Skeletal Muscle最新文献

Background: Skeletal muscle is a plastic tissue that adapts to increased mechanical loading/contractile activity through fusion of muscle stem cells (MuSCs) with myofibers, a physiological process referred to as myonuclear accretion. However, it is still unclear whether myonuclear accretion is driven by increased mechanical loading per se, or occurs, at least in part, in response to muscle injury/regeneration. Here, we developed a non-damaging protocol to evaluate contractile activity-induced myonuclear accretion/hypertrophy in physiological conditions.

Methods: Contractile activity was generated by applying repeated electrical stimuli over the mouse plantar flexor muscles. This method is commonly referred to as NeuroMuscular Electrical Simulation (NMES) in Human. Each NMES training session consisted of 80 isometric contractions delivered at ∼15% of maximal tetanic force to avoid muscle damage. C57BL/6J male mice were submitted to either a short (i.e., 6 sessions) or long (i.e., 12 sessions) individualized NMES training program while unstimulated mice were used as controls. Histological investigations were performed to assess the impact of NMES on MuSC number and status, myonuclei content and muscle tissue integrity, typology and size.

Results: NMES led to a robust proliferation of MuSCs and myonuclear accretion in the absence of overt signs of muscle damage/regeneration. NMES-induced myonuclear accretion was specific to type IIB myofibers and was an early event preceding muscle hypertrophy inasmuch as a mild increase in myofiber cross-sectional area was only observed in response to the long-term NMES training protocol.

Conclusion: We conclude that NMES-induced myonuclear accretion and muscle hypertrophy are driven by a mild increase in mechanical loading in the absence of overt signs of muscle injury.

Background: Duchenne muscular dystrophy (DMD) is a prevalent, fatal degenerative muscle disease with no effective treatments. Mdx mouse model of DMD exhibits impaired muscle performance, oxidative stress, and dysfunctional autophagy. Although antioxidant treatments may improve the mdx phenotype, the precise molecular mechanisms remain unclear. This study investigates the effects of aminoguanidine hemisulfate (AGH), an inhibitor of reactive oxygen species (ROS), on mitochondrial autophagy, oxidative stress, and muscle force in mdx mice.

Methods: Male wild-type (WT) and mdx mice were divided into three groups: WT, mdx, and AGH-treated mdx mice (40 mg/kg intraperitoneally for two weeks) at 6 weeks of age. Gene expression, western blotting, H&E staining, immunofluorescence, ROS assays, TUNEL apoptosis, glutathione activity, and muscle force measurements were performed. Statistical comparisons used one-way ANOVA.

Results: AGH treatment significantly reduced the protein levels of LC3, and p62 in mdx mice, indicating improved autophagy activity and the ability to clear damaged mitochondria. AGH restored the expression of mitophagy-related genes Pink1 and Parkin and increased Mfn1, rebalancing mitochondrial dynamics. It also increased Pgc1α and mtTFA levels, promoting mitochondrial biogenesis. ROS levels were reduced, with higher Prdx3 and MnSOD expression, improving mitochondrial antioxidant defenses. AGH normalized the GSSG/GSH ratio and decreased glutathione reductase and peroxidase activities, further improving redox homeostasis. Additionally, AGH reduced apoptosis, shown by fewer TUNEL-positive cells and lower caspase-3 expression. Histological analysis revealed decreased muscle damage and fewer embryonic and neonatal myosin-expressing fibers. AGH altered fiber composition, decreasing MyH7 while increasing MyH4 and MyH2. Muscle force improved significantly, with greater twitch and tetanic forces. Mechanistically, AGH modulated the AKT/FOXO1 pathway, decreasing myogenin and Foxo1 while increasing MyoD.

Conclusions: AGH treatment restored mitochondrial autophagy, reduced oxidative stress, apoptosis, and altered muscle fiber composition via the AKT/FOXO1 pathway, collectively improving muscle force in mdx mice. We propose AGH as a potential therapeutic strategy for DMD and related muscle disorders.

