Ethical approval
All experimental procedures were approved by the Committee on Animal Experiments of Sapporo Medical University (No. 18-030). Animal care was in accordance with institutional guidelines.
Induction of experimental autoimmune myositis
Female BALB/c mice (8-week-old, n=28) and male Wistar rats (9-week-old, n=1) were supplied by Sankyo Lab Service (Sapporo, Japan). Mice were given food and water ad libitum and housed in an environmentally controlled room (24 ± 2°C) with a 12-h light-dark cycle. Health was monitored by weight and general assessment of animal activity (every other day). EAM was induced by immunizing mice with partially purified myosin, including myosin-binding protein C, as reported previously [30, 33]. Briefly, skeletal muscle (30 g) obtained from a Wistar rat was minced and washed four times in 30 mM KCl/150 mM sodium phosphate buffer (pH 7.5), 1 mM EDTA, and 1mM DTT. Myosin was extracted by incubation of muscle sample with 90 ml chilled 300 mM KCl/150 mM phosphate buffer containing 5 mM MgCl2, 5 mM ATP, 1 mM DTT, and 1 mM EDTA on ice for 45 min with constant agitation. The homogenate was centrifuged for 30 min at 4°C at 2200 g. For myosin precipitation, the supernatant was collected, filtered, and diluted with 15 volumes of chilled ultrapurewater. The precipitate was recovered via centrifugation for 10 min at 4°C at 10,000 g, dissolved in 500 mM KCl, and stored at −80°C. Purified rat myosin (10 mg/ml) was emulsified with an equal amount of complete Freund's adjuvant (Difco) with 3.3 mg/ml Mycobacterium butyricum (Difco). BALB/c mice were each immunized intracutaneously with 50-100 μl of an emulsion into three to four locations (a total of 200 μl) on the back on days 0, 7, and 14. One hour after the first immunization, pertussis toxin (500 ng in 100 μl saline; List Biological Laboratories) was intraperitoneally injected into each animal. In the present study, all treated animals underwent successful EAM, defined by a significant increase in spleen weight. Further details can be found in the Supplementary Materials and Methods.
Experimental design
To assess the molecular and physiological adaptations induced by HIIT in skeletal muscle from EAM mice, we performed two separate experiments. The primary outcome of this study will be fatigue resistance. Secondary outcomes constitute mitochondrial enzyme activity, the amount of mitochondrial respiratory complexes and ER stress-related proteins, myosin heavy chain (MyHC) isoforms, and the phosphorylation levels of signaling proteins.
Experiment 1.
We first examined the effect of HIIT on muscle fatigability and ER/mitochondrial adaptation in EAM mice. Female BALB/c mice (n=12) were randomly assigned to CNT (n=6) and EAM (n=6) groups. Random numbers were generated using the standard = RAND() function in Microsoft Excel. In the EAM group, HIIT was performed on the left leg (referred to as the EAM + HIIT group), and the right leg served as a non-training EAM control. HIIT was started 24 hours after the last immunization and was carried out every other day for a total of 14 sessions (Figure 1A). The training order was randomized daily, with each animal trained at a different time each training day. Under isoflurane anesthesia, mice were placed supine on a platform with the foot secured to a footplate connected to a torque sensor (S-14154, Takei Scientific Instruments) at an angle of 0° dorsiflexion (i.e., 90° relative to the tibia). The plantar flexor muscles were activated by supramaximal (45 V, 0.5-ms) monophasic rectangular current pulses via a pair of surface electrodes. The stimulation scheme was designed to mimic the activation pattern during all-out cycling bouts, i.e. 0.25 s contractions produced every 0.5 s [23, 34]. Each session consisted of six sets of 60 contractions at 4-min intervals. Twenty-four hours after the last HIIT session, in vivo fatigue resistance of the plantar flexor muscles in each group was measured by 80 repeated 350 ms, 70 Hz tetani given at an interval of 3 s. This was done by an investigator unaware of treatment side. Twenty-four hours after the measurement of fatigue resistance (i.e., 48 hours after the last HIIT session), mice were killed by cervical dislocation under isoflurane anesthesia and the gastrocnemius (GAS) and plantaris muscles were used for skinned muscle fiber experiments and for biochemical analyses (see below).
Experiment 2.
To investigate cellular signaling that underlies the HIIT-induced physiological adaptations, female BALB/c mice (n=16) were randomly divided into the same groups as in Experiment 1 (n=8 in each group). Immediately after one HIIT session, mice were killed by rapid cervical dislocation under isoflurane anesthesia and muscles were subsequently isolated. The phosphorylation levels of AMPKα Thr172, CaMKII Thr286, ACC Ser79, and p38 MAPK The180/Tyr182 were investigated in GAS muscles from each animal.
Myosin heavy chain isoforms separation
Aliquots of GAS muscle extracts containing 5 µg protein were used for myosin heavy chain (MyHC) electrophoresis as previously described [35]. Using a 6.8% polyacrylamide slab gel, electrophoresis was run at 4ºC for 24 h at 160 V and stained with Coomassie brilliant blue. Images of gels were densitometrically evaluated with ImageJ.
Measurement of Ca2+-activated force in skinned muscle fibers
Chemically skinned muscle fibers were prepared and Ca2+-activated force was measured as described previously [36]. The gastrocnemius (GAS) muscle was pinned out at resting length under paraffin oil kept at 4°C. Single muscle fibers were dissected under a stereomicroscope. Four to six skinned fibers were obtained from one whole muscle. A segment of the skinned fiber was connected to a force transducer (Muscle tester, World Precision Instruments) and then incubated with a N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffered solution (see below) containing 1% (vol/vol) Triton X-100 for 10 min in order to remove membranous structures. Fiber length was adjusted to optimal length (2.5 μm) by laser diffraction as described previously [37] and the contractile properties were measured at room temperature (24°C).
