Myogenic Regulatory Factors MyoD Research Articles

Ellagic acid can mitigate chronic ammonia stress-induced adverse effects through the augmentation of liver autophagy in yellow catfish (Pelteobagrus fulvidraco)

The toxicity of ammonia to the majority of fish species is well-known, however, its harmful impact can be mitigated through nutritional intervention. The aim of this study was to investigate whether dietary supplementation of ellagic acid (EA) can mitigate the toxicity of chronic ammonia stress on yellow catfish. Three groups of yellow catfish were fed diets containing different concentrations of EA (0.00, 0.02, and 0.20%) for a duration of 56 days. Additionally, the fish were exposed to three different levels of ammonia (0.00, 2.50, and 25.00 mg/L total ammonia nitrogen) in order to assess any potential interactions. The results showed that ammonia toxicity could affect growth (weight gain rate and specific growth rate were decreased but feed conversion ratio declined), leads to oxidative stress (total antioxidant capacity, superoxide dismutase, and catalase decreased but H2O2 content increased), and physiological function damage (total cholesterol, total protein, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, acid phosphatase increased). Additionally, autophagy (becn1, atg3, atg4a, atg7, lc3a, and sqstm1), apoptosis (tgf-β, smad 2, caspase 3, caspase 9, and baxa), inflammation (TNF-α, IL-1β, and Myd88) related genes were up-regulated while myogenic regulatory factors (myod, myf5, myog, and igf) were down-regulated by ammonia. The inclusion of dietary EA supplementation has the potential to alleviate the detrimental impact of ammonia poisoning on fish growth, oxidative damage, physiological function impairment, and apoptotic processes by activating autophagy.

Formoterol Induces Muscle Regeneration Through Myogenic Regulatory Factor MyoD in Rat Model of Statin-Induced Myopathy

<p><strong>ABSTRACT</strong></p>
 <p><strong>Background and Objective:</strong> Statins can induce myopathy; however, the impact on muscle regeneration remains unclear. β2-agonists may mitigate statin myotoxicity. This study investigated the effects of simvastatin and formoterol on skeletal muscle regeneration and injury using a rat model.<br /><strong>Methods:</strong> Adult Sprague-Dawley rats (n = 90) were divided into three groups: control (A), simvastatin treatment (B), and simvastatin plus formoterol treatment (C). The rats were kept at room temperature for 3 months where an animal house diet was available ad libitum. Simvastatin was administered orally once a day, by oral gavage for 12 weeks; formoterol was concurrently administered to one group. Extensor digitorum longus muscles were analyzed for the myogenic differentiation factor (MyoD) by immunohistochemistry. Serum lactate dehydrogenase (LDH) levels were quantified as a marker of muscle damage.<br /><strong>Results:</strong> MyoD-positive myonuclei were significantly increased in the C group (simvastatin plus formoterol group) compared to the A and B Groups (control and simvastatin-only groups) (p < 0.05). Serum LDH levels were significantly lower in group C than in groups A and B (p < 0.05).<br /><strong>Conclusion:</strong> Formoterol alleviates statin-associated muscle injury in a rat model as evidenced by increased MyoD expression and decreased LDH levels. The activation of dormant satellite cells and increased expression of MyoD indicate the potential of formoterol to promote muscle regeneration.</p>

Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells.

Culture temperatures for broiler chicken cells are largely based on those optimized for mammalian species, although normal broiler body temperature is typically more than 3°C higher. The objective was to evaluate the effects of simulating broiler peripheral muscle temperature, 41°C, compared with standard temperature, 38°C, on the in vitro proliferation and differentiation of primary muscle-specific stem cells (satellite cells; SC) from the pectoralis major (PM) of broiler chickens. Primary SC cultures were isolated from the PM of 18-day-old Ross 708 × Yield Plus male broilers. SC were plated in triplicate, 1.8-cm2, gelatin-coated wells at 40,000 cells per well. Parallel plates were cultured at either 38°C or 41°C in separate incubators. At 48, 72, and 96h post-plating, the culture wells were fixed and immunofluorescence-stained to determine the expression of the myogenic regulatory factors Pax7 and MyoD as well as evaluated for apoptosis using a TUNEL assay. After 168h in culture, plates were immunofluorescence-stained to visualize myosin heavy chain and Pax7 expression and determine myotube characteristics and SC fusion. Population doubling times were not impacted by temperature (p ≥ 0.1148), but culturing broiler SC at 41°C for 96h promoted a more rapid progression through myogenesis, while 38°C maintained primitive populations (p ≤ 0.0029). The proportion of apoptotic cells increased in primary SC cultured at 41°C (p ≤ 0.0273). Culturing at 41°C appeared to negatively impact fusion percentage (p < 0.0001) and tended to result in the formation of thinner myotubes (p = 0.061) without impacting the density of differentiated cells (p = 0.7551). These results indicate that culture temperature alters primary broiler PM SC myogenic kinetics and has important implications for future in vitro work as well as improving our understanding of how thermal manipulation can alter myogenesis patterns during broiler embryonic and post-hatch muscle growth.

237 Evaluation of Thermal Variation During Early-Stage Incubation on Broiler Chicken Pectoralis Major Muscle Satellite Cell Mitotic Activity and Heterogeneity at 28 Days Post-Hatch

Abstract Muscle satellite cells (SC) have an essential role in post-hatch skeletal muscle growth. To evaluate the effects of thermal variation (TV) during early-stage incubation (ESI) on Pectoralis major (PM) muscle SC heterogeneity of broiler chickens reared to 28 d post-hatch a randomized complete block design experiment with a 3 × 2 factorial treatment structure was conducted. Yield Plus × Ross 708 broiler breeder eggs (n = 2,160) ranging from 54 to 59 g were allotted equally by weight into 90-egg trays (n = 4 trays per incubator). All eggs were pre-warmed at 24 °C for 8 h and incubated at 37.5 °C from embryonic day (ED) 0 to 4. From ED 4 to 11, eggs were incubated at 1 of 3 temperatures (n = 2 incubators per TV treatment); 37.5 °C (CTL), 36.4 °C (COLD), or 38.6 °C (HOT). On ED 11, all incubators were returned to the CTL (37.5 °C) set point until ED 18. On ED 18, all eggs were transferred to baskets in hatchers set to 36.7 °C. After 514 h of incubation, all chicks were removed simultaneously, vent sexed, and weighed. Chicks were randomly allotted to floor pens (6 pens per treatment per sex) blocked by location and reared on a common diet. On d 28, birds (n = 10 per TV x sex treatment) were injected with bromodeoxyuridine (BrdU) before PM sample collection. Cryosections were immunofluorescence stained to facilitate taxonomy of SC populations expressing the myogenic regulatory factors Pax7 and MyoD as well as the proliferation marker BrdU by fluorescence microscopy. Data were analyzed as a 2-way ANOVA using SAS PROC GLIMMIX with TV treatment and broiler sex as main effects. Means were separated with the PDIFF option at P ≤ 0.05. Tendencies were declared when 0.0501 ≤ P ≤ 0.10. A TV × sex interaction was observed only for the MyoD+: Pax7+ SC density (P = 0.0232); however, none of the pairwise mean comparisons were significant at P &amp;lt; 0.05. Though CTL male and COLD female chicks had twice the density of MyoD+:Pax7+ SC as CTL females (P = 0.0535) and HOT males (P = 0.0645), respectively. There is no clear explanation for this observation. There were no interactive or main effects on density of MyoD+, Pax7+, MyoD+:BrdU+, Pax7+:BrdU+, MyoD+:Pax7+:BrdU+ SC (P ≥ 0.1258) or nuclear density (P = 0.1017) in 28-d-old broilers. These data suggest that if TV during ESI impacts SC myogenic regulatory factor heterogeneity and mitotic activity in broilers, the effects have concluded by d 28 post-hatch in commercial broilers. Further exploration of earlier time points will be necessary to determine whether the previously observed effects of TV during ESI on growth performance and meat yield are SC-mediated post-hatch.

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.

