title: Real-Time Fluorescence Measurements of ROS and [Ca2+] in Ischemic / Reperfused Rat Hearts: Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning.
abstract: Mitochondrial permeability transition pore (mPTP) opening is critical for ischemia / reperfusion (I/R) injury and is associated with increased [Ca2+] and reactive oxygen species (ROS). Here we employ surface fluorescence to establish the temporal sequence of these events in beating perfused hearts subject to global I/R. A bespoke fluorimeter was used to synchronously monitor surface fluorescence and reflectance of Langendorff-perfused rat hearts at multiple wavelengths, with simultaneous measurements of hemodynamic function. Potential interference by motion artefacts and internal filtering was assessed and minimised. Re-oxidation of NAD(P)H and flavoproteins on reperfusion (detected using autofluorescence) was rapid (t0.5 < 15 s) and significantly slower following ischemic preconditioning (IP). This argues against superoxide production from reduced Complex 1 being a critical mediator of initial mPTP opening during early reperfusion. Furthermore, MitoPY1 (a mitochondria-targeted H2O2-sensitive fluorescent probe) and aconitase activity measurements failed to detect matrix ROS increases during early reperfusion. However, two different fluorescent cytosolic ROS probes did detect ROS increases after 2-3 min of reperfusion, which was shown to be after initiation of mPTP opening. Cyclosporin A (CsA) and IP attenuated these responses and reduced infarct size. [Ca2+]i (monitored with Indo-1) increased progressively during ischemia, but dropped rapidly within 90 s of reperfusion when total mitochondrial [Ca2+] was shown to be increased. These early changes in [Ca2+] were not attenuated by IP, but substantial [Ca2+] increases were observed after 2-3 min reperfusion and these were prevented by both IP and CsA. Our data suggest that the major increases in ROS and [Ca2+] detected later in reperfusion are secondary to mPTP opening. If earlier IP-sensitive changes occur that might trigger initial mPTP opening they are below our limit of detection. Rather, we suggest that IP may inhibit initial mPTP opening by alternative mechanisms such as prevention of hexokinase 2 dissociation from mitochondria during ischemia.
authors.affiliation: School of Biochemistry and Bristol Cardiovascular, Biomedical Sciences Building, University of Bristol, Bristol, United Kingdom.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Andrienko
authors.affiliation: School of Biochemistry and Bristol Cardiovascular, Biomedical Sciences Building, University of Bristol, Bristol, United Kingdom.|INSERM U1045-L'Institut de Rythmologie et Modélisation Cardiaque (LIRYC), Université de Bordeaux, Bordeaux, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Pasdois
authors.affiliation: School of Biochemistry and Bristol Cardiovascular, Biomedical Sciences Building, University of Bristol, Bristol, United Kingdom.
authors.forename: Andreas
authors.initials: A
authors.lastname: Rossbach
authors.affiliation: School of Biochemistry and Bristol Cardiovascular, Biomedical Sciences Building, University of Bristol, Bristol, United Kingdom.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Halestrap
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004195:Disease Models, Animal; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D006861:Hydrogen Peroxide / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D019194:Ischemic Preconditioning; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D057054:Molecular Imaging / Q000379:methods; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D002118:Calcium