Search Results (445)

Cryotherapy is a commonly used strategy for skeletal muscle recovery, although the efficacy of its use has been controversial. Therefore, more research is needed to understand under what circumstances it should be used. This study aimed to examine the cell-autonomous effects of acute cold exposure on primary mouse myoblasts, focusing on metabolic signaling through the AMPK/mTORC1 pathway. In it, we hypothesized that cold exposure (COLD) would impair myoblast proliferation, differentiation, and protein synthesis in an AMPK-dependent manner. Wild-type (WT) and AMPK double-knockout (dKO) myoblast cultures were treated at 37 °C or 26 °C to evaluate AMPK-dependent effects. As expected, 30 min of cold exposure activated AMPK and decreased mTORC1 activity and protein synthesis; however, mTORC1 and protein synthesis were downregulated independently of AMPK activation. Additionally, cold exposure suppressed proliferation 6 h post-treatment in WT, but not dKO, myoblasts. On the other hand, in differentiated WT and dKO cells, cold treatment did not influence myotube size, although dKO myotubes exhibited decreased fusion index and increased size compared to WT. These findings offer new insights into the cell-autonomous metabolic effects of cryotherapy in skeletal muscle and indicate that while COLD-induced AMPK activation contributes to impaired myoblast proliferation, AMPK is not necessary for the COLD-induced inhibition of the mTORC1 pathway and protein synthesis. Full article

Biomimetic scaffolds are required in tissue engineering to provide structural support as well as promote cell adhesion, proliferation, and differentiation. Fibrous scaffolds composed of micro- and nanofibers replicate the architecture of the native extracellular matrix. Electrospinning is widely used for fabricating nanofibers; however, constructing fibrous scaffolds that integrate multiple fiber scales into a single structure is difficult. We addressed this issue by developing a fiber-spinning device using two pairs of counter-facing syringes that simultaneously produce micro- and nanofibers under different processing conditions. Poly(ε-caprolactone) solutions are ejected through needle-type nozzles via centrifugal force, and fiber diameter is controlled by adjusting the polymer concentration and nozzle diameter. We fabricated scaffolds with the proposed device, which exhibited a random three-dimensional fibrous network in which microfibers and nanofibers were homogeneously integrated. C2C12 myoblasts cultured on the composite scaffolds strongly adhered to the fibrous network, remained viable, and extended along the fibers to form multinucleated cells within the structure. The developed system produced composite micro/nanofiber scaffolds with tunable morphology and biocompatibility, providing a platform for fibrous tissue engineering applications. Full article

The basic helix-loop-helix (bHLH) transcription factor ‘Atoh8’ is involved in the regulation of several developmental processes and pathologies. It regulates organogenesis, reprogramming, stem cell fate determination, and cancer development. However, the mechanisms underlying these observations remain unclear. Unlike many tissue-specific bHLH factors, Atoh8 is ubiquitously expressed during development as well as in adult tissues. In this study, we explored whether Atoh8 modulates basic cellular functions, which may reveal a common mechanism that could explain the diverse observations reported in the literature. Our findings demonstrate that the loss of Atoh8 impairs autophagy. In both primary myoblasts and mouse embryonic stem cells lacking Atoh8, we observed differential expression of LC3B-II, TFEB, and accumulation of p62, indicating impairment of autophagy. Furthermore, mass spectrometric analysis performed on C2C12 and Atoh8 overexpressing C2C12 myoblasts revealed significant alterations in the expression of proteins associated with mitochondrial and lysosomal functions. Finally, Cut&Tag sequencing performed in Atoh8 overexpressing C2C12 cells revealed that Atoh8 binds to multiple genes involved in autophagosome assembly. Overall, this study underscores that Atoh8 is a critical regulator of macroautophagy, and its reduction disrupts the autophagic process, whereas its overexpression results in increased autophagic flux. Full article

Gelatin methacryloyl (GelMA) hydrogels are widely used in tissue engineering because of their tunable mechanical and biological properties. However, many studies have arbitrarily selected key synthesis parameters, such as methacrylic anhydride (MA) concentration, (lithium phenyl-2 4 6-trimethyl-benzoyl phosphinate) LAP concentration, GelMA content, UV exposure time, and reaction duration, without clear justification. This study aimed to systematically optimize GelMA hydrogel fabrication and evaluate the mechanical and biological performances of the resulting hydrogels for craniofacial muscle tissue engineering. Hydrogels were synthesized following a standardized protocol, and the reaction progress was confirmed via proton nuclear magnetic resonance (¹H-NMR). The swelling ratio, degradation behavior, compressive strength, and metabolic activity (AlamarBlue assay using C2C12 myoblasts) were assessed. Statistical analysis was performed using independent t-tests and one-way ANOVA with Tukey’s post hoc test (p < 0.05). The results showed that small variations in MA concentration and reaction time significantly affected the hydrogel properties. Higher GelMA concentrations (10–20%) enhanced the mechanical strength but reduced the biological activity. LAP ≥ 0.5% and prolonged UV exposure lowered metabolic activity, whereas 0.1% LAP with 1–2 min of UV exposure provided an optimal balance. These findings provide a reproducible framework for GelMA fabrication and establish a foundation for developing tailored biomaterials for muscle tissue engineering. Full article