Comparative evaluation of soybean meal vs. extruded soybean meal as a replacer for fishmeal in diets of olive flounder (Paralichthys olivaceus): Effects on growth performance and muscle quality

A 56-day feeding trial was performed to compare the impacts of replacement of dietary fishmeal by soybean meal (SM) or extruded soybean meal (ESM) on the growth performance and muscle quality of olive flounder (Paralichthys olivaceus) (initial weight: 6.32 ± 0.01 g). Nine isonitrogenous and isolipidic diets were prepared by substituting 0% (FM, control), 12% (SM12/ESM12), 24% (SM24/ESM24), 36% (SM36/ESM36) and 48% (SM48/ESM48) of fishmeal with SM or ESM respectively. Results indicated that contents of anti-nutritional factors (trypsin inhibitors, glycinin and β-conglycinin) in ESM were significantly lower than those in SM. The weight gain rate, specific growth rate, feed efficiency ratio, protein efficiency ratio, activities of intestinal trypsin and lipase, as well as expressions of myogenic regulatory factors (myf5, myod and mrf4) and protein synthesis-related genes (akt, pi3k, tor and s6) in fish muscle decreased linearly and quadratically with increasing replacement levels of dietary fishmeal by SM or ESM. Expressions of mstn and protein degradation-related genes (foxo1, murf-1, mafbx, capn2 and ctsl) in muscle showed a trend opposite to growth parameters responding to dietary SM or ESM levels. Significant differences in all the above parameters were observed in the SM36, SM48 and ESM48 groups, accompanied by the declines in contents of most muscle free amino acids. The increase of dietary fishmeal replacement level by SM linearly and quadratically decreased muscle hardness, water holding capacity (WHC), pH and total collagen content. Markedly lower values were found in the SM36 and SM48 groups. However, ESM could replace up to 36% of dietary fishmeal without significantly impacts on above muscle quality parameters. Additionally, reduced myofiber density and sarcomere length were observed in the SM36, SM48, ESM36 and ESM48 groups. However, at the same substitution level, dietary ESM relieved the effects of SM on myofiber morphology to some extent. In conclusion, according to broken-line regression on SGR, the optimal replacing level of fishmeal by SM and ESM for olive flounder was 22.07% and 34.39% respectively. The improved replacement level of fishmeal by ESM, in terms of fish growth, muscle hardness and WHC, may be due to the fact that dietary ESM alleviated the adverse impacts of SM on digestive enzyme activity, muscle growth-related genes expression, muscle pH, collagen content and myofiber morphology of fish.

Platelet-rich plasma promotes skeletal muscle regeneration and neuromuscular functional reconstitution in a concentration-dependent manner in a rat laceration model

Abnormal function of injured muscle with innervation loss is a challenge in sports medicine. The difficulty of rehabilitation is regenerating and reconstructing the skeletal muscle tissue and the neuromuscular junction (NMJ). Platelet-rich plasma (PRP) releases various growth factors that may provide an appropriate niche for tissue regeneration. However, the specific mechanism of the PRP's efficacy on muscle healing remains unknown. In this study, we injected PRP with different concentration gradients (800, 1200, 1600 × 109 pl/L) or saline into a rat gastrocnemius laceration model. The results of histopathology and neuromyography show that PRP improved myofibers regeneration, facilitated electrophysiological recovery, and reduced fibrosis in a concentration-dependent manner. Furthermore, we found that PRP promotes the activity of satellite cells by upregulating the expression of the myogenic regulatory factor (MyoD, myogenin). Meanwhile, PRP promotes the regeneration and maturation of acetylcholine receptor (AChR) clusters of the Neuromuscular junction (NMJ) on the regenerative myofibers. Finally, we found that the expression of the Agrin, LRP4, and MuSK was upregulated in the PRP-treated groups, which may contribute to AChR cluster regeneration and functional recovery. The conclusions proposed a hypothesis for PRP treatment's efficacy and mechanism in muscle injuries, indicating promising application prospects.

Vitamin D3 decreases myoblast fusion during the growth and increases myogenic gene expression during the differentiation phase in muscle satellite cells from Korean native beef cattle.

