Abstract 1418: Distinct Cardioprotective Mechanism of Cyclosporin A and Ischemic Preconditioning Against Ischemia/Reperfusion Revealed by Real-Time Two-Photon Imaging of Perfused Rat Hearts
Background: We recently established a real-time imaging system to monitor mitochondrial function in the perfused heart subjected to ischemia/reperfusion, using two-photon laser scanning microscopy. The loss of mitochondrial membrane potential (ΔΨm) is a critical step of cardiomyocyte death during ischemia/reperfusion, which is mediated by the opening of the mitochondrial permeability transition pore (MPTP). We tested the effect of a MPTP blocker, cyclosporin A (CsA), and that of ischemic preconditioning (IPC) on ΔΨm loss.
Methods and Results: The rat heart was cannulated and perfused with Tyrode’s solution in Langendorff mode. After loading with a fluorescent indicator of ΔΨm, tetramethylrhodamine ethyl-ester, it was transferred onto the microscope stage. Under the two-photon excitation with 810 nm line of a Ti:Sapphire laser, the heart was subjected to ischemia/reperfusion by clamping the perfusion line and releasing the clamp. Spatio-temporal changes of ΔΨm in response to ischemia/reperfusion were monitored at subcellular level. During ischemia/reperfusion, cells maintained a constant ΔΨm for the cell-to-cell specific period of latency, followed by a rapid and irreversible ΔΨm loss. CsA (0.2 μmol/L) did not affect the latency period, but slowed the process of ΔΨm loss and blunted its severity. In contrast, IPC (3 cycles of 5 min ischemia and 5 min reperfusion) not only decreased the number of cells undergoing ΔΨm loss but also delayed the onset of ΔΨm loss, whereas it did not change the duration of ΔΨm loss in unprotected cells. Moreover, ΔΨm level was fully polarized in the protected cells. Although CsA and IPC achieved the similar level of protection, the mechanism of action was suggested to be distinct, as evidenced by the differential kinetics of ΔΨm loss in each individual cell.
Conclusions: This novel two-photon imaging provides deeper insights into anti-ischemia/reperfusion therapy targeting mitochondria.