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

AIMS The cardiac ryanodine receptor (RyR) Ca(2+) release channel homotetramer harbours approximately 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 Ca(2+) signalling. In cardiac myocytes, arrayed RyRs or Ca(2+) 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 Ca(2+) 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 Ca(2+) signals was performed using confocal microscopy in conjunction with the indicators 5-(-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (for ROS) and rhod-2 (for Ca(2+)). Photoactivated or antimycin A (AA, 5 microg/mL)-induced mitochondrial ROS production elicited a transient increase in Ca(2+) 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 Ca(2+) stores in the sarcoplasmic reticulum contributed in part to the gradual, but not the phasic, spark depression. H(2)O(2) at 200 microM elicited a bidirectional effect on sparks and produced sustained spark activation at 50 microM. Lowering basal mitochondrial ROS production, scavenging baseline ROS, and applying the sulphydryl-reducing agent dithiothreitol diminished the incidence of spontaneous Ca(2+) sparks and abolished the Ca(2+) spark responses to mitochondrial ROS. CONCLUSION Mitochondrial ROS exert bidirectional regulation of Ca(2+) sparks in a dose- and time (history)-dependent manner, and basal ROS constitute a hitherto unappreciated determinant for the production of spontaneous Ca(2+) sparks. As such, ROS signalling may play an important role in Ca(2+) homeostasis as well as Ca(2+) dysregulation in oxidative stress-related diseases.