*2.6. Measurement of Sarcoplasmic Reticulum Ca2*<sup>+</sup>

We used magnesium-Fluo-4-acetoxymethylester (mag-Fluo-4/AM) (M14206, Thermo Fisher Scientific, Eugene, OR, USA), which demonstrates increased fluorescence upon Ca2<sup>+</sup> binding, to indicate SR free Ca2+, as per Park et al. (2000) [26]. Briefly, after washing samples twice with fresh PBS, dye (5 mM mag-Fluo-4/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 mag-Fluo-4/AM-loaded fibers were washed with fresh PBS three times (20 s/time, 1-min process). The slide was then 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 (Japan) under 488-nm krypton/argon laser illumination, with fluorescence detected at 526 nm. Analysis and statistical methods were similar to those used for the measurement of cytoplasmic Ca2<sup>+</sup> mentioned above.
