Ca2+ spark and Ca2+ transient detection and contraction measurement

Isolated ventricular myocytes loaded with Ca²⁺ indicator Fluo-4 AM (5 μmol/liter at room temperature for 8 min) (Invitrogen) were placed in a recording chamber. Ca²⁺ sparks and transients were recorded as reported previously (19). For Ca²⁺ spark recording, confocal line-scan imaging was carried out in resting cells at 488-nm excitation and 505-nm collection with a Zeiss 710 inverted confocal microscope (Carl Zeiss, Oberkochen, Germany) with a ×40 oil immersion lens (numerical aperture 1.3). Line-scan images were acquired at a sampling rate of 3.84 ms/line, along the longitudinal axis of the cell. For the detection of systolic Ca²⁺ transient, after the cells were stimulated with field stimulation (1 Hz) to reach a steady state, confocal line-scan imaging was performed with the same confocal parameters used for Ca²⁺ spark recording under field stimulation (1 Hz). Myocyte contraction was measured by detecting the length of two edges of the cell along with the time of stimulation. Myocytes were superfused with HEPES-buffered external solution during the experiment.