Ethical approval
All experiments were approved by the Animal Experimentation Committee of Japan Sport Sciences University. All experiments complied with the policies and regulations of the “Basic Guidelines for the Appropriate Conduct of Animal Experiments and Related Activities in Academic Research Institutions” issued by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The study was conducted in accordance with ARRIVE guidelines.
Animals
10-week-old male Sprague–Dawley rats were purchased from CLEA, Japan. Rats were kept in cages with a 12 h/12 h light/dark cycle (dark time 18:00‒06:00) at 23 °C. All rats received a standard solid diet (CE-7; CLEA Japan,Tokyo,Japan ) and water ad libitum. Rats were acclimatized for 1 week prior to the experiment.
Belt or pad electrode electrical stimulation
After overnight fasting, the right and left ankle joints were shaved under isoflurane anesthesia (anesthetic aspiration rate: 250‒300 mL/min, concentration: ~2.0%‒2.2%). The rats were placed on their backs on a platform, belt-type electrodes (Hormer Ion Corp., Tokyo, Japan) were attached to both ankle joints, and 6 mm × 6 mm pad-type electrodes (Hormer Ion Corp.) were attached to the posterior side of both ankle joints (Fig. 1).
The muscles of the lower extremity were stimulated simultaneously on both sides by electrical stimulation at 7‒8 Hz, eliciting a continuous twitch for 30 min. The stimulation intensity was preliminarily tested with a belt electrode, and the minimum current run value of 3.0 mA for maximum torque in 60 Hz stimulation was set to 1.20 mA when the peak value of the current was unified at 7‒8 Hz stimulation. The pad electrode was stimulated at an intensity of 0.27 mA with a unified current density, considering the area difference with the belt electrode (Pad electrode area 28.26 mm2 / Belt electrode area 125 mm2) x 1.2 mA = 0.27 mA). No immobilization of the lower extremity was performed during contraction, which was conducted in the naturally extended position.
Acute response to belt electrode electrical stimulation
For acute response analysis, muscles were sampled immediately after a single (30 min × 1 set, Fig. 1) exercise with a belt electrode to evaluate glycogen consumption, energy expenditure, and mitochondrial biosynthesis due to muscle contraction by phosphorylated AMPK.
Chronic response to pad electrode or belt electrode electrical stimulation
Chronic response analysis was performed in three groups: CONT, DEN, and DEN + ES. For electrical stimulation, the TA and GAS muscles were harvested 24 h after 1 week of chronic stimulation (30 min × 1 set, 1 time/day, 7 times) in sciatic denervated rats (Fig. 1), and the effects of suppressing muscle atrophy by muscle weight (n = 8‒11), muscle fiber cross-sectional area (n = 4‒6), muscle proteolytic signals, mitochondrial biosynthesis signals, COX IV protein expression and mitochondrial enzyme activity (n = 6), indicators of mitochondrial content, were evaluated.
Similarly, pad electrodes were used for rats with denervated transection. 24 h after 1 week of chronic stimulation (30 min × 1 set, 1 time/day, 7 times), the TA and GAS muscles were sampled to evaluate the effect of muscle weight on inhibition of muscle atrophy (n = 8‒11).
Muscle
The MG and TA were used in all the experiments.The resulting muscles were cut in two for biochemical analysis and muscle fiber CSA measurements.
For CSA measurements, MG and TA muscles were embedded in O.C.T compound (Sakura Finetek Japan, Tokyo, Japan) and flash frozen in cooled isopentane (166-00615, Fuji Film Wako Pure Chemicals Corporation, Osaka, Japan). The other MG and TA muscles were rapidly frozen in liquid nitrogen for western blotting and muscle glycogen content analysis. All muscle samples were stored at -80°C until analysis.
Muscle glycogen content
Frozen muscle (10‒20 mg) was powdered and diluted in homogenization buffer containing 300 µL 30% KOH saturated with 100 µL 1M Na2SO4. The samples were boiled at 95 °C and mixed every 10 min for 30 min. After incubation, 480 μl of ethanol was added and centrifugaed at 1000 × g for 5 min at 4 °C. The supernatant was discarded and the pellet was dried for 1 h.
Next, 200 µL Tris-HCl (pH 6.8) was added, dissolved using a sonicator, and the samples were incubated at 95°C for 2 h. Finally, glycogen content was determined by measuring the absorbance at 505 nm using a LabAssayTM Glucose Kit (298-65701, Fujifilm Wako Pure Chemicals Corp.). The data obtained were corrected by the weight of each muscle in powder form.
Muscle fiber cross-sectional area
Experimental methods and CSA analysis were modified from previous studies[12–14].The medial gastrocnemius and TA muscles were cut into 10-μm-thick frozen sections using a cryostat (CM-502, Sakura Finetek Japan).
Samples were blocked with 5% goat serum (PCN5000, Thermo Fisher Scientific Japan, Tokyo, Japan) for 1 h and incubated with a primary anti-laminin antibody (L8271, 1:1000, Sigma Aldrich Japan, Tokyo, Japan) for 2 h. The primary antibody was diluted with a blocking reagent at room temperature. After incubation, the samples were washed with 0.1 M phosphate buffer (5 min × 3 times) and incubated with Alexa fluor 488 conjugated secondary antibody (1:2000, A-11008, Thermo Fisher Scientific, MA, USA) at room temperature for 2 h. After incubation, samples were washed with 0.1 M phosphate buffer (5 min × 3 times) and mounted with fluorescence anti-fading reagent (12593-64, Nacalai Tesque, Kyoto, Japan). Images were captured using a confocal laser microscope (FV-3000; Olympus, Tokyo, Japan) and quantified using MyoVision (University of Kentucky, Kentucky, USA).
