Carl Johan Sundberg

Exercise-induced expression of angiogenesis-related transcription and growth factors in human skeletal muscle

mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and hypoxia-inducible factor (HIF) subunits HIF-1alpha and HIF-1beta 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 (approximately 15-20% lower) at the same absolute workload. Exercise increased VEGF mRNA expression by 178% and HIF-1beta by 340%, but not HIF-1alpha 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-1alpha and HIF-1beta mRNA. The changes in VEGF, HIF-1alpha, and HIF-1beta 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-1alpha and HIF-1beta mRNA.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.

Physiological activation of hypoxia inducible factor-1 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‐1α protein levels and a more prevalent nuclear staining of HIF‐1α. 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‐1α. 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‐1α can be activated during physiologically relevant conditions.

A transcriptional map of the impact of endurance exercise training on skeletal muscle phenotype

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.

Human muscle gene expression responses to endurance training provide a novel perspective on Duchenne muscular dystrophy

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 ~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 ~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 α7β1 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.—Timmons, J. A., Larsson, O., Jansson, E., Fischer, H., Gustafsson, T., Greenhaff, P. L., Ridden, J., Rachman, J., Peyrard‐Janvid, M., Wahlestedt, C., Johan, C. Sundberg Human muscle gene expression responses to endurance training provide a novel perspective on Duchenne muscular dystrophy. FASEB J. 19, 750–760 (2005)

Muscle acetyl group availability is a major determinant of oxygen deficit in humans during submaximal exercise

The delay in skeletal muscle mitochondrial ATP production at the onset of exercise is thought to be a function of a limited oxygen supply. The delay, termed the oxygen deficit, can be quantified by assessing the above baseline oxygen consumption during the first few minutes of recovery from exercise. During submaximal exercise, the oxygen deficit is reflected by the extent of muscle phosphocreatine (PCr) breakdown. In the present study, nine male subjects performed 8 min of submaximal, single leg knee extension exercise after saline (Control) and dichloroacetate (DCA) infusion on two separate occasions. Administration of DCA increased resting skeletal muscle pyruvate dehydrogenase complex activation status threefold (Control = 0.4 +/- 0.1 vs. DCA = 1.3 +/- 0.1 mmol acetyl-CoA.min-1.kg wet muscle-1 at 37 degrees C, P < 0.01) and elevated acetylcarnitine concentration fivefold (Control = 2.2 +/- 0.5 vs. DCA = 10.9 +/- 1.2 mmol/kg dry mass, P < 0.01). During exercise, PCr degradation was reduced by approximately 50% after DCA (Control = 33.2 +/- 7.1 vs. DCA = 18.4 +/- 7.1 mmol/kg dry mass, P < 0.05). It would appear, therefore, that in humans acetyl group availability is a major determinant of the rate of increase in mitochondrial respiration at the onset of exercise and hence the oxygen deficit.

An evaluation of analysis pipelines for DNA methylation profiling using the Illumina HumanMethylation450 BeadChip platform

The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.

Modulation of extracellular matrix genes reflects the magnitude of physiological adaptation to aerobic exercise training in humans

BackgroundRegular exercise reduces cardiovascular and metabolic disease partly through improved aerobic fitness. The determinants of exercise-induced gains in aerobic fitness in humans are not known. We have demonstrated that over 500 genes are activated in response to endurance-exercise training, including modulation of muscle extracellular matrix (ECM) genes. Real-time quantitative PCR, which is essential for the characterization of lower abundance genes, was used to examine 15 ECM genes potentially relevant for endurance-exercise adaptation. Twenty-four sedentary male subjects undertook six weeks of high-intensity aerobic cycle training with muscle biopsies being obtained both before and 24 h after training. Subjects were ranked based on improvement in aerobic fitness, and two cohorts were formed (n = 8 per group): the high-responder group (HRG; peak rate of oxygen consumption increased by +0.71 ± 0.1 L min-1; p < 0.0001) while the low-responder group (LRG; peak rate of oxygen consumption did not change, +0.17 ± 0.1 L min-1, ns). ECM genes profiled included the angiopoietin 1 and related genes (angiopoietin 2, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1) and 2 (TIE2), vascular endothelial growth factor (VEGF) and related receptors (VEGF receptor 1, VEGF receptor 2 and neuropilin-1), thrombospondin-4, α2-macroglobulin and transforming growth factor β2.Resultsneuropilin-1 (800%; p < 0.001) and VEGF receptor 2 (300%; p < 0.01) transcript abundance increased only in the HRG, whereas levels of VEGF receptor 1 mRNA actually declined in the LRG (p < 0.05). TIE1 and TIE2 mRNA levels were unaltered in the LRG, whereas transcription levels of both genes were increased by 2.5-fold in the HRG (p < 0.01). Levels of thrombospondin-4 (900%; p < 0.001) and α2-macroglobulin (300%, p < 0.05) mRNA increased substantially in the HRG. In contrast, the amount of transforming growth factor β2 transcript increased only in the HRG (330%; p < 0.01), whereas it remained unchanged in the LRG (-80%).ConclusionWe demonstrate for the first time that aerobic training activates angiopoietin 1 and TIE2 genes in human muscle, but only when aerobic capacity adapts to exercise-training. The fourfold-greater increase in aerobic fitness and markedly differing gene expression profile in the HRG indicates that these ECM genes may be critical for physiological adaptation to exercise in humans. In addition, we show that, without careful demonstration of physiological adaptation, conclusions derived from gene expression profiling of human skeletal muscle following exercise may be of limited value. We propose that future studies should (a) investigate the mechanisms that underlie the apparent link between physiological adaptation and gene expression and (b) use the genes profiled in this paper as candidates for population genetic studies.

International olympic committee consensus statement on the health and fitness of young people through physical activity and sport

The International Olympic Committee (IOC) recognises the health and fitness benefits of physical activity (PA) and sport as stated in recommendation #51 from the Olympic Movement in Society Congress held in Copenhagen, 2009: Everyone involved in the Olympic Movement must become more aware of the fundamental importance of Physical Activity and sport for a healthy lifestyle, not least in the growing battle against obesity, and must reach out to parents and schools as part of a strategy to counter the rising inactivity of young people.1 The IOC assembled an expert group (January 2011) to discuss the role of PA and sport on the health and fitness of young people and to critically evaluate the scientific evidence as a basis for decision making. Specifically, the purpose of this consensus paper is to identify potential solutions through collaboration between sport and existing programmes and to review the research gaps in this field. The ultimate aim of the paper is to provide recommendations for young peoples sport and PA stakeholders. After an introduction to the scope of the problem, issues addressed include how best to define the current state of …

Substrate availability limits human skeletal muscle oxidative ATP regeneration at the onset of ischemic exercise.

We have demonstrated previously that dichloroacetate can attenuate skeletal muscle fatigue by up to 35% in a canine model of peripheral ischemia (Timmons, J.A., S.M. Poucher, D. Constantin-Teodosiu, V. Worrall, I.A. Macdonald, and P.L. Greenhaff. 1996. J. Clin. Invest. 97:879-883). This was thought to be a consequence of dichloroacetate increasing acetyl group availability early during contraction. In this study we characterized the metabolic effects of dichloroacetate in a human model of peripheral muscle ischemia. On two separate occasions (control-saline or dichloroacetate infusion), nine subjects performed 8 min of single-leg knee extension exercise at an intensity aimed at achieving volitional exhaustion in approximately 8 min. During exercise each subjects lower limbs were exposed to 50 mmHg of positive pressure, which reduces blood flow by approximately 20%. Dichloroacetate increased resting muscle pyruvate dehydrogenase complex activation status by threefold and elevated acetylcarnitine concentration by fivefold. After 3 min of exercise, phosphocreatine degradation and lactate accumulation were both reduced by approximately 50% after dichloroacetate pretreatment, when compared with control conditions. However, after 8 min of exercise no differences existed between treatments. Therefore, it would appear that dichloroacetate can delay the accumulation of metabolites which lead to the development of skeletal muscle fatigue during ischemia but does not alter the metabolic profile when a maximal effort is approached.

An integrative analysis reveals coordinated reprogramming of the epigenome and the transcriptome in human skeletal muscle after training

Regular endurance exercise training induces beneficial functional and health effects in human skeletal muscle. The putative contribution to the training response of the epigenome as a mediator between genes and environment has not been clarified. Here we investigated the contribution of DNA methylation and associated transcriptomic changes in a well-controlled human intervention study. Training effects were mirrored by significant alterations in DNA methylation and gene expression in regions with a homogeneous muscle energetics and remodeling ontology. Moreover, a signature of DNA methylation and gene expression separated the samples based on training and gender. Differential DNA methylation was predominantly observed in enhancers, gene bodies and intergenic regions and less in CpG islands or promoters. We identified transcriptional regulator binding motifs of MRF, MEF2 and ETS proteins in the proximity of the changing sites. A transcriptional network analysis revealed modules harboring distinct ontologies and, interestingly, the overall direction of the changes of methylation within each module was inversely correlated to expression changes. In conclusion, we show that highly consistent and associated modifications in methylation and expression, concordant with observed health-enhancing phenotypic adaptations, are induced by a physiological stimulus.