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

Aims The cardiac ryanodine receptor (RyR) Ca2þ 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 Ca2þ signalling. In cardiac myocytes, arrayed RyRs or Ca2þ release units are packed in the close proximity of mitochondria, the primary source of intracellular ROS production. Thepresent study investigatedwhether and howmitochondria-derived ROS regulate Ca2þ spark activity in intact cardiac myocytes. Methods and results Bidirectional manipulation of mitochondrial ROS production in intact rat cardiac myocytes was achieved by photostimulation and pharmacological means. Simultaneous measurement of intracellular ROS and Ca2þ signals was performed using confocal microscopy in conjunction with the indicators 5-(–6)-chloromethyl-20,70-dichlorodihydrofluorescein diacetate (for ROS) and rhod-2 (for Ca2þ). Photoactivated or antimycin A (AA, 5 mg/mL)-induced mitochondrial ROS production elicited a transient increase in Ca2þ 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 Ca2þ stores in the sarcoplasmic reticulum contributed in part to the gradual, but not the phasic, spark depression. H2O2 at 200 mM elicited a bidirectional effect on sparks and produced sustained spark activation at 50 mM. Lowering basal mitochondrial ROS production, scavenging baseline ROS, and applying the sulphydryl-reducing agent dithiothreitol diminished the incidence of spontaneous Ca2þ sparks and abolished the Ca2þ spark responses to mitochondrial ROS. Conclusion Mitochondrial ROS exert bidirectional regulation of Ca2þ sparks in a doseand time (history)dependent manner, and basal ROS constitute a hitherto unappreciated determinant for the production of spontaneous Ca2þ sparks. As such, ROS signalling may play an important role in Ca2þ homeostasis as well as Ca2þ dysregulation in oxidative stress-related diseases.