Store-operated calcium entry activity revealed by confocal live cell calcium imaging in isolated mouse pacemaker cells

J. Liu, D.G. Allen and Y.K. Ju, School of Medical Sciences (F13), University of Sydney, NSW 2006, Australia.

Aims: To determine whether and to what extent store-operated calcium entry (SOCE) activity contributes to pacemaker automaticity and regulation in isolated mouse sinoatrial node (SAN) cells.

Methods and Results: Hearts were quickly removed from adult male Balb/c mice after deep anaesthesia and the SAN regions were micro-dissected. SAN pacemaker cells were isolated with a mixture of enzymes, loaded with calcium indicator Fluo-4 AM and examined with a Zeiss 510 confocal microscope. Store depletion in pacemaker cells were induced by 0 calcium perfusion, and significant reduction in store calcium content was verified by a reduction in caffeine-induced calcium transient. When extracellular Ca²⁺ was replenished after store depletion, a characteristic intracellular Ca²⁺ rise was seen in single pacemaker cells concomitant with recovery of spontaneous firing. SOCE blockers SKF-96365 and BTP-2 both inhibited the rise in intracellular Ca²⁺, indicating functional involvement of SOCE after store depletion. Under physiological conditions, SOCE blocker BTP-2 decreased firing rate (16.0±1.4%), amplitude of spontaneous calcium transients (21.8±4.0%), and caffeine-induced calcium transients (14.6±4.1%). Moreover, BPT-2 significantly delayed pacemaker firing recovery (half recovery from ∼2.8 min to ∼6.5 min) after inhibition by cholinergic stimulation with carbachol, indicating SOCE contributes to recovery of pacemaker activity after cholinergic-induced quiescence.

Conclusion: This study provides evidence that SOCE may contribute to the maintenance and regulation of pacemaker activity in mouse pacemaker cells.