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

Lan Wei-Lapierre,Guohua Gong,Brent J Gerstner,Sylvie Ducreux,David I Yule,Sandrine Pouvreau,Xianhua Wang,Shey-Shing Sheu,Heping Cheng,Robert T Dirksen,Wang Wang

doi:10.1074/jbc.m113.455709

Abstract

Superoxide flashes are transient bursts of superoxide production within the mitochondrial matrix that are detected using the superoxide-sensitive biosensor, mitochondria-targeted circularly permuted YFP (mt-cpYFP). However, due to the pH sensitivity of mt-cpYFP, flashes were suggested to reflect transient events of mitochondrial alkalinization. Here, we simultaneously monitored flashes with mt-cpYFP and mitochondrial pH with carboxy-SNARF-1. In intact cardiac myocytes and purified skeletal muscle mitochondria, robust mt-cpYFP flashes were accompanied by only a modest increase in SNARF-1 ratio (corresponding to a pH increase of <0.1), indicating that matrix alkalinization is minimal during an mt-cpYFP flash. 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 NH4Cl only minimally influenced flash frequency and failed to alter flash amplitude. However, matrix acidification modulated superoxide flash frequency in a bimodal manner. Low concentrations of nigericin (< 100 nM) that resulted in a mild dissipation of the mitochondrial pH gradient increased flash frequency, whereas a maximal concentration of nigericin (5 μm) collapsed the pH gradient and abolished flash activity. These results indicate that mt-cpYFP flash events reflect a burst in electron transport chain-dependent superoxide production that is coincident with a modest increase in matrix pH. Furthermore, flash activity depends strongly on a combination of mitochondrial oxidation and pH gradient.

Highlights

Mitochondrial superoxide flashes measured with mt-cpYFP were suggested to reflect mitochondrial matrix alkalinization
In intact cardiac myocytes and purified skeletal muscle mitochondria, robust mt-cpYFP flashes were accompanied by only a modest increase in SNARF-1 ratio, indicating that matrix alkalinization is minimal during an mt-cpYFP flash
We set out to determine the relative contribution of mitochondrial superoxide and pH to mt-cpYFP flash activity in cardiac myocytes and skeletal muscle Flexor Digitorum Brevis (FDB) fibers

Summary

Background

Mitochondrial superoxide flashes measured with mt-cpYFP were suggested to reflect mitochondrial matrix alkalinization. This study reported that global mt-cpYFP fluorescence in purified Arabidopsis mitochondria increased upon application of mitochondrial substrates and was not altered by conditions expected to either enhance (e.g. menadione, manganese-superoxide dismutase knockdown) or reduce (Tiron, TEMPOL) superoxide production, consistent with the events reflecting ETC-dependent proton efflux rather than superoxide production. Effects of these interventions on spontaneous flash activity in Arabidopsis mitochondria were not reported. To directly address the issue of whether mt-cpYFP flashes reflect bursts in superoxide production and/or mitochondrial matrix alkalinization, we used a red-shifted pH-sensitive dye, carboxy-SNARF-1, to simultaneously monitor changes in matrix pH during mSOF events in mitochondria from both cardiac and skeletal muscle. The results demonstrate that mitochondrial mt-cpYFP flash activity reflects ETC- and pHdependent superoxide production, with only a minor component due to a concurrent alkalinization of the mitochondrial matrix during each event

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION