Abstract 17699: Inositol-1,4,5-Trisphosphate-Mediated Pacemaker Activity in NCX KO Mouse Sinoatrial Nodal Cells

Abstract

Sinoatrial node (SAN) automaticity is due to the interplay of several membrane currents, including the current produced by Na-Ca exchanger (NCX) in response to Ca cycling. Several lines of evidence suggest that inositol-1,4,5-triphosphate receptors (IP 3 Rs) that mobilize [Ca] i are implicated in generation of automaticity in embryonic and adult cardiomyocytes. Increased IP 3 R expression observed in failing heart may predispose to arrhythmias and could contribute to the elevated heart rates associated with systolic heart failure. However, whether IP 3 R signaling influences SAN pacemaking is still controversial, in part due to the confounding influence of periodic Ca flux through the sarcolemma during every beat. To address this concern, we used NCX knockout (KO) SAN cells to study the role of IP 3 signaling on pacemaker activity. In these cells Ca flux across the sarcolemmal membrane is practically eliminated due to NCX ablation and no Ca flux through L-type Ca channels due to lack of action potentials, yet periodic spontaneous waves are still generated by the localized Ca releases (LCRs) of the “Ca clock”. Thus these waves are “uncoupled” from the membrane because there is no NCX to contribute to the diastolic depolarization in response to the release of [Ca] i . We recorded spontaneous Ca oscillations using line scan confocal microscopy in WT and NCX KO SAN cells in the presence of an IP 3 R blocker or during inhibition of phospholipase C (PLC). We found that superfusion with the IP 3 R blocker, 2-APB (2 μm) decreased the frequency of Ca transients in WT SAN cells by 82.6% (n=9, p<0.05) and Ca waves in NCX KO cells by 66% (n=10, p<0.05). Similar results were also obtained on superfusion with the PLC antagonist, U73122 (1 μm). Alternatively, an increase in IP 3 production using the α-1 adrenergic receptor agonist phenylephrine (10 μm) led to an increase in the frequency of Ca transients in WT SAN cells (n=8. P< 0.05) and Ca waves in NCX KO cells (n=9, p <0.05). This effect was blocked on the subsequent additional application of 2-APB. In summary our results indicate that Ca release from IP 3 Rs can modulate Ca oscillation frequency in NCX KO SAN cells and support our hypothesis that IP 3 signaling modulates the Ca cycling processes that regulate pacemaker frequency in the SAN.