SESN2 prevents the slow-to-fast myofiber shift in denervated atrophy via AMPK/PGC-1α pathway

Animal procedures

Male 10-week-old C57BL/6 J mice, kept in a 24 h light/dark period environment, were provided by SPF (Beijing) Biotechnology Co., Ltd. Denervation was completed via surgery on the right hind legs as delineated previously [16]. The mice were then randomly divided into four groups (six mice per group): sham operation group (control); denervation group; denervation + AAV-shSESN2 (adeno-associated virus, U6-MCS-CAG-EGFP; Genochem, China) group and denervation + AAV-SESN2 group (CMV-betaGlobin-MCS-SV40 PolyA, Genochem). The sequence of shSESN2 primers was 5′-GCGTCTTTGGCATCAGATACG-3′. AAV9 injections of gastrocnemius (GAS) were completed 3 weeks in advance to construct SESN2 knockout or overexpression models. In particular, 10 µl of virus (1.0 × 1012 viral genomes (vg)/ml) were injected into each point around the gastrocnemius. To prevent the backflow of viral particles, the syringe was left in place for an extra 5 min after injection. Four to six injections were conducted on every limb. As for preparation of AMPK activation models, AICAR (an AMPK activator; MedChemExpress, USA, HY-13417) was administered to the mice via an intraperitoneal injection at a dose of 500 mg/kg once a day for 7 days prior to operation. An additional 7 days of AICAR treatment was performed until muscles were collected. Mice were euthanized by cervical dislocation at the indicated time, and GAS, extensor digitorum longus (EDL), and soleus (SOL) were removed, weighed, and frozen for the next experiments. For cervical dislocation, a trained person grasped the skin on the back of the neck by the thumb and forefinger and immediately pulled on the base of the tail in an opposite upward direction from the head. The immediate dislocation of the spinal column from the brain ensured death within a few seconds.

All animal experiments were completed as per the guidelines of the Chinese National Institutes of Health and obtained the approval of the Ethical Committee on Animal Experiments (Huazhong University of Science and Technology, no. 2021-S2789).

Muscle mass measurements, H&E staining, and fiber diameter quantification

Muscles (GAS, EDL, and SOL) of the denervated and control sides were collected and weighed at the proper temporal points. The wet weight ratio (muscular weight of the denervated side divided by the weight of the control side) was utilized to assess muscle atrophy.

For hematoxylin and eosin (H&E) staining, muscle samples were subjected to fixation in paraformaldehyde (4%) for 24 h, and afterwards subjected to dehydration and paraffin embedment. The 4-μm cross-cutting muscle sections were acquired before H&E staining (Bioyear, PRC) as per the instructions.

For quantitative analysis of fiber diameters, muscular transection was stained for wheat germ agglutinin (WGA) to visualize the fibers. ImageJ program was leveraged to quantify the minimum Feret’s diameter of myofibers stochastically selected within each group.

Western blot

The primary antibodies below were utilized: myosin heavy chain (MHC; R&D Systems, USA, MAB4470), slow MHC (Abcam, ab185967), fast MHC (Abcam, ab91506), MHC IIa (Abcam, ab124937), MHC IIb (Abcam, ab221149), myoglobin (Santa Cruz, USA, sc-393020), troponin I-ss (Santa Cruz, sc-514899), TOM20 (Abcam, UK, ab186735), TIM23 (Abcam, ab230253), SESN2 (Abcam, ab178518), AMPK (Cell Signaling Technology [CST], USA, 5831), phospho-AMPK (CST, 50081), PGC-1α (Abcam, ab106814), HIF-2α (Abcam, ab109616), GAPDH (Protein Technology, PRC, 60004-1-Ig). The primary antibody dilution factors were 1:3000 (R&D Systems), 1:5000 (Protein Technology), or 1:1000 (Abcam and CST).

In short, the protein was abstracted via RIPA lysis buffering solution to which 1% protease suppressor was added (Roche, USA, 11206893001). The same quantity of protein (10–50 μg) was isolated via 10% SDS–PAGE, and afterwards moved onto nitrocellulose films (Merck Millipore, USA, Z358657). The films were subjected to blockade with 5% w/v BSA prior to cultivation with the first antibodies under 4 °C overnight. Subsequently, the films were cultivated with second antibodies conjugated with antirabbit or antimouse IgG (Abcam) for 60 min under ambient temperature and visualized via the Immobilon ECL matrix tool (Merck Millipore, WBKLS0050).

Immunofluorescence

Paraffin-embedded GAS underwent cross-sectional cuts, which were made into 4-μm sections as previously stated [16]. Following antigen retrieval, permeabilization, and goat serum blocking, primary antibody (mouse anti-slow MHC antibody and rabbit-anti-fast MHC antibody; Abcam) incubation was implemented at 4 °C. Next, samples were stained for 1 h using secondary Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies (Abcam, ab150113 and ab150079), followed by 5 min DAPI/PI (Sigma, USA, D9542) staining. The samples were then imaged with a fluorescence microscope. Similar processes were oriented with C2C12 cells.

ATP level

ATP contents in mouse GAS were identified via ATP analysis tool (Beyotime, China, S0026). Briefly, GAS tissue was homogenized in ATP assay lysis buffering solution, and the supernatant was harvested via centrifugation at 12,000g for 300 s under 4 °C and subjected to quantification via BCA analysis. Subsequently, 100 μl ATP identification reagent was supplemented into 100 μl supernatant, and the firefly luciferase (FFL) activity was identified and studied via luminescent spectrometric analysis (EnSpire, USA). The ATP content was normalized to cell protein level and presented as proportion (%) in contrast to the controls. Similar procedures were performed in C2C12 cells.

Metabolic enzyme activity analysis

The activities of LDH and SDH were identified via commercially available tools bought from Nanjing Jiancheng Biological Engineering Institution (China, A020-1-2 and A022-1-1) as per the supplier’s specification. Specific enzyme activity was presented as U/mg protein.

Cell culture and transfection

C2C12 cells were obtained from iCell Bioscience Inc. and cultivated in DMEM (Gibco, USA) including 10% FBS (Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin solution (Sangon Biotech, China) in 5% CO2 at 37 ℃ in a cell culture incubator. Cells were differentiated into myotubes posterior to the cultivation with differentiation medium (2% horse serum in DMEM) for 1 week as delineated previously [11]. For genetic knockout, three short interference RNAs for murine SESN2 (siSESN2) and negative controls (siNC) were synthesized by RiboBio Company (PRC). The transfection process was completed as per the specification of Lipofectamine 2000 (Invitrogen, USA, 11668019). Cells were collected at 48 h posterior to the transfection process for the subsequent assays. siRNAs with maximal suppressive effect were utilized for assays later on. The siRNA sequences are presented in Additional file 1: Table S1.

Detection of mitochondrial ROS level

Identification of mitochondrial ROS (mtROS) was completed via the fluorogenic stain MitoSOX Red (Thermo Fisher, USA, M36008), which was targeted to mitochondria in alive cells. In short, 5 mM stock solution and 5 μM work solution of MitoSOX were produced as per the supplier’s specification. Cells were cultivated in work solution for 600 s without light under 37 °C and afterwards cleaned three times in a warm buffering solution. The pictures of alive cells were collected via a confocal laser scan microscope. Finally, more than 30 cells in every group were chosen stochastically to produce the eventual outcomes.

Statistical analysis

Statistical analysis of three independent experiments with at least three technical repetitions was conducted using GraphPad Prism 9.0.0 (GraphPad, USA), and the statistical data were expressed as mean ± standard deviation. Differences were evaluated by one-way or two-way analysis of variance (ANOVA) with Tukey post-hoc test. P-value less than 0.05 was considered statistically significant.

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