Fatigue-induced changes in force production can be analyzed in terms of a generally decreased ability of cross-bridges formation to generate force, decreased myofibrillar Ca2+ sensitivity, decreased sarcoplasmic reticulum (SR) Ca2+ release, or combination of these . Previous studies that used skinned or intact fibers indicated superoxide-dependent protein modifications  , glycogen depression   , and protein degradation  as the probable causes of decreased SR Ca2+ release .
Unaccustomed eccentric contraction (ECC) induces skeletal muscle damage characterized by a long-lasting decrease in muscle strength and delayed onset muscle soreness . Muscles damaged by unaccustomed ECC are characterized by sarcolemmal disruption and myofibrillar disorganization during ECC  and crystallized structures within the Z disk and SR swelling immediately following ECC  ; consequently, overstretched sarcomeres act as the origin of such muscle damage . Reportedly, impaired excitation-contraction coupling occurs in eccentrically damaged mammalian muscles . It has been suggested that sarcomeres within myofibrils, transverse tubules (t-tubules), and SR are susceptible to damage in the overstretched regions, possibly marking the basis of excitation-contraction coupling damage .
Recently, the presence of nitrates within the diet and their potential as a source of nitric oxide (NO) has gained increasing attention. NO itself plays an important regulatory role in several physiological processes, such as vasodilatation, blood pressure regulation, mitochondrial respiration, cell signaling, and mitochondrial biogenesis   . The classic mechanism for NO generation via the oxidation of L-arginine in a reaction catalyzed by nitric oxide synthase (NOS) has been well-documented .
Dietary supplementation with inorganic nitrate an NO donor, enhances NO bioavailability, reduces oxygen cost of exercise, and increases exercise performance in endurance exercise . Further, supplemented nitrate enhances endurance performance  and reduces the PCr cost of force production . Removal of dietary nitrate supplementation from the diet has been shown to reduce running distance and speed to the control level, despite the gain of improved endurance during dietary nitrate supplementation . Recent studies have shown that dietary nitrate supplementation enhances SR Ca2+ release in mouse fast-twitch muscles  and that NO, synthesized from L-arginine injection, increases Ca2+ regulatory protein concentrations .
Based on these findings, we designed a hypothesis that dietary nitrate supplementation inhibits ECC-induced alterations in the SR function. The main objective of the present study was to examine whether dietary nitrate supplementation prior to ECC would alleviate the decline in muscle contractile properties and SR Ca2+ handling in rat fast-twitch skeletal muscle immediately following ECC.
2. Materials and Methods
2.1. Animal Care and Nitrate Ingestion
Thirty 9-wk-old male Wistar rats were housed in a thermally controlled room maintained between 20˚C and 24˚C under a 12-h light/dark cycle. Water and food were provided ad libitum. All study procedures were approved by the Animal Care Committee of Hiroshima University. The rats were randomly divided into three nitrate-treated groups (n = 10 for each group) and were administered dietary nitrate supplementation for 0 (non-treated), 3, and 6 days. The period of dietary nitrate supplementation used in this study is similar to that utilized in the study by Hernández et al. . The rats were administered 1 mmol kg−1 day−1 NaNO3 diluted with 4 mL tap water. We used the nitrate dose described by Ferguson et al. .
2.2. Exercise Procedures
Throughout the experiment, the rats were deeply anesthetized with an intraperitoneal injection of a mixture of medetomidine (0.4 mg kg body wt−1), midazolam (2.0 mg kg body wt−1), and butorphanol (2.5 mg kg body wt−1). ECC was performed as described previously . Briefly, an animal was placed in the supine position on a supporting platform, with the left foot secured in a foot holder attached to the rim of a servomotor. Further, the knee was secured using a strap such that the foot was positioned perpendicular to the lower leg. A pair of sterilized needle electrodes was inserted through aseptically prepared skin to stimulation of the peroneal nerve in the left leg that innervates the left extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. The correct location of the needles was confirmed by the dorsiflexion of the ankle joints and extension of the toes in response to the electrical stimulation of the common peroneal nerve. Repetitive contractions of the EDL and TA muscles were induced by electrical stimuli applied to the common peroneal nerve. In addition, muscle contractions were elicited by stimulating the peroneal nerve using a 1000-ms train of 1-ms pulse at 50 Hz and supramaximal voltage. For the ECC protocol, the experimental leg was forcibly extended with the servomotor at an angular velocity of 150˚ s−1 from the ankle joint, from 30˚ to 180˚, in synchrony with the electrical stimulation of the nerve over a 1-s period. The ECC was repeated every 4 s for a total of 200 repetitions.
