Abstract 1398: Effects Of Cardioprotective Drugs On Mitochondrial Functions During Myocardial Ischemia/reperfusion Revealed By Real-time Two-photon Imaging Of Perfused Rat Hearts.
Background: We recently established a real-time imaging system to monitor the mitochondrial functions in perfused hearts subjected to ischemia/reperfusion, using two-photon laser scanning microscopy. The opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨm) and mitochondrial dysfunction, is a critical step in cardiomyocyte death during ischemia/reperfusion. We tested the effects of various drugs on Δ Ψm loss in perfused rat hearts using this novel system.
Methods and Results: Langendorff-perfused rat hearts were loaded with a fluorescent indicator of ΔΨm, tetramethylrhodamine ethyl-ester, and was pretreated with the drugs for 30 minutes. The spatio-temporal changes of ΔΨm in response to ischemia/reperfusion were monitored at a subcellular level, under excitation with a two-photon laser. Ischemia/reperfusion elicits stereotyped responses in cardiomyocytes; cells maintained a constant ΔΨm for the cell-to-cell specific period of latency, followed by a rapid and irreversible ΔΨm depolarization and subsequent cytolysis. The mitochondrial ATP-sensitive potassium channel opener diazoxide (100 μmol/L) and the mitochondrial peripheral benzodiazepine receptor ligand 4′-chlorodiazepam (50 μmol/L) markedly suppressed the latency and thus decreased the likelihood that cells would undergo ΔΨm depolarization; they 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 depolarization in unprotected cells. While this mimicked the effects of ischemic preconditioning to suppress latency, it did not reflect that of cyclosporin A, a MPTP blocker which rather slowed the process of depolarization. In addition, neither a free radical scavenger 2-mercaptopropionyl glycine nor an iron chelator desferroxamine, which reduces the generation of hydroxyl radical through inhibiting the iron-mediated Fenton reaction, was protective. Simply scavenging oxygen radicals or reducing radical generation may not be a feasible strategy to achieve cardioprotection.
Conclusions: This novel two-photon imaging provides deeper insight into anti-ischemia/reperfusion therapy targeting mitochondria.