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.

A single bout of exhaustive exercise signals expression of vascular endothelial growth factor (VEGF) in the exercising muscle. Previous studies have reported that mice with life-long deletion of skeletal myofiber VEGF have fewer capillaries and a severe reduction in endurance exercise. However, in adult mice, VEGF gene deletion conditionally targeted to skeletal myofibers limits exercise capacity without evidence of capillary regression. To explain this, we hypothesized that adult skeletal myofiber VEGF acutely regulates skeletal muscle perfusion during muscle contraction. A tamoxifen-inducible skeletal myofiber-specific VEGF gene deletion mouse (skmVEGF-/-) was used to reduce skeletal muscle VEGF protein by 90% in adult mice. Three weeks after inducing deletion of the skeletal myofiber VEGF gene, skmVEGF-/- mice exhibited diminished maximum running speed (-10%, P < 0.05) and endurance capacity (-47%; P < 0.05), which did not persist after 8 wk. In skmVEGF-/- mice, gastrocnemius complex time to fatigue measured in situ was 71% lower than control mice. Contraction-induced perfusion measured by optical imaging during a period of electrically stimulated muscle contraction was 85% lower in skmVEGF-/- than control mice. No evidence of capillary rarefication was detected in the soleus, gastrocnemius, and extensor digitorum longus (EDL) up to 8 wk after tamoxifen-induced VEGF ablation, and contractility and fatigue resistance of the soleus measured ex vivo were also unchanged. The force-frequency of the EDL showed a small right shift, but fatigue resistance did not differ between EDL from control and skmVEGF-/- mice. These data suggest myofiber VEGF is required for regulating perfusion during periods of contraction and may in this manner affect endurance capacity.

VEGF plays an essential role in regulating angiogenesis and clearance of apoptotic cells. A loss of skeletal myofiber expressed VEGF inhibits exercise‐induced angiogenesis. We hypothesized that skeletal myofiber VEGF deficient mice would increase fibroblast proliferation in hind limb muscles; especially following repeated bouts of exercise.MethodsUsing an HSA‐Cre‐ERT2 X VEGFLoxP (skmVEGF−/−) mouse model, VEGF levels are reduced by >;80% in skeletal muscles of adult mice compared to control littermates (WT). 8 week exercise trained (EX) and untrained (UT) mice were administered BrdU daily during the final week. Proliferating cells (BrdU+) and fibroblasts (vimentin+) were detected by confocal microscopy.ResultsTotal BrdU+ cells/myofiber increased in UT and EX skmVEGF−/− mice compared to the WT‐UT and WT‐EX groups, respectively, in the plantaris (WT‐UT, 6%, skmVEGF−/−UT, 32%, WT‐EX, 37%, skmVEGF−/−EX, 47%) and gastrocnemius (WT‐UT, 2%, skmVEGF−/−UT, 43%, WT‐EX, 25%, skmVEGF−/−EX, 43%). Exercise training, alone, also increased BrdU incorporation in WT muscles. Gastrocnemius sections from exercise trained skmVEGF−/− mice displayed fibrotic foci containing numerous vimentin+/BrdU+ cells. These data suggest that VEGF may prevent muscle injury and fibrosis during exercise training.

In adult mice, the importance of vascular endothelial growth factor (VEGF) for maintaining skeletal muscle capillaries is unclear. We hypothesized that deletion of the VEGF gene, targeted simultaneously to both endothelial cells and myofibers, would lead to a loss of capillaries in locomotor muscles.MethodsCapillary to fiber ratio (C:F), fiber‐cross sectional area (FCSA), and fiber‐type composition were measured in soleus, plantaris and gastrocnemius cryo‐sections from wild type (WT) and HSA‐Cre‐ERT2 X VEGFLoxP x PDGFb‐iCre‐ERT2 (EC+MyoVEGF−/−) mice. C:F was decreased in soleus from EC+MyoVEGF−/− mice compared to WT (WT, 1.64 ± 0.19, EC+MyoVEGF−/− 1.20 ± 0.19, n=3–9, p < 0.05). FCSA increased in both EC+MyoVEGF−/− soleus and plantaris (soleus: WT, 1953 ± 347 μm2, EC+MyoVEGF−/−, 3148 ± 543 μm2, plantaris: WT, 1731 ± 189 μm2, EC+MyoVEGF−/−, 3261 ± 803 μm2, n=3–9, p < 0.05). EC+MyoVEGF−/− mice also revealed a shift from type IIB to type IIA fibers in the plantaris (type IIB: WT, 66 ± 7 %, EC+MyoVEGF−/−, 48 ± 8 %, type IIA: WT, 19 ± 4%, EC+MyoVEGF−/−, 36 ± 16%, n=3–9, p < 0.05). Unlike previously reported studies in adult skeletal myofiber VEGF‐deficient mice, capillary regression does occur in adult skeletal muscle when VEGF expression is inhibited in both endothelial cells and myofibers.

Functional overload (FO) stimulates muscle growth and increases strength and endurance. Vascular endothelial growth factor (VEGF) is essential for exercise‐induced angiogenesis. We hypothesized VEGF expressed by skeletal myofibers is critical for muscle growth and improvements in force production and fatigue resistance. To test this hypothesis, adult mice with conditional skeletal myofiber specific VEGF gene deletion (skmVEGF‐/‐) and wild type (WT) littermates underwent functional overload (FO) of the plantaris (removal of gastrocnemius and soleus) or sham surgery. In vivo plantaris isometric force and fatigue resistance (% of maximal force after 10 contractions) were measured 7 or 14d after FO or sham surgery with a dual mode foot plate system in anesthetized skmVEGF‐/‐ or WT mice. Mass increased ~ 35% in both 14d‐FO groups compared to sham (p<0.01). In WT mice subjected to FO, maximal isometric force was unchanged at 7d and increased at 14d (sham, 0.37±0.03; FO, 0.46±0.04 g/mg body mass, p<0.05). In skmVEGF‐/‐ mice, FO was associated with a decrease in maximal force after 7d (sham, 0.39±0.0; FO, 0.23±0.03 g/mg body mass, p<0.05) and 14d (sham, 0.39±0.03; FO, 0.27±0.04 g/mg body mass, p<0.05). Fatigue resistance was not different between groups at 7d. After 14d of FO, fatigue resistance increased from 58±3%‐sham to 73±2%‐FO of maximal force in WT mice (p<0.05) and was unchanged in skmVEGF‐/‐ mice. These data suggest that VEGF‐dependent functions are essential for improving muscle strength and fatigue resistance, independent of muscle growth, in response to a hypertrophic stimulus. Grant Funding Source: Supported by NIH R15AR060469