(-)-Epicatechin combined with 8 weeks of treadmill exercise is associated with increased angiogenic and mitochondrial signaling in mice

Abstract

The purpose of this study was to conduct an 8 week endurance training program with and without (-)-epicatechin treatment and to determine whether there is a possible cumulative effect on protein markers of angiogenesis and mitochondrial biogenesis. Thirty-four 14-month old male mice (C57BL/6N) were randomized into four groups: control (C); (-)-epicatechin only ((-)-Epi); control with endurance training (CE); and (-)-epicatechin with endurance training ((-)-Epi-Ex). Mice in the training groups performed treadmill exercise for 8 weeks (5 × /week for 60 min/session), whereas mice in the (-)-epicatechin group received 1.0 mg/kg of body mass twice daily during the training period. At 8 weeks, distance ran on the treadmill increased by 46, 69, and 84% in the (-)-Epi, CE, and (-)-Epi-Ex groups, respectively compared to the control group (p < 0.001 for all comparisons). Furthermore, the (-)-Epi-Ex group had significantly higher exercise capacity than the (-)-Epi and CE group. For angiogenic regulators, the (-)-Epi-Ex group had significantly higher VEGF-R2 protein expression with a concomitant reduction in TSP-1 protein expression than the exercise group. Interestingly, FoxO1 protein expression was significantly reduced for all three experimental groups compared to the control group. Protein markers such as PGC-1β and TFAM were significantly higher in the (-)-Epi-Ex group compared to the three other groups. These findings suggest that (-)-epicatechin treatment combined with 8 weeks of endurance training provide a cumulative effect on a number of angiogenic and mitochondrial signaling which functionally translates to enhanced exercise tolerance.

Keywords: exercise tolerance; fatigue; mitochondrial proteins; mouse model; muscle; skeletal.

FIGURE 1

Measurements of capillarity for the plantaris muscle. (A) (N_CAF): *significantly different than control group (p < 0.0001); ^†significantly different than Epi and CE groups (p < 0.0001). (B) (CD): *significantly different than control group (p ≤ 0.018); ^†significantly different than Epi (p = 0.043) and CE (p = 0.005) groups. (C) (C/F_i): *significantly different than control group (p < 0.001); ^†significantly different than Epi and CE groups (p = 0.001). (D) (CFPE): *significantly different than control group (p ≤ 0.013); ^†significantly different than Epi (p = 0.037) and CE (p = 0.006) groups. (n = 3 per group; mean ± SEM).

FIGURE 2

Basal pro- and anti-angiogenic responses in the quadriceps femoris muscle following 8 weeks of endurance training with and without (–)–epicatechin. (A) are representative Western blots. (B) (VEGF-A) there was no significant difference between groups. (C) (VEGF-R2): *significantly (p < 0.03) different than control; **significantly (p = 0.018) different than CE; and ^†significantly (p = 0.002) different than CE. (D) (ADAMTS-1): *significantly (p ≤ 0.03) different than control. (E) (TSP-1): *significantly (p < 0.001) different than control; ^††significantly (p < 0.04) different than (–)–Epi and CE groups. (F) (CD47) there was no different between groups for TSP-1 receptor. (G) (FOXO1): *significantly (p < 0.04) different than control. (n = 4–5 animals per group; mean ± SEM).

FIGURE 3

Basal protein expression of mitochondrial biogenesis regulators in the quadriceps femoris muscle following 8 weeks of endurance training with and without (–)–epicatechin. (A) are representative Western blots. (B) (PGC-1α and PGC-1β): no significant differences between groups for PGC-1α, whereas for PGC-1β *significantly (p ≤ 0.014) different than control group; ^††significantly (p ≤ 0.02) different than (–)–Epi and CE groups. (C) (TFAM): *significantly (p < 0.04) different than control group; and ^††significantly (p ≤ 0.015) different than (–)–Epi and CE groups. (n = 4–5 animals per group; mean ± SEM).

FIGURE 4

Citrate synthase and cytochrome c oxidase activity is increased in the hindlimb muscles after (–)–epicatechin treatment. (A) is citrate synthase activity in the lateral gastrocnemius muscle normalized to the control group *significantly different than control [p-values ranged from ≤ 0.007 to 0.048; ^††different than (–)–Epi and CE, p < 0.0001; n = 4 animals per group]. (B) is CcO activity which was determined in the muscle of mice in the control group incubated in the presence (20 μM final concentration; triangle) or absence (vehicle, circle) of (–)–epicatechin for 25 min using the polarographic method by increasing the amount of substrate cytochrome c. CcO activity is defined as consumed [O₂ (μM)/min/protein mg] (*p ≤ 0.001 significantly different from placebo group, n = 4–5 animals per group, mean ± SEM).