All procedures used in this study received approval from the Institutional Animal Care and Use Committee of Kawasaki University of Medical Welfare (No. 17-012). Eighteen male Wistar rats (6 weeks old) were purchased from Charles River Laboratory (Yokohama, Japan). They were given water and standard chow (MF; Oriental Yeast Corporation, Tokyo, Japan) containing 1.53% (mass/mass) ARG ad libitum and housed in a thermally controlled room at 22–26 °C with a 12/12 h light/dark cycle for at least 1 week before the experiment began. At the end of the experiments, the rats were euthanized with an overdose of isoflurane, followed by cervical dislocation.

Rats aged 7–8 weeks old (~ 290 g) were randomly assigned to a control (CON) or ARG group (n = 9 in each group). ARG rats were received water containing 0.4% (mass/vol.) ARG (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) ad libitum for 7 days, starting 3 days before and continuing for 3 days after the ECC protocol. The mean ARG intake in this study (584 mg/kg body wt/day) aligned with previous studies using similar protocols (Lomonosova et al. 2014; Kanzaki et al. 2018). CON rats received water without ARG.

Under anesthesia, achieved with an intraperitoneal injection of medetomidine (0.4 mg/kg body wt), midazolam (2.0 mg/kg body wt), and butorphanol (2.5 mg/kg body wt), rats were placed in a supine position, and the left foot was secured in a homemade foot holder attached to the rim of a servomotor. ECC was induced using an electrical stimulator (SEN-3401; Nihon Kohden, Tokyo, Japan) as previously described (Kanzaki et al. 2017, 2018). Briefly, ECCs were elicited in the anterior crural muscles by stimulating the peroneal nerve with a 1/s train of 1/ms pulses at 50 Hz and supramaximal voltage during forced plantar flexion (150° angular movement at 150°/s from 30° ankle flexion). ECC was repeated every 4 s for 200 cycles. Anesthetized rats were recovered using an intraperitoneal injection of the medetomidine antagonist atipamezole (0.8 mg/kg body wt).

Three days after ECC induction, the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles were excised from both legs under anesthesia. Muscles from the contralateral limb served as resting controls. Tail vein blood was collected, drawn into a tube containing EDTA, and centrifuged at 1000 g and 4 °C for 10 min. Both the obtained plasma and minced TA muscles were stored at −80 °C until analyses.

To eliminate potential influences of acute muscle contractions on subsequent analyses, EDL and TA muscles were used for force measurements and biochemical analyses, respectively. Isolated EDL muscles were mounted between two platinum plate stimulation electrodes (Iwashiya Kishimoto Medical Instruments, Kyoto, Japan) and connected to an isometric force transducer (TB-611 T; Nihon Kohden). The muscles were placed in Krebs–Ringer solution [in mM: 115 NaCl, 20 NaHCO3, 11 glucose, 5 KHCO3, 5 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 2 CaCl2, 1 MgCl2, 0.4 glutamine, and 0.3 glutamic acid] with continuous bubbling of 95% O2-5% CO2, maintaining an extramuscular pH of 7.4. Isometric contractions were elicited via direct stimulation at 10 and 80 Hz with supramaximal voltage, 1/ms pulses, and 1/s trains. Force output was recorded on a personal computer and analyzed using LabChart version 8 (ADInstruments, CO, USA). All measurements were performed in a temperature-controlled room at 25 °C. Absolute force was normalized to cross-sectional area, calculated as wet muscle weight divided by the product of muscle length and density (1.06 mg/mm3).

Nitrite and nitrate (NOx) concentrations were determined following established protocols (Kanzaki et al. 2018, 2019). Frozen TA muscles (~ 150 mg) were pulverized under liquid nitrogen, vortexed with 4 vol. (vol./mass) of ice-cold phosphate-buffered saline (pH 7.4) containing 10 mM N-ethylmaleimide and 2.5 mM EDTA, and centrifuged at 10,000 g and 4 °C for 5 min. The resultant supernatant or thawed plasma was mixed with Griess reagent, and absorbance at 540 nm was measured after a 30-min incubation at 37 °C.

