title: Reactive oxygen species contribute to the development of arrhythmogenic Ca²⁺ waves during β-adrenergic receptor stimulation in rabbit cardiomyocytes.
abstract: While β-adrenergic receptor (β-AR) stimulation leads to positive inotropic effects, it can also induce arrhythmogenic Ca2+ 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 Ca2+ waves during β-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 SRCa2+ 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 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 Ca2+ waves and partially prevented augmentation of SRCa2+ 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 β-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.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA.
authors.forename: Elisa
authors.initials: E
authors.lastname: Bovo
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Lipsius
authors.forename: Aleksey V
authors.initials: AV
authors.lastname: Zima
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002316:Cardiotonic Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology; D007545:Isoproterenol / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D000318:Adrenergic beta-Agonists; D002316:Cardiotonic Agents; D017382:Reactive Oxygen Species; D011943:Receptors, Adrenergic, beta; D019837:Ryanodine Receptor Calcium Release Channel; D007545:Isoproterenol; D002118:Calcium