Causes and consequences of succinate accumulation during cardiac ischemia.

Abstract

Ischemia occurs when restricted blood flow prevents oxygen delivery to cells and can result in cell injury or death, especially for organs with high oxygen consumption demand, such as the heart. When oxygen demand exceeds supply, the TCA cycle intermediate succinate accumulates in the myocardium. If blood flow is restored, termed reperfusion, oxidation of the accumulated succinate leads to the production of high levels of reactive oxygen species (ROS) putting oxidative stress on cardiomyocytes. Identification of therapeutic targets for ischemia-reperfusion injury requires determining and quantifying the mechanisms of how succinate is produced and oxidized under these conditions. In vitro experiments on suspensions of purified mitochondria were used to quantify routes of succinate production during hypoxia and succinate oxidation under reperfusion conditions. A computer model of mitochondrial metabolism, accounting for mitochondrial substrate transport, oxidative phosphorylation, and the TCA cycle was developed to aid our research by analyzing data, identifying novel hypotheses, and designing experiments to test identified hypotheses. Results suggest that during hypoxia, succinate is primarily generated by the reversal of succinate dehydrogenase. We also found evidence that following a shift from hypoxic to oxygenated conditions, respiration on succinate causes a rapid accumulation of toxic levels of oxaloacetate (OAA), which in turn inhibits complex II and impairs ATP production. Thus, we hypothesize that, following ischemia and reperfusion in the heart, excess OAA must be cleared away before normal energy metabolism can be restored. Potential pathways of OAA clearance include canonical TCA cycle activity via citrate synthase, as well as via malic enzyme, glutamic oxaloacetic transaminase, and enzymes with oxaloacetate decarboxylase activity. Experiments with and without pharmacological inhibition of these pathways were conducted to quantify their relative contributions to OAA clearance under reperfusion conditions.