Immediately following ECC, the experimental EDL and TA muscles (left hindlimb) as well as contralateral resting muscles (right hindlimb) were quickly excised. The amount of EDL or TA muscle obtained was considered too small for physiological or biochemical analysis. Therefore, EDL and TA muscles were used to measure the force production and biochemical analyses, respectively. As reported by Kanzaki et al.  , EDL and TA muscles have almost the same composition of rat fast-twitch fibers and exhibit similar functional deficits following ECC. Some previous studies assumed that these muscles are similarly affected by ECC . At the end of the experiments, the rats were euthanized with pentobarbital sodium (200 mg kg body wt−1), followed by cervical dislocation.
2.3. Measurement of Isometric Force Production
Isometric force production of the EDL muscles was recorded at 30˚C in a chamber filled with a solution of the following composition (as previously described  ): 115 mM NaCl, 5 mM KHCO3, 1 mM MgCl2, 20 mM NaHCO3, 2 mM CaCl2, 5 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 11 mM glucose, 0.3 mM glutamic acid, and 0.38 mM glutamine. The solution was continuously bubbled with 95% O2/5% CO2, yielding a pH of 7.4. The EDL muscles were connected to an isometric force transducer. The stimulation pulses were applied via two platinum plate electrodes placed on each side of the muscle. The muscles were allowed to equilibrate for 10 min, during which, the optimal length was determined. Tetanic forces were elicited via direct stimulation at 20, 40, 60, and 80 Hz using a supramaximal voltage, 1-ms pulses, and 1.5-s trains. Force was recorded on a personal computer, analyzed using dedicated software (Lab Chart; ADInstruments, Nagoya, Japan), and normalized to the cross-sectional area, where the cross-sectional area was computed as the muscle wet weight divided by the product of the muscle length and density (1.07 g mL−1).
2.4. Homogenate Preparation
The TA muscle pieces were diluted in a ratio of 1:9 (mass vol−1) in ice-cold homogenizing buffer (pH 7.4) composed of 300 mM sucrose, 20 mM MOPS/KOH, 0.0014 mM pepstatin, 0.83 mM benzamidine, 0.0022 mM leupeptin, and 0.2 mM phenylmethanesulfonyl fluoride . They were mechanically homogenized thrice with a hand-held glass homogenizer (Asone, Osaka, Japan) at 5000 rpm for 30-s bursts separated by 30-s breaks. Then, the homogenate was centrifuged at 5000× g for 10 min. The obtained supernatant was quickly frozen in liquid nitrogen and stored at −80˚C. The measurements of SR Ca2+-ATPase activity and Ca2+-uptake and -release rate were performed using the supernatant. The protein concentrations were determined using the method described by Bradford .
2.5. SR Ca2+-ATPase Activity
The SR Ca2+-ATPase activity in the presence of 1 µg mL−1 Ca2+ ionophore A23187 (Sigma) was spectrophotometrically measured in muscle homogenates in triplicate at 37˚C as per the methods described by Simonides & van Hardeveld . The assay mixture (pH 7.1) comprised 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 1 mM EGTA, 200 mM KCl, 15 mM MgCl2, 0.8 mM CaCl2, 10 mM sodium azide (NaN3), 0.4 mM NADH, 10 mM phosphoenolpyruvate, 12.1 U mL−1 pyruvate kinase, and 20.2 U mL−1 lactate dehydrogenase. The reaction was initiated by adding Mg-ATP at a final concentration of 4 mM. Finally, the CaCl2 concentration was increased to 20 mM to selectively inhibit SR Ca2+-ATPase activity. The remaining activity was considered as the background ATPase activity. The activity of SR Ca2+-ATPase was calculated as the difference between the total and background ATPase activities.
