Validation of the bespoke surface fluorescence apparatus.

<p><b>A,</b> Time course of NAD(P)H (top) and flavoprotein (bottom) surface fluorescence and the reflectance signals at the corresponding excitation and emission wavelengths together with data for the left ventricular pressure (LVP). <b>B,</b> Example of Indo-1 fluorescence transients recorded at 405 nm (in blue) and at 485 nm (in red), and Indo-1 ratio calculated after subtracting background autofluorescence. The top trace shows corresponding LVP data.</p

Infarct size and hemodynamic recovery of hearts in all experiments.

<p>Infarct size and hemodynamic recovery of hearts in all experiments.</p

Surface fluorescence spectra of hearts at different stages of dye loading and ischemia-reperfusion protocols.

<p><b>A</b>, PO1 perfused hearts; <b>B</b>, Calcein loaded hearts; <b>C</b>, Indo-1 loaded hearts. The coloured shaded areas indicate the band passes of the filters used for excitation and emission in the filter wheel mode of operation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167300#pone.0167300.s005" target="_blank">S1 Table</a> for details).</p

Measurement of mitochondrial H₂O₂ following ischemia / reperfusion in the perfused heart using MitoPY1 and aconitase activity.

<p><b>A</b>, MitoPY1 fluorescence (535 nm) in control (black) and IP (grey) in Langendorff-perfused hearts subjected to 30 min ischemia + 30 min reperfusion. Fluorescence was normalized to the 1 min pre-iscaemic value. MitoPY1 was successfully loaded into the hearts as shown by the increase in fluorescence upon addition of H₂O₂ to the perfusion medium. <b>B,</b> Mean 535 nm fluorescence (± SEM as error bars) was normalized to the average value between 20.5 and 21.5 min obtained on reperfusion after 30 min global ischemia in control (black, n = 10) and IP hearts (grey, n = 8). <b>C</b>, Corresponding autofluorescence data for hearts subject to a mock loading protocol (n = 6 in both control and IP hearts). Corresponding infarct sizes are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167300#pone.0167300.t001" target="_blank">Table 1</a>. <b>D</b>, Aconitase activity in mitochondria isolated from normoxic control hearts (Cont) and both control (CP Rep) and ischemic preconditioned (IP Rep) hearts subjected to 30 min ischemia and 90 s reperfusion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167300#pone.0167300.s001" target="_blank">S1 Fig</a> The grey bars confirm the reduction in aconitase activity following treatment of the mitochondrial extract with 200 μmol/L H₂O₂. Data are given as means ± SEM (error bars) of 4 hearts in each group.</p

Measurement of mitochondrial H₂O₂ in cardiomyocytes using MitoPY1.

<p>Confocal imaging of cardiomyoytes loaded with MitoTracker Deep Red and MitoPY1 before and after H₂O₂-challenge. H₂O₂ (100 μmol/L) was added directly to the coverslips and incubated for 10 min before re-imaging again. Scale bars: 20 μm, top panels and 5 μm, bottom panels. As expected, the MitoPY1 signal was low in the basal state but increased substantially upon addition of H₂O₂. The high resolution image (bottom panel) reveals individual interfibrillar mitochondria and the overlay of green MitoPY1 signal and red Mitotracker signal (right panels) confirms the dye’s mitochondrial localisation.</p

Schematic to show proposed sequence of events relating ischemia and reperfusion to mPTP opening and increases in ROS and [Ca²⁺].

<p>Events confirmed in the present paper are shown in lilac and those previously reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167300#pone.0167300.ref021" target="_blank">21</a>] are shown in mauve.</p

Validation of the bespoke surface fluorescence apparatus.

A, Time course of NAD(P)H (top) and flavoprotein (bottom) surface fluorescence and the reflectance signals at the corresponding excitation and emission wavelengths together with data for the left ventricular pressure (LVP). B, Example of Indo-1 fluorescence transients recorded at 405 nm (in blue) and at 485 nm (in red), and Indo-1 ratio calculated after subtracting background autofluorescence. The top trace shows corresponding LVP data.</p

Perfusion protocols used for fluorescence measurements.

<p><b>A,</b> A 30 min dye loading protocol was used for loading the heart with 5 μmol/L 5-cH₂DCFDA, diAM, 0.4 μmol/L calcein-AM or 3 μmol/L MitoPY1. <b>B,</b> A 45 min dye loading protocol with recirculation was used to load the heart with 3 μmol/L Indo-1. <b>C,</b> Hearts were perfused with 5 μmol/L PO1 for 30 min before index ischemia and 30 min on reperfusion. Further details are given in Supporting Information.</p

H₂O₂ production measured using PO1 fluorescence in control and IP hearts during ischemia / reperfusion.

<p><b>A</b>, Averaged fluorescence data for control (black) or IP (grey) hearts perfused with 5 μmol/L PO1. PO1 was present in the perfusion solution from 30 min (Control) or 40 min (IP) before starting global ischemia. PO1 was excluded from the perfusion solution after 30 min of reperfusion. The fluorescence was normalized using the value obtained at -11 min for control and -21 min for IP hearts and then averaged for each group. <b>B</b>, Mean data (± SEM as error bars; n = 11 for control and n = 9 for IP hearts) for PO1 fluorescence during the reperfusion period. <b>C,</b> Mean data (± SEM as error bars; n = 17 for control and n = 16 for CsA-treated hearts) for PO1 fluorescence during reperfusion of control hearts (black) and CsA-treated hearts (grey). Data were normalized to the fluorescence value at -16 min. Where present, CsA (0.2 mmol/L) was added to the PO1 perfusion solution 15 min before ischemia and during 30 min of reperfusion. Statistically significant effects of IP and CsA are shown by the horizontal lines (* p ≤ 0.05; ** p ≤ 0.01). Corresponding infarct sizes are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167300#pone.0167300.t001" target="_blank">Table 1</a>.</p

Rapid re-oxidation of NAD(P)H and flavoproteins upon reperfusion of control and IP hearts following 30 min global ischemia.

<p>Data are presented as means ± SEM (error bars) of 8 control and 9 IP hearts (* p<0.05; ** p<0.01). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167300#pone.0167300.s006" target="_blank">S2 Table</a> presents mean data for the corresponding values at 1 min before ischemia and at 2, 10 and 30 min of reperfusion, while a full representative trace for each parameter is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167300#pone.0167300.s002" target="_blank">S2 Fig</a></p