The process of myogenesis, which involves the growth and differentiation of muscle cells, is a crucial determinant of meat yield and quality in beef cattle. Essential nutrients, such as vitamins D and A, play vital roles in the development and maintenance of various tissues, including muscle. However, limited knowledge exists regarding the specific effects of vitamins A and D in bovine muscle. Therefore, the aim of this study was to investigate the impact of vitamins A and D treatment on myogenic fusion and differentiation in bovine satellite cells (BSC). BSC were isolated from Korean native beef cattle, specifically from four female cows approximately 30 mo old. These individual cows were used as biological replicates (n = 3 or 4), and we examined the effects of varying concentrations of vitamins A (All-trans retinoic acid; 100 nM) and D (1,25-dihydroxy-vitamin D3; 1 nM, 10 nM, and 100 nM), both individually and in combination, on myoblast fusion and myogenic differentiation during the growth phase (48 h) or differentiation phase (6 d). The results were statistically analyzed using GLM procedure of SAS with Tukey's test and t-tests or one-way ANOVA where appropriate. The findings revealed that vitamin A enhanced the myoblast fusion index, while vitamin D treatment decreased the myoblast fusion index during the growth phase. Furthermore, vitamin A treatment during the differentiation phase promoted terminal differentiation by regulating the expression of myogenic regulatory factors (Myf5, MyoD, MyoG, and Myf6) and inducing myotube hypertrophy compared to the control satellite cells (P < 0.01). In contrast, vitamin D treatment during the differentiation phase enhanced myogenic differentiation by increasing the mRNA expression of MyoG and Myf6 (P < 0.01). Moreover, the combined treatment of vitamins A and D during the growth phase increased myoblast fusion and further promoted myogenic differentiation and hypertrophy of myotubes during the differentiation phase (P < 0.01). These results suggest that vitamin A and D supplementation may have differential effects on muscle development in Korean native beef cattle during the feeding process.

PREFACE: A SPECIAL SELECTION ON EMERGING TECHNIQUES FOR BIOMECHANICS–PART II

PREFACE: A SPECIAL SELECTION ON EMERGING TECHNIQUES FOR BIOMECHANICS–PART II

205 Exposure to Heat Stress Stimulates Cell Proliferation and Heat Shock Protein Expression in Bovine Primary Satellite Cells

Abstract Understanding the myogenic and self-renewal capability of satellite cells under heat stress (HS) is fundamental to coping with global warming in the beef cattle industry. Study objectives were to determine transcriptomic changes and proliferation capability of bovine satellite cells (BSCs) under moderate versus acute levels of HS and exposure times. Satellite cells were isolated from four three-month-old Holstein calves (semitendinosus) and cultured with 10% FBS/DMEM. Following 24h of attachment period, BSCs were incubated under the following temperature conditions 1) control (38°C; Con), 2) moderate (39.5°C; MHS) and extreme (41°C; EHS) and for various exposure times (0, 1, 3, 6, 12, 24, and 48 h). Cells were harvested and used for mRNA gene expression via RT-qPCR at each time point. Cell proliferation was measured using the ELISA kit. Data were analyzed by two-way ANOVA and Tukey HSD test. The expression of HSP70 was greatest (P &amp;lt; 0.05) in cells treated EHS at 1 and 3 h. Cells treated EHS overexpressed (P &amp;lt; 0.05) HSPB1 at 3, 6, 12, and 24 h. A similar pattern was observed (P &amp;lt; 0.05) for MyoD expression at 24 h after heat exposure. Contrarily, Myf5 and PAX3 were decreased (P &amp;lt; 0.05) by EHS compared to the Con at 3 h. Cell proliferation rate was enhanced (P &amp;lt; 0.01) in both MHS and EHS treated BSCs compared withthe Con at 48 h. Our results suggest that extreme heat exposure stimulates the HSPs (HSP70 and HSPB1) expression while modulating muscle regeneration by regulating myogenic regulatory factors (MyoD, Myf5) and PAX3. The transcriptional effect peaked after 3h of heat exposure, but the long-term (48h) exposure may activate the proliferation of BSCs.

Myogenic regulatory factors Myod and Myf5 are required for dorsal aorta formation and angiogenic sprouting

The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development.

