Myotube Diameter Research Articles

The mechanosensitive gene arrestin domain containing 2 regulates myotube diameter with direct implications for disuse atrophy with aging.

Arrestin domain containing 2 and 3 (Arrdc2/3) are genes whose mRNA contents are decreased in young skeletal muscle following mechanical overload. Arrdc3 is linked to the regulation of signaling pathways in nonmuscle cells that could influence skeletal muscle size. Despite a similar amino acid sequence, Arrdc2 function remains undefined. The purpose of this study was to further explore the relationship of Arrdc2/Arrdc3 expression with changes in mechanical load in young and aged muscle and define the effect of Arrdc2/3 expression on C2C12 myotube diameter. In young and aged mice, mechanical load was decreased using hindlimb suspension whereas mechanical load was increased by reloading previously unloaded muscle or inducing high-force contractions. Arrdc2 and Arrdc3 mRNAs were overexpressed in C2C12 myotubes using adenoviruses. Myotube diameter was determined 48-h posttransfection, and RNA sequencing was performed on those samples. Arrdc2 and Arrdc3 mRNA content was higher in the unloaded muscle within 1 day of disuse and remained higher up through 10 days. The induction of Arrdc2 mRNA was more pronounced in aged muscle than young muscle in response to unloading. Reloading previously unloaded muscle of young and aged mice restored Arrdc2 and Arrdc3 levels to ambulatory levels. Increasing mechanical load beyond normal ambulatory levels lowered Arrdc2 mRNA, but not Arrdc3 mRNA, in young and aged muscle. Arrdc2 overexpression only was sufficient to lower myotube diameter in C2C12 cells in part by altering the transcriptome favoring muscle atrophy. These data are consistent with Arrdc2 contributing to disuse atrophy, particularly in aged muscle.NEW & NOTEWORTHY We establish Arrdc2 as a novel mechanosensitive gene highly induced in response to mechanical unloading, particularly in aged muscle. Arrdc2 induction in C2C12 myotubes is sufficient to produce thinner myotubes and a transcriptional landscape consistent with muscle atrophy and disuse.

Gromwell ameliorates glucocorticoid-induced muscle atrophy through the regulation of Akt/mTOR pathway

BackgroundMuscle atrophy is characterized by decreased muscle mass, function, and strength. Synthetic glucocorticoids, including dexamethasone (Dexa), are commonly used to treat autoimmune diseases. However, prolonged exposure of Dexa with high dose exerts severe side effects, including muscle atrophy. The purpose of this study was to investigate whether Gromwell root extract (GW) can prevent Dexa-induced muscle atrophy in C2C12 cells and mice and to characterize the composition of GW to identify bioactive compounds.MethodsFor in vitro experiments, GW (0.5 and 1 µg/mL) or lithospermic acid (LA, 5 and 10 µM) was added to C2C12 myotubes on day 4 of differentiation and incubated for 24 h, along with 50 µM Dexa. For in vivo experiment, four-week-old male C57BL/6 mice were randomly divided into the four following groups (n = 7/group): Con group, Dexa group, GW0.1 group, and GW0.2 group. Mice were fed experimental diets of AIN-93 M with or without 0.1 or 0.2% GW for 4 weeks. Subsequently, muscle atrophy was induced by administering an intraperitoneal injection of Dexa at a dose of 15 mg/kg/day for 38 days, in conjunction with dietary intake.ResultsIn Dexa-induced myotube atrophy, treatment with GW increased myotube diameter, reduced the expression of muscle atrophy markers, and enhanced the expression of myosin heavy chain (MHC) isoforms in C2C12 cells. Supplementation with the GW improved muscle function and performance in mice with Dexa-induced muscle atrophy, evidenced in the grip strength and running tests. The GW group showed increased lean body mass, skeletal muscle mass, size, and myosin heavy chain isoform expression, along with reduced skeletal muscle atrophy markers in Dexa-injected mice. Supplementation with GW increased protein synthesis and decreased protein degradation through the Akt/mammalian target of rapamycin and glucocorticoid receptor/forkhead box O3 signaling pathways, respectively. We identified LA as a potential bioactive component of the GW. LA treatment increased myotube diameter and decreased the expression of muscle atrophy markers in Dexa-induced C2C12 cells.ConclusionsThese findings underscore the potential of the GW in preventing Dexa-induced skeletal muscle atrophy and highlight the contribution of LA to its effects.