2.6. SR Ca2+ Uptake and Release Rate
SR Ca2+ uptake and release rates were measured at 37˚C in triplicate using the Ca2+ fluorescent dye indo-1, as previously described . Aliquots of the homogenate were incubated for 3 min at 37˚C in an assay buffer (pH 7.0) composed of 100 mM KCl, 20 mM N-2-hydroxyethylpiperazine-N''-2-ethanesulfonic acid, 10 mM NaN3, 6.8 mM potassium oxalate, 0.5 mM MgCl2 and 0.001 mM indo-1. SR Ca2+ uptake was initiated by the addition of 1 mM Mg-ATP, which was continued until little or no change in the Ca2+ concentration ([Ca2+]) was observed. Then, Ca2+ release was initiated by adding 10 mM 4-chloro-m-cresol. [Ca2+] was monitored using a spectrofluorometer (FB-8300ST; Nihon-Bunko, Tokyo, Japan) and computed as per the ratiometric method .
2.7. Statistical Analysis
Statistical analyses were conducted using the SigmaPlot statistical software (version 14; Systat Software, San Jose, CA). All data are presented as mean ± standard error values of the mean (SE) values. Two-way ANOVA was used to investigate the effects of the contractile protocol (ECC vs. Rest) and period of dietary nitrate supplementation. When significant differences were detected, Holm-Sidak post hoc test was performed. Statistical significance was set at P-value < 0.05.
3.1. Isometric Force Production
There were no significant differences among the measurement results of the three resting groups. ECC-induced force production by 80-Hz stimulation in the 0-, 3-, and 6-day nitrate-treated muscles declined to 71.9%, 73.9%, and 83.5%, respectively, compared with that in the contralateral resting muscles (Figure 1). Regarding force production, compared with the contralateral resting muscles, the 0- and 3-day nitrate-treated muscles showed a significant decline (P = 0.005 and P = 0.001, respectively), whereas the 6-day nitrate-treated muscles showed no significant difference (P = 0.125).
3.2. SR Ca2+ Handling Function
The Ca2+-ATPase activity following ECC in the 0-, 3-, and 6-day nitrate-treated muscles was 86.8%, 97.6%, and 96.5%, respectively, compared with that of in the contralateral resting muscles (Figure 2). Further, the rate of Ca2+ uptake was 90.5%, 92.3%, and 93.2% in the 0-, 3-, and 6-day nitrate-treated muscles, respectively, compared with that in the contralateral resting muscles (Figure 3).
Figure 1. Effect of dietary nitrate on force production immediately following eccentric contraction. The rats were orally administered nitrate (1 mmol kg−1 day−1) prior to the ECC protocol. ECC was repeated in the anterior muscles of the left hind−limb for a total of 200 cycles. The rested muscles of the contralateral (right) legs were used as controls. Immediately following ECC, the extensor digitorum longus muscles were excised and used to measure of isometric force production. Isometric forces were evoked via direct electrical stimulation at 80 Hz. The values represent mean ± standard error of mean values (SE) (n = 8 for each muscle). *P < 0.05, versus rested muscle within rats. ECC, eccentric contraction.
Figure 2. Effect of dietary nitrate on SR Ca2+-ATPase activity immediately following eccentric contraction. The rats were orally administered nitrate (1 mmol kg−1 day−1) prior to the ECC protocol. Immediately following ECC of a total of 200 cycles, the tibialis anterior muscles were excised. Activities were measured on muscle homogenates. The values represent mean ± standard error of mean values (SE) (n = 8 for each muscle). #P < 0.05, significant main effect for ECC (rest > ECC). ECC, eccentric contraction; SR, sarcoplasmic reticulum.
Regarding Ca2+-ATPase activity and Ca2+ uptake, a main effect was observed between ECC and Rest (Rest > ECC: P = 0.047 and P = 0.036, respectively).
Although the rate of ECC-induced Ca2+ release significantly declined to 78.6% and 77.6% in the 0- and 3-day nitrate-treated muscles, respectively, compared with that in the contralateral resting muscles (Figure 4, P = 0.036 and P = 0.022, respectively), no significant difference in this regard was found between the 6-day nitrate-treated and contralateral resting muscles (102.9%, P = 0.797).
