Bidirectional regulation of Ca2+ sparks by mitochondria-derived reactive oxygen species in cardiac myocytes

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Abstract
The cardiac ryanodine receptor (RyR) Ca2+ release channel homotetramer harbours ∼21 potentially redox-sensitive cysteine residues on each subunit and may act as a sensor for reactive oxygen species (ROS), linking ROS homeostasis to the regulation of Ca2+ signalling. In cardiac myocytes, arrayed RyRs or Ca2+ release units are packed in the close proximity of mitochondria, the primary source of intracellular ROS production. The present study investigated whether and how mitochondria-derived ROS regulate Ca2+ spark activity in intact cardiac myocytes. Bidirectional manipulation of mitochondrial ROS production in intact rat cardiac myocytes was achieved by photostimulation and pharmacological means. Simultaneous measurement of intracellular ROS and Ca2+ signals was performed using confocal microscopy in conjunction with the indicators 5-(–6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (for ROS) and rhod-2 (for Ca2+). Photoactivated or antimycin A (AA, 5 µg/mL)-induced mitochondrial ROS production elicited a transient increase in Ca2+ spark activity, followed by gradual spark suppression. Intriguingly, photoactivated mitochondrial ROS oscillations subsequent to the initial peaks mirrored phasic depressions of the spark activity, suggesting a switch of ROS modulation from spark-activating to spark-suppressing. Partial deletion of Ca2+ stores in the sarcoplasmic reticulum contributed in part to the gradual, but not the phasic, spark depression. H2O2 at 200 µM elicited a bidirectional effect on sparks and produced sustained spark activation at 50 µM. Lowering basal mitochondrial ROS production, scavenging baseline ROS, and applying the sulphydryl-reducing agent dithiothreitol diminished the incidence of spontaneous Ca2+ sparks and abolished the Ca2+ spark responses to mitochondrial ROS. Mitochondrial ROS exert bidirectional regulation of Ca2+ sparks in a dose- and time (history)-dependent manner, and basal ROS constitute a hitherto unappreciated determinant for the production of spontaneous Ca2+ sparks. As such, ROS signalling may play an important role in Ca2+ homeostasis as well as Ca2+ dysregulation in oxidative stress-related diseases.