All solutions were prepared as described in detail elsewhere [38]. They contained (in mM) 36 Na+, 126 K+, 90 HEPES, 8 ATP and 10 creatine phosphate, and had a pH of 7.09–7.11 and a free Mg2+ concentration set at 1.0 mM. The maximum Ca2+ solution contained 49.5 mM Ca-EGTA and 0.5 mM free EGTA, whereas the relaxation solution contained 50 mM free EGTA. Various pCa (-log free Ca2+ concentration) solutions (pCa 6.4, 6.2, 6.0, 5.8, 5.6, 5.4, and 4.7) were prepared by mixing the maximum Ca2+ solution and the relaxation solution in appropriate proportions [39]. The contractile apparatus was directly activated by exposing the skinned fiber to the various pCa solutions and force was measured. Isometric force produced at each pCa was expressed as a percentage of the corresponding maximum force and analysed by fitting a Hill curve using SigmaPlot 13.0 software to establish the pCa50 (pCa at half-maximum force). The cross-sectional area of fibers was calculated from measurements of their diameters. The maximum Ca2+-activated force per cross-sectional area (Fmax) is expressed as mN/mm2.
Mitochondrial enzyme activity
The maximal activities of citrate synthase (CS) and cytochrome c oxidase (COX) were determined in whole muscle homogenates. In brief, whole plantaris muscles were homogenized in ice-cold 100 mM potassium phosphate buffer (100 μl/mg wet wt) and maximal CS and COX activities were measured spectrophotometrically as described previously [40, 41].
Immunoblotting
Immunoblots were performed as previously described [42] using: anti-PGC-1α (ab54481, Abcam), anti-total OXPHOS rodent WB antibody cocktail (ab110413, Abcam), anti-glucose-regulated protein (Grp) 78 (ADI-SPA-826, Enzo Life Sciences), anti-Grp94 (ADI-SPA-851, Enzo Life Sciences), anti-inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) (#3294, Cell signaling), anti-PKR-like endoplasmic reticulum kinase (PERK) (#5683, Cell signaling), anti-phospho-AMPKα Thr172 (#2531, Cell signaling), anti-AMPKα (#2532, Cell signaling), anti-phospho-CaMKII Thr286 (#12716, Cell signaling), anti-CaMKII (611292, BD Biosciences, San Jose, CA), anti-phospho-ACC Ser79 (#3661, Cell signaling), anti-ACC (#3662, Cell signaling), anti-phospho-p38 MAPK (#4511, Cell signaling), anti-p38 MAPK (#9212, Cell signaling).
Muscle pieces were homogenized in ice-cold homogenizing buffer (40 μl/mg wet wt) consisting of (mM): Tris maleate, 10; NaF, 35; NaVO4, 1; 1% Triton X 100 (vol/vol), and 1 tablet of protease inhibitor cocktail (Roche) per 50 ml. The protein content was determined using the Bradford assay [43]. Aliquots of the whole muscle homogenates (20 μg) were diluted with Laemmli buffer (mM): Urea, 4000, Tris/HCl, 250; SDS, 3.5; 20% glycerol (vol/vol); 0.0005% bromophenol blue (wt/vol). Proteins were applied to a 4–15% Criterion Stain Free gel (BioRad). Gels were imaged (BioRad Stain Free imager) and then proteins were transferred onto polyvinylidine fluoride membranes and were blocked in 3% (wt/vol) non-fat milk, Tris-buffered saline containing 0.05% (vol/vol) Tween 20, followed by incubation with primary antibody overnight at 4°C. Membranes were then washed and incubated for 1 h at room temperature with secondary antibody (1:5000, donkey-anti-rabbit or donkey-anti-mouse, BioRad). Images of membrane were collected following exposure to chemiluminescence substrate (Millipore) using a charge-coupled device camera attached to ChemiDOC MP (BioRad), and Image Lab Software (BioRad) was used for detection as well as densitometry. The levels of protein expression were normalized to the total proteins from stain-free image.
Statistics
Data are presented as mean ± SEM. Data normality was examined with the Shapiro-Wilk test. Power calculations were performed to estimate an adequate sample size in order to detect a meaningful difference. In Experiment 1, for normally distributed data (the distribution of the MyHC isoforms, CS activity, COX activity, the expression levels of PGC-1α, NDUFB8, SDHB, UQCRC, MTC01, ATP5, Grp78, Grp94, IRE1α, and PERK, Fmax, pCa50), one-way ANOVA was used to determine the mean differences among the three groups (CNT, EAM, and EAM+IT group). Fatigue resistance (group x repetitions) and specific force-pCa relationship (group x pCa) were assessed by two-way repeated measures ANOVA. In Experiment 2, for normally distributed data (the phosphorylation levels of AMPK, CaMKII, and p38MAPK), one-way ANOVA was used to determine the mean differences between the groups. When these ANOVA tests showed significance, Bonferroni or Tukey post hoc test was performed. If data exhibited a non-normal distribution (the phosphorylation levels of ACC), a Kruskal-Wallis one-way ANOVA was used on ranks. A P value less than 0.05 was regarded as statistically significant. Statistical testing was performed with SigmaPlot (version 13, Systat Software, Inc).