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.

Transcriptional memory in skeletal muscle. Don't forget (to) exercise.

Transcriptional memory describes an ancient and highly conserved form of cellular learning that enables cells to benefit from recent experience by retaining a mitotically inheritable but reversible memory of the initial transcriptional response when encountering an environmental or physiological stimulus. Herein, we will review recent progress made in the understanding of how cells can make use of diverse constituents of the epigenetic toolbox to retain a transcriptional memory of past states and perturbations. Specifically, we will outline how these mechanisms will help to improve our understanding of skeletal muscle plasticity in health and disease. We describe the epigenetic road map that allows skeletal muscle fibers to navigate through training-induced adaptation processes, and how epigenetic memory marks can preserve an autobiographical history of lifestyle behavior changes, pathological challenges, and aging. We will further consider some key findings in the field of exercise epigenomics to emphasize major challenges when interpreting dynamic changes in the chromatin landscape in response to acute exercise and training.

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.

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.

Exercise, skeletal muscle and inflammation: ARE-binding proteins as key regulators in inflammatory and adaptive networks.

The role of inflammation in skeletal muscle adaptation to exercise is complex and has hardly been elucidated so far. While the acute inflammatory response to exercise seems to promote skeletal muscle training adaptation and regeneration, persistent, low-grade inflammation, as seen in a multitude of chronic diseases, is obviously detrimental. The regulation of cytokine production in skeletal muscle cells has been relatively well studied, yet little is known about the compensatory and anti-inflammatory mechanisms that resolve inflammation and restore tissue homeostasis. One important strategy to ensure sequential, timely and controlled resolution of inflammation relies on the regulated stability of mRNAs encoding pro-inflammatory mediators. Many key transcripts in early immune responses are characterized by the presence of AU-rich elements (AREs) in the 3'-untranslated regions of their mRNAs, allowing efficient fine-tuning of gene expression patterns at the post-transcriptional level. AREs exert their function by recruiting particular RNA-binding proteins, resulting, in most cases, in de-stabilization of the target transcripts. The best-characterized ARE-binding proteins are HuR, CUGBP1, KSRP, AUF1, and the three ZFP36 proteins, especially TTP/ZFP36. Here, we give a general introduction into the role of inflammation in the adaptation of skeletal muscle to exercise. Subsequently, we focus on potential roles of ARE-binding proteins in skeletal muscle tissue in general and specifically exercise-induced skeletal muscle remodeling. Finally, we present novel data suggesting a specific function of TTP/ZFP36 in exercise-induced skeletal muscle plasticity.

Neutrophil extracellular traps: a walk on the wild side of exercise immunology.

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. While the potential value of cf-DNA for the prediction of disease outcome and therapeutic response is well documented, the release mechanisms and biological relevance of cf-DNA have long remained enigmatic. The discovery of neutrophil extracellular traps (NETs) provided a novel mechanistic explanation for increased cf-DNA levels. Now there is increasing evidence that NETs may contribute to cf-DNA in diverse infectious, non-infectious and autoinflammatory conditions, as well as in response to acute exercise. NETs have now been firmly established as a fundamental immune mechanism used by neutrophils to respond to infection and tissue injury. On the other side, aberrant formation of NETs appears to be a driving force in the pathogenesis of autoimmunity and cardiovascular disease. Thus, the emergence of NETs in the 'exercising vasculature' raises important questions considering beneficial effects, as well as occasional adverse effects, of exercise on immune homeostasis. This review gives an overview of the current state of research into the mechanisms of how NETs are released, contribute to host defence and participate in inflammatory disorders. We discuss the impact of exercise-induced NETs, considering a potentially beneficial role in the prevention of lifestyle-related diseases, as well as putative detrimental effects that may arise in elite sports. Finally, we propose that exercise-induced cf-DNA responses could be exploited for diagnostic/prognostic purposes to identify individuals who are at increased risk of cardiovascular events or autoimmunity.

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.

Direct quantification of cell-free, circulating DNA from unpurified plasma.

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

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.

Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

Nanovesicular vaccines: exosomes.

Exosomes are small membrane vesicles derived from late endosome. They are about 30--100 nm in diameter. The secretion of exosomes is a process in which multivesicular bodies fuse with the cell membrane, and all cells that contain multivesicular endocytic compartments could theoretically secrete exosomes. The surprising biological functions of exosomes are only slowly being unveiled, but it is already clear that they serve to remove obsolete membrane proteins and act as messages of inter-cellular communication. Exosomes derived from tumor or antigen-presenting cells have been extensively investigated. They are released into the extracellular environment and fuse with the membranes of neighboring cells, delivering membrane and cytoplasmic proteins from one cell to another. Exosomes carry immunorelevant structures which play important roles in immune response, such as MHC molecules, costimulatory molecules, heat shock proteins, and naive tumor antigens. Therefore they have been suggested as potential vaccines. Consequently, exosomes have shown considerable anti-tumor effect in several studies and are in phase I clinical trials.

Experimental autoimmune neuritis induces differential microglia activation in the rat spinal cord.

The reactive spatial and temporal activation pattern of parenchymal spinal cord microglia was analyzed in rat experimental autoimmune neuritis (EAN). We observed a differential activation of spinal cord microglial cells. A significant increase in ED1(+) microglia predominantly located in the dorsal horn grey matter of lumbar and thoracic spinal cord levels was observed on Day 12. As revealed by morphological criteria and by staining with further activation markers [allograft inflammatory factor 1 (AIF-1), EMAPII, OX6, P2X(4)R], reactive microglia did not reach a macrophage-like state of full activation. On Day 12, a significant proliferative response could be observed, affecting all spinal cord areas and including ED1(+) microglial cells and a wide range of putative progenitor cells. Thus, in rat EAN, a reactive localized and distinct microglial activation correlating with a generalized proliferative response could be observed.

Examination of tumorigenesis of precursor lesions in bladder cancer by in situ hybridization.

Through examinations using fluorescence in situ hybridization (FISH) of chromosomes 1 and 9, we tried to obtain more information on dysplasia and carcinoma in situ (Cis) in relation to the oncogenesis of bladder cancer. 63 paraffin sections (dysplasia grades I-III and Cis) were evaluated, and 8 negative sections functioned as a control group. For FISH, DNA samples of CEP 1 and 9 (alpha satellites) were chosen. Gains (aneuploidy) or losses (monosomy) of chromosomal material were determined microscopically. Dysplasia grades I-III showed a 5-18% aberration in chromosome 1 aneuploidy and a 19-29% aberration in monosomy 9. Cis revealed 27% aneuploidy of chromosomes 1 and 9. Although at present dysplasia grade III and Cis of the bladder are viewed as histopathologically identical, we examined both molecular genetic differences in chromosome 9. As referred to in the literature we found the same genetic aberrations for dysplasias (grades I-III) and noninvasive papillary bladder tumors as well as for Cis and solid invasive bladder cancer.