RESUMO
In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including contractility, relaxation and automatism. cAMP cascade leads to the activation of the classic protein kinase A but also to the stimulation of the recently discovered exchange protein directly activated by cAMP (Epac). The role of Epac in the regulation of intracellular Ca2+ homeostasis and contractility in cardiac myocytes is still matter of debate. In this study we showed that the selective Epac activator, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (8-CPT), produced a positive inotropic effect when adult rat cardiac myocytes were stabilized at low [Ca2+]o (0.5mM), no changes at 1mM [Ca2+]o and a negative inotropic effect when [Ca2+]o was increased to 1.8mM. These effects were associated to parallel variations in sarcoplasmic reticulum (SR) Ca2+ content. At all [Ca2+]o studied, 8-CPT induced an increase in Ca2+ spark frequency and enhanced CaMKII autophosphorylation and the CaMKII-dependent phosphorylation of SR proteins: phospholamban (PLN, at Thr17 site) and ryanodine receptor (RyR2, at Ser2814 site). We used transgenic mice lacking PLN CaMKII phosphorylation site (PLN-DM) and knock-in mice with an inactivated CaMKII site S2814 on RyR2 (RyR2-S2814A) to investigate the involvement of these processes in the effects of Epac stimulation. In PLN-DM mice, 8-CPT failed to induce the positive inotropic effect at low [Ca2+]o and RyR2-S2814A mice showed no propensity to arrhythmic events when compared to wild type mice myocytes. We conclude that stimulation of Epac proteins could have either beneficial or deleterious effects depending on the steady-state Ca2+ levels at which the myocyte is functioning, favoring the prevailing mechanism of SR Ca2+ handling (uptake vs. leak) in the different situations.
Assuntos
Sinalização do Cálcio , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Arritmias Cardíacas/patologia , Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
KEY POINTS: 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. ABSTRACT: Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under ß-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.
Assuntos
Arritmias Cardíacas/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Fosforilação/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca(2+)-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31±6 to 5±1 beats/3min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0±14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca(2+) channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca(2+) channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca(2+) channels might contribute to reperfusion-induced PBs, by increasing SR Ca(2+) content and Ca(2+) influx.