*2.5. Measurement of Cytoplasmic Ca2*<sup>+</sup>

We used fluo-3-acetoxymethylester (Fluo-3/AM) (Invitrogen, Carlsbad, CA, USA), which demonstrates increased fluorescence upon Ca2<sup>+</sup> binding, to determine cytoplasmic free Ca2<sup>+</sup>. Briefly, after washing the samples three times with fresh PBS, dye (5 mM Fluo-3/AM) was slowly added along the sides of the single muscle fibers, followed by incubation in the dark at 37 ◦C for 30 min. After incubation, the glass slide-mounted Fluo-3/AM-loaded fibers were washed with fresh PBS three times (20 s/time, 1-min process). The slide was quickly placed on the microscope stage, with the fibers focused in the bright field (20-s process) and scanned via laser confocal microscopy in combination with an Olympus FV10-ASW system (Tokyo, Japan) under 488-nm krypton/argon laser illumination, with fluorescence detected at 526 nm. According to their length, three to five pictures were captured at 10× objective magnification for each muscle fiber (10-s capture process for each picture). In consideration of the influence of muscle fiber size on the fluorescence intensity, the average fluorescence intensity (total fluorescence intensity/total area of selected region) was used to measure the Ca2<sup>+</sup> levels. Specifically, the average fluorescence intensity of 10 different regions in each picture was measured using Olympus Fluoview v4.2 software. All pictures (3–5 pictures, depending on the muscle fiber length) of each fiber, with 10 muscle fibers per sample, were used for statistical analysis.
