RESUMEN
Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) by Ca(2+) calmodulin-dependent kinase II (CaMKII) was a critical but not the unique event responsible for the production of reperfusion-induced arrhythmogenesis, suggesting the existence of other mechanisms cooperating in an additive way to produce these rhythm alterations. Oxidative stress is a prominent feature of ischemia/reperfusion injury. Both CaMKII and RyR2 are proteins susceptible to alteration by redox modifications. This study was designed to elucidate whether CaMKII and RyR2 redox changes occur during reperfusion and whether these changes are involved in the genesis of arrhythmias. Langendorff-perfused hearts from rats or transgenic mice with genetic ablation of CaMKII phosphorylation site on RyR2 (S2814A) were subjected to ischemia-reperfusion in the presence or absence of a free radical scavenger (mercaptopropionylglycine, MPG) or inhibitors of NADPH oxidase and nitric oxide synthase. Left ventricular contractile parameters and monophasic action potentials were recorded. Oxidation and phosphorylation of CaMKII and RyR2 were assessed. Increased oxidation of CaMKII during reperfusion had no consequences on the level of RyR2 phosphorylation. Avoiding the reperfusion-induced thiol oxidation of RyR2 with MPG produced a reduction in the number of arrhythmias and did not modify the contractile recovery. Conversely, selective prevention of S-nitrosylation and S-glutathionylation of RyR2 was associated with higher numbers of arrhythmias and impaired contractility. In S2814A mice, treatment with MPG further reduced the incidence of arrhythmias. Taken together, the results suggest that redox modification of RyR2 synergistically with CaMKII phosphorylation modulates reperfusion arrhythmias.
Asunto(s)
Arritmias Cardíacas/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Contracción Miocárdica/genética , Daño por Reperfusión Miocárdica/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Potenciales de Acción , Animales , Arritmias Cardíacas/metabolismo , Western Blotting , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/efectos de los fármacos , Electroforesis , Depuradores de Radicales Libres/farmacología , Glutatión/metabolismo , Preparación de Corazón Aislado , Masculino , Ratones , Ratones Transgénicos , Contracción Miocárdica/efectos de los fármacos , Daño por Reperfusión Miocárdica/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Tiopronina/farmacologíaRESUMEN
UNLABELLED: Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca²âº-handling alterations are known to occur in SHR. However, the putative modifications of Ca²âº-handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial. We investigated intracellular Ca²âº, Ca²âº-handling proteins and apoptosis in SHR vs. control Wistar rats (W) from 3 to 15 months (mo). Changes associated with the transition to HF (i.e. lung edema and decrease in midwall fractional shortening), occurred at 15 mo in 38% of SHR (SHRF). In SHRF, twitch and caffeine-induced Ca²âº transients, significantly decreased relative to 6/9 mo and 15 mo without HF signs. This decrease occurred in association with a decrease in the time constant of caffeine-Ca²âº transient decay and an increase in Naâº/Ca²âº exchanger (NCX) abundance (p<0.05) with no changes in SERCA2a expression/activity. An increased Ca²âº-calmodulin-kinase II activity, associated with an enhancement of apoptosis (TUNEL and Bax/Bcl2) was observed in SHR relative to W from 3 to 15 mo. CONCLUSIONS: 1. Apoptosis is an early and persistent event that may contribute to hypertrophic remodeling but would not participate in the contractile impairment of SHRF. 2. The increase in NCX expression/activity, associated with an increase in Ca²âº efflux from the cell, constitutes a primary alteration of Ca²âº-handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.