(−)-Epicatechin maintains endurance training adaptation in mice after 14 days of detraining

Abstract

The purpose of this study was to determine whether (−)-epicatechin (mainly found in cocoa) could attenuate detraining effects in the hindlimb muscles of mice. Thirty-two male mice were randomized into 4 groups: control, trained, trained with 14 d of detraining and vehicle (DT-14-W), and trained with 14 d of detraining and (−)-epicatechin [DT-14-(−)-Epi]. DT-14-(−)-Epi received (−)-epicatechin (1.0 mg/kg 2×/d), whereas water was given to the DT-14-W group. The latter 3 groups performed 5 wk of endurance training 5×/wk. Hindlimb muscles were harvested, and Western blots, as well as enzyme analyses, were performed. Training significantly increased capillary-to-fiber ratio (≈78.8%), cytochrome-c oxidase (≈35%), and activity (≈144%) compared to controls. These adaptations returned to control levels for the DT-14-W group, whereas the DT-14-(−)-Epi group was able to maintain capillary-to-fiber ratio (≈44%), CcO protein expression (≈45%), and activity (≈108%) above control levels. In addition, the increase in capillarity was related to decreased protein expression of thrombospondin-1, an antiangiogenic regulator. Furthermore, there were no significant differences in endurance capacity between the trained and DT-14-(−)-Epi groups. Our data suggest that (−)-epicatechin may be a suitable compound to maintain exercise-induced improved capillarity and mitochondrial capacity, even when exercise regimens are discontinued.—Hüttemann, M., Lee, I., Malek, M. H. (−)-Epicatechin maintains endurance training adaptation in mice after 14 d of detraining.

Although the ability to maintain aerobic exercise is multifactorial (e.g., cardiac function, blood flow, and mitochondrial function), it is primarily dependent on oxygen supply and delivery to the working muscle (1). The diffusion of oxygen from the red blood cells to the muscle is facilitated by capillaries and ultimately used by the mitochondria to produce energy. Since skeletal muscle is a dynamic tissue, various perturbations, such as endurance training/detraining (2) or genetic deletion (3, 4) can affect oxygen delivery to the working muscle and, therefore, influence aerobic capacity.

Although no individual actively seeks to become detrained, numerous scenarios, such as onset of illness or injury, may impede the individual's ability to maintain their habitual exercise regimen. Studies have shown that the effects of detraining or immobilization are observable within the first 7–14 d after termination of an endurance training regimen (2, 5, 6). For example, Malek et al. (2) reported that 7 d of detraining partly reversed the increases in skeletal muscle capillarity observed from 10 wk of endurance training in the soleus and plantaris muscles of rats. In addition, the researchers found that resting vascular endothelial growth factor (VEGF-A) protein expression returned to control levels in the detrained group (2). Roudier et al. (7) found that 9 d of hindlimb suspension resulted in decreased capillary-to-fiber ratio in the soleus muscle of rats with a concomitant increase in the antiangiogenic growth factor thrombospondin-1 (TSP-1). More recently, Lehnen et al. (8) reported that muscle GLUT4 protein returned to baseline values after 14 d of detraining in both wild-type and hypertensive rats. Taken together, these studies suggest a critical window (7–14 d) in which the effects of detraining or immobilization are prominent in skeletal muscle.

In addition to alterations in muscle capillarity it is well established in exercise physiology that endurance training increases mitochondrial energy production (9, 10). An important marker of enhanced oxidative phosphorylation is the increase in protein expression of electron transport chain (ETC) complexes, specifically cytochrome-c oxidase (CcO; complex IV) activity (9, 10). For example, Chabi et al. (11) reported that 8 wk of voluntary wheel running increased protein expression of CcO by ≈55% in the plantaris muscle compared to controls, whereas denervation for 7–14 d resulted in ≈30% decrease in CcO protein expression (11). Geng et al. (12) found, in the plantaris muscle of mice, that 4 wk of voluntary wheel running increased CcO protein expression by 80%. These studies collectively indicate that CcO protein expression and activity are dynamic, increasing with endurance training, but diminishing with disuse.

A number of natural compounds have been reported to provide an exercise mimetic effect with limited success. For example, GW1516, a peroxisome proliferator-activated receptor δ modulator that increases glucose uptake in skeletal muscle, was found to require an exercise stimulus to initiate its cellular action (13), but it did not induce mitochondrial gene expression and function (14). Chronic treatment with resveratrol has been shown to lead to nephrotoxicity (15) and to not attenuate aging-associated muscular fatigue in 28-mo-old rats (16). Moreover, Pae et al. (17) reported that epigallocatechin-3-gallate supplementation resulted in significant increases in inflammatory markers. In addition, a recent meta-analysis revealed that quercetin, a natural flavanol, has trivial to very small effects on improving aerobic capacity (18).

