Date Published: October 24, 2004
Publisher: Public Library of Science
Author(s): Steven D Mason, Richard A Howlett, Matthew J Kim, I. Mark Olfert, Michael C Hogan, Wayne McNulty, Reed P Hickey, Peter D Wagner, C. Ronald Kahn, Frank J Giordano, Randall S Johnson
Abstract: The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1α (HIF-1α). To determine the role of HIF-1α in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1α knockout mice (HIF-1α KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1α KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1α KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1α KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1α KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function.
Partial Text: During exercise in normoxia, the partial pressure of oxygen in muscle tissue has been shown to dip to as low as 3.1 mm Hg, whereas in the capillary, it remains at 38 mm Hg (Hoppeler et al. 2003). In order to maintain effort, skeletal muscle exertion must be able to rely on pathways designed to help the tissue cope with oxygen stress after oxygen delivery capacity is exceeded. A switch between aerobic and nonaerobic metabolism during strenuous exertion requires mechanisms to adjust metabolic function, and this need is acute in extended exertion in skeletal muscle. It is clear that the transcription factor hypoxia-inducible factor 1α (HIF-1α) is an essential factor in maintenance of ATP levels in cells (Seagroves et al. 2001). In fact, although HIF-1α is typically thought of as acting only during hypoxia, its loss has an effect on both normoxic and hypoxic ATP levels in a number of tissue types (Seagroves et al. 2001; Cramer et al. 2003), and this implicates the factor in regulation of metabolic function even during conditions of normal physiologic oxygenation.
In 4-mo–old mice with the skeletal-muscle HIF-1α gene knocked out (HIF-1α KOs), the frequency of excision was evaluated through real-time PCR techniques. We saw deletion frequencies consistent with those described previously for this cre recombinase transgene (Bruning et al. 1998) with some variation in penetration; mean frequency of deletion was 54.9%, with the highest frequency of muscle-specific deletion of HIF-1α being 72% in the gastrocnemius of 4-mo–old mice homozygous for the loxP-flanked allele (Table 1). This transgene is expressed at a lower level in cardiac tissue, and cardiac deletion was detected (Table 1); however, none of the phenotypes described below were seen in cardiac myocyte-specific deletions of HIF-1α (Figure 1A). Gross muscle sections were evaluated histologically to evaluate both vascularization and fiber type (Tables 2 and 3), and ultrastructurally to determine number of mitochondria (Figure 1B). No changes were detected in any of these features in HIF-1α KOs, except for a slight but statistically significant decrease in type IIA fibers in the soleus muscles (Table 3). Similar hematocrit and blood hemoglobin levels were seen in HIF-1α KOs and wild-type (WT) mice (Figure 2).