Muscle-specific VEGF deficiency greatly reduces exercise endurance in mice

Abstract

Vascular endothelial growth factor (VEGF) is required for vasculogenesis and angiogenesis during embryonic and early postnatal life. However the organ-specific functional role of VEGF in adult life, particularly in skeletal muscle, is less clear. To explore this issue, we engineered skeletal muscle-targeted VEGF deficient mice (mVEGF-/-) by crossbreeding mice that selectively express Cre recombinase in skeletal muscle under the control of the muscle creatine kinase promoter (MCKcre mice) with mice having a floxed VEGF gene (VEGFLoxP mice). We hypothesized that VEGF is necessary for regulating both cardiac and skeletal muscle capillarity, and that a reduced number of VEGF-dependent muscle capillaries would limit aerobic exercise capacity. In adult mVEGF-/- mice, VEGF protein levels were reduced by 90 and 80% in skeletal muscle (gastrocnemius) and cardiac muscle, respectively, compared to control mice (P < 0.01). This was accompanied by a 48% (P < 0.05) and 39% (P < 0.05) decreases in the capillary-to-fibre ratio and capillary density, respectively, in the gastrocnemius and a 61% decrease in cardiac muscle capillary density (P < 0.05). Hindlimb muscle oxidative (citrate synthase, 21%; beta-HAD, 32%) and glycolytic (PFK, 18%) regulatory enzymes were also increased in mVEGF-/- mice. However, this limited adaptation to reduced muscle VEGF was insufficient to maintain aerobic exercise capacity, and maximal running speed and endurance running capacity were reduced by 34% and 81%, respectively, in mVEGF-/- mice compared to control mice (P < 0.05). Moreover, basal and dobutamine-stimulated cardiac function, measured by transthoracic echocardiography and left ventricular micromanomtery, showed only a minimal reduction of contractility (peak +dP/dt) and relaxation (peak -dP/dt, tau(E)). Collectively these data suggests adequate locomotor muscle capillary number is important for achieving full exercise capacity. Furthermore, VEGF is essential in regulating postnatal muscle capillarity, and that adult mice, deficient in cardiac and skeletal muscle VEGF, exhibit a major intolerance to aerobic exercise.

Figure 1. Protein expression of VEGF in skeletal muscle, heart and other body organs in 5-month old muscle VEGF-deficient (mVEGF−/−) and wild-type (WT) control mice

Data are means ±s.e.m.n= 5–7/group. *Significantly different compared to WT (P < 0.05).

Figure 2. Protein expression of VEGF receptor 1 & 2

Data are means ±s.e.m. A, protein expression of VEGF receptor 1, i.e. flt-1, measured from the samples in Fig. 1A. B, protein expression of VEGF receptor 2, i.e. flk-1, measured from the samples in Fig. 1A. *Significantly different compared to WT (P < 0.05).

Figure 3. Alkaline phosphatase staining of muscle sections obtained from deep and superficial areas within the gastrocnemius muscle of wild-type (WT) control and muscle VEGF-deficient (mVEGF−/−) mice

A, representative images showing alkaline phosphatase stained muscle sections obtained from deep and superficial areas within the gastrocnemius muscle of wild-type (WT) control and muscle VEGF deficient (mVEGF−/−) mice. Images depicted are transverse sections magnified at 40× (bar = 50 μm). Capillaries appear dark purple (or black) and muscle fibres are yellow stained. B, morphometric analysis of pooled (deep and superficial) data from gastrocnemius muscle of mVEGF−/− and WT mice. Data show differences in capillary-to-fibre ratio and capillary density, but no difference in the average muscle fibre cross-sectional area between mVEGF−/− and WT mice. Data are means ±s.e.m. *Significantly different compared to WT (P < 0.05).

Figure 5. Haemodynamic data obtained from left ventricular micromanometer catheterization of muscle VEGF-deficient (mVEGF−/−) and wild-type (WT) control mice under general anaesthesia

Data are means ±s.e.m. mVEGF−/−, n= 7; WT, n= 5. ANOVA results identify P-valve for main effect of genotype obtained from two-way ANOVA (comparing genotype and drug dose).

Figure 4. Alkaline phosphatase staining of cardiac muscle sections and average capillary density for wild-type (WT) control and muscle VEGF-deficient (mVEGF−/−) mice

A, representative images showing alkaline phosphatase stained cardiac muscle sections from WT and mVEGF−/− mice. Images shown at 12.5× magnification (Bar = 5 μm). Capillaries appear as dark purple stained. B, average capillary density (number of capillary per mm²) in WT and VEGF deficient (mVEGF−/−) mice (n= 3/group). Data are means ±s.e.m. *Significantly different compared to WT (P < 0.05).