Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease

Abstract

Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin-D-mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3 ± 2.0 flashes/1000 μm²·100 s before and after 5 tetani) and markedly decreased (to 7.7 ± 1.6 flashes/1000 μm²·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature-dependent increase in mSOF frequency (11.9 ± 0.8 and 19.8 ± 2.6 flashes/1000 μm²·100 s at 23°C and 37°C) was observed in fibers from RYR1(Y522S/WT) mice, a mouse model of malignant hyperthermia and heat-induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress

Pulsing of Membrane Potential in Individual Mitochondria: A Stress-Induced Mechanism to Regulate Respiratory Bioenergetics in Arabidopsis

“…The observation that matrix alkalinization is attenuated by the buffering effect of free phosphate may also resolve the contradictory explanations that have been put forward for mt-cpYFP flashes in animal mitochondria Ma et al, 2011;Wei et al, 2011). When characterizing mt-cpYFP flashes in living cardiomyocytes, Wang et al (2008) and Fang et al (2011) observed that each flash coincided with a membrane potential pulse.…”

Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle – pivotal roles in Ca2+ and reactive oxygen species signaling

“…Here, ROS waves do not depend on mitochondrial Ca 2+ uptake. Spatially and temporally confined bursts of superoxide anion production ('flashes') by mitochondria have also been documented by means of a mitochondria-targeted circularly permuted YFP (cpYFP) and a superoxide-sensitive fluorophore, mitoSOX, in cardiomyocytes , skeletal muscle fibers (Pouvreau, 2010;Wei et al, 2011) and living animals (Fang et al, 2011). The reported flashes are not dependent on the transfer of Ca 2+ from the SR to mitochondria.…”

Respective Contribution of Mitochondrial Superoxide and pH to Mitochondria-targeted Circularly Permuted Yellow Fluorescent Protein (mt-cpYFP) Flash Activity

“…Individual flashes were also accompanied by stepwise increases of MitoSOX signal and decreases of NADH autofluorescence, supporting the superoxide origin of mt-cpYFP flashes. Transient matrix alkalinization induced by NH 4 Cl only minimally influenced flash frequency and failed to alter flash amplitude. However, matrix acidification modulated superoxide flash frequency in a bimodal manner.…”