People go to high altitudes for several reasons. Miners work at altitudes of up to 6000 m above sea level, travelers climb high altitudes for mountaineering, trekking, and skiing, and military personnel are sometimes stationed in high-altitude locations. The rapid ascent to high altitudes is associated with a reduction in partial pressure of oxygen, contributing to reduced physical performance as a result of reduced oxygen availability at decreased atmospheric pressures (9), i.e., hypobaric hypoxia (HH). HH is associated with significant alterations in the cardiovascular (13) and musculoskeletal energetic systems (6).
While there are medications for HH-induced changes in the cardiovascular system, e.g., hypoxic pulmonary vasoconstriction (9,24,25,31), there are few available treatments for the musculoskeletal energetic system, which is associated with mitochondrial biogenesis. Mitochondrial density is consistently and substantially decreased in individuals acclimatizing to high altitudes (7,14). Therefore, improving mitochondrial function in the skeletal muscle would reduce the HH-induced changes in the musculoskeletal energetic system.
Severe high-altitude hypoxia exposure is considered a triggering stimulus for redox disturbances in cells. HH increases oxidative stress and impairs mitochondrial function in the skeletal muscle (21). In addition, HH-induced changes in mitochondrial biogenesis are partially attributed to mitochondrial redox distress (4). Dihydromyricetin (DHM), i.e., ampelopsin, is the main bioactive polyphenol in rattan tea; recent studies have reported that DHM has excellent antioxidant, hepatoprotective, and neuroprotective properties (20,30). Phytochemicals, such as Rhodiola rosea, represent a common preventive medication for high-altitude sickness; resveratrol, a well-known bioactive compound, increases endurance to exercise (17,23). Given the notable antioxidant and cytoprotective properties of DHM, which are similar to Rhodiola and resveratrol, we assumed that natural polyphenols may exert positive effects on mitochondrial function and biogenesis under HH and consequently improve physical performance. However, such role has not yet been reported. A study has shown that epicatechin, a polyphenolic component of green tea, with a biological composition similar to that of DHM, could promote resistance to fatigue and antioxidant capacity in muscle cells (27). Therefore, it would be of considerable interest to assess the effects of DHM on exercise capacity and mitochondrial function under HH conditions.
In this study, we hypothesize that DHM attenuates the HH-induced exercise intolerance after rapid ascent to high altitudes. To test this hypothesis, we administered three doses of DHM (50, 75, and 100 mg·kg−1) to rats for 7 d and then exposed them to acute HH conditions. Physical performance, metabolic biomarkers, mitochondrial morphology, and mitochondrial biogenesis-related indexes (e.g., activity of respiratory chain complexes, mitochondrial DNA (mtDNA) content, mitochondrial biogenesis, and dynamics markers) were assessed.
MATERIALS AND METHODS
All surgical and experimental procedures were performed in accordance with the Third Military Medical University Institutional Animal Care and Use Committee-approved protocols and the animal care standards of the American College of Sports Medicine.
Animals and reagents
Male Sprague–Dawley rats (180–220 g) were purchased and transferred to the Experimental Animal Center of the Third Military University. Rats were housed in cages maintained at constant temperature and light–dark cycles (light cycle, 0800–1800 h). Rats had access to food and water ad libitum. DHM and R. rosea were purchased from Nanjing Zelang Biotechnology Co. Ltd., China.
Drug treatment and simulated high-altitude model
Rats were randomly divided into six groups with 12 rats per group: normoxia group (NOR), control group (CON), R. rosea group (500 mg·kg−1·d−1), and three DHM groups (50, 75, and 100 mg·kg−1·d−1; DHM-50, DHM-75, and DHM-100, respectively). Each dose of DHM and R. rosea was dissolved in 0.5 mL of distilled water; NOR and CON groups were given 0.5 mL of distilled water as placebo. Dosages were administered intragastrically everyday for 7 d. With the exception of the NOR group, all groups were subjected to a hypobaric hypoxic chamber after the drug treatment, where the atmospheric pressure was reduced to 408 mm Hg, simulating high-altitude (5000 m) with 10.9% oxygen under HH conditions. The partial pressure of nitrogen decreased as the total pressure decreased on ascent; the nitrogen content did not change. The ascending and descending rates for the hypobaric hypoxic chambers were 3–4 m·s−1. After the 24-h HH treatment, their endurance capacity was measured as described in the next section.