Figure 3. Effect of dietary nitrate on SR Ca2+-uptake rate immediately following eccentric contraction. The rats were orally administered nitrate (1 mmol kg−1 day−1) prior to the ECC protocol. Immediately following ECC of a total of 200, the tibialis anterior muscles were excised. SR Ca2+-uptake rate was measured on muscle homogenates. The values represent mean ± standard error of mean values (SE) (n = 8 for each muscle). #P < 0.05, significant main effect for ECC (rest > ECC). ECC, eccentric contraction; SR, sarcoplasmic reticulum.
Figure 4. Effect of dietary nitrate on SR Ca2+-release rate immediately following eccentric contraction. The rats were orally administered nitrate (1 mmol kg−1 day−1) prior to the ECC protocol. Immediately following ECC of a total of 200, the tibialis anterior muscles were excised. SR Ca2+-release rate was measured on muscle homogenates. The values represent mean ± standard error of mean values (SE). *P < 0.05, versus rested muscle within rats. ECC, eccentric contraction; SR, sarcoplasmic reticulum.
Dietary nitrate supplementation has been shown to exert a variety of effects on physiological function, with recent evidence that short-term supplementation lowers the resting blood pressure    , reduces the energetic cost of exercise    , activates muscle contraction    , and enhances endurance   and intense intermittent exercise performance . However, to the best of our knowledge, there is no sufficient evidence regarding the effects of nitrate ingestion before ECC on muscle contractile properties. The following remarkable results were noted in this study. First, 6-day dietary nitrate supplementation, but not 3-day supplementation, mitigated ECC induced decreases in force production, demonstrating that 6-day dietary nitrate supplementation prior to the ECC protocol markedly improved force production in rat fast-twitch muscles following ECC.
ECC results in an inability to produce the desired force characterized by triad deformation  , sarcomere inhomogeneity  , increased membrane permeability  , inflammation  , and proteolysis  . It has been well documented that modified intracellular Ca2+ handling induced by dietary nitrate supplementation may enhance muscle performance  . Recently, an enhancement in SR Ca2+ handling function by dietary nitrate supplementation in mouse fast-twitch muscle was reported . Second, 6-day nitrate supplementation prior to the ECC protocol inhibited ECC-induced decreases in the SR Ca2+ release rate in rat fast-twitch muscles. We noted that loss of ECC-induced contractile activity can be attributed to a failure of SR Ca2+ release . Nitrate ingestion enhanced SR Ca2+ release and tetanic force production via modifications of the cellular Ca2+ handling components in mouse fast-twitch muscle . NO is primarily synthesized from L-arginine by neuronal NOS and can enhance Ca2+ regulatory proteins concentration  . In the skeletal muscles, L-arginine-driven NO is moderately generated in the resting state, and its production markedly increases with contractile activity . Considering these observations, it can be hypothesized that enhanced SR Ca2+ release following dietary nitrate supplementation can alleviate ECC-induced loss of contractile function.
Ca2+-regulated cysteine proteases (calpains) comprise a proteolytic system in the skeletal muscles. Previous in vitro studies on the effect of NO on calpains demonstrated that the use of NO donors can inhibit the activation of calpains mediated via S-nitrosylation . In a recent study, it was demonstrated that treatment with a calpain inhibitor could attenuate ECC-elicited force deficits, proteolysis of proteins regulated Ca2+ release from SR in fast-twitch muscles of rats  , and L-arginine ingestion can attenuate ECC-induced proteolysis of Ca2+ regulatory proteins by decreasing calpain activation via S-nitrosylation . Considering these findings, it was suggested that the attenuation of the ECC induced decline of SR Ca2+ release in this study is attributable to the decreased calpain activation via S-nitrosylation induced by the 6-day ingestion of nitrate, an NO donor, although calpain was not analyzed in this study. Thus effects dietary nitrate supplementation on contractile function and calpain activity following ECC should be explored in future investigations.
In conclusion, the present results indicated that nitrate ingestion is capable of alleviating ECC-related decreases in muscle force production. These findings suggest that a supplemental ingestion of nitrate exerts beneficial effects, such as muscle performance restoration following physical activity.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (18K10817).
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