Markers Of Myogenesis Research Articles

Preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model

We previously demonstrated that transcutaneous CO2 application promotes muscle fiber-type switching, fracture healing, and osteogenesis by increasing blood flow and angiogenesis. Here, we aimed to investigate the preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model. Eleven-week-old male Sprague-Dawley rats were divided into hindlimb suspension (HS), HS with transcutaneous CO2 application (HSCO2), and control groups. HSCO2 rats were administered transcutaneous 100 % CO2 gas in their bilateral hindlimbs, five times a week for 20 min. After 3 weeks, we harvested the gastrocnemius, femur, and tibia for assessment. Histological analysis revealed a significant decrease in the gastrocnemius myofiber cross-sectional area in HS rats compared to the control rats, whereas HSCO2 rats exhibited a significant increase compared to HS rats. Micro-computed tomography showed significant bone atrophy in the trabecular and cortical bones of the femur in HS rats compared to those of the control rats, whereas significant improvement was noted in HSCO2 rats. Histological analysis of the proximal tibia revealed more marrow adipose tissue in the HS rats than in the control rats. However, in the HSCO2 rats, fewer marrow adipose tissue and osteoclasts were observed. Moreover, HSCO2 rats had more osteoblasts and higher expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and vascular endothelial growth factor (VEGF) than the HS rats. The gastrocnemius and distal femur of HSCO2 rats also exhibited elevated PGC-1α and VEGF expression and upregulation of the myogenesis markers and osteogenesis markers compared to those of HS rats. This treatment effectively prevented disuse osteoporosis and muscle atrophy by promoting local angiogenesis and blood flow. PGC-1α is crucial for promoting this angiogenic pathway. Transcutaneous CO2 application may be a novel preventive procedure for disuse osteoporosis and muscle atrophy, complementing medication and rehabilitation.

Melatonin Regulates Rheumatoid Synovial Fibroblasts-Related Inflammation: Implications for Pathological Skeletal Muscle Treatment.

Melatonin has been reported to regulate circadian rhythms and have anti-inflammatory characteristics in various inflammatory autoimmune diseases, but its effects in diseases-associated muscle atrophy remain controversial. This study is aimed to determine the evidence of melatonin in rheumatoid arthritis (RA)-related pathological muscle atrophy. We used initially bioinformatics results to show that melatonin regulated significantly the correlation between pro-inflammation and myogenesis in RA synovial fibroblasts (RASF) and myoblasts. The conditioned medium (CM) from melatonin-treated RASF was incubated in myoblasts with growth medium and differentiated medium to investigate the markers of pro-inflammation, atrophy, and myogenesis. We found that melatonin regulated RASF CM-induced pathological muscle pro-inflammation and atrophy in myoblasts and differentiated myocytes through NF-κB signaling pathways. We also showed for the first time that miR-30c-1-3p is negatively regulated by three inflammatory cytokines in human RASF, which is associated with murine-differentiated myocytes. Importantly, oral administration with melatonin in a collagen-induced arthritis (CIA) mouse model also significantly improved arthritic swelling, hind limb grip strength as well as pathological muscle atrophy. In conclusion, our study is the first to demonstrate not only the underlying mechanism whereby melatonin decreases pro-inflammation in RA-induced pathological muscle atrophy but also increases myogenesis in myoblasts and differentiated myocytes.

RBP4 promotes denervation-induced muscle atrophy through STRA6-dependent pathway.

Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear. Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed. The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7days and sustained for 28days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice. In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.

Systematic Identification of Long Noncoding RNAs during Three Key Organogenesis Stages in Zebrafish.

Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related functions have not yet been elucidated. In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos that are important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2), and 36 hpf (T3). LncRNA gas5, associated with muscle development, was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied through qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: (1) A total of 1486 differentially expressed lncRNAs were identified between T2 and T1, among which 843 lncRNAs were upregulated and 643 were downregulated. The comparison with T3 and T2 resulted in 844 differentially expressed lncRNAs, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. A total of 2137 differentially expressed lncRNAs were found between T3 and T1, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. (2) The results of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebrafish. (3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were more highly affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. (4) The results of the expression of the skeletal myogenesis marker myod showed that the expression of myod in myotomes was abnormal, suggesting that skeletal myogenesis was affected after lncRNA gas5 knockout. The results of this study provide an experimental basis for further studies on the role of lncRNA gas5 in the embryonic skeletal muscle development of zebrafish.

