Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII-constitutive phosphorylation of RyR2 at site S2814

Abstract

Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D+/+) have increased propensity to arrhythmias under β-adrenergic stress conditions. Although abnormal Ca2+ release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca2+ uptake remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to rescue the increased arrhythmia propensity of S2814D+/+ mice. We generated phospholamban (PLN)-deficient/S2814D+/+ knock-in mice by crossing two colonies, S2814D+/+ and PLNKO mice (SD+/+/KO). SD+/+/KO myocytes exhibited both increased SR Ca2+ uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca2+ load (relative to PLNKO), a characteristic of S2814D+/+ myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D+/+ mice in vivo or programmed electric stimulation and high extracellular Ca2+ in S2814D+/- hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca2+ waves evoked by high extracellular Ca2+ provocation in S2814D+/+ mice into non-propagated Ca2+ mini-waves on confocal microscopy. Myocyte Ca2+ waves, typical of S2814D+/+ mice, could be evoked in SD+/+/KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca2+ uptake required to prevent the arrhythmias induced by a Ca2+-calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca2+ leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca2+-triggered arrhythmias.