TA muscles (~ 150 mg) were homogenized in 9 vol. (vol./mass) of an ice-cold homogenization buffer [100 mM KCl, 20 mM Tris/HCl (pH 7.4), 5 mM EDTA, 5 mM N-ethylmaleimide, 0.05% (vol./vol.) Triton X-100, and protease inhibitor cocktail (25,955-11; Nacalai Tesque, Kyoto, Japan)]. In preliminary experiments, we noted that the eNOS monomer was identified solely in the supernatant, not in the whole muscle homogenate. Therefore, one third of the homogenate was centrifuged at 10,000 g and 4 °C for 15 min, and the resulting supernatant was collected. Protein content of the supernatant and homogenate was determined using the Bradford assay, with bovine serum albumin serving as a standard (Bradford 1976).

Samples were diluted with a sample buffer [62.5 mM Tris/HCl (pH 6.8), 2% (mass/vol.) SDS, 10% (vol./vol.) glycerol, 10% (vol./vol.) 2-mercaptoethanol, and 0.02% (mass/vol.) bromophenol blue] and heated for 5 min at 95 °C. Subsequently, they were subjected to either 6% (for nNOS and eNOS monomer) or 10% (for CD68, NADPH oxidase 2, and protein carbonyl) SDS-PAGE. For analyses of nNOS dimer, eNOS dimer, and 3-nitrotyrosine, samples were diluted in the sample buffer without 2-mercaptoethanol but not heated before undergoing 6% low-temperature SDS-PAGE (for nNOS and eNOS dimer) (Klatt et al. 1995) or 10% SDS-PAGE (for 3-nitrotyrosine). Proteins were transferred to polyvinylidene difluoride membranes, which were blocked with Tris-buffered saline (pH 7.4) containing 5% (mass/vol.) skim-milk and 0.1% (vol./vol.) Tween-20 for 60 min at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-CD68 (1:1,000 dilution; ab31630, Abcam, Cambridge, UK), anti-eNOS (1:150 dilution; SC-376751, Santa Cruz Biotechnology, TX, USA), anti-3-nitrotyrosine (1:500 dilution; ab52309, Abcam), anti-nNOS (1:500 dilution; sc-5302, Santa Cruz Biotechnology), and anti-NADPH oxidase 2 (NOX2) catalytic subunit gp91phox (1:500 dilution; sc-130543, Santa Cruz Biotechnology). For protein carbonyl analysis, the membranes were reacted with dinitrophenylhydrazine before the blocking step, followed by a 60 min incubation at room temperature with an antidinitrophenyl antibody (1:2000 dilution; #ROIK03, Shima Laboratories, Tokyo, Japan). The membranes were then incubated for 60 min at room temperature with anti-mouse (1:7500 dilution; P0260, Dako, Glostrup, Denmark) or anti-rabbit (1:7500 dilution; sc-2357, Santa Cruz Biotechnology) secondary antibodies.

Finally, target proteins were visualized using ImmunoStar Zeta (FUJIFILM Wako Pure Chemical Corporation), and images were captured using LumiCube Plus (Liponics, Tokyo, Japan). To quantify total proteins on membranes, they were stained with Coomassie Blue R or Ponceau S. Densitometric analysis of immunoreactive and total proteins was performed using Image J (National Institute of Health, MD, USA), and the target protein amount was normalized to total proteins.

Values were represented as means ± SD. Student’s t-tests were used to compare changes in plasma NOx content. Effects of ARG ingestion (CON vs. ARG rats) and ECC (rested vs. ECC muscles) were assessed using two-way repeated-measures ANOVA. Regarding CD68, NOX2, and 3-nitrotyrosine, rank transformation was applied before ANOVA owing to nonnormality and heteroscedasticity in the data. When significant differences were found, Holm–Sidak post hoc tests were performed. The significance level was set at P < 0.05. Statistical analyses were performed using SigmaPlot version 14.5 (Systat software, CA, USA).