Diversity of atrial local Ca2+ signalling: evidence from 2‐D confocal imaging in Ca2+‐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 Ca2+ current (ICa)‐gated central Ca2+ release has a fast component that is followed by a slower and delayed rising phase. The mechanisms that regulate the central Ca2+ releases remain poorly understood. The fast central release component is highly resistant to dialysed Ca2+ buffers, while the slower, delayed component is completely suppressed by such exogenous buffers. Here we used dialysis of Ca2+ buffers (EGTA) into voltage‐clamped rat atrial myocytes to isolate the fast component of central Ca2+ release and examine its properties using rapid (240 Hz) two‐dimensional confocal Ca2+ imaging. We found two populations of rat atrial myocytes with respect to the ratio of central to peripheral Ca2+ release (Rc/p). In one population (‘group 1’, ∼60% of cells), Rc/p converged on 0.2, while in another population (‘group 2’, ∼40%), Rc/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 Ca2+ sparks on activation of ICa. 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 Ca2+ pools consistently showed larger central Ca2+ stores in group 2 and larger peripheral stores in group 1 cells. The Rc/p was larger at more positive and negative voltages in group 1 cells. In contrast, in group 2 cells, the Rc/p was constant at all voltages. In group 1 cells the gain of peripheral Ca2+ release sites (Δ[Ca2+]/ICa) 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 Ca2+ 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 ICa in both cell groups. Removal of extracellular Ca2+ or application of Ni2+ (5 mm) suppressed equally ICa and Ca2+ release from the central release sites at +60 mV. Depolarization to +100 mV, where ICa is absent and the Na+–Ca2+ exchanger (NCX) acts in reverse mode, did not trigger the fast central Ca2+ releases in either group, but brief reduction of [Na+]o to levels equivalent to [Na+]i facilitated fast peripheral and central Ca2+ releases in group 2 myocytes, but not in group 1 myocytes. In group 2 cells, long‐lasting (> 1 min) exposures to caffeine (10 mm) or ryanodine (20 μm) significantly suppressed ICa‐triggered central and peripheral Ca2+ releases. Our data suggest significant diversity of local Ca2+ signalling in rat atrial myocytes. In one group, ICa‐triggered peripheral Ca2+ release propagates into the interior triggering central Ca2+ release with significant delay. In a second group of myocytes ICa triggers a significant number of central sites as rapidly and effectively as the peripheral sites, thereby producing more synchronized Ca2+ releases throughout the myocytes. The possible presence of vestigial t‐tubules and larger Ca2+ content of central sarcoplasmic reticulum (SR) in group 2 cells may be responsible for the rapid and strong activation of central release of Ca2+ in this subset of atrial myocytes.