Background: Maintaining the connection between skeletal muscle fibers and the surrounding basement membrane is essential for muscle function. Dystroglycan (DG) serves as a basement membrane extracellular matrix (ECM) receptor in many cells, and is also expressed in the outward-facing membrane, or sarcolemma, of skeletal muscle fibers. DG is a transmembrane protein comprised of two subunits: alpha-DG (α-DG), which resides in the peripheral membrane, and beta-DG (β-DG), which spans the membrane to intracellular regions. Extensive post-translational processing and O-mannosylation are required for α-DG to bind ECM proteins, which is mediated by a glycan structure known as matriglycan. O-mannose glycan biosynthesis is initiated by the protein O-mannosyltransferase 1 (POMT1) and POMT2 enzyme complex and leads to three subtypes of glycans called core M1, M2, and M3. The lengthy core M3 is capped with matriglycan. Genetic defects in post-translational O-mannosylation of DG interfere with its receptor function and result in muscular dystrophy with central nervous system and skeletal muscle pathophysiology.

Methods: To evaluate how the loss of O-mannosylated DG in skeletal muscle affects the development and progression of myopathology, we generated and characterized mice in which the Pomt1 gene was specifically deleted in skeletal muscle (Pomt1^skm) to interfere with POMT1/2 enzyme activity. To investigate whether matriglycan is the primary core M glycan structure that provides the stabilizing link between the sarcolemma and ECM, we generated mice that retained cores M1, M2, and M3, but lacked matriglycan (conditional deletion of like-acetylglucosaminyltransferase 1; Large1^skm). Next, we restored Pomt1 using gene transfer via AAV2/9-MCK-mPOMT1 and determined the effect on Pomt1^skm pathophysiology.

Results: Our data showed that in Pomt1^skm mice O-mannosylated DG is required for sarcolemma resilience, remodeling of muscle fibers and muscle tissue, and neuromuscular function. Notably, we observed similar body size limitations, sarcolemma weakness, and neuromuscular weakness in Large1^skm mice that only lacked matriglycan. Furthermore, our data indicate that genetic rescue of Pomt1 in Pomt1^skm mice limits contraction-induced sarcolemma damage and skeletal muscle pathology.

Conclusions: Collectively, our data indicate that DG modification by Pomt1/2 results in core M3 capped with matriglycan, and that this is required to reinforce the sarcolemma and enable skeletal muscle health and neuromuscular strength.

Background: INTER- and INTRAmuscular fat (IMF) is elevated in high metabolic states and can promote inflammation. While magnetic resonance imaging (MRI) excels in depicting IMF, the lack of reproducible tools prevents the ability to measure change and track intervention success.

Methods: We detail an open-source fully-automated iterative threshold-seeking algorithm (ITSA) for segmenting IMF from T1-weighted MRI of the calf and thigh within three cohorts (CaMos Hamilton (N = 54), AMBERS (N = 280), OAI (N = 105)) selecting adults 45-85 years of age. Within the CaMos Hamilton cohort, same-day and 1-year repeated images (N = 38) were used to evaluate short- and long-term precision error with root mean square coefficients of variation; and to validate against semi-automated segmentation methods using linear regression. The effect of algorithmic improvements to fat ascertainment using 3D connectivity and partial volume correction rules on analytical precision was investigated. Robustness and versatility of the algorithm was demonstrated by application to different MR sequences/magnetic strength and to calf versus thigh scans.

Results: Among 439 adults (319 female(89%), age: 71.6 ± 7.6 yrs, BMI: 28.06 ± 4.87 kg/m², IMF%: 10.91 ± 4.57%), fully-automated ITSA performed well across MR sequences and anatomies from three cohorts. Applying both 3D connectivity and partial volume fat correction improved precision from 4.99% to 2.21% test-retest error. Validation against semi-automated methods showed R² from 0.92 to 0.98 with fully-automated ITSA routinely yielding more conservative computations of IMF volumes. Quality control shows 7% of cases requiring manual correction, primarily due to IMF merging with subcutaneous fat. A full workflow described methods to export tags for manual correction.

Conclusions: The greatest challenge in segmenting IMF from MRI is in selecting a dynamic threshold that consistently performs across repeated imaging. Fully-automated ITSA achieved this, demonstrated low short- and long-term precision error, conducive of use within RCTs.

