Materials
Dulbecco’s modified essential medium (DMEM) was purchased from Nikken Bio Med Lab (Kyoto, Japan), fetal bovine serum (FBS) from Biowest (Nuaillé, France), horse serum and penicillin-streptomycin from Thermo Fisher Scientific (Waltham, MA, USA). Human insulin and the primary antibodies targeting β-actin and β-tubulin were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). The primary antibodies targeting p-Akt, Akt, p-p70, p70, p-mTOR, mTOR, p-FoxO1, p-AMPK, and AMPK were purchased from Cell Signaling (Beverly, MA, USA). The secondary antibodies, anti-rabbit IgG, and anti-mouse IgG were purchased from GE Healthcare (Bucks, UK). The C2C12 cells were obtained from the American Type Culture Collection (Manassas, VA, USA).
The dried extract preparation of TJ-41 (lot no. 2,140,041,010) was kindly provided by Tsumura & Co. (Tokyo, Japan). For in vitro experiments, one gram of TJ-41 was sonicated in 10 mL of dimethyl sulfoxide (DMSO), centrifuged, and the supernatant was harvested and diluted with DMSO to the appropriate concentration. For in vivo experiments, TJ-41 was mixed with water, sonicated, and diluted with water to a specific concentration.
In vitro experiment
The C2C12 cells, a murine myoblast cell line, were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere (5% CO2 and 95% air).
Myoblasts fuse into multinucleated fibers called myotubes, and matured myotubes form muscle fibers [26]. To examine the effects of TJ-41, therefore, we differentiated C2C12 myoblast into myotubes in all the in vitro experiments. When cell confluency reached 80–90%, the medium was replaced with differentiation medium consisting of DMEM supplemented with 2% horse serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. The differentiation medium (DM) was changed every 2 days. Four days later, the C2C12 cells differentiated into myotubes and were then used for the experiments.
Serum-free medium (FM) consisted of DMEM supplemented with 2% 100 U/mL penicillin and 100 µg/mL streptomycin but without serum. Atrophy was induced by replacing the DM of the differentiated C2C12 myotubes with the FM and culturing them for another 24 h (serum starvation). The control C2C12 myotubes were incubated for another 24 h after the replenishment of the DM. Then RNA was collected from the control group and serum-starved group using a Qiashredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) and subjected to RNA analysis.
Previous studies have shown that TJ-41 concentration-dependently reduced IL-6 production from macrophages and restored autophagy in HEK293 cells in the concentration range of 10–500 µg/mL [24, 27]. TJ-41 concentration in the present study was determined based on these studies. The presence of 0.1% DMSO (v/v) in the medium affected the viability of C2C12 only slightly [28], and the concentration has been commonly adopted in the C2C12 experiments [29]. Therefore, we set the final DMSO concentration to 0.1% (v/v) in all the experiments.
To examine the effects of TJ-41 on atrogene expression, the DM of the differentiated C2C12 myotubes was changed to FM containing vehicle (0.1% DMSO v/v) or TJ-41 (final concentrations of 1, 10, or 100 µg/mL in medium, containing 0.1% DMSO v/v). The DM of the control group was replaced with DM containing DMSO (vehicle, 0.1% DMSO v/v). After 24 h incubation, RNA was collected from these groups using a Qiashredder and RNeasy Mini Kit (Qiagen) and subjected to RNA analysis.
To examine the effects of TJ-41 on the signaling pathway related to atrogenes, the DM of the C2C12 myotubes was replaced with the DM containing vehicle (0.1% DMSO v/v) or TJ-41 (final concentrations of 1, 10, or 100 µg/mL in medium, containing 0.1% DMSO v/v). After additional 24 h incubation, protein samples were collected and subjected to Western blot.
In all these experiments, three biological replicates and two technical replicates were performed.
Animal experiments
Male C57BL/6J mice were purchased from Nippon CLEA (Tokyo, Japan). They were housed individually in similarly-designed cages and were maintained in a controlled environment (temperature, 24 ± 1 °C; humidity, 55 ± 5%) with a 12 h:12 h light:dark cycle. The mice were provided ad libitum access to standard chow and water. They were acclimated for 2 weeks in the aforementioned conditions and were used for the experiments at 11 weeks of age.
TJ-41 was mixed with water, sonicated, and orally administered by gavage. Control mice were administered water as a vehicle. The low-dose and high-dose groups received 0.3 and 1.0 g TJ-41 per kg body weight, respectively. Gavage feeding was performed daily during the experiments.
To examine the effects of TJ-41 on skeletal muscles of the wildtype mice, mice were administered water (vehicle), low-dose of TJ-41, or high-dose of TJ-41 by gavage daily for 21 days (n = 4 in each group, total number is 12). Then muscle samples were collected and subjected to Western blot.
The mice were subjected to tail-suspension, which induced hindlimb muscle atrophy. To achieve this, the entire tail of a mouse was covered with medical adhesive tape. The distal end of the tape was attached to a paperclip, which was then attached to a swivel on a cross-bar. The cross-bar was positioned approximately 15 cm above the cage floor. Using this device, the mouse was suspended such that the hindlimbs did not touch the floor, but the forelimbs were free to move. During this time, the mouse had ad libitum access to food and water. To examine the effects of tail-suspension on atrogene expression and muscle weight,
To examine the effects of tail-suspension on the expression of atrogin-1 and MuRF1 in the muscles, mice were subjected to 24 h of tail-suspension, and RNA was collected from the gastrocnemius muscles. To examine the effects of tail-suspension on muscle weight, mice were subjected to 14 days of tail-suspension and the gastrocnemius muscles were weighed. These data were compared with that of the control group (n = 4 in each group, total number is 12).
To examine changes in atrogenes expression in response to TJ-41, mice were divided into six groups: (1) non-suspended, vehicle (water), (2) non-suspended, low-dose TJ-41, (3) non-suspended, high-dose TJ-41, (4) suspended, vehicle, (5) suspended, low-dose TJ-41, (6) suspended, high-dose TJ-41 (n value is 10, 10, 10, 9, 10, 9 in each group, respectively, and total number is 58). The mice were subjected to 7 days of everyday treatment with vehicle (water) or TJ-41, followed by 24 h of non-suspended state or tail-suspension, then sacrificed. They were sacrificed 24 h after the last gavage. RNA was collected from the gastrocnemius muscles.
To examine the effects of TJ-41 on tail-suspension-induced muscle atrophy, another set of mice were divided into six groups: (1) non-suspended, vehicle, (2) non-suspended, low-dose TJ-41, (3) non-suspended, high-dose TJ-41, (4) suspended, vehicle, (5) suspended, low-dose TJ-41, (6) suspended, high-dose TJ-41 n value is 9, 10, 10, 9, 10, 8 in each group, respectively, and total number is 56). Mice were subjected to pretreatment for 7 days, then non-suspended state or tail-suspension for 14 days. During the time course, the mice were administered vehicle or TJ-41 every day. Then they were sacrificed and the gastrocnemius muscles were collected and weighed. They were sacrificed 24 h after the last gavage.
In all the animal experiments, the mouse was euthanized using carbon dioxide. Mice which had accidentally escaped from the suspension before the end of the experiments were excluded. In animal experiments, including and excluding criteria were not set a priori.
RNA analysis
RNA was extracted from C2C12 myotubes, using a Qiashredder and RNeasy Mini Kit (Qiagen). Harvested gastrocnemius muscles were immersed in RNAlater (Qiagen) to stabilize RNA in tissues. RNA extraction was performed using an RNeasy Fibrous Tissue Mini Kit (Qiagen). DNase I treatment was performed using an RNase-Free DNase Set (Qiagen).
The cDNA was synthesized from the RNA samples using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) and used according to the manufacturer’s instructions. The cDNA was mixed with SYBR Green Master Mix (Applied Biosystems, CA, USA) and specific primers for each gene, and then subjected to quantitative real-time PCR using a StepOnePlus Real-Time PCR System (Applied Biosystems). The cycle threshold for each gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as the internal control. The primer sequences were:
GAPDH: 5’-AGGTCGGTGTGAACGGATTTG-3’ (forward) and 5’-TGTAGACCAGTAGTTGAGGTCA-3’ (reverse);
MuRF1: 5’-TGCCTGGAGATGTTTACCAAGC-3’ (forward) and 5’-AAACGACCTCCAGACATGGACA-3’ (reverse);
atrogin-1: 5’-AAGGCTGTTGGAGCTGATAG CA-3’ (forward) and 5’-CACCCACATGTTAATGTTGCCC-3’ (reverse).
Western blot
The C2C12 myotubes were lysed in radioimmunoprecipitation assay buffer combined with a protease inhibitor cocktail (cOmplete Mini, Roche Applied Science, Penzberg, Germany) and a phosphatase inhibitor cocktail (PhosSTOP, Roche Applied Science). Harvested muscle tissue was immersed in ice-cold T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) with cOmplete Mini and PhosSTOP, and pulverized using a CellDestroyer (Bio Medical Science Inc, Tokyo, Japan). The proteins in these lysates were separated using SDS-PAGE and were electro-transferred onto a polyvinylidene difluoride membrane. The membranes were blocked using Blocking One (Nacalai Tesque Inc, Kyoto, Japan) and probed using appropriate primary and secondary antibodies. After immunoblotting, the proteins were visualized using an ECL detection system (GE Healthcare) or LumiGLO Reserve Chemiluminescent Substrate (SeraCare Life Sciences, Inc. MA, USA). The chemiluminescence images were scanned using a LuminoGraph I (Atto Corp, Tokyo, Japan). Blots were quantified using Image J software 1.44.
Cell morphology
To examine the effects of TJ-41 on serum-starved C2C12 myotube width, the DM of the differentiated C2C12 myotubes was changed to FM containing vehicle (0.1% DMSO v/v) or TJ-41 (final concentrations of 10 or 100 µg/mL in medium, containing 0.1% DMSO v/v). The DM of the control was replaced with DM containing DMSO (vehicle, 0.1% DMSO v/v). After 24 h incubation, the C2C12 myotubes were fixed using 4% paraformaldehyde in PBS solution (Wako, Osaka, Japan). To visualize cell morphology, the myotubes were treated with Actin Green 488 ReadyProbes reagent (Thermo Fisher Scientific). The images were scanned using a BZ-X810 fluorescence microscope (Keyence, Tokyo, Japan). Myotube width was determined using ImageJ (National Institutes of Health, Bethesda, MD, USA). Three independent biological replicates were performed, and three different fields were randomly selected from each experiment. Up to 20 representative fibers were selected from each field, and the width of each fiber was measured.
Statistical analysis
The results were expressed as mean ± standard error of the mean values. Between-group comparisons were performed using Student’s t-tests. Multiple-group comparisons were performed using ANOVA, then post-hoc comparison was performed using the Tukey-Kramer method. Data analysis was conducted using R software. A value of p < 0.05 was considered significant.