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Characterizing Substrate Dependent Differential Regulation of Mitochondrial Respiration in the Heart and Kidney Using Computational Modeling

It is recognized that the efficiency of mitochondrial ATP synthesis depends on the choice of respiratory substrates, which differentially generate reducing equivalents NADH and FADH2 through tricarboxylic acid (TCA) cycle that feed electrons to electron transport chain (ETC) for oxidative phosphorylation (OxPhos). Specifically, various substrate combinations represent differential contributions from different substrate oxidation pathways in mitochondria towards OxPhos and ATP synthesis. However, the relevance of these substrates and their combinations has not been quantitatively or systematically characterized in neither the heart nor the kidney. We determined the respiratory responses of mitochondria isolated from the heart ventricle and kidney outer medulla (OM) and cortex to different substrate combinations and different ADP concentrations. State 3 O2 consumption rates (OCR) were determined comparing different substrates for complexes I & II (pyruvate+malate, glutamate+malate, α‐ketoglutarate+malate, succinate, succinate+rotenone) in response to increasing concentrations of ADP. Comparisons of the state 3 OCR of the heart and kidney (OM & cortex) mitochondria yielded dramatically different values, which were also distinct for different substrates. State 3 OCR of the heart mitochondria was significantly greater than that found in the kidney mitochondria (OM & cortex). Heart mitochondria energized with succinate without rotenone (blocker of complex I) and saturating concentrations of ADP failed to stimulate state 3 respiration. This was in contrast to the kidney where responses to succinate alone and succinate + rotenone were similar indicating that the reverse electron transfer via complex I (blocked by rotenone) may play a less prominent regulatory role in the kidney than in the heart. Alternately, oxaloacetate, a TCA cycle intermediate, may accumulate faster in the heart compared to the kidney, thereby inhibiting succinate oxidation. To better understand these substrate and tissue specific effects of mitochondrial respiration, thermodynamically‐constrained mechanistic computational models were developed integrating the kinetics of TCA cycle, ETC/OxPhos, and substrate transport/oxidation, incorporating published mitochondrial bioenergetic data along with our respirometry data. Intrinsic model parameters such as Michaelis‐Menten constants were assumed identical and fixed for the kidney and heart, while extrinsic model parameters such as maximal enzymatic rates were separately estimated based on available experimental data for the heart and kidney mitochondrial bioenergetics. Simulations generated using these computational models have provided us with a deeper quantitative understanding of the key determinants of the kinetic and molecular mechanisms involved in the differential regulation of substrate‐dependent mitochondrial respiration in the heart and kidney (OM & cortex). These models will be essential for understanding the regulation of substrate utilization and ATP synthesis in different tissues under normal and pathological conditions.