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Dr. Gary A. Dudley 1952-2006
Introduction
One unique feature of skeletal muscle is the ability to adapt to various external stimuli (6). For example, endurance training increases skeletal muscle capillarity and mitochondrial volume, whereas detraining results in the opposite response (45). Detraining, however, may come in different forms such as cessation of an exercise regimen because of injury, immobilization of a limb, or exposure to microgravity (i.e., space flight). The hindlimb suspension paradigm with rodents is a research model used to study skeletal muscle adaptation when the muscle is unloaded (22). This approach, therefore, may be ideal for human translation particularly as it relates to clinical populations who have limited mobility or are bedridden (26).
Findings from hindlimb suspension studies indicate that the mechanisms associated with muscle degradation may be due, in part, to changes in the Akt/mTOR (mammalian target of rapamycin) and FoxO (forkhead box O) signaling pathways (36). Moreover, studies examining the use of various interventions to attenuate muscle degradation reported conflicting results (4,23). The hindlimb suspension paradigm presents a unique opportunity to examine components of the oxygen delivery and utilization pathways. The 2 main components of this pathway are the capillaries, which allow oxygen to be delivered from the blood to the muscle, and the mitochondria, which use the oxygen to generate energy (13,44). Studies have shown that capillary development and/or maintenance may be under the control of angiogenic stimulators, such as vascular endothelial growth factor isoform A (VEGF-A), and angiogenic inhibitors, such as thrombospondin-1 (TSP-1) (11,13,35). Moreover, markers of oxidative capacity are associated with peroxisome proliferator-activated receptor-γ coactivator 1 (PGC-1) proteins (46), mitochondrial transcription factor A (TFAM) (20), and cytochrome c oxidase (CcO), the proposed rate-limiting enzyme in the electron transport chain (16,42). In oxidative muscle, it has been shown that hindlimb suspension reduces skeletal muscle capillarity and oxidative capacity. Roudier et al. (34) reported that 9 days of hindlimb suspension resulted in increased protein expression of TSP-1 and decreased VEGF-R2 (VEGF receptor) in the soleus muscle of rats. Wagatsuma et al. (43) reported that 7 days of hindlimb suspension significantly reduced mRNA expression of PGC-1 and TFAM, which corresponded to deceased oxidative enzyme activity in the gastrocnemius muscle. Moreover, Cassano et al. (3) reported that 14 days of hindlimb suspension significantly reduced mitochondrial protein expression in rodent oxidative muscle.
Our laboratory has shown that the flavanol (−)-epicatechin, which is found in cacao beans (38), may promote angiogenic and oxidative capacity in glycolytic hindlimb muscles of mice (13,27) and rats (14). In addition, we have shown that (−)-epicatechin maintains endurance training adaptations such as enhanced capillarity and oxidative capacity in the hindlimb muscles of mice after 14 days of detraining (13). Moreover, we have reported that 30 days of (−)-epicatechin treatment increase markers of mitochondrial biogenesis in the plantaris muscle of rats with innate low running capacity (14). Taken together, these studies suggest that in glycolytic muscle, (−)-epicatechin supplementation may attenuate loss of capillarity and oxidative capacity in detrained muscle (13) or increase capillarity and mitochondrial function in a model of congenital muscle dysfunction (14). What remains unknown, however, is the effect of (−)-epicatechin supplementation on oxidative skeletal muscle. This information would be important to know because various diseases such as stroke (24) or chronic obstructive pulmonary disease (COPD) (40) are associated with a reduction in oxidative fibers, which manifests in muscle fatigue from activities of daily living.
The purposes of this study, therefore, were to conduct a 14-day hindlimb suspension protocol with and without (−)-epicatechin supplementation to determine whether (−)-epicatechin treatment can attenuate the loss in muscle degradation, angiogenesis, and mitochondrial signaling in oxidative skeletal muscle. Based on our previous studies with (−)-epicatechin in primarily glycolytic tissue (13,14,27), we hypothesized that mice treated with (−)-epicatechin during the hindlimb suspension period would exhibit less muscle degradation than mice receiving vehicle only. Secondarily, we hypothesized that (−)-epicatechin supplementation would attenuate the loss in capillarity and mitochondrial signaling after 14 days of hindlimb suspension.
Methods
Experimental Approach to the Problem
Mice were randomized into 3 groups: (a) control (C); (b) hindlimb suspension for 14 days with vehicle (HS-V); and (c) hindlimb suspension for 14 days with (−)-epicatechin (HS-(−)-Epi). Animals were killed after the experimental period and the hindlimb muscles harvested.