Recently, Nogueira et al. (19) reported that 15 consecutive days of low-dose (−)-epicatechin treatment resulted in significant increases in skeletal muscle capillarity, mitochondrial biogenesis, and oxidative enzyme activity, which corresponded to lower muscle fatigue and higher endurance capacity when compared to the placebo group (19). In addition, the improvements observed in the (−)-epicatechin-treated animals were significantly higher than those from exercise alone (19). Therefore, given the possible exercise-mimetic properties of (−)-epicatechin, this compound may potentially attenuate the effects of detraining in skeletal muscle.

No studies, to date, have examined the role of (−)-epicatechin in attenuating the effects of detraining. The purposes of this study, therefore, were to conduct a 5-wk endurance training regimen and determine whether treadmill exercise capacity can be maintained following 14 d of detraining with (−)-epicatechin treatment; (−)-epicatechin can regulate protein expression of VEGF-A and TSP-1 and, therefore, skeletal muscle capillarity following detraining; and (−)-epicatechin can maintain the training-induced increases in oxidative capacity during the detraining period. We hypothesized that (−)-epicatechin would attenuate the detraining losses in skeletal muscle capillarity when compared to vehicle and that this would be related to protein expression of VEGF-A and TSP-1. In addition, we hypothesized that the (−)-epicatechin-treated group would maintain oxidative capacity during the detraining period via maintaining mitochondrial protein complexes and CcO activity. Furthermore, we hypothesized that endurance capacity would be better maintained in (−)-epicatechin-treated mice after 14 d of detraining, whereas vehicle-treated mice would have endurance capacity returning to control levels.

MATERIALS AND METHODS

Animals and ethical approval

We studied 5-mo-old, C57BL/6 male mice (n=32, Harlan Laboratories, Frederick, MD, USA) that were randomized into 4 groups. Animals were placed 4/cage and fed a standard diet without limitations. The room temperature was kept at 21°C with 12-h light-dark cycles. All animal care and experimental procedures were approved by the Wayne State University Institutional Animal Care and Use Committee.

Experimental design and approach

A between-subjects design was used to determine whether (−)-epicatechin treatment attenuates the detraining losses in the oxidative capacity of the hindlimb muscles. Mice performed an incremental treadmill test to exhaustion and were then randomized into 4 groups: group 1, control; group 2, trained; group 3, trained with 14 d of detraining and vehicle (water) treatment (DT-14-W); and group 4, trained with 14 d of detraining and (−)-epicatechin treatment (DT-14-(−)-Epi). Groups 2–4 performed endurance training on a rodent treadmill 5×/wk for 5 wk. All groups then performed a second incremental treadmill test to compare pretraining and posttraining exercise capacity. Animals in the trained group were sacrificed 48 h after the exercise test, whereas animals in the DT-14-W and DT-14-(−)-Epi groups received either the vehicle or (−)-epicatechin for 14 consecutive days. After 14 d, animals in the control, DT-14-W, and DT-14-(−)-Epi groups performed a third incremental treadmill test to exhaustion and were then sacrificed 48 h later. The plantaris and quadriceps femoris muscles were harvested and used for morphometric, biochemical, and molecular analyses.

Incremental treadmill test

On ≥2 occasions prior to the test, all mice were familiarized with the treadmill (1055MSD Exer-6M; Columbus Instruments, Columbus, OH, USA) at a slow speed (≈5 m/min) at 0° incline for ∼5–10 min. The incremental test consisted of warm-up at 4 m/min for 2 min followed by an increase of 2 m/min every minute thereafter. A shock grid (0.2 mA) at the back of the treadmill was used to discourage the mice from stopping while the treadmill belt was moving. Exhaustion was determined when the mouse was no longer able to maintain its normal running position on the treadmill and/or was unwilling to run, as indicated by the frequent contact (i.e., touching the shock grid with each stride) or sitting on the shock grid, consistent with previous studies from our laboratory (2, 3, 19),

Endurance training and detraining intervention

For 5 wk, mice in groups 2–4 underwent moderate treadmill endurance training, which began at 7.5 m/min at 5° incline for ∼12 min 5×/wk and progressively reached ∼60% of the maximum work rate for 60 min by the end of the training period. A shock grid (0.2 mA) at the back of the treadmill was used to discourage the mice from stopping while the treadmill was moving. Animals in group 1 were placed on a nonmoving treadmill during the training sessions. Following the training intervention period, mice in groups 3 and 4 were cage confined for 14 d.

(−)-Epicatechin administration

Mice in the DT-14-(−)-Epi group were given 1.0 mg/kg body mass 2×/d (morning and evening) for 14 d, whereas animals in the DT-14-W group received vehicle (water) for the same period of time. Both (−)-epicatechin (Sigma Aldrich, St. Louis, MO, USA) and vehicle were administered via oral gavage, consistent with our previous study (19).

