Recording of Ca2+ sparks in atrial myocytes using confocal imaging
Atrial myocytes were bathed in an internal solution composed of (in mM; Sigma, USA) 135 NaCl, 5.4 KCl, 1.0 MgCl2, 1.8 CaCl2, 10 glucose, 0.33 NaH2PO4, and 10 HEPES, pH=7.4, with NaOH and incubated in 10 µM Fluo-4 AM for 20 minutes (Thermo Fisher Scientific; USA). A laser-scanning confocal microscope system (TCS SP5; Leica; Germany) with a 20× water-immersion objective was applied to acquire Ca2+ sparks. Fluo-4 AM was excited at 488 nm with an Ar laser. Myocytes were placed with their long axes within ±10 degrees of the cell and approximately equidistant between the outer edge of the cell and the nuclei to ensure that the nuclear area was not included in the scan line. All experiments were performed at room temperature (22-24 °C). Sparks were analyzed with SparkMaster12, and the number, frequency (sparks/100 µm/s, Ca2+ release events), amplitude (ΔF/F0), full width at half-maximum (FWHM, µm) and full duration at half-maximum (FDHM, ms) of the detected sparks were obtained. To exclude false positive events, a threshold criterion for spark detection of 3.8 was chosen for data analysis. F0 was the initial fluorescence recorded under steady-state conditions. Spark amplitudes and widths were determined based on the Ca2+ released during individual sparks.