Subjects
We used 6-month-old male C57BL/6N mice (N = 25, Harlan Laboratories, Inc.), which were randomized into 3 groups with 1 group having an extra animal. Animals were placed 1 per cage and provided a standard diet (DietGel 76A; ClearH2O, Portland, ME, USA) without restrictions. Furthermore, the room temperature was maintained at 21° C with 12-hour light-dark cycles. All animal care and experimental procedures were approved by the University Institutional Animal Care and Use Committee.
Procedures
(−)-Epicatechin Dosage
Consistent with our previous work on (−)-epicatechin in mice (13,27) and rats (14), animals in the (−)-epicatechin groups (2 and 3) were given a dosage of 1.0 mg·kg−1 of body mass twice a day (morning and evening). Animals in group 2 received the vehicle (water), whereas animals in group 3 received (−)-epicatechin (Sigma-Aldrich, St. Louis, MO, USA) mixed with water for 14 consecutive days. Delivery of vehicle or (−)-epicatechin was through oral gavage by experienced personnel.
Hindlimb Suspension Protocol
The hindlimb suspension paradigm used here was consistent with the protocol recommended by Ferreira et al. (7). Briefly, animals in groups 2 and 3 were anesthetized with 2% isoflurane, and the fifth and sixth intervertebral space was located. Thereafter, a pilot hole was made with a 25-G needle between the 2 vertebrae into the intervertebral space (7). A sterile steel wire suture (Steelex Monofilament; Jorgensen Laboratory, Inc., Loveland, CO, USA) was inserted into the space and twisted to form a loop (7). A second smaller loop was then formed above the larger loop. After the procedure, animals were given 7 days to recover (7).
On the first day of hindlimb suspension, animals were placed in a large rodent cage with a slotted steel bar extending the full length of the cage (7). The small second loop was connected to a fishing swivel, which was connected to a sewing machine bobbin. This approach allows the animal full rotational movement in a stationary location (7). The animal's hindlimb was raised so as not to make contact with the bottom of the cage. The tail was covered with sterile gauze and secured with tape to prevent dropping downward and becoming necrotic (7). All mice had water and food ad libitum during the suspension period (Figure 1).
Figure 1: Depiction of the hindlimb suspension using in the study. Each mouse is suspended according to the protocol recommended by Ferreira et al. (
7). In addition, mice were provided diet gel and water in which they could reach without placing their hindlimb on the edge of the ceramic crocks. Animals were monitored daily by both veterinarian technicians and investigator (MHM). Diet gel and water was replaced as needed during the experimental period.
Tissue Removal Procedure
After the suspension period, mice in all 3 groups were overdosed with sodium pentobarbital (200 mg·kg−1, intraperitoneally). The soleus and medial gastrocnemius muscles from both hindlimb muscles were removed. These 2 muscles were selected for this study because they have predominantly oxidative fibers (1). The muscles were flash frozen in liquid nitrogen and stored at −80° C for biochemical analyses. The right medial gastrocnemius muscles were sliced from the widest point of the middle belly and frozen in precooled isopentane (−140° C) and stored at −80° C. Transverse sections (10 µm) were cut on a cryotome (Leica CM 1950; Leica Microsystems, Buffalo Grove, IL, USA) at −20° C and mounted on slides for histochemical analyses of capillarity. This approach is routinely used in our laboratory (13,14,18).
Quantification of Capillaries
The Rosenblatt capillary staining method (33) was used for the medial gastrocnemius muscle. In addition, muscle sections were viewed under a digital microscope (×20 magnification, Leica DMD108; Leica Microsystems). Capillaries were quantified from the digital image of the cross section by experienced personnel. The following indices were measured as recommended by Hepple et al. (9,10): (a) the number of capillaries around a fiber (NCAF), (b) the capillary-to-fiber ratio on an individual-fiber basis (C/Fi), and (c) capillary density (CD), which was calculated using the fiber area as the reference space. Capillary-to-fiber perimeter exchange (CFPE) index was estimated from the capillary-to-fiber surface area. Fiber cross-sectional area (FCSA) and fiber perimeter (FP) were measured with the image analysis system and commercial software (SigmaScan Pro version 5.0; Systat Software, Inc., Point Richmond, CA, USA).