Tissue preparation

All mice were anesthetized with pentobarbital sodium (60 mg/kg, i.p.) and the quadriceps femoris and plantaris muscles were removed, along with the heart. For the plantaris muscle, an entire transverse slice from the widest point of the middle belly portion of the muscles was excised and frozen in precooled isopentane (−140°C) and stored at −80°C until further processing. Transverse 10-μm serial sections were cut on a cryotome (Leica CM 1950; Leica Microsystems, Buffalo Grove, IL, USA) at −20°C and mounted on slides for histochemical analysis of capillary number. Great care was taken to ensure that the widest part of the muscle was sectioned and that sectioning was perpendicular to the orientation of the fibers. The quadriceps femoris muscle was prepared for enzyme/metabolite and molecular analyses.

Capillary staining and indices

Sections from the plantaris muscle from all 4 groups were stained using the method of Rosenblatt (20). Briefly, the sections were first fixed for 5 min in a Guth and Samaha (21) fixative and then incubated for 1 h at 36°C in a lead-adenosinetriphosphatase staining medium to stain capillaries. It should be noted that there are no differences in the number of capillaries visualized with frozen biopsy samples using the Rosenblatt technique and the number visualized in tissue sections prepared from perfusion-fixed muscle (22).

Consistent with our previous studies (2, 3, 19), muscle sections were viewed under a digital microscope (×20 view; Leica DMD108; Leica Microsystems). Capillaries were quantified manually from the digital image on individual fibers by a single individual. The following indices were measured: the number of capillaries around a fiber (N_CAF), the capillary-to-fiber ratio on an individual-fiber basis (C/F_i), and capillary density (CD), which was calculated by using the fiber area as the reference space (22). Capillary-to-fiber perimeter exchange index (CFPE) was calculated as an estimate of the capillary-to-fiber surface area (23). Quantification of the capillary supply was performed randomly selecting a fiber in an artifact-free region (no holes due to freeze fracture and fibers intact; ref. 24). Fiber cross-sectional area (FCSA) and fiber perimeter were measured with the image analysis system and commercial software (SigmaScan Pro 5.0; Systat Software, Point Richmond, CA, USA).

Muscle metabolic enzyme activity

The quadriceps femoris muscle was separated into 10-mg pieces and pulverized using a pestle for 60 s on ice buffer (175 mM KCl and 2 mM EDTA; pH 7.4) for citrate synthase, consistent with our previous approach (3, 19). Homogenates were frozen in liquid nitrogen and underwent 3 freeze/thaw cycles using liquid nitrogen. Before use, the homogenates were thawed a final time and centrifuged at 8000 g for 1 min to remove particulate matter.

Using the supernatant, we measured citrate synthase activity via the technique of Srere (25). The activity of citrate synthase was measured by the rate of production of the mercaptide ion based on conversion of acetyl-CoA and oxaloacetate into citrate synthase and CoA-SH. CoA-SH in the presence of 5,5′-disthiobis-2-nitrobenzoic acid produces mercaptide ion. Samples were analyzed by spectrophotometry (DU 730; Beckman, Fullerton, CA, USA) at 412 nm. All samples were tested in triplicate and measured at 37°C.

CcO-specific activity measurements

CcO activity was analyzed in the quadriceps femoris muscle with a micro-Clark-type oxygen electrode in a closed chamber (Oxygraph System; Hansatech, Norfolk, UK) at 25°C. Frozen tissue was solubilized in 10 mM K-HEPES (pH 7.4), 40 mM KCl, 1% Tween 20, 1 μM oligomycin, 1 mM PMSF, 10 mM KF, 2 mM EGTA, and 1 mM sodium vanadate, as described previously (26). CcO activity was measured in the presence of 20 mM ascorbate by the addition of increasing amounts of cytochrome c from cow heart (Sigma-Aldrich). Oxygen consumption was recorded on a computer and analyzed with the Oxygraph software. Protein concentration was determined with the DC protein assay kit (Bio-Rad, Hercules, CA, USA). CcO-specific activity was defined as consumed O₂ (nanomoles) per minute per milligram total protein.

Western blot analysis

The Western blot analysis procedure was consistent with our previous work (19), with slight modifications as recommended by Baltes-Breitwisch et al. (27). Briefly, 50 mg of the quadriceps femoris muscle was homogenized in a glass tissue grinder with RIPA buffer (Sigma-Aldrich) and protease and phosphatase inhibitor cocktails (P2714 and P2850; Sigma-Aldrich). Homogenates were passed through an insulin syringe 5 times and centrifuged for 10 min at 4°C, and the supernatant was collected, portioned into aliquots, and stored at −80°C. Total protein was measured by the bicinchoninic acid method (BCA protein assay kit, Bio-Rad).

