Calcium homeostasis of orbicularis oculi–derived myotubes compared with quadriceps- and EOM-derived myotubes. (A) Representative traces showing changes in [Ca²⁺]_i induced by stimulation with KCl. Individual cells were perfused in Krebs-Ringer containing 100 µM La³⁺ (light gray bar) and then stimulated with 60 mM KCl for 10 s (black bar). Ratiometric images (340 nm/380 nm) were acquired and converted into [Ca²⁺] as described in Materials and methods. Continuous line, quadriceps-derived myotubes; dashed line, orbicularis oculi–derived myotubes; dotted line, EOM-derived myotubes. (B and C) KCl dose–response curves (B) and 4-cmc dose–response curves (C). Closed circles and continuous lines, quadriceps-derived myotubes; open circles and dashed lines, orbicularis oculi–derived myotubes; open squares and dotted lines, EOM-derived myotubes. Curves were generated using the sigmoidal fit function of the Origin software and show the changes in peak calcium, expressed as [Ca²⁺] in nM. Each point represents the mean (±SEM) of 5–12 different cells. For KCl- and 4-cmc–induced Ca²⁺ release, individual cells were perfused with Krebs-Ringer plus 100 µM La³⁺, and the indicated concentration of agonist was applied using a microperfusion system. (D) Mean (±SEM) resting [Ca²⁺]. *, P < 0.05; **, P < 0.001. (E) Total amount of Ca²⁺ in the SR. Values represent the mean (±SEM) calculated area under the curve. Student’s t test: ***, P < 0.0001. For quadriceps (QU)-derived myotubes, cells from six donors were measured; for orbicularis oculi (OO)–derived myotubes, cells from five donors were measured; and for EOM-derived myotubes, cells from four donors were measured.