Background/Objectives: Plasma glutamine levels in skeletal muscle change in response to exercise intensity and duration, both in physiological and pathological states. Glutamine contributes to muscle differentiation and regeneration; however, the mechanisms underlying this process remain unclear. This study investigated the role of glutamine glutaminolysis in myogenic differentiation, with a focus on epigenetic regulation of myogenin gene expression. Methods: C2C12 myoblasts were differentiated into myotubes using media containing various concentrations of glutamine, glutamate, or dimethyl 2-oxoglutarate (DM-α-KG), a cell-permeable analog of α-ketoglutarate. Results: Glutamine, glutamate, and DM-α-KG promoted C2C12 myoblast differentiation in a concentration-dependent manner, whereas the glutaminase inhibitor CB-839 suppressed differentiation. 4 mM glutamine increased myogenin mRNA expression by about 5-fold. CB-839 also inhibited glutamine-induced expression of myogenin but did not influence the effects of glutamate or DM-α-KG. Furthermore, glutamine increased histone H3 lysine 27 acetylation (H3K27ac) by about two-fold, whereas CB-839 (200 nM) and A-485 (10 µM), a CBP/p300 histone acetyltransferase inhibitor, reduced H3K27ac levels by about half. These results indicate that glutamine not only serves as a structural amino acid for muscle formation but also enhances myogenin transcription through epigenetic mechanisms. Conclusions: This report demonstrates glutaminolysis-dependent histone H3 acetylation, which induces myogenin transcription in myoblasts. These results, connecting glutamine supplementation during resistance training, may make it an effective strategy to accelerate muscle regeneration. Full article

Background: Piper nigrum L. (PNL) is a rich source of piperine, a bioactive alkaloid with pharmaceutical, cosmetic, nutritional supplement, and agricultural applications, yet efficient and sustainable extraction methods remain underexplored. Methods: This study compared ultrasonic bath extraction (UBE) and ultrasonic probe extraction (UPE) using natural deep eutectic solvents (NADES) for isolating piperine from PNL fruits. Six NADES formulations were screened, with NADES-5 (choline chloride:glycerin:urea, 1:1:1) showing superior performance. Response surface methodology with a Box–Behnken design optimized extraction parameters, including liquid-to-solid ratio, extraction time, temperature, and water content, for both UBE and UPE. Results: Optimized UPE consistently outperformed UBE, yielding 49.97 mg/g of piperine versus 25.67 mg/g under identical NADES conditions. Comprehensive characterization using TLC, HPTLC, UV, FTIR, Raman, HPLC, NMR, XRD, SEM, and EDX confirmed the successful isolation and structural integrity of piperine, with samples obtained via UPE exhibiting higher purity (98.7% vs. 95.2%) and enhanced crystallinity. In vitro cytotoxicity assays demonstrated that piperine extracted by UPE showed stronger activity against C2C12 myoblasts (IC₅₀: 24.3 μg/mL vs. 40.6 μg/mL) and greater anticancer effects in MCF-7 and HT-29 cells compared to piperine extracted by UBE. Antioxidant evaluation via DPPH, ABTS, FRAP, and TAC assays, along with intracellular reactive oxygen and nitrogen species suppression in THP-1 and RAW 264.7 macrophages, further confirmed the superior biological potential of the UPE-derived piperine sample. Conclusions: These findings indicate that UPE using NADES is a sustainable approach for high-yield piperine extraction with enhanced purity and bioactivity, supporting its potential for pharmaceutical applications. Full article

