Angio-adaptation in unloaded skeletal muscle: new insights into an early and muscle type-specific dynamic process

Abstract

With a remarkable plasticity, skeletal muscle adapts to an altered functional demand. Muscle angio-adaptation can either involve the growth or the regression of capillaries as respectively observed in response to endurance training or muscle unloading. Whereas the molecular mechanisms that regulate exercise-induced muscle angiogenesis have been extensively studied, understanding how muscle unloading can in contrast lead to capillary regression has received very little attention. Here we have investigated the consequences of a 9 day time course hindlimb unloading on both capillarization and expression of angio-adaptive molecules in two different rat skeletal muscles. Both soleus and plantaris muscles were atrophied similarly. In contrast, our results have shown different angio-adaptive patterns between these two muscles. Capillary regression occurred only in the soleus, a slow-twitch and oxidative postural muscle. Conversely, the level of capillarization was preserved in the plantaris, a fast-twitch and glycolytic muscle. We have also measured the time course protein expression of key pro- and anti-angiogenic signals (VEGF-A, VEGF-B, VEGF-R2, TSP-1). Our results have revealed that the angio-adaptive response to unloading was muscle-type specific, and that an integrated balance between pro- and anti-angiogenic signals plays a determinant role in regulating this process. In conclusion, we have brought new evidence that measuring the ratio between pro- and anti-angiogenic signals in order to evaluate muscle angio-adaptation was a more accurate approach than analysing the expression of molecular factors taken individually.

Figure 1. Muscle-type differences in capillarization and angio-adaptive proteins

Relative levels of capillarization and angio-adaptive proteins in the plantaris muscle compared to the soleus muscle, under control conditions (i.e. day 0). Values have been normalized to soleus levels. Data are expressed as means ± s.e.m. (n = 6 rats/group). Significant difference between soleus and plantaris muscles: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 2. Hindlimb unloading and skeletal muscle capillarization

A, representative images of capillaries staining in soleus (left) and plantaris (right) muscles from control animals (day 0, top panel) and after 9 days of hindlimb unloading (day 9, bottom panel). Scale bar, 50 μm. B–C, quantitative measurement of capillary density (B) and capillary-to-fibre ratio (C) in soleus (left panel) and plantaris (right panel) muscles following time course hindlimb unloading (HU, from days 0 to 9). Data are expressed as means ± s.e.m. (n = 6 rats/group). Significantly different from day 0: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Significantly different from day 1: ¶P ≤ 0.05; ¶¶P ≤ 0.01; ¶¶¶P ≤ 0.001. Significantly different from day 3: †P ≤ 0.05; ††P ≤ 0.01; †††P ≤ 0.001. Significantly different from day 5: #P ≤ 0.05. Percentage of muscle atrophy (D) and capillary regression (E) based on mean FCSA and C/F values, respectively. Data are expressed as means ± s.e.m. (n = 6 rats/group). FCSA, mean fibre cross-sectional area; C/F, capillary-to-fibre ratio.

Figure 3. VEGF-A, VEGF-B and VEGF receptor-2 protein expression in response to hindlimb unloading

Animals were subjected to a 9-day time course hindlimb unloading. Protein levels were measured by western blotting in soleus and plantaris muscles, left and right panels respectively. β-Tubulin protein was used as a loading control. Top-panels of A, B and C show images of VEGF-A, VEGF-B and VEGF-R2 protein expressions, respectively. Bottom panels of A, B and C show densitometric analyses of VEGF-A, VEGF-B or VEGF-R2 proteins relative to β-tubulin. Densitometric analyses show means ± s.e.m. (n = 6 rats/group). Significant differences: vs. day 0 **P ≤ 0.01; ***P ≤ 0.001; vs. day 0.5: ¶P ≤ 0.05; ¶¶P ≤ 0.01; ¶¶¶P ≤ 0.001; vs. day 1: †P ≤ 0.05; ††P ≤ 0.01; †††P ≤ 0.001; vs. day 3 #P ≤ 0.05; ##P ≤ 0.01; ##P ≤ 0.001; vs. day 7 ‡‡P ≤ 0.01.

Figure 4. TSP-1, p53 and Mdm2 protein expressions in response to hindlimb unloading

Animals were subjected to a 9 day time course hindlimb unloading. Protein levels were measured by Western blotting in soleus and plantaris muscles. β-Tubulin protein was used as a loading control. Top-panels of A, B and C show representative pictures of TSP-1, p53 and Mdm2 protein expressions, respectively. Bottom panels of A, B and C show densitometric analyses of TSP-1, p53 or Mdm2 proteins relative to β-tubulin. Data are means ± s.e.m. (n = 6 rats/group). Significant differences are vs. day 0 *P≤0.05; **P ≤ 0.01; ***P ≤ 0.001; vs. day 0.5 ¶P ≤ 0.05; ¶¶P ≤ 0.01; ¶¶¶P ≤ 0.001; vs. day 1 †P ≤ 0.05; ††P ≤ 0.01; †††P ≤ 0.001; vs. day 3 #P ≤ 0.05; ###P ≤ 0.001; and vs. day 5, $P ≤ 0.05.

Figure 5. Hindlimb unloading and the angioadaptive balance in soleus and plantaris muscles

The ratios between the relative expression of pro-angiogenic proteins (VEGF-A, VEGF-B and VEGF-R2) and the anti-angiogenic TSP-1 proteins were calculated during the time course of hindlimb unloading. A shows VEGF-A to TSP-1 ratio over the time of hindlimb unloading in soleus and plantaris muscles (left and right panels, respectively). Data are means ± s.e.m. (n = 6 rats/group). Significant differences are vs. day 0.5, ¶P ≤ 0.05; and vs. day 1 †P ≤ 0.05. B illustrates the time course of changes in muscle capillarization and in the ratios VEGF-A, VEGF-B, VEGF-R2, or the sum of all pro-angiogenic proteins measured (VEGF-A+VEGF-B+VEGF-R2), relative to TSP-1 in soleus and plantaris (left and right panels, respectively). Ratios at different HU time points were normalized to day 0 (equal to 1.0 arbitrary unit). Muscle capillarization at different HU time points is expressed as a percentage of capillarization at day 0. C, schematic illustration of how the angio-adaptative balance is differently regulated between soleus and plantaris muscles in response to hindlimb unloading.