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bioRxiv · 10.1101/2022.04.27.489760

Inhibiting succinate release worsens cardiac reperfusion injury by enhancing mitochondrial reactive oxygen species generation

Milliken, A. S.·Nadtochiy, S. M.·Brookes, P. S.
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Abstract

The metabolite succinate accumulates during cardiac ischemia. Within 5 min. of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II (Cx-II). The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia-reperfusion (IR) injury. It has been proposed that blocking monocarboxylate transporter 1 (MCT-1) may be beneficial in IR, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT-1 would retain succinate in cells, exacerbating ROS generation and IR injury. Using the mitochondrial ROS probe mitoSOX, and a custom-built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in-situ during the first moments of reperfusion, and found that acute MCT-1 inhibition enhanced mitochondrial ROS generation at reperfusion, and worsened IR injury (recovery of function and infarct size). Both these effects were abrogated by tandem inhibition of Cx-II, suggesting that succinate retention worsens IR due to driving more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator TMRE, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT-1 inhibition. Together, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation, as key determinants of PT pore opening and IR injury outcomes.

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Milliken, A. S., Nadtochiy, S. M., Brookes, P. S.. 2022-04-27. Inhibiting succinate release worsens cardiac reperfusion injury by enhancing mitochondrial reactive oxygen species generation. https://doi.org/10.1101/2022.04.27.489760

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