Two-dimensional confocal images of organization, density, and gating of focal Ca(2+) release sites in rat cardiac myocytes

In cardiac myocytes Ca(2+) cross-signaling between Ca(2+) channels and ryanodine receptors takes place by exchange of Ca(2+) signals in microdomains surrounding dyadic junctions, allowing first the activation and then the inactivation of the two Ca(2+)-transporting proteins. To explore the details of Ca(2+) signaling between the two sets of receptors we measured the two-dimensional cellular distribution of Ca(2+) at 240 Hz by using a novel confocal imaging technique. Ca(2+) channel-triggered Ca(2+) transients could be resolved into dynamic “Ca(2+) stripes” composed of hundreds of discrete focal Ca(2+) releases, appearing as bright fluorescence spots (radius ≅ 0.5 μm) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca(2+) releases triggered stochastically by Ca(2+) current (I(Ca)) changed little in duration (≅7 ms) and size (≅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 Ca(2+) release sites per μm(3) in highly Ca(2+)-buffered cells, where diffusion of Ca(2+) is limited to 50 nm, suggests the presence of about one independent, functional Ca(2+) release site per half sarcomere. The density and distribution of Ca(2+) release sites suggest they correspond to dyadic junctions. The abrupt onset and termination of focal Ca(2+) releases indicate that the cluster of ryanodine receptors in individual dyadic junctions may operate in a coordinated fashion.