Electrochemical Na⁺ and Ca²⁺gradients drive coupled-clock regulation of automaticity of isolated rabbit sinoatrial nodal pacemaker cells

Coupling of an intracellular Ca²⁺ clock to surface membrane ion channels, i.e., a “membrane clock,” via coupling of electrochemical Na⁺ and Ca²⁺ gradients (E_Na and E_Ca, respectively) has been theorized to regulate sinoatrial nodal cell (SANC) normal automaticity. To test this hypothesis, we measured responses of [Na⁺] i, [Ca²⁺] i, membrane potential, action potential cycle length (APCL), and rhythm in rabbit SANCs to Na⁺ /K⁺ pump inhibition by the digitalis glycoside, digoxigenin (DG, 10–20 μmol/l). Initial small but significant increases in [Na⁺] i and [Ca²⁺] i and reductions in E Na and E Ca in response to DG led to a small reduction in maximum diastolic potential (MDP), significantly enhanced local diastolic Ca²⁺ releases (LCRs), and reduced the average APCL. As [Na⁺] i and [Ca²⁺] i continued to increase at longer times following DG exposure, further significant reductions in MDP, E_Na, and E_Ca occurred; LCRs became significantly reduced, and APCL became progressively and significantly prolonged. This was accompanied by increased APCL variability. We also employed a coupled-clock numerical model to simulate changes in E_Na and E_Ca simultaneously with ion currents not measured experimentally. Numerical modeling predicted that, as the E Na and E Ca monotonically reduced over time in response to DG, ion currents (I_CaL, I_CaT, I_f, I_Kr, and I_bNa) monotonically decreased. In parallel with the biphasic APCL, diastolic I_NCX manifested biphasic changes; initial I_NCX increase attributable to enhanced LCR ensemble Ca²⁺ signal was followed by I_NCX reduction as E_NCX (E_NCX = 3E_Na-2E_Ca) decreased. Thus SANC automaticity is tightly regulated by E_Na, E_Ca, and E_NCX via a complex interplay of numerous key clock components that regulate SANC clock coupling.