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

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.

Chronology of critical events in neonatal rat ventricular myocytes occurring during reperfusion after simulated ischemia

“…Almost simultaneously, there was a sharp increase in LAR-GECO1.2 signal, reflecting an abrupt increase in [Ca 2+ ] Mi . These three events are consistent with an almost simultaneous SP and MPT, reminiscent of our observations in whole adult hearts [7]. Lastly, Fig 7C shows a case when there was a moderate increase in Fluo-4 signal during “ischemia”, followed by a poor recovery during”reperfusion” (just touching 50% level), followed by a blunted secondary increase, followed by a very slow decrease in Fluo-4 signal to the level of full disappearance.…”

“…We randomly selected 5 to 7 NRVMs per plate which met the criteria of (1) being a myocyte (exhibiting rhythmic Ca 2+ transients at baseline); (2) having clearly demarcated cell boundaries to allow for accurate cell segmentation; (3) having overt signs of “ischemic” effects including at least 50% loss in the ΔΨ m and cell shrinking, and (3) surviving within 6 minutes of “reperfusion”, as demonstrated by at least 50% recovery in the ΔΨ m during MitoView633 restaining combined with the lack of SP (i.e., retainment of Fluo-4 inside cells). These criteria were imposed due to the fact that these characteristics are observed in the whole heart model [7], and the aim here was to replicate whole heart events so the findings could be applied in entirety and not as a model specific phenomenon. Once acceptable myocytes were identified, they were manually segmented across all images of a time-series, and the area and mean fluorescence for each selected cell in each frame was calculated using ImageJ software.…”

“…Also, the pathway from MPT to SP remains obscure [6]. A recent study from our lab found that in a whole rabbit heart model, SP and MPT are virtually simultaneous, but inhibition of the MPT pore by Cyclosporine A (CsA) creates a detectable separation between events, such that SP occurs ahead of MPT [7]. We speculated that SP is the primary event in cardiomyocyte death, and provides the source of excessive Ca 2+ influx, leading to mitochondrial Ca 2+ concentrations at which MPT is unavoidable even in the presence of CsA.…”

“…Our study mentioned above [7] lacked information about the dynamics of either cytoplasmic or intra-mitochondrial Ca 2+ in cells undergoing critical transitions. Another relevant study conducted in the whole mouse heart by Davidson et al [9] monitored cytoplasmic Ca 2+ and revealed very slow and long-standing Ca 2+ waves followed by MPT in a subset of cells exhibiting Ca 2+ waves.…”

“…Through the process of eliminating potential Ca 2+ sources and analysis of cellular Zn 2+ uptake we posit that the initial cytoplasmic Ca 2+ overload occurs through a non-selective channel/pore whose size and/or permeability expands with time of reperfusion, only eventually permitting passage of ‘canonical’ SP indicators such as TO-PRO3. We discuss the possibility that variations in the putative channel/pore evolution can explain the variability in the timing of critical events observed in different studies [7, 9], or even in different myocytes from the same tissue or monolayer.…”