Abstract
The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase A (PKA) signaling. The mean AP cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine (ado) binds to adenosine receptors that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates an membrane-clock outward current, IKACh. Ado also inhibits adenylyl cyclase activity via Giα signaling, impacting cAMP-mediated PKA-dependent protein phosphorylation and intracellular Ca2+ cycling. We hypothesize that in addition to IKAdo activation, ado signaling impacts Ca2+ via Giα signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 uM ado substantially increased both the mean APCL (on average by 43%, n=10) and AP beat-to-beat variability from 5.1+/-1.7% to 7.2+/-2.0% (n=10) measured via membrane potential and 5.0+/-2.2 to 10.6+/-5.9 (n=40) measured via Ca2+ (assessed as the coefficient of variability, CV=SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic spontaneous, local Ca2+ releases (LCRs) (Ca2+ clock effect): as LCR size distributions shifted from larger to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR Ca2+ signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of ado drifted upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher ado concentrations (>100 uM): small stochastic LCRs of the Ca2+ clock failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of ado to activate Gβγ and IKACh, Ado and to activate Giα, suppressing adenylyl cyclase activity, both contribute to the ado-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.
Competing Interest Statement
The authors have declared no competing interest.
