*2.2. Muscle Sample Collection and Preparation*

Muscle sample collection was carried out at 9:00 am for the SA, PRE, LT, and POST groups. Due to the specific sampling procedures in the IBA and ET groups, it could not be guaranteed that sample collection in these two groups always occurred at 9:00 am. Animals were anesthetized with sodium pentobarbital (90 mg/kg). The two distinct skeletal muscles (PL and AM) were carefully isolated and surgically removed. Subsequently, left leg muscles were treated with embedding medium for frozen section cutting and staining and right leg muscles were used for all other experiments. Following surgical intrusion, the squirrels were euthanized via sodium pentobarbital overdose injection.

#### *2.3. Skeletal Muscle Fiber Cross-Sectional Area (CSA) Determination*

As described previously [25], immunofluorescence and confocal analyses were used to measure the muscle fiber cross-sectional area (CSA) in frozen sections. Briefly, 10-μm thick frozen cross-sections were cut from the muscle mid-belly at −20 ◦C with a CM1850 cryostat (Leica, Wetzlar, Germany) and stored at −80 ◦C until further staining. After fixing in 4% paraformaldehyde for 30 min, slices were permeabilized in 0.1% Triton X-100 for 30 min, blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 60 min, and then incubated at 4 ◦C overnight with an anti-laminin antibody (1:500, Boster, BA1761-1, Wuhan, China) to visualize muscle fiber CSA. Subsequently, after washing them three times with PBS (10 min/time), the sections were incubated at 37 ◦C for 2 h with a 647-labeled IgG secondary antibody (1:400, Thermo Fisher Scientific, A-21235, Eugene, OR, USA). Finally, the slices were treated with anti-fade mounting medium (Life Technologies, 1427588, Eugene, OR, USA). Images were visualized via confocal laser scanning microscopy (Olympus, FV1000, Tokyo, Japan) at a 40× objective magnification. Image-Pro Plus 6.0 was used to measure muscle fiber CSA. In detail, eight images were captured from each sample, and the CSA of all complete muscle fibers (about 30) within each picture was then analyzed. Therefore, the CSA of ~250 muscle fibers per skeletal muscle sample was determined.

#### *2.4. Single Muscle Fiber Isolation*

We anaesthetized the squirrels with 90 mg/kg sodium pentobarbital, after which muscle samples (including the tendons) were carefully removed from the neighboring tissues and sarcolemma, ensuring that the blood and nerve supply remained intact. The muscle samples were subsequently separated into two full-length strips along the longitudinal axis using a pair of tweezers. The muscle strips, which were obtained from the same middle region, were then washed with 20 mL of PBS (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium chloride, 1.4 mM monopotassium phosphate, pH 7.4) and digested with 3 mL of enzymatic digestion solution containing 0.35% collagenase I (Sigma-Aldrich, C0130-1G, Saint Quentin Fallavier, France), followed by orbital shaker incubation at 37 ◦C for 2 h and saturation with 95% O2 and 5% CO2 to ensure complete digestion of the muscle fiber samples. Finally, the digestion solution was removed with a PBS rinse and the muscle samples were added to Dulbecco's modified Eagle's medium (DMEM) (Hyclone, AC10221937, Pittsburgh, PA, USA) containing 10% fetal bovine serum (FBS) (Everygreen, 11011-8611, Hangzhou, China), 25 μM of N-benzyl-p-toluenesulfonamide (BTS) (TCI, B3082, Shanghai, China), and 0.1 M HEPES (Guoan, H0082, Xi'an, China), and were carefully stirred with a pipette. The digested single muscle fiber samples were finally plated on culture chamber slides and viewed via inverted microscopy (Olympus, IX2-ILL100, Tokyo, Japan).
