Yan Yuan, Liu Jie, Wei Chaoliang, Li Kaitao, Xie Wenjun, Wang Yanru, Cheng Heping
Institute of Molecular Medicine, National Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871, China.
Cardiovasc Res. 2008 Jan 15;77(2):432-41. doi: 10.1093/cvr/cvm047. Epub 2007 Oct 25.
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.
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.
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.
心肌兰尼碱受体（RyR）钙释放通道同四聚体每个亚基含有约21个潜在的氧化还原敏感半胱氨酸残基，可能作为活性氧（ROS）的传感器，将ROS稳态与钙信号调节联系起来。在心肌细胞中，排列的RyRs或钙释放单元紧密排列在线粒体附近，线粒体是细胞内ROS产生的主要来源。本研究调查了线粒体衍生的ROS是否以及如何调节完整心肌细胞中的钙火花活性。
通过光刺激和药理学方法对完整大鼠心肌细胞中线粒体ROS的产生进行双向调控。使用共聚焦显微镜结合5-（-6）-氯甲基-2'，7'-二氯二氢荧光素二乙酸酯（用于检测ROS）和罗丹明-2（用于检测钙）指示剂同时测量细胞内ROS和钙信号。光激活或抗霉素A（AA，5μg/mL）诱导的线粒体ROS产生引起钙火花活性短暂增加，随后火花逐渐受到抑制。有趣的是，初始峰值后光激活的线粒体ROS振荡反映了火花活性的阶段性抑制，表明ROS调节从火花激活转变为火花抑制。肌浆网中钙储存的部分缺失部分导致了火花的逐渐抑制，但不是阶段性抑制。200μM的过氧化氢对火花产生双向作用，在50μM时产生持续的火花激活。降低基础线粒体ROS产生、清除基线ROS以及应用巯基还原剂二硫苏糖醇可降低自发钙火花的发生率，并消除钙火花对线粒体ROS的反应。
线粒体ROS以剂量和时间（历史）依赖性方式对钙火花发挥双向调节作用，基础ROS是自发钙火花产生的一个迄今未被重视的决定因素。因此，ROS信号可能在钙稳态以及氧化应激相关疾病中的钙失调中起重要作用。
