Fura 2

Intracellular Ca²⁺ measurements were obtained from Fura-2 (Invitrogen) AM-loaded myotubes as described previously (Goonasekera et al., 2007 (link)). Briefly, myotubes were differentiated for 5–6 days on glass bottom dishes and loaded with 5 μM Fura-2 AM for 45 min at 37°C in a normal rodent Ringer’s solution consisting of 145 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.4. Coverslips of Fura-2–loaded cells were then mounted in a tissue chamber on the stage of an epifluorescence-equipped inverted microscope (Zeiss). Cells were sequentially excited at 340- and 380 nm wavelength and fluorescence emission at 510 nm was collected using a high-speed CCD camera (Hamamatsu). The results are presented as the ratio of 340/380 nm. Maximal increase or peak change in intracellular Ca²⁺ by induction of 10 mM caffeine was defined as the difference between peak and 10 s of baseline fluorescence ratios prior to addition of caffeine. To better visualize differences in peak change across multiple treatment groups, 340/380 ratios for each individual myofiber were normalized to their own 10 s of baseline 340/380 ratios prior to addition of caffeine. Data are also presented without normalization in Figure 5—figure supplement 1D–F.

Calcium measurements were performed as previously described (Gailly et al., 1993 (link); De Backer et al., 2002 (link)). Briefly, calcium imaging was performed on DIV13-15 hippocampal cultures. Cells were loaded with 1 μM Fura-2-AM (Molecular Probes) in Krebs solution [contained (mM): 135 NaCl, 5.9 KCl, 2 CaCl₂, 1.2 MgCl₂, 10.6 Hepes Na⁺, and 10 glucose (pH 7.3)] for 30 min at room temperature. Fura-2 loaded cells were excited alternatively at 340 and 380 nm and fluorescence emission was monitored at 510 nm using an AxioCam MRm camera (Zeiss) coupled to an inverted microscope (Axiovert 200 M Zeiss, objective 20X Fluar N.A. 0.75). Concentration of intracellular calcium ([Ca²⁺]_i) was calculated from the ratio of the fluorescence intensities excited at the two wavelengths and after cell calibration, Δ[Ca²⁺] measurements were calculated by subtracting the resting [Ca²⁺]_i value from the [Ca²⁺]_i peak amplitude. For each experiment, the [Ca²⁺]_i values measured on individual cells were averaged for all cells studied on one coverslip.

Hippocampal neurons were pre-incubated for 30 min with Fura-2, AM (2.5 μM, Thermo Fisher Scientific) and the non-ionic detergent Pluronic F-127 (0.05% (w/v) in DMSO, Merck), and imaged in ECS containing 1 µM TTX and 10 µM bicuculine. The Fura-2 signal was captured using an inverted AxioObserver D1 microscope controlled with ZEN 2012 software (Zeiss) and equipped with a CCD camera and Lambda-DG4 fast illumination system (Sutter Instruments, Novato) for excitation at 340 and 380 nm. The fluorescence intensity of the Fura-2 emission was measured at 510 nm as a ratio of signals obtained after excitation at 340 and 380 nm. Data was sampled every 500 ms during the Ca²⁺ imaging. The cells were continuously perfused with ECS at 37 °C, and solutions were exchanged using a multiple capillary perfusion system consisting of a computer-controlled multichannel peristaltic pump (Reglo ICC, Ismatec). Traces of individual cells expressed as the F340/F380 ratio were horizontally aligned by their baseline and analysed using Matlab 2019b (MathWorks). An average trace was calculated as the mean of all recorded cells, with the shaded area corresponding to the standard error of the mean (SEM).

The method for the measurement of intracellular Ca²⁺ concentration has been described previously [31 (link)]. In brief, the cells were preloaded with 5 μM of Fura-2 AM (Beyotime, Shanghai, China) in HEPES buffer for 1 h at room temperature. Images of the Fura-2-loaded cells were captured using a laser confocal microscope (LSM 510, Carl Zeiss, Oberkochen, Germany) and analyzed using Image-Pro Plus v6.0 software (Media Cybernetics, Crofton, MA, USA). Background-subtracted fluorescent images for excitation at 340 nm and 380 nm were captured. The intracellular Ca²⁺ concentration was estimated from the ratio of Fura-2 fluorescence emitted at 510 nm after excitation at 340 nm to that after excitation at 380 nm, according to the Grynkiewicz equation [32 (link)].

The method for the measurement of intracellular Ca²⁺ concentration has been described previously [13 (link)]. In brief, the cells were preloaded with 5 μM Fura-2 AM (Beyotime) in HEPES buffer for 1 h at room temperature. Images of the Fura-2-loaded cells were captured using a laser confocal microscope (Carl Zeiss) and analyzed using Image-Pro Plus v6.0 software. Background-subtracted fluorescent images for excitation at 340 and 380 nm were captured. The intracellular Ca²⁺ concentration was estimated from the ratio of Fura-2 fluorescence emitted at 510 nm after excitation at 340 nm to that after excitation at 380 nm, according to the Grynkiewicz equation [23 (link)].

Samples were washed with phosphate-buffered saline (PBS), and 1 μM Fura-2 [acetoxymethyl (AM) ester form] (Life Technologies, Eugene, Oregon), prepared in PBS, was loaded for 1 h at 37 °C in the dark. The dye was replaced with dye-free PBS for 30 min in the dark at RT. Images were taken at the 380/510 and 340/510 nm excitation/emission wavelengths of calcium-free and calcium-bound Fura-2 dye, respectively, using a fluorescence microscope (Carl Zeiss Apotome 2, Germany). The fluorescence intensity was measured using ImageJ software, and the 340/380 ratio was calculated using Equation (1) to determine the relative calcium intensity:

Fibroblasts from the five patients and one control were plated at a density of 3x10⁴ cells on 12 mm glass cover slips disposed into P24 plates the day before the experiment. Cells were loaded with 5 μM Fura-2AM (Invitrogen) and 0.06% pluronic acid F.127 (Invitrogen) for 30 min at 37°C in Ca²⁺-free HCSS (2.5 mM glucose, 120 mM NaCl, 0.8 mM MgCl₂, 25 mM Hepes, 5.4 mM KCl, pH 7.4), and washed for 30 min in HCSS (2 mM CaCl₂, 2.5 mM glucose, 120 mM NaCl, 0.8 mM MgCl₂, 25 mM Hepes, 5.4 mM KCl, pH 7.4). Then, cover slips were placed into a heated chamber mounted on the microscope stage equipped and Fura-2 fluorescence was imaged ratiometrically using alternate excitation at 340 and 380 nm, and a 510 nm emission filter with a Neofluar 20X/0.75 objective in an Axiovert 75M inverted fluorescence microscope (Zeiss). For single-cell analysis of [Ca²⁺]_i the fluorescence ratio intensity at 340 nm (F₍₃₄₀₎) and 380 nm (F₍₃₈₀₎) (F₍₃₄₀₎/ F₍₃₈₀₎) was obtained. Bradykinin (10 μM; Sigma) and thapsigargin (1 μM; Sigma) were added to induce Ca²⁺ release of ER in the absence of extracellular Ca²⁺. Image acquisition was performed with the Aquacosmos 2.5 software (Hamamatsu).