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 molecular pathways that are activated and contribute to physiological remodeling of skeletal muscle in response to endurance exercise have not been fully characterized. We previously reported that ∼800 gene transcripts are regulated following 6 wk of supervised endurance training in young sedentary males, referred to as the training-responsive transcriptome (TRT) (Timmons JA et al. J Appl Physiol 108: 1487-1496, 2010). Here we utilized this database together with data on biological variation in muscle adaptation to aerobic endurance training in both humans and a novel out-bred rodent model to study the potential regulatory molecules that coordinate this complex network of genes. We identified three DNA sequences representing RUNX1, SOX9, and PAX3 transcription factor binding sites as overrepresented in the TRT. In turn, miRNA profiling indicated that several miRNAs targeting RUNX1, SOX9, and PAX3 were downregulated by endurance training. The TRT was then examined by contrasting subjects who demonstrated the least vs. the greatest improvement in aerobic capacity (low vs. high responders), and at least 100 of the 800 TRT genes were differentially regulated, thus suggesting regulation of these genes may be important for improving aerobic capacity. In high responders, proangiogenic and tissue developmental networks emerged as key candidates for coordinating tissue aerobic adaptation. Beyond RNA-level validation there were several DNA variants that associated with maximal aerobic capacity (Vo(₂max)) trainability in the HERITAGE Family Study but these did not pass conservative Bonferroni adjustment. In addition, in a rat model selected across 10 generations for high aerobic training responsiveness, we found that both the TRT and a homologous subset of the human high responder genes were regulated to a greater degree in high responder rodent skeletal muscle. This analysis provides a comprehensive map of the transcriptomic features important for aerobic exercise-induced improvements in maximal oxygen consumption.

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.

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.

BackgroundDiagnostics of the human ageing process may help predict future healthcare needs or guide preventative measures for tackling diseases of older age. We take a transcriptomics approach to build the first reproducible multi-tissue RNA expression signature by gene-chip profiling tissue from sedentary normal subjects who reached 65 years of age in good health.ResultsOne hundred and fifty probe-sets form an accurate classifier of young versus older muscle tissue and this healthy ageing RNA classifier performed consistently in independent cohorts of human muscle, skin and brain tissue (n = 594, AUC = 0.83–0.96) and thus represents a biomarker for biological age. Using the Uppsala Longitudinal Study of Adult Men birth-cohort (n = 108) we demonstrate that the RNA classifier is insensitive to confounding lifestyle biomarkers, while greater gene score at age 70 years is independently associated with better renal function at age 82 years and longevity. The gene score is ‘up-regulated’ in healthy human hippocampus with age, and when applied to blood RNA profiles from two large independent age-matched dementia case–control data sets (n = 717) the healthy controls have significantly greater gene scores than those with cognitive impairment. Alone, or when combined with our previously described prototype Alzheimer disease (AD) RNA ‘disease signature’, the healthy ageing RNA classifier is diagnostic for AD.ConclusionsWe identify a novel and statistically robust multi-tissue RNA signature of human healthy ageing that can act as a diagnostic of future health, using only a peripheral blood sample. This RNA signature has great potential to assist research aimed at finding treatments for and/or management of AD and other ageing-related conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0750-x) contains supplementary material, which is available to authorized users.

Physical activity and molecular ageing presumably interact to precipitate musculoskeletal decline in humans with age. Herein, we have delineated molecular networks for these two major components of sarcopenic risk using multiple independent clinical cohorts. We generated genome-wide transcript profiles from individuals (n = 44) who then undertook 20 weeks of supervised resistance-exercise training (RET). Expectedly, our subjects exhibited a marked range of hypertrophic responses (3% to +28%), and when applying Ingenuity Pathway Analysis (IPA) up-stream analysis to ∼580 genes that co-varied with gain in lean mass, we identified rapamycin (mTOR) signaling associating with growth (P = 1.4×10−30). Paradoxically, those displaying most hypertrophy exhibited an inhibited mTOR activation signature, including the striking down-regulation of 70 rRNAs. Differential analysis found networks mimicking developmental processes (activated all-trans-retinoic acid (ATRA, Z-score = 4.5; P = 6×10−13) and inhibited aryl-hydrocarbon receptor signaling (AhR, Z-score = −2.3; P = 3×10−7)) with RET. Intriguingly, as ATRA and AhR gene-sets were also a feature of endurance exercise training (EET), they appear to represent “generic” physical activity responsive gene-networks. For age, we found that differential gene-expression methods do not produce consistent molecular differences between young versus old individuals. Instead, utilizing two independent cohorts (n = 45 and n = 52), with a continuum of subject ages (18–78 y), the first reproducible set of age-related transcripts in human muscle was identified. This analysis identified ∼500 genes highly enriched in post-transcriptional processes (P = 1×10−6) and with negligible links to the aforementioned generic exercise regulated gene-sets and some overlap with ribosomal genes. The RNA signatures from multiple compounds all targeting serotonin, DNA topoisomerase antagonism, and RXR activation were significantly related to the muscle age-related genes. Finally, a number of specific chromosomal loci, including 1q12 and 13q21, contributed by more than chance to the age-related gene list (P = 0.01–0.005), implying possible epigenetic events. We conclude that human muscle age-related molecular processes appear distinct from the processes regulated by those of physical activity.

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.

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.