Mitochondrial flashes: From indicator characterization to in vivo imaging

Abstract

Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and contributes to human disease. Superoxide is the primary reactive oxygen species (ROS) produced in mitochondria. In vivo detection of superoxide has been a challenge in biomedical research. Here we describe the methods used to characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH dynamics. In vitro characterization reveals the high selectivity of cpYFP to superoxide over other ROS species and its dual sensitivity to pH. Confocal and two-photon imaging in conjunction with transgenic expression of the biosensor cpYFP to the mitochondrial matrix detects mitochondrial flash events in living cells, perfused intact hearts, and live animals. The mitochondrial flashes are discrete and stochastic single mitochondrial events triggered by transient mitochondrial permeability transition (tMPT) and composed of a bursting superoxide signal and a transient alkalization signal. The real-time monitoring of single mitochondrial flashes provides a unique tool to study the integrated dynamism of mitochondrial respiration, ROS production, pH regulation and tMPT kinetics under diverse physiological and pathophysiological conditions.

Reduction of Elevated Proton Leak Rejuvenates Mitochondria in the Aged Cardiomyocyte

“…Cell permeabilization and pH stress. Rat cardiomyocytes were cultured with mt-cpYFP adenovirus (Wang et al, 2016b) for 3 days in M199 medium. After incubation in Ca 2+ -free Tyrode's solution for 30 min, the medium was changed to a solution of 100 mM potassium aspartate, 20 mM KCl, 10 mM glutathione, 10 mM KH2PO4, 0.1 mM EGTA, 8% dextran 40,000, pH 7.5, with 50 μg/ml saponin for 30 s and then maintained in saponin-free internal solution (Lukyanenko and Gyorke, 1999).…”

“…SS-31 attenuates the excessive mitochondrial flash (mitoflash) activity of aged cardiomyocytes, while normalizing membrane potential and ROS. The mitoflash Hou et al, 2014;Shen et al, 2014;Wang et al, 2008;Wang et al, 2016b;Zhang et al, 2015), is triggered by nanodomain proton influx into the mitochondrial matrix (Wang et al, 2016c). Thus, we wondered whether the increased proton leak in the old cells triggered excessive mitoflash activity.…”

Physiological Ca2+ Transients Versus Pathological Steady-State Ca2+ Elevation, Who Flips the ROS Coin in Skeletal Muscle Mitochondria

“…Transgenic mouse model expressing a mitochondria-targeted biosensor (mt-cpYFP) allowed a real-time measurement of a ROS-related mitochondrial signal, termed "mitoflash" (Wang et al, 2008;Ding et al, 2015). Although mitoflash activities could be composed of multifaceted signals including matrix alkalization, superoxide, and arguably some other oxidants (Wang et al, 2008(Wang et al, , 2016Schwarzländer et al, 2012;Wei-LaPierre et al, 2013), the mechanism underlying mitoflash events is believed to be linked to transitory opening of mPTP due to the high spatiotemporal correlations between mitoflash events and IMM depolarization (Wang et al, 2008(Wang et al, , 2016. Besides transitory opening of mPTP, there are also other potential causes of IMM depolarization during mitoflash events, such as the activation of uncoupling proteins UCP2 and UCP3.…”

Mitochondrial Flashes: Dump Superoxide and Dance with Protons Now

“…Wang et al attribute this disconnect ( 5 ) to potential differences in cpYFP sequence and Escherichia coli strains, although sequence identity was verified and several strains consistently gave negative results. Their superoxide assay starts after oxygenation, yet they insist that cpYFP must be fully reduced to reveal its response to oxidation and superoxide ( 5 ), implying that a protein reversibly and repeatedly modified by superoxide in cells must be fully reduced to reveal its superoxide sensitivity when purified. The disturbing truth is that in >8 years, nobody has been able to obtain graded and reversible cpYFP responses to superoxide.…”

“…( 3 )]. They now reinterpret cpYFP flashes as multifaceted signals including a superoxide or ROS signal and an alkalinisation signal ( 5 ). This belatedly acknowledges flashes as pH events but adds new confusion by blending the invisible superoxide with undefined reactive oxygen species (ROS), despite their evidence that cpYFP fluorescence is unaffected by various reactive species ( 3 ).…”