Temporal response of positive and negative regulators in response to acute and chronic exercise training in mice

Abstract

Angiogenesis is controlled by a balance between positive and negative angiogenic factors, but temporal protein expression of many key angiogenic regulators in response to exercise are still poorly defined. In C57BL/6 mice, we evaluated the temporal protein expression of several pro-angiogenic and anti-angiogenic factors in response to (1) a single acute bout of exercise and (2) chronic exercise training resulting from 3, 5, 7, 14 and 28 days of voluntary wheel running. Following acute exercise, protein levels of vascular endothelial growth factor-A (VEGF), endostatin and nucleolin were increased at 2-4 h (P < 0.05), whereas matrix metalloproteinase (MMP)-2 was elevated within a 12-24 h window (P < 0.05). Training increased muscle capillarity 11%, 15% and 22% starting with 7, 14 and 28 days of training, respectively (P < 0.01). Basal VEGF and MMP-2 were increased by 31% and 22%, respectively, compared to controls (P < 0.05) after 7 days (7d) training, but decreased to back to baseline after 14d training. After 28d training VEGF fell 49% below baseline control (P < 0.01). Basal muscle expression of thrombospondin 1 (TSP-1) was ∼900% greater in 14d- and 28d-trained mice compared to either 5d- and 7d-trained mice (P < 0.05), and tended to increase by ∼180-258% compared to basal control levels (P < 0.10). The acute responsiveness of VEGF to exercise in untrained mice (i.e. 161% increase, P < 0.001) was lost with capillary adaptation occurring after 7, 14 and 28d training. Taken together, these data support the notion that skeletal muscle angiogenesis is controlled by a balance between positive and negative mitogens, and reveals a complex, highly-coordinated, temporal scheme whereby these factors can differentially influence capillary growth in response to acute versus chronic exercise.

Figure 1. Protein time course expression of VEGF, MMP-2, MMP-9, TSP-1, endostatin and nucleolin following a single bout of acute exercise measured by ELISA or Western blot analysis

Control mice did not exercise; all others ran for 1 h (at 20 m min⁻¹, 10 deg incline) and were killed immediately (0), 2, 4, 6, 12 or 24 h post exercise (n= 4 mice each group). A.U. represents arbitrary densitometry units representing Western blot analysis for which each factor has been normalized to the housekeeping gene GAPDH and/or β-tubulin. *Significantly different compared to basal control group, P < 0.05; $ signifies P≤ 0.10 compared to basal control.

Figure 2

Data showing group mean (±SEM) data for daily distance, daily running speed (km h⁻¹) and daily maximal running speed (km h⁻¹) voluntarily performed by mice with 24 h access to in-cage running wheels.

Figure 3

Gastrocnemius muscle capillary-to-fibre ratio and capillary density in control, untrained, mice and in mice that voluntarily trained for 3, 5, 7, 14 or 28 days (n= 6 per group). *P < 0.05 and **P < 0.01 compared to control.

Figure 4. Time course of protein expression of VEGF, MMP-2, MMP-9, nucleolin, endostatin and TSP-1 following training measured by ELISA or Western blot analysis

Control mice are untrained; all others are voluntarily trained using a running wheel for 3, 5, 7, 14 or 28 days (n= 12 per group). Following training, half of the mice per group (n= 6) were maintained under basal conditions, and the other half (n= 6) performed a single bout of exercise (1 h, 20 m min⁻¹, 10 deg incline) with muscle harvested 4 h post exercise. A.U., arbitrary densitometry units representing Western blot analysis for which each respective factor has been normalized to the housekeeping gene GAPDH. *Significantly different compared to Basal control group, P < 0.05; ‡significant difference between Basal and Acute exercise within the same training group, P < 0.05; †significantly different compared to 3-day-trained Basal levels, P < 0.05; #significantly different compared to 14-day-trained Basal levels, P < 0.05; & significantly different compared to 28-day-trained Basal levels, P < 0.05; $ signifies P≤ 0.10 compared to Basal control.