The Coupled-Pacemaker Clock System of Sinoatrial Nodal Cells Regulates Both the Action Potential Rate and Rhythm

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Normal automaticity of the sinatrial-node cells (SANC) is regulated by integrated molecular functions within a system of two clock-like oscillators: the sarcoplasmic reticulum, acting as a “Ca2+ clock,” rhythmically discharges diastolic local-Ca2+ releases (LCRs) beneath the cell surface membrane; LCRs activate an inward Na+/Ca2+ exchanger current (INCX) that prompts the “Membrane clock,” the ensemble of other sarcolemmal-electrogenic molecules, to generate action potentials (APs). Crosstalk between the two clocks regulates SANC spontaneous AP cycle length (CL). We determine whether clock crosstalk also regulates the rhythm of AP CL in response to perturbation that induce bradycardia via specific inhibition of either membrane or Ca2+ clock functions.We employed ivabradine (IVA, 3, 10 and 30µM) which directly inhibits membrane clock ion channel functions, but has no direct effect on Ca2+ clock proteins functions. The IVA-induced increase in the AP CL, however, lowers Ca2+ influx, which reduces Ca2+-activated calmodulin-AC-cAMP/PKA signaling, affecting Ca2+ and phosphorylation-dependent functions that deive the coupled clock system. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA, 0.5 and 5µM) reduces the magnitude and delays the occurrence of the LCR-Ca2+ signal, leading to a reduction in Ca2+-activated calmodulin-AC-cAMP/PKA signaling, and delayed and reduced Ca2+ activation of INCX and a prolongation of the AP CL. Importantly, prolongation of LCR period (IVA by 16±2 to 46±5%, CPA by 17±2 to 53±5%), AP CL (IVA by 13±4 to 36±8%, CPA by 16±3 to 44±8%) and the increase in AP CL variability (IVA by 38±10 to 190±50%, CPA by 36±17 to 183±50%) are proportional to each other. The tight inter-relationships among these variables in response to specific perturbation of either the M or the Ca2+ clock, therefore, reflect clock crosstalk.