Western Blot Analysis
The entire soleus and left medial gastrocnemius muscles from each group were pulverized and homogenized separately in lysis buffer (Radioimmunoprecipitation assay; Sigma-Aldrich, St. Louis, MO, USA) in the presence of protease and phosphatase inhibitor cocktails (cOmplete and PhosSTOP; Roche Diagnostics Corporation, Indianapolis, IN, USA). Homogenates were passed through an insulin syringe 3 times and centrifuged for 10 minutes 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, Hercules, CA, USA).
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 minutes in a thermal cycler (S1000; Bio-Rad). Samples were loaded onto 7.5% (TSP-1, PGC-1α, PGC-1β, myosin heavy chain type I [MHC-I], and mTOR/FRAP) or 12% TGX precast gels (Bio-Rad). The transfer, blocking, and primary and secondary antibody incubation procedures were consistent with our previous studies (13,14).
The monoclonal primary antibodies used were TSP-1 (1:500, sc-59886; Santa Cruz Biotechnology, Inc, Dallas, TX, USA), PGC-1β (1:100; sc-373771; Santa Cruz Biotechnology, Inc), mTOR/FRAP (1:500, 6858-1; Epitomics, Abcam Company, Burlingame, CA, USA), Slow-MHC (1:1,000, AB11083; Abcam), α-tubulin (1:2,000, ab11304; Abcam, Cambridge, MA, USA), and complex IV (1:1,000, MS404; MitoSciences). The polyclonal primary antibodies used were Akt (1:500, 9272; Cell Signaling), VEGF (1:500, sc-507; Santa Cruz Biotechnology, Inc), GSK-3β (1:500; 9315; Cell Signaling), FoxO1 (1:200; 2880; Santa Cruz Biotechnology, Inc), anti-TFAM (1:1,000; ab131607; Abcam), and PGC-1α (1:1,000; AB3242; Millipore). The secondary antibodies used were goat anti-mouse and goat anti-rabbit IRDyes (1:30,000) purchased from Li-Cor Biosciences. Loading control for target proteins was normalized to α-tubulin. Quantification of bands was performed with the Odyssey software program (Li-Cor Biosciences).
Statistical Analyses
To determine changes in body mass, a 3 [group: C, HS-V, or HS-(−)-Epi] × 3 [time: before surgery, 7 days after surgery, and 14 days after hindlimb suspension] mixed factorial analysis of variance (ANOVA) was performed. In addition, separate 1-way ANOVAs were conducted for comparison of groups for each outcome variable. For all significant overall F-ratios, Tukey HSD post hoc was conducted to identify statistically significant mean differences among groups. The data are presented as mean ± SEM. For all analyses, a p value of p ≤ 0.05 was considered significant.
Results
Anthropometric and Muscle Mass
The 3 × 3 mixed factorial ANOVA revealed a significant interaction (F4,44 = 65.91; p < 0.0001) for body mass. After the significant interaction, simple main effects' testing was performed to examine mean differences for each time point within each group. As shown in Table 1, there were no significant differences between the 3 time points within the control group. For the HS-V group, however, there were no differences in body mass before the surgery and 7 days after the surgery, but there was a significant decrease after 14 days of hindlimb suspension. These patterns of responses for body mass were also observed for the HS-(−)-Epi group (Table 1).
Table 1: Anthropometric measurements for each group (mean ± SEM).*
We also examined the soleus and medial gastrocnemius muscle masses. Fourteen days of hindlimb suspension significantly decreased both absolute and relative muscle mass for the soleus muscle in both experimental groups compared to the control group (Table 1). For the medial gastrocnemius muscle, however, there was only a statistically significant decrease in muscle mass for the 2 experimental conditions for the absolute value compared with controls.
Capillary Assessment
When capillarity and fiber dimensions were examined for the medial gastrocnemius muscle, we found that NCAF and C/Fi were significantly reduced in the HS-V group compared with controls (Figure 2). A similar decrease was observed for the HS-(−)-Epi when compared with the control group, but NCAF and C/Fi were significantly higher compared with the HS-V group. There were no statistical differences between the 3 groups for CD and CFPE. As shown in Table 2, when compared to the control group, FCSA was significantly smaller in the HS-V and HS-(−)-Epi groups. The HS-(−)-Epi group did have a significantly larger FCSA than the HS-V group. For FP, however, the HS-V group was significantly lower than the control but not statistically different from the HS-(−)-Epi group.
