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 <i>in vivo</i> or programmed electric stimulation and high extracellular Ca²⁺ in S2814D⁺^/⁻ hearts <i>ex vivo</i> 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.