Abstract
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 (ΔΨm)-sensitive dye, TMRM, was used as the time of MPT (TMPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (TSP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (TSP-TMPT = 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 (TSP -TMPT = -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.
Highlights
Ischemic insult to the myocardium is a major cause of acute or delayed cardiac dysfunction
This study addresses the relationship between the events of sarcolemmal permeabilization (SP) and mitochondrial permeability transition (MPT) in isolated perfused rabbit hearts after prolonged episodes of global no-flow ischemia
The most important novel findings of this study are threefold: (1) in control hearts the apparent event of MPT overlaps with the onset of SP; (2) Cyclosporine A (CsA) separates SP from MPT (SP occurs first); (3) mild permeabilization of myocytes with digitonin reasonably reproduces coupled MPT/SP events that naturally occur during reperfusion, whereas induction of MPT by H2O2 does not reproduce these events
Summary
Ischemic insult to the myocardium is a major cause of acute or delayed cardiac dysfunction. Reperfusion in a timely fashion is the most effective way to limit infarct size. This phenomenon, termed myocardial ischemia-reperfusion (I/R) injury, has been a topic of intensive research for several decades, motivated by strong experimental and (still limited) clinical evidence that pharmacological interventions upon reperfusion, aimed at specific mechanisms in the pathophysiological cascade leading to cell death, can significantly limit the infarct size (see for review [1]). Older theories postulated that the primary event is the loss of sarcolemmal integrity (we shall use term sarcolemmal permeabilization or SP), presumably a result of sarcolemmal rupture due to hypercontracture, secondary to cellular calcium (Ca2+) overload [2] and/or cell swelling [3] upon reperfusion. Electron microscopy evidence suggested that SP is an early event in reperfusion [4]
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