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Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects.

ABSTRACT: BACKGROUND: Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1-10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased. CONCLUSIONS/SIGNIFICANCE: Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.

SUBMITTER: Christensen B

PROVIDER: S-EPMC3285196 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects.

Christensen Britt B Lundby Carsten C Jessen Niels N Nielsen Thomas S TS Vestergaard Poul F PF Møller Niels N Pilegaard Henriette H Pedersen Steen B SB Kopchick John J JJ Jørgensen Jens Otto L JO

PloS one 20120222 2

<h4>Background</h4>Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.<h4>Methodology/principal findings</h4>The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1-10 hours, and 2) a separate study with prolonged administration for 16 ...[more]

PMID: 22384088

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Project description:Aims/hypothesesReduced mitochondrial capacity in skeletal muscle has been observed in obesity and type 2 diabetes. In humans, the aetiology of this abnormality is not well understood but the possibility that it is secondary to the stress of nutrient overload has been suggested. To test this hypothesis, we examined whether sustained overfeeding decreases skeletal muscle mitochondrial content or impairs function.MethodsTwenty-six healthy volunteers (21 men, 5 women, age 25.3 ± 4.5 years, BMI 25.5 ± 2.4 kg/m2) underwent a supervised protocol consisting of 8 weeks of high-fat overfeeding (40% over baseline energy requirements). Before and after overfeeding, we measured systemic fuel oxidation by indirect calorimetry and performed skeletal muscle biopsies to measure mitochondrial gene expression, content and function in vitro. Mitochondrial function in vivo was measured by 31P NMR spectroscopy.ResultsWith overfeeding, volunteers gained 7.7 ± 1.8 kg (% change 9.8 ± 2.3). Overfeeding increased fasting NEFA, LDL-cholesterol and insulin concentrations. Indirect calorimetry showed a shift towards greater reliance on lipid oxidation. In skeletal muscle tissue, overfeeding increased ceramide content, lipid droplet content and perilipin-2 mRNA expression. Phosphorylation of AMP-activated protein kinase was decreased. Overfeeding increased mRNA expression of certain genes coding for mitochondrial proteins (CS, OGDH, CPT1B, UCP3, ANT1). Despite the stress of nutrient overload, mitochondrial content and mitochondrial respiration in muscle did not change after overfeeding. Similarly, overfeeding had no effect on either the emission of reactive oxygen species or on mitochondrial function in vivo.Conclusions/interpretationSkeletal muscle mitochondria are significantly resilient to nutrient overload. The lower skeletal muscle mitochondrial oxidative capacity in human obesity is likely to be caused by reasons other than nutrient overload per se.Trial registrationClinicalTrials.gov NCT01672632.

Project description:Context and objectiveThe myokine irisin has been proposed to regulate energy homeostasis. Little is known about its association with metabolic parameters and especially with parameters influencing pathways of lipid metabolism. In the context of a clinical trial, an exploratory post hoc analysis has been performed in healthy subjects to determine whether simvastatin and/or ezetimibe influence serum irisin levels. The direct effects of simvastatin on irisin were also examined in primary human skeletal muscle cells (HSKMCs).Design and participantsA randomized, parallel 3-group study was performed in 72 men with mild hypercholesterolemia and without apparent cardiovascular disease. Each group of 24 subjects received a 14-day treatment with either simvastatin 40 mg, ezetimibe 10 mg, or their combination.ResultsBaseline irisin concentrations were not significantly correlated with age, BMI, estimated GFR, thyroid parameters, glucose, insulin, lipoproteins, non-cholesterol sterols, adipokines, inflammation markers and various molecular markers of cholesterol metabolism. Circulating irisin increased significantly in simvastatin-treated but not in ezetimibe-treated subjects. The changes were independent of changes in LDL-cholesterol and were not correlated with changes in creatine kinase levels. In HSKMCs, simvastatin significantly increased irisin secretion as well as mRNA expression of its parent peptide hormone FNDC5. Simvastatin significantly induced cellular reactive oxygen species levels along with expression of pro- and anti-oxidative genes such as Nox2, and MnSOD and catalase, respectively. Markers of cellular stress such as atrogin-1 mRNA and Bax protein expression were also induced by simvastatin. Decreased cell viability and increased irisin secretion by simvastatin was reversed by antioxidant mito-TEMPO, implying in part that irisin is secreted as a result of increased mitochondrial oxidative stress and subsequent myocyte damage.ConclusionsSimvastatin increases irisin concentrations in vivo and in vitro. It remains to be determined whether this increase is a result of muscle damage or a protective mechanism against simvastatin-induced cellular stress.Trial registrationClinicalTrials.gov NCT00317993 NCT00317993.

Project description:BackgroundAs the interest in manned spaceflight increases, so does the requirement to understand the transcriptomic mechanisms that underlay the detrimental physiological adaptations of skeletal muscle to microgravity. While microgravity-induced differential gene expression (DGE) has been extensively investigated, the contribution of differential alternative splicing (DAS) to the plasticity and functional status of the skeletal muscle transcriptome has not been studied in an animal model. Therefore, by evaluating both DGE and DAS across spaceflight, we set out to provide the first comprehensive characterization of the transcriptomic landscape of skeletal muscle during exposure to microgravity.MethodsRNA-sequencing, immunohistochemistry, and morphological analyses were conducted utilizing total RNA and tissue sections isolated from the gastrocnemius and quadriceps muscles of 30-week-old female BALB/c mice exposed to microgravity or ground control conditions for 9 weeks.ResultsIn response to microgravity, the skeletal muscle transcriptome was remodeled via both DGE and DAS. Importantly, while DGE showed variable gene network enrichment, DAS was enriched in structural and functional gene networks of skeletal muscle, resulting in the expression of alternatively spliced transcript isoforms that have been associated with the physiological changes to skeletal muscle in microgravity, including muscle atrophy and altered fiber type function. Finally, RNA-binding proteins, which are required for regulation of pre-mRNA splicing, were themselves differentially spliced but not differentially expressed, an upstream event that is speculated to account for the downstream splicing changes identified in target skeletal muscle genes.ConclusionsOur work serves as the first investigation of coordinate changes in DGE and DAS in large limb muscles across spaceflight. It opens up a new opportunity to understand (i) the molecular mechanisms by which splice variants of skeletal muscle genes regulate the physiological adaptations of skeletal muscle to microgravity and (ii) how small molecule splicing regulator therapies might thwart muscle atrophy and alterations to fiber type function during prolonged spaceflight.

Dataset Information

Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects.

Publications

Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects.

Similar Datasets

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