Figure 2: Capillarity measurements for the medial gastrocnemius muscle. Comparison of NCAF, C/Fi, CD, and CFPE between the three groups (n = 3 per group; mean ± SEM). Note: Panel A = *HS-V (p = 0.001) and HS-(−)-Epi (p = 0.016) significantly different than control group; †significantly (p = 0.025) different than HS-V group. Panel B = No significant differences between groups. Panel C = *HS-V (p = 0.02) and HS-(−)-Epi (p = 0.043) significantly different than control group; †significantly (p = 0.045) different than HS-V group. Panel D = No significant differences between groups.
Table 2: Morphological measurements of medial gastrocnemius muscle (mean ± SEM).*
Protein Quantification
The results of the 1-way ANOVAs revealed significant mean differences in protein expression for signaling pathways related to angiogenesis, muscle degradation, and mitochondrial biogenesis. Specifically, although pro-angiogenic factor VEGF-A was reduced in both the HS-V and HS-(−)-Epi groups compared with controls, anti-angiogenic factor TSP-1 was increased twofold in the HS-V group but did not change in the HS-(−)-Epi group (Figure 3). Myosin heavy chain type I levels were significantly reduced in the HS-V group but unchanged in the HS-(−)-Epi group compared with controls (Figure 4A) as were anabolic complex mTOR, Akt, and the key mitochondrial transcriptional activator TFAM (Figure 4C, D, Figure 5B). In contrast, FoxO1 and GSK-3β showed the opposite pattern and were only induced in the HS-V group (Figure 4B and E). Peroxisome proliferator-activated receptor-γ coactivator 1–α was significantly downregulated in both HS-V and HS-(−)-Epi groups, whereas PGC-1β was strongly induced in the HS-(−)-Epi group only (Figure 5A). Finally, CcO, the proposed rate-limiting enzyme of the electron transport chain was significantly reduced in the HS-V group but maintained similar levels as the controls in the HS-(−)-Epi group (Figure 5C).
Figure 3: Protein expression for pro- and antiangiogenic signaling factors in soleus muscle. Representative Western blot from soleus muscle for all three groups. (n = 4–6 per group; mean ± SEM). Note: VEGF: *significantly less (p ≤ 0.014) than the control group. TSP-1: *significantly greater (p < 0.0001) than control and HS-(−)-Epi groups.
Figure 4: Protein expression for MHC I, FoxO1, and mTOR/Akt signaling in the medial gastrocnemius muscle. Representative Western blot results from the medial gastrocnemius muscle for all three groups. (n = 4–6 per group; mean ± SEM). MHC I (panel A): *significantly less than control (p = 0.028) and HS-(−)-Epi (p = 0.04) groups. FoxO1 (panel B), *significantly greater than control (p = 0.013) and HS-(−)-Epi (p = 0.038) groups. mTOR (panel C), *significantly greater than HS-V (p = 0.002) group. Akt (panel D), *significantly less than control (p = 0.026) and HS-(−)-Epi (p = 0.001) groups. GSK-3b (panel E): **significantly greater than control (p = 0.023) and HS-(−)-Epi (p < 0.0001) groups; Control and HS-(−)-Epi were not significantly different (p = 0.069).
Figure 5: Protein expression for mitochondrial biogenesis signaling in the medial gastrocnemius muscle. Representative Westerns blots from the medial gastrocnemius muscle for all three groups. (n = 4–6 per group; mean ± SEM). Note: PGC-1a (panel A), *significantly less (p ≤ 0.010) than control group; PGC-1b (panel A), **significantly greater than control (p = 0.015) and HS-V (p = 0.005) group. TFAM (panel B), *significantly less than control (p = 0.038) and HS-(−)-Epi (p = 0.012) groups. CcO (panel C), *significantly less than control (p = 0.032) and HS-(−)-Epi (p = 0.011) groups.
Discussion
The main findings of this investigation are that 14 days of hindlimb suspension with (−)-epicatechin supplementation resulted in an attenuation of the signaling pathways associated with muscle degradation. For protein markers of angiogenesis, we found that hindlimb suspension reduced VEGF-A in the groups receiving the vehicle or (−)-epicatechin supplementation compared with controls. Importantly, TSP-1 protein expression significantly increased in the hindlimb suspension group receiving the vehicle, but not in the group receiving (−)-epicatechin.
Studies have suggested that decreases in the soleus muscle mass may be observed as early as 3 days after hindlimb suspension (29). In this study, we found that 14 days of hindlimb suspension significantly decreased FCSA (Table 2) in the 2 experimental groups compared with controls. Animals supplemented with (−)-epicatechin, however, had significantly higher FCSA than animals that received the vehicle. For FP, we observed that animals receiving the vehicle during hindlimb suspension had significantly smaller values compared with controls. Moreover, animals receiving (−)-epicatechin had a small, but nonsignificant reduction in FP compared to the control group.