Maternal rodent exposure to di-(2-ethylhexyl) phthalate decreases muscle mass in the offspring by increasing myostatin.

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolites can cross the placenta and may cause birth defects and developmental disorders. However, whether maternal DEHP exposure affects skeletal muscle development in the offspring and the pathways involved are unknown. This study investigated the effects of maternal DEHP exposure and the contribution of myostatin (MSTN) to skeletal muscle development in the offspring. Pregnant wild-type and muscle-specific myostatin knockout (MSTN KO) C57BL/6 mice were randomized to receive vehicle (corn oil) or 250mg/kg DEHP by gavage every other day until their pups were weaned (postnatal day 21 [PND21]). Body weights of the offspring mice were measured longitudinally, and their hindleg muscles were harvested at PD21. Also, C2C12 cells were treated with mono-2-ethylhexyl phthalate (MEHP), the primary metabolite of DEHP, and proteolysis, protein synthesis, and myogenesis markers were measured. The contribution of myostatin to maternal DEHP exposure-induced muscle wasting in the offspring was determined. Maternal DEHP exposure reduced body weight growth, myofibre size, and muscle mass in the offspring compared to controls (Quad: 2.70±0.1 vs. 3.38±0.23, Gastroc: 2.29±0.09 vs. 2.81±0.14, Tibialis: 1.01±0.07 vs. 1.25±0.11, mg/tibial length in mm, all P<0.01, n=35). Maternal DEHP exposure significantly increased Myostatin expression (2.45±0.41 vs. 0.03±0.00 DEHP vs. controls, P<0.01, n=5), Atrogin-1(2.68±0.65 vs. 0.63±0.01, P<0.05, n=5), MuRF1 (1.56±0.51 vs. 0.31±0.01, P<0.05, n=5), and Smad2/3 phosphorylation (4.12±0.35 vs. 0.49±0.18, P<0.05), and decreased MyoD (0.27±0.01 vs. 1.52±0.01, P<0.05, n=5), Myogenin (0.25±0.03 vs. 1.95±0.56, P<0.05, n=5), and AKT phosphorylation (4.12±0.35 vs. 1.00±0.06, P<0.05, n=5), in skeletal muscle of the offspring in MSTNflox/flox , but not in MSTN KO mice. Maternal DEHP exposure resulted in up-regulation of CCAAT/enhancer-binding protein δ (C/EBPδ, 4.12±0.35 vs. 1.00±0.19, P<0.05, n=5) in skeletal muscle of the offspring in MSTNflox/flox and MSTN KO mice (4.12±0.35 vs. 4.35±0.28, P>0.05, n=5). In vitro, C/EBPδ silencing abrogated the MEHP-induced increases in Myostatin, MuRF-1, and Atrogin-1 and decreases in MyoD and Myogenin expression. Maternal DEHP exposure impairs skeletal muscle development in the offspring by enhancing the C/EBPδ-myostatin pathway in mice.

Pyruvate dehydrogenase kinase 4 promotes ubiquitin-proteasome system-dependent muscle atrophy.

Muscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism underlying this detrimental condition is currently unclear. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscle and has been implicated in the pathogenesis of several diseases. The current study was designed to investigated and delineate the role of PDK4 in the context of muscle atrophy, which could be identified as a potential therapeutic avenue to protect against dexamethasone-induced muscle wasting. The dexamethasone-induced muscle atrophy in C2C12 myotubes was evaluated at the molecular level by expression of key genes and proteins involved in myogenesis, using immunoblotting and qPCR analyses. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with dexamethasone (25mg/kg body weight, i.p., 10days). Body weight, grip strength, muscle weight and muscle histology were assessed. The expression of myogenesis markers were analysed using qPCR, immunoblotting and immunoprecipitation. The study was extended to in vitro human skeletal muscle atrophy analysis. Knockdown of PDK4 was found to prevent glucocorticoid-induced muscle atrophy and dysfunction in C2C12 myotubes, which was indicated by induction of myogenin (0.3271±0.102 vs 2.163±0.192, ****P<0.0001) and myosin heavy chain (0.3901±0.047 vs. 0.7222±0.082, **P<0.01) protein levels and reduction of muscle atrophy F-box (10.77±2.674 vs. 1.518±0.172, **P<0.01) expression. In dexamethasone-induced muscle atrophy model, mice with genetic ablation of PDK4 revealed increased muscle strength (162.1±22.75 vs. 200.1±37.09g, ***P<0.001) and muscle fibres (54.20±11.85% vs. 84.07±28.41%, ****P<0.0001). To explore the mechanism, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. PDK4 phosphorylates myogenin at S43/T57 amino acid residues, which facilitates the recruitment of muscle atrophy F-box to myogenin and leads to its subsequent ubiquitination and degradation. Finally, overexpression of non-phosphorylatable myogenin mutant using intramuscular injection prevented dexamethasone-induced muscle atrophy and preserved muscle fibres. We have demonstrated that PDK4 mediates dexamethasone-induced skeletal muscle atrophy. Mechanistically, PDK4 phosphorylates and degrades myogenin via recruitment of E3 ubiquitin ligase, muscle atrophy F-box. Rescue of muscle regeneration by genetic ablation of PDK4 or overexpression of non-phosphorylatable myogenin mutant indicates PDK4 as an amenable therapeutic target in muscle atrophy.

VP.62 DUX4 expression turn on the myogenic program in MSC

Muscle tissue degeneration is accompanied by fibrosis in patients with the facioscapulohumeral dystrophy (FSHD). In skeletal muscles, MSCs are present and they can participate in muscles regeneration. When a muscle is injured, MSCs can migrate towards damaged site from blood circulation and neighboring intact tissues and participate in the recovery process also. Since FSHD is a genetic disease associated with aberrant <i>DUX4</i> expression, we hypothesized that MSC involved in the regeneration process may also have overexpressed level of <i>DUX4</i>, and created a model of MSC with inducible <i>DUX4</i> expression (MSC-DUX4) using the Jagannathan's approach and construct pCW57.1-DUX4-WT from Addgen. MSCs were obtained from the Cell culture collection of the IDB RAS. <i>DUX4</i> expression was confirmed in MSC-DUX4 cells by immunofluorescent staining. We conducted a comparative analysis of the differentiation potencies of MSCs and MSCs with inducible <i>DUX4</i> expression (MSCs were from the same donor). It was shown that MSCs with induced <i>DUX4</i> expression have an increased potential for myogenic differentiation, which is not characteristic of normal MSCs. These cells can fuse and form mature myotubes expressing the main markers of late myogenesis (Troponin T, MF20). At the same time, MSCs with induced expression of <i>DUX4</i> have reduced potencies for main MSC differentiations - adipogenic and osteogenic, that was shown using biochemical, immunocytochemical and RT-PCR assays. Thus, upon induction of DUX4 expression, MSCs turn on the myogenic program, while losing the capacity for their main differentiations. This does not seem so counterintuitive, since the genes involved in myogenesis are <i>DUX4</i>-downstream target genes. However, the real contribution of MSC-DUX4 to the regeneration of muscles affected by FSHD remains to be analyzed.

Effects of PGC-1α overexpression on the myogenic response during skeletal muscle regeneration

The ability of skeletal muscle to regenerate from injury is crucial for locomotion, metabolic health, and quality of life. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1A) is a transcriptional coactivator required for mitochondrial biogenesis. Increased mitochondrial biogenesis is associated with improved muscle cell differentiation, however PGC1A's role in skeletal muscle regeneration following damage requires further investigation. The purpose of this study was to investigate the role of skeletal muscle-specific PGC1A overexpression during regeneration following damage. 22 C57BL/6J (WT) and 26 PGC1A muscle transgenic (A1) mice were injected with either phosphate-buffered saline (PBS, uninjured control) or Bupivacaine (MAR, injured) into their tibialis anterior (TA) muscle to induce skeletal muscle damage. TA muscles were extracted 3- or 28-days post-injury and analyzed for markers of regenerative myogenesis and protein turnover. Pgc1a mRNA was ∼10–20 fold greater in A1 mice. Markers of protein synthesis, AKT and 4EBP1, displayed decreases in A1 mice compared to WT at both timepoints indicating a decreased protein synthetic response. Myod mRNA was ∼75% lower compared to WT 3 days post-injection. WT mice exhibited decreased cross-sectional area of the TA muscle at 28 days post-injection with bupivacaine compared to all other groups. PGC1A overexpression modifies the myogenic response during regeneration.

Skeletal muscle RUNX1 is related to insulin sensitivity through its effect on myogenic potential.

Skeletal muscle is the major site of insulin action. There are limited data on the relationship between insulin action and skeletal muscle myogenic/regenerative potential. RUNX1 is a transcription factor which plays a role in muscle development and regeneration. The aim of our study was to assess the role of skeletal muscle myogenic/regenerative potential in the development of insulin resistance through the studies on RUNX1 transcription factor. This study is a cross-sectional study. Experimental part with myoblast cell line culture. We examined 41 young healthy volunteers, 21 normal weight and 20 with overweight or obesity. Hyperinsulinemic-euglycemic clamp and vastus lateralis muscle biopsy were performed. In L6 myoblast and human skeletal muscle myoblasts (hSkMM) cell cultures, RUNX1 was silenced at two stages of development. Cell growth, the expression of markers of myogenesis, nuclei fusion index, Akt phosphorylation and glucose uptake were measured. Skeletal muscle RUNX1 expression was decreased in overweight/obese individuals in comparison with normal-weight individuals and was positively related to insulin sensitivity, independently of BMI. Runx1 loss-of-function at the stage of myoblast inhibited myoblast proliferation and differentiation and reduced insulin-stimulated Akt phosphorylation and insulin-stimulated glucose uptake. In contrast, Runx1 knockdown in myotubes did not affect Akt phosphorylation, glucose uptake and other parameters studied. Myogenic/regenerative potential of adult skeletal muscle may be an important determinant of insulin action. Our data suggest that muscle RUNX1 may play a role in the modulation of insulin action through its effect on myogenesis.

Changes in Elastic Moduli of Fibrin Hydrogels Within the Myogenic Range Alter Behavior of Murine C2C12 and Human C25 Myoblasts Differently.

Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates (“2D”) or embedded in fibrin hydrogels (“3D”) with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels’ fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7–30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.

Effects of heart failure and diabetes on invasive biomarkers of skeletal muscle wasting

Abstract Funding Acknowledgements Type of funding sources: None. Background Muscle wasting is common in patients with diabetes mellitus (DM) and chronic heart failure (HF), which exacerbates cachexia and poor quality of life. However, a gap in clinical translation remains as the mechanisms underlying muscle wasting in DM and HF remain poorly validated, with most evidence assumed from experimental small-animal models. Purpose Validate if markers of muscle wasting (e.g., autophagy, proteolysis, myogenesis) were correlated to muscle phenotype and clinical indices in humans with DM and HF. Method Male patients with DM (n=4), HF (n=4; NYHA=2), DMHF (n=7; NYHA=2) were included. DM was classified as a HbA1c level ≥48mmol/mol and HF classified as a left ventricular ejection fraction (LVEF) ≤40%, with patients on optimised medication. Muscle biopsies of pectoralis major were excised during routine pacemaker surgery and frozen at -80°C for both histological and gene expression analysis. Muscle samples were sectioned, stained, and imaged for fibre cross-sectional area and fibre type. RT-PCR gene expression was conducted for markers of muscle regeneration (myod, myogenin), protein degradation (myostatin and autophagy-related LC3B, ATG7, CTSL), and anabolic signalling (IGF-1). Statistical analysis included ANOVAs and correlation analysis to identify relationships between variables. Values of p&amp;lt;0.05 were accepted as significant. Results Groups (DM, HF, DMHF) were not different (P&amp;gt;0.05) in relation to age (79±9, 72±8, 72±10 years) or BMI (24.5±3.6, 28.5±4.2, 29.5±6.0) respectively. Fibre size or fibre type were not different (P&amp;gt;0.05) between groups. In line with this, gene expression for markers of myogenesis and anabolism as well as myostatin were not different (P&amp;gt;0.05) between groups, but a trend in downregulation of the proteolytic autophagy-related genes (LC3B, ATG7, CTSL) in DMHF vs DM or HF was found on average by 21, 36, and 30% respectively. Autophagy-related genes LC3B, ATG7, CTSL were positively correlated (P&amp;lt;0.05) to type IIa fibre numerical density (R=0.82, 0.86, 0.89) and LVEF (R=0.85, 0.92, 0.83), respectively. Conclusion Our findings indicate that gene expression of autophagy could represent a robust marker of muscle wasting and cardiac dysfunction in humans with DM and HF. Muscle biopsies from patients with DM and HF identified the proteolytic system of autophagy, important for cellular homeostasis, may be inhibited and this was correlated to fibre phenotype and LVEF. However, the process of muscle wasting in DM and HF within the clinical setting may not necessarily reflect those reported in animal models given various other markers were not changed.