Protein samples (40 μg) were mixed with sample buffer (4× sample buffer, Li-Cor Biosciences, Lincoln, NE, USA), and ultrapure water was added to the final desired volume. Samples were then incubated at 95°C for 5 min and loaded onto 7.5% (TSP-1) or 12% TGX precast gels (Bio-Rad) and run for 1 h at 160 V. Thereafter, proteins were electrotransferred onto polyvinylidene fluoride membranes (PVDF-FL, Immobilon transfer membranes; Millipore, Billerica, MA, USA) with a semidry blotting apparatus (12 V, 50 min; Bio-Rad). Prior to transfer, the gel was soaked in transfer buffer (48 mM Tris-HCl, 39 mM glycine, 0.0375% SDS, and 20% methanol) for 10 min, the blotting paper (Thermo Fisher Scientific, Rockford, IL, USA) was soaked in transfer buffer for 5 min, and the membrane was soaked in methanol for 2–3 min. The membrane was blocked, after the transfer, with Odyssey blocking buffer (Li-Cor Biosciences) for 1 h at room temperature, and then incubated overnight with primary antibody at 4°C with gentle shaking. The following day, the membrane was washed 4 times for 5 min at room temperature in Tris-buffered saline with Tween (TBST) wash solution. The secondary antibody was added, and the membrane was incubated at room temperature for 1 h. After incubation with the secondary antibody, the membrane received 4 additional 5-min washes in TBST at room temperature. Care was taken not to expose the membrane to light.

The mouse monoclonal primary antibodies used were TSP-1 (1:500; sc-59886; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MitoProfile Total OXPHOS (1:1,000; MS601; MitoSciences, Eugene, OR, USA), and GAPDH (1:2000; ab9484; Abcam, Cambridge, MA, USA). The polyclonal primary antibodies used were VEGF (1:500; sc-507; Santa Cruz Biotechnology), and α-tubulin (1:2000; 2144; Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies used were goat anti-mouse IRDye (1:30,000) and goat anti-rabbit IRDye (1:30,000) purchased from Li-Cor Biosciences. All images of blots were acquired with the Odyssey infrared imaging system (Li-Cor Biosciences), and quantitative analysis was performed with the Li-Cor software.

Statistical analyses

All data are presented as means ± se. Separate 1-way ANOVAs were performed to compare the relevant group means for each dependent variable. Separate 2 (time: pretraining and post-training) × 4 [group: control, treatment, DT-14-W, and DT-14-(−)-Epi] mixed factorial ANOVAs were performed for body mass, as well as exercise indices. In addition, separate 3 (time: pretraining, post-training, and 15-d detraining) × 3 [group: control, DT-14-W, and DT-14-(−)-Epi] mixed factorial ANOVAs were performed for exercise indices. For the CcO activity, a 4 [group: control, training, DT-14-W, and DT-14-(−)-Epi] × 10 (cytochrome c concentration: 0, 1, 2, 3, 5, 10, 15, 20, 25, and 30 μM) mixed factorial ANOVA was performed. When appropriate, post hoc Tukey's HSD was used to determine which means were significantly different from each other (28). An α level at P ≤ 0.05 was considered significant for all statistical comparisons. The analyses were conducted using the Statistical Package for the Social Sciences software (SPSS 18.0; SPSS Inc., Chicago, IL, USA).

RESULTS

Animals

As shown in Table 1, body and muscle masses were not statistically different (P>0.05) between the 4 groups. In addition, there were no statistical differences when the data were normalized to body mass.

Table 1.

Body and muscle mass for each group

Parameter	Control, n = 8		Trained, n = 8		DT-14-W, n = 8		DT-14-(−)-Epi, n = 8
	Pre	Post	Pre	Post	Pre	Post	Pre	Post
Body mass (g)	31.5 ± 0.7	31.7 ± 1.1	30.8 ± 0.7	29.9 ± 1.1	31.3 ± 0.7	30.3 ± 1.1	31.3 ± 0.7	31.1 ± 1.1
Quadriceps femoris mass (mg)	−	245.0 ± 10.6	−	223.8 ± 7.1	−	233.5 ± 3.5	−	240.8 ± 6.4
Quadriceps femoris/body mass (mg · g−1)	−	7.43 ± 0.28	−	7.53 ± 0.15	−	7.53 ± 0.18	−	7.95 ± 0.19
Plantaris (mg)	−	22.0 ± 1.4	−	19.6 ± 1.2	−	20.5 ± 1.3	−	18.6 ± 1.4
Plantaris/body mass (mg · g−1)	−	0.67 ± 0.02	−	0.67 ± 0.04	−	0.66 ± 0.05	−	0.61 ± 0.05

Values are expressed as means ± se. No significant difference (P>0.05) between groups for any of the indices.

Incremental treadmill test after 5 wk of training

The 2 × 4 mixed factorial ANOVA revealed a significant time × group interaction for time [F (3,28)=31.57, P<0.0001], speed [F (3,28)=39.15, P<0.0001], and distance [F (3,28)=41.64, P < 0.0001]. In addition, there were main effects for time and group for all 3 exercise indices, but these main effects were not interpreted because of the significant interaction (28). As shown in Table 2, the follow-up analyses indicated that the 5 wk of treadmill endurance training increased exercise performance by ≈20% (exercise time) to ≈40% (distance) when compared to the control group. Furthermore, there were no statistically significant differences (P>0.05) in exercise capacity between the 3 exercise groups at the end of the 5-wk training regimen (Table 2).

Table 2.

