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. 2016 Jul 1;311(1):R192-9.
doi: 10.1152/ajpregu.00533.2015. Epub 2016 May 25.

Skeletal myofiber VEGF regulates contraction-induced perfusion and exercise capacity but not muscle capillarity in adult mice

Amy E Knapp  1 , Daniel Goldberg  1 , Hamid Delavar  1 , Breanna M Trisko  1 , Kechun Tang  1 , Michael C Hogan  1 , Peter D Wagner  1 , Ellen C Breen  2
Affiliations

Skeletal myofiber VEGF regulates contraction-induced perfusion and exercise capacity but not muscle capillarity in adult mice

Amy E Knapp et al. Am J Physiol Regul Integr Comp Physiol. .

Abstract

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.

Keywords: exercise; fatigue; perfusion; skeletal myofiber; vascular endothelial growth factor.

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Figures

Fig. 1.
Fig. 1.
Vascular endothelial growth factor (VEGF) protein levels in hind limb skeletal muscles. VEGF levels were measured by ELISA in muscles collected from skmVEGF−/− and VEGF+/+ mice. A: 23 days post-tamoxifen. B: 58 days post-tamoxifen. Values are expressed as means ± SE; n = 11–16 for gastrocnemius, n = 5–6 for soleus, and n = 3–6 for extensor digitorum longus (EDL). *Significant difference compared with VEGF+/+ mice (P < 0.001).
Fig. 2.
Fig. 2.
Post-tamoxifen VEGF protein levels in heart, lung, kidney, and brain. As expected, there were no differences in VEGF levels between the two groups in heart, lung, kidney, or brain. Values are expressed as means ± SE; n = 6.
Fig. 3.
Fig. 3.
Pre- and post-tamoxifen maximum running speeds, showing a small but significant reduction after VEGF gene deletion. A: 21 days post-tamoxifen. B: 56 days post-tamoxifen. Values are expressed as means ± SE; n = 6–10. *Significant difference between VEGF+/+ and the skeletal myofiber-specific VEGF gene deletion mouse (skmVEGF−/−) at the post-tamoxifen time point (P < 0.01).
Fig. 4.
Fig. 4.
Impaired endurance exercise capacity in skeletal myofiber VEGF gene-deleted mice. A: 22 days following tamoxifen. B: 57 days following tamoxifen. Values are expressed as means ± SE; n = 6–10. *Significant difference between VEGF+/+ and skmVEGF−/− at the post-tamoxifen time point (P < 0.05).
Fig. 5.
Fig. 5.
Loss of myofiber VEGF does not alter the skeletal muscle capillary-to-fiber ratio. A: 23 days post-tamoxifen. B: 58 days post-tamoxifen. Values are expressed as means ± SE; n = 4–6. No differences were found in any muscle at either time.
Fig. 6.
Fig. 6.
Contractile properties in isolated EDL and soleus muscles from skeletal myofiber-specific gene-ablated mice. Force was measured as a function of stimulation frequency to assess the contractile function of each muscle. A: soleus 21 days following tamoxifen (n = 4–6 muscles per group). B: EDL 21 days following tamoxifen (n = 5 or 6 muscles per group). C: soleus 56 days following tamoxifen (n = 5–7 muscles per group). D: EDL 56 days following tamoxifen (n = 6–7 muscles per group). The EDL muscle, 56 days after tamoxifen delivery, showed a significant right shift in the force-frequency curve. There were no significant differences in any other muscle between skmVEGF−/− and VEGF+/+. Values are expressed as means ± SE. *Significant difference (P < 0.05) between skmVEGF−/− and VEGF +/+ muscles.
Fig. 7.
Fig. 7.
Gastrocnemius complex fatigue and perfusion monitored in situ is inhibited in mice with skeletal myofiber-targeted VEGF gene deletion. A: time to fatigue was measured in the gastrocnemius complex electrically stimulated to contract via the sciatic nerve. Values are expressed as means ± SE; n = 5 or 6. *P < 0.02. B: real-time measurements of muscle perfusion were collected simultaneously by optically imaging electrically stimulated (ES) and resting (R) gastrocnemius complexes in mice delivered near-infrared fluorescent nanospheres during (ES) and after (Post-ES) periods of muscle contraction. Nanospheres were delivered at a constant rate over the first 2 min. The muscle was stimulated to contract 30 s into the delivery period (arrow). Data are represented as the average over 30-s intervals of the ratio of fluorescence in the stimulated (ES) hind limb muscles to the nonstimulated (Rest) in the same mouse over time. Values are expressed as means ± SD; n = 5 VEGF+/+ mice, and n = 5 skmVEGF−/− mice. *P < 0.0001 between genotypes.
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