Pathway In Skeletal Muscle Cells Research Articles

Eliminating elevated p53 signaling fails to rescue skeletal muscle defects or extend survival in lamin A/C-deficient mice.

Lamins A and C, encoded by the LMNA gene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation. LMNA mutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which many LMNA mutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragile Lmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts of Lmna mutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity in Lmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.

The cellular bases of mobility from the Study of Muscle, Mobility and Aging (SOMMA).

Findings from the Study of Muscle, Mobility and Aging (SOMMA) in this issue of Aging Cell show that several biological pathways in skeletal muscle cells play an important role in determining mobility in older adults. These are based on assays in skeletal muscle biopsies obtained from participants, aged 70 years and older in SOMMA tested for association with assessments related to mobility, including muscle mass, strength, power, cardiopulmonary fitness, and 400 m walking speed. The papers show that, using mass spectrometry, oxidative modifications of proteins essential to myocellular function are associated with poorer mobility. Using RNA-seq to quantify gene expression, lower levels of expression of antioxidant enzymes located in mitochondria, autophagy, patterns of expression of genes involved in autophagy, and higher levels of RNA transcripts that increase with denervation were associated with poorer performance on tests of mobility. These results extend previous research from the Baltimore Longitudinal Study of Aging and recent studies from SOMMA showing the importance of mitochondrial energetics in mobility. Together, these findings are painting a picture of how fundamental cellular processes influence the loss of mobility with aging. They may also be a window on aging in other cells, tissues, and systems. The data collected in SOMMA are publicly available and SOMMA welcomes collaborations with scientists who are interested in research about human aging.

Optogenetic decoding of Akt2-regulated metabolic signaling pathways in skeletal muscle cells using transomics analysis.

Insulin regulates various cellular metabolic processes by activating specific isoforms of the Akt family of kinases. Here, we elucidated metabolic pathways that are regulated in an Akt2-dependent manner. We constructed a transomics network by quantifying phosphorylated Akt substrates, metabolites, and transcripts in C2C12 skeletal muscle cells with acute, optogenetically induced activation of Akt2. We found that Akt2-specific activation predominantly affected Akt substrate phosphorylation and metabolite regulation rather than transcript regulation. The transomics network revealed that Akt2 regulated the lower glycolysis pathway and nucleotide metabolism and cooperated with Akt2-independent signaling to promote the rate-limiting steps in these processes, such as the first step of glycolysis, glucose uptake, and the activation of the pyrimidine metabolic enzyme CAD. Together, our findings reveal the mechanism of Akt2-dependent metabolic pathway regulation, paving the way for Akt2-targeting therapeutics in diabetes and metabolic disorders.

A Novel Partial PPARγ Agonist Has Weaker Lipogenic Effect in Adipocytes and Stimulates GLUT4 Translocation in Skeletal Muscle Cells via AMPK-Dependent Signaling

Introduction: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are highly effective in treating insulin resistance. However, associated side effects such as weight gain due to increase in adipogenesis and lipogenesis hinder their clinical use. The aim of the study was to design and synthesize novel partial PPARγ agonists with weaker lipogenic effect in adipocytes and enhanced glucose transporter 4 (GLUT4) translocation stimulatory effect in skeletal muscle cells. Methods: Novel partial PPARγ agonists (GS1, GS2, and GS3) were designed and screened to predict their binding interactions with PPARγ by molecular docking. The stability of the docked ligand-PPARγ complex was studied by molecular dynamics (MD) simulation. The cytotoxicity of synthesized compounds was tested in 3T3-L1 adipocytes and L6 myoblasts by MTT assay. The lipogenic effect was investigated in 3T3-L1 adipocytes using oil red O staining and GLUT4 translocation stimulatory effect in L6-GLUT4myc myotubes by an antibody-coupled colorimetric assay. Results: The molecular docking showed the binding interactions between designed agonists and PPARγ. MD simulation demonstrated good stability between the GS2-PPARγ complex. GS2 and GS3 did not show any significant effect on cell viability up to 80 or 100 μM concentration. Pioglitazone treatment significantly increased intracellular lipid accumulation in adipocytes compared to control. However, this effect was significantly less in GS2- and GS3-treated conditions compared to pioglitazone at 10 μM concentration, indicating weaker lipogenic effect. Furthermore, GS2 significantly stimulated GLUT4 translocation to the plasma membrane in a dose-dependent manner via the AMPK-dependent signaling pathway in skeletal muscle cells. Conclusion: GS2 may be a promising therapeutic agent for the treatment of insulin resistance and type 2 diabetes mellitus without adiposity.

Caffeine increases myoglobin expression via the cyclic AMP pathway in L6 myotubes.

Myoglobin is an important regulator of muscle and whole‐body metabolism and exercise capacity. Caffeine, an activator of the calcium and cyclic AMP (cAMP)/protein kinase A (PKA) pathway, enhances glucose uptake, fat oxidation, and mitochondrial biogenesis in skeletal muscle cells. However, no study has shown that caffeine increases the endogenous expression of myoglobin in muscle cells. Further, the molecular mechanism underlying the regulation of myoglobin expression remains unclear. Therefore, our aim was to investigate whether caffeine and activators of the calcium signaling and cAMP/PKA pathway increase the expression of myoglobin in L6 myotubes and whether the pathway mediates caffeine‐induced myoglobin expression. Caffeine increased myoglobin expression and activated the cAMP/PKA pathway in L6 muscle cells. Additionally, a cAMP analog significantly increased myoglobin expression, whereas a ryanodine receptor agonist showed no significant effect. Finally, PKA inhibition significantly suppressed caffeine‐induced myoglobin expression in L6 myotubes. These results suggest that caffeine increases myoglobin expression via the cAMP/PKA pathway in skeletal muscle cells.

α-Amyrin induces GLUT4 translocation mediated by AMPK and PPARδ/γ in C2C12 myoblasts.

α-Amyrin, a natural pentacyclic triterpene, has an antihyperglycemic effect in mice and dual PPARδ/γ action in 3T3-L1 adipocytes, and potential in the control of type 2 diabetes (T2D). About 80% of glucose uptake occurs in skeletal muscle cells, playing a significant role in insulin resistance (IR) and T2D. Peroxisome-proliferator activated receptors (PPARs), in particular PPARδ and PPARγ, are involved in the regulation of lipids and carbohydrates and, along with adenosine-monophosphate (AMP) - activated protein kinase (AMPK) and protein kinase B (Akt), are implicated in translocation of glucose transporter 4 (GLUT4); however, it is still unknown whether α-amyrin can affect these pathways in skeletal muscle cells. Our objective was to determine the action of α-amyrin in PPARδ, PPARγ, AMPK, and Akt in C2C12 myoblasts. The expression of PPARδ, PPARγ, fatty acid transporter protein (FATP), and GLUT4 was quantified using reverse transcription quantitative PCR and Western blot. α-Amyrin increased these markers along with phospho-AMPK (p-AMPK) but not p-Akt. Molecular docking showed that α-amyrin acts as an AMPK-allosteric activator, and may be related to GLUT4 translocation, as evidenced by confocal microscopy. These data support that α-amyrin could have an insulin-mimetic action in C2C12 myoblasts and should be considered as a bioactive molecule for new multitarget drugs with utility in T2D and other metabolic diseases.

A milk casein hydrolysate-derived peptide enhances glucose uptake through the AMP-activated protein kinase signalling pathway in skeletal muscle cells.

What is the central question of this study? How do common active ingredients contained in both Lactobacillus helveticus-fermented milk and milk casein hydrolysate (MCH) enhance glucose metabolism by skeletal muscle? What is the main finding and its importance? MCH enhanced glucose uptake in skeletal muscle cells by stimulating AMP-activated kinase, but not insulin, signalling. Moreover, the MCH-derived specific peptide Ile-Pro-Pro mimicked this effect, suggesting a mechanism for MCH-induced metabolic improvement. Improvement of glucose metabolism in the skeletal muscle has a key role in exercise performance and prevention of metabolic diseases. In our previous study, we showed that intake of milk casein hydrolysate improves glucose metabolism in humans, but the mechanism of action was not elucidated. In this study, we aimed to investigate the mechanism of action of milk casein hydrolysate and its derived peptides on glucose uptake and glucose metabolic signalling in cultured skeletal muscle cells. Differentiated C2C12 myotubes were used for the experiments. The differentiated cells were incubated with milk casein hydrolysate, valine-proline-proline and isoleucine-proline-proline. Subsequently, the rate of 2-deoxy-glucose uptake and the phosphorylation levels of insulin-dependent and -independent signalling factors were examined. We found that the rate of 2-deoxy-glucose uptake in both milk casein hydrolysate and isoleucine-proline-proline-treated cells was higher than that in the control cells. Immunoblotting assays showed that the phosphorylation levels of AMP-activated protein kinase, a rate-limiting factor in insulin-independent signalling, and of liver kinase B1, an upstream factor of AMP-activated protein kinase, in both milk casein hydrolysate and isoleucine-proline-proline-treated cells were higher than those in the control cells. Such significant effects were not observed after treatment with valine-proline-proline. Moreover, the insulin-dependent signalling was not significantly affected under the different conditions. The findings of our study suggest that milk casein hydrolysate enhances glucose uptake by activating insulin-independent AMP-activated protein kinase signalling in skeletal muscle cells, which might be mediated by a milk casein hydrolysate-derived peptide, namely, isoleucine-proline-proline.

Puerarin ameliorates skeletal muscle wasting and fiber type transformation in STZ-induced type 1 diabetic rats

Puerarin is an isoflavonoid extracted from Pueraria lobate with extensive pharmacological effects in traditional Chinese medicine. The evidence implicates that puerarin mitigates hyperglycemia and various relevant complications. Here, the effect of puerarin on skeletal muscle wasting induced by type 1 diabetes (T1D) was explored. Streptozotocin (STZ)-induced T1D male Sprague Dawley (SD) rats were used in this study. Muscle strength, weight and size were measured. L6 rat skeletal muscle cells were applied for in vitro study. Our results showed that eight-week oral puerarin administration (100 mg/kg) increased muscle strengths and weights accompanied by enhanced skeletal muscle cross-sectional areas in diabetic rats. Simultaneously, puerarin also reduced expressions of several muscle wasting marker genes including F-box only protein 32 (Atrogin-1) and muscle-specific RING-finger 1 (Murf-1) in diabetic group both in vitro and in vivo. Transformation from type I fibers (slow muscle) to type II fibers (fast muscle) were also observed under puerarin administration in diabetic rats. Puerarin promoted Akt/mTOR while inhibited LC3/p62 signaling pathway in skeletal muscle cells. In conclusion, our study showed that puerarin mitigated skeletal muscle wasting in T1D rats and closely related with Akt/mTOR activation and autophagy inhibition. Whether this effect in murine applies to humans remains to be determined.

Prokineticin receptor 1 ameliorates insulin resistance in skeletal muscle.

Type 2 diabetes mellitus may result from insulin resistance in skeletal muscle. Prokineticin receptor 1 (Prokr1) improves metabolic phenotype in adipose tissue and the cardiovascular system; however, its effects on skeletal muscle have not been investigated. We investigated the Prokr1 signaling pathways and its metabolic function in murine myoblast, satellite cells, and their differentiated myotubes. We measured the expression levels of Prokr1 in the skeletal muscle of mice as well as human skeletal muscle cell-derived myotubes. Prokineticin 2 (PROK2), a ligand of PROKR1, induced calcium mobilization in a dose-dependent manner and altered the mRNA levels of 578 genes in PROKR1-overexpressed HEK293T cells. Functional enrichment of differentially expressed genes revealed that PROKR1 activated Gq-mediated PI3K/AKT and MAPK/ERK signaling pathways in skeletal muscle cells. Prokr1 significantly activated the PI3K/AKT signaling pathway in myotubes derived from C2C12 and satellite cells, regardless of the presence or absence of insulin. Prokr1 also promoted the translocation of glucose transporter 4 (GLUT4) into the plasma membrane. In palmitate-induced insulin-resistant myotubes, Prokr1 enhanced insulin-stimulated AKT phosphorylation, GLUT4 translocation, and glucose uptake. mRNA and protein levels of Prokr1 were significantly decreased in skeletal muscle and white adipose tissue of diet-induced obese mice, and the amount of PROKR1 protein was significantly decreased in human skeletal muscle cell-derived myotubes under insulin resistance conditions. Taken together, these results demonstrate that Prokr1 plays an important role in insulin sensitivity and is a potential therapeutic target to ameliorate insulin resistance in skeletal muscle.

Zinc Modulates Several Transcription-Factor Regulated Pathways in Mouse Skeletal Muscle Cells.

Zinc is an essential metal ion involved in many biological processes. Studies have shown that zinc can activate several molecules in the insulin signalling pathway and the concomitant uptake of glucose in skeletal muscle cells. However, there is limited information on other potential pathways that zinc can activate in skeletal muscle. Accordingly, this study aimed to identify other zinc-activating pathways in skeletal muscle cells to further delineate the role of this metal ion in cellular processes. Mouse C2C12 skeletal muscle cells were treated with insulin (10 nM), zinc (20 µM), and the zinc chelator TPEN (various concentrations) over 60 min. Western blots were performed for the zinc-activation of pAkt, pErk, and pCreb. A Cignal 45-Reporter Array that targets 45 signalling pathways was utilised to test the ability of zinc to activate pathways that have not yet been described. Zinc and insulin activated pAkt over 60 min as expected. Moreover, the treatment of C2C12 skeletal muscle cells with TPEN reduced the ability of zinc to activate pAkt and pErk. Zinc also activated several associated novel transcription factor pathways including Nrf1/Nrf2, ATF6, CREB, EGR1, STAT1, AP-1, PPAR, and TCF/LEF, and pCREB protein over 120 min of zinc treatment. These studies have shown that zinc’s activity extends beyond that of insulin signalling and plays a role in modulating novel transcription factor activated pathways. Further studies to determine the exact role of zinc in the activation of transcription factor pathways will provide novel insights into this metal ion actions.

Acetic acid stimulates G-protein-coupled receptor GPR43 and induces intracellular calcium influx in L6 myotube cells.

Short chain fatty acids (SCFAs) produced endogenously in the gut by bacterial fermentation of dietary fiber have been studied as nutrients that act as signaling molecules to activate G-protein coupled receptors (GPCRs) such as GPR41 and GPR43. GPR43 functioning involves the suppression of lipid accumulation and maintaining body energy homeostasis, and is activated by acetic acid or propionic acid. Previously, we reported that the orally administered acetic acid improves lipid metabolism in liver and skeletal muscles and suppresses obesity, thus improving glucose tolerance. Acetic acid stimulates AMP-activated protein kinase (AMPK) through its metabolic pathway in skeletal muscle cells. We hypothesized that acetic acid would stimulate GPR43 in skeletal muscle cells and has function in modulating gene expression related to muscle characteristics through its signal pathway. The objective of the current study was to clarify this effect of acetic acid. The GPR43 expression, observed in the differentiated myotube cells, was increased upon acetic acid treatment. Acetic acid induced the intracellular calcium influx in the cells and this induction was significantly inhibited by the GPR43-specific siRNA treatment. The calcineurin molecule is activated by calcium/calmodulin and is associated with proliferation of slow-twitch fibers. Calcineurin was activated by acetic acid treatment and inhibited by the concomitant treatment with GPR43-siRNA. Acetic acid induced nuclear localization of myocyte enhancer factor 2A (MEF2A), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and nuclear factor of activated t cells c1 (NFATc1). However, these localizations were abolished by the treatment with GPR43-siRNA. It was concluded that acetic acid plays a role in the activation of GPR43 and involves the proliferation of slow-twitch fibers in L6 skeletal muscles through the calcium-signaling pathway caused by induction of intracellular calcium influx.

Regulation of Lin28a-miRNA let-7b-5p pathway in skeletal muscle cells by peroxisome proliferator-activated receptor delta.

Lin28a/miRNA let-7b-5p pathway has emerged as a key regulators of energy homeostasis in the skeletal muscle. However, the mechanism through which this pathway is regulated in the skeletal muscle has remained unclear. We have found that 8 wk of aerobic training (Tr) markedly decreased let-7b-5p expression in murine skeletal muscle, whereas high-fat diet (Hfd) increased its expression. Conversely, Lin28a expression, a well-known inhibitor of let-7b-5p, was induced by Tr and decreased by Hfd. Similarly, in human muscle biopsies, Tr increased LIN28 expression and decreased let-7b-5p expression. Bioinformatics analysis of LIN28a DNA sequence revealed that its enrichment in peroxisome proliferator-activated receptor delta (PPARδ) binding sites, which is a well-known metabolic regulator of exercise. Treatment of primary mouse skeletal muscle cells or C2C12 cells with PPARδ activators GW501516 and AICAR increased Lin28a expression. Lin28a and let-7b-5p expression was also regulated by PPARδ coregulators. While PPARγ coactivator-1α (PGC1α) increased Lin28a expression, corepressor NCoR1 decreased its expression. Furthermore, PGC1α markedly reduced the let-7b-5p expression. PGC1α-mediated induction of Lin28a expression was blocked by the PPARδ inhibitor GSK0660. In agreement, Lin28a expression was downregulated in PPARδ knocked-down cells leading to increased let-7b-5p expression. Finally, we show that modulation of the Lin28a-let-7b-5p pathway in muscle cells leads to changes in mitochondrial metabolism in PGC1α dependent fashion. In summary, we demonstrate that Lin28a-let-7b-5p is a direct target of PPARδ in the skeletal muscle, where it impacts mitochondrial respiration.

Preclinical insights into the gut-skeletal muscle axis in chronic gastrointestinal diseases.

Muscle wasting represents a constant pathological feature of common chronic gastrointestinal diseases, including liver cirrhosis (LC), inflammatory bowel diseases (IBD), chronic pancreatitis (CP) and pancreatic cancer (PC), and is associated with increased morbidity and mortality. Recent clinical and experimental studies point to the existence of a gut‐skeletal muscle axis that is constituted by specific gut‐derived mediators which activate pro‐ and anti‐sarcopenic signalling pathways in skeletal muscle cells. A pathophysiological link between both organs is also provided by low‐grade systemic inflammation. Animal models of LC, IBD, CP and PC represent an important resource for mechanistic and preclinical studies on disease‐associated muscle wasting. They are also required to test and validate specific anti‐sarcopenic therapies prior to clinical application. In this article, we review frequently used rodent models of muscle wasting in the context of chronic gastrointestinal diseases, survey their specific advantages and limitations and discuss possibilities for further research activities in the field. We conclude that animal models of LC‐, IBD‐ and PC‐associated sarcopenia are an essential supplement to clinical studies because they may provide additional mechanistic insights and help to identify molecular targets for therapeutic interventions in humans.

Anti-Inflammatory and General Glucocorticoid Physiology in Skeletal Muscles Affected by Duchenne Muscular Dystrophy: Exploration of Steroid-Sparing Agents.

In Duchenne muscular dystrophy (DMD), the activation of proinflammatory and metabolic cellular pathways in skeletal muscle cells is an inherent characteristic. Synthetic glucocorticoid intake counteracts the majority of these mechanisms. However, glucocorticoids induce burdensome secondary effects, including hypertension, arrhythmias, hyperglycemia, osteoporosis, weight gain, growth delay, skin thinning, cushingoid appearance, and tissue-specific glucocorticoid resistance. Hence, lowering the glucocorticoid dosage could be beneficial for DMD patients. A more profound insight into the major cellular pathways that are stabilized after synthetic glucocorticoid administration in DMD is needed when searching for the molecules able to achieve similar pathway stabilization. This review provides a concise overview of the major anti-inflammatory pathways, as well as the metabolic effects of glucocorticoids in the skeletal muscle affected in DMD. The known drugs able to stabilize these pathways, and which could potentially be combined with glucocorticoid therapy as steroid-sparing agents, are described. This could create new opportunities for testing in DMD animal models and/or clinical trials, possibly leading to smaller glucocorticoids dosage regimens for DMD patients.

