Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII‐constitutive phosphorylation of RyR2 at site S2814
Authors: Mazzocchi G. , Sommese L. , Palomeque J. , Felice J. I. , Di Carlo M. N. , Fainstein D. , Gonzalez P. , Contreras P. , Skapura D. , McCauley M. D. , Lascano E. C. , Negroni J. A. , Kranias E. G. , Wehrens X. H. T. , Valverde C. A. , Mattiazzi A. 
Published: December 2015

Key points
Mice with Ca2+–calmodulin‐dependent protein kinase (CaMKII) constitutive pseudo‐phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D+/+ mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca2+ leak in diastole and increased propensity to arrhythmias under stress conditions.
We generated phospholamban (PLN)‐deficient S2814D+/+ knock‐in mice by crossing two colonies, S2814D+/+ and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D+/+ mice.
PLN ablation partially rescues the altered intracellular Ca2+ dynamics of S2814D+/+ hearts and myocytes, but enhances SR Ca2+ sparks and leak on confocal microscopy.
PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2.
PLN ablation aborts the arrhythmogenic SR Ca2+ waves of S2814D+/+ and transforms them into non‐propagating events.
A mathematical human myocyte model replicates these results and predicts the increase in SR Ca2+ uptake required to prevent the arrhythmias induced by a CaMKII‐dependent leaky RyR2.
AbstractMice 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.