Fura 2

Cardiomyocytes were loaded with 1 μM Fura-2 (Beyotime, S1502) for 20 min, and fluorescence intensity was recorded with a dual-excitation fluorescence photomultiplier system (Ionoptix). Cells were exposed to light emitted by a 75W lamp and passed through either a 360 or a 380 nm filter. Fluorescence emissions were captured between 480 and 520 nm by a photomultiplier tube after first illuminating cells at 360 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 nm excitation scan was then repeated at the end of the recording and qualitative changes in intracellular Ca²⁺ concentration were inferred from the ratio of Fura-2 fluorescence intensity (FFI) at two wavelengths (360/380). This “interpolated numerator” method provides better signal-to-noise ratio than the "interleaved" method (rapidly switching between 340 nm and 380 nm wavelengths [29 (link)]. Fluorescence decay time (single exponential) was derived as an indicator of intracellular Ca²⁺ clearance [40 (link)]. Caffeine-induced release of intracellular Ca²⁺ from SR was used to assess SR Ca²⁺ load capacity. Cardiomyocytes were initially stimulated to contract at 0.5 Hz to ensure steady state. Electrical stimulus was stopped and a caffeine puff (10 mM for 10 s) was applied to proximity of cells through a micropipette to trigger SR Ca²⁺ depletion [41 (link)].