A selectively suppressing amino acid transporter: Sodium-coupled neutral amino acid transporter 2 inhibits cell growth and mammalian target of rapamycin complex 1 pathway in skeletal muscle cells.

Sodium-coupled neutral amino acid transporter 2 (SNAT2), also known as solute carrier family 38 member 2 (SLC38A2), is expressed in the skeletal muscle. Our research previously indicated that SNAT2 mRNA expression level in the skeletal muscle was modulated by genotype and dietary protein. The aim of this study was to investigate the key role of the amino acid transporter SNAT2 in muscle cell growth, differentiation, and related signaling pathways via SNAT2 suppression using the inhibitor α-methylaminoisobutyric acid (MeAIB). The results showed that SNAT2 suppression down-regulated both the mRNA and protein expression levels of SNAT2 in C2C12 cells, inhibited cell viability and differentiation of the cell, and regulated the cell distribution in G0/G1 and S phases (P < 0.05). Meanwhile, most of the intercellular amino acid content of the cells after MeAIB co-culturing was significantly lower (P < 0.05). Furthermore, the mRNA expression levels of system L amino acid transporter 1 (LAT1), silent information regulator 1, and peroxisome proliferator-activated receptor-gamma co-activator 1 alpha, as well as the protein expression levels of amino acid transporters LAT1 and vacuolar protein sorting 34, were all down-regulated. The phosphorylated protein expression levels of mammalian target of rapamycin (mTOR), regulatory-associated protein of mTOR, 4E binding protein 1, and ribosomal protein S6 kinase 1 after MeAIB treatment were also significantly down-regulated (P < 0.05), which could contribute to the importance of SNAT2 in amino acid transportation and skeletal muscle cell sensing. In conclusion, SNAT2 suppression inhibited C2C12 cell growth and differentiation, as well as the availability of free amino acids. Although the mTOR complex 1 signaling pathway was found to be involved, its response to different nutrients requires further study.

The myokine meteorin-like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2.

Meteorin‐like (metrnl) is a recently identified adipomyokine that beneficially affects glucose metabolism; however, its underlying mechanism of action is not completely understood. We here show that the level of metrnl increases in vitro under electrical pulse stimulation and in vivo in exercised mice, suggesting that metrnl is secreted during muscle contractions. In addition, metrnl increases glucose uptake via the calcium‐dependent AMPKα2 pathway in skeletal muscle cells and increases the phosphorylation of HDAC5, a transcriptional repressor of GLUT4, in an AMPKα2‐dependent manner. Phosphorylated HDAC5 interacts with 14‐3‐3 proteins and sequesters them in the cytoplasm, resulting in the activation of GLUT4 transcription. An intraperitoneal injection of recombinant metrnl improved glucose tolerance in mice with high‐fat‐diet‐induced obesity or type 2 diabetes, but not in AMPK β1β2 muscle‐specific null mice. Metrnl improves glucose metabolism via AMPKα2 and is a promising therapeutic candidate for glucose‐related diseases such as type 2 diabetes.

MiR-9-5p Inhibits Skeletal Muscle Satellite Cell Proliferation and Differentiation by Targeting IGF2BP3 through the IGF2-PI3K/Akt Signaling Pathway.

MicroRNAs are evolutionarily conserved, small non-coding RNAs that play critical post-transcriptional regulatory roles in skeletal muscle development. We previously found that miR-9-5p is abundantly expressed in chicken skeletal muscle. Here, we demonstrate a new role for miR-9-5p as a myogenic microRNA that regulates skeletal muscle development. The overexpression of miR-9-5p significantly inhibited the proliferation and differentiation of skeletal muscle satellite cells (SMSCs), whereas miR-9-5p inhibition had the opposite effect. We show that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is a target gene of miR-9-5p, using dual-luciferase assays, RT-qPCR, and Western Blotting, and that it promotes proliferation and differentiation of SMSCs. In addition, we found that IGF2BP3 regulates IGF-2 expression, using overexpression and knockdown studies. We show that Akt is activated by IGF2BP3 and is essential for IGF2BP3-induced cell development. Together, our results indicate that miR-9-5p could regulate the proliferation and differentiation of myoblasts by targeting IGF2BP3 through IGF-2 and that this activity results in the activation of the PI3K/Akt signaling pathway in skeletal muscle cells.

A Mathematical Model Linking Ca2+-Dependent Signaling Pathway and Gene Expression Regulation in Human Skeletal Muscle

The physiological adaptation to aerobic endurance exercises is provided by activation of signaling pathways in skeletal muscle cells. Training-induced activation of specific signaling pathways results in significant transcriptional responses. Despite the ongoing endeavours to experimentally investigate regulatory mechanisms and signal transduction pathways involved in the contraction-induced adaptation, quantitative contribution of certain signal molecules in expression regulation of genes responsible for intracellular response has not been studied comprehensively yet. The paper presents novel developed model linking Ca2+-dependent signaling pathway and downstream transcription regulation of early and late response genes in human skeletal muscle during exercise. Numerical analysis of the model enabled to reveal crucial steps in this signal transduction pathway for the adaptation and demonstrated the necessity of consideration of additional transcription factors regulating transcription of late response genes in order to adequately reproduce gene expression data that were taken in human vastus lateralis muscle during and after acute cycling exercise.

Nitrate uptake and metabolism in human skeletal muscle cell cultures

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear. We hypothesized that exogenous nitrate can be taken up and metabolized to nitrite/NO inside the skeletal muscle. We found rapid uptake of exogeneous nitrate in both myoblasts and myotubes, increasing nitrite levels in myotubes, but not myoblasts. During differentiation we found increased expression of molybdenum containing proteins, such as xanthine oxidoreductase (XOR) and the mitochondrial amidoxime-reducing component (MARC); nitrate and nitrite reductases. Sialin, a known nitrate transporter, was detected in myoblasts; nitrate uptake decreased after sialin knockdown. Inhibition of chloride channel 1 (CLC1) also led to significantly decreased uptake of nitrate. Addition of exogenous nitrite, which resulted in higher intracellular nitrite levels, increased intracellular cGMP levels in myotubes. In summary, our results demonstrate for the first time the presence of the nitrate/nitrite/NO pathway in skeletal muscle cells, namely the existence of strong uptake of exogenous nitrate into cells and conversion of intracellular nitrate to nitrite and NO. Our results further support our previously formulated hypothesis about the importance of the nitrate to nitrite to NO intrinsic reduction pathways in skeletal muscles, which likely contributes to improved exercise tolerance after nitrate ingestion.

Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2.

Trace elements such as copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) function as enzyme cofactors and second messengers in cell signaling. Trace elements are emerging as key regulators of differentiation and development of mammalian tissues including blood, brain, and skeletal muscle. We previously reported an influx of Cu and dynamic expression of metal transporters during differentiation of skeletal muscle cells. Here, we demonstrate that during differentiation of skeletal myoblasts an increase of Mn, Fe and Zn also occurs. Interestingly the Mn increase is concomitant with increased Mn-dependent SOD2 levels. To better understand the Mn import pathway in skeletal muscle cells, we probed the functional relevance of the closely related proteins ZIP8 and ZIP14, which are implicated in Zn, Mn, and Fe transport. Partial depletion of ZIP8 severely impaired growth of myoblasts and led to cell death under differentiation conditions, indicating that ZIP8-mediated metal transport is essential in skeletal muscle cells. Moreover, knockdown of Zip8 impaired activity of the Mn-dependent SOD2. Growth defects were partially rescued only by Mn supplementation to the medium, suggesting additional functions for ZIP8 in the skeletal muscle lineage. Restoring wild type Zip8 into the knockdown cells rescued the proliferation and differentiation phenotypes. On the other hand, knockdown of Zip14, had only a mild effect on myotube size, consistent with a role for ZIP14 in muscle hypertrophy. Simultaneous knockdown of both Zip8 and Zip14 further impaired differentiation and led cell death. This is the first report on the functional relevance of two members of the ZIP family of metal transporters in the skeletal muscle lineage, and further supports the paradigm that trace metal transporters are important modulators of mammalian tissue development.