Abstract

Automaticity of Sino-atrial node (SAN) pacemaker cells is regulated by integrated functions within a system of two coupled clocks: the so-called "membrane clock", dependent mainly on the funny current (If), and the denominated "Ca2+ clock", dependent mainly on the RyR2. During exercise or under stress, the pacemaker accelerates its rate (positive chronotropic effect) due to β-adrenergic stimulation. As a result, cAMP levels increase in the SAN cells and PKA is activated, phosphorylating different targets. While cAMP shifts the If activation curve increasing its availability, it is less clear whether phosphorylation of RyR2 affects SAN modulation by β-adrenergic stimulation. S2808 and S2030 are considered the major RyR2 phosphorylation sites that are substrates of PKA but few studies have addressed the role of S2030 in the regulation of RyR2. The main objective is to investigate the involvement of RyR2 phosphorylation by PKA in SAN modulation by β-adrenergic stimulation. Mice with phospho-ablation at the PKA sites (S2030A and S2808A) and mimetic phosphorylation (S2030D) of the RyR2 were compared to WT. Pacemaker activity was evaluated in vivo and ex vivo by EGC telemetry and confocal microscopy respectively. ECG was recorded at basal conditions and after stress (isoproterenol, epinephrine + caffeine injection). SAN were dissected from mice, loaded with intracellular calcium dye and visualised by confocal microscopy in basal conditions and under isoproterenol (ISO). In vivo, lack of PKA phosphorylation on Serine2030 (S2030A) induced an impaired positive chronotropic response after ISO injection compared to WT and S2030D mice. Moreover, both, S2030A and the phosphomimetic S2030D, presented more arrhythmias following epinephrine plus caffeine injection compared to WT. Ex-vivo, the [Ca2+]i transients rate of pacemaker cells failed to accelerate in S2030A compared to WT and S2030D when perfused with 20 nM isoproterenol. These results show that RyR2 phosphorylation at S2030 is involved in pacemaker response to beta-adrenergic stimulation.