Intracellular Ca²⁺ measurements

Intracellular Ca²⁺ transients were measured in isolated adult ventricular cardiomyocytes using the fluorescent Ca²⁺ indicator Fura Red™, AM (Thermo Fisher Scientific). Freshly isolated myocytes were plated on laminin-coated glass coverslips and allowed to adhere for 10–15 min. Cells were loaded with Fura Red™, AM (5 µM) prepared in imaging buffer containing 0.02% Pluronic F-127 for 20 min at room temperature in the dark. Following dye loading, cells were washed and incubated in dye-free buffer for an additional 15 min to allow complete de-esterification. Coverslips were transferred to a temperature-controlled recording chamber and superfused with Tyrode solution containing 1.8 mM CaCl₂. Fura Red fluorescence was recorded using alternating excitation at 405 nm and 488 nm, with emission collected in the red channel (>600 nm). Ratiometric Ca²⁺ signals were calculated as the ratio of fluorescence intensity obtained at 405 nm to that at 488 nm excitation (F405/F488), which is inversely related to intracellular Ca²⁺ concentration. For experiments measuring Ca²⁺ transients, cells were field-stimulated at 1 Hz using suprathreshold voltage. Data were analyzed as changes in fluorescence ratio (ΔR/R₀) relative to baseline.