The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics, post-translational modifications and optical spectroscopy. One of the major hypothesizes in this program is that the activity of the multi-protein Complexes that perform Oxidative Phosphorylation are coordinated in some fashion to balance the rate of ATP production with utilization in the cell. This results in the observed metabolic homeostasis where the potential energy for doing work is maintained near constant in the cell even during major alterations in workload. The following major findings were made over the last year: 1) To broaden our analysis of metabolic regulation in the mitochondria we have expanded our studies to study the ancestors of mitochondria, simple bacteria. We have initiated studies on isolated bacteria believed to be closest to the mitochondrial origins, paracoccus denitrificans. Our initial studies have demonstrated that the respiratory rate can be acutely modulated using the volume regulatory processes in these bacteria. Using this approach we have demonstrated that the bacteria up-regulate metabolic capacity acutely with increases in work demand based on the increase in mitochondrial NADH and net reduction of FAD, cyto b and cytochrome oxidase. Most notably cytochrome c did not change its redox state suggesting the cytochrome oxidase was directly stimulated since it clearly was operating at a higher flux rate with no change in primary substrate, reduced cytochrome c. These initial studies are promising in that they demonstrate that the bacterial energy conversion system is not working in a simple feedback mechanism, as we have hypothesized for the mammalian mitochondria. We are continuing to characterize the basic energy metabolism of paracoccus during growth and volume regulatory processes using our proteomic and non-invasive optical approaches. It is hoped that this simplified system will provide some new insights into the regulation of mitochondrial oxidative phosphorylation, in the coming year. 2) Our previous work on the regulation of oxidative phosphorylation has concentrated on isolated mitochondria that we have extrapolated to in vivo conditions. We are now moving our non-invasive optical studies of the chromophores of oxidative phosphorylation into the study of the isolated perfused heart. We have demonstrated that a homemade artificial oxygen delivery system, based on perflurocarbons, works in the perfused heart increasing oxygen delivery by more than 3 fold. Optical spectroscopy techniques are being developed for the heart, these include the use of a catheter based white light LED to transmit light from the ventricular cavity across the heart wall. Spectroscopic studies demonstrate that the perflurocarbons supported increase in oxygen delivery to the perfused hearts of rat and rabbits as witnessed by increases the oxygenation of both the myoglobin as well as a decrease in reduced cytochrome oxidase. These data suggest that using the classical saline perfused heart is hypoxic. Initial studies suggest that this preparation will be appropriate for conducting work jump experiments in the intact heart where the optical spectroscopy will give us some insight into the distribution of reducing equivalents in the respiratory chain of the mitochondrial supporting this work. 3) We completed and published our studies on the role of mitochondrial cAMP in regulating oxidative phosphorylation. We have reached the conclusion that mitochondrial matrix cAMP does not impact the capability of the mitochondria to generate ATP nor change the redox poise of the cytochrome chain. We believe this make matrix cAMP a very unlikely candidate in the acute regulation of mitochondrial ATP production.

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U.S. National Heart Lung and Blood Inst
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Kuzmiak-Glancy, Sarah; Covian, Raúl; Femnou, Armel N et al. (2018) