Exercise-induced expression of vascular endothelial growth factor mRNA in rat skeletal muscle is dependent on fibre type

Abstract

In this study, we quantified the expression of the vascular endothelial growth factor (VEGF) gene in individual muscle fibres at the end of a single 90 min run of 20-25 m min-1, at 10 % incline. In addition, we evaluated the co-ordinated expression of several hypoxia-sensitive genes, including the ORP-150 gene. Individual fibres were taken from rat plantaris muscle, either at the end of a single bout of exercise or at rest, and classified as Type I, IIa, IIx or IIb, according to the expression of myosin heavy chain (MHC) isoforms. VEGF mRNA levels increased by 90 % in exercising whole plantaris in comparison with those in control muscle (P < 0.001), while the VEGF protein content increased by 72 % (P < 0.05). Using real-time PCR analysis, an accurate and reproducible method for quantification of mRNA levels, a marked rise in VEGF transcript levels was observed at the end of exercise in individual myofibres (P < 0.05), providing the first direct evidence that VEGF transcripts increase in muscle cells after a single bout of exercise. This exercise-induced increase in VEGF transcript levels was specifically observed in type IIb myofibres, which are predominantly glycolytic and more susceptible to local hypoxia than oxidative myofibres such as type I or IIa fibres (110 %, P < 0.05). Moreover, treadmill exercise increased the expression of two hypoxia-sensitive genes. The levels of mRNA for Flt-1, a VEGF-specific receptor, and those for ORP-150, a chaperone essential for the secretion of mature VEGF, increased in whole plantaris muscles (108 and 92 %, respectively, P < 0.05). Taken together, these findings are consistent with the suggestion that hypoxia could be one of the mechanisms involved in exercise-induced capillary growth.

Figure 1. Quantitative densitometry of VEGF protein in whole muscle and individual myofibres

A, representative set of Western blots of VEGF protein in rat plantaris muscle. VEGF protein was detected after 10 % SDS-PAGE. a, b and c, samples from plantaris muscle corresponding to 35, 25, and 15 μg of skeletal muscle protein, respectively. d, sample from left ventricle of hypoxic rats intended to produce intense immunoreactive signals needed for determination of the standard curves (15 μg of total protein). e, sample of human recombinant VEGF (15 ng). B, representative set of Western blots of VEGF protein in plantaris muscles taken from control and active rats. C, detection of VEGF protein in pooled myofibres. The immunoreactive band was compared with that obtained in whole muscle. D, quantitative densitometry of VEGF protein in whole plantaris muscle. VEGF is quantified by measuring both the optical density and the surface area of the immunoreactive bands. Data are means ± s.e.m. * Significantly different from control non-exercised plantaris muscles, P < 0.05.

Figure 2. Quantitative analysis of VEGF gene expression by real-time RT-PCR

VEGF gene expression in whole plantaris muscle (A) and in individual myofibres (B) from control non-exercised (n = 6) and active rats (n = 6). Data are means ± s.e.m. * Significantly different from control values, P < 0.05, ** Significantly different from control values, P < 0.001.

Figure 3. Quantitative analysis of VEGF gene expression by real-time RT-PCR in single myofibres from control non-exercised and active rats

Individual fibres were negative for PECAM mRNA signal and were categorised into four types, type I, IIa, IIx and IIb, according to their MHC isoform content (Delp & Duan, 1996). Data are means ± s.e.m. * Significantly different from control values, P < 0.05.

Figure 4. Quantitative densitometry for the ratios of Flt-1/cycA and Flk-1/cycA mRNA

Quantitative densitometry for Flt-1/cycA (A) and Flk-1/cycA (B) mRNA ratio in whole plantaris muscle of rats at rest (control, n = 6) and following 90 min of treadmill exercise (active, n = 6). Gene expression was examined by real-time RT-PCR. Data are means ± s.e.m. * Significantly different from control values, P < 0.05.

Figure 5. Quantitative densitometry for the ratios of ORP-150/cycA and HIF-1α/cycA mRNA

Quantitative densitometry for ORP-150/cycA and HIF-1α/cycA mRNA ratios in whole plantaris muscle of rats at rest (control, n = 6) and following 90 min of treadmill exercise (active, n = 6). Gene expression was examined by real-time RT-PCR. Data are means ± s.e.m. * Significantly different from control values, P < 0.05.