In this report, we showed that oral administration of Dendrobium Taiseed Tosnobile (DTT, also known as Taiwan Emperor No.1) allowed Lewis Lung Carcinoma (LLC) tumor-bearing mice to maintain body weight and grip strength in a dose-dependent manner. Histological analysis showed that treatment with DTT water extract significantly reduced muscle fiber damage by inducing muscle regeneration and improved the cross-sectional area of the rectus femoris, soleus, and gastrocnemius of LLC tumor-bearing C57BL/6 female mice. Further studies revealed that DTT water extract also reduced the expression of inflammatory cytokines such as IL-6 and TNF-α, both in vitro and in vivo. Other analyses showed that DTT water extract promoted the differentiation of C2C12 myoblasts with or without IL-6 by maintaining Myosin Heavy Chain (MyHC) levels. This suggests that DTT water extract acts against muscle wasting via multiple mechanisms. Interestingly, vitamin B1 was identified as an ingredient in DTT water extract through an HPLC analysis. Vitamin B1 was shown to ameliorate IL-6 but not TNF-α generation in active THP-1 cells and protected C2C12 myotubes against IL-6. Further studies showed that DTT and vitamin B1 promoted the multi-nucleus fusion step of C2C12 differentiation by inducing E-cadherin-β-catenin expression with or without IL-6 treatment. In summary, DTT water extract protects muscle cells under cancer conditions through direct and indirect mechanisms, with vitamin B1 being a key functional ingredient that reduces IL-6 generation and aids muscle cell fusion against IL-6 treatment. Full article

Background/Objectives: Palmitoylethanolamide (PEA) is an endogenous lipid mediator with endocannabinoid-like activity. Despite its therapeutic potential in muscle-related inflammatory disorders, including sarcopenia, its clinical use is limited by poor solubility and bioavailability. To overcome these issues, we developed hybrid nanoparticles combining poly(lactic-co-glycolic acid) (PLGA) and lipids to enhance PEA encapsulation and ok delivery. Methods: PEA-loaded hybrid nanoparticles (PEA-Hyb-np) were produced via a modified single-emulsion solvent evaporation method using stearic acid and Gelucire^® 50/13 as lipid components. Characterization included particle size, morphology, PDI, and zeta potential, as well as DSC, FT-IR, and XRD analyses. For the biological evaluation in a C2C12 myoblasts cell culture, coumarin-6-labeled nanoparticles were employed. Results: PEA-Hyb-np showed mean particle sizes of ~150 nm, with internal lipid–polymer phase separation. This structure enabled high encapsulation efficiency (79%) and drug loading (44.2 mg/g). Drug release in physiological and non-physiological media was enhanced due to drug amorphization, confirmed by DSC, FT-IR, and XRD analyses. Cytocompatibility studies showed no toxicity and improved cell viability compared to unloaded nanoparticles. Cellular uptake studies by confocal microscopy and flow cytometry demonstrated efficient and time-dependent internalization. Conclusions: PEA-Hyb-np represent a promising delivery platform to improve the solubility, bioavailability, and therapeutic efficacy of PEA for muscle-targeted applications. Full article

Background/Objective: Cellular senescence is increasingly recognized as a key mechanism underlying sarcopenia, an age-related muscle disorder with no effective therapeutic. 6,4′-Dihydroxy-7-methoxyflavanone (DMF), a flavonoid isolated from Dalbergia odorifera T. Chen, has shown anti-senescence potential. This study aimed to investigate the protective effects of DMF against myoblasts senescence and elucidate the underlying molecular mechanisms. Method: A cellular model of senescence was established in C2C12 myoblasts using D-galactose (D-gal). The effects of DMF pretreatment were evaluated by assessing senescence phenotypes, myogenic differentiation, and mitochondrial function. The role of Sirtuin3 (SIRT3) was confirmed using siRNA-mediated knockdown. Results: DMF Pre-treatment effectively attenuated D-gal-induced senescence, as indicated by restored proliferation, reduced senescence-associated β-galactosidase activity, decreased DNA damage, and the downregulation of p53, p21^Cip1/WAF1 and p16^INK4a. Furthermore, DMF rescued myogenic differentiation capacity, enhancing the expression of Myoblast determination protein 1, Myogenin, Myosin heavy chain and Muscle-specific regulatory factor 4, and promoting myotube formation. Mechanistically, DMF was identified as a SIRT3 activator. It enhanced SIRT3 expression and activity, leading to the deacetylation and activation of the mitochondrial antioxidant enzyme superoxide dismutase 2. This consequently reduced mitochondrial reactive oxygen species, improved mitochondrial membrane potential and ATP production, and suppressed the NF-κB pathway by inhibiting IκBα phosphorylation and p65 acetylation/nuclear translocation. Crucially, all the beneficial effects of DMF—including oxidative stress reduction, mitochondrial functional recovery, anti-inflammatory action, and ultimately, the attenuation of senescence and improvement of myogenesis—were abolished upon SIRT3 knockdown. Conclusions: Our findings demonstrate that DMF alleviates myoblasts senescence and promotes myogenic differentiation by activating the SIRT3-SOD2 pathway, thereby reducing oxidative stress and NF-κB-driven inflammation responses. DMF emerges as a promising therapeutic candidate for sarcopenia. Full article

