Reactive oxygen species contribute to the development of arrhythmogenic Ca2+waves during ß-adrenergic receptor stimulation in rabbit cardiomyocytes

Key point beta-Adrenergic receptor (beta-AR) stimulation is the most important positive inotropic effect on the heart, but it can also induce cardiac arrhythmias. In rabbit ventricular myocytes, short-term beta-AR stimulation induced a positive inotropic effect that was associated with increased ryanodine receptor phosphorylation. However, prolonged beta-AR stimulation increased the occurrence of calcium waves during diastole. This effect was associated with an increase in the reactive oxygen species production and oxidation of thiol groups on ryanodine receptors. These results suggest that phosphorylation combined with oxidation of ryanodine receptors during beta-AR stimulation increases the receptor activity to a critical level leading to the generation of arrhythmogenic calcium waves. Thus, attenuating reactive oxygen species production during beta-AR stimulation may be a promising therapeutic strategy to prevent the occurrence of arrhythmias, while at the same time preserving cardiac positive inotropy. Abstract While beta-adrenergic receptor (beta-AR) stimulation leads to positive inotropic effects, it can also induce arrhythmogenic Ca2+ waves. beta-AR stimulation increases mitochondrial oxygen consumption and, thereby, the production of reactive oxygen species (ROS). We therefore investigated the role of ROS in the generation of Ca2+ waves during beta-AR stimulation in rabbit ventricular myocytes. Isoproterenol (ISO) increased Ca2+ transient amplitude during systole, sarcoplasmic reticulum (SR) Ca2+ load and the occurrence of Ca2+ waves during diastole. These effects, however, developed at different time points during ISO application. While SR Ca2+ release and load reached a maximum level after 3 min, Ca2+ waves occurred at the highest frequency only after 6 min of ISO application. Measurement of intra-SR-free Ca2+ concentration ([Ca2+]SR) showed an initial increase of SR Ca2+ load followed by a gradual decline over time during ISO application. This decline of [Ca2+]SR was not due to decreased SR Ca2+ uptake, but instead was the result of increased SR Ca2+ leak mainly in the form of Ca2+ waves. ISO application led to significant RyR phosphorylation at the protein kinase A (PKA)-specific site, which remained relatively stable throughout beta-AR activation. Moreover, beta-AR stimulation significantly increased ROS production after 46 min of ISO application. The ROS scavenger Tiron and the superoxide dismutase mimetic MnTBPA abolished the ISO-mediated ROS production. The mitochondria-specific antioxidant Mito-Tempo and an inhibitor of the electron transport chain, rotenone, also effectively prevented the ISO-mediated ROS production. Scavenging ROS during ISO application decreased the occurrence of Ca2+ waves and partially prevented augmentation of SR Ca2+ leak, but did not affect the increase of Ca2+ transient amplitude. Treatment of myocytes with ISO for 15 min significantly reduced the free thiol content in RyRs. These data suggest that increased mitochondrial ROS production during beta-AR stimulation causes RyR oxidation. Together with RyR phosphorylation, oxidation of RyRs increases diastolic SR Ca2+ leak to a critical level leading to the generation of arrhythmogenic Ca2+ waves.