Cyclosporine-insensitive mode of cell death after prolonged myocardial ischemia: Evidence for sarcolemmal permeabilization as the pivotal step

<div><p>A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60–80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨ_m)-sensitive dye, TMRM, was used as the time of MPT (T_MPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (T_SP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (T_SP-T_MPT = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 μM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (T_SP -T_MPT = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5–20 μM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H₂O₂ (200–800 μM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence.</p></div

The summary of the time difference between the MPT and SP events (T_SP-T_MPT) in individual myocytes from the three experimental groups as indicated.

<p>Thick horizontal bars show average values. Note that CsA increases separation between MPT and SP, such that T_SP-T_MPT is significantly negative. *, p < 0.01.</p

Wave-like propagation of MPT caused by H₂O₂.

<p>The time is shown according to the onset of observation starting 6 min after the end of H₂O₂ application (200 μM). <b>A</b> to <b>D</b>, consecutive snapshots taken every 2 minutes. Green, fluorescence of TMRM (F_TMRM); orange, fluorescence of YO-PRO1 (F_YO-PRO1). The nature of objects brightly stained with YO-PRO1 is unknown, but they may represent dead fibroblasts or other cell types which are usually abundant in the most superficial layers and are visible immediately after YO-PRO1 delivery to the heart. White arrows point to two such bright objects, which were used as fiducial points to track myocytes amid slight changes in the field of view and the focal plane. Note that unlike the naturally occurring MPT/SP events, H₂O₂-induced MPT propagated as a wide front (white line), easily crossing cell boundaries, and was not associated with immediate cellular YO-PRO1 uptake. <b>E</b> and <b>F</b>, Different structure of the propagating front of _FTMRM loss during naturally occurring MPT (<b>E</b>; same cell as #13 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s005" target="_blank">S4 Fig</a> as well as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s014" target="_blank">S5 Movie</a>) and H₂O₂-induced MPT (<b>F</b>). In each Panel, F_TMRM was computed as the function of the distance along the long axis of the rectangular region of interest (ROI) approximating the selected cell (white rectangle). The magnified image of the ROI is shown at the top of each Panel. For each x-position along the cell, F_TMRM was averaged over all pixels in the respective column (across the width) of the rectangle. This yielded an instantaneous profile of the propagating wave (green curve). The minimal value on the y-axis of mean F_TMRM represents the minimal value in the field of view (extracellular level, or background). Note that during naturally occurring MPT (<b>E</b>) the level of F_TMRM in the wake of the MPT front is very close to the background level indicating complete loss of the TMRM in that part of the cell. In contrast, during H₂O₂-induced MPT (<b>F</b>) the level of F_TMRM in the wake of the MPT front remains at about 50% of the dynamic range.</p

Assessment of infarct size using TTC staining.

<p><b>A</b>, <i>left to right</i>, examples of original scanned images of TTC staining, the respective green channel signal, and the thresholded regions, respectively (see detailed description of TTC staining procedure in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s002" target="_blank">S1 Fig</a>). Rows from top to bottom in <b>A</b> represent <i>No_BDM</i>, <i>Control</i>, and <i>CsA</i> groups, respectively. <b>B</b>, average percent of severe infarct (white), intermediate infarct (pink) and viable tissue (red) in the three experimental groups. The error bars are omitted for clarity, but none of the regions were significantly different between the groups by 2-way ANOVA. At least in part this is due to the large variation in TTC staining data between hearts in each group (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s007" target="_blank">S6 Fig</a>).</p

Detailed spatiotemporal analysis of F_TMRM loss and F_YO-PRO1 gain during reperfusion in a representative myocyte from a control heart.

<p><b>A</b>, each row shows F_TMRM (green), F_YO-PRO1 (orange) and the merged image of the cell undergoing the critical transition (outlined with white) for different time points (<i>a</i> to <i>e</i>) indicated in <b>B</b>. <b>B</b>, the cell-averaged F_TMRM (green) and F_YO-PRO1 (orange) as the function of time shown as 0–100% of the dynamic range of each signal. The vertical green and orange dashed lines indicate T_MPT and T_SP, respectively. Note the spatiotemporal overlap between the processes of F_TMRM loss (indicator of MPT) and F_YO-PRO1 gain (indicator of SP), such that at time point <i>b</i> there is a clear uptake of YO-PRO1 while the majority of the cell interior still shows polarized mitochondria.</p

Detailed spatiotemporal analysis of F_TMRM loss and F_YO-PRO1 gain during reperfusion in a myocyte from a heart treated with CsA (0.2 μM).

<p>Notations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.g001" target="_blank">Fig 1</a>. Note that compared to a myocyte from a control heart shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.g001" target="_blank">Fig 1</a>, the earliest detectable F_YO-PRO1 gain (indicator of SP), which is observed at the time point <i>b</i>, clearly occurs before the processes of F_TMRM loss (indicator of MPT).</p