Kinetic analysis of isolated mitochondrial function

Quantitative understanding of cardiac energy metabolism requires a mechanistic biophysical model of mitochondria. We have developed such a model that includes oxidative phosphorylation, proton handling and adenine nucleotide translocase systems. Here we compare model simulations with experimental observations on suspensions of isolated cardiac mitochondria and found them agree under various conditions. The model simulates kinetics of mitochondrial respiration, ATP synthesis, and the steady state relationship between oxygen consumption rate and membrane potential under increasingly uncoupled conditions. The kinetic response of mitochondria to substrate availability and other external perturbations was monitored with the fluorescent probe Rhodamine‐123, a commonly used membrane potential‐sensitive dye. Deconvolution of the dynamic membrane potential signal from the measured fluorescence was achieved by devising a model of the electrogenic transport of the dye across the mitochondrial inner membrane. Analysis of our data reveals that the time course of the mitochondrial membrane potential is significantly different in shape and magnitude from that of the fluorescence. The combined model of oxidative phosphorylation and dye transport allows more accurate estimation of transient changes in membrane potential from the fluorescence signal. This is the first report of a detailed biophysical model of mitochondria quantitatively accounting for the experimentally observed dynamics of mitochondrial function. This work was supported by NIH grant HL072011 (to DAB).