title: Detection of mitochondrial depolarization/recovery during ischaemia--reperfusion using spectral properties of confocally recorded TMRM fluorescence.
abstract: Timing and pattern of mitochondrial potential (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 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 (FTMRM) 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 FTMRM 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 μm), becoming undetectable at 5-20 μm FCCP. During ischaemia, a dramatic decrease in MPA, reaching the low/undetectable level comparable to that induced by 5-20 μm 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 FTMRM in the presence of 5 μm 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 FTMRM is not a reliable indicator of m changes during I-R.
authors.affiliation: Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, 95 S 2000 E, Salt Lake City, UT 84112-5000, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Venable
authors.forename: Tyson G
authors.initials: TG
authors.lastname: Taylor
authors.forename: Katie J
authors.initials: KJ
authors.lastname: Sciuto
authors.forename: Jerry
authors.initials: J
authors.lastname: Zhao
authors.forename: Junko
authors.initials: J
authors.lastname: Shibayama
authors.forename: Mark
authors.initials: M
authors.lastname: Warren
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Spitzer
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Zaitsev
mesh_terms: D000818:Animals; D019540:Area Under Curve; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D005583:Fourier Analysis; D053078:Membrane Potential, Mitochondrial; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008929:Mitochondria, Heart / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D015427:Reperfusion Injury / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry / Q000494:pharmacology; D013057:Spectrum Analysis; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods*
chemical_list: D012235:Rhodamines; C401833:tetramethylrhodamine methyl ester; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone