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Detection of mitochondrial depolarization/recovery during ischaemia–reperfusion using spectral properties of confocally recorded TMRM fluorescence
Author(s) -
Venable Paul W.,
Taylor Tyson G.,
Sciuto Katie J.,
Zhao Jerry,
Shibayama Junko,
Warren Mark,
Spitzer Kenneth W.,
Zaitsev Alexey V.
Publication year - 2013
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2012.248153
Subject(s) - depolarization , confocal , biophysics , protonophore , confocal microscopy , fluorescence , mitochondrion , chemistry , ischemia , membrane potential , fluorescence microscope , nuclear magnetic resonance , biochemistry , biology , microbiology and biotechnology , medicine , physics , optics
Key points• Mitochondrial inner membrane potential (ΔΨ m ) collapse during myocardial ischaemia is one of the key events determining the physiological consequences of ischaemic attack in terms of post‐ischaemic arrhythmias and cell survival. • Timing and pattern of ΔΨ m collapse during ischaemia remain controversial, in part due to difficulties in interpreting the fluorescence of potentiometric cationic probes commonly used for assessment of ΔΨ m in cellular and multicellular experimental models. • This manuscript presents a new method for monitoring ΔΨ m in whole hearts based on the regular arrangement of mitochondria in cardiac myocytes, thus permitting detection of ΔΨ m collapse using spectral analysis of fluorescence. • The proposed method will help to ascertain the role of mitochondrial function in acute cardiovascular conditions, such as acute myocardial infarction or sudden cardiac arrest.Abstract Timing and pattern of mitochondrial potential (ΔΨ m ) depolarization during no‐flow ischaemia–reperfusion (I–R) remain controversial, at least in part due to difficulties in interpreting the changes in the fluorescence of ΔΨ m ‐sensitive dyes such as TMRM. The objective of this study was to develop a new approach for interpreting confocal TMRM signals during I–R based on spatial periodicity of mitochondrial packaging in ventricular cardiomyocytes. TMRM fluorescence ( F TMRM ) was recorded from Langendorff‐perfused rabbit hearts immobilized with blebbistatin using either a confocal microscope or an optical mapping system. The hearts were studied under normal conditions, during mitochondrial uncoupling using the protonophore FCCP, and during I–R. Confocal images of F TMRM were subjected to spatial Fourier transform which revealed distinct peaks at a spatial frequency of ∼2 μm −1 . The area under the peak (MPA) progressively decreased upon application of increasing concentrations of FCCP (0.3–20 μ m ), becoming undetectable at 5–20 μ m FCCP. During ischaemia, a dramatic decrease in MPA, reaching the low/undetectable level comparable to that induced by 5–20 μ m FCCP, was observed between 27 and 69 min of ischaemia. Upon reperfusion, a heterogeneous MPA recovery was observed, but not a de novo MPA decrease. Both confocal and wide‐field imaging registered a consistent decrease in spatially averaged F TMRM in the presence of 5 μ m FCCP, but no consistent change in this parameter during I–R. We conclude that MPA derived from confocal images provides a sensitive and specific indicator of significant mitochondrial depolarization or recovery during I–R. In contrast, spatially averaged F TMRM is not a reliable indicator of ΔΨ m changes during I–R.