Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 15, Pages 10567-10577Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.455709
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Funding
- National Institutes of Health [AR44657, HL114760, HL093671]
- American Heart Association
- Universite Lyon 1
- CNRS
- Academia Dei Lincea Fund
- National Basic Research Program of China [2013CB531201, 2011CB809102]
- National Science Foundation of China [31221002, 31130067]
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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. How-ever, 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 mu M) 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.
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