Myogenesis is a tightly regulated process essential for embryonic development, postnatal growth, and muscle regeneration. We recently identified that cytokinins (CTKs), a class of adenine-derived signaling molecules originally characterized in plants, are present in cultured skeletal muscle cells. The most abundant type of cytokinins detected within cultured muscle cells was isopentenyladenine (iP) in its nucleotide, riboside, and free base derivatives. The purpose of this study was to determine whether CTKs are also present in regenerating muscle tissue in vivo and to characterize the effects of iP and its riboside form, isopentenyladenosine (iPR), on muscle cell proliferation and differentiation. These effects were observed relative to adenine and adenosine, and to a second class of cytokinins with a large aromatic side chain, kinetin (the free base), and kinetin riboside. Cardiotoxin was used to induce muscle injury and repair processes in the gastrocnemius of 3- and 12-month-old mice. Samples were collected 3- and 7 days post-injury for ultra high-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-(ESI+)-HRMS/MS). Four CTKs (N⁶-benzyladenine (BA), dihydrozeatin-9-N-glucoside (DZ9G), isopentenyladenosine (iPR), and 2-methylthio-isopentenyladenosine (2-MeSiPR) were detected. 2-MeSiPR levels were significantly influenced by aging, as this CTK was increased in response to injury only in the younger mice. Treatment of C2C12 myoblasts with 10 µM of isopentenyladenosine (iPR) or kinetin riboside reduced cell proliferation, whereas iP (the free base) increased proliferation in a biphasic response. During differentiation, both iPR and kinetin riboside impaired myotube formation, while the free-base forms of iP and kinetin had no effect. Our data establishes that CTKs are present within muscle tissue and highly responsive to injury and aging. Furthermore, the biological activities of CTKs in muscle cells are influenced by structural modifications, including riboside conjugation and side chain composition. Understanding these differences provides insight into the distinct roles of CTKs in muscle cell metabolism and differentiation, offering potential implications for the use of exogenous CTKs in muscle biology and regenerative medicine. Full article

Background: L-arginine is a conditionally essential amino acid that serves as a substrate for nitric oxide synthase and regulates energy metabolism. While its ergogenic effects have been proposed, the mechanisms underlying its anti-fatigue properties are not fully understood. Methods: Male ICR mice were orally administered L-arginine (300, 600, or 1200 mg/kg bw/day) for 28 days. Fatigue was chronically induced using twice-weekly forced swimming or treadmill running, and fatigue resistance was then assessed under these paradigms. Blood, skeletal muscle, and liver were analyzed for biomarkers including glucose, lactate, LDH, CPK, NEFA, ammonia, glycogen, nitric oxide, cortisol, and antioxidant enzymes. In parallel, C2C12 myoblasts were treated with L-arginine under proliferative and differentiated conditions to assess hexokinase (HK) activity, myogenin expression, and ROS generation. Results: In vivo, L-arginine decreased serum LDH, CPK, NEFA, ammonia, nitric oxide, and cortisol, while enhancing blood glucose and glycogen storage in both muscle and liver. Forced swimming reduced serum lactate, whereas treadmill exercise elevated intramuscular lactate, suggesting context-dependent lactate regulation. Importantly, L-arginine did not significantly improve forced-swimming immobility time, whereas treadmill time-to-exhaustion increased at the highest dose. Antioxidant responses were improved, as reflected by normalized hepatic catalase activity. In vitro, L-arginine increased HK activity, promoted myogenin expression, and reduced ROS levels, supporting improved glucose utilization, muscle differentiation, and oxidative stress resistance. Conclusions: These findings demonstrate that L-arginine supplementation under chronic fatigue-inducing paradigms improves endurance and alleviates fatigue by enhancing energy metabolism, preserving glycogen, reducing muscle injury, and attenuating oxidative stress. L-arginine shows potential as a functional ingredient for promoting exercise performance and recovery. Full article