Direct reprogramming of dermal fibroblasts derived from Northern elephant seals into muscle cells

Ex vivo tissue culture allows the study of complex cellular mechanisms that are relevant to physiological responses while overcoming the challenges presented by studying animals that are not tractable. In a primary cell culture system, certain proliferating cells can be functionally reprogrammed into other cell types via overexpression of key genes. Dermal fibroblasts can be reprogrammed into muscle cells, which are often challenging to obtain but offer a unique system to study metabolic responses, by overexpression of the myogenic transcription factor myod. We isolated cells from Northern elephant seal (NES) skin samples and propagated them in primary culture. NES cells respire, stain positive for fibroblast markers (vimentin and PDGFR), and are amenable to electroporation. We overexpressed GFP‐myod in NES fibroblasts and conducted antibiotic selection and purification by FACS. As expected, expression of myod was higher in transfected cells than in controls according to qPCR analysis (t‐test p&lt; 0.05). Treatment with small molecules (CHIR99021, Forskolin and Repsox) enhanced myod expression. Furthermore, fibroblasts overexpressing myod expressed downstream markers of myogenesis (myogenin, myosin heavy chain 1 and myosin heavy chain 8) and the effect was enhanced when myod‐overexpressing cells were supplemented with small molecules. We are currently evaluating the capacity of myod‐overexpressing cells to differentiate into myotubes and comparing metabolic profiles with primary NES myoblasts. Establishing differentiated muscle fibers from other mature cell types could provide a unique platform to conduct mechanistic studies in species where muscle tissue samples cannot be obtained from live animals.

Modulatory effects of cell–cell interactions between porcine skeletal muscle satellite cells and fibroblasts on the expression of myogenesis-related genes

ABSTRACT Co-culture is mimicked in vivo condition, which is influenced by adjacent cells. This study aimed to determine different myogenic genes in mono- and co-culture of satellite cells and fibroblasts. Fibroblasts and satellite cells were isolated from the piglet’s thigh muscles. The expression of PAX-7, MyoD, MyoG, and PAX-3 was downregulated in 24 h co-culture satellite cells than in monoculture. The expression of Myf-5 and CAV-1 was upregulated in 24 h co-culture satellite cells than in 24 h monoculture. The expression of PAX-7, MyoD, and Myf-5 was downregulated in 24 and 48 h co-culture fibroblasts than 24 h monoculture. The expression of MyoG and PAX-3 was upregulated in 24 and 48 h co-culture than 48 h monoculture fibroblasts. The Cyto-C was downregulated in 24 and 48 h co-culture fibroblast than 24 h monoculture. The expression of MyoG and CAV-1 was upregulated in 48 h incubation period co-culture fibroblasts than 24 h monoculture. The MyoD and CAV-1 expression were upregulated in 24 h co-culture than 24 h satellite cells and fibroblasts. The Bcl-2 and BAX were downregulated in 48 h co-culture than 24 h. This result recommended that the satellite cells and fibroblasts interaction enhance the myogenesis marker expression without cellular morphology change.

Effect of High-Protein Diets on Integrated Myofibrillar Protein Synthesis before Anterior Cruciate Ligament Reconstruction: A Randomized Controlled Pilot Study.