Cyclin C's Role in Myoblast Differentiation‐Induced Mitochondrial Fragmentation

One of the largest and most dynamic tissues in the body, skeletal muscle, requires constant regeneration and upkeep. Dysregulation of this regeneration process has been implicated in many neuromuscular diseases and myotonic dystrophies. Regeneration requires the differentiation of myogenic lineages including exiting the cell cycle, gene expression changes, and fusing of myoblasts into multinucleate myotubes. Part of this reconstruction requires the breakdown and repopulation of mitochondrial networks. At the early onset of myoblast differentiation, there is an upregulation of dynamin‐related protein, Drp1, and an increase in mitophagy mediated by sequestosome (SQSTM1) removal of mitochondria. Previously, our lab has shown that mitochondrial fragmentation following stress requires the transcriptional regulator cyclin C, the regulatory subunit for cyclin‐dependent kinase 8 (Cdk8). Preliminary data indicate that cyclin C is required for mitochondrial fragmentation during myoblast differentiation. At the early onset, cyclin C co‐localizes with the mitochondria, as visualized with indirect immunofluorescence. Cells were additionally treated with PFTμ, a cytosolic chaperone inhibitor that blocks translocation of cyclin C to the mitochondria, and in turn inhibits cyclin C‐mediated mitochondrial fragmentation. This treatment resulted in lack of mitochondrial fragmentation typically seen during the differentiation process. In addition, efficiency of differentiation was quantified using gene expression of myogenic regulatory factors (MRFs) MyoD and Myosin Heavy Chain (MyHC), which are normally expressed in a temporal manner throughout differentiation. Treatment of cells with PFTμ significantly decreased expression of MyoD by 1.2 fold compared to endogenous controls, as well as a 1.6 fold decrease in MyHC expression. Future studies aim to elucidate the role of cyclin C‐mediated mitochondrial fragmentation during myoblast differentiation and how this relates to the homeostasis of muscle regeneration processes.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

SummaryGenetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

L-Arginine Supplementation for Nulliparous Sows during the Last Third of Gestation.

Simple SummaryNutrition of gestating sows plays an important role in the uteroplacental efficiency, muscle development, birth weight and viability of the piglets. This is especially important for nulliparous sows, since the reproductive performance of nulliparous sows is lower compared to multiparous ones. Previous studies have shown that arginine enhances angiogenesis and placental growth, being beneficial to fetus growth. Because during its formation process skeletal muscle tissue compete for nutrients with other organs also in formation during the intrauterine development, the increase of nutrient flow by enhancing angiogenesis may benefit the skeletal muscle formation during the fetal stage. In this sense, arginine supplementation for sows seems to be a promising nutritional strategy to maximize myogenesis and muscle development of piglets. In the present study, piglets born from sows fed diet with 1.0% L-arginine (ARG) had greater mRNA expression of the gene encoding myoblast determination protein (MYOD) and myogenin (MYOG) compared to the piglets born from sows at the control group. The mRNA expression of IGF-2 gene tended to be greater in piglets born from ARG sows compared to those born from control sows. Despite differences in gene expression, no differences in the histomorphometric variables of skeletal muscle were observed between piglets from arginine-supplemented and control sows. In conclusion, supplementation of 1.0% L-arginine for nulliparous sows from 85 to 114 days of gestation increased mRNA expression of the myogenic regulatory factors MYOD and MYOG and IGF-2 in skeletal muscle of piglets.We evaluated the effects of L-arginine supplementation during the last third of gestation on molecular mechanisms related to skeletal muscle development of piglets and litter traits at birth. Twenty-three nulliparous sows averaging 205.37 ± 11.50 kg of body weight were randomly assigned to the following experimental treatments: control (CON), where pregnant sows were fed diets to meet their nutritional requirements; arginine (ARG), where sows where fed CON + 1.0% L-arginine. Skeletal muscle from piglets born from sows from ARG group had greater mRNA expression of MYOD (p = 0.043) and MYOG (p ≤ 0.01), and tended to present greater mRNA expression (p = 0.06) of IGF-2 gene compared to those born from CON sows. However, there were no differences (p > 0.05) in the histomorphometric variables of fetuses’ skeletal muscle. The total weight of born piglets, total weight of born alive piglets, piglet weight at birth, coefficient of variation of birth weight, and the incidence of intrauterine growth restriction (IUGR) piglets did not differ between groups. No stillborn piglets (p < 0.01) were verified in the ARG sows compared to CON group. The blood levels of estradiol (p = 0.035) and urea (p = 0.03) were higher in ARG sows compared to those from the CON group. In summary, our data show that arginine supplementation of nulliparous sows at late gestation enhance mRNA expression of key myogenic regulatory factors, which likely contribute to improve animal growth rates in later stages of development.