Results of incremental treadmill test for all groups

Parameter	Control, n = 7			Trained, n = 8			DT-14-W, n = 8			DT-14-(−)-Epi, n = 8
	Pre	Post	14-d DT	Pre	Post	14-d DT	Pre	Post	14-d DT	Pre	Post	14-d DT
Time (s)	662 ± 18	656 ± 10	647 ± 10	699 ± 16	811 ± 11$,&	−	686 ± 17	788 ± 10$,&	667 ± 10#,@	692 ± 17	816 ± 10$,&	794 ± 10*,#
Speed (m · min−1)	23.4 ± 0.6	23.1 ± 0.4	22.9 ± 0.3	24.8 ± 0.5	28.5 ± 0.3$,&	−	24.3 ± 0.5	27.5 ± 0.3$,&	23.8 ± 0.3#,@	24.5 ± 0.5	28.5 ± 0.3$,&	27.7 ± 0.3*,#
Distance (m)	193 ± 7	190 ± 5	186 ± 4	210 ± 6	262 ± 5$,&	−	204 ± 7	247 ± 5$,&	197 ± 4#,@	207 ± 7	262 ± 5$,&	251 ± 4*,#

Values are expressed as means ± se.

^*P < 0.05 vs. corresponding control and DT-14-W groups;

^#P < 0.05 vs. pretest group;

^@P < 0.05 vs. posttest group;

^$P < 0.05 vs. pretest group;

^&P < 0.05 vs. control posttest group.

Incremental treadmill test after 14 d of detraining

The 3 × 3 mixed factorial ANOVA revealed a significant time × group interaction for time [F (4,40)= 20.54, P<0.0001], speed [F(4,40)=22.76, P <0.0001], and distance [F (4,40)=23.66, P<0.0001]. In addition, there were main effects for time and group for all 3 exercise indices, but these main effects were not interpreted because of the significant interaction. As shown in Table 2, the follow-up analyses indicated that the DT-14-(−)-Epi was the only group in which exercise indices did not return to pretraining values.

Hindlimb capillarity

The 1-way ANOVA for fiber area and perimeter measurements for the plantaris muscle revealed significant F ratios. To further examine the differences among the group means, pairwise Tukey's HSD follow-up tests were conducted. As shown in Table 3, the fiber area for the trained and DT-14-(−)-Epi groups was significantly larger when compared to the control group, whereas no statistical difference was found between the control and DT-14-W groups. In addition, there were no statistically significant differences (P>0.05) between groups for fiber perimeter (Table 3).

Table 3.

Muscle morphometric assessments for the plantaris

Parameter	Group
	Control	Trained	DT-14-W	DT-14-(−)-Epi
Area (μm2)	2687 ± 59	3307 ± 275*	2903 ± 160	3254 ± 207*
Perimeter (μm)	223 ± 4	252 ± 18	234 ± 12	243 ± 3

Values are expressed as means ± se.

^*P < 0.05 vs. control group.

The 1-way ANOVA for capillary indices (N_CAF, C/F_i, CD, and CFPE) resulted in significant F ratios. To further examine the differences among the group means, pairwise Tukey's HSD follow-up tests were conducted. As shown in Fig. 1A, N_CAF was significantly greater for the trained and DT-14-(−)-Epi groups when compared to the control group. In addition, N_CAF for the DT-14-W group was significantly lower when compared to the trained and DT-14-(−)-Epi groups. A similar pattern was also found for C/F_i (Fig. 1B). For CD (Fig. 1C) and CFPE (Fig. 1D), however, the trained group had statistically significant higher values than the control and DT-14-W groups (Fig. 1). The DT-14-(−)-Epi group had significantly higher CFPE than the control and DT-14-W, but it was similar (P>0.05) to the trained group.

Figure 1.

Capillary measurements for the hindlimb muscle. Plantaris comparison (mean±se) of N_CAF (A), C/F_i (B), CD (C), and CFPE (D) between control, trained, DT-14-W, and DT-14-(−)-Epi groups (n=3/group). *P < 0.05 vs. control group; **P < 0.05 vs. trained group; ^†P < 0.05 vs. DT-14-W group.

Detraining and (−)-epicatechin on VEGF and TSP-1 protein expression

As shown in Fig. 2, we examined protein expression of VEGF-A and TSP-1 following training and 14 d of detraining. The protein content of TSP-1 was significantly higher in the control and DT-14-W groups compared to the trained and DT-14-(−)-Epi groups, whereas VEGF-A protein expression was higher (P<0.05) in the DT-14-W and DT-14-(−)-Epi groups compared to the control and trained groups.

Figure 2.

Protein expression of antiangiogenic and proangiogenic responses. Representative Western blot results for TSP-1 and VEGF-A from quadriceps femoris muscle (means±se; n=4–5/group). *P < 0.05 vs. control and DT-14-W groups; ^†P < 0.05 vs. trained group.

