PK11195 Protects From Cell Death Only When Applied During Reperfusion: Succinate-Mediated Mechanism of Action.

: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury. : Rabbit ventricular myocytes underwent simulated acute ischemia (20 min) and reperfusion (at 15 min, 1 h, and 3 h) in the absence and presence of 50 μM PK11195, a TSPO inhibitor. Cell death was measured by lactate dehydrogenase (LDH) assay, while changes in mitochondrial Ca, membrane potential (ΔΨ), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates. : Cell death was increased by ~200% following I/R compared to control untreated ventricular myocytes. Incubation with 50 μM PK11195 during both ischemia and reperfusion did not reduce cell death but increased mitochondrial Ca uptake and ROS generation. However, application of 50 μM PK11195 only at the onset and during reperfusion effectively protected against cell death. The large-scale oscillations in ΔΨ observed after ~1 h of reperfusion were significantly delayed by 1 μM cyclosporin A and almost completely prevented by 50 μM PK11195 applied during 3 h of reperfusion. After an initial increase, mitochondrial Ca, measured with Myticam, rapidly declined during 3 h of reperfusion after the initial transient increase. This decline was prevented by application of PK11195 at the onset and during reperfusion. PK11195 prevented a significant increase in succinate utilization following I/R and succinate-induced forward-mode ROS generation. Treatment with PK11195 was also associated with a significant increase in glutamate and a decrease in leucine utilization. : PK11195 administered specifically at the moment of reperfusion limited ROS-induced ROS release and cell death, likely in part, by a shift from succinate to glutamate utilization. These data demonstrate a unique mechanism to limit cardiac injury after I/R.