A low maximal oxygen consumption (VO2max) is a strong risk factor for premature mortality. Supervised endurance exercise training increases VO2max with a very wide range of effectiveness in humans. Discovering the DNA variants that contribute to this heterogeneity typically requires substantial sample sizes. In the present study, we first use RNA expression profiling to produce a molecular classifier that predicts VO2max training response. We then hypothesized that the classifier genes would harbor DNA variants that contributed to the heterogeneous VO2max response. Two independent preintervention RNA expression data sets were generated (n=41 gene chips) from subjects that underwent supervised endurance training: one identified and the second blindly validated an RNA expression signature that predicted change in VO2max ("predictor" genes). The HERITAGE Family Study (n=473) was used for genotyping. We discovered a 29-RNA signature that predicted VO2max training response on a continuous scale; these genes contained approximately 6 new single-nucleotide polymorphisms associated with gains in VO2max in the HERITAGE Family Study. Three of four novel candidate genes from the HERITAGE Family Study were confirmed as RNA predictor genes (i.e., "reciprocal" RNA validation of a quantitative trait locus genotype), enhancing the performance of the 29-RNA-based predictor. Notably, RNA abundance for the predictor genes was unchanged by exercise training, supporting the idea that expression was preset by genetic variation. Regression analysis yielded a model where 11 single-nucleotide polymorphisms explained 23% of the variance in gains in VO2max, corresponding to approximately 50% of the estimated genetic variance for VO2max. In conclusion, combining RNA profiling with single-gene DNA marker association analysis yields a strongly validated molecular predictor with meaningful explanatory power. VO2max responses to endurance training can be predicted by measuring a approximately 30-gene RNA expression signature in muscle prior to training. The general approach taken could accelerate the discovery of genetic biomarkers, sufficiently discrete for diagnostic purposes, for a range of physiological and pharmacological phenotypes in humans.

This description of some of the present knowledge on skeletal muscle fibers, their metabolic potentials, and their interplay with the degree of physical activity has revealed that skeletal muscle of man has a very large capacity for adaptation. Moreover, this adaptability appears to be of utmost importance for the metabolic response as well as for performance. Although all this is true, it should not distract us from the fact that we are lacking the most important information. The questions that need to be answered are: What triggers the changes to take place? Which are the regulatory mechanisms?

mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and hypoxia-inducible factor (HIF) subunits HIF-1α and HIF-1β in human skeletal muscle was studied during endurance exercise at different degrees of oxygen delivery. Muscle biopsies were taken before and after 45 min of one-legged knee-extension exercise performed under conditions of nonrestricted or restricted blood flow (∼15–20% lower) at the same absolute workload. Exercise increased VEGF mRNA expression by 178% and HIF-1β by 340%, but not HIF-1α and FGF-2. No significant differences between the restricted and nonrestricted groups were observed. The exercise-induced increase in VEGF mRNA was correlated to the exercise changes in HIF-1α and HIF-1β mRNA. The changes in VEGF, HIF-1α, and HIF-1β mRNAs were correlated to the exercise-induced increase in femoral venous plasma lactate concentration. It is concluded that 1) VEGF but not FGF-2 gene expression is upregulated in human skeletal muscle by a single bout of dynamic exercise and that there is a graded response in VEGF mRNA expression related to the metabolic stress and 2) the increase in VEGF mRNA expression correlates to the changes in both HIF-1α and HIF-1β mRNA.

The human hypoxia inducible factor 1 (HIF-1) system is activated under various pathological conditions, yet less is known about its physiological regulation in healthy human tissue. We have studied the effect of exercise on the activation of HIF-1 in human skeletal muscle. Employing a model where oxygen consumption increases and oxygen tension can be manipulated, nine healthy male subjects performed 45 min of one-legged knee-extension exercise. Biopsies were taken before, directly after, and 30, 120, and 360 min after exercise. Exercise led to elevated HIF-1alpha protein levels and a more prevalent nuclear staining of HIF-1alpha. Interestingly, a concurrent decrease in von Hippel-Lindau tumor suppressor protein (VHL) levels was detected in some subjects. Moreover, exercise induced an increase in the DNA binding activity of HIF-1alpha. Characterization of gene expression by real-time PCR demonstrated that the HIF-1 target genes VEGF and EPO were activated. VEGF mRNA was further increased when blood flow to the exercising leg was restricted. In conclusion, these data clearly demonstrate that physical activity induces the HIF-1-mediated signaling pathway in human skeletal muscle, providing the first evidence that human HIF-1alpha can be activated during physiologically relevant conditions.

Endurance training leads to many adaptational changes in several tissues. In skeletal muscle, fatty acid usage is enhanced and mitochondrial content is increased. The exact molecular mechanisms regulating these functional and structural changes remain to be elucidated. Contractile activity-induced metabolic perturbation has repeatedly been shown to be important for the induction of mitochondrial biogenesis. Recent reports suggest that the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha)/mitochondrial transcription factor A (Tfam) pathway is involved in exercise-induced mitochondrial biogenesis. In the present study, nine healthy men performed two 45-min bouts of one-legged knee extension exercise: one bout with restricted blood flow, and the other with nonrestricted blood flow to the working muscle. Muscle biopsies were obtained from the vastus lateralis muscle before exercise and at 0, 30, 120, and 360 min after the exercise bout. Biopsies were analyzed for whole muscle, as well as fiber-type specific mRNA expression of myocyte-enriched calcineurin interacting protein (MCIP)-1, PGC-1alpha, and downstream mitochondrial transcription factors. A novel finding was that, in human skeletal muscle, PGC-1alpha mRNA increased more after exercise with restricted blood flow than in the nonrestricted condition. No changes were observed for the mRNA of NRF-1, Tfam, mitochondrial transcription factor B1, and mitochondrial transcription factor B2. Muscle fiber type I and type II did not differ in the basal PGC-1alpha mRNA levels or in the expression increase after ischemic training. Another novel finding was that there was no difference between the restricted and nonrestricted exercise conditions in the increase of MCIP-1 mRNA, a marker for calcineurin activation. This suggests that calcineurin may be activated by exercise in humans and does not exclude that calcineurin could play a role in PGC-1 transcription activation in human skeletal muscle.

Global gene expression profiling is used to generate novel insight into a variety of disease states. Such studies yield a bewildering number of data points, making it a challenge to validate which genes specifically contribute to a disease phenotype. Aerobic exercise training represents a plausible model for identification of molecular mechanisms that cause metabolic-related changes in human skeletal muscle. We carried out the first transcriptome-wide characterization of human skeletal muscle responses to 6 wk of supervised aerobic exercise training in 8 sedentary volunteers. Biopsy samples before and after training allowed us to identify approximately 470 differentially regulated genes using the Affymetrix U95 platform (80 individual hybridization steps). Gene ontology analysis indicated that extracellular matrix and calcium binding gene families were most up-regulated after training. An electronic reanalysis of a Duchenne muscular dystrophy (DMD) transcript expression dataset allowed us to identify approximately 90 genes modulated in a nearly identical fashion to that observed in the endurance exercise dataset. Trophoblast noncoding RNA, an interfering RNA species, was the singular exception-being up-regulated by exercise and down-regulated in DMD. The common overlap between gene expression datasets may be explained by enhanced alpha7beta1 integrin signaling, and specific genes in this signaling pathway were up-regulated in both datasets. In contrast to these common features, OXPHOS gene expression is subdued in DMD yet elevated by exercise, indicating that more than one major mechanism must exist in human skeletal muscle to sense activity and therefore regulate gene expression. Exercise training modulated diabetes-related genes, suggesting our dataset may contain additional and novel gene expression changes relevant for the anti-diabetic properties of exercise. In conclusion, gene expression profiling after endurance exercise training identified a range of processes responsible for the physiological remodeling of human skeletal muscle tissue, many of which were similarly regulated in DMD. Furthermore, our analysis demonstrates that numerous genes previously suggested as being important for the DMD disease phenotype may principally reflect compensatory integrin signaling.

It has not been established which physiological processes contribute to endurance training-related changes (Delta) in aerobic performance. For example, the relationship between intramuscular metabolic responses at the intensity used during training and improved human functional capacity has not been examined in a longitudinal study. In the present study we hypothesized that improvements in aerobic capacity (Vo(2max)) and metabolic control would combine equally to explain enhanced aerobic performance. Twenty-four sedentary males (24 +/- 2 yr; 1.81 +/- 0.08 m; 76.6 +/- 11.3 kg) undertook supervised cycling training (45 min at 70% of pretraining Vo(2max)) 4 times/wk for 6 wk. Performance was determined using a 15-min cycling time trial, and muscle biopsies were taken before and after a 10-min cycle at 70% of pretraining Vo(2max) to quantify substrate metabolism. Substantial interindividual variability in training-induced adaptations was observed for most parameters, yet "low responders" for DeltaVo(2max) were not consistently low responders for other variables. While Vo(2max) and time trial performance were related at baseline (r(2) = 0.80, P < 0.001), the change in Vo(2max) was completely unrelated to the change in aerobic performance. The maximal parameters DeltaVe(max) and DeltaVeq(max) (DeltaVe/Vo(2max)) accounted for 64% of the variance in DeltaVo(2max) (P < 0.001), whereas Deltaperformance was related to changes in the submaximal parameters Veq(submax) (r(2) = 0.33; P < 0.01), muscle Deltalactate (r(2) = 0.32; P < 0.01), and Deltaacetyl-carnitine (r(2) = 0.29; P < 0.05). This study demonstrates that improvements in high-intensity aerobic performance in humans are not related to altered maximal oxygen transport capacity. Altered muscle metabolism may provide the link between training stimulus and improved performance, but metabolic parameters do not change in a manner that relates to aerobic capacity changes.

The aims of this study were 1) to characterize changes in matrix metalloproteinase (MMP), endostatin, and vascular endothelial growth factor (VEGF)-A expression in skeletal muscle in response to a single bout of exercise in humans; and 2) to determine if any exchange of endostatin and VEGF-A between circulation and the exercising leg is associated with a change in the tissue expression or plasma concentration of these factors. Ten healthy males performed 65 min of cycle exercise, and muscle biopsies were obtained from the vastus lateralis muscle at rest and immediately and 120 min after exercise. In the muscle biopsies, measurements of mRNA expression levels of MMP-2, MMP-9, MMP-14, and tissue inhibitor of metalloproteinase; VEGF and endostatin protein levels; and MMP activities were performed. Femoral arterial and venous concentrations of VEGF-A and endostatin were determined before, during, and 120 min after exercise. A single bout of exercise increased MMP-9 mRNA and activated MMP-9 protein in skeletal muscle. No measurable increase of endostatin was observed in the skeletal muscle or in plasma following exercise. A concurrent increase in skeletal muscle VEGF-A mRNA and protein levels was induced by exercise, with no signs of peripheral uptake from the circulation. However, a decrease in plasma VEGF-A concentration occurred following exercise. Thus 1) a single bout of exercise activated the MMP system without any resulting change in tissue endostatin protein levels, and 2) the increased VEGF-A protein levels are due to changes in the skeletal muscle tissue itself. Other mechanisms are responsible for the observed exercise-induced decrease in VEGF-A in plasma.