Resting citrate synthase activity

The one-way ANOVA for citrate synthase for the quadriceps femoris muscle resulted in a significant F ratio. Five weeks of endurance training resulted in a 6% increase (P=0.001) in resting citrate synthase activity in the quadriceps femoris muscle compared to the control group. After 14 d of detraining, resting citrate synthase activity values were similar to control levels, whereas the (−)-epicatechin-treated mice were able to maintain resting citrate synthase activity similar to the trained group despite detraining (not shown).

Mitochondrial oxidative phosphorylation protein complex in hindlimb

As shown in Fig. 3, the protein expression of the mitochondrial complexes I (subunit NDUFB8; ≈20 kDa), III (core 2 subunit; ≈47 kDa), IV (CcO, subunit II; ≈24 kDa), and V (ATP-synthase-α subunit; ≈53 kDa) were examined for all 4 groups. For complex I, expression in the DT-14-(−)-Epi group was significantly (P<0.01) greater than in the control group, but not statistically different from the other 3 groups, whereas for complex III protein, expression in the DT-14-(−)-Epi group was significantly greater than in all other groups (Fig. 3). We were, however, unable to measure the band for complex II (FeS subunit; ≈30 kDa) because of the proximity to our loading control protein (GAPDH; ≈38 kDa), despite several attempts to achieve separation between the two bands, so as to confidently and reliably measure the band. Nevertheless, as shown in Fig. 3, for complex IV, the DT-14-(−)-Epi group had significantly higher protein expression than the control and DT-14-W groups, but was similar to the trained group. Lastly, the DT-14-(−)-Epi group had significantly higher protein expression for complex V when compared to the control and DT-14-W groups, whereas the trained group had significantly more complex V protein expression than the control group.

Figure 3.

Protein expression of oxidative phosphorylation complexes. Representative Western blot results for mitochondrial protein complexes from the quadriceps femoris muscle (means±se, n=4–5/group). *P < 0.05 vs. corresponding control group; **P < 0.05 vs. corresponding control and DT-14-W groups; ***P < 0.05 vs. corresponding control group; ^†P < 0.05 vs. corresponding control, trained, and DT-14-W groups; ^††P < 0.05 vs. corresponding control and DT-14-W groups; ^#P < 0.05 vs. corresponding control group.

CcO-specific activity

We analyzed the effects of detraining on mitochondrial energy metabolism by measuring CcO-specific activity. As shown in Fig. 4, training increases CcO-specific activity by ≈144% compared to control. Furthermore, 14 d of detraining with water returned CcO-specific activity to control levels, whereas the (−)-epicatechin-treated group was able to maintain CcO-specific activity by ≈108% above control levels (Fig. 4).

Figure 4.

CcO-specific activity determined using the polarographic method by increasing the substrate cytochrome c concentrations from 1 to 30 μM. At maximal turnover (at 30 μM cytochrome c), CcO activity increased 125% in the trained group. Detraining following the training regimen resulted in complete loss of the exercise-induced increased CcO activity back to control levels. In contrast, (−)-epicatechin treatment for 14 d after exercise cessation maintained increased CcO activity at levels approaching those of the trained group, which showed a significant 85% increase compared with controls. Values are means ± se. ns, not significant (P>0.05) between groups indicated by brackets. *P < 0.05 vs. control and DT-14-W groups; ^†P < 0.05 vs. control and DT-14-W groups.

DISCUSSION

The principle and unique findings of the present study are that 14 d of detraining with (−)-epicatechin treatment resulted in maintenance of incremental treadmill performance and hindlimb capillarity via changes in protein expression of VEGF-A and TSP-1. In addition, some proteins of the oxidative phosphorylation system were preserved in the quadriceps femoris muscle. Notably, protein expression of CcO and specific activity were maintained in detrained mice treated with (−)-epicatechin compared to those animals treated with the vehicle. This is the first study that has comprehensively examined the interaction between (−)-epicatechin and detraining in skeletal muscle. These results suggest that (−)-epicatechin may preserve two critical components of the oxygen transport pathway, capillaries and mitochondria.

Role of (−)-epicatechin in preserving skeletal muscle angiogenesis

It has been suggested that VEGF-A and TSP-1 may regulate angiogenesis and, therefore, result in capillary maintenance or regression (7, 29). Thus, in a resting muscle that is primarily oxidative (slow-twitch), the ratio between VEGF-A and TSP-1 would be high, whereas in a glycolytic (fast-twitch) muscle, this ratio would be reversed. Malek and Olfert (3) have previously reported that TSP-1-null mice have higher CD in both oxidative and glycolytic hindlimb muscles compared to controls. In addition, the researchers found that basal VEGF-A levels in the hindlimb muscles were ≈55% greater in the TSP-1-null mice than in controls (3). Recently, Roudier et al. (7) reported a higher proportion of VEGF-A than TSP-1 protein expression in the soleus muscle of rats. In these same animals, the researchers reported higher TSP-1 expression in the plantaris muscle, which is highly glycolytic (7). Furthermore, they examined the ratio between VEGF-A and TSP-1 in the soleus and plantaris muscles of rats following 9 d of hindlimb suspension (7). The researchers reported that TSP-1 protein expression was significantly higher at d 7 and 9 of hindlimb suspension compared to the control group for the soleus muscle (7), whereas no change in TSP-1 protein expression from control levels was observed for the plantaris muscle. VEGF-A protein expression, however, remained unchanged from control levels in soleus muscle, but it was significantly higher at d 7 and 9 from control levels in the plantaris muscle (7). Roudier et al. (7) concluded that capillary maintenance or regression is a result of an integrated balance between proangiogenic and antiangiogenic factors.

