Systems Analysis of Carboxylate Transport and Oxidation Pathways in Cardiac Mitochondria

Abstract

Experimental assessment and computational modeling were used to analyze substrate transport, tricarboxylic acid cycle kinetics, and oxidative phosphorylation in suspensions of purified cardiac mitochondria. The kinetics of ATP synthesis and carbohydrate oxidation, including during hypoxia and reoxygenation, were investigated using various substrate combinations and conditions. Model simulations fit to transient respiration and NAD(P)H measurements reveal novel insights into pyruvate dehydrogenase regulation, regulation of mitochondrial leak, and the clearance of oxaloacetate during respiration on succinate. High concentrations of succinate induced increased mitochondrial leak respiration driven in part by ROS-activated uncoupling. Oxidative phosphorylation under succinate-fueled respiration was inhibited by rapid buildup of oxaloacetate, inhibiting succinate dehydrogenase. Malic enzyme and oxaloacetate decarboxylase activities represent potenital pathways for removal of oxaloacetate, with glutamate further enhancing clearance. The developed model captures the observed transient behaviors as well as steady-state relationships between ATP synthesis rate and phosphate metabolite levels, lending a new systems-level understanding of mitochondrial energy metabolism. In sum, these findings offer a framework for simulating and interpreting mitochondrial function in vitro and in vivo. Key PointsThis study uses experiments and computer simulations to probe the interactions between substrate transport processes, TCA cycle kinetics, redox state, and oxidative ATP synthesis in cardiac mitochondria. The developed kinetic model simulates mitochondrial metabolism in vitro and represents a framework for integrative modeling of cardiac energy metabolism. Model-based analysis identifies a kinetic model of pyruvate dehydrogenase (PDH) deactivation during leak-state respiration and activation during oxidative phosphorylation. High levels of cation leak during respiration on succinate are explained by a ROS-dependent activation of uncoupling.