Abstract C062: Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting

Abstract Background: Patients with pancreatic cancer (PC) often experience cachexia, a multifactorial syndrome that includes weight loss, malnutrition, appetite suppression, and tissue wasting. Unfortunately, the factors secreted from the pancreas or other tissues that drive cachexia are still poorly defined. Lipocalin 2 (LCN2) is a cytokine involved in innate immunity that we have previously shown to be increased in the pancreas and circulation of patients with PC and mouse models of PC. Recent studies suggest that LCN2 contributes to appetite suppression in cancer cachexia in orthotopic models of PC. However, a reduction in muscle regeneration is also commonly seen in cachexia that is not repaired by increased nutrient intake. In a non-cancer setting, LCN2 has been implicated as an important factor in mediating skeletal muscle repair. Therefore, we hypothesized that elevated LCN2 in PC contributes to cachexia by inhibiting muscle progenitor cell proliferation and differentiation into new muscle fibers. Methods: Muscles from a syngeneic orthotopic mouse model of PC were assessed for muscle RING-finger protein-1 (MuRF1, an E3 ubiquitin ligase highly expressed in muscle during wasting) and LCN2 by qPCR. C2C12 mouse muscle myoblasts were cultured in proliferation or differentiation media with recombinant mouse LCN2. Proliferation across 3 days was assessed by MTS assay. Differentiation was assessed by imaging myotubes and western blotting for myosin heavy chain (MyHC) expression which increases during differentiation. PC genetically engineered mice ± a whole body LCN2 were assessed for pancreatic tumor and gastroc muscle size. One-way and Two-way ANOVAs were used to determine statistical significance. Results: LCN2 and MuRF1 expression are elevated in muscles of mice with PC. C2C12 cells treated with LCN2 decreased proliferation in a dose-dependent manner up to concentrations of LCN2 commonly observed in PC. LCN2 treatments at a physiologic concentration observed in PC delayed differentiation of C2C12 cells into myotubes by reducing myotube diameter and delaying MyHC expression. Deleting LCN2 in a PC genetic mouse model prevented body weight and gastroc weight loss but did not affect pancreas weight. Conclusions: Here, we show a relationship between muscle wasting and LCN2 expression during PC. Elevated circulating LCN2 may be contributing to decreased muscle regeneration observed in PC-associated cachexia by directly inhibiting muscle progenitor cell proliferation and differentiation. Targeting LCN2 in PC may be a viable method of slowing or preventing muscle wasting in PC to improve patient outcomes. Citation Format: Kristyn Gumpper-Fedus, Valentina Pita Grisanti, Ericka Velez Bonet, Martha A. Belury, Christopher Coss, Zobeida Cruz-Monserrate. Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C062.

Remedial effects of tilapia skin peptides against dexamethasone-induced muscle atrophy in mice by modulation of AKT/FOXO3a and Sirt1/PGC-1α signaling pathways

Tilapia skin peptides (TSP) possess a range of physiological activities. This study aimed to explore the effects of TSP on Dexamethasone (DEX)-induced muscle atrophy. In vitro, C2C12 myotube myotube diameter and expression levels of the muscle-specific E3 ubiquitin ligases F-box only protein 32 (Atrogin-1) and muscle ring finger protein 1 (MuRF1) challenged with DEX were reversed by TSP. In vivo, DEX was injected subcutaneously to build muscle atrophy model mice. TSP enhanced grip strength, running distance, body lean muscle content, cross-sectional area of the gastrocnemius and tibialis anterior muscles of DEX-induced mice. Moreover, TSP inhibited the expression levels of Atrogin-1 and MuRF1. Mechanically, TSP improved DEX-induced muscle atrophy by regulating the Protein Kinase B α (AKT)/Forkhead box O3 protein (FOXO3a), Sirtuin 1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling pathway, and downstream factors such as nuclear respiratory factor (NRF)1/2 and mitochondrial transcription factor A (TFAM).

