Institute of Molecular Medicine and State Key Laboratory of Biomembrane and Membrane Biotechnology, Center for Life Sciences, Peking University, Beijing, China.
Biophys J. 2012 Mar 7;102(5):1011-21. doi: 10.1016/j.bpj.2012.01.044. Epub 2012 Mar 6.
Superoxide flash represents quantal and bursting production of mitochondrial reactive oxygen species (ROS) instigated by transient opening of the mitochondrial permeability transition pore (mPTP). Given their critical role in metabolism, ischemia-reperfusion injury, and apoptosis, characterization of flash properties would be valuable to further mechanistic and physiological studies of this newly discovered mitochondrial phenomenon. Here we developed the flash detector FlashSniper based on segmentation of two-dimensional feature maps extracted from time-lapse confocal image stacks, and on the theory for correcting optical distortion of flash-amplitude histograms. Through large-scale analysis of superoxide flashes in cardiomyocytes, we demonstrated uniform mitochondrial ROS excitability among subsarcolemmal and intermyofibrillar mitochondria, and exponential distribution of intervals between consecutive flash events. Flash ignition displayed three different patterns: an abrupt rise from quiescence (44%), a rise with an exponential foot (27%), or a rise occurring after a pedestal precursor (29%), closely resembling action-potential initiation in excitable cells. However, the optical blurring-corrected amplitudes of superoxide flashes were highly variable, as were their durations, indicating stochastic automaticity of single-mitochondrion ROS excitation. Simultaneous measurement of mitochondrial membrane potential revealed that graded, rather than all-or-none, depolarization mirrored the precursor and the primary peak of the flash. We propose that superoxide flash production is a regenerative process dominated by stochastic, autonomous recruitment of a limited number of units (e.g., mPTPs) in single mitochondria.
超氧 flash 代表线粒体活性氧物质 (ROS) 的量子爆发和突发产生，这是由线粒体通透性转换孔 (mPTP) 的短暂开放引发的。鉴于它们在代谢、缺血再灌注损伤和细胞凋亡中的关键作用，对 flash 特性进行特征描述将有助于进一步研究这一新发现的线粒体现象的机制和生理学。在这里，我们开发了基于从延时共聚焦图像堆栈中提取的二维特征图的分割以及对 flash 幅度直方图的光学失真校正理论的 FlashSniper flash 检测器。通过对心肌细胞中超氧 flash 的大规模分析，我们证明了在肌小节下和肌纤维间的线粒体之间存在均匀的 ROS 兴奋性，并且连续 flash 事件之间的间隔呈指数分布。Flash 点火显示出三种不同的模式：从静止状态的突然上升 (44%)、带有指数脚的上升 (27%) 或在基座前体之后的上升 (29%)，与兴奋细胞中的动作电位起始非常相似。然而，超氧 flash 的光学模糊校正幅度及其持续时间高度可变，表明单个线粒体 ROS 激发的随机性自动性。同时测量线粒体膜电位表明，渐变而不是全有或全无的去极化反映了 flash 的前体和主要峰值。我们提出，超氧 flash 的产生是一个再生过程，由单个线粒体中有限数量的单元 (例如 mPTP) 的随机、自主募集主导。
