Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice

Abstract

Temporal expression of positive and negative angiogenic factors in response to detraining is poorly understood. We report the protein expression of anti-angiogenic peptides (thrombospondin-1, TSP-1; and endostatin) as well as pro-angiogenic factors (vascular endothelial growth factor, VEGF; matrix metalloproteinases-2 and -9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) in response to detraining in triceps surae muscles of C57BL/6 mice. Male mice were allowed to exercise voluntarily for 21 days, and then basal and acute response to exercise were evaluated at 1, 7, 14 and 28 days detraining (D1, D7, D14, D28, respectively, n = 12/group). As seen in the D1 mice, training resulted in the increased muscle capillary-to-fibre ratio (C/F), increased maximal running time and elevated basal expression of VEGF and matrix metalloproteinase-9 (P < 0.05). After 7 days of detraining (D7), C/F levels were similar to control levels, but both basal VEGF and TSP-1 were elevated (P < 0.05). At D14 and D28, TSP-1 protein was not different compared to baseline levels; however, VEGF was elevated in gastrocnemius (GA), but not the soleus (SOL) or plantaris (PLT) muscles, of D14 mice. Endostatin tended to decrease in D14 and D28 compared to controls. Timing of nucleolin protein expression differed between muscle groups, with increases at D1, D7 and D14 in the PLT, SOL and GA muscles, respectively. The response of VEGF and nucleolin to acute exercise was blunted with training, and remained blunted in the PLT and SOL even after 28 days of detraining, at a time point long after muscle capillarization was observed to be similar to pre-training levels. These data suggest that TSP-1 may be a mediator of capillary regression with detraining, even in the face of elevated VEGF, suggesting that pro-angiogenic regulators may not be able to prevent the regression of skeletal muscle capillaries under physiological conditions. The responses of matrix metalloproteinases, endostatin and nucleolin poorly correlated with detraining-induced capillary regression.

Figure 1

Time line depicting study design Acute ex = 1 h acute exercise on rodent treadmill at 20 m min⁻¹, 10° incline. Following the acute exercise bout, mice were killed 4 h later to assess expression of angioregulatory factors in response to an exercise stimulus. Control mice (not shown in figure) did not have access to running wheels and therefore served as untrained controls.

Figure 2

Maximal running test showing aerobic exercise capacity before training (pre-training), post-training and after detraining in male mice The maximal run test is measured using a standardized incremental maximal exercise protocol on a rodent treadmill. *P < 0.05 compared to pre-training. All data are n = 12/group and are represented as mean ± s.e.m.

Figure 3

Basal gastrocnemius muscle protein expression of VEGF, TSP-1, MMP-2, MMP-9, endostatin and nucleolin Control mice did not train, all other groups of mice voluntarily trained on running wheels for 21 days and subsequently detrained for 1, 7, 14 or 28 days. *P < 0.05 compared to control. All data are n = 6 male mice per group and are represented as mean ± s.e.m., arbitary densitometry units (au) from Western blot analysis; D, days detrained; MMP, matrix metalloproteinase; TSP, thrombospondin; VEGF, vascular endothelial growth factor.

Figure 4

Basal plantaris muscle protein expression of VEGF, TSP-1, MMP-2, MMP-9, endostatin and nucleolin Control mice did not train, all other groups of mice voluntarily trained on running wheels for 21 days and subsequently detrained for 1, 7, 14 or 28 days. *P < 0.05 compared to control. All data are n = 6 male mice per group and are represented as mean ± s.e.m., arbitary densitometry units (au) from Western blot analysis; D, days detrained; MMP, matrix metalloproteinase; TSP, thrombospondin; VEGF, vascular endothelial growth factor.

Figure 5

Basal soleus muscle protein expression of VEGF, TSP-1, MMP-2, MMP-9, endostatin and nucleolin Control mice did not train, all other groups of mice voluntarily trained on running wheels for 21 days and subsequently detrained for 1, 7, 14 or 28 days. *P < 0.05 compared to control. All data are n = 6 male mice per group and are represented as mean ± s.e.m., arbitary densitometry units (au) from Western blot analysis; D, days detrained; MMP, matrix metalloproteinase; TSP, thrombospondin; VEGF, vascular endothelial growth factor.

Figure 6

Protein expression in response to acute exercise for VEGF, endostatin and nucleolin in the gastrocnemius, plantaris and soleus compared to basal expression reported in Figs 3–5, respectively Data reflect protein expression 4 h postacute exercise. *P < 0.05 compared to basal in same detrained. VEGF, vascular endothelial growth factor .