Predetermined recruitment of calcium release sites underlies excitation-contraction coupling in rat atrial myocytes.
Excitation-contraction coupling (E-C coupling) was studied in isolated fluo-3-loaded rat atrial myocytes at 22 and 37 degrees C using rapid confocal microscopy. Within a few milliseconds of electrical excitation, spatially discrete subsarcolemmal Ca2+ signals were initiated. Twenty to forty milliseconds after stimulation the spatial overlap of these Ca2+ signals gave a 'ring' of elevated Ca2+ around the periphery of the cells. However, this ring was not continuous and substantial Ca2+ gradients were observed. The discrete subsarcolemmal Ca2+-release sites, which responded in a reproducible sequence to repetitive depolarisations and displayed the highest frequencies of spontaneous Ca2+ sparks in resting cells, were denoted 'eager sites'. Immunostaining atrial myocytes for type II ryanodine receptors (RyRs) revealed both subsarcolemmal 'junctional' RyRs, and also 'non-junctional' RyRs in the central bulk of the cells. A subset of the junctional RyRs comprises the eager sites. For cells paced in the presence of 1 mM extracellular Ca2+, the response was largely restricted to a subsarcolemmal 'ring', while the central bulk of the cell displayed a approximately 5-fold lower Ca2+ signal. Under these conditions the non-junctional RyRs were only weakly activated during E-C coupling. However, these channels are functional and the Ca2+ stores were at least partially loaded, since substantial homogeneous Ca2+ signals could be stimulated in the central regions of atrial myocytes by application of 2.5 mM caffeine. Neither the location nor activation order of the eager sites was affected by increasing the trigger Ca2+ current (by increasing extracellular Ca2+ to 10 mM) or the sarcoplasmic reticulum (SR) Ca2+ load (following 1 min incubation in 10 mM extracellular Ca2+), although with increased SR Ca2+ load, but not greater Ca2+ influx, the delay between the sequential activation of eager sites was reduced. In addition, increasing the trigger Ca2+ current or the SR Ca2+ load changed the spatial pattern of the Ca2+ response, in that the Ca2+ signal propagated more reliably from the subsarcolemmal initiation sites into the centre of the cell. Due to the greater spatial spread of the Ca2+ signals, the averaged global Ca2+ transients increased by approximately 500 %. We conclude that rat atrial myocytes display a predetermined spatiotemporal pattern of Ca2+ signalling during early E-C coupling. A consistent set of eager Ca2+ release sites with a fixed location and activation order on the junctional SR serve to initiate the cellular response. The short latency for activation of these eager sites suggests that they reflect clusters of RyRs closely coupled to voltage-operated Ca2+ channels in the sarcolemma. Furthermore, their propensity to show spontaneous Ca2+ sparks is consistent with an intrinsically higher sensitivity to Ca2+-induced Ca2+ release. While the subsarcolemmal Ca2+ response can be considered as stereotypic, the central bulk of the cell grades its response in direct proportion to cellular Ca2+ load and Ca2+ influx.