Unresolved intramuscular inflammation, not diminished skeletal muscle regenerative capacity, is at the root of rheumatoid cachexia: insights from a rat CIA model

Rheumatoid arthritis targets numerous organs in patients, including the skeletal muscle, resulting in rheumatoid cachexia. In the muscle niche, satellite cells, macrophages, and myofibroblasts may be affected and the factors they release altered. This study aimed to assess these cell types, cytokines, and growth factors and their relationships to muscle fiber size and number in a rodent collagen‐induced arthritis (CIA) model, in order to identify new therapeutic targets. Fiber cross‐sectional area (CSA) was 57% lower in CIA than controls (p < 0.0001), thus smaller but more fibers visible per field of view. Immunostaining indicated the increased presence of satellite cells, macrophages, myofibroblasts, and myonuclei per field of view in CIA (p < 0.01), but this finding was not maintained when taking fiber number into consideration. Western blots of gastrocnemius samples indicated that tumor necrosis factor‐α was significantly elevated (p < 0.01) while interleukin‐10 (IL‐10) was decreased (p < 0.05) in CIA. This effect was maintained (and heightened for IL‐10) when expressed per fiber number. Myogenic regulatory factors (MyoD and myogenin), transforming growth factor‐β and inhibitor of differentiation were significantly elevated in CIA muscle and levels correlated significantly with CSA. Several of these factors remained elevated, but bone morphogenetic protein‐7 decreased when considering fiber number per area. In conclusion, CIA‐muscle demonstrated a good regenerative response. Myoblast numbers per fiber were not elevated, suggesting their activity results from the persistent inflammatory signaling which also significantly hampered maintenance of muscle fiber size. A clearer picture of signaling events at cellular level in arthritis muscle may be derived from expressing data per fiber.

The Essential Role of Stathmin in Myoblast C2C12 for Vertical Vibration-Induced Myotube Formation.

Vertical vibration (VV) is a type of whole body vibration, which induces muscle contraction through vibration to improve muscle strength and bone density. However, the mechanism of VV on muscle cell myotube formation is still unclear. In the current study, we aim to clarify the mechanism involved in VV’s stimulation of myotube formation. In order to identify the molecules regulated by VV, we performed proteomics analysis including 2D electrophoresis combined with MALDI-TOF/TOF Mass. Stathmin was identified as a high potential molecule responding to VV stimulation, and we found that under VV stimulation, the expression of stathmin gene and protein increased in a time-dependent manner. In addition, we also confirmed that the increase of stathmin stimulated by VV is mediated through the PI3K/Akt pathway. Furthermore, stathmin siRNA significantly down-regulated the expression of myogenic regulatory factor (MRF) MyoD, decorin, and type I collagen (Col-I), and down-regulated the cellular process regulators such as FGF7, TGFBr1 and PAK3. Taken together, our results confirm that under the stimulation of VV, PI3K/Akt and stathmin would be activated, as well as the up-regulation of MRFs, such as FGF7, TGFBr1 and PAK3 to initiate myogenesis. It also showed that the response of MRF to VV stimulation was significantly related to stathmin expression, which also confirmed the importance of stathmin in the entire myotube formation process. This study may provide evidence of stathmin as a biological indicator of VV to increase muscle strength.

Modulation of Ki67 and myogenic regulatory factor expression by tocotrienol-rich fraction ameliorates myogenic program of senescent human myoblasts.

IntroductionReplicative senescence results in dysregulation of cell proliferation and differentiation, which plays a role in the regenerative defects observed during age-related muscle atrophy. Vitamin E is a well-known antioxidant, which potentially ameliorates a wide range of age-related manifestations. The aim of this study was to determine the effects of tocotrienol-rich fraction (TRF) in modulating the expression of proliferation- and differentiation-associated proteins in senescent human myoblasts during the differentiation phase.Material and methodsHuman skeletal muscle myoblasts were cultured until senescence. Young and senescent cells were treated with TRF for 24 h before and after differentiation induction, followed by evaluation of cellular morphology and efficiency of differentiation. Expression of cell proliferation marker Ki67 protein and myogenic regulatory factors MyoD and myogenin were determined.ResultsOur findings showed that treatment with TRF significantly improved the morphology of senescent myoblasts. Promotion of differentiation was observed in young and senescent myoblasts with TRF treatment as shown by the increased fusion index and larger size of myotubes. Increased Ki67 and myogenin expression with TRF treatment was also observed in senescent myoblasts, suggesting amelioration of the myogenic program by TRF during replicative senescence.ConclusionsTRF modulates the expression of regulatory factors related to proliferation and differentiation in senescent human myoblasts and could be beneficial for ameliorating the regenerative defects during aging.

