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

Key point β‐Adrenergic receptor (β‐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 β‐AR stimulation induced a positive inotropic effect that was associated with increased ryanodine receptor phosphorylation. However, prolonged β‐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 β‐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 β‐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 β‐adrenergic receptor (β‐AR) stimulation leads to positive inotropic effects, it can also induce arrhythmogenic Ca 2+ waves. β‐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 Ca 2+ waves during β‐AR stimulation in rabbit ventricular myocytes. Isoproterenol (ISO) increased Ca 2+ transient amplitude during systole, sarcoplasmic reticulum (SR) Ca 2+ load and the occurrence of Ca 2+ waves during diastole. These effects, however, developed at different time points during ISO application. While SR Ca 2+ release and load reached a maximum level after 3 min, Ca 2+ waves occurred at the highest frequency only after 6 min of ISO application. Measurement of intra‐SR‐free Ca 2+ concentration (Ca 2+ SR ) showed an initial increase of SR Ca 2+ load followed by a gradual decline over time during ISO application. This decline of Ca 2+ SR was not due to decreased SR Ca 2+ uptake, but instead was the result of increased SR Ca 2+ leak mainly in the form of Ca 2+ waves. ISO application led to significant RyR phosphorylation at the protein kinase A (PKA)‐specific site, which remained relatively stable throughout β‐AR activation. Moreover, β‐AR stimulation significantly increased ROS production after 4–6 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 Ca 2+ waves and partially prevented augmentation of SR Ca 2+ leak, but did not affect the increase of Ca 2+ 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 β‐AR stimulation causes RyR oxidation. Together with RyR phosphorylation, oxidation of RyRs increases diastolic SR Ca 2+ leak to a critical level leading to the generation of arrhythmogenic Ca 2+ waves.