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.