Protein extraction and western blotting
Muscle samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer (188-02453, Fujifilm Wako Pure Chemicals Co.) containing protease and phosphatase inhibitor cocktail (169-26063/167-24381, Fujifilm Wako Pure Chemicals Co.). Protein concentrations of the samples were determined using the BCA method (297-73101, Fujifilm Wako Pure Chemicals Corp.). Equal amounts (40 µg) of protein were separated using SDS-PAGE (NW04125BOX, Thermo Fisher Scientific) and transferred to a polyvinylidene fluoride (PVDF) membrane (IB24001, Thermo Fisher Scientific). Protein transfer was confirmed by staining with Ponceau S (33427.01; SERVA Electrophoresis GmbH, Heidelberg, Germany). Membranes were blocked with blocking reagent (NYPBR01, Toyobo, Osaka, Japan) for 1 h, and primary antibodies (2531/2532/4844/4259, Cell Signaling Technology, MA, USA) were diluted with the dilution reagent (NKB-101, Toyobo). After incubation, cells were washed with Tris-buffered saline containing 0.01% Tween-20 (TBST; T9142, Takara Bio Inc.). The membrane was then incubated with a secondary antibody (7074, Cell Signaling Technology) diluted with reagent (NKB-101, Toyobo) for 1 h at room temperature and washed again with TBST. Protein bands were visualized using fluorescent reagents (SuperSignal West Pico chemiluminescent substrate; Thermo Fisher Scientific). iBright 1500 (FL1500, Thermo Fisher Scientific) and iBright Analysis Software (windows, Thermo Fisher Scientific) were used to scan and quantify the blots. Ponceau S signal intensity was used as a loading control.
RT-PCR
Muscle samples were homogenized using TRIzol (356203, Thermo Fisher Scientific ). Chloroform (163-20145, Fujifilm Wako Pure Chemicals Corp.) was added to the homogenized samples, mixed, and allowed to stand for 15 min. Muscle samples were centrifuged at 4°C and 12,000 × g for 15 min, and after collecting the supernatant, ethanol was added and mixed. Total RNA was extracted using an RNA extraction kit (74106; QIAGEN, Hilden, Germany).
Total RNA concentration was measured using NANODROP ONE (Thermo Fisher Scientific), and 1,500 ng total RNA was extracted using a High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA). Reverse transcription was performed using cDNA. Real-time PCR was performed using the SYBR gene expression assay (Applied Biosystems) in an optical reaction module with a thermal cycler (CFX96, Bio-Rad, California,USA), with the following primers: MuRF1 (Forward: GACATCTTCCACGCTGCCAA/Reverse: TGCCGGTCCATGATCACTTC); Atrogin1 (Forward: AAGGAGCGCCATGGATACTG/Reverse: AGCTCCAACAGCCTACTACG); and β-actin (Forward: CACCCGCGAGTACAACCTTC/Reverse: CCCATACCCACCATCACACACC).
Mitochondrial enzyme activity
The method described by Spinazzi et al.[15] was used to measure citrate synthase activity. Tissues were crushed in homogenate buffer (sucrose/Tris/MgCl/015-21274/207-06275/132-001751, Fujifilm Wako Pure Chemicals Corp.) using a biomasher and further crushed using a sonicator. The tissue was then centrifuged at 500 × g for 5 min at 4°C and the supernatant was collected. Protein concentration of the collected supernatant was determined using the BCA method (297-73101, Fujifilm Wako Pure Chemicals Co.). 5μl of sample solution was diluted to an equal volume (1 mg/mL) of protein and 50 µL DW 1 mM DTNB (043-16403, Fujifilm Wako Pure Chemicals Co.), 10 µL 3 mM acetyl CoA (00546-96, Nacalai Tesque, Kyoto, Japan), 9 µL 10% TritonX-100, 1 µL 1 M Tris-HCl (pH 8.0) and 20 µL 10 mM oxaloacetic acid (00546-96, Nacalai Tesque) were added. After mixing, changes in absorbance at 412 nm were immediately measured in a multimode plate reader (Tecan, Menendorf, Switzerland) at 37°C for 15 min at 5 s intervals. The results were analyzed using SparkControl Magellan 2.2, and the activity of CS was evaluated by analyzing five consecutive changes in absorbance with the largest slope per minute.
Statistical analysis
Data are presented as mean ± SE. For acute stimulation, muscle drive was evaluated using an unresponsive non-parametric Wilcoxon signed-rank test to compare the two groups. In chronic stimulation, changes in muscle wet weight, CSA measurements, muscle proteolytic signals, mitochondria-related signals, and enzyme activity were evaluated using the non-parametric Kruskal–Wallis test. Statistical significance was defined as p < 0.05, and statistical evaluation was performed using the statistical analysis software Graph. GraphPad Prism (version 8.3.0, GraphPad Software, San Diego, CA, USA) was used for statistical analysis.