Dietary supplementation with Lacticaseibacillus rhamnosus IDCC3201 alleviates sarcopenia by modulating the gut microbiota and metabolites in dexamethasone-induced models.

Probiotics can exert direct or indirect influences on various aspects of health claims by altering the composition of the gut microbiome and producing bioactive metabolites. The aim of this study was to examine the effect of Lacticaseibacillus rhamnosus IDCC3201 on skeletal muscle atrophy in dexamethasone-induced C2C12 cells and a mouse animal model. Dexamethasone treatment significantly reduced C2C12 muscle cell viability, myotube diameter, and levels of muscle atrophic markers (Atrogin-1 and MuRF-1). These effects were alleviated by conditioned media (CM) and cell extract (EX) derived from L. rhamnosus IDCC3201. In addition, we assessed the in vivo therapeutic effect of L. rhamnosus IDCC3201 in a mouse model of dexamethasone (DEX)-induced muscle atrophy. Supplementation with IDCC3201 resulted in significant enhancements in body composition, particularly in lean mass, muscle strength, and myofibril size, in DEX-induced muscle atrophy mice. In comparison to the DEX-treatment group, the normal and DEX + L. rhamnosus IDCC3201 groups showed a higher transcriptional level of myosin heavy chain family genes (MHC1, MHC1b, MHC2A, 2bB, and 2X) and a reduction in atrophic muscle makers. These analyses revealed that L. rhamnosus IDCC3201 supplementation led to increased production of branched-chain amino acids (BCAAs) and improved the Allobaculum genus within the gut microbiota of muscle atrophy-induced groups. Taken together, our findings suggest that L. rhamnosus IDCC3201 represents a promising dietary supplement with the potential to alleviate sarcopenia by modulating the gut microbiome and metabolites.

Beef peptides mitigate skeletal muscle atrophy in C2C12 myotubes through protein degradation, protein synthesis, and the oxidative stress pathway.

This study aimed to investigate the potential of beef peptides (BPs) in mitigating muscle atrophy induced by dexamethasone (DEX) with underlying three mechanisms in vitro (protein degradation, protein synthesis, and the oxidative stress pathway). Finally, the anti-atrophic effect of BPs was enhanced through purification and isolation. BPs were generated using beef loin hydrolyzed with alcalase/ProteAX/trypsin, each at a concentration of 0.67%, followed by ultrafiltration through a 3 kDa cut-off. BPs (10-100 μg mL-1) dose-dependently counteracted the DEX-induced reductions in myotube diameters, differentiation, fusion, and maturation indices (p < 0.05). Additionally, BPs significantly reduced FoxO1 protein dephosphorylation, thereby suppressing muscle-specific E3 ubiquitin ligases such as muscle RING-finger containing protein-1 and muscle atrophy F-box protein in C2C12 myotubes at concentrations exceeding 25 μg mL-1 (p < 0.05). BPs also enhanced the phosphorylation of protein synthesis markers, including mTOR, 4E-BP1, and p70S6K1, in a dose-dependent manner (p < 0.05) and increased the mRNA expression of antioxidant enzymes. Fractionated peptides derived from BPs, through size exclusion and polarity-based fractionation, also demonstrated enhanced anti-atrophic effects compared to BPs. These peptides downregulated the mRNA expression of primary muscle atrophy markers while upregulated that of antioxidant enzymes. Specifically, peptides GAGAAGAPAGGA (MW 924.5) and AFRSSTKK (MW 826.4) were identified from fractionated peptides of BPs. These findings suggest that BPs, specifically the peptide fractions GAGAAGAPAGGA and AFRSSTKK, could be a potential strategy to mitigate glucocorticoid-induced skeletal muscle atrophy by reducing the E3 ubiquitin ligase activity.

Effects of resistance training on alleviating hypoxia-induced muscle atrophy: Focus on acetylation of FoxO1.

This study aims to explore the role of FoxO1 and its acetylation in the alleviation of hypoxia-induced muscle atrophy by resistance training. Forty male Sprague-Dawley rats were randomly divided into four groups: normoxic control group (C), normoxic resistance training group (R), hypoxic control group (H) and hypoxic resistance training group (HR). Rats in R and HR groups were trained on an incremental weight-bearing ladder every other day, while those in H and HR groups were kept in an environment containing 12.4% O2 . After 4 weeks, muscles were collected for analysis. Differentiated L6 myoblasts were analysed invitro after hypoxia exposure and plasmids transfection (alteration in FoxO1 acetylation). The lean body mass loss, wet weight and fibre cross-sectional area of extensor digitorum longus of rats were decreased after 4 weeks hypoxia, and the adverse reactions above was reversed by resistance training. At the same time, the increase in hypoxia-induced autophagy was suppressed, which was accompanied by a decrease in the expression of nuclear FoxO1 and cytoplasmic Ac-FoxO1 by resistance training. The L6 myotube diameter increased and the expression of autophagic proteins were inhibited under hypoxia via intervening by FoxO1 deacetylation. Overall, resistance training alleviates hypoxia-induced muscle atrophy by inhibiting nuclear FoxO1 and cytoplasmic Ac-FoxO1-mediated autophagy.

Eldecalcitol prevents muscle loss and osteoporosis in disuse muscle atrophy via NF-κB signaling in mice

We investigated the effect of eldecalcitol on disuse muscle atrophy. C57BL/6J male mice aged 6 weeks were randomly assigned to control, tail suspension (TS), and TS-eldecalcitol–treated groups and were injected intraperitoneally twice a week with either vehicle (control and TS) or eldecalcitol at 3.5 or 5 ng for 3 weeks. Grip strength and muscle weights of the gastrocnemius (GAS), tibialis anterior (TA), and soleus (SOL) were determined. Oxidative stress was evaluated by malondialdehyde, superoxide dismutase, glutathione peroxidase, and catalase. Bone microarchitecture was analyzed using microcomputed tomography. The effect of eldecalcitol on C2C12 myoblasts was analyzed by measuring myofibrillar protein MHC and the atrophy markers Atrogin-1 and MuRF-1 using immunofluorescence. The influence of eldecalcitol on NF-κB signaling pathway and vitamin D receptor (VDR) was assessed through immunofluorescence, (co)-immunoprecipitation, and VDR knockdown studies. Eldecalcitol increased grip strength (P < 0.01) and restored muscle loss in GAS, TA, and SOL (P < 0.05 to P < 0.001) induced by TS. An improvement was noted in bone mineral density and bone architecture in the eldecalcitol group. The impaired oxidative defense system was restored by eldecalcitol (P < 0.05 to P < 0.01 vs. TS). Eldecalcitol (10 nM) significantly inhibited the expression of MuRF-1 (P < 0.001) and Atrogin-1 (P < 0.01), increased the diameter of myotubes (P < 0.05), inhibited the expression of P65 and P52 components of NF-κB and P65 nuclear location, thereby inhibiting NF-κB signaling. Eldecalcitol promoted VDR binding to P65 and P52. VDR signaling is required for eldecalcitol-mediated anti-atrophy effects. In conclusion, eldecalcitol exerted its beneficial effects on disuse-induced muscle atrophy via NF-κB inhibition.

Angiotensin-(1-7) improves skeletal muscle regeneration.

Skeletal muscle possesses regenerative potential via satellite cells, compromised in muscular dystrophies leading to fibrosis and fat infiltration. Angiotensin II (Ang-II) is commonly associated with pathological states. In contrast, Angiotensin (1-7) [Ang-(1-7)] counters Ang-II, acting via the Mas receptor. While Ang-II affects skeletal muscle regeneration, the influence of Ang-(1-7) remains to be elucidated. Therefore, this study aims to investigate the role of Ang-(1-7) in skeletal muscle regeneration. C2C12 cells were differentiated in the absence or presence of 10 nM of Ang-(1-7). The diameter of myotubes and protein levels of myogenin and myosin heavy chain (MHC) were determined. C57BL/6 WT male mice 16-18 weeks old) were randomly assigned to injury-vehicle, injury-Ang-(1-7), and control groups. Ang-(1-7) was administered via osmotic pumps, and muscle injury was induced by injecting barium chloride to assess muscle regeneration through histological analyses. Moreover, embryonic myosin (eMHC) and myogenin protein levels were evaluated. C2C12 myotubes incubated with Ang-(1-7) showed larger diameters than the untreated group and increased myogenin and MHC protein levels during differentiation. Ang-(1-7) administration enhances regeneration by promoting a larger diameter of new muscle fibers. Furthermore, higher numbers of eMHC (+) fibers were observed in the injured-Ang-(1-7), which also had a larger diameter. Moreover, eMHC and myogenin protein levels were elevated, supporting enhanced regeneration due to Ang-(1-7) administration. Ang-(1-7) effectively promotes differentiation in vitroand improves muscle regeneration in the context of injuries, with potential implications for treating muscle-related disorders.

Alcalase-Assisted Mytilus edulis Hydrolysate: A Nutritional Approach for Recovery from Muscle Atrophy.

Muscle atrophy is a complex physiological condition caused by a variety of reasons, including muscle disuse, aging, malnutrition, chronic diseases, immobilization, and hormonal imbalance. Beyond its effect on physical appearance, this condition significantly reduces the quality of human life, thus warranting the development of preventive strategies. Although exercising is effective in managing this condition, it is applicable only for individuals who can engage in physical activities and are not bedridden. A combination of exercise and nutritional supplementation has emerged as a more advantageous approach. Here, we evaluated the effects of enzyme-assisted hydrolysates of Mytilus edulis prepared using Protamex (PMH), Alcalase (AMH), or Flavourzyme (FMH) in protecting against muscle atrophy in a dexamethasone (Dex)-induced muscular atrophy model in vitro and in vitro. Alcalase-assisted M. edulis hydrolysate (AMH) was the most efficient among the tested treatments and resulted in higher protein recovery (57.06 ± 0.42%) and abundant amino acid composition (43,158 mg/100 g; 43.16%). AMH treatment also escalated the proliferation of C2C12 cells while increasing the total number of nuclei, myotube coverage, and myotube diameter. These results were corroborated by a successful reduction in the levels of proteins responsible for muscle atrophy, including E3 ubiquitin ligases, and an increase in the expression of proteins associated with muscle hypertrophy, including myogenin and MyHC. These results were further solidified by the successful enhancement of locomotor ability and body weight in zebrafish following AMH treatment. Thus, these findings highlight the potential of AMH in recovery from muscle atrophy.

Steamed Ginseng Berry Powder Ameliorates Skeletal Muscle Atrophy via Myogenic Effects.

Sarcopenia is an age-related loss of muscle mass and function for which there is no approved pharmacological treatment. We tested direct efficacy by evaluating grip strength improvement in a sarcopenia mouse model rather than drug screening, which inhibits specific molecular mechanisms. Various physiological functions of ginseng berries are beneficial to the human body. The present study aimed to evaluate the efficacy and safety of steamed ginseng berry powder (SGBP). SGBP administration increased myotube diameter and suppressed the mRNA expression of sarcopenia-inducing molecules. SGBP also reduced the levels of inflammatory transcription factors and cytokines that are known to induce sarcopenia. Oral administration of SGBP improved muscle mass and physical performance in a mouse model of sarcopenia. In summary, our data suggest that SGBP is a novel therapeutic candidate for the amelioration of muscle weakness, including sarcopenia.

Cancer-Cachexia-Induced Human Skeletal Muscle Myotube Degeneration Is Prevented via Cannabinoid Receptor 2 Agonism In Vitro.

Cachexia syndrome, leading to reduced skeletal muscle and fat mass, is highly prevalent in cancer patients, resulting in further negative implications for these patients. To date, there is no approved therapy for cachexia syndrome. The objective of this study was to establish an in vitro model of cancer cachexia in mature human skeletal muscle myotubes, with the intention of exploiting the cell model to assess potential cachexia therapeutics, specifically cannabinoid related drugs. Having cultured and differentiated primary human muscle myoblasts to mature myotubes, we successfully established two cancer cachexia models using conditioned media (CM) from human colon adenocarcinoma (SW480) and from non-small-cell lung carcinoma (H1299) cultured cells. The cancer-CM-induced extensive myotube degeneration, demonstrated by a significant reduction in mature myotube diameter, which progressed over the period studied. Myotube degeneration is a characteristic feature of cancer cachexia and was used in this study as an index of cachexia. Expression of cannabinoid 1 and 2 receptors (CB1R and CB2R) was confirmed in the mature human skeletal muscle myotubes. Subsequently, the effect of cannabinoid compounds on this myotube degeneration were assessed. Tetrahydrocannabinol (THC), a partial CB1R/CB2R agonist, and JWH133, a selective CB2R agonist, proved efficacious in protecting mature human myotubes from the deleterious effects of both (SW480 and H1299) cancer cachexia conditions. ART27.13, a full, peripherally selective CB1R/CB2R agonist, currently being trialled in cancer cachexia (IRAS ID 278450, REC 20/NE/0198), was also significantly protective against myotube degeneration in both (SW480 and H1299) cancer cachexia conditions. Furthermore, the addition of the CB2R antagonist AM630, but not the CB1R antagonist Rimonabant, abolished the protective effect of ART27.13. In short, we have established a convenient and robust in vitro model of cancer-induced human skeletal muscle cachexia. The data obtained using the model demonstrate the therapeutic potential of ART27.13 in cancer-induced cachexia prevention and provides evidence indicating that this effect is via CB2R, and not CB1R.

Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control) or with 50, 100, or 250 ng/mL of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function, as well as immunohistochemical staining, to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/mL significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/mL of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle.

Preventive Effects of Collagen-Derived Dipeptide Prolyl-Hydroxyproline against Dexamethasone-Induced Muscle Atrophy in Mouse C2C12 Skeletal Myotubes.

Glucocorticoids, commonly used to manage inflammatory diseases, can induce muscle atrophy by accelerating the breakdown of muscle proteins. This research delves into the influence of Prolyl-hydroxyproline (Pro-Hyp), a collagen-derived peptide, on muscle atrophy induced with dexamethasone (DEX), a synthetic glucocorticoid, in mouse C2C12 skeletal myotubes. Exposure to DEX (10 μM) for 6 days resulted in a decrease in myotube diameter, along with elevated mRNA and protein levels of two muscle-atrophy-related ubiquitin ligases, muscle atrophy F-box (MAFbx, also known as atrogin-1) and muscle ring finger 1 (MuRF-1). Remarkably, treatment with 0.1 mM of Pro-Hyp mitigated the reduction in myotube thickness caused by DEX, while promoting the phosphorylation of Akt, mammalian target of rapamycin (mTOR), and forkhead box O3a (Foxo3a). This led to the inhibition of the upregulation of the ubiquitin ligases atrogin-1 and MuRF-1. These findings indicate the potential significance of Pro-Hyp as a promising therapeutic target for countering DEX-induced muscle atrophy.

Protective effect of Luffa cylindrica Roemer against dexamethasone-induced muscle atrophy in primary rat skeletal muscle cells

Glucocorticoids (GCs) are commonly used in the treatment of chronic inflammatory conditions. However, the administration of high doses and long-term use of GCs can induce muscle atrophy (MA) in patients, leading to a decline in quality of life and increased mortality. MA leads to protein degradation in skeletal muscle, resulting in a reduction of muscle mass. This process is triggered by GCs like dexamethasone (DEX), which induce the expression of E3 ubiquitin ligases, namely Atrogin-1 and muscle RING-finger protein-1 (MuRF1). In this study, we examined the anti-MA potential of Luffa cylindrica Roemer (LCR) on DEX-treated primary skeletal myotubes. Primary skeletal myotubes stimulated with LCR alone resulted in a significant upregulation of myotube development, characterized by an increase in both the number and diameter of myotubes. Contrastingly, combined treatment with LCR and DEX reduced the expression of Atrogin-1, while treatment with DEX alone induced the expression of MuRF1. Furthermore, LCR treatment successfully restored the number and diameter of myotubes that had been diminished by DEX treatment. These findings suggest that LCR holds potential for treating MA, as an accelerating effect on muscle development and anti-MA effects on primary skeletal muscle cells were observed.

Myogenesis Effects of RGX365 to Improve Skeletal Muscle Atrophy.

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.

Hexavalent chromium inhibits myogenic differentiation and induces myotube atrophy

Hexavalent chromium [Cr(VI)] is extensively used in many industrial processes. Previous studies reported that Cr(VI) exposures during early embryonic development reduced body weight with musculoskeletal malformations in rodents while exposures in adult mice increased serum creatine kinase activity, a marker of muscle damage. However, the impacts of Cr(VI) on muscle differentiation remain largely unknown. Here, we report that acute exposures to Cr(VI) in mouse C2C12 myoblasts inhibit myogenic differentiation in a dose-dependent manner. Exposure to 2 μM of Cr(VI) resulted in delayed myotube formation, as evidenced by a significant decrease in myotube formation and expression of muscle-specific markers, such as muscle creatine kinase (Mck), Myocyte enhancer factor 2 (Mef2), Myomaker (Mymk) and Myomixer (Mymx). Interestingly, exposure to 5 μM of Cr(VI) completely abolished myotube formation in differentiating C2C12 cells. Moreover, the expression of key myogenic regulatory factors (MRFs) including myoblast determination protein 1 (MyoD), myogenin (MyoG), myogenic factor 5 (Myf5), and myogenic factor 6 (Myf6) were significantly altered in Cr(VI)-treated cells. The inhibitory effect of Cr(VI) on myogenic differentiation was further confirmed in freshly isolated mouse satellite cells, a stem cell population essential for adult skeletal muscle regeneration. Furthermore, Cr(VI) exposure to fully differentiated C2C12 myotubes resulted in a decrease in myotube diameter, which was exacerbated upon co-treatment with dexamethasone. Together, our results demonstrate that Cr(VI) inhibits myogenic differentiation and induces myotube atrophy in vitro.

Effect of Cucumis melo L. peel extract supplemented postbiotics on reprograming gut microbiota and sarcopenia in hindlimb-immobilized mice

Postbiotics made from lactic acid bacteria may ameliorate sarcopenia via the metabolic reprogramming of gut dysbiosis. This study investigated the anti-sarcopenic effect of postbiotics (WDK) produced from polyphenol-rich melon peel extract (Cucumis melo L. var. makuwa, KEE) and whey with Lentilactobacillus kefiri DH5 (DH5) in C2C12 skeletal muscle cells and hindlimb-immobilized mice. WDK significantly ameliorated palmitate-induced atrophy of C2C12 cells, restoring myotube length and diameter. It also upregulated the expression of myogenic genes including Atrogin-1, Igf-1, and MyoD. Hindlimb-immobilized C57BL/6J mice were randomly divided and orally administered 10 mL/kg body weight of saline (CON), Whey, Whey + DH5 (WD), DH5 + KEE, Whey + DH5 + KEE postbiotic (WDK) for three weeks (n = 10/group). Interestingly, WDK significantly improved muscle function in hindlimb-immobilized mice by restoring both the grip strength and the mass of the soleus muscle, which was closely related to the upregulation of the myoD gene. WDK increased microbial diversity and modulated the distribution of intestinal bacteria, particularly those involved in protein synthesis and the production of butyrate. There was a significant correlation between myogenic biomarkers and butyrate producing gut microbiota. Restoration of muscle mass and function following postbiotic WDK is strongly related to the regulation of myogenic genes by in part remodulating gut microbiota. In conclusion, these findings suggest that polyphenol- and whey-based postbiotics WDK may have potential as an effective manner to combat the progression of sarcopenia.

