β-Adrenergic Receptor Stimulation of ROS Production Generates Spontaneous Ca 2+ Waves in Rabbit Ventricular Myocytes

Abstract

Stimulation of β-adrenergic receptors (β-AR) leads to positive inotropic effects, but also can generate pro-arrhythmogenic spontaneous Ca2+ waves. We investigated the role of reactive oxygen species (ROS) production in the generation of Ca2+ waves during β-AR stimulation in rabbit ventricular myocytes. In electrically stimulated myocytes, isoproterenol (ISO; 0.1 μM) increased Ca2+ transient amplitude during systole, sarcoplasmic reticulum (SR) Ca2+ load and the occurrence of spontaneous Ca2+ waves during diastole. These effects, however, developed at different time points during ISO application. While SR Ca2+ release and load reached maximum after 3 min, Ca2+ waves did not appear until 6-12 min after ISO application. Measurements of intra-SR free Ca2+ ([Ca2+]SR) with Fluo-5N showed an initial increase of SR Ca2+ load from 0.9 to 2.1 mM followed by a gradual decline to 1.4 mM after 12 min of ISO application. This decline of [Ca2+]SR was not due to decreased SERCA activity, but instead was the result of increased SR Ca2+ leak in the form of Ca2+ waves. SR Ca2+ leak, measured as a decline of [Ca2+]SR after SERCA inhibition, was increased by 30% after 6-12 min of ISO application. Moreover, ISO significantly increased ROS production. ROS scavenger Tiron and superoxide dismutase mimetic MnTBPA abolished the ISO-mediated ROS production. Tiron (10 mM) or MnTBPA (20 μM) significantly decreased the occurrence of Ca2+ waves during ISO application and partially prevented ISO-mediated SR Ca2+ leak, but did not affect ISO-mediated increase in SR Ca2+ load or Ca2+ transient amplitude. ROS donor t-butyl peroxide (100 μM) elicited Ca2+ waves that were dependent on elevated SR Ca2+ load. These results demonstrate that β-AR-mediated ROS production acts in conjunction with elevated SR Ca2+ load to generate spontaneous Ca2+ waves in rabbit cardiomyocytes.