Physical performance was assessed with the run-to-fatigue model using a motorized treadmill. Before the experiment, animals were acclimatized to the motorized treadmill (speed, 9 m·min−1) for 7 d (10 min·d−1) at normobaric conditions. After 7 d of drug treatment and 24-h HH treatment, all rats ran on the motorized treadmill at 9 m·min−1 until they reached fatigue, which was defined as the inability to run despite continuous electrode slice prodding for 20 s. The run-to-fatigue time was recorded. Rats with signs of injury were immediately removed from the motorized treadmill.
Tissue collection and blood assays
Immediately after the run-to-fatigue assessment test, the rats were sacrificed by cervical dislocation. Blood was collected by retro-orbital venous plexus sampling, transferred to heparinized tubes, and centrifuged at 5000 rpm for 5 min. The resulting plasma was stored at −80°C. Serum blood urea nitrogen (BUN), creatine kinase (CK), and lactate dehydrogenase (LDH) were measured in an automatic analyzer (Olympus AU5400; Olympus, Japan). Gastrocnemius muscles were carefully isolated; parts of the muscles were immediately processed for transmission electron microscopy (TEM). The rest was frozen and stored at −80°C.
Mitochondrial morphology in the gastrocnemius muscle was assessed by TEM. Gastrocnemius muscles from three animals in each group were cut into pieces of approximately 1 mm3, fixed in 2.5% glutaraldehyde and subsequently in 1% osmium tetroxide, dehydrated, and embedded in Epon in a longitudinal or transverse orientation. Ultrathin sections (60 nm) were stained with 2% uranyl acetate and lead citrate. In each biopsy, at least 10 longitudinal and transverse sections were examined using a JEOL 1400 (JEOL Ltd., Tokyo, Japan) transmission electron microscope at an accelerating voltage of 80 kV. A minimum of 10 micrographs were taken at 17,500× and 50,000× magnification.
Quantification of mtDNA
DNA was isolated from gastrocnemius muscle tissues using the Mitochondrial DNA Extraction Kit (Genmed Scientifics, Inc.). DNA was quantified spectrophotometrically (260 nm) and subjected to quantitative real-time polymerase chain reaction (RT-PCR) (100 ng per reaction) (SYBR® Green I fluorescent RT-PCR protocol; PE Biosystems) in an ABI Prism 7000 Fast RT-PCR System (PerkinElmer). Relative amounts of mtDNA were obtained by comparing their amplification products with those of β-actin (forward, 5′-CCACCATGTACCCAGGCATT-3′; reverse, 5′-CGGACTCATCGTACTCCTGC-3′) and ND1 (forward, 5′-TTAATTGCCATGGCCTTCCTCACC-3′; reverse, 5′-TGGTTAGAGGGCGTATGGGTTCTT-3′).
Enzyme-linked immunosorbent assay
An enzyme-linked immunosorbent assay (MitoSciences, Eugene, OR) was used to assess the activity of mitochondrial electron transport chain complex I, II, IV, and V.
For protein analysis, western blotting was performed with antibodies against mitochondrial transcription factor A (TFAm) (1:2000, ab131607; Abcam), peroxisome proliferator-activated receptor-γcoactivator 1α (PGC-1α) (1:500, ab106814; Abcam), mitofusin 1 (MFN-1) (1:130, ab126575; Abcam), mitochondrial fission 1 (FIS1) (1:500, ab71498; Abcam), AMP-activated protein kinase (AMPK) (1:2000, ab32047; Abcam), AMPK phosphorylation (p-AMPK) (1:1000, ab72845; Abcam), sirtuin 1 (SIRT1) (1:1000, #9475; Cell Signaling Technology), dynamin-related protein 1 (DRP1) (1:1000, #8570; Cell Signaling Technology), MFN-2 (1:1000, #9482; Cell Signaling Technology), and nuclear respiratory factor 1 (1:250, MAB5306; R&D Systems). Gastrocnemius muscles were homogenized in a glass homogenizer with a sodium dodecyl sulfate lysis buffer (Beyotime Institute of Biotechnology) and protease inhibitors (Roche). The muscle homogenates were centrifuged for 5 min at 13,000 rpm at room temperature. The resulting supernatant was transferred to a new tube. Protein concentration of the supernatant was quantified with the BCA Protein Assay Reagent Kit. Samples were separated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline Tween-20 for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary antibodies. The membrane was washed with Tris-buffered saline Tween-20 (0.1%) and incubated with peroxidase-conjugated secondary antibodies for 2 h. Protein was immune-detected using the Fusion FX5 molecular imager (Vilber Lourmat, France) and FUSION-CAPT software. The intensities of the bands were quantified with the BIO-1D analysis software.
Data were analyzed using one-way ANOVA with an appropriate post hoc test or by unpaired Student’s t-test using SPSS 11.0 (SPSS Inc., Chicago, IL). Data were expressed as mean ± SEM. Statistical significance was set at P < 0.05.
DHM improves physical performance under high-altitude conditions
To assess the effects of DHM on physical performance under high-altitude conditions, Sprague–Dawley rats were treated with DHM (50, 75, or 100 mg·kg−1) or placebo for 7 d and then subjected to a run-to-fatigue model under simulated high-altitude conditions. As shown in Figure 1A, the average run-to-fatigue time of the CON group was significantly lower than that of the NOR group, indicating that HH decreased physical performance. The DHM-75 and DHM-100 groups had longer run-to-fatigue time than that of the CON group (P < 0.05). The DHM-100 group had 3 times higher run-to-fatigue time than that of the CON group, indicating that DHM effectively attenuated the HH-induced exercise intolerance under high-altitude conditions. In addition, R. rosea had a prophylactic effect on physical performance, which was equal to the effect of DHM at the dose of 75 mg·kg−1, and DHM at the dose of 100 mg·kg−1 was more effective.
We also assessed metabolic status by quantifying several biomarkers. The results revealed that plasma LDH, BUN, and CK of the CON group were significantly higher than those of the NOR group (Fig. 2) (P < 0.05). The DHM-75 and DHM-100 groups had lower LDH and BUN levels than those of the CON group (Fig. 2) (P < 0.05). All three DHM groups had significantly lower plasma CK levels, which are associated with muscle damage after strenuous exercise (16) (Fig. 2) (P < 0.05).
DHM attenuates HH-induced mitochondrial injuries and promotes mitochondrial proliferation
To assess the effects of DHM on mitochondrial morphology under HH, gastrocnemius muscles were examined by TEM. As shown in Figure 3, the CON group had decreased mitochondrial density and more swollen mitochondria with disorganized and fragmented cristae than those of the NOR group, indicating that HH resulted in significant damage to skeletal muscle cell mitochondria. However, The DHM-100 group had higher mitochondrial density, with less swollen and more intact cristae structures compared with those of the CON group.
We also quantified mtDNA, an indicator of mitochondrial proliferation and density, by quantitative RT-PCR. Using β-actin as an endogenous control for nuclear DNA, mitochondrial density was determined by the copy number of mtDNA per diploid nuclear genome. The relative amount of mtDNA in gastrocnemius muscle cells was significantly lower (Fig. 1B) (P < 0.05) in the CON group than that in the NOR group. Both DHM-75 and DHM-100 had higher mtDNA content than that of the CON group (Fig. 1B) (P < 0.05), indicating that DHM could effectively restore the reduction in mitochondrial proliferation in the skeletal muscle under high-altitude conditions.
DHM improves activity of mitochondrial respiratory chain complexes
The activities of mitochondrial respiratory chain complexes (I, II, IV, and V) in muscle cells were measured. The results revealed significantly lower activity in the CON group than that in the NOR group. The DHM-100 group had higher complex I, II, and IV activities than those in the CON group (Fig. 4) (P < 0.05); complex II activity was 67.6% higher. Compared with that in the CON group, the R. rosea (500 mg·kg−1·d−1) group had higher complex I and II, but not V, activities in muscle cells (Fig. 4). DHM was more effective than R. rosea in restoring the activities of mitochondrial respiratory chain complexes.
DHM up-regulates the expression of genes involved in mitochondrial biogenesis
PGC-1α is a key regulator of mitochondrial biogenesis, which is enhanced by its interaction with SIRT1. NRF-1 and TFAm induce the expression of nuclear-coded mitochondrial proteins (10,18). In this study, we assessed the expression of these four mitochondrial biogenesis-related proteins in skeletal muscle cells. Western blot results revealed that these proteins were significantly lower in the CON group compared with those in the NOR group. However, the DHM-75 and DHM-100 groups had higher protein levels than those in the CON group (Fig. 5) (P < 0.05). In addition, AMPK activation can induce increased mitochondrial biogenesis in skeletal muscle cells by the phosphorylation of PGC-1α (12). Our results revealed that DHM effectively restored the downregulation of AMPK and p-AMPK in muscle cells under high-altitude conditions.
DHM modulates mitochondrial dynamics of fusion/fission
We assessed the effects of DHM on mitochondrial dynamics in muscle cells under HH. As shown in Figure 6, mitochondrial fusion-related proteins (MFN1 and MFN2) were significantly decreased (P < 0.05) and the fission-related proteins (DRP1 and FIS1) were increased in the CON group, indicating that HH altered the mitochondrial dynamics by promoting fission and suppressing fusion. However, in the DHM-75 and DHM-100 groups, the protein levels of MFN1 and MFN2 were up-regulated, whereas DRP1 and FIS1 were downregulated when compared with those in the CON group. Our findings revealed that DHM modulates mitochondrial dynamics of fusion/fission in muscle cells under HH.
HH decreases physical performance as a result of reduced oxygen availability due to decreased atmospheric pressure (2,5,9). Acute exposure to severe environmental hypoxia (i.e., high altitude) is an aggressive physiological stress that contributes to mitochondrial oxidative damage and dysfunction. Mitochondria are the major sites of energy generation that lead to approximately 80% of cellular adenosine triphosphate (ATP) in mammalian cells (28). It has been reported that the mitochondrial content of a muscle is a major determinant of endurance capacity (11). Studies have shown that HH consistently reduces mitochondrial density and decreases the oxidative phosphorylation cycle, resulting in a reduction of aerobic ATP production and poor exercise tolerance. Therefore, mitochondria play important roles during the adaptation to high-altitude hypoxia by counteracting the HH-induced mitochondrial distress and maintaining mitochondrial density and biogenesis, especially in muscle cells. Rattan tea is a popular beverage in China and other Asian countries. DHM in rattan tea is an excellent antioxidant flavonoid that enhances cellular antioxidant capacity through the activation of ERK and Akt signaling pathways. Moreover, DHM possesses hepatoprotective and neuroprotective activities (15). The results of this study revealed that DHM exerts effective cytoprotection in skeletal muscle cells under HH condition, counteracting HH-induced mitochondrial injury, maintaining mitochondrial biogenesis and dynamics, preserving mitochondrial oxidative phosphorylation and ATP synthesis, and improving physical performance. These findings reveal that DHM may be a novel and safe agent for preventing exercise intolerance and, potentially, other altitude-related illnesses attributed to HH.
Mitochondrial biogenesis plays a crucial role in maintaining mitochondrial content, dynamics, and functions. The main regulator of mitochondrial biogenesis in the skeletal muscle is the transcriptional coactivator PGC-1α (32), which is involved in the synthesis of mitochondria-rich Type I muscle fibers (19). The in vivo up-regulation of PGC-1α in muscles improves physical performance (3); however, PGC-1α may decrease in a hypoxic muscle, resulting in decreased mitochondrial biogenesis (33). SIRT1 functions with PGC-1α by de-acetylating it at multiple lysine sites to promote mitochondrial biogenesis (29). NRF-1 and TFAm, which are downstream transcription factors of PGC-1α, induce the expression of mitochondrial transcripts (18). AMPK also regulates mitochondrial content by sensing the energy status of the muscle cell. AMPK activity is increased in the skeletal muscle during exercise, and endurance exercises induce the AMPK–PGC-1α signaling pathway (1). Our results revealed that the expressions of these factors were significantly downregulated in HH exposure, whereas DHM pretreatment significantly restored the downregulation of these factors, indicating a concomitant protection of mitochondrial biogenesis. Further studies should be conducted to understand the underlying mechanism.
Mitochondria are morphologically dynamic organelles that involve two principal processes called fusion and fission (26,34). Mitochondrial fusion and fission control the length, shape, size, and number of mitochondria. Therefore, both processes contribute to the maintenance of mitochondrial function and optimize bioenergetic capacity. Mitochondrial fusion depends on two members of the MFN family: MFN 1 and 2. Mitochondrial fission requires the presence of DRP1 and the adaptor protein FIS1 from the cytosol (8,34). In this study, DHM up-regulated the fusion-related genes MFN1 and MFN2 in skeletal muscle cells under HH exposure and downregulated the fission-related genes DRP1 and FIS1. It is possible that these modifications in fusion/fission could restore the loss of mitochondrial biogenesis under HH exposure, leading to increased physical performance.
In addition, parameters such as BUN, LDH, and CK are biomarkers of the metabolic status of the body (17). In addition, plasma CK is one of the most commonly used biomarkers of muscle damage (16). After exercise, plasma levels of BUN, LDH, and CK increase (22). DHM reduced these biomarkers of fatigue and increased physical performance during HH. In addition, the reduction in CK by DHM suggests that this flavonoid might reduce muscle damage caused by strenuous physical exercise.
In conclusion, our study suggests that DHM improves physical performance under simulated high altitude by maintaining mitochondrial biogenesis and dynamics in skeletal muscle cells. DHM is a novel and safe compound that prevents exercise intolerance and, potentially, other altitude-related illnesses attributed to HH.
This work was funded by research grants from the key projects of the “12th Five-Year Plan” for Medical Science Development of the People’s Liberation Army, China (BWS12J034).
D. Z., K. C., and P. L. contributed equally in this article.
The authors have no conflicts of interest to declare.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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