Muscle Enriched Lamin Interacting Protein (Mlip) Binds Chromatin and Is Required for Myoblast Differentiation.

Muscle-enriched A-type lamin-interacting protein (Mlip) is a recently discovered Amniota gene that encodes proteins of unknown biological function. Here we report Mlip’s direct interaction with chromatin, and it may function as a transcriptional co-factor. Chromatin immunoprecipitations with microarray analysis demonstrated a propensity for Mlip to associate with genomic regions in close proximity to genes that control tissue-specific differentiation. Gel mobility shift assays confirmed that Mlip protein complexes with genomic DNA. Blocking Mlip expression in C2C12 myoblasts down-regulates myogenic regulatory factors (MyoD and MyoG) and subsequently significantly inhibits myogenic differentiation and the formation of myotubes. Collectively our data demonstrate that Mlip is required for C2C12 myoblast differentiation into myotubes. Mlip may exert this role as a transcriptional regulator of a myogenic program that is unique to amniotes.

Antioxidant effects of lebanese Crocus sativus L. and its main components, crocin and safranal, on human skeletal muscle cells

Introduction- Crocus sativus L. (C. sativus L.) has gained interest as a potential source of pharmacologically bioactive compounds and is believed to possess antioxidant properties. This study used human myoblast cells to evaluate the protective effects of C. sativus L. extracts, (crocin, and safranal) on hydrogen peroxide (H2O2)-induced oxidative stress using human myoblast cells. Methods- HPLC was used to detect the presence of the active compounds safranal/crocin in the Lebanese C. sativus L. extracts. Their cellular antioxidant property was examined by trypan blue, cell adhesion, immunostaining and cell cycle assays. Transcriptional expression and activities of SOD1/2, GPX1, and catalase were also determined. The ability of C. sativus L. and its main components to modulate myogenic regulatory factors (MyoD, Myf5) was evaluated using RT-PCR assay. Results- The water/methanol (50:50, v/v) extract of C. sativus L. stigmas were found to contain safranal and crocin, with a high concentration of trans-crocin 3/4 in particular. In vitro, pretreatment (24 h) with safranal exhibited the lowest antioxidant effect whereas pretreatment with C. sativus L. extracts (0.3 µg/ml) and more notably with crocin (0.3 µM) attenuated the toxic impact of H2O2 (50 µM, 24 h) and also restored the capacity of adhesion among LHCN-M2 cells. This protective effect was associated with both a reduction of intracellular reactive oxygen species generation and an up-regulation of antioxidant enzyme activities. Furthermore, crocin pretreatment significantly improved the transcriptional expression of myogenic regulatory genes. Conclusion- C. sativus L., particularly its main component crocin, was shown to have therapeutic potential against oxidative stress-associated skeletal muscle diseases.

The protective effect of rosmarinic acid on myotube formation during myoblast differentiation under heat stress.

High ambient temperature is one of the most important environmental factors that caused the reduction of livestock productivity and the increase of mortality. It has been shown that heat stress could affect the meat quality characteristics by physiological and metabolic perturbations in live livestock. Rosmarinic acid (RA) is a natural polyphenolic phytochemical compound that has many important biological activities, such as antioxidant, antimutagenic, and antitumor. The purpose of this study was to investigate the possible function and mechanism of RA on myoblast proliferation and differentiation under heat stress condition. The results showed that heat stress reduced the viability of myoblast and increased the percentage of apoptotic cells, and it also disrupted myotube formation by altering the expression of myogenic regulatory factors MyoD, myogenin, and MyHC. However, pretreatment of RA can protect C2C12 cells from heat stress-induced apoptosis, and it also increased the expression level of MyoD, myogenin, and MyHC under heat stress, which indicated that RA have protective effect on heat stress-caused failure of myotube formation during myoblast differentiation. Above all, our finding demonstrated that RA can promote the differentiation of C2C12 myoblast and maintain the formation of myotubes even under heat stress condition.