Skeletal myofiber VEGF is essential for the exercise training response in adult mice

Abstract

Vascular endothelial growth factor (VEGF) is exercise responsive, pro-angiogenic, and expressed in several muscle cell types. We hypothesized that in adult mice, VEGF generated within skeletal myofibers (and not other cells within muscle) is necessary for the angiogenic response to exercise training. This was tested in adult conditional, skeletal myofiber-specific VEGF gene-deleted mice (skmVEGF-/-), with VEGF levels reduced by >80%. After 8 wk of daily treadmill training, speed and endurance were unaltered in skmVEGF-/- mice, but increased by 18% and 99% (P < 0.01), respectively, in controls trained at identical absolute speed, incline, and duration. In vitro, isolated soleus and extensor digitorum longus contractile function was not impaired in skmVEGF-/- mice. However, training-induced angiogenesis was inhibited in plantaris (wild type, 38%, skmVEGF-/- 18%, P < 0.01), and gastrocnemius (wild type, 43%, P < 0.01; skmVEGF-/-, 7%, not significant). Capillarity was maintained (different from VEGF gene deletion targeted to multiple cell types) in untrained skmVEGF-/- mice. Arteriogenesis (smooth muscle actin+, artery number, and diameter) and remodeling [vimentin+, 5'-bromodeoxycytidine (BrdU)+, and F4/80+ cells] occurred in skmVEGF-/- mice, even in the absence of training. skmVEGF-/- mice also displayed a limited oxidative enzyme [citrate synthase and β-hydroxyacyl CoA dehydrogenase (β-HAD)] training response; β-HAD activity levels were elevated in the untrained state. These data suggest that myofiber expressed VEGF is necessary for training responses in capillarity and oxidative capacity and for improved running speed and endurance.

Keywords: angiogenesis; exercise; metabolism; peripheral vascular disease.

Fig. 1.

VEGF levels in hindlimb muscles from wild-type and skeletal myofiber VEGF gene-ablated mice. VEGF protein in the soleus (A), gastrocnemius (B), plantaris (C), and extensor digitorum longus (EDL) from VEGF+/+ and skeletal myofiber-specific VEGF gene-deleted (skmVEGF−/−) mice (D) that were either exercise trained (TRAINED) or remained cage-confined (UNTRAINED). Values are means ± SE, P < 0.01. *P indicates a significant difference between genotype within the same UNTRAINED or TRAINED group, #P indicates a significant difference between exercise condition within the same genotype.

Fig. 2.

Treadmill exercise capacity. Maximal speed (A) and endurance (B) were tested in VEGF+/+ and skmVEGF−/− mice at the following time points: pre-tamoxifen (TAM) (day 0), 21 days post-tamoxifen to conditionally delete the VEGF gene, and 8 wk after cage confinement or exercise training (day 78). Values are means ± SE, P < 0.01. *P indicates a significant difference between genotype within the same untrained or exercissed group, #P indicates a significant difference after exercise training within the same genotype (n = 9–10).

Fig. 3.

Ex vivo soleus and EDL contractile muscle function. Total (A, C) and relative (B, D) force-frequency measurements from the soleus (A and B) and EDL (C and D). Values are means ± SE, n = 6–9 P < .05. *P indicating significant difference between skmVEGF−/− exercised mice and all groups. E: time to fatigue (seconds) was recorded in soleus and EDL. Values are the means ± SE, n = 6–9 *Difference between the skmVEGF−/− exercised mice and all other groups (P < 0.01).

Fig. 4.

Vascular remodeling in skmVEGF−/− mice. Confocal images of 5′-bromodeoxycytidine (BrdU)+ and vimentin+ (VIM) cells (A), α-smooth muscle actin+ (SMA) vessels (B), and F4/F80+ (F4/80) macrophages (C) in the gastrocnemius. The total number of SMA+ arteries per cross-sectional area (mm²) in the gastrocnemius were counted in each group (D). The distribution of arterial diameters (μm) are represented as the percentage of the total number counted (E). DAPI, 4′,6-diamidino-2-phenylindole. Values are means ± SE, P < 0.01. *P indicates a significant difference between genotype within the same UN or EX group, #P indicates a significant difference after exercise training within the same genotype (n = 4).

Fig. 5.

Metabolic enzyme activity. Citrate synthase (CS) (A), β-hydroxyacyl CoA dehydrogenase (β-HAD) (B), and phosphofructokinase (PFK) (C) activites (μmol·mg⁻¹·min⁻¹) in the soleus, plantaris, gastrocnemius, and EDL are shown. Values are means ± SE, P < 0.01. *P indicates a significant difference between genotype within the same unexercised or exercised group, #P indicates a significant difference after exercise training within the same genotype (n = 4–9).