Magnesium (Mg²⁺) is a key regulator of cellular biochemical processes and an essential cofactor in skeletal muscle physiology. Although Mg²⁺ deficiency has been linked to reduced muscle strength, its role in the regulation of calcium (Ca²⁺) signaling and in inflammation remains incompletely understood. In this study, we examined the effects of Mg²⁺ availability using the murine myoblast cell line C2C12. Cells were differentiated under low, normal, or high Mg²⁺ conditions, and myotube formation, intracellular Ca²⁺ fluxes, and resistance to inflammatory stimuli were assessed. Mg²⁺ deficiency impaired myotube differentiation, while Mg²⁺ supplementation preserved Ca²⁺ response during stimulation and contributed to protect myotubes against inflammation-induced damage. Collectively, these findings highlight a dual role of Mg²⁺ in sustaining functional performance under repeated stress and protecting myotubes against inflammatory injury. This study supports the importance of adequate dietary Mg²⁺ intake as a potential strategy to mitigate muscle loss associated with aging and chronic inflammation. Full article

Mechanistically, OPs inhibit acetylcholinesterase (AChE), an enzyme that terminates cholinergic transmission, triggering a sustained activation of acetylcholine receptors. A component of the treatment for OP intoxication is oximes as AChE reactivators. However, oximes may not be efficacious and could worsen OP effects. Further, dealkylation of the AChE-OP adducts prevents oxime reactivation. Therefore, other approaches are needed to rescue AChE activity. We propose that replacing aged extracellular AChE with active intracellular enzymes may be an effective approach. Thus, molecular screening was used to identify small molecules that could displace aged AChE. C2C12 myoblasts were treated with 20 μM of diisopropylfluorophosphate (DFP) for one hour, followed by a drug panel. AChE activity and surface abundance were measured after 6 h. From the chemical screen, a promising hit, Pz-1 (a tyrosine kinase inhibitor), was identified, which decreased surface AChE on DFP-exposed C2C12 myoblasts in a dose-dependent manner without impacting viability. Additionally, AChE presence and activity were recovered after washing and supplementing the media with 100 nM of acetylcholine. Biochemically, Pz-1 inhibits muscle-specific protein kinase (MuSK), a kinase that interacts with AChE. These results suggest that altering MuSK activity may disrupt protein–protein interactions, destabilizing AChE, which may lead to the discovery of new countermeasures for OP exposures. Full article

This study aimed to evaluate the regenerative effects of various microcurrent waveforms in cast-induced gastrocnemius muscle atrophy in rabbits, integrating both in vitro and in vivo analyses. After two weeks of enforced hindlimb immobilization via casting, twenty-four rabbits were divided into four groups and treated for two weeks: Group-1 (control) received sham microcurrent, Group-2 was treated with a square waveform microcurrent, Group-3 with a sine waveform, and Group-4 with a triangular waveform. Treatments were administered daily for one hour. Calf circumference, muscle thickness (via ultrasound), tibial nerve CMAP, muscle fiber CSA, and protein expression (via Western blot analysis) were assessed. Among the groups, the sine waveform microcurrent resulted in significantly enhanced recovery across all measured parameters (p < 0.05), showing superior improvements in muscle thickness, CMAP amplitude, and fiber CSA. Immunohistochemical analysis revealed increased expression of proliferation and angiogenesis markers, including BrdU, PCNA, VEGF, and PECAM-1, while Western blotting demonstrated robust upregulation of myogenic regulatory factors such as MyoD and myogenin. Furthermore, levels of inflammatory and apoptotic markers, including TNF-α, NF-κB, and cleaved caspase-3, and stress response proteins, including p-CHK1 and p-CHK2, were markedly reduced. Collectively, these findings indicate that sine waveform microcurrent stimulation most effectively promotes muscle regeneration in both dexamethasone-induced C2C12 myoblasts and cast-induced muscle atrophy, underscoring its therapeutic potential and warranting further studies to optimize clinical application parameters. Full article

We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the differentiation and fusion of skeletal myoblasts. We have also analyzed the genomic programs underlying rexinoid-enhanced myogenic differentiation to identify novel regulatory pathways. As such, we observed a significant upregulation of a transcript encoding a predicted transmembrane protein, Tmem182, during C2C12 myoblast differentiation. Despite the documentation of Tmem182 expression in skeletal muscles, its regulation had yet to be explored. Here, we show that Tmem182 gene expression is markedly augmented in early myoblast differentiation and further enhanced by RXR signaling. In addition, Tmem182 expression is specific to muscle tissues and related to muscle master regulator MyoD. We found that MyoD and histone acetyltransferase p300 are bound to the Tmem182 promoter, and Tmem182 expression is p300-dependent. Thus, our data display a putative epigenetic signature associated with p300 and histone acetylation in rexinoid-responsive locus activation and transcription of myogenic targets. Full article