Sarcoplasmic reticulum Ca²⁺ pumping kinetics regulates timing of local Ca²⁺releases and spontaneous beating rate of rabbit sinoatrial node pacemaker cells

Rationale: Sinoatrial node cells (SANCs) generate local, subsarcolemmal Ca²⁺ releases (LCRs) from sarcoplasmic reticulum (SR) during late diastolic depolarization. LCRs activate an inward Na⁺-Ca²⁺ exchange current (INCX), which accelerates diastolic depolarization rate, prompting the next action potential (AP). The LCR period, ie, a delay between AP-induced Ca²⁺ transient and LCR appearance, defines the time of late diastolic depolarization INCX activation. Mechanisms that control the LCR period, however, are still unidentified. Objective: To determine dependence of the LCR period on SR Ca²⁺ refilling kinetics and establish links between regulation of SR Ca²⁺ replenishment, LCR period, and spontaneous cycle length. Methods and Results: Spontaneous APs and SR luminal or cytosolic Ca²⁺ were recorded using perforated patch and confocal microscopy, respectively. Time to 90% replenishment of SR Ca²⁺ following AP-induced Ca²⁺ transient was highly correlated with the time to 90% decay of cytosolic Ca²⁺ transient (T-90C). Local SR Ca²⁺ depletions mirror their cytosolic counterparts, LCRs, and occur following SR Ca²⁺ refilling. Inhibition of SR Ca²⁺ pump by cyclopiazonic acid dose-dependently suppressed spontaneous SANCs firing up to ≈50%. Cyclopiazonic acid and graded changes in phospholamban phosphorylation produced by β-adrenergic receptor stimulation, phosphodiesterase or protein kinase A inhibition shifted T-90_C and proportionally shifted the LCR period and spontaneous cycle length (R²=0.98). Conclusions: The LCR period, a critical determinant of the spontaneous SANC cycle length, is defined by the rate of SR Ca²⁺ replenishment, which is critically dependent on SR pumping rate, Ca²⁺ available for pumping, supplied by L-type Ca²⁺ channel, and ryanodine receptor Ca²⁺ release flux, each of which is modulated by cAMP-mediated protein kinase A-dependent phosphorylation.