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.
Highlights
Reperfusion of the heart following prolonged ischemia causes irreversible damage through myocyte death and resulting infarct formation
It should be noted that Chouchani et al [20] did detect an increase in H2O2 using the mitochondrial-targeted boronate cage reagent MitoB and aconitase activity, this was measured at 15 min of reperfusion, again after mitochondrial permeability transition pore (mPTP) opening has peaked [2] and when our own data confirm that a significant increase in reactive oxygen species (ROS) has occurred
One such established mechanism involves hexokinase 2 (HK2) whose binding to mitochondria is associated with resistance to mPTP opening [22,56,57] and with stabilisation of contact sites between the inner and outer membrane whose breakage may enhance cytochrome c release [56,58]
Summary
Reperfusion of the heart following prolonged ischemia causes irreversible damage through myocyte death and resulting infarct formation. The exact molecular composition of the mPTP remains uncertain [8,9,10], it is well established that its opening is triggered by elevated matrix [Ca2+], while the sensitivity to [Ca2 +] is greatly increased by oxidative stress, elevated phosphate and decreased matrix adenine nucleotides [3] These are all conditions associated with ischemia / reperfusion (I/R), and we, like many others, have proposed that the main triggers for mPTP opening during early reperfusion are an increased matrix [Ca2+] together with mitochondrial ROS production [3,8]. We wished to monitor ROS and [Ca2+] dynamics in the perfused beating heart subject to I/R
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