Intrasarcomere [Ca2+] gradients and their spatio-temporal relation to Ca2+ sparks in rat cardiomyocytes

1. Line-scan analyses of spontaneous Ca2+ sparks, non-propagating local rises in Ca2+ concentration, and the early phase of Ca2+ transients in cardiomyocytes were performed with a rapid-scanning laser confocal microscope (Nikon RCM8000) and fluo-3. 2. On electrical stimulation, points at which rise in Ca2+ began earliest were observed at regular spacings of 1.82 +/- 0.26 mu m (mean +/- S.D.) along the longitudinal axis of the cell. The points were heavily stained with di-2-ANEPEQ, which stains the T-tubules, indicating that they were at the Z-line. 3. The points where spontaneous Ca2+ sparks originated coincided with the points which showed faster Ca2+ elevation, i.e. the Z-line. 4. In some cases where a Ca2+ spark had occurred within about 30 ms before the evoked Ca2+ transient, fast elevation of Ca2+ was not observed at the corresponding Z-line, indicating the presence of a refractory period in Ca2+ release from the SR. 5. The present results provide visual evidence for Ca2+ release from the junctional sarcoplasmic reticulum in cardiomyocytes. The presence of a refractory period in Ca2+ release after Ca2+ sparks provided new evidence that the normal Ca2+ transient may be the summation of Ca2+ sparks.