Animal experiments

C57BL/6J male mice aged 6 weeks were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (China). The mice were acclimatized and maintained in a temperature-controlled environment, humidity of 55% to 70%, and a 12-h/12-h dark/light cycle, with access to water and standard rodent chow ad libitum as recommended by the Animal Welfare Act regulations and the Guide for the Care and Use of Laboratory Animals [24]. According to their body weights, the mice were randomized into 4 groups (n = 8): control group (vehicle), TS group (vehicle), TS + eldecalcitol low dose (3.5 ng twice per week), and TS + eldecalcitol high dose (5 ng twice per week). The mice were administered with either vehicle (0.8% ethanol, 0.1% Tween 80 in phosphate-buffered saline [PBS]) or eldecalcitol by intraperitoneal injection twice a week, as per the group they were assigned, for 3 weeks [23]. This study was approved by the Animal Ethics and Experimental Safety Committee of Fudan University (SYXK2020-0032).

Tail suspension in mice

To induce disuse muscle atrophy, the mice were suspended individually in special cages for 21 consecutive days, as elaborated in the previous reports [20, 39]. Briefly, one end of the hinge was connected to the tail by sticking the medical tape, and the other end was connected to the top of the cage. The length of the rope was adjusted to allow free movement of the animal on its forelegs and tilt its body 30 °C to 40 °C horizontally. All mice drank freely and received 8 g of standard rodent food every day. The mice were treated as described earlier for 21 days. The following days after the last treatment, the mice were euthanized and dissected to collect tissues for further analysis.

Body weight, hind limb muscle weights, and grip strength

Wet muscle weights were recorded immediately after tissue excision. Body weight and muscle weights of the gastrocnemius (GAS), tibialis anterior, and soleus of each mouse were measured by a digital balance. The muscle grip strength of the hind limb was measured by a grip strength meter (KW-ZL Grip Strength Meter, KEW BASIS, Nanjing, China) every week. Forepaws of mice were grasped by a wire grid while being lifted by their tails and their posture was kept parallel to the table surface. A gentle pull at the rate of 2 cm/s was continuously applied to the mice’s tails until the grip was released. The maximum force exerted was measured in grams. The measurements were repeated to collect replicates of data per mouse, and the grip strength was analyzed for each mouse based on the maximum value.

Histological analysis

The GAS muscles collected from the hind limb of the mice were fixed using 4% paraformaldehyde in PBS, pH 7.4 for 24 h at 4 °C. Following this, the samples were embedded in paraffin and cut into 10 μm sections. These tissue sections were subjected to a series of regular histological processes of deparaffinizing, rehydrating, and counterstaining with hematoxylin/eosin (H&E). The tissue slides from all groups were screened under a microscope (E800; Canon, Tokyo, Japan), and the images were captured using a CCD camera. ImageJ software (NIH ImageJ system, Bethesda, MD, USA) was used to analyze the images and to calculate cross-sectional areas (CSAs) of myofibers.

Micro-computed tomography

Micro-computed tomography (CT) was performed as described in our previous study [41]. Femurs were fixed in 4% paraformaldehyde for 12 h and transferred to 75% ethanol at 4 °C for analyzing bone microarchitecture by micro-CT at the resolution of 8.96 μm, voltage of 50 kV, and current of 450 mA. The scanned images were analyzed by SCANCO evaluation software to determine the density and distance parameters, including bone volume (BV), tissue volume (TV), bone/tissue volume (BV/TV), bone surface/total tissue volume (BS/TV), bone surface/total bone volume (BS/BV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular pattern factor (Tb.Pf), and structure model index (SMI).

Oxidative stress makers analysis

We determined the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities as indicators of oxidative stress in GAS muscle and serum after 21 days of treatment with vehicle or eldecalcitol. The mice were killed by cervical dislocation. The GAS muscle of the hind limb was dissected, weighed, and immediately frozen in liquid nitrogen. The GAS homogenate was centrifuged at 12,000 rpm at 4 °C for 15 min, and the supernatant was collected and frozen at − 20°C until the day of assay. For serum preparation, blood samples were collected and centrifuged at 3000 rpm for 15 min. Using the respective detection kits (MDA: A003-1; SOD: A00-1; GSH-Px: A005; CAT: A007, Jiancheng Bioengineering Ltd., Nanjing, China), the assays were performed strictly according to the manufacturer’s instructions.

Cell culture

The C2C12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). To induce differentiation, C2C12 cells were plated in a 6-well plate with Dulbecco’s modified containing 4.5 g/L glucose (DMEM) (Gibco) growth media supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. When C2C12 cells reached 70% to 80% confluency, the medium was changed to DMEM with 2% horse serum (Thermo Fisher Scientific, CA, USA) in the differentiation medium. After 5 days of treatment with DMEM containing 2% horse serum, the C2C12 myoblasts were differentiated into myotubes. Cells were incubated with eldecalcitol at 0.1, 1, and 10 nM, whereas control and tumor necrosis factor (TNF)-α control were incubated with the vehicle. To induce muscle atrophy in vitro, the TNF-α control and eldecalcitol cells were incubated with 100 ng/mL TNF-α in differentiation medium for 24 h. Cells were harvested or used for morphological analysis after incubation.

C2C12 myotubes diameter measurement

Differentiation in C2C12 myoblasts cultures was induced by 2% horse serum (Hyclone, Chicago, IL, USA) in DMEM. After respective treatment and atrophy induction in vitro, the cells were washed and harvested for Western blotting or fixed in ethanol for immunostaining. The images of cells stained for myotubes were scanned under a microscope using a digital camera mounted on a Nikon Ti microscope. The myotubes diameters were measured using NIS Elements BR 3.00 software (Nikon, Tokyo, Japan). The myotube diameters were quantified from the diameters of 3 randomly selected sites of at least 100 myotubes by ImageJ software.

Western blot

A radioimmunoprecipitation assay (RIPA) lysis buffer and a Nuclear and Cytoplasmic Protein Extraction Kit (P0027) purchased from Beyotime were used to lyse the total protein and extract nuclear proteins supplemented with protease inhibitors. The protein concentration in the supernatant was measured using a piccinic acid assay kit (Pierce, Rockford, IL, USA). An equal amount of protein (20–30 μg) was used to electrophoresis each sample on the 10–15% sodium dodecyl sulfate-polyacrylamide gel and transferred to the polyvinylidene fluoride membrane. Before incubating with the first antibody, first seal the membrane with BSA (5%) under RT for 1 h. The membrane was washed with Tris-buffered saline containing 0.1% Tween 20 detergent and then reacted with the corresponding secondary antibody for 45 to 60 min. The immunoblots were developed with enhanced chemiluminescence plus reagent, and the results were quantified with laboratory image version 2.7.1.

Immunofluorescence

After 48 h of treatment with eldecalcitol 10 nM, C2C12 myotubes were exposed to 100 ng/mL of TNF-α for 24 h. A 30-min permeabilization with PBS containing 0.2% Triton X-100 was applied to the spheroids after washing 2 times with PBS. The cells were again rinsed 3 times with PBS for 5 min and blocked for 30 min in PBS with 5% bovine serum albumin. Subsequently, the C2C12 cells were incubated with the primary antibody overnight, and then incubated with the fluorescent secondary antibody for 2 h. In addition, the nuclei were counterstained with a blue DAPI stain.

Co-immunoprecipitation

After the indicated treatments, the collected cells were lysed on ice for 15 min in radioimmunoprecipitation assay lysis buffer. The supernatant was collected after centrifugation at 14,000 rpm for 10 min and incubated with protein agarose A/G beads (abs955, absin, Shanghai, China) at 4 °C for 60 min to reduce nonspecific binding. Clarified lysate (500-μg protein) was incubated overnight at 4 °C with nonspecific Immunoglobulin G(2 g), mouse anti-VDR antibody (Santa Cruz) overnight on a rotating plate. Afterward, protein A/G agarose beads were added to each sample and incubated at 4 °C for another 2 h. Following this, the precipitate was washed 3 times with a washing buffer, and an equal amount of protein from each sample was subjected to Western blot analysis.

RNA interference

Individual RNA siRNAs specific for VDR were obtained from RiboBio (Guangzhou, China) as negative controls. C2C12 cells were maintained in a differentiation medium for 4 days in 6-well plates. The myotubes were transfected with Lipofectamine RNAiMAX transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. The base sequence for si-VDR: GGCAGCCAAGACTACAAT.

Statistical analysis

Statistical analysis was performed using SPSS 21.0 software (Chicago, IL, United States). The data were expressed as the mean ± standard error of the mean. In vitro cell experiments were performed in replicates of 4 to reduce error in sampling. One-way ANOVA was used to analyze the significance of differences between experimental groups. P < 0.05 was considered as statistically significant.