Human Skeletal Muscle Satellite Cells Research Articles

Infectivity of three Mayaro Virus geographic isolates in human cell lines

Mayaro virus (MAYV) is an emergent arthropod-borne virus that causes an acute febrile illness accompanied by arthralgia, similar to chikungunya virus. Increasing urbanization of MAYV outbreaks in the Americas has led to concerns for geographic expansion and spillover. Given the potential importance of this pathogen, we sought to fill critical gaps in knowledge regarding MAYV infectivity and geographic variation. This study describes the cytopathogenicity of MAYV in human dermal fibroblasts, human skeletal muscle satellite cells, human embryonic kidney cells (HEK), peripherally derived human macrophages, and Vero cells. We found that regional differences between these viruses do not affect replication kinetics, with high titers peaking at 37 h post infection. MAYV-U, did however, cause the most cytopathic effect in a time-dependent manner. Compared to the other two prototypic isolates, MAYV-U harbors unique mutations in the E2 protein, D60G and S205F, that are likely to interact with the host cell receptor and could affect infectivity. We further demonstrate that pre-treatment of cells with interferon-β inhibited viral replication in a dose-dependent manner. Together, these findings advance our understanding of MAYV infection of human target cells and provide initial data regarding variation according to geography.

Identification of Potential circRNA-microRNA-mRNA Regulatory Network in Skeletal Muscle.

Circular RNAs (circRNAs) are a newly discovered family of regulatory RNAs generated through backsplicing. Genome-wide profiling of circRNAs found that circRNAs are ubiquitously expressed and regulate gene expression by acting as a sponge for RNA-binding proteins (RBPs) and microRNAs (miRNAs). To identify circRNAs expressed in mouse skeletal muscle, we performed high-throughput RNA-sequencing of circRNA-enriched gastrocnemius muscle RNA samples, which identified more than 1,200 circRNAs. In addition, we have identified more than 14,000 and 15,000 circRNAs in aging human skeletal muscle tissue and satellite cells, respectively. A subset of abundant circRNAs was analyzed by RT-PCR, Sanger sequencing, and RNase R digestion assays to validate their expression in mouse skeletal muscle tissues. Analysis of the circRNA-miRNA-mRNA regulatory network revealed that conserved circNfix might associate with miR-204-5p, a suppressor of myocyte enhancer factor 2c (Mef2c) expression. To support the hypothesis that circNfix might regulate myogenesis by controlling Mef2c expression, silencing circNfix moderately reduced Mef2c mRNA expression and inhibited C2C12 differentiation. We propose that circNfix promotes MEF2C expression during muscle cell differentiation in part by acting as a sponge for miR-204-5p.

Cultured human skeletal muscle satellite cells exhibit characteristics of mesenchymal stem cells and play anti-inflammatory roles through prostaglandin E2 and hepatocyte growth factors.

Skeletal muscle satellite cells (SkMSCs) play crucial roles in muscle fiber maintenance, repair, and remodeling; however, it remains unknown if these properties are preserved in cultured SkMSCs. In this study, we investigated the characteristics of cultured SkMSCs and their ability to regulate the activity of M1 macrophages. SkMSCs grew well with an average population doubling time of 26.26 ± 6.85 h during 10 passages (P). At P5, Pax7, MyoD, cluster of differentiation (CD)34, and CD56 were not expressed in SkMSCs, but the MSC markers CD73, CD105, and CD90 were expressed and the cells were differentiated into adipocytes and osteoblasts. When SkMSCs were cocultured with macrophages, interleukin (IL)-1β secretion was decreased, prostaglandin (PG)E2 was produced in coculture, and cyclooxygenase-2 protein was induced in an SkMSC-dependent manner. Hepatocyte growth factor (HGF) was highly secreted by monocultured SkMSCs; interferon-γ and lipopolysaccharide reduced its expression level. However, HGF expression recovered when SkMSCs and macrophages were cocultured. Although exogenous PGE2 upregulated macrophage pro-IL-1β expression, it suppressed the secretion of cleaved IL-1β. In contrast, HGF decreased active IL-1β secretion without affecting pro-IL-1β expression. Co-treatment of macrophages with HGF and PGE2 reduced pro-IL-1β expression level and active IL-1β secretion. Our results suggest that SkMSCs lose their satellite cell properties during serial passaging but acquire mesenchymal stem cell properties including the ability to exert an anti-inflammatory response for macrophages through PGE2 and HGF.

Factors secreted from high glucose treated endothelial cells impair expansion and differentiation of human skeletal muscle satellite cells.

Cellular communication occurs between endothelial cells and skeletal muscle satellite cells and is mitogenic for both cell types under normal conditions. Skeletal muscle atrophy and endothelial cell dysfunction occur in tandem in cardiovascular disease, type II diabetes and ageing. The present study investigated how induction of endothelial cell dysfunction via high glucose treatment impacts growth and differentiation of human skeletal muscle satellite cells in vitro. Secreted factors from high glucose treated endothelial cells impaired satellite cell expansion and differentiation via decreased proliferation and dysregulation of p38 mitogen-activated protein kinase in satellite cells committed to myogenesis. These findings highlight a novel potential role for endothelial cells in the development and pathology of skeletal muscle atrophy, which is common in patients with endothelial dysfunction related pathologies. Cross-talk between endothelial cells (ECs) and skeletal muscle satellite cells (MuSC) has been identified as an important regulator of cellular functions in both cell types. In healthy conditions, EC secreted factors promote MuSC growth and differentiation. Endothelial and satellite cell dysfunction occur in tandem in many disease states; however, no data exist examining the impact of dysfunctional EC signalling on satellite cells. Therefore, the present study aimed to evaluate the effect that factors secreted from high glucose (HG) treated ECs have on the growth and differentiation of human satellite cells (HMuSC) using a conditioned medium (CM) cell culture model. Satellite cells were isolated from human skeletal muscle and grown in CM from normal or HG treated human umbilical vein ECs (HUVECs). Satellite cells grown in CM from HG treated HUVECs reduced growth (25%), differentiation (25%) and myonuclear fusion (35%). These responses were associated with increased superoxide (50%) and inflammatory cytokines (25-50%) in HG treated HUVECs and HG-CM. Decreased expansion of HG-CM treated HMuSCs was driven by a decrease in proliferation. Impaired gene expression and protein content of myogenic differentiation factors were preceded by decreased phosphorylation of p38 mitogen-activated protein kinase in HMuSC treated with CM from HG treated HUVECs. The results obtained in the present study are the first to show that factors secreted from HG treated ECs cause impairments in human muscle satellite cell growth and differentiation in vitro, highlighting endothelial cell health and secretion as a potential target for treating vascular disease-associated skeletal muscle dysfunction.

Retained differentiation capacity of human skeletal muscle satellite cells from spinal cord-injured individuals.

Despite the well‐known role of satellite cells in skeletal muscle plasticity, the effect of spinal cord injury on their function in humans remains unknown. We determined whether spinal cord injury affects the intrinsic ability of satellite cells to differentiate and produce metabolically healthy myotubes. We obtained vastus lateralis biopsies from eight spinal cord‐injured and six able‐bodied individuals. Satellite cells were isolated, grown and differentiated in vitro. Gene expression was measured by quantitative PCR. Abundance of differentiation markers and regulatory proteins was determined by Western blotting. Protein synthesis and fatty acid oxidation were measured by radioactive tracer‐based assays. Activated satellite cells (myoblasts) and differentiated myotubes derived from skeletal muscle of able‐bodied and spinal cord‐injured individuals expressed similar (P > 0.05) mRNA levels of myogenic regulatory factors. Myogenic differentiation factor 1 expression was higher in myoblasts from spinal cord‐injured individuals. Desmin and myogenin protein content was increased upon differentiation in both groups, while myotubes from spinal cord‐injured individuals contained more type I and II myosin heavy chain. Phosphorylated and total protein levels of Akt‐mechanistic target of rapamycin and forkhead box protein O signalling axes and protein synthesis rate in myotubes were similar (P > 0.05) between groups. Additionally, fatty acid oxidation of myotubes from spinal cord‐injured individuals was unchanged (P > 0.05) compared to able‐bodied controls. Our results indicate that the intrinsic differentiation capacity of satellite cells and metabolic characteristics of myotubes are preserved following spinal cord injury. This may inform potential interventions targeting satellite cell activation to alleviate skeletal muscle atrophy.

Serial Passaging Reduces Replication and Fusion Capacity of Primary Human Skeletal Muscle Satellite Cells

Replication and fusion of skeletal muscle satellite cells (SkMSCs) are essential for skeletal muscle maintenance and repair. Advancing age impairs SkMSCs replication potential and fusion capacity, however SkMSCs isolated from older human skeletal muscle do not consistently demonstrate these defects, making it difficult to study SkMSC isolated from aged human muscle. PURPOSE: To investigate if serial passaging of SkMSCs can mimic aging associated defects in replication and fusion. We hypothesized that serial passaging of primary human SkMSCs induces replicative senescence and poor fusion capacity in part through inhibition of the cell cycle regulator cyclin-dependent kinase 4 (CDK4) through the activation of CDK inhibitor p16Ink4a. METHODS: SkMSCs were isolated from vastus lateralis biopsies of young men. SkMSCs were serially passaged every five days and passaging continued until SkMSCs were unable to replicate. Population doubling level (PDL) was calculated using passage specific final and starting SkMSC counts. Expression of the myogenic regulator, myogenin (MyoG) and the regeneration regulator, paired box 7 (Pax7) were analyzed via rt-PCR. Senescence was determined by SA-B-gal staining and fusion capacity determined by immunohistochemical staining. RESULTS: Primary human SkMSCs failed to replicate at passage 16 (Pass16) and Pass16 SkMSCs exhibited decreased fusion. PDL was decreased from passage 4 to 16 (Pass4: 3.9 vs. Pass16: 0.6, AU). CDK4 mRNA decreased (Pass4: 1.0 vs. Pass16: 0.4, AU) and p16Ink4a mRNA was increased (Pass4: 1.0 vs. Pass16: 4.9, AU). Pax7 mRNA was unchanged (Pass4: 1.0 vs. Pass16: 1.3, AU), while myoG mRNA was increased (Pass4: 1.0 vs Pass16: 8.6, AU). CONCLUSIONS: Serially passaging SkMSCs isolated from young humans mimics an aged phenotype evidenced by impaired replication and fusion. Our findings suggest serial passaging of primary human SkMSC may be used to study aged SkMSCs.

Protein Availability and Satellite Cell Dynamics in Skeletal Muscle.

Human skeletal muscle satellite cells are activated in response to both resistance and endurance exercise. It was initially proposed that satellite cell proliferation and differentiation were only required to support resistance exercise-induced hypertrophy. However, satellite cells may also play a role in muscle fibre remodelling after endurance-based exercise and extracellular matrix regulation. Given the importance of dietary protein, particularly branched chain amino acids, in supporting myofibrillar and mitochondrial adaptations to both resistance and endurance-based training, a greater understanding of how protein intake impacts satellite cell activity would provide further insight into the mechanisms governing skeletal muscle remodelling with exercise. While many studies have investigated the capacity for protein ingestion to increase post-exercise rates of muscle protein synthesis, few investigations have examined the role for protein ingestion to modulate satellite cell activity. Here we review the molecular mechanisms controlling the activation of satellite cells in response to mechanical stress and protein intake in both in vitro and in vivo models. We provide a mechanistic framework that describes how protein ingestion may enhance satellite activity and promote exercise adaptations in human skeletal muscle.

2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls

BackgroundChronic obstructive pulmonary disease (COPD) is associated with several extra-pulmonary effects of which skeletal muscle wasting is one of the most common and contributes to reduced quality of life, increased morbidity and mortality. The molecular mechanisms leading to muscle wasting are not fully understood. Proteomic analysis of human skeletal muscle is a useful approach for gaining insight into the molecular basis for normal and pathophysiological conditions.MethodsTo identify proteins involved in the process of muscle wasting in COPD, we searched differentially expressed proteins in the vastus lateralis of COPD patients with low fat free mass index (FFMI), as a surrogate of muscle mass (COPDL,n = 10) (FEV1 33 ± 4.3% predicted, FFMI 15 ± 0.2 Kg.m−2), in comparison to patients with COPD and normal FFMI (COPDN,n = 8) and a group of age, smoking history, and sex matched healthy controls (C, n = 9) using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, combined with mass spectrometry (MS). The effect of silencing DOT1L protein expression on markers of cell arrest was analyzed in skeletal muscle satellite cells (HSkMSCs) in vitro and assessed by qPCR and Western blotting.ResultsA subset of 7 proteins was differentially expressed in COPDL compared to both COPDN and C. We found an increased expression of proteins associated with muscle homeostasis and protection against oxidative stress, and a decreased expression of structural muscle proteins and proteins involved in myofibrillogenesis, cell proliferation, cell cycle arrest and energy production. Among these was a decreased expression of the histone methyltransferase DOT1L. In addition, silencing of the DOT1L gene in human skeletal muscle satellite cells in vitro was significantly related to up regulation of p21 WAF1/Cip1/CDKN1A, a marker of cell arrest and ageing.Conclusions2D-DIGE coupled with MS identified differences in the expression of several proteins in the wasted vastus lateralis that are relevant to the disease process. Down regulation of DOT1L in the vastus lateralis of COPDL patients may mediate the muscle wasting process through up regulation of markers of cell arrest and senescence.

