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Physiological activation of hypoxia inducible factor-1 in human skeletal muscle

Helene Ameln^*,,1,2, Thomas Gustafsson^*,,1,2, Carl Johan Sundberg^*, Kensaku Okamoto^,, Eva Jansson, Lorenz Poellinger and Yuichi Makino^,

^* Department of Physiology and Pharmacology,
Department of Cell and Molecular Biology, and
Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; and
Division of Clinical immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

² Correspondence: Department of Physiology and Pharmacology, Section of Molecular Exercise Physiology, Karolinska Institutet, Stockholm 171 77, Sweden. E-mail: helene.ameln{at}fyfa.ki.se

SPECIFIC AIMS

We have tested the hypothesis that hypoxia-mediated transcriptional regulatory pathways play a role in adaptation of the human skeletal muscle to exercise. Therefore, we measured protein levels and subcellular localization of the hypoxia inducible factor 1 (HIF-1) as well as mRNA levels of its target genes vascular endothelial growth factor (VEGF) and erythropoietin (EPO) in biopsy specimens from human skeletal muscle before and after 45 min of exercise.

To clarify the correlation between the activation of HIF-1 and the reduction of oxygen tension in muscular cells during exercise, we employed a blood flow restricting model in which oxygen delivery to the exercising leg is reduced by 15–20%.

In these experiments we have also measured the levels of the von Hippel Lindau (VHL) protein, a key component of the complex regulating HIF-1 protein stability.

PRINCIPAL FINDINGS

1. Exercise elevates HIF-1 protein levels
Acute 45 min exercise resulted in elevated HIF-1 protein levels in human vastus lateralis muscle. Figure 1 B shows a representative blot from one subject, for all five time points. Before exercise, HIF-1 immunoreactivity was very low (Fig. 1B , lane 1). Immediately after exercise, the HIF-1 signal was markedly increased, and this was sustained until 360 min after exercise (Fig. 1B , lanes 2–5). The median relative increase in HIF-1 protein levels after 45 min of exercise was 83% (n=8, P<0.05). Figure 1C shows HIF-1 protein levels in two subjects before and immediately after exercise in both the nonrestricted condition (NR-cond) and restricted condition (R-cond). No significant difference in exercise-induced changes was found between the R-cond (+73%) and the NR-cond (+97%). There was no exercise-induced change in HIF-1 mRNA levels in either of the two conditions (Fig. 1A ) supporting previous cell findings of regulation at the protein level. Analyses failed to detect any Hif-2 related immunoreactivity in human muscle homogenate (Fig. 1D , lanes 2–5), whereas the antibodies recognized transiently overexpressed HIF-2 in whole cell extracts (Fig. 1D , lane 8).

View larger version (30K): [in this window] [in a new window]   Figure 1. HIF-1 mRNA and HIF-1 and HIF-2 protein in human skeletal muscle. A) Real time PCR analyses of Hif-1 mRNA levels before (Pre) and 0, 30,120, and 360 min after a 45 min bout of one-legged knee extension exercise, under nonrestricted (NR) and restricted (R) blood flow conditions, n = 9. B) Immunoblot analyses of HIF-1 protein levels before (Pre) and 0, 30,120, and 360 min after (Post) exercise (lanes 1–5). Whole cell extract from hypoxic (1% O2) HeLa cells was used as a positive control (lane 6). The lower molecular weight band represents nonspecific immunoreactivity. C) Immunoblot analyses of HIF-1 protein levels from 2 subjects, before (Pre) and immediately after (Post) exercise with and without blood flow restriction (R or NR cond). D) Immunoblot analyses of HIF-2 protein levels before (Pre) and 0, 30,120, and 360 min after (Post) exercise (lanes 1–5). Whole cell extract from COS7 cells transiently overexpressing hHIF-2 was used as a positive control (lanes 6–8).

2. Exercise increases the DNA-binding activity of the HIF-1-ARNT complex and translocates HIF-1 to the nucleus
Before exercise, the myonuclear HIF-1 specific staining signal was low. Immediately, as well as 30 min after exercise, a markedly stronger myonuclear staining was observed. Gel shift analysis of a ³²P-labeled HRE probe incubated with cellular extracts revealed an enhanced DNA binding activity after exercise. The specificity of the band was confirmed by antibody-induced super shift analysis. The observed nuclear translocation and DNA binding further support HIF-1 activation in response to exercise.

3. Exercise increases mRNA expression of HIF-1 target genes VEGF and EPO
In both conditions, an increase in VEGF mRNA expression was detected (RT-PCR) 30 min after the end of exercise, reaching peak expression at 120 min after exercise. At this time point, VEGF mRNA was three times higher in the R- than in the NR-condition (Fig. 2 B, P<0.01). There were no changes in VEGF mRNA steady-state levels in the control group subjected to biopsies only (Fig. 2B ). EPO mRNA was significantly increased 360 min after exercise (Fig. 2C ) with no difference between the two conditions. These findings show that exercise induces expression of HIF-1-regulated target genes.

View larger version (18K): [in this window] [in a new window]   Figure 2. A) Venous plasma lactate levels (mean±SE) in antecubitan vein during exercise with and without blood flow restriction. P < 0.05 = difference (preexercise vs. 15’) between restricted (R) and nonrestricted (NR) conditions, n = 9. B) Real time PCR analyses. Exercise-induced increase in VEGF mRNA levels (mean±SE) in nonrestricted (NR) and restricted (R) blood flow conditions. P < 0.05, difference (preexercise vs. 2 h) between R and NR conditions. *P < 0.05 = difference between preexercise and exercise within the same conditions (preexercise vs. 2 h), n= 9. C) Real time PCR analyses. Exercise-induced increase in Epo mRNA levels (mean±SE) in nonrestricted (NR) and restricted (R) blood flow conditions. **P < 0.05 = difference (preexercise vs. 6 h) independent of exercise conditions, n = 9.

4. von Hippel-Lindau tumor suppressor protein (pVHL) decreases after exercise in some subjects
VHL protein decreased markedly in two subjects immediately after exercise in both conditions. In four additional subjects, the effect was not as pronounced but was lower after exercise when compared with before exercise (data not shown). An interesting observation was that those subjects who demonstrated the largest increase in HIF-1 protein levels demonstrated the strongest down-regulation of pVHL protein levels. There was no exercise-induced change in VHL mRNA levels in either of the two conditions (Fig. 2A ).

To the best of our knowledge, this is the first data showing that VHL can be regulated at the protein level.

CONCLUSIONS

Despite a growing number of studies demonstrating HIF-1 activation under pathological conditions such as solid tumor development and ischemic organ damage, the physiological responsiveness of the human HIF-1 system has not been thoroughly explored. Here we show, for the first time, an up-regulation of the HIF-1 protein and its target gene expression in human skeletal muscle during physical exercise. These observations demonstrate a possible involvement of the HIF-1 system in the physiological response to acute changes in oxygen demands in human tissue.

We detected no changes in HIF-1 mRNA levels in response to exercise, suggesting that the observed acute up-regulation in HIF-1 protein levels mainly depended on post-transcriptional mechanisms. In skeletal muscle it is well known that exercise produces a substantial reduction in extracellular (venous) and intracellular PO₂. It is therefore plausible that an up-regulation of HIF-1 protein in human skeletal muscle might be induced by exercise-dependent reduction in oxygen tension. HIF-1 postexercise protein levels did not differ between the two conditions despite the differences in lactate levels indicating a lower oxygen tension in the R condition compared with the NR condition. This finding may suggest that hypoxia is already low enough to fully stabilize HIF-1 in the NR condition. On the other hand, there are several other regulatory mechanisms that may help to modulate HIF-1 stabilization. Therefore, other exercise-induced alterations in intramuscular adenine nucleotide metabolism, pH, and/or redox potential may have been involved.

An interesting observation was that those subjects who demonstrated the largest increase in HIF-1 protein levels demonstrated a clear down-regulation of VHL protein levels, although no changes in VHL mRNA levels were detected. This raises the possibility that VHL may be down-regulated as part of a negative feedback mechanism. Such a mode of regulation further illustrates the complexity in hypoxia signaling and may significantly impact HIF-1 protein levels and function in cells where VHL is limiting.

In the present study, we observed under physiological conditions not only stabilization of HIF-1 protein levels but also nuclear translocation of the protein as well as induced HIF-1 DNA binding activity. This is a novel finding in human tissue. These data clearly demonstrate that exercise activates HIF-1 activity and downstream function in human skeletal muscle.

In agreement with this conclusion, we observed that exercise-induced expression of HIF-1-regulated target genes. Thus, we observed exercise-induced expression of VEGF and EPO mRNA levels. The increased levels of VEGF mRNA expression peaked at 2 h postexercise, which agrees well with the time frame that is usually required for exercise-induced transcriptional activation. Repetitive biopsies taken in a control experiment did not influence VEGF mRNA. VEGF mRNA expression increases after acute exercise have been reported earlier. The present study represents, however, the first study in humans showing significantly greater VEGF mRNA expression in skeletal muscle when blood flow and oxygen delivery are reduced. Since the HIF-1 postexercise protein levels did not differ between the two conditions, the enhanced VEGF expression in the ischemic condition may depend on mechanisms unrelated to HIF-1. Human skeletal muscle EPO mRNA levels were induced by exercise. However, there was no difference between the elevated EPO mRNA levels in the two models of exercise, arguing that the observed difference in VEGF mRNA levels is specific for this particular gene product. The role of EPO in skeletal muscle physiology is unknown. Local production of EPO outside the kidney and liver has recently been reported in ischemic CNS preconditioning models as well as following angiogenic stimulation of the uterus. In a similar fashion, EPO may induce these effects in skeletal muscle.

The current study is the first to show that several components of the HIF-1 pathway are activated in response to acute changes in oxygen demand in healthy human skeletal muscle. Our results support the hypothesis that oxygen sensitive pathways could be important for physical activity adaptation processes (such as angiogenesis) in human skeletal muscle. This report provides the first evidence that human HIF-1 can be activated under physiological conditions, supporting the general importance of this pathway.

View larger version (20K): [in this window] [in a new window]   Figure 3. HIF-1 activation in exercising human skeletal muscle. In normoxia/rest (A), we found low HIF-1 levels, lending support to the idea that HIF-1 is hydroxylated by a prolyl hydroxylase recognized by VHL, a ubiquitin-protein ligase, and targeted for degradation by the proteasome. Exercise (B) increases oxygen consumption and reduces oxygen tension to levels that inhibit prolyl hydroxylase, resulting in accumulation of the HIF-1 protein and translocation into the nucleus. In the nucleus, HIF-1 and ARNT (HIF-1ß) dimerized and activated target genes such as VEGF and EPO. No further activation of HIF-1 was observed when oxygen delivery to the exercising muscle was reduced (C). mRNA levels for VEGF (but not EPO) were significantly higher when blood flow to the exercising leg was restricted.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2304fje; doi: 10.1096/fj.04-2304fje

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