The Anti-Muscle Atrophy Effects of Ishige sinicola in LPS-Induced C2C12 Myotubes through Its Antioxidant and Anti-Inflammatory Actions

Inflammation and oxidative stress are known to be major factors in muscle atrophy. The objective of this study was to evaluate whether the antioxidant activity of Ishige sinicola ethanol extract (ISE) and fractions from ISE could prevent lipopolysaccharide (LPS)-induced muscle atrophy in C2C12 myotubes. IS was extracted with ethanol and fractionated with five organic solvents. Then, ISE and five fractions from ISE were used to evaluate the total antioxidant activity and the protective effect of LPS-induced muscle atrophy in C2C12 myotubes. The ISE and butanol (BuOH) fraction showed higher total antioxidant activity and higher total phenol content than other fractions of ISE. The ISE and BuOH fraction significantly attenuated the LPS-induced diameter of C2C12 myotubes as well as the mRNA and protein expression levels of the muscle-specific E3 ubiquitin ligases. The mRNA expression of forkhead box O type 3α was stimulated by LPS, which was suppressed by the BuOH fraction but not ISE. Furthermore, ISE and the BuOH fraction significantly reduced LPS-stimulated gene expression of pro-inflammatory cytokines and inflammation-inducible enzymes, which was mediated by through the inhibition of the p38/extracellular signal-regulated kinase signaling pathway. Thus, ISE exerts a protective effect against muscle atrophy in LPS-induced C2C12 myotubes through the antioxidant activity and anti-inflammatory effects of ISE.

Human milk extracellular vesicles enhance muscle growth and physical performance of immature mice associating with Akt/mTOR/p70s6k signaling pathway

Extracellular vesicles (EVs) play an important role in human and bovine milk composition. According to excellent published studies, it also exerts various functions in the gut, bone, or immune system. However, the effects of milk-derived EVs on skeletal muscle growth and performance have yet to be fully explored. Firstly, the current study examined the amino acids profile in human milk EVs (HME) and bovine milk EVs (BME) using targeted metabolomics. Secondly, HME and BME were injected in the quadriceps of mice for four weeks (1 time/3 days). Then, related muscle performance, muscle growth markers/pathways, and amino acids profile were detected or measured by grip strength analysis, rotarod performance testing, Jenner-Giemsa/H&E staining, Western blotting, and targeted metabolomics, respectively. Finally, HME and BME were co-cultured with C2C12 cells to detect the above-related indexes and further testify relative phenomena. Our findings mainly demonstrated that HME and BME significantly increase the diameter of C2C12 myotubes. HME treatment demonstrates higher exercise performance and muscle fiber densities than BME treatment. Besides, after KEGG and correlation analyses with biological function after HME and BME treatment, results showed L-Ornithine acts as a “notable marker” after HME treatment to affect mouse skeletal muscle growth or functions. Otherwise, L-Ornithine also significantly positively correlates with the activation of the AKT/mTOR pathway and myogenic regulatory factors (MRFs) and can also be observed in muscle and C2C12 cells after HME treatment. Overall, our study not only provides a novel result for the amino acid composition of HME and BME, but the current study also indicates the advantage of human milk on skeletal muscle growth and performance.