Mitochondrial ROS Production Contributes to Generation of Ca Waves During Beta-Adrenergic Receptor Stimulation in Rabbit Ventricular Myocytes

Abstract

Beta-adrenergic receptor (beta-AR) activation leads to positive inotropic effect but can also increase diastolic sarcoplasmic reticulum (SR) Ca release in the form of pro-arrhythmic Ca waves. Beta-AR activation increases cellular energy demand and mitochondrial reactive oxygen species (ROS) production. We investigated the role of mitochondrial ROS in the generation of Ca waves during beta-AR stimulation. In electrically stimulated myocytes, application of ISO (0.1 microM) led to the occurrence Ca waves during diastole. The frequency of Ca waves increased over time during ISO application, with a particular steep increase after 6 minutes. Frequency of ISO-mediated Ca waves positively correlated with mitochondrial ROS production measured with Mito-SOX (a dye specifically targeted to mitochondria). The mitochondria specific antioxidant Mito-Tempo (5 microM) effectively prevented the ISO-mediated ROS production, as well as ISO-mediated Ca waves. Inhibition of the mitochondrial respiration with rotenone (0.3 microM) produced similar preventive effects on mitochondrial ROS production and Ca wave generation. Measurements of intra-SR free Ca ([Ca]SR) showed an initial increase of [Ca]SR (SR Ca load) followed by a gradual decline over time during ISO application. This decline of [Ca]SR was the result of increased SR Ca leak particularly in the form of Ca waves. Mito-Tempo did not affect the initial increase in SR Ca load, however, the ROS scavenger significantly prevented the [Ca]SR decline in the presence of ISO. Furthermore, Mito-Tempo significantly reduced ISO-mediated augmentation of SR Ca leak. These results suggest that mitochondrial ROS production play an important role in the generation of Ca waves during beta-AR stimulation.