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 (I-Ca)-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 (R-c/p). In one population ('group 1', similar to 60% of cells), R-c/p converged on 0.2, while in another population ('group 2', similar to 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 Ca2+ 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 Ca2+ pools consistently showed larger central Ca2+ 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 Ca2+ release sites (Delta[Ca2+]/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 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 I-Ca in both cell groups. Removal of extracellular Ca2+ or application of Ni2+ (5 mm) suppressed equally I-Ca and Ca2+ release from the central release sites at +60 mV. Depolarization to +100 mV, where I-Ca 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+](0) 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 mu M) significantly suppressed I-Ca-triggered central and peripheral Ca2+ releases. Our data suggest significant diversity of local Ca2+ signalling in rat atrial myocytes. In one group, I-Ca-triggered peripheral Ca2+ release propagates into the interior triggering central Ca2+ release with significant delay. In a second group of myocytes I-Ca triggers a significant number of central sites as rapidly nd 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 maybe responsible for the rapid and strong activation of central release of Ca2+ in this subset of atrial myocytes.