Increasing dietary protein intake during periods of muscle disuse may mitigate the resulting decline in muscle protein synthesis (MPS). The purpose of this randomized pilot study was to determine the effect of increased protein intake during periods of disuse before anterior cruciate ligament (ACL) reconstruction on myofibrillar protein synthesis (MyoPS), and proteolytic and myogenic gene expression. Six healthy, young males (30 ± 9 y) were randomized to consume a high-quality, optimal protein diet (OP; 1.9 g·kg−1·d−1) or adequate protein diet (AP; 1.2 g·kg−1·d−1) for two weeks before ACL reconstruction. Muscle biopsies collected during surgery were used to measure integrated MyoPS during the intervention (via daily deuterium oxide ingestion) and gene expression at the time of surgery. MyoPS tended to be higher, with a large effect size in OP compared to AP (0.71 ± 0.1 and 0.54 ± 0.1%·d−1; p = 0.076; g = 1.56). Markers of proteolysis and myogenesis were not different between groups (p > 0.05); however, participants with greater MyoPS exhibited lower levels of MuRF1 gene expression compared to those with lower MyoPS (r = −0.82, p = 0.047). The data from this pilot study reveal a potential stimulatory effect of increased daily protein intake on MyoPS during injury-mediated disuse conditions that warrants further investigation.

Ganoderma lucidum Induces Myogenesis Markers to Avert Damage to Skeletal Muscles in Rats Exposed to Hypobaric Hypoxia.

Jatwani, Arti, and Rajkumar Tulsawani. Ganoderma lucidum induces myogenesis markers to avert damage to skeletal muscles in rats exposed to hypobaric hypoxia. High Alt Med Biol. 24:287-295, 2023. Background: Hypobaric hypoxia (HH) has been reported to induce skeletal muscle loss and impair myogenesis. Aqueous extract of G. lucidum (AqGL) contains bioactive metabolites attributed to various pharmacological effects. In this study, protective effect of AqGL in ameliorating muscle mass loss following acute HH has been reported. Materials and Methods: Male Sprague-Dawley rats were divided into following five groups of six rats in each group: unexposed control (Group 1), 6 hours of HH exposure (Group 2), 6 hours of HH exposure+AqGL extract 50 mg/kg body weight (BW) (Group 3), 6 hours of HH exposure+AqGL extract 100 mg/kg BW (Group 4), and 6 hours of HH exposure+AqGL extract 200 mg/kg BW (Group 5). Experimental animals from all groups, except Group, 1 were exposed to HH, simulated altitude of 25,000 ft for 6 hours. After exposure period, gastrocnemius muscle was collected, weighed, and morphological, biochemical, and molecular markers were analyzed. Results: HH-exposed rat muscle showed significant (p < 0.05) increase in oxidative stress markers (reactive oxygen species & malondialdehyde), which was concomitant with decrease in its mass compared to controls. AqGL treatment significantly (p < 0.05) prevented muscle oxidative stress, restored reduced glutathione content, reduced protein carbonyl content and advanced oxidation protein product, and restored muscle mass loss at effective dose of 100 mg/kg BW. Furthermore, AqGL supplementation enhanced Myf5 (p < 0.01), MyoD (p < 0.01), MyoG (p < 0.05), and Mrf4 (nonsignificantly), brain-derived neurotrophic factor (p < 0.01), and interleukin 6 (p < 0.01) expression along with restoration of tumor necrosis factor alpha (p < 0.001) and myostatin (p < 0.05) in hypoxia-exposed muscle, evidencing induction of myogenesis markers. Moreover, histological analysis showed increased myocyte number; nuclei shifted toward the periphery in the treatment group supporting muscle regeneration. Conclusion: AqGL supplementation attenuates muscle mass loss by preventing oxidative stress and inducing modulation in myogenesis markers under HH environment.

EFFECT OF OBESE FACTORS FREE FATTY ACID AND LIPOPOLYSACCHARIDE ON EXPRESSION OF METABOLIC AND MYOGENIC GENES CPT1b AND Myogenin IN MUSCLE CELLS

Carnitine palmitoyl transferase 1b (CPT1b) and Myogenin are important markers of metabolism and myogenesis, respectively. The present study aimed to research the expression of CPT1b and Myogenin mRNAs in skeletal muscle C2C12 cells incubated with obese factors, free fatty acid (FFA), or lipopolysaccharide (LPS), to support the mechanism related to obesity and metabolic disorders. The result indicated that FFA significantly induced upregulation of CPT1b and Myogenin mRNAs in the skeletal muscle cells. In contrast, LPS treatment did not affect on expression of CPT1 mRNA and tendency reduced expression of Myogenin mRNA in the skeletal muscle cells. These data support the evidence FFA is an energy and nutrient source for metabolism and myogenesis in the skeletal muscle cells whereas LPS seemingly suppresses myogenesis. Therefore, the mechanisms linking FFA or LPS to obesity induced skeletal muscle metabolic dysfunctions could be different.

