Intracellular pH (pHi)

Myoplasmic pH was measured with the ratiometric fluorescent probe BCECF (2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein) (Westerblad and Allen, 1992). Fibers from the flexor digitorum brevis were loaded with AM-BCECF (10 µM, ThermoFisher Scientific) in HEPES buffered saline (in mM): 135 NaCl, 5 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 0.4 NaH₂PO₄, 5.5 glucose, 20 HEPES, pH 7.4 with NaOH for 30 min at 37°C. Fibers were then imaged using an Andor Neo sCMOS spinning-disc confocal microscope (dual excitation at 445 nm and 488 nm and emission filter 535 nm) in the UT Southwestern Live Cell Imaging Core Facility. Images were acquired every 2 min over a 1-h session during which a 25% CO₂ challenge was applied for 20 min. Calibration was then performed at the end of every experimental run by applying the H⁺ ionophore nigericin (10 µM) and then superfusing the fibers with high K⁺ solutions (in mM: 140 KCl, 0.5 MgCl₂, 1.2 KHPO₄, 20 pH buffer, pH with KOH) buffered with Mes (pH 4.5, 5.5), a 50/50 mixture of Mes and HEPES (pH 6.5, 7.5) or Tris (pH 8.5, 9.36) in a high K solution (Westerblad and Allen, 1992). After subtraction of background fluorescence, the fluorescence ratio, R = F488/F445, was calculated using ImageJ and the calibration data were fit to the following equation: