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Predetermined recruitment of calcium release sites underlies excitation-contraction coupling in rat atrial myocytes

Lauren Mackenzie*, Martin D. Bootman*†, Michael J. Berridge*† and Peter Lipp*

1. Excitation-contraction coupling (E-C coupling) was studied in isolated fluo-3-loaded rat atrial myocytes at 22 and 37°C using rapid confocal microscopy.

2. 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.

3. 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'.

4. 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.

5. 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 ~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.

6. 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 ~500 %.

7. 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.

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