Ca2+ sparks involving multiple Ca2+ release sites along Z-lines in rat heart cells

1. High spatial resolution confocal imaging was used to investigate the fundamental nature of "Ca2+ sparks' in rat cardiac myocytes loaded with the fluorescent calcium indicator, fluo-3. 2. The sites at which calcium sparks occurred (Ca2+ release sites) were packed closely and irregularly in transverse planes along Z-lines (mean spacing between sites of 0.76 microns). In contrast, sites were spaced more regularly in the longitudinal direction, at intervals of 1.8 microns (i.e. the sarcomere length). 3. Diffusion of released Ca2+ was slower transversely (apparent diffusion coefficient, D, 7.9 microns 2 s-1) than longitudinally (D, 17.1 microns 2 s-1). 4. Frequently, discrete sites several hundred nanometres apart transversely activated in near synchrony. The probability of transverse synchronous activity fell to low levels (< 20%) at sites separated by more than 1.0 micron. Synchronous activation was not observed between sites on different Z-lines (i.e. separated longitudinally by 1.8 microns). 5. High temporal resolution confocal microscopy (stationary spot) revealed Ca2+ sparks with "stepped' rises, consistent with multiple sites of origin. 6. We conclude that the Ca2+ spark as originally described is usually not an "elementary' event, in the sense of being indivisible, but is often comprised of yet smaller, triggered units of Ca2+ release.