Histamine H3 Receptor Signaling Regulates the NLRP3 Inflammasome Activation in C2C12 Myocyte During Myogenic Differentiation.

NLRP3 inflammasome has been implicated in impaired post-injury muscle healing and in muscle atrophy. Histamine receptors play an important role in inflammation, but the role of histamine H3 receptor (H3R) in myocyte regeneration and in the regulation of NLRP3 inflammasome is not known. We studied the effects of H3R signaling on C2C12 myocyte viability, apoptosis, and tumor necrosis factor alpha (TNFα)-induced NLRP3 inflammasome activation during striated myogenic differentiation at three time points (days 0, 3, and 6). Expression of Nlrp3, interleukin-1β (IL-1β), and myogenesis markers were determined. TNFα reduced overall viability of C2C12 cells, and exposure to TNFα induced apoptosis of cells at D6. Activation of H3R had no effect on viability or apoptosis, whereas inhibition of H3R increased TNFα-induced apoptosis. Stimulation of C2C12 cells with TNFα increased Nlrp3 mRNA expression at D3 and D6. Moreover, TNFα reduced the expression of myogenesis markers MyoD1, Myogenin, and Myosin-2 at D3 and D6. H3R attenuated TNFα-induced expression of Nlrp3 and further inhibited the myogenesis marker expression; while H3R -blockage enhanced the proinflammatory effects of TNFα and increased the myogenesis marker expression. TNFα-induced secretion of mature IL-1β was dependent on the activation of the NLRP3 inflammasome, as shown by the reduced secretion of mature IL-1β upon treatment of the cells with the small molecule inhibitor of the NLRP3 inflammasome (MCC950). The activation of H3R reduced TNFα-induced IL-1β secretion, while the H3R blockage had an opposite effect. In conclusion, the modulation of H3R activity regulates the effects of TNFα on C2C12 myocyte differentiation and TNFα-induced activation of NLRP3 inflammasome. Thus, H3R signaling may represent a novel target for limiting postinjury muscle inflammation and muscle atrophy.

Direct reprogramming of dermal fibroblasts derived from Northern elephant seals into muscle cells

Ex vivo tissue culture allows the study of complex cellular mechanisms that are relevant to physiological responses in animals while overcoming the challenges presented by studying animals that are not tractable. In a primary cell culture system, certain proliferating cells can be functionally reprogrammed into other cell types via overexpression of key genes. Dermal fibroblasts can be reprogrammed into muscle progenitor cells (myoblasts), which are often challenging to obtain but offer a unique system to study metabolic responses, by overexpression of the myogenic transcription factor myod. We isolated fibroblasts from Northern elephant seal (NES) skin samples and propagated them in primary culture. We overexpressed myod in NES fibroblasts and conducted antibiotic selection with 2 ug/mL puromycin for 3 days. As expected, expression of myod was significantly higher in transfected cells according to qPCR analysis (p< 0.05). Treatment with small molecules (CHIR99021, Forskolin and Repsox) enhanced myod expression. Furthermore, fibroblasts overexpressing myod expressed downstream markers of myogenesis (myogenin, myosin heavy chain 1 and myosin heavy chain 8) and the effect was enhanced when myod-overexpressing cells were supplemented with small molecules. We are currently evaluating the capacity of myod-overexpressing dermal fibroblasts to differentiate into myotubes and comparing metabolic profiles with primary NES myoblasts. Establishing differentiated muscle fibers from other mature cell types could provide a unique platform to conduct mechanistic studies in species where muscle tissue samples cannot be obtained from live animals.

Role of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 in Chicken Embryo Osteogenesis, Adipogenesis, Myogenesis, and Vitamin D3 Metabolism.

A study was conducted to understand the effects of 25-hydroxyvitamin D3 (25OHD) and 1,25-dihydroxyvitamin D3 (1,25OHD) administration on the expression of key genes related to osteogenesis, adipogenesis, myogenesis, and vitamin D3 metabolism in the chicken embryo. A total of 120 fertilized Cobb 500 eggs were used in the current study and were reared under standard incubation conditions. On embryonic day 3 (ED 3), PBS (C), PBS with 40ng 1,25OHD (1,25D-L), 200ng 1,25OHD (1,25D-H), 40ng 25OHD (25D-L), or 200ng 25OHD (25D-H) were injected into the dorsal vein of developing embryos. Whole embryos were harvested at 1, 3, and 6h post-injection for gene expression analyses (n=8). Gene expression for key osteogenesis markers (RUNX2: runt-related transcription factor 2; BMP2: bone morphogenetic protein 2; COL1A2: collagen type I alpha 2 chain; BGLAP: bone gamma-carboxyglutamate protein; SPP1: secreted phosphoprotein 1; and ALP: alkaline phosphatese), adipogenesis markers (PPAR-γ: peroxisome proliferator-activated receptor gamma; FASN: fatty acid synthase; and FABP4: fatty acid binding protein 4), myogenesis markers (MYOG: myogenin; MYOD1: myogenic differentiation 1; and MYF5: myogenic factor 5), and the enzyme responsible for vitamin D3 inactivation (CYP24A1: cytochrome P450 family 24 subfamily A member 1) were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data were normalized by the ΔΔCT method and analyzed using a one-way ANOVA. Results indicated that at 1h post-injection, no differences were found among treatments. At 3h, the early osteogenesis differentiation marker, ALP, was increased by 1,25D-H and 25D-H, and 25D-H also stimulated the expression of adipogenesis markers (FAPB4 and FASN). In contrast, the expression of myogenesis markers (MYOD1 and MYF5) was suppressed by 25OHD or 1,25OHD treatments, respectively. At 6h, a late osteogenic differentiation marker, SPP1, was increased by 25D-H. MYOD1 and MYF5 were continuously suppressed by 25OHD treatments or 1,25D-H. The evidence of vitamin D3 metabolite retention was assessed by measuring CYP24A1 expression. At 1h, there were no differences in CYP24A1 expression. At 3h, all treatments upregulated CYP24A1 expression relative to control (PBS) embryos. However, at 6h, only the 25D-H group retained higher CYP24A1 expression compared to the other treatments. In conclusion, the results suggested both 1,25OHD and 25OHD induced chicken embryo osteogenesis and adipogenesis, but inhibited myogenesis during early chicken embryo development. The higher dosage of 25OHD showed a possibility of a longer retention time in the embryos.

Initiating aerobic exercise with low glycogen content reduces markers of myogenesis but not mTORC1 signaling

BackgroundThe effects of low muscle glycogen on molecular markers of protein synthesis and myogenesis before and during aerobic exercise with carbohydrate ingestion is unclear. The purpose of this study was to determine the effects of initiating aerobic exercise with low muscle glycogen on mTORC1 signaling and markers of myogenesis.MethodsEleven men completed two cycle ergometry glycogen depletion trials separated by 7-d, followed by randomized isocaloric refeeding for 24-h to elicit low (LOW; 1.5 g/kg carbohydrate, 3.0 g/kg fat) or adequate (AD; 6.0 g/kg carbohydrate, 1.0 g/kg fat) glycogen. Participants then performed 80-min of cycle ergometry (64 ± 3% VO2peak) while ingesting 146 g carbohydrate. mTORC1 signaling (Western blotting) and gene transcription (RT-qPCR) were determined from vastus lateralis biopsies before glycogen depletion (baseline, BASE), and before (PRE) and after (POST) exercise.ResultsRegardless of treatment, p-mTORC1Ser2448, p-p70S6KSer424/421, and p-rpS6Ser235/236 were higher (P < 0.05) POST compared to PRE and BASE. PAX7 and MYOGENIN were lower (P < 0.05) in LOW compared to AD, regardless of time, while MYOD was lower (P < 0.05) in LOW compared to AD at PRE, but not different at POST.ConclusionInitiating aerobic exercise with low muscle glycogen does not affect mTORC1 signaling, yet reductions in gene expression of myogenic regulatory factors suggest that muscle recovery from exercise may be reduced.