It has been suggested that the Akt/mTOR pathway may play a critical role in muscle atrophy (2). Nagatomo et al. (25) found that 3 weeks of hindlimb suspension upregulated FoxO1 mRNA in the soleus muscle with a concomitant loss of muscle mass. Lang et al. (17) reported that mTOR heterozygous knockout mice showed impaired muscle mass recovery in the soleus muscle after 10 days of hindlimb immobilization compared with controls. The results of these studies indicate that hindlimb suspension may activate muscle degradation signaling.
In this study, we examined protein expression of various regulators of muscle degradation signaling. We observed a significant reduction in MHC I, mTOR, and Akt protein expression in mice treated with the vehicle compared with controls and (−)-epicatechin-treated groups. For regulators associated with muscle atrophy such as FoxO1 and GSK-3β, there were significant increases in the group receiving the vehicle (Figure 4), but not in the (−)-epicatechin-treated group.
Related to the signaling pathways associated with muscle degradation, Reed et al. (32) found that inhibition of basal FoxO activity promoted muscle fiber hypertrophy. Liu et al. (21) reported that FoxO3 protein expression increased ∼55% in the gastrocnemius of rodent muscle after 4 weeks of hindlimb suspension, whereas Léger et al. (19) reported that 8 weeks of detraining after 2 months of resistance training resulted in decreased protein expression of Akt-1 and GSK-3β in the vastus lateralis of healthy men. Moreover, the authors found that training significantly reduced protein expression of FoxO1 compared with controls (19). Doucet et al. (5) reported significantly higher protein expression of FoxO1 in patients with COPD compared to controls. Moreover, when patients with COPD were subdivided into those with preserved muscle mass and low muscle mass, the latter group had significantly higher protein expression of phosphorylated GSK-3β. In a series of experiments using cultured C2C12 myotubes, Verhees et al. (41) concluded that GSK-3β is a critical regulator of skeletal muscle atrophy. The results in this investigation, therefore, suggest that (−)-epicatechin may attenuate signaling pathways associated with muscle degradation.
Oxidative muscle fibers have an abundance of capillaries, and it has been suggested that this is controlled, in part, by a balance between angiogenic stimulators (i.e., VEGF) and inhibitors (TSP-1) (30,35). Studies related to hindlimb suspension indicate an induction of capillary rarefaction in the muscle. For example, Kanazashi et al. (15) reported significant reductions in capillary-to-fiber ratio and NCAF after 7-day hindlimb suspension in the soleus muscle of rats, which corresponded to significant reductions in protein expression of VEGF. Moreover, the ratio of VEGF:TSP-1 mRNA was significantly reduced in the hindlimb suspension group compared with controls. Roudier et al. (34) reported increased CD in the soleus muscle of rats after 9 days of hindlimb suspension, but a significant decrease in capillary-to-fiber ratio. Furthermore, the investigators found no change in basal VEGF-A protein expression in the hindlimb suspension group when compared with controls. There was, however, a significant decrease observed in VEGF-B and VEGF-R2 protein expression (34). Roudier et al. (34) reported that TSP-1 protein expression was significantly increased compared to controls and therefore an observed reduction in the VEGF-A:TSP-1 protein expression ratio in the soleus muscle.
When angiogenic stimulators and inhibitors were examined, our data indicated that there was ∼20–23% decrease in basal VEGF-A protein expression for both HS-V and HS-(−)-Epi groups compared with the control group. For TSP-1 protein expression, however, we found a 3-fold increase in basal levels for the HS-V group, whereas the HS-(−)-Epi group had similar levels to the control group. The attenuation of TSP-1 by (−)-epicatechin is consistent with the findings of Hüttemann et al. (13) who also reported that (−)-epicatechin maintained the endurance training-induced reduction of TSP-1 in the hindlimb muscle after 14 days of detraining. In another study, Hüttemann et al. (14) reported that 30 days of (−)-epicatechin supplementation in a rodent model of congenital skeletal muscle dysfunction increased VEGF-A protein expression in the plantaris (i.e., glycolytic) muscle, which then returned to control levels after 15 days of stopping (−)-epicatechin supplementation. Conversely, these studies reported that TSP-1 protein expression significantly decreased after 30 days of (−)-epicatechin supplementation and remained at that same level after 15 days of cessation (14). Future studies, therefore, are needed to determine the time course of VEGF-A and TSP-1 protein expression with (−)-epicatechin supplementation in both glycolytic and oxidative muscle fibers.
The lack of consistent results across studies for PGC-1α expression may be, in part, due to different suspension periods, strain and species of rodents, and the hindlimb muscle that was examined (oxidative vs. glycolytic vs. mixed). For example, Wagatsuma et al. (43) reported a significant increase in PGC-1α mRNA expression in the gastrocnemius muscle of mice after 7 days of hindlimb suspension with a concomitant decrease in PGC-1β expression, whereas Nagatomo et al. (25) reported that 3 weeks of hindlimb suspension increased PGC-1α mRNA expression in the soleus muscle of rats. Conversely, Oishi et al. (28) reported that PGC-1α protein expression in the soleus muscle of rats was significantly reduced compared with controls after 2 weeks of hindlimb suspension. Liu et al. (21) also found a significant decrease in PGC-1α protein expression in the deep-red region of the gastrocnemius (i.e., medial gastrocnemius) muscle after 4 weeks of hindlimb suspension. The authors did not examine PGC-1β protein expression in their sample.
Here, we examined the protein expression of various regulators of mitochondrial biogenesis. Peroxisome proliferator-activated receptor-γ coactivator 1 transcriptional coactivators integrate metabolic signaling and skeletal muscle fiber-type switching (31). Both PGC-1α and PGC-1β strongly increase total mitochondrial respiration and are expressed in tissues that rely heavily on mitochondrial oxidative phosphorylation (8). We also found that PGC-1α protein expression in the soleus muscle was significantly reduced in both HS-V and HS-(−)-Epi groups compared with the control group (Figure 5). For PGC-1β, however, we found that 15 days of hindlimb suspension did not change basal protein expression in the HS-V group, whereas (−)-epicatechin supplementation resulted in a significant increase.
Peroxisome proliferator-activated receptor-γ coactivator 1 family of regulated coactivators also controls the expression of TFAM, which plays an essential role in both transcription and replication of mitochondrial DNA (37). Liu et al. (21) reported decreased TFAM protein expression for the hindlimb muscle in their study. In addition, the authors found significant reductions in complex I protein expression, but not complex IV (21). In this investigation, we found a significant reduction in TFAM protein expression in the group receiving the vehicle during hindlimb suspension, whereas the group receiving (−)-epicatechin did not have reduced TFAM expression. Furthermore, we found that CcO was not reduced after 15 days of hindlimb suspension with (−)-epicatechin supplementation. Interestingly, Liu et al. (21) reported a significant reduction in complex IV only in the tibialis anterior, which is predominantly a glycolytic muscle. Our findings suggest that (−)-epicatechin supplementation during hindlimb suspension attenuates mitochondrial loss.
In summary, results of this investigation, in conjunction with the results from our previous work, on glycolytic skeletal muscle (13,14,27) indicate that (−)-epicatechin counteracts muscle degradation, while maintaining angiogenesis and mitochondrial biogenesis when the muscle is unloaded. Future studies, however, are needed to determine whether (−)-epicatechin can attenuate the negative effects of hindlimb suspension for extended periods (i.e., 30–60 days).
Practical Applications
Although this study was conducted with an animal model, there are potential applications to human models. To our knowledge, pure (−)-epicatechin whether in powder or tablet form is not available within the United States. Therefore, human studies that examine the role of (−)-epicatechin on various physiological outcomes may use a cocoa powder that is fortified with a specific dosage of (−)-epicatechin. In a pilot study, Taub et al. (39) supplemented 5 patients with heart failure and type 2 diabetes for 3 months with (−)-epicatechin rich cocoa (100 mg·d−1). Using muscle biopsies from the vastus lateralis muscle, the investigators reported significant pre-post increases in protein expression for markers associated with muscle development and maintenance such as myocyte enhancer factor 2, myogenic regulatory factor 5, and myogenic differentiation (39). Moreover, the results of 2 meta-analyses indicate that moderate consumption of cocoa, by human participants, is effective in preventing cardiovascular disease (47) because it improves flow-mediated dilatation and insulin resistance (12). Future studies, however, are still needed to determine the optimal dosage and timing of (−)-epicatechin supplementation to attenuate the deleterious effects in skeletal muscle derived from various perturbations such as space travel, injury, and/or disease.
Acknowledgments
This study was supported by start-up funds to MHM from Wayne State University.
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