Diversity of atrial local Ca 2+ signalling: evidence from 2‐D confocal imaging in Ca 2+ ‐buffered rat atrial myocytes

Atrial myocytes, lacking t‐tubules, have two functionally separate groups of ryanodine receptors (RyRs): those at the periphery colocalized with dihydropyridine receptors (DHPRs), and those at the cell interior not associated with DHPRs. We have previously shown that the Ca 2+ current ( I Ca )‐gated central Ca 2+ release has a fast component that is followed by a slower and delayed rising phase. The mechanisms that regulate the central Ca 2+ releases remain poorly understood. The fast central release component is highly resistant to dialysed Ca 2+ buffers, while the slower, delayed component is completely suppressed by such exogenous buffers. Here we used dialysis of Ca 2+ buffers (EGTA) into voltage‐clamped rat atrial myocytes to isolate the fast component of central Ca 2+ release and examine its properties using rapid (240 Hz) two‐dimensional confocal Ca 2+ imaging. We found two populations of rat atrial myocytes with respect to the ratio of central to peripheral Ca 2+ release ( R c/p ). In one population (‘group 1’, ∼60% of cells), R c/p converged on 0.2, while in another population (‘group 2’, ∼40%), R c/p had a Gaussian distribution with a mean value of 0.625. The fast central release component of group 2 cells appeared to result from in‐focus Ca 2+ sparks on activation of I Ca . In group 1 cells intracellular membranes associated with t‐tubular structures were never seen using short exposures to membrane dyes. In most of the group 2 cells, a faint intracellular membrane staining was observed. Quantification of caffeine‐releasable Ca 2+ pools consistently showed larger central Ca 2+ stores in group 2 and larger peripheral stores in group 1 cells. The R c/p was larger at more positive and negative voltages in group 1 cells. In contrast, in group 2 cells, the R c/p was constant at all voltages. In group 1 cells the gain of peripheral Ca 2+ release sites (Δ[Ca 2+ ]/ I Ca ) was larger at −30 than at +20 mV, but significantly dampened at the central sites. On the other hand, the gains of peripheral and central Ca 2+ releases in group 2 cells showed no voltage dependence. Surprisingly, the voltage dependence of the fast central release component was bell‐shaped and similar to that of I Ca in both cell groups. Removal of extracellular Ca 2+ or application of Ni 2+ (5 m m ) suppressed equally I Ca and Ca 2+ release from the central release sites at +60 mV. Depolarization to +100 mV, where I Ca is absent and the Na + –Ca 2+ exchanger (NCX) acts in reverse mode, did not trigger the fast central Ca 2+ releases in either group, but brief reduction of [Na + ] o to levels equivalent to [Na + ] i facilitated fast peripheral and central Ca 2+ releases in group 2 myocytes, but not in group 1 myocytes. In group 2 cells, long‐lasting (> 1 min) exposures to caffeine (10 m m ) or ryanodine (20 μ m ) significantly suppressed I Ca ‐triggered central and peripheral Ca 2+ releases. Our data suggest significant diversity of local Ca 2+ signalling in rat atrial myocytes. In one group, I Ca ‐triggered peripheral Ca 2+ release propagates into the interior triggering central Ca 2+ release with significant delay. In a second group of myocytes I Ca triggers a significant number of central sites as rapidly and effectively as the peripheral sites, thereby producing more synchronized Ca 2+ releases throughout the myocytes. The possible presence of vestigial t‐tubules and larger Ca 2+ content of central sarcoplasmic reticulum (SR) in group 2 cells may be responsible for the rapid and strong activation of central release of Ca 2+ in this subset of atrial myocytes.