Electrically Dormant Sinoatrial Nodal Cells (SANC) are Awakened by Increased Camp-Dependent Phosphorylation of Coupled-Clock Proteins

Abstract

SANC firing rate and rhythm is controlled via the degree of coupling between rhythmic local Ca2+ releases (LCRs) emerging beneath the cell membrane (‘Ca2+clock’) and sarcolemmal electrogenic mechanisms (‘membrane clock’). Dormant SANC (ones that don’t fire spontaneous action potentials (APs)) may therefore represent the extreme of clock uncoupling. We hypothesized that increased cAMP-mediated, PKA-dependent phosphorylation of the coupled-clock system proteins would restore rhythmic AP firing in dormant SANC. We recorded membrane potential (Vm) and intracellular Ca2+ kinetics in enzymatically isolated single, dormant guinea pig SANC prior to and during β-adrenoceptor stimulation (BARs, isoproterenol 0.1-1 µM) or during exposure to cell-permeant cAMP (CPT-cAMP, 300 µM). Dormant SANC that did not fire still maintained a resting membrane potential of −35+/-3mV (n=17), and exhibited small, random LCRs. ∼40% responded to BARs/CPT-cAMP. This initial response involved increases in LCR size and rhythmicity, and hyperpolarization of Vm, accompanied by small amplitude, spontaneous APs (rate ∼1Hz) triggering whole-cell Ca2+ transients. During the subsequent transition to steady-state AP firing, average LCR size increased while average LCR period shortened, partially synchronizing LCRs to late diastole, (rate ∼2Hz); this was paralleled by MDP hyperpolarization to ∼-60 mV. AP amplitude increased, and average AP cycle length shortened similar to LCR period. When BARs/CPT-cAMP was washed out, all changes reversed in order, and SANC again exhibited small random LCRs, with Vm ∼-40 mV. In conclusion, at least in some SANC, dormancy represents membrane & Ca2+ clock uncoupling that is rescuable through increased cAMP and PKA-dependent phosphorylation of both membrane and Ca2+ clock proteins. Specifically, rescuing impaired clock coupling occurs through: (1) increased spatial LCR synchronization, yielding larger LCRs; (2) increased temporal LCR synchronization, decreasing average LCR period and shifting larger LCR occurrence to late diastole.