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The coordinated release and reuptake of calcium into the sarcoplasmic reticulum (SR) is critical to maintain an adequate heart function. Reactive oxygen species (ROS) and reactive nitrogen species (RNS), generated in the heart under normal basal condition, modulate the function of different proteins via the reversible oxidation of critical cysteine residues. Excess ROS/RNS generation has been shown to impair heart function, but extensive evidence indicates that the controlled production of these molecules increases cardiac contractility by targeting SR calcium proteins. Ryanodine receptors (RyR2) are endogenously S-nitrosylated and S-glutathionylated and both redox modifications increase the activity of these channels in vitro. Moreover, exercise or rapid pacing increases the RyR2 S-glutathionylation, a modification that depends on the activation of NADPH oxidase (NOX2). In isolated cardiomyocytes, this enzyme is rapidly activated by stretch, generating an immediate burst of ROS which increases calcium release by RyR2. Nitroxyl, a particular ROS/RNS, increases cardiac inotropy in vivo, by targeting critical thiols in RyR2, the SR Ca2+-ATPase and phospholamban, allowing the simultaneous increase in calcium release and reuptake, required to produce a sustained increase in the calcium transient. In this minireview we present some of the recent work on the redox regulation of SR calcium cycling proteins., Sociedad Argentina de Fisiología

Antecedentes de la investigación: El preacondicionamiento hipóxico se ha utilizado como medida de protección para el miocardio, sin embargo es clínicamente inaplicable. Las especies reactivas de oxígeno (ERO), estarían involucradas en la producción de modificaciones estructurales y eléctricas, que hacen susceptible al tejido auricular a arritmias y disfunción contráctil. Por lo tanto, un reforzamiento del sistema de defensa antioxidante protegería al corazón del daño hipóxico. Objetivo: Determinar el efecto de la suplementación de Omega 3 y vitaminas antioxidantes C y E, en el estado antioxidante de pacientes sometidos a cirugía cardíaca con circulación extracorpórea (CEC). Métodos: Un total de 50 pacientes se incluyó en el protocolo. Se randomizaron 15 pacientes a recibir Omega 3, siete días antes de la cirugía, y vitaminas C y E, dos días antes de la cirugía, ambos hasta el post-operatorio y 15 a placebo. Los valores controles se obtuvieron en 20 pacientes sin intervención farmacológica preacondicionante. Se obtuvieron muestras de sangre en condiciones básales, antes del inicio de las vitaminas, 6-8 h post-operatorio y al alta para la determinación del estado antioxidante (capacidad antioxidante del plasma, FRAP; relación glutatión reducido (GSH)Zoxidado (GSSG) y enzimas antioxidantes en eritrocito y daño oxidativo (malondialdehído y F2 - isoprostanos en plasma). En tejido se midió por Western Blot la expresión de la subunidad catalítica de la NADPH oxidasa (NOX-2) y la citosólica p47phox. La localización de la subunidad de membrana NOX-2/gp91phox se evidenció mediante inmunofluorescencia. Como índice de daño oxidativo en tejido se utilizaron los niveles de malondialdehído. Se utilizó análisis multivariado ANOVA con post-test de una vía de Bonferroni. Resultados: Al momento de la cirugía, los pacientes que habían recibido tratamiento, presentaban niveles de GSH/GSSG un 56% mayor (p

We aimed to define the relative contribution of both PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca2+ influx to the cell by low [Ca]o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [3H]-ryanodine binding and fast Ca2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca2+. Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βAR stimulation by PKA and CaMKII, respectively. The isoproterenolinduced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the β-induced increase in the channel activity as indicated by the enhancement of the [3H]-ryanodine binding and the velocity of fast SR Ca2+ release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca2+ release during βAR stimulation., Centro de Investigaciones Cardiovasculares