Two-dimensional confocal images of organization, density, and gating of focal Ca2+ release sites in rat cardiac myocytes

In cardiac myocytes Ca2+ cross-signaling between Ca2+ channels and ryanodine receptors takes place by exchange of Ca2+ signals in microdomains surrounding dyadic junctions, allowing first the activation and then the inactivation of the two Ca2+-transporting proteins. To explore the details of Ca2+ signaling between the two sets of receptors we measured the two-dimensional cellular distribution of Ca2+ at 240 Hz by using a novel confocal imaging technique, Ca2+ channel-triggered Ca2+ transients could be resolved into dynamic "Ca2+ stripes" composed of hundreds of discrete focal Ca2+ releases, appearing as bright fluorescence spots (radius congruent to 0.5 mu m) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca2+ releases triggered stochastically by Ca2+ current (I-Ca) changed little in duration (congruent to 7 ms) and size (congruent to 100,000 Ca ions) between -40 and +60 mV, but their frequency of activation and first latency mirrored the kinetics and voltage dependence of I-Ca. The resolution of 0.95 +/- 0.13 reproducible focal Ca2+ release sites per mu m(3) in highly Ca2+-buffered cells, where diffusion of Ca2+ is limited to 50 nm, suggests the presence of about one independent, functional Ca2+ release site per half sarcomere, The density and distribution of Ca2+ release sites suggest they correspond to dyadic junctions. The abrupt onset and termination of focal Ca2+ releases indicate that the cluster of ryanodine receptors in individual dyadic junctions may operate in a coordinated fashion.