmacological restoration of α-SG function using cystic fibrosis transmembrane conductance regulator (CFTR) correctors, such as C17, can rescue the SG-complex, improving muscle strength in an LGMDR3 mouse model. Our initial aim was to generate 3D diaphragm-like models of LGMDR3 by seeding patient-derived myoblasts onto a decellularised diaphragm scaffold, thereby mimicking the disease environment and enabling drug screening beyond the limitations of 2D cultures. While the models did not behave as anticipated, the unexpected outcome led us to uncover a previously underappreciated role of α-SG. Specifically, we found that α-SG expressed by immature myoblasts is crucial for cell adhesion and migration, key processes for muscle development, regeneration, and successful engraftment into a decellularized extracellular matrix. These processes, compromised in LGMDR3 cells, can be rescued through CFTR correctors, further supporting their potential therapeutic application in LGMDR3.

Non-peripheral (displaced) myonuclei are characteristic of skeletal muscle pathology and severe injury but also appear after exercise and with aging. Displaced myonuclei are typically attributed to the activity of muscle stem cells, or satellite cells. We sought to address whether displaced myonuclei in adult skeletal muscle are exclusively from an exogenous source such as satellite cells or can result from resident myonuclear migration. To address this question, we used a murine recombination-independent muscle fibre-specific doxycycline-inducible fluorescent myonuclear labelling approach, EdU stem cell fate tracking, two durations of plantaris muscle mechanical overload (MOV, 3 days and 7 days), and fluorescent histology. Our findings show that: 1) displaced myonuclei emerge early during MOV in adult mice, 2) resident myonuclear movement occurs rapidly during MOV, and 3) the contribution of resident versus exogenous displaced myonuclei depends on the preferential effects of MOV for specific fibre types or fibre sizes with a given MOV duration. These observations provide fundamental insights on myonuclear motility in response to stress in vivo and reframe our understanding of how a recognized feature of mammalian skeletal muscle can emerge in response to stressors such as mechanical loading.

Skeletal muscle is a highly plastic tissue that plays a crucial role in overall metabolic health, and in diseases such as obesity and type 2 diabetes. Recently, the importance of preserving muscle mass during weight loss has gained appreciation, especially with significant weight loss observed from incretin therapies, which includes loss of both fat and lean mass (Conte et al, JAMA 332:9-10, 2024). This has prompted investigation into pharmacological candidates that can prevent the loss of muscle mass seen during weight loss. Urocortins and their cognate receptors pose an interesting target, as recent evidence shows that they play a role in diseases such as heart failure, diabetes, and obesity. Urocortin treatment results in decreased food intake, muscle hypertrophy and improved skeletal muscle glucose uptake. However, the molecular mechanisms by which urocortins act have yet to be elucidated. The aim of this review is to highlight our current understanding of the effects of urocortins on metabolic adaptations.

Background: Immune-mediated necrotizing myopathy (IMNM) is a subgroup of idiopathic inflammatory myopathies associated with anti-signal recognition particle (SRP) or anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) autoantibodies. The direct pathogenic effects of IMNM patient autoantibodies on skeletal muscle contractile force, independent of the downstream activation of the complement pathway, remain understudied.

Methods: This study leverages a custom 3-D human skeletal muscle microtissue (hMMT) culture platform that encourages muscle cell contractile apparatus maturation and enables analysis of contractile function. Force generation competent hMMTs were treated with total immunoglobulins (IgGs) isolated from the plasma of IMNM patients with amplification of anti-SRP⁺ (n = 7) or anti-HMGCR⁺ (n = 7) autoantibodies and delivered in complement inactivated media for 4 days. hMMT function was then evaluated by quantifying the peak force and contraction kinetics in response to electrical field stimulation, followed by histological analysis of muscle cell gross morphology and sarcomere structure. To determine whether IgG from IMNM patients can enter muscle cells, 2-D myotube cultures were treated with donor total IgG delivered in complement replete media for 36 h, and then analyzed using immunostaining and confocal microscopy.

Results: Exposure to total IgGs isolated from a subset of IMNM patients induced a decline in hMMT twitch and tetanus contractile force and were associated with sarcomere fragility and slowed muscle cell contraction and relaxation rates. Pathogenic influences on hMMT force generation were observed at a greater frequency in response to total IgGs isolated from IMNM patients with anti-HMGCR + autoantibodies. Substantial intracellular human IgG staining was observed in conditions where myotubes were treated with total IgGs from IMNM patients.

Conclusions: This study demonstrates that total IgGs isolated from IMNM patients have the aberrant capacity to enter muscle cells in the absence of complement. Further, a subset of patient IgGs exert direct pathogenic influences on engineered muscle contractile function that are independent of the complement system. Together, these findings have important implications for the advancement of IMNM precision medicine therapies.

Background: Skeletal muscle is a dynamic tissue capable of structural and metabolic remodeling in response to physiological and pathological stimuli. These adaptations are central to understanding the mechanisms underlying conditions such as genetic myopathies, cancer, aging, and recovery from injury. Muscle fiber characterization-assessing fiber type, size, and metabolic profile-is essential for such studies. However, conventional histological methods often rely on serial tissue sections and multiple staining protocols, which are time-consuming, require significant biological material, and introduce methodological bias.

Methods: We developed FLASH (Fluorescence-based Labeling for Assessing Skeletal muscle Histology), a novel methodology combining enzymatic (SDH or GPDH) and quadruple fluorescent labeling (Laminin, MYH4, MYH2, MYH7) on a single muscle section. The resulting images were analyzed using a custom macro in Fiji/ImageJ, integrating the Cellpose segmentation algorithm. This automated pipeline detects individual muscle fibers, quantifies their cross-sectional area (CSA), identifies fiber types based on myosin isoform expression, and measures enzymatic staining intensity. Batch analysis was implemented to process entire image folders automatically. Validation was performed by comparing automated fiber detection with expert manual segmentation using correlation analysis and Bland-Altman plots.

Results: The FLASH method allowed simultaneous assessment of both contractile and metabolic properties within individual fibers on the same section, removing the need for serial cuts. The automated image analysis achieved high accuracy in fiber detection (r > 0.95 compared to manual annotation) and produced consistent CSA and fiber-type quantification, even under suboptimal staining conditions. The macro enabled significant time savings by automating the complete analysis workflow, including ROI generation and Excel data export for each image.

Conclusions: FLASH provides an efficient and robust tool for high-throughput skeletal muscle histology. By combining enzymatic and fluorescent co-labeling with machine learning-based image analysis, this method improves reproductibility, reduces experimental complexity, and minimizes user bias. FLASH is particularly well-suited for large-scale or longitudinal studies investigating muscle adaptation in health and disease.

Chronic kidney disease (CKD) and peripheral artery disease (PAD) frequently coexist and synergistically exacerbate skeletal muscle dysfunction, contributing to limb function impairment and increased risk of amputation and mortality. Both diseases independently promote fibrotic remodeling in muscle, suggesting that anti-fibrotic therapies may improve muscle health in this high-risk population. We tested whether targeting the fibrotic niche with batimastat, a matrix metalloprotease inhibitor, or pirfenidone, an approved anti-fibrotic medication, would improve ischemic limb function in a mouse model of CKD and PAD. Male mice (n = 21) were fed an adenine diet to induce CKD and subsequently underwent surgical femoral artery ligation to induce hindlimb ischemia, an experimental model of PAD. Pirfenidone significantly improved ischemic muscle absolute force (P < 0.0001) and specific force (P = 0.0027), and increased time-tension integral during a muscle fatigue test (P < 0.0001), while batimastat significantly reduced these parameters compared to placebo. Surprisingly, neither treatment altered muscle fibrosis, perfusion recovery, capillary density, or myofiber regeneration, indicating that functional improvements with pirfenidone occurred independently of structural remodeling. These findings suggest that pirfenidone preserves muscle strength and quality and may have therapeutic potential to improve limb function in patients with CKD and PAD. Further investigation is warranted to define pirfenidone's mechanism of action in skeletal muscle and evaluate its efficacy in the clinical setting.

Background: Enzymes of the Ten-Eleven Translocation family are responsible for 5-methylcytosine (5mC) oxidation and play a key role in regulating DNA demethylation during various developmental processes, including myogenesis. However, they also exhibit 5mC-independent functions whose importance for muscle development remains unexplored. As the Drosophila genome lacks the enzymes required for 5mC deposition but contains a single Tet gene essential for viability, we analyzed its role in flight muscle development.

Methods: Using a combination of genetics, imaging techniques, transcriptomic analysis and functional assays, we assessed the impact of Tet loss of function (using either Tet null or Tet catalytic inactive mutants, as well as Tet knockdown) on indirect flight muscle development from the larval to adult stages and during aging in Drosophila melanogaster.

Results: We found that Tet loss leads to a decrease in the number of adult muscle progenitors in the larva, dysregulation of the myogenic expression program in the pupa and disrupted flight muscle organization in the adult. Interestingly, our data reveal that these phenotypes are largely independent of TET enzymatic activity. However, analysis of TET-catalytic inactive flies also highlights the enzyme's critical role in adult fly mobility and its ability to prevent premature muscle aging. Further experiments demonstrate that TET expression in muscle progenitors and the central nervous system is essential for maintaining adult mobility.

Conclusions: These results highlight the crucial role of TET beyond 5mC DNA oxidation, suggesting that both catalytic-dependent and catalytic-independent functions of TET are essential for muscle development and function in vivo.

Background: Skeletal muscle is an important organ for health and movement, largely driven by specific muscle fibres. However, the comparison of fibre-type-specific DNA methylation and protein abundance from the same sample presents challenges. By combining previous methodological approaches we were able to directly compare the methylome and proteome in Type I and Type II human skeletal muscle fibres in males and females.

Methods: We assessed the methylome using the EPICv2 Infinium array and the proteome using liquid chromatography tandem mass spectrometry (LC-MS/MS) from Type I and Type II fibre pools from both males ( $n=7$ ) and females ( $n=5$ ).

Results: We identified 5,689 robust differentially methylated regions (Fisher P-value $<0.001$ ) and found strong relationships between methylation and protein abundance in key contractile and metabolic genes. Further, we generated a reference matrix of Type I and Type II fibres and leveraged deconvolution algorithms to accurately estimate fibre-type proportions using whole-muscle DNA methylation data, providing a method to correct for fibre-type in future studies. These results are presented primarily as a resource for others to utilise.

Conclusion: We provide integrated methylome and proteome profiles of human muscle fibre-types generalisable to both male and females as a freely accessible interactive repository, MyoMETH ( https://myometh.net ), allowing further investigation into fibre regulation. Data are available via ProteomeXchange with identifier PXD066393 and the Gene Expression Omnibus at GSE304045 .