Skeletal Muscle Cell Cultures Research Articles

Activation of the hypoxia-inducible factor pathway by roxadustat improves glucose metabolism in human primary myotubes from men

Aims/hypothesisHypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes.MethodsPrimary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser.ResultsExposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg−1 min−1) and type 2 diabetes (25.3 ± 4.4 pmol mg−1 min−1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g−1 h−1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g−1 h−1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345).Conclusions/interpretationRoxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.Graphical

Small extracellular vesicle microRNAs in pediatric myasthenia gravis plasma and skeletal muscle.

The diagnosis of myasthenia gravis (MG) in children remains difficult. Circulating small extracellular vesicle (sEV)-derived miRNAs (sEV-miRNAs) have been recognized as biomarkers of various diseases and can be excreted by different cell types. These biomarker candidates also play a vital role in autoimmune diseases via intercellular communication. In the present study, we used sEV isolation and purification methods to extract the plasma-derived sEV-miRNAs from children with MG and healthy controls. A small RNA sequencing analysis confirmed the miRNA expression features in plasma-derived sEVs from MG patients. The miRNA expression analysis in vitro was determined using microarray analysis. The enrichment and network analyses of altered sEV-miRNAs were performed using miRNA databases and Database for Annotation, Visualization, and Integrated Discovery website. Quantitative real-time polymerase chain reaction was performed for validation of sEV-miRNA. The diagnostic power of altered sEV-miRNAs was evaluated using receiver operating characteristic curve analyses. Twenty-four sEV-miRNAs with altered expression level were identified between groups by DESeq2 method. The miRNAs were extracted from the sEVs, which were isolated from human primary skeletal muscle cell culture treated with mAb198. The target genes and enriched pathways of sEV-miRNAs partially overlapped between cell supernatant and plasma samples. The significantly downregulated miR-143-3p was validated in quantitative real-time polymerase chain reaction analysis. For the first time, we report that plasma-derived sEV-miRNAs may act as novel circulating biomarkers and therapeutic targets in pediatric MG.

Effect of Short-Chain Fatty Acids on Inflammatory and Metabolic Function in an Obese Skeletal Muscle Cell Culture Model.

The fermentation of non-digestible carbohydrates produces short-chain fatty acids (SCFAs), which have been shown to impact both skeletal muscle metabolic and inflammatory function; however, their effects within the obese skeletal muscle microenvironment are unknown. In this study, we developed a skeletal muscle in vitro model to mimic the critical features of the obese skeletal muscle microenvironment using L6 myotubes co-treated with 10 ng/mL lipopolysaccharide (LPS) and 500 µM palmitic acid (PA) for 24 h ± individual SCFAs, namely acetate, propionate and butyrate at 0.5 mM and 2.5 mM. At the lower SCFA concentration (0.5 mM), all three SCFA reduced the secreted protein level of RANTES, and only butyrate reduced IL-6 protein secretion and the intracellular protein levels of activated (i.e., ratio of phosphorylated-total) NFκB p65 and STAT3 (p < 0.05). Conversely, at the higher SCFA concentration (2.5 mM), individual SCFAs exerted different effects on inflammatory mediator secretion. Specifically, butyrate reduced IL-6, MCP-1 and RANTES secretion, propionate reduced IL-6 and RANTES, and acetate only reduced RANTES secretion (p < 0.05). All three SCFAs reduced intracellular protein levels of activated NFκB p65 and STAT3 (p < 0.05). Importantly, only the 2.5 mM SCFA concentration resulted in all three SCFAs increasing insulin-stimulated glucose uptake compared to control L6 myotube cultures (p < 0.05). Therefore, SCFAs exert differential effects on inflammatory mediator secretion in a cell culture model, recapitulating the obese skeletal muscle microenvironment; however, all three SCFAs exerted a beneficial metabolic effect only at a higher concentration via increasing insulin-stimulated glucose uptake, collectively exerting differing degrees of a beneficial effect on obesity-associated skeletal muscle dysfunction.

Extracellular vesicles from obese and diabetic mouse plasma alter C2C12 myotube glucose uptake and gene expression.

Recent studies have indicated a role for circulating extracellular vesicles (EVs) in the pathogenesis of multiple diseases. However, most invitro studies have used variable and arbitrary doses of EVs rather than interpreting EVs as an existing component of standard skeletal muscle cell culture media. The current study provides an initial investigation into the effects of circulating EVs on the metabolic phenotype of C2C12 myotubes by replacing EVs from fetal bovine serum with circulating EVs from control mice or mice with obesity and type 2 diabetes (OT2D). We report that EVs associated with OT2D decrease 2-NBDG uptake (a proxy measure of glucose uptake) in the insulin-stimulated state compared to controls. OT2D associated EV treatment also significantly decreased myosin heavy chain type 1 (MHCI) mRNA abundance in myotubes but had no effect on mRNA expression of any other myosin heavy chain isoforms. OT2D-associated circulating EVs also significantly increased lipid accumulation within myotubes without altering the expression of a selection of genes important for lipid entry, synthesis, or catabolism. The data indicate that, in a severely diabetic state, circulating EVs may contribute to insulin resistance and alter gene expression in myotubes in a manner consistent with the skeletal muscle phenotype observed in OT2D.

1643-P: Unique Signature of Circular RNA in Skeletal Muscle of Different Insulin Sensitivity

Diabetes pathologies are complex, affecting numerous organs such as liver, pancreas, and skeletal muscle. Circular RNAs (circRNA) are a class of non-coding RNAs that are characterized by a covalently closed loop structure. Functionally, they can act on cell physiology by sponging microRNAs and thus regulating gene and protein expression. The emerging function of these circRNA is not fully understood, but initial studies have recently shown that circRNA are involved in insulin secretion regulation. Therefore, deregulation of this class of RNAs may lead to metabolic disorders in pancreatic β-cells and thus be involved in the pathogenesis of type 1 (T1DM) and type 2 diabetes (T2DM). Our research is focused on the unique signature by circular RNA and the impact on skeletal muscle metabolism. We demonstrated that human skeletal muscle cells in culture maintain their gene expression phenotype. A different gene signature of the cells according to their different muscular fiber type sources (oxidative versus glycolytic versus mixt) was confirmed. Moreover, an independent microRNA composition has also been highlighted. As CircRNA can regulate microRNAs and then gene expression and proteins, we wondered whether these observed signatures could be explained by circRNA. We therefore focused on key circRNAs involved in myogenesis (such as circZNF609, circHIPK3) and we were able to see that there is a difference in expression of circRNA according to the muscle type. Moreover, we analyzed correlations between circRNA levels and insulin sensitivity (measured by PKB phosphorylation). Finally, we are currently completing our analysis with in-depth sequencing techniques to identify all expressed circRNA in muscle fiber types to potentially identify new targets in insulin metabolic pathways. Disclosure M.Yepmo: None. E.Meugnier: None. M.Pinget: Consultant; Novo Nordisk, Stock/Shareholder; ILONOV. K.Bouzakri: Consultant; Adocia, Stock/Shareholder; ILONOV.

Effects of Simulated Microgravity on the Internalization of Cerium Oxide Nanoparticles by Proliferating Human Skeletal Myoblasts

Spaceflight typically exerts detrimental effects on living organisms by promoting/accelerating some degenerative processes associated with aging and pathology onset on Earth, like muscle degeneration. As possible countermeasures to spaceflight effects, a few recent studies successfully tested the administration of nanomaterials for tuning of cellular activities and for promotion of certain cell phenotypes in microgravity, but largely missed investigation of early interactions of these nanomaterials with their cellular targets under altered gravity conditions. This study aims at filling this gap by elucidation of early interactions of a selected typology of redox-active nanoparticles (cerium oxide nanoparticles, also termed nanoceria, NC) with proliferating human skeletal myoblasts (HSkM) by concomitant exposure to simulated microgravity (achieved by a random positioning machine, RPM). To this purpose, NC were synthesized by a direct precipitation method and were chemically, structurally, and functionally characterized by several independent techniques prior to administration to skeletal muscle cell cultures and loading on the RPM. Confocal and electron microscopy evidence of nanoparticle internalization by several mechanisms (mostly involving macropinocytosis) in HSkM is provided, along with evidence of transcriptional regulation of key antioxidant response markers (Nos1, Sod2, and Sod3), promising oxidative stress alleviation and muscle protection under mechanical unloading conditions.

Ectopic PLAG1 induces muscular dystrophy in the mouse

Even though various genetic mutations have been identified in muscular dystrophies (MD), there is still a need to understand the biology of MD in the absence of known mutations. Here we reported a new mouse model of MD driven by ectopic expression of PLAG1. This gene encodes a developmentally regulated transcription factor known to be expressed in developing skeletal muscle, and implicated as an oncogene in certain cancers including rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma composed of myoblast-like cells. By breeding loxP-STOP-loxP-PLAG1 (LSL-PLAG1) mice into the MCK-Cre line, we achieved ectopic PLAG1 expression in cardiac and skeletal muscle. The Cre/PLAG1 mice died before 6 weeks of age with evidence of cardiomyopathy significantly limiting left ventricle fractional shortening. Histology of skeletal muscle revealed dystrophic features, including myofiber necrosis, fiber size variation, frequent centralized nuclei, fatty infiltration, and fibrosis, all of which mimic human MD pathology. QRT-PCR and Western blot revealed modestly decreased Dmd mRNA and dystrophin protein in the dystrophic muscle, and immunofluorescence staining showed decreased dystrophin along the cell membrane. Repression of Dmd by ectopic PLAG1 was confirmed in dystrophic skeletal muscle and various cell culture models. In vitro studies showed that excess IGF2 expression, a transcriptional target of PLAG1, phenocopied PLAG1-mediated down-regulation of dystrophin. In summary, we developed a new mouse model of a lethal MD due to ectopic expression of PLAG1 in heart and skeletal muscle. Our data support the potential contribution of excess IGF2 in this model. Further studying these mice may provide new insights into the pathogenesis of MD and perhaps lead to new treatment strategies.

Mimicking exercise in vitro - effects of myotube contractions and mechanical stretch on omics.

The number of studies using skeletal muscle (SkM) cell culture models to study exercise in vitro are rapidly expanding. Progressively, more comprehensive analysis methods, such as different omics approaches including transcriptomics, proteomics and metabolomics have been used to examine the intra- and extracellular molecular responses to exercise mimicking stimuli in cultured myotubes. Among other techniques, exercise-like electrical pulse stimulation (EL-EPS) and mechanical stretch of SkM cells are the two most commonly used methods to mimic exercise in vitro. In this mini-review we focus on these two approaches and their effects on the omics of myotubes and/or cell culture media. Furthermore, besides traditional two-dimensional (2D) methods, the use of three-dimensional (3D) SkM approaches are increasing in the field of in vitro exercise mimicry. Our aim with this mini-review is to provide the reader with an up-to-date overview of the 2D and 3D models and the use of omics approaches to study the molecular response to exercise in vitro.

Improving quantification of myotube width and nuclear/cytoplasmic ratio in myogenesis research

Background and Objective: The culture of skeletal muscle cells is particularly relevant to basic biomedical research and translational medicine. The incubation of dissociated cells under controlled conditions has helped to dissect several molecular mechanisms associated with muscle cell differentiation, in addition to contributing for the evaluation of drug effects and prospective cell therapies for patients with degenerative muscle pathologies. The formation of mature multinucleated myotubes is a stepwise process involving well defined events of cell proliferation, commitment, migration, and fusion easily identified through optical microscopy methods including immunofluorescence and live cell imaging. The characterization of each step is usually based on muscle cell morphology and nuclei number, as well as the presence and intracellular location of specific cell markers. However, manual quantification of these parameters in large datasets of images is work-intensive and prone to researcher's subjectivity, mostly because of the extremely elongated cell shape of large myotubes and because myotubes are multinucleated. Methods: Here we provide two semi-automated ImageJ macros aimed to measure the width of myotubes and the nuclear/cytoplasmic localization of molecules in fluorescence images. The width measuring macro automatically determines the best angle, perpendicular to most cells, to draw a profile plot and identify and measure individual myotubes. The nuclear/cytoplasmic ratio macro compares the intensity values along lines, drawn by the user, over cytoplasm and nucleus. Results: We show that the macro measurements are more consistent than manual measurements by comparing with our own results and with the literature. Conclusions: By relying on semi-automated muscle specific ImageJ macros, we seek to improve measurement accuracy and to alleviate the laborious routine of counting and measuring muscle cell features.

Morphological and Molecular Responses of Lateolabrax maculatus Skeletal Muscle Cells to Different Temperatures.

Temperature strongly modulates muscle development and growth in ectothermic teleosts; however, the underlying mechanisms remain largely unknown. In this study, primary cultures of skeletal muscle cells of Lateolabrax maculatus were conducted and reared at different temperatures (21, 25, and 28 °C) in both the proliferation and differentiation stages. CCK-8, EdU, wound scratch and nuclear fusion index assays revealed that the proliferation, myogenic differentiation, and migration processes of skeletal muscle cells were significantly accelerated as the temperature raises. Based on the GO, GSEA, and WGCNA, higher temperature (28 °C) induced genes involved in HSF1 activation, DNA replication, and ECM organization processes at the proliferation stage, as well as HSF1 activation, calcium activity regulation, myogenic differentiation, and myoblast fusion, and sarcomere assembly processes at the differentiation stage. In contrast, lower temperature (21 °C) increased the expression levels of genes associated with DNA damage, DNA repair and apoptosis processes at the proliferation stage, and cytokine signaling and neutrophil degranulation processes at the differentiation stage. Additionally, we screened several hub genes regulating myogenesis processes. Our results could facilitate the understanding of the regulatory mechanism of temperature on fish skeletal muscle growth and further contribute to utilizing rational management strategies and promoting organism growth and development.

Effects of Maca on Muscle Hypertrophy in C2C12 Skeletal Muscle Cells.

With aging, sarcopenia and the associated locomotor disorders, have become serious problems. The roots of maca contain active ingredients (triterpenes) that have a preventive effect on sarcopenia. However, the effect of maca on muscle hypertrophy has not yet been investigated. The aim of this study was to examine the effects and mechanism of maca on muscle hypertrophy by adding different concentrations of yellow maca (0.1 mg/mL and 0.2 mg/mL) to C2C12 skeletal muscle cell culture. Two days after differentiation, maca was added for two days of incubation. The muscle diameter, area, differentiation index, and multinucleation, were assessed by immunostaining, and the expression levels of the proteins related to muscle protein synthesis/degradation were examined by Western blotting. Compared with the control group, the muscle diameter and area of the myotubes in the maca groups were significantly increased, and the cell differentiation index and multinucleation were significantly higher in the maca groups. Phosphorylation of Akt and mTOR was elevated in the maca groups. Maca also promoted the phosphorylation of AMPK. These results suggest that maca may promote muscle hypertrophy, differentiation, and maturation, potentially via the muscle hypertrophic signaling pathways such as Akt and mTOR, while exploring other pathways are needed.

Antioxidant effects of bis-indole alkaloid indigo and related signaling pathways in the experimental model of Duchenne muscular dystrophy.

Indigo is a bis-indolic alkaloid that has antioxidant and anti-inflammatory effects reported in literature and is a promissory compound for treating chronic inflammatory diseases. This fact prompted to investigate the effects of this alkaloid in the experimental model of Duchenne muscular dystrophy. The main aim of this study was to evaluate the potential role of the indigo on oxidative stress and related signaling pathways in primary skeletal muscle cell cultures and in the diaphragm muscle from mdx mice. The MTT and Neutral Red assays showed no indigo dose-dependent toxicities in mdx muscle cells at concentrations analyzed (3.12, 6.25, 12.50, and 25.00 μg/mL). Antioxidant effect of indigo, in mdx muscle cells and diaphragm muscle, was demonstrated by reduction in 4-HNE content, H2O2 levels, DHE reaction, and lipofuscin granules. A significant decrease in the inflammatory process was identified by a reduction on TNF and NF-κB levels, on inflammatory area, and on macrophage infiltration in the dystrophic sample, after indigo treatment. Upregulation of PGC-1α and SIRT1 in dystrophic muscle cells treated with indigo was also observed. These results suggest the potential of indigo as a therapeutic agent for muscular dystrophy, through their action anti-inflammatory, antioxidant, and modulator of SIRT1/PGC-1α pathway.

The effect of metformin on autophagy by LC3 expression in Type 2 Diabetes Mellitus (T2DM) human skeletal muscle cell culture

Background: Skeletal muscle is an essential tissue in glucose metabolism. Reduced autophagic capacity to remove damaged contractile proteins in skeletal muscle cells will contribute to the loss of muscle mass. Metformin is the first-line agent for treating type 2 diabetes mellitus (T2DM) patients. This study aims to investigate the effects of metformin on autophagy through LC3 expression in human skeletal muscle cell culture (SkMC). Methods: The T2DM human SkMC was obtained from T2DM treatment naïve patients, purchased from AcceGen Biotech®. Fully differentiated myotubes were randomized into the control and treatment groups. The treatment group was given Metformin in three doses (1 mM, 2 mM, and 3 mM). An immunoblotting assay of AMPKα and LC3 was performed using electrochemiluminescence (ECL). The quantitative expression of AMPKα and LC3 were measured at baseline, after 24-hour, 48-hour, and 72-hour. Data were analyzed using SPSS version 22 for Windows.Results: AMPKα and LC3 expression were higher in the treatment group compared to the control group. The levels of AMPKα and LC3 expression in the treatment group increased dose-dependent. Linear regression analysis demonstrated a significant correlation between metformin administration and LC3 expression levels (p&lt;0.0001). Conclusion: Metformin administration on T2DM human SkMC resulted in increased autophagic activity, marked by increased LC3 expression.

Development of a primary skeletal muscle cell culture model from human muscle biopsies

Purpose: This work aimed to develop and validate a culture model of primary human skeletal muscle cells. Methods: Muscle biopsies were obtained post-mortem from the vastus lateralis. The biopsies were enzymatically digested with collagenase 1A (1mg/mL) and dispase II (4,8mg/mL) for 60 minutes at 37°C in order to isolate fibroblasts and satellite cells. Then, the satellite cells (CD56+) were separated from fibroblasts (CD56-) by two methods: pre-plating or immunomagnetic cell sorting using an anti-CD56 antibody, a transmembrane glycoprotein expressed on the surface of satellite cells. Indeed, some human muscle biopsies contain a high proportion of fibroblasts that can contaminate the myoblasts cell culture if the separation method is not efficient. The performance of these cell separation methods was evaluated by FACS. We also compared the effects of two culture media on myoblasts proliferation (10%FBS vs. 10%FBS + 2%Ultroser G (UG)) using cell doubling time as parameter. Two differentiation media (2%FBS vs. 0.5%UG) were tested on the myogenic index, which corresponds to the proportion of cells that have fused to form myotubes. This index was determined through the detection of the myosin heavy chain (MYH) by immunofluorescence. Finally, we studied the expression of several muscle cell markers during myogenesis by qRT-PCR and immunofluorescence. Results: The cells obtained from the enzymatic digestion of the biopsy contained a varying proportion of CD56+ satellite cells (86,15 ± 12,09%; 95%CI:73,45 to 98,84). The cell population obtained after immunomagnetic cell sorting method contained more than 92% of CD56+ cells (96,11 ± 3,27%; 95%CI:92,68 to 99,54). In comparison, the ratio of CD56+ obtained by the preplating method was lower (84,19 ± 9,94%; 95%CI:73,76 to 94,62), but no significant difference was observed between both methods. The cell doubling time was significantly shorter with culture media containing 10%FBS and 2%UG than with the media containing only 10%FBS (25.70 ± 3,96h vs. 70.64 ± 21,68h, p = 0.0313). The myogenic index was slightly higher with the 0.5%UG than 2%FBS differentiating medium (62,75% ± 8,57% vs. 56,39 ± 16,45%). Finally, the increase of MyoD1, myogenin and MYH, three markers of differentiation, indicated the phenotypic transition of myoblasts to a myogenic phenotype. Conclusions: Immunomagnetic cell sorting allows better satellite cells and fibroblasts separation than the conventionally used preplating technique. Ultoser G serum substitute accelerates cell proliferation and improves the differentiation of myoblasts into myotubes. The markers MyoD1, myogenin and MYH are good markers of the myogenic differentiation of myoblasts. This in vitro model makes it possible to purify efficiently muscle satellite cells from human muscle biopsies. These cells can be used in in vitro studies to study the pathophysiological mechanisms of muscle disease and the mechanisms of action of treatments.

Light Cross-Linkable Marine Collagen for Coaxial Printing of a 3D Model of Neuromuscular Junction Formation.

Collagen is a major component of the extracellular matrix (ECM) that modulates cell adhesion, growth, and migration, and has been utilised in tissue engineering applications. However, the common terrestrial sources of collagen carry the risk of zoonotic disease transmission and there are religious barriers to the use of bovine and porcine products in many cultures. Marine based collagens offer an attractive alternative and have so far been under-utilized for use as biomaterials for tissue engineering. Marine collagen can be extracted from fish waste products, therefore industry by-products offer an economical and environmentally sustainable source of collagen. In a handful of studies, marine collagen has successfully been methacrylated to form collagen methacrylate (ColMA). Our work included the extraction, characterization and methacrylation of Red Snapper collagen, optimisation of conditions for neural cell seeding and encapsulation using the unmodified collagen, thermally cross-linked, and the methacrylated collagen with UV-induced cross-linking. Finally, the 3D co-axial printing of neural and skeletal muscle cell cultures as a model for neuromuscular junction (NMJ) formation was investigated. Overall, the results of this study show great potential for a novel NMJ in vitro 3D bioprinted model that, with further development, could provide a low-cost, customizable, scalable and quick-to-print platform for drug screening and to study neuromuscular junction physiology and pathogenesis.

Identification of mRNA Degradome Variation Dependent on Divergent Muscle Mass in Different Pig Breeds

Abstract The search is still on for the molecular processes associated with the development and metabolism of skeletal muscles. Selection conducted in farm animals is focused on high muscle mass because it delivers higher economic profit. The present study aimed to shed light on mRNA degradome signals that could be characteristic for molecular processes associated with an abundance of muscle mass and to identify miRNA regulatory networks controlling these processes in pigs applying next-generation-sequencing (NGS). In the study, over 10,000 degraded transcripts were identified per sample, with the highest abundance for genes encoding mitochondrial proteins (COXs, NDs, CYTB, ATP6 and ATP8). Moreover, only 26% of the miRNA targets were found within this degraded transcript pool, which suggested for miRNAs other molecular mechanism at different level of gene expression than mRNA degradation. On the other hand, a small share of the identified degraded transcripts associated with miRNA regulation suggests a different mechanism of mRNA degradation for identified degraded transcropts. Subsequently, most of the miRNA gene degraded targets, such as ENO3, CKM, CRYAB and ADAM19 encode proteins involved in the muscle mass control. The present study showed an interesting dependence between miRNAs and their targets. Nevertheless, the complete view of the miRNA regulatory network could be a subject of further advanced research, which would employ a miRNA transfection procedure in skeletal muscle cell cultures.

Transcriptomic Profile of Primary Culture of Skeletal Muscle Cells Isolated from Semitendinosus Muscle of Beef and Dairy Bulls.

The aim of the study was to identify differences in the transcriptomic profiles of primary muscle cell cultures derived from the semitendinosus muscle of bulls of beef breeds (Limousin (LIM) and Hereford (HER)) and a dairy breed (Holstein-Friesian (HF)) (n = 4 for each breed). Finding a common expression pattern for proliferating cells may point to such an early orientation of the cattle beef phenotype at the transcriptome level of unfused myogenic cells. To check this hypothesis, microarray analyses were performed. The analysis revealed 825 upregulated and 1300 downregulated transcripts similar in both beef breeds (LIM and HER) and significantly different when compared with the dairy breed (HF) used as a reference. Ontological analyses showed that the largest group of genes were involved in muscle organ development. Muscle cells of beef breeds showed higher expression of genes involved in myogenesis (including erbb-3, myf5, myog, des, igf-1, tgfb2) and those encoding proteins comprising the contractile apparatus (acta1, actc1, myh3, myh11, myl1, myl2, myl4, tpm1, tnnt2, tnnc1). The obtained results confirmed our hypothesis that the expression profile of several groups of genes is common in beef breeds at the level of proliferating satellite cells but differs from that observed in typical dairy breeds.

β-Hydroxybutyrate is reduced in humans with obesity-related NAFLD and displays a dose-dependent effect on skeletal muscle mitochondrial respiration in vitro.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation and impaired insulin sensitivity. Reduced hepatic ketogenesis may promote these pathologies, but data are inconclusive in humans and the link between NAFLD and reduced insulin sensitivity remains obscure. We investigated individuals with obesity-related NAFLD and hypothesized that β-hydroxybutyrate (βOHB; the predominant ketone species) would be reduced and related to hepatic fat accumulation and insulin sensitivity. Furthermore, we hypothesized that ketones would impact skeletal muscle mitochondrial respiration in vitro. Hepatic fat was assessed by 1H-MRS in 22 participants in a parallel design, case control study [Control: n = 7, age 50 ± 6 yr, body mass index (BMI) 30 ± 1 kg/m2; NAFLD: n = 15, age 57 ± 3 yr, BMI 35 ± 1 kg/m2]. Plasma assessments were conducted in the fasted state. Whole body insulin sensitivity was determined by the gold-standard hyperinsulinemic-euglycemic clamp. The effect of ketone dose (0.5-5.0 mM) on mitochondrial respiration was conducted in human skeletal muscle cell culture. Fasting βOHB, a surrogate measure of hepatic ketogenesis, was reduced in NAFLD (-15.6%, P < 0.01) and correlated negatively with liver fat (r2 = 0.21, P = 0.03) and positively with insulin sensitivity (r2 = 0.30, P = 0.01). Skeletal muscle mitochondrial oxygen consumption increased with low-dose ketones, attributable to increases in basal respiration (135%, P < 0.05) and ATP-linked oxygen consumption (136%, P < 0.05). NAFLD pathophysiology includes impaired hepatic ketogenesis, which is associated with hepatic fat accumulation and impaired insulin sensitivity. This reduced capacity to produce ketones may be a potential link between NAFLD and NAFLD-associated reductions in whole body insulin sensitivity, whereby ketone concentrations impact skeletal muscle mitochondrial respiration.

Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat

Cell-based meat (CBM) production is a promising technology that could generate meat without the need of animal agriculture. The generation of tissue requires a three-dimensional (3D) scaffold to provide support to the cells and mimic the extracellular matrix (ECM). For CBM, the scaffold needs to be edible and have suitable nutritional value and texture. Here, we demonstrate the use of textured soy protein—an edible porous protein-based biomaterial—as a novel CBM scaffold that can support cell attachment and proliferation to create a 3D engineered bovine muscle tissue. The media composition was optimized for 3D bovine satellite cell (BSC) proliferation and differentiation by adding myogenic-related growth factors. Myogenesis of several cell combinations was compared, and elevated myogenesis and ECM deposition were shown in co-culture of BSCs with bovine smooth muscle cells and tri-cultures of BSCs, bovine smooth muscle cells and bovine endothelial cells. The expression of proteins associated with ECM gene sets was increased in the co-culture compared with BSC monoculture. Volunteers tasted the product after cooking and noted its meaty flavour and sensorial attributes, achieving the goal of replicating the sensation and texture of a meat bite. This approach represents a step forward for the applied production of CBM as a food product. Animal skeletal muscle cell culture has the potential to provide new protein sources without the need for conventional animal agriculture. Ben-Ayre et. al. address one of the challenges of growing cell-based meat in vitro by using textured soy protein as an extracellular matrix scaffold. This approach allowed three-dimensional skeletal muscle cell development to produce a meat-like product for human consumption.

Screening of by-products from the food industry as growth promoting agents in serum-free media for skeletal muscle cell culture.

The most significant cost driver for efficient bio-production of edible animal proteins is the cell culture media, where growth factors account for up to 96% of the total cost. The culture media must be serum-free, affordable, contain only food-grade ingredients, be efficient to promote cell growth and available in massive quantities. The commercially available serum substitutes are expensive and not necessarily food-grade. Identifying inexpensive food-safe alternatives to serum is crucial. By-products from food production are available in massive quantities, contain potential factors that can promote growth and are promising ingredients for serum replacement. The main goal of this study was to explore if food-grade by-product materials can be used as growth promoting agents in skeletal muscle cell culture to develop a tailor-made serum free media. Different by-products, including chicken carcass, cod backbone, eggshell membrane, egg white powder and pork plasma were enzymatically or chemically hydrolyzed. The hydrolysates in addition to lyophilized pork plasma and yeast extract were further characterized by size-exclusion chromatography, elemental combustion analysis and degree of hydrolysis. The materials were used as supplement to or replacement of commercial serum and further evaluated for their effect on metabolic activity, cell proliferation and cell cytotoxicity in muscle cells cultured in vitro. Our results indicate that none of the materials were cytotoxic to the skeletal muscle cells. Hydrolysates rich in peptides with approximately 2-15 amino acids in length were shown to improve cell growth and metabolic activity. Of all the materials tested pork plasma hydrolysates and yeast extract were the most promising. Pork plasma hydrolysates increased metabolic activity by 110% and cell proliferation with 48% when cultured in serum-free conditions for 3 days compared with control cells cultured with full serum conditions. Most interestingly, this response was dependent on both material and choice of enzyme used. We suggest that these materials have the potential to replace serum during cultivation and as such be included in a tailor-made serum-free media.