In the present study, the effects of 14 d of (−)-epicatechin treatment in conjunction with detraining following 5 wk of moderate intensity endurance training was evaluated in the quadriceps muscle of mice. This muscle was selected, in part, because we wanted to remain consistent with our previous study (19), which indicated significant improvements in the oxidative capacity of the quadriceps femoris muscle with (−)-epicatechin treatment. As shown in Fig. 2, resting protein expression of VEGF-A in the trained group was not statistically different (P>0.05) from the control group, whereas TSP-1 protein expression was reduced by 70% in the trained group compared to control levels. Following 14 d of detraining with the vehicle (water), VEGF-A levels were greater by 35%, whereas TSP-1 levels returned to control levels (Fig. 2). For the detrained group that received (−)-epicatechin, however, VEGF-A protein expression was similar to the DT-14-W group, whereas TSP-1 levels remained similar to the trained group (Fig. 2). Malek et al. (2) reported that 7 d of detraining resulted in increases in resting protein concentration of VEGF-A for both plantaris and soleus muscles of rats when compared to trained animals. Therefore, elevated VEGF-A protein expression observed in the detrained groups is consistent with our previous study (2) and may be an attempt by the muscle to preserve training-induced angiogenesis.

Although the mechanism of (−)-epicatechin-induced increase in vascular proliferation is still under investigation, it has been suggested that (−)-epicatechin is a selective nitration inhibitor (30, 31). El-Remessy et al. (30, 31) showed that (−)-epicatechin selectively inhibits peroxynitrite-mediated tyrosine nitration in the retina. More recently, Al-Gayyar et al. (32) reported that (−)-epicatechin blocked diabetes-induced peroxynitrite production in Müller cells. It should be noted, however, that the researchers used a large daily dose of (−)-epicatechin (100 mg/kg, orally) for 4 wk (32). In the present study, we used a low-dose (−)-epicatechin treatment (1.0 mg/kg, 2×/d). Nevertheless, we observed that VEGF-A protein expression in the detrained groups was ≈35% higher than in the trained group (Fig. 2).

The effects of (−)-epicatechin on TSP-1 protein expression are unknown. To our knowledge, only one study has examined the interaction between (−)-epicatechin and TSP-1 (33). Using a paradigm of type 2 diabetes mellitus, Ryu et al. (33) reported that isolated rat aortic vascular smooth muscle cells (VSMCs) incubated in high glucose concentrations for 48 h had significantly greater TSP-1 protein expression compared to controls. When VSMCs were incubated in glucose and (−)-epicatechin, however, TSP-1 protein expression was reduced by ≈40% (33). Ryu et al. (33) concluded that (−)-epicatechin inhibited the high-glucose-induced TSP-1 expression, which may improve vascular health in patients with type 2 diabetes.

In addition to examining VEGF-A and TSP-1 protein expression, we examined skeletal muscle capillarity in the plantaris muscle using established indices (2, 3, 19, 34). The plantaris muscle is primarily a glycolytic muscle fiber, which typically has low capillarity (35). As shown in Fig. 1), 5 wk of endurance training resulted in greater capillarity in the plantaris muscle by ≈75%. Conversely, when 14 d of detraining was introduced, N_CAF and C/F_i returned to control levels in the group that received the vehicle. The (−)-epicatechin-treated group, however, also showed reduced N_CAF and C/F_i, but these indices were 50 and 44% greater (P<0.05) than in the control group. We also found changes in FCSA, which were 23% greater with 5 wk of moderate intensity endurance training and remained greater than in the control group with 14 d of detraining and (−)-epicatechin treatment (Table 3). These findings were consistent with our previous detraining study in the plantaris muscle of rats (2) and similar to the findings of Ishihara et al. (36) for the same muscle. It is interesting to note, however, that CD for the DT-14-(−)-Epi was not statistically different from the DT-14-(−)-W group in the current study (Table 3). This may be due, in part, to the decrease in N_CAF of the DT-14-(−)-Epi compared to the trained group despite no change in fiber area between the two groups (Table 3).

In the current investigation, we also calculated CFPE, which is an estimate of the proximity between capillaries and muscle cells (22). Therefore, an increase in CFPE results in improved oxygen flux potential from the red blood cells to the myocyte (3). Functionally, this may translate to increased exercise performance. For example, Malek and Olfert (3) reported that TSP-1-null mice had greater CFPE than littermate controls, which corresponded to an 80% improvement in running distance for the treadmill run-to-exhaustion test. In the present study, CFPE was higher in the trained (56%) and DT-14-(−)-Epi (31%) groups compared to the control group. The effects of (−)-epicatechin on angiogenesis have been observed in regions of the brain. Van Praag et al. (37) reported ≈64% increase in vasculature in the hippocampus after 2 wk of (−)-epicatechin treatment compared to controls, which corresponded to increased memory. Taken together, these results indicate that (−)-epicatechin may mediate angiogenic remodeling in the brain and skeletal muscle, two highly aerobic tissues.

(−)-Epicatechin preserves mitochondria function despite detraining

One of the hallmarks of endurance training is an increase in oxidative capacity (38). In the present study, we examined the effects of detraining and (−)-epicatechin treatment on protein expression of ETC complexes, as well as CcO-specific activity in the quadriceps femoris muscle. This approach was taken because we wanted to determine the potential mechanism by which (−)-epicatechin-treated mice were able to maintain their exercise capacity similar to the trained group despite 14 d of detraining (Table 2).

CcO protein expression and specific activity

One of the adaptations to endurance training is increased CcO and resting citrate synthase in the working muscle (39), whereas detraining and/or disease result in a reverse effect (40). Notably, we showed that complex IV protein expression was greater in the trained group by ≈35% compared to control levels. This increase indicates that our 5-wk moderate intensity training protocol provided enough stress to the working muscles to stimulate an increase in oxidative capacity. Furthermore, we showed that 14 d of detraining with (−)-epicatechin resulted in a preservation of CcO (Fig. 3) that did not differ significantly from the trained group. In addition, we found that 14 d of detraining returned CcO protein expression to near-control levels in the DT-14-W group. Another common marker of increased mitochondrial mass is citrate synthase. Our results indicated that resting citrate synthase activity was not statistically different between the trained and DT-14-(−)-Epi groups, and 6% greater than in the control and DT-14-W groups. The results of the current investigation indicate that 14 d of (−)-epicatechin treatment following cessation of endurance training results in preservation of CcO amount and resting citrate synthase activity.

In addition to examining the protein expression of mitochondrial complexes, we also determined the activity of CcO. The mitochondrial ETC and ATP synthase together comprise the oxidative phosphorylation system, which generates >90% of cellular energy (41, 42). As electron transfer occurs, CcO pumps protons across the inner mitochondrial membrane, which, together with ETC complexes I and III, generates the mitochondrial membrane potential (41, 42). The mitochondrial membrane potential is then used by ATP synthase to produce ATP from ADP and phosphate. In intact mammalian cells, CcO is the proposed rate-limiting step of the ETC under physiological conditions (41, 42). Changes in CcO activity, therefore, will affect overall flux in the ETC. Studies have shown that CcO activity and protein expression are reduced in skeletal muscle with various perturbations, such as aging or disease, which then leads to reduced energy production and thus decreased functional activity (43, 44).

In the current investigation, we observed that exercise alone increased CcO-specific activity by >2-fold compared to controls (Fig. 4). The increase in CcO activity is dependent on the intensity and duration of the training period (9, 45). For example, Soussi et al. (46) reported that CcO activity improved ≈45% in endurance-trained rats compared to controls. In the current investigation, 5 wk of moderate intensity (60% of maximal treadmill speed) exercise resulted in significant improvements in resting CcO activity at maximal turnover (30 μM; Fig. 4) compared to controls. Notably, the training-induced improvements in resting CcO activity in the quadriceps femoris muscle were maintained by (−)-epicatechin, with an almost 2-fold increase compared to the detrained group receiving the vehicle (Fig. 4). Our data suggest that (−)-epicatechin may be a suitable compound to maintain exercise-induced improvements in mitochondrial capacity and aerobic activity, even when exercise regimens are discontinued.

In summary, the current study examined the role of (−)-epicatechin on attenuating the effects of 14 d of detraining in skeletal muscle using an integrative approach ranging from the level of the mitochondria (ETC complexes and CcO activity) to the muscle (protein expression of proangiogenic and antiangiogenic regulators and capillarity) to the functional level (treadmill testing). We found that a low-dose (−)-epicatechin treatment attenuated the detraining losses in skeletal muscle capillarity and bioenergetics attained following 5 wk of endurance training. In addition, it was shown that (−)-epicatechin may potentially regulate the protein expression between angiogenic and antiangiogenic factors, such as VEGF-A and TSP-1, respectively. Furthermore, skeletal muscle oxidative capacity was preserved for mitochondrial CcO protein levels and specific activity. Therefore, these effects seen at the cellular level were able to manifest in the integrative response (treadmill exercise), such that the detrained group receiving (−)-epicatechin had similar exercise capacity compared to the trained group. The findings of the present study, in conjunction with those from our previous investigation with (−)-epicatechin (19), suggest that (−)-epicatechin has the potential to maintain skeletal muscle capillarity and oxidative capacity during 14 d of detraining.