ROS Flashes in Mitochondria Occur Concomitantly with Inner Mitochondrial Membrane Depolarization and Mitochondrial Calcium Sparks

Objectives:  Study of complex ROS dynamics and ROS mito-flashes in various living cells using fluorescent confocal imaging. Simultaneous analysis of the mitochondrial and extra-mitochondrial ROS dynamics. Analysis of the mitochondrial membrane potential and depolarization. Simultaneous analysis of mitochondrial calcium and ROS kinetics. It is well documented that mitochondria can produce a major amount of reactive oxygen species (ROS) which are involved in many physiological and pathological processes. The complex interrelationships, however, between ROS, mitochondrial inner membrane potential (\(\Delta\Psi\)m) and mitochondrial Ca2+ were not entirely investigated. In this work, we further underline biphasic ROS dynamics, demonstrating initial and continuing ROS expansion, followed by mitochondrial ROS flashes. Additionally, a huge heterogeneity in the rates of mitochondrial ROS production and ROS flashes start times has been shown. Comparing mitochondrial and extra mitochondrial fluorescence signals, we demonstrated that the mechanisms of ROS flashes may be a triggering of flashes by certain amounts of external ROS. These mechanisms involve mitochondrial permeability transition pore opening (collapse of mitochondrial membrane potential, \(\Delta\Psi\)m) and mitochondrial calcium sparks. Furthermore, mitochondria to mitochondria interactions can be seen as a wave propagation of mitochondrial ROS flashes and \(\Delta\Psi\)m collapses similar to the phenomenon of ROS-induced ROS release first demonstrated for cardiomyocytes. Our data show that mechanisms of mitochondrial ROS flashes activation and simultaneous depolarization can entail involvement of extra mitochondrial ROS produced either by individual mitochondrion or by adjacent mitochondria. This could represent common processes in ROS-ROS and mitochondria-mitochondria signaling, playing thus an important role in the cellular and mitochondrial physiology.