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

Lea K Seidlmayer; Benjamin J Hanson; Phung N Thai; Saul Schaefer; Donald M Bers; Elena N Dedkova

doi:10.3389/fphys.2021.628508

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

Front Physiol. 2021 May 4:12:628508. doi: 10.3389/fphys.2021.628508. eCollection 2021.

Authors

Lea K Seidlmayer¹, Benjamin J Hanson², Phung N Thai³, Saul Schaefer³, Donald M Bers⁴, Elena N Dedkova²

Affiliations

¹ Department of Cardiology, University Hospital Olbenburg, Olbenburg, Germany.
² Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, United States.
³ Department of Internal Medicine, Division of Cardiovascular Medicine, School of Medicine, University of California, Davis, Davis, CA, United States.
⁴ Department of Pharmacology, School of Medicine, University of California, Davis, Davis, CA, United States.

Abstract

Aim: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca²⁺ handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca²⁺ handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury. Methods: 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 Ca²⁺, membrane potential (ΔΨ_m), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates. Results: 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 Ca²⁺ 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_m 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 Ca²⁺, 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. Conclusion: 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.

Keywords: Biolog Mitoplates; PK11195; TSPO; cell death; ischemia-reperfusion; mitochondrial F1F0-ATPase; mitochondrial calcium uptake; mitochondrial permeability transition pore.

Abstract

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