Background: Muscle stem cells (MuSCs) undergo numerous state transitions throughout life, which are critical for supporting normal muscle growth and regeneration. Epigenetic modifications in skeletal muscle play a significant role in influencing the niche and cellular states of MuSCs. Mixed-lineage leukemia 4 (Mll4) is a histone methyltransferase critical for activating the transcription of various target genes and is highly expressed in skeletal muscle. This raises the question of whether Mll4 has a regulatory function in modulating the state transitions of MuSCs, warranting further investigation.

Methods: To assess if myofiber-expressed Mll4, a histone methyltransferase, contributes to the maintenance of MuSCs, we crossed MCK^Cre/+ or HSA^MerCreMer/+ mice to Mll4^f/f mice to generate myofiber-specific Mll4-deleted mice. Investigations were conducted using 8-week-old and 4-week-old MCK^Cre/+;Mll4^f/f mice, and adult HSA^MerCreMer/+;Mll4^f/f mice between the ages of 3 months and 6 months.

Results: During postnatal myogenesis, Mll4 deleted muscles were observed with increased number of cycling MuSCs that proceeded to a differentiation state, leading to MuSC deprivation. This phenomenon occurred independently of gender. When Mll4 was ablated in adult muscles using the inducible method, adult MuSCs lost their quiescence and differentiated into myoblasts, also causing the depletion of MuSCs. Such roles of Mll4 in myofibers coincided with decreased expression levels of distinct Notch ligands: Jag1 and Dll1 in pubertal and Jag2 and Dll4 in adult muscles.

Conclusions: Our study suggests that Mll4 is crucial for maintaining MuSCs in both pubertal and adult muscles, which may be accomplished through the modulation of distinct Notch ligand expressions in myofibers. These findings offer new insights into the role of myofiber-expressed Mll4 as a master regulator of MuSCs, highlighting its significance not only in developmental myogenesis but also in adult muscle, irrespective of sex.

Despite its notoriously mild phenotype, the dystrophin-deficient mdx mouse is the most common model of Duchenne muscular dystrophy (DMD). By mimicking a human DMD-associated metabolic comorbidity, hyperlipidemia, in mdx mice by inactivating the apolipoprotein E gene (mdx-ApoE) we previously reported severe myofiber damage exacerbation via histology with large fibro-fatty infiltrates and phenotype humanization with ambulation dysfunction when fed a cholesterol- and triglyceride-rich Western diet (mdx-ApoE^W). Herein, we performed comparative lipidomic and metabolomic analyses of muscle, liver and serum samples from mdx and mdx-ApoE^W mice using solution and high-resolution-magic angle spinning (HR-MAS) ¹H-NMR spectroscopy. Compared to mdx and regular chow-fed mdx-ApoE mice, we observed an order of magnitude increase in lipid deposition in gastrocnemius muscle of mdx-ApoE^W mice including 11-fold elevations in -CH₃ and -CH₂ lipids, along with pronounced elevations in serum cholesterol, fatty acid, triglyceride and phospholipids. Hepatic lipids were also elevated but did not correlate with the extent of muscle lipid infiltration or differences in serum lipids. This study provides the first lipometabolomic signature of severe mdx lesions exacerbated by high circulating lipids and lends credence to claims that the liver, the main regulator of whole-body lipoprotein metabolism, may play only a minor role in this process.

Intramuscular injection of Wnt7a has been shown to accelerate and augment skeletal muscle regeneration and to ameliorate dystrophic progression in mdx muscle, a model for Duchenne muscular dystrophy (DMD). Here, we assessed muscle regeneration and function in wild type (WT) and mdx mice where Wnt7a was deleted in muscle using a conditional Wnt7a floxed allele and a Myf5-Cre driver. We found that both WT and mdx mice lacking Wnt7a in muscle, exhibited marked deficiencies in muscle regeneration at 21 d following cardiotoxin (CTX) induced injury. Unlike WT, deletion of Wnt7a in mdx resulted in decreased force generation prior to CTX injury. However, both WT and mdx muscle lacking Wnt7a displayed decreased force generation following CTX injection. Notably the regeneration deficit in mdx mice was rescued by a single tail vein injection of extracellular vesicles containing Wnt7a (Wnt7a-EVs). Therefore, we conclude that the regenerative capacity of muscle in mdx mice is highly dependant on the upregulation of endogenous Wnt7a following injury, and that systemic delivery of Wnt7a-EVs represents a therapeutic strategy for treating DMD.

Background: Hip osteoarthritis patients display higher levels of fatty infiltration (FI) in the gluteus minimus (GM) compared to other hip muscles. We investigated specific histological factors such as fiber type composition and collagen deposition, and functional outcomes like muscle strength and activation associated with FI in these patients.

Methods: In twelve men (67 ± 6 y) undergoing total hip replacement (THR), hip and knee muscle strength and activation (electromyography, EMG) were assessed bilaterally. Magnetic resonance imaging (MRI) was used to compare the relative FI area and muscle cross sectional area (CSA) of the GM, rectus femoris (RF), tensor fascia latae (TFL) and vastus lateralis (VL). Adipocyte content, fiber type composition, grouping, fiber size, centrally nucleated fiber frequency, collagen deposition, satellite cell density and capillarization were assessed in intraoperative biopsies of the four muscles. Differences between GM and other muscles were assessed with repeated-measures one-way ANOVA followed by Dunnett's post-hoc test. Pearson coefficients were calculated for the correlations between FI measurements and the other histological and functional parameters.

Results: Strength was lower in the affected limb. Knee extensor weakness was accompanied by lower VL muscle activation. Muscle CSA and FI did not differ between sides. In the affected limb, GM displayed larger relative FI area (MRI) compared to RF and VL. Biopsy adipocyte content was higher in GM than RF and TFL. Compared to the other hip muscles, GM displayed higher type 1 fiber content while its type 2X fiber content was lower. Fiber grouping levels were higher in GM than the other muscles. Collagen content was higher in GM than TFL and VL.  FI in GM was associated with type 1 (r = 0.43) and type 2X (r = -0.34) fiber content, fiber grouping (r = 0.39), and collagen deposition (r = 0.37). FI in VL was negatively associated with maximal knee extension strength (r = -0.65).

Conclusions: In patients undergoing THR, the higher FI levels of GM compared to other hip muscles were associated with fiber type composition and grouping, and with higher collagen deposition. Experimental studies exploring these associations could potentially uncover new targets for the treatment of intramuscular FI and related impairments in muscle function.

Trial registration: KEK number: 2016-01852, date of registration: 12-4-2017.

Background: Insulin resistance and type 2 diabetes impair cellular regeneration in multiple tissues including skeletal muscle. The molecular basis for this impairment is largely unknown. Glucose uptake via glucose transporter GLUT4 is impaired in insulin resistance. In healthy muscle, acute injury stimulates glucose uptake. Whether decreased glucose uptake via GLUT4 impairs muscle regeneration is presently unknown. The goal of this study was to determine whether GLUT4 regulates muscle glucose uptake and/or regeneration following acute injury.

Methods: Tibialis anterior and extensor digitorum longus muscles from wild-type, control, or muscle-specific GLUT4 knockout (mG4KO) mice were injected with the myotoxin barium chloride to induce muscle injury. After 3, 5, 7, 10, 14, or 21 days (in wild-type mice), or after 7 or 14 days (in control & mG4KO) mice, muscles were isolated to examine [³H]-2-deoxyglucose uptake, GLUT4 levels, extracellular fluid space, fibrosis, myofiber cross-sectional area, and myofiber centralized nuclei.

Results: In wild-type mice, muscle glucose uptake was increased 3, 5, 7, and 10 days post-injury. There was a rapid decrease in GLUT4 protein levels that were restored to baseline at 5-7 days post-injury, followed by a super-compensation at 10-21 days. In mG4KO mice, there were no differences in muscle glucose uptake, extracellular fluid space, muscle fibrosis, myofiber cross-sectional areas, or percentage of centrally nucleated myofibers at 7 days post-injury. In contrast, at 14 days injured muscles from mG4KO mice exhibited decreased glucose uptake, muscle weight, myofiber cross sectional areas, and centrally nucleated myofibers, with no change in extracellular fluid space or fibrosis.

Conclusions: Collectively, these findings demonstrate that glucose uptake via GLUT4 regulates skeletal myofiber regeneration following acute injury.

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.