title: Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII-constitutive phosphorylation of RyR2 at site S2814.
abstract: Mice with Ca(2+) -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) Ca(2+) 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 Ca(2+) dynamics of S2814D(+/+) hearts and myocytes, but enhances SR Ca(2+) 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 Ca(2+) waves of S2814D(+/+) and transforms them into non-propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a CaMKII-dependent leaky RyR2.
  
  
  
  Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under β-adrenergic stress conditions. Although abnormal Ca(2+) release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca(2+) uptake remains controversial. We tested the hypothesis that an increase in SR Ca(2+) 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 Ca(2+) uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca(2+) 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 Ca(2+) in S2814D(+) /(-) hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca(2+) waves evoked by high extracellular Ca(2+) provocation in S2814D(+/+) mice into non-propagated Ca(2+) mini-waves on confocal microscopy. Myocyte Ca(2+) 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 Ca(2+) uptake required to prevent the arrhythmias induced by a Ca(2+) -calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca(2+) uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca(2+) leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca(2+) -triggered arrhythmias.
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: G
authors.initials: G
authors.lastname: Mazzocchi
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: L
authors.initials: L
authors.lastname: Sommese
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: J
authors.initials: J
authors.lastname: Palomeque
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: J I
authors.initials: JI
authors.lastname: Felice
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: M N
authors.initials: MN
authors.lastname: Di Carlo
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: D
authors.initials: D
authors.lastname: Fainstein
authors.affiliation: Cátedra de Patología, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: P
authors.initials: P
authors.lastname: Gonzalez
authors.affiliation: Departamento de Fisiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
authors.forename: P
authors.initials: P
authors.lastname: Contreras
authors.affiliation: Departments of Molecular Physiology and Biophysics, Medicine (in Cardiology), and Pediatrics, Baylor College of Medicine, Cardiovascular Research Institute, Houston, TX, 77030, USA.
authors.forename: D
authors.initials: D
authors.lastname: Skapura
authors.affiliation: Departments of Molecular Physiology and Biophysics, Medicine (in Cardiology), and Pediatrics, Baylor College of Medicine, Cardiovascular Research Institute, Houston, TX, 77030, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: McCauley
authors.affiliation: Departamento de Biología Comparada, Celular y Molecular, Universidad Favaloro, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: E C
authors.initials: EC
authors.lastname: Lascano
authors.affiliation: Departamento de Biología Comparada, Celular y Molecular, Universidad Favaloro, Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: J A
authors.initials: JA
authors.lastname: Negroni
authors.affiliation: Department of Pharmacology, University of Cincinnati College of Medicine, Cincinnati, OH, 45267.
authors.forename: E G
authors.initials: EG
authors.lastname: Kranias
authors.affiliation: Departments of Molecular Physiology and Biophysics, Medicine (in Cardiology), and Pediatrics, Baylor College of Medicine, Cardiovascular Research Institute, Houston, TX, 77030, USA.
authors.forename: X H T
authors.initials: XH
authors.lastname: Wehrens
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: C A
authors.initials: CA
authors.lastname: Valverde
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs Médicas, UNLP, La Plata, Argentina.
authors.forename: A
authors.initials: A
authors.lastname: Mattiazzi
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000172:deficiency* / Q000235:genetics; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism*; D055879:Gene Knock-In Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D019837:Ryanodine Receptor Calcium Release Channel; D000099211:Phospholamban; C000592748:ryanodine receptor 2. mouse