Functionalized recombinant spider silk as support for muscle engineering

Event Abstract Back to Event Functionalized recombinant spider silk as support for muscle engineering Ulrika Johansson1, Lubna Al-Khalili2, Veronique Chotteau2 and My Hedhammar1 1 School of Biotechnology, KTH Royal Institute of Technology, Division of Protein Technology, Sweden 2 School of Biotechnology, KTH Royal Institute of Technology, Division of Industrial Biotechnology, Sweden Introduction: Satellite cells are myogenic precursors that reside as a quiescent cells in every muscle fiber. Upon minor muscle injury the satellite cells are able to proliferate and give rise to a regenerated muscle and new satellite cells. Severe injury with extensive loss of skeletal muscle results in unrepaired muscle tissue and malfunction. The regeneration of myogenic tissue needs, besides precursor cells, a direction by specific extracellular matrix (ECM)[1]. The ECM directs the muscle regeneration into fibers with a unique alignment to connect with the surrounding tissue for restoration of function[2]. We have developed methods to functionalize recombinant spider silk proteins with peptides from the ECM[3],[4]. The properties of the functionalized spider silk may offer a scaffold with appropriate strength, elasticity, fiber guidance and adequate porosity. Herein we investigate if functionalized recombinant spider silk can act as support for muscle regeneration. Materials and Methods: Human skeletal muscle satellite cells (HSkMSC) were seeded and cultured on a coated layer of recombinant spider silk with functionalized ECM motifs from fibronectin (FN), vitronectin (VN) and RGD. As controls we used non-functionalized recombinant spider silk (WT), hydrophobic plates (HP) as well as tissue culture treated (TCT) plates without recombinant spider silk coating. The quick adhesion of HSkMSC on the different ECM coated wells were studied using crystal violet. The proliferation capacities were followed over 7 days using Alamar Blue. In parallel, HSkMSC were cultured until cell density reached a confluency of 90% and thereafter a myogenic differentiation protocol was initiated using DMEM with low glucose and 2% FBS. After 7 days of differentiation the cells were fixed in 4% PFA and stained for the myogenic marker Desmin. Furthermore, HSkMSC were seeded and cultured on FN-silk in fiber format. The cell growth was followed over a 2 weeks’ time period, using Alamar Blue. The cell viability at the endpoint was analyzed as well. Results and Discussion: The HSkMSC were able to adhere to all silk variants of ECM motifs. The most pronounced adherent effect was seen on the FN-silk where HSkMSC were able to proliferate to the same degree as in the TCT-control. Moreover, FN-silk increased the myogenic capacity of HSkMSC (74 %) compared to TCT-control (50%, Fig. 1A). The HSkMSC formed myotubes on all silk variants with ECM motifs; on FN-silk multi nucleated myotubes were formed (Fig. 1B). The HSkMSC aligned, survived and proliferated also in the flexible and permeable 3D fiber format of FN-silk (Fig.1C). Conclusion: We propose that the use of spider silk matrices with ECM motifs could be useful for development of a therapeutic option for muscle engineering due to its ability to adhere and align satellite cells along an elastic, permeable scaffold which support differentiation. Spiber Technologies AB is acknowledged for providing recombinant spider silk proteins. The Swedish Research Council, FORMAS and Knut and Alice Wallenberg Foundation supported this work.

Serum from human burn victims impairs myogenesis and protein synthesis in primary myoblasts.

The pathophysiological response to a severe burn injury involves a robust increase in circulating inflammatory/endocrine factors and a hypermetabolic state, both of which contribute to prolonged skeletal muscle atrophy. In order to characterize the role of circulating factors in muscle atrophy following a burn injury, human skeletal muscle satellite cells were grown in culture and differentiated to myoblasts/myotubes in media containing serum from burn patients or healthy, age, and sex-matched controls. While incubation in burn serum did not affect NFκB signaling, cells incubated in burn serum displayed a transient increase in STAT3 phosphorlyation (Tyr705) after 48 h of treatment with burn serum (≈ + 70%; P < 0.01), with these levels returning to normal by 96 h. Muscle cells differentiated in burn serum displayed reduced myogenic fusion signaling (phospho-STAT6 (Tyr641), ≈−75%; ADAM12, ≈-20%; both P < 0.01), and reduced levels of myogenin (≈−75%; P < 0.05). Concomitantly, myotubes differentiated in burn serum demonstrated impaired myogenesis (assessed by number of nuclei/myotube). Incubation in burn serum for 96 h did not increase proteolytic signaling (assessed via caspase-3 and ubiquitin levels), but reduced anabolic signaling [p-p70S6k (Ser421/Thr424), −30%; p-rpS6 (Ser240/244), ≈-50%] and impaired protein synthesis (−24%) (P < 0.05). This resulted in a loss of total protein content (−18%) and reduced cell size (−33%) (P < 0.05). Overall, incubation of human muscle cells in serum from burn patients results in impaired myogenesis and reduced myotube size, indicating that circulating factors may play a significant role in muscle loss and impaired muscle recovery following burn injury.

Human skeletal muscle satellite cells are a manageable tool for gene therapy (P2.009)

Human skeletal muscle satellite cells are a manageable tool for gene therapy (P2.009)

Temporal analysis of reciprocal miRNA-mRNA expression patterns predicts regulatory networks during differentiation in human skeletal muscle cells.

microRNAs (miRNAs) are short noncoding RNAs that regulate gene expression through posttranscriptional repression of target genes. miRNAs exert a fundamental level of control over many developmental processes, but their role in the differentiation and development of skeletal muscle from myogenic progenitor cells in humans remains incompletely understood. Using primary cultures established from human skeletal muscle satellite cells, we performed microarray profiling of miRNA expression during differentiation of myoblasts (day 0) into myotubes at 48 h intervals (day 2, 4, 6, 8, and 10). Based on a time-course analysis, we identified 44 miRNAs with altered expression [false discovery rate (FDR) < 5%, fold change > ±1.2] during differentiation, including the marked upregulation of the canonical myogenic miRNAs miR-1, miR-133a, miR-133b, and miR-206. Microarray profiling of mRNA expression at day 0, 4, and 10 identified 842 and 949 genes differentially expressed (FDR < 10%) at day 4 and 10, respectively. At day 10, 42% of altered transcripts demonstrated reciprocal expression patterns in relation to the directional change of their in silico predicted regulatory miRNAs based on analysis using Ingenuity Pathway Analysis microRNA Target Filter. Bioinformatic analysis predicted networks of regulation during differentiation including myomiRs miR-1/206 and miR-133a/b, miRNAs previously established in differentiation including miR-26 and miR-30, and novel miRNAs regulated during differentiation of human skeletal muscle cells such as miR-138-5p and miR-20a. These reciprocal expression patterns may represent new regulatory nodes in human skeletal muscle cell differentiation. This analysis serves as a reference point for future studies of human skeletal muscle differentiation and development in healthy and disease states.

S143 Premature Ageing And Skeletal Muscle Dysfunction In Copd Patients: Development Of A Cell Culture Model

Introduction COPD is a disease of accelerated ageing, as increased cellular-ageing (senescence) occurs in the lungs of these patients. We aim at developing a primary skeletal muscle cell culture (Human skeletal muscle satellite cells [HSKMC]) model of muscle ageing to study the cascade of events that occur in muscle senescence in vitro and to explore the effect of inflammation (TNF alpha) and oxidative stress (H 2 O 2 ), two of the putative mechanisms related to muscle dysfunction and/wasting, on muscle differentiation and on protein loss in differentiated cells. Methods and results HSKMC were cultured to senescence when the cells stopped replicating. DNA was isolated from cells in serial passages of culture. Telomere length, a marker of biological ageing, was measured by qPCR and expressed as the ratio of telomere repeat copy number to single gene copy number in the experimental sample relative to a control sample (relative T/S ratio) (n = 3). Preliminary results show a progressive shortening of telomere length with cellular ageing when comparing early (passage 2, 1.433 ± 0.05 relative T/S ratio) with a later passage (passage 15, 0.340 ± 0.2 relative T/S ratio) (Fig 1). Conclusion We have developed a novel in vitro model of ageing skeletal muscle cells, which will help us to assess the role of accelerated ageing in muscle dysfunction and/wasting in COPD patients. Dr Lakhdar was funded by an LTERS fellowship grant.

Whey protein supplementation accelerates satellite cell proliferation during recovery from eccentric exercise.

Human skeletal muscle satellite cells (SCs) are essential for muscle regeneration and remodeling processes in healthy and clinical conditions involving muscle breakdown. However, the potential influence of protein supplementation on post-exercise SC regulation in human skeletal muscle has not been well investigated. In a comparative human study, we investigated the effect of hydrolyzed whey protein supplementation following eccentric exercise on fiber type-specific SC accumulation. Twenty-four young healthy subjects received either hydrolyzed whey protein+carbohydrate (whey, n=12) or iso-caloric carbohydrate (placebo, n=12) during post-exercise recovery from 150 maximal unilateral eccentric contractions. Prior to and 24, 48 and 168h post-exercise, muscle biopsies were obtained from the exercise leg and analyzed for fiber type-specific SC content. Maximal voluntary contraction (MVC) and serum creatine kinase (CK) were evaluated as indices of recovery from muscle damage. In type II fiber-associated SCs, the whey group increased SCs/fiber from 0.05 [0.02; 0.07] to 0.09 [0.06; 0.12] (p<0.05) and 0.11 [0.06; 0.16] (p<0.001) at 24 and 48h, respectively, and exhibited a difference from the placebo group (p<0.05) at 48h. The whey group increased SCs/myonuclei from 4 % [2; 5] to 10 % [4; 16] (p < 0.05) at 48 h, whereas the placebo group increased from 5 % [2; 7] to 9 % [3; 16] (p < 0.01) at 168 h. MVC decreased (p<0.001) and muscle soreness and CK increased (p<0.001), irrespective of supplementation. In conclusion, whey protein supplementation may accelerate SC proliferation as part of the regeneration or remodeling process after high-intensity eccentric exercise.

In vitro palmitate treatment of myotubes from postmenopausal women leads to ceramide accumulation, inflammation and affected insulin signaling.

Menopause is associated with an increased incidence of insulin resistance and metabolic diseases. In a chronic palmitate treatment model, we investigated the role of skeletal muscle fatty acid exposure in relation to the metabolic deterioration observed with menopause. Human skeletal muscle satellite cells were isolated from premenopausal (n = 6) and postmenopausal (n = 5) women. In an in vitro model, the myotubes were treated with palmitate (300 µM) for one-, two- or three days during differentiation. Effects on lipid accumulation, inflammation and insulin signaling were studied. Palmitate treatment led to a 108% (CI 95%: 50%; 267%) increase in intramyocellular ceramide in the myotubes from the postmenopausal women (post-myotubes) compared with a 26% (CI 95%: −57%; 96%) increase in myotubes from the premenopausal women (pre-myotubes), (p<0.05). Furthermore, post-myotubes had a 22% (CI 95%: 4%; 34%) increase in pJNK (p = 0.04) and a 114% (CI 95%: 50%; 177%) increase in Hsp70 protein expression (p = 0.03) after three days of palmitate treatment, compared with pre-myotubes, in which no increase in either pJNK (−12% (CI 95: −26%; 2%)) or Hsp70 (7% (CI 95: −78%; 91%)) was detected. Furthermore, post-myotubes showed a blunted insulin stimulated phosphorylation of AS160 in response to chronic palmitate treatment compared with pre-myotubes (p = 0.02). The increased intramyocellular ceramide content in the post-myotubes was associated with a significantly higher mRNA expression of Serine Palmitoyltransferase1 (SPT1) after one day of palmitate treatment (p = 0.03) in post-myotubes compared with pre-myotubes. Our findings indicate that post-myotubes are more prone to develop lipid accumulation and defective insulin signaling following chronic saturated fatty acid exposure as compared to pre-myotubes.

Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.

Large‐scale isolation of human skeletal muscle satellite cells from post‐mortem tissue and development of quantitative assays to evaluate modulators of myogenesis

BackgroundDuring aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis.MethodsSatellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56+ cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised.ResultsLarge-scale isolation of CD56+ cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers.ConclusionsWe have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function.Electronic supplementary materialThe online version of this article (doi:10.1007/s13539-012-0097-z) contains supplementary material, which is available to authorized users.

Up-regulation of calcium-dependent proteolysis in human myoblasts under acute oxidative stress

The reduced regenerative potential of muscle fibres, most likely due to a decreased number and/or function of satellite cells, could play a significant role in the progression of muscle ageing. Accumulation of reactive oxygen species has been clearly correlated to sarcopenia and could contribute to the impairment of satellite cell function. In this work we have investigated the effect of oxidative stress generated by hydrogen peroxide in cultured human skeletal muscle satellite cells. We specifically focused on the activity and regulation of calpains. These calcium-dependent proteases are known to regulate many transduction pathways including apoptosis and play a critical role in satellite cell function. In our experimental conditions, which induce an increase in calcium concentration, protein oxidation and apoptotic cell death, a significant up-regulation of calpain expression and activity were observed and ATP synthase, a major component of the respiratory chain, was identified as a calpain target. Interestingly we were able to protect the cells from these H 2O 2-induced effects and prevent calpain up-regulation with a natural antioxidant extracted from pine bark (Oligopin ®). These data strongly suggest that oxidative stress could impair satellite cell functionality via calpain-dependent pathways and that an antioxidant such as Oligopin ® could prevent apoptosis and calpain activation.

Activation of the erythropoietin receptor in human skeletal muscle

Erythropoietin receptor (EPOR) expression in non-hematological tissues has been shown to be activated by locally produced and/or systemically delivered EPO. Improved oxygen homeostasis, a well-established consequence of EPOR activation, is very important for human skeletal muscle performance. In the present study we investigate whether human skeletal muscle fibers and satellite cells express EPOR and if it is activated by exercise. Ten healthy males performed 65 min of cycle exercise. Biopsies were obtained from the vastus lateralis muscle and femoral arterio-venous differences in EPO concentrations were estimated. The EPOR protein was localized in areas corresponding to the sarcolemma and capillaries. Laser dissection identified EPOR mRNA expression in muscle fibers. Also, EPOR mRNA and protein were both detected in human skeletal muscle satellite cells. In the initial part of the exercise bout there was a release of EPO from the exercising leg to the circulation, possibly corresponding to an increased bioavailability of EPO. After exercise, EPOR mRNA and EPOR-associated JAK2 phosphorylation were increased. Interaction with JAK2 is required for EPOR signaling and the increase found in phosphorylation is therefore closely linked to the activation of EPOR. The receptor activation by acute exercise suggests that signaling through EPOR is involved in exercise-induced skeletal muscle adaptation, thus extending the biological role of EPO into the skeletal muscle.

Molecular basis of the myogenic profile of aged human skeletal muscle satellite cells during differentiation

Sarcopenia is the age-related loss of muscle mass, strength and function. Human muscle proteins are synthesized at a slower rate in the elderly than in young adults, leading to atrophy and muscle mass loss with a decline in the functional capability. Additionally, aging is accompanied by a decrease in the ability of muscle tissue to regenerate following injury or overuse due to the impairment of intervening satellite cells, in which we previously reported oxidative damage evidences. The aim of the present study was to determine the effects of aging on myoblasts and myotubes obtained from human skeletal muscle, and characterize the transcriptional profile as molecular expression patterns in relation to age-dependent modifications in their regenerative capacity. Our data show that the failure to differentiate does not depend on reduced myogenic cell number, but difficulty to complete the differentiation program. Data reported here suggested the following findings: (i) oxidative damage accumulation in molecular substrates, probably due to impaired antioxidant activity and insufficient repair capability, (ii) limited capability of elderly myoblasts to execute a complete differentiation program; restricted fusion, possibly due to altered cytoskeleton turnover and extracellular matrix degradation and (iii) activation of atrophy mechanism by activation of a specific FOXO-dependent program.