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

Abstract

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.