Local, stochastic release of Ca2+ in voltage-clamped rat heart cells: visualization with confocal microscopy

1. Confocal microscopy and the fluorescent Ca2+ indicator fluo-3 (K+ salt) were used to measure cytosolic free calcium ion concentration ([Ca2+]) during excitation-contraction (E-C) coupling in single, voltage-clamped, rat cardiac ventricular cells. 2. Local [Ca2+]i transients were measured nearly simultaneously in different, separate, subcellular volumes of approximately 2.0 microns 3. During depolarization, local [Ca2+]i transients were distinctly different from each other and from whole-cell [Ca2+]i transients. These differences were particularly apparent during small depolarizations, and were substantially reduced by ryanodine. 3. Components of the local [Ca2+]i transients, particularly those evoked by small depolarizations, were closely similar, in time course and amplitude, to spontaneous local [Ca2+]i transients, or 'sparks' (which have been shown previously to be Ca2+ released from sarcoplasmic reticulum). 4. Analysis of local [Ca2+]i transients in the spatial frequency domain (power spectrum) revealed that high power at spatial frequencies of 0.05-0.2 microns-1 was always associated with spontaneous calcium 'sparks' and with local [Ca2+]i transients evoked by small depolarizing pulses (e.g. to -31 mV). Evoked local [Ca2+]o transients in the presence of ryanodine, and those evoked by depolarization to very positive clamp-pulse potentials (+45 mV), were associated with considerably lower power at this frequency. 5. The results suggest that whole-cell [Ca2+]i transients evoked by voltage-clamp depolarization, and thus by L-type Ca2+ current, are comprised of local [Ca2+]i transients that are similar to the spontaneous calcium 'sparks'. At very positive clamp-pulse potentials, however, the electrically evoked local [Ca2+]i transients may be smaller, perhaps as a result of smaller unitary L-type Ca2+ current.