The Acute, Short-, and Long-Term Effects of Endurance Exercise on Skeletal Muscle Transcriptome Profiles.

A better understanding of the cellular and molecular mechanisms that are involved in skeletal muscle adaptation to exercise is fundamentally important to take full advantage of the enormous benefits that exercise training offers in disease prevention and therapy. The aim of this study was to elucidate the transcriptional signatures that distinguish the endurance-trained and untrained muscles in young adult males (24 ± 3.5 years). We characterized baseline differences as well as acute exercise-induced transcriptome responses in vastus lateralis biopsy specimens of endurance-trained athletes (ET; n = 8; VO2max, 67.2 ± 8.9 mL/min/kg) and sedentary healthy volunteers (SED; n = 8; VO2max, 40.3 ± 7.6 mL/min/kg) using microarray technology. A second cohort of SED volunteers (SED-T; n = 10) followed an 8-week endurance training program to assess expression changes of selected marker genes in the course of skeletal muscle adaptation. We deciphered differential baseline signatures that reflected major differences in the oxidative and metabolic capacity of the endurance-trained and untrained muscles. SED-T individuals in the training group displayed an up-regulation of nodal regulators of oxidative adaptation after 3 weeks of training and a significant shift toward the ET signature after 8 weeks. Transcriptome changes provoked by 1 h of intense cycling exercise only poorly overlapped with the genes that constituted the differential baseline signature of ETs and SEDs. Overall, acute exercise-induced transcriptional responses were connected to pathways of contractile, oxidative, and inflammatory stress and revealed a complex and highly regulated framework of interwoven signaling cascades to cope with exercise-provoked homeostatic challenges. While temporal transcriptional programs that were activated in SEDs and ETs were quite similar, the quantitative divergence in the acute response transcriptomes implicated divergent kinetics of gene induction and repression following an acute bout of exercise. Together, our results provide an extensive examination of the transcriptional framework that underlies skeletal muscle plasticity.

Effects of Training Status and Exercise Mode on Global Gene Expression in Skeletal Muscle.

The aim of this study was to investigate differences in skeletal muscle gene expression of highly trained endurance and strength athletes in comparison to untrained individuals at rest and in response to either an acute bout of endurance or strength exercise. Endurance (ET, n = 8, VO2max 67 ± 9 mL/kg/min) and strength athletes (ST, n = 8, 5.8 ± 3.0 training years) as well as untrained controls (E-UT and S-UT, each n = 8) performed an acute endurance or strength exercise test. One day before testing (Pre), 30 min (30'Post) and 3 h (180'Post) afterwards, a skeletal muscle biopsy was obtained from the m. vastus lateralis. Skeletal muscle mRNA was isolated and analyzed by Affymetrix-microarray technology. Pathway analyses were performed to evaluate the effects of training status (trained vs. untrained) and exercise mode-specific (ET vs. ST) transcriptional responses. Differences in global skeletal muscle gene expression between trained and untrained were smaller compared to differences in exercise mode. Maximum differences between ET and ST were found between Pre and 180'Post. Pathway analyses showed increased expression of exercise-related genes, such as nuclear transcription factors (NR4A family), metabolism and vascularization (PGC1-α and VEGF-A), and muscle growth/structure (myostatin, IRS1/2 and HIF1-α. The most upregulated genes in response to acute endurance or strength exercise were the NR4A genes (NR4A1, NR4A2, NR4A3). The mode of acute exercise had a significant effect on transcriptional regulation Pre vs. 180'Post. In contrast, the effect of training status on human skeletal muscle gene expression profiles was negligible compared to strength or endurance specialization. The highest variability in gene expression, especially for the NR4A-family, was observed in trained individuals at 180'Post. Assessment of these receptors might be suitable to obtain a deeper understanding of skeletal muscle adaptive processes to develop optimized training strategies.

Quality Control of Serum and Plasma by Quantification of (4E,14Z)-Sphingadienine-C18-1-Phosphate Uncovers Common Preanalytical Errors During Handling of Whole Blood.

BACKGROUND: Nonadherence to standard operating procedures (SOPs) during handling and processing of whole blood is one of the most frequent causes affecting the quality of serum and plasma. Yet, the quality of blood samples is of the utmost importance for reliable, conclusive research findings, valid diagnostics, and appropriate therapeutic decisions. METHODS: UHPLC-MS-driven nontargeted metabolomics was applied to identify biomarkers that reflected time to processing of blood samples, and a targeted UHPLC-MS analysis was used to quantify and validate these biomarkers. RESULTS: We found that (4E,14Z)-sphingadienine-C18-1-phosphate (S1P-d18:2) was suitable for the reliable assessment of the pronounced changes in the quality of serum and plasma caused by errors in the phase between collection and centrifugation of whole blood samples. We rigorously validated S1P-d18:2, which included the use of practicality tests on >1400 randomly selected serum and plasma samples that were originally collected during single- and multicenter trials and then stored in 11 biobanks in 3 countries. Neither life-threatening disease states nor strenuous metabolic challenges (i.e., high-intensity exercise) affected the concentration of S1P-d18:2. Cutoff values for sample assessment were defined (plasma, ≤0.085 µg/mL; serum, ≤0.154 µg/mL). CONCLUSIONS: Unbiased valid monitoring to check for adherence to SOP-dictated time for processing to plasma or serum and/or time to storage of whole blood at 4 °C is now feasible. This novel quality assessment step could enable scientists to uncover common preanalytical errors, allowing for identification of serum and plasma samples that should be excluded from certain investigations. It should also allow control of samples before long-term storage in biobanks.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

TGF-ß Contributes to Impaired Exercise Response by Suppression of Mitochondrial Key Regulators in Skeletal Muscle.

A substantial number of people at risk of developing type 2 diabetes could not improve insulin sensitivity by physical training intervention. We studied the mechanisms of this impaired exercise response in 20 middle-aged individuals at high risk of developing type 2 diabetes who performed 8 weeks of controlled cycling and walking training at 80% individual Vo2 peak. Participants identified as nonresponders in insulin sensitivity (based on the Matsuda index) did not differ in preintervention parameters compared with high responders. The failure to increase insulin sensitivity after training correlates with impaired upregulation of mitochondrial fuel oxidation genes in skeletal muscle, and with the suppression of the upstream regulators PGC1α and AMPKα2. The muscle transcriptomes of the nonresponders are further characterized by the activation of transforming growth factor (TGF)-ß and TGF-ß target genes, which is associated with increases in inflammatory and macrophage markers. TGF-ß1 as inhibitor of mitochondrial regulators and insulin signaling is validated in human skeletal muscle cells. Activated TGF-ß1 signaling downregulates the abundance of PGC1α, AMPKα2, the mitochondrial transcription factor TFAM, and mitochondrial enzymes. Thus, the data suggest that increased TGF-ß activity in skeletal muscle can attenuate the improvement of mitochondrial fuel oxidation after training and contribute to the failure to increase insulin sensitivity.

Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time.

Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n = 19) and untrained (SED, n = 17) individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

Basal and exercise induced label-free quantitative protein profiling of m. vastus lateralis in trained and untrained individuals.

Morphological and metabolic adaptations of the human skeletal muscle to exercise are crucial to improve performance and prevent chronic diseases and metabolic disorders. In this study we investigated human skeletal muscle protein composition in endurance trained (ET) versus untrained individuals (UT) and its modulation by an acute bout of endurance exercise. Participants were recruited based on their VO2max and subjected to a bicycle exercise test. M. vastus lateralis biopsies were taken before and three hours after exercise. Muscle lysates were analyzed using off-gel LC-MS/MS. Relative protein abundances were compared between ET and UT at rest and after exercise. Comparing UT and ET, we identified 92 significantly changed proteins under resting conditions. Specifically, fiber-type-specific and proteins of the oxidative phosphorylation and tricarboxylic acid cycle were increased in ET. In response to acute exercise, 71 proteins in ET and 44 in UT were altered. Here, a decrease of proteins involved in energy metabolism accompanied with alterations of heat shock and proteasomal proteins could be observed. In summary, long-term endurance training increased the basal level of structural and mitochondrial proteins in skeletal muscle. In contrast, acute exercise resulted in a depletion of proteins related to substrate utilization, especially in trained athletes. BIOLOGICAL SIGNIFICANCE: The investigation of the human skeletal muscle proteome in response to exercise may provide novel insights into the process of muscular plasticity. It is of importance in the development of exercise-based strategies in the prevention and therapy of many chronic inflammatory and degenerative diseases which are often accompanied by muscular deconditioning. Up to date, proteomic investigations of the human muscle proteome in adaptation to exercise are mainly focused on untrained individuals and often restricted to animal studies. In the present study we compare the protein composition in endurance trained athletes and untrained individuals in the resting muscle and its modulation in response to acute exercise. To our knowledge, we present the first comprehensive analysis of skeletal muscle proteome alterations in response to acute and long-term exercise intervention.

Neutrophils release extracellular DNA traps in response to exercise.

Intense exercise evokes a rapid and transient increase in circulating cell-free DNA (cf-DNA), a phenomenon that is commonly observed in a variety of acute and chronic inflammatory conditions. In this study, we aimed to shed new light on the release and clearance mechanisms of cf-DNA in response to exercise. We hypothesized that activated neutrophils may primarily contribute to exercise-evoked cf-DNA levels by releasing neutrophil extracellular traps (NETs). Analysis of plasma and/or serum samples from male athletes at rest and in response to exhaustive treadmill exercise revealed an immediate and transient increase in cf-DNA that was concomitantly counterbalanced by an increase in serum DNase activity. Consistently, rapid release and clearance kinetics for cf-DNA could also be observed in response to intensive cycling exercise, with no significant differences between endurance-trained (V̇o2max >57 ml·min(-1)·kg(-1)) and healthy (V̇o2max <49 ml·min(-1)·kg(-1)) sedentary individuals. In postexercise blood smear samples, we detected seemingly intact neutrophils displaying morphological signs of NET release, as indicated by abnormal swollen nuclei and emanating DNA fibers. In support, we observed a striking correlation of postexercise cf-DNA concentrations with plasma levels of the granule-derived enzyme myeloperoxidase. Our study indicates that intense exercise induces liberation of NETs, which is sufficiently counterbalanced in healthy individuals by a concomitant rise in serum DNase activity. As aberrant release of NETs has been linked to diverse disease states, monitoring of cf-DNA/DNase levels or activities in response to standardized exercise testing could provide a valuable tool to identify people who are at increased risk for cardiac ischemia, thrombosis, autoimmunity, or chronic fatigue.

Exhaustive exercise modifies different gene expression profiles and pathways in LPS-stimulated and un-stimulated whole blood cultures.

Exhaustive exercise can interfere with immunity, causing transient immunosuppression and infections/inflammation in athletes. We used microarray technology to analyze the gene expression profiles of whole blood in short time (1h) LPS-stimulated and un-stimulated cultures drawn before, 30min after, 3h after and 24h after a half-marathon run. Four male and 4 female athletes participated. Exercise induced differential expression of genes known to be involved in innate immunity/inflammatory response, metabolic response, DNA methylation, apoptosis and regulation of brain function. Several genes with prominent anti-inflammatory function were up-regulated in un-stimulated cultures, including ARG-1, SOCS3, DUSP-1, ORMs, IRAK3, and GJB6. Some of these genes were also strongly up-regulated in LPS-stimulated cultures (ARG-1, ORM2, and GJB6). Some genes were strongly up-regulated through exercise in LPS-stimulated cultures, but not in un-stimulated cultures (TNIP3, PLAU, and HIVEP1). There was also a row of genes, which were strongly down-regulated by exercise in LPS-stimulated cultures, notably IFN-ß1 and CXCL10. Exercise also significantly changed the expression of genes (OLIG2, TMEM106B) which are known to be related to brain function and expression of which has never been documented in peripheral blood. In summary, exhaustive exercise, in addition to modifying gene expression in un-stimulated cells, could also interfere with the early gene expression response to endotoxin. There was an anti-inflammatory bias of gene regulation by exercise, including genes involved in the negative regulation of TLRs signalling. The results of the present study demonstrate that some potentially important effects of exercise can only be detected in relation to pathogen stimulation.

Electroretinographic assessment of retinal function during acute exposure to normobaric hypoxia.

BACKGROUND: The current study aimed to investigate retinal function during exposure to normobaric hypoxia. METHODS: Standard Ganzfeld ERG equipment (Diagnosys LLC, Cambridge, UK) using an extended ISCEV protocol was applied to explore intensity-response relationship in dark- and light- adapted conditions in 13 healthy volunteers (mean age 25 ± 3 years). Baseline examinations were performed under atmospheric air conditions at 341 meters above sea level (FIO2 of 21 %), and were compared to hypoxia (FIO2 of 13.2 %) by breathing a nitrogen-enriched gas mixture for 45 min. All subjects were monitored using infrared oximetry and blood gas analysis. RESULTS: The levels of PaCO2 changed from 38.4 ± 2.7 mmHg to 36.4 ± 3.0 mmHg, PaO2 from 95.5 ± 1.9 mmHg to 83.7 ± 4.6 mmHg, and SpO2 from 100 ± 0 % to 87 ± 4 %, from baseline to hypoxia respectively. A significant decrease (p < 0.05) was found for saturation amplitude of the dark-adapted b-wave intensity-response function (Vmax), dark-adapted a- and b-wave amplitudes of combined rod and cone responses (3 and 10 cd.s/m(2)), light-adapted b-wave amplitudes of single flash (3 and 10 cd.s/m(2)), and flicker responses (5-45 Hz) during hypoxia compared to baseline, without changes in implicit times. The a-wave slope of combined rod and cone responses (3 and 10 cd.s/m(2)) and the oscillatory potentials were significantly lower during hypoxia (p < 0.05). A isolated light-adapted ON response (250 ms flash) showed a reduction of amplitudes at hypoxia (p < 0.05), but no changes were observed for the OFF response. CONCLUSIONS: The results show significant impairment of retinal function during simulated normobaric short-term hypoxia affecting specific retinal cells of rod and cone pathways.

Changes in spontaneous and LPS-induced ex vivo cytokine production and mRNA expression in male and female athletes following prolonged exhaustive exercise.

PURPOSE: The capacity of whole blood cultures to produce cytokines in response to endotoxin (LPS) was studied in athletes before, 30 min after, 3 h after and 24 h after a half-marathon run. METHODS: Eight well trained men and 8 well trained women (6 of them in the late luteal phase of their cycle) participated. EDTA blood was incubated with or without LPS for 1 h, and cytokine concentration and gene expression were determined. To quantify LPS-dependent release on a per monocyte basis (LDR), the mean values of the difference (delta) between cytokine concentration in stimulated and unstimulated cultures, normalized to monocyte numbers, were calculated. RESULTS: LDR of TNF-alpha was significantly reduced by exercise with identical kinetic in men and women. TNF-alpha mRNA expression was slightly down-regulated following exercise (P < 0.05), but significantly so only in women. LDR of IL-6 was also reduced, but with a faster kinetic in women than in men. Similarly, 30 min post-exercise; LDR and spontaneous release of IL-1ra were significantly less in women than men. Concomitantly, IL-Ira mRNA was significantly elevated in unstimulated and in stimulated cultures in men only. IL-10 and IL-10 mRNA were significantly induced 30 min following exercise in absence of any detectable LDR. Women showed significantly lower levels than men. LDR and spontaneous release of IL-8 was enhanced in men and TGF-beta1 in women. A significant up-regulation was seen in unstimulated IL-8 mRNA for women and LPS-stimulated IL-8 mRNA expression for men following exercise. CONCLUSION: Altogether, LPS-dependent ex vivo cytokine release was strongly influenced by exercise and these changes could only in part be attributed to changes in messenger RNA. Results for IL-1ra, IL-6 and IL-10 pointed to a less pronounced anti-inflammatory response in women as compared with men. Our results also indicate an early production of IL-10 by peripheral blood cells in response to exercise.

Short-term treadmill running as a model for studying cell-free DNA kinetics in vivo.

BACKGROUND: Increased plasma concentrations of cell-free DNA (cf-DNA) are considered a hallmark of various clinical conditions. Despite intensive research in this field, limited data are available concerning the time course of release and clearance of cf-DNA in vivo. METHODS: We extracted cf-DNA from plasma samples taken before and immediately after a 10-km cross-country run, and from samples taken before, immediately after, and 30 min after exhaustive short-term treadmill exercise. The contribution of nuclear (nDNA) and mitochondrial DNA (mtDNA) was measured by quantitative real-time PCR. The incremental treadmill exercise setup was exploited to delineate the precise sequencing and timing of cf-nDNA, lactate, and high-mobility group box 1 protein (HMGB1) release during the exercise and recovery phases. RESULTS: Postexercise plasma cf-nDNA concentrations in cross-country and treadmill runners were significantly increased, by 7.6-fold and 9.9-fold, respectively (P < 0.001). cf-nDNA concentrations were not correlated with age, sex, or body mass index. Plasma concentrations of cf-nDNA and HMGB1 in postexercise samples of treadmill runners were significantly correlated (r = 0.84; P = 0.004). cf-mtDNA concentrations were not affected by treadmill exercise. Time-course analyses demonstrated that cf-nDNA is released within minutes after the onset of exercise and is rapidly cleared from the circulation after the cessation of exercise. Nearly congruent kinetics for cf-nDNA, lactate, and HMGB1 were observed during the exercise phase. CONCLUSIONS: A single bout of exhaustive short-term treadmill exercise constitutes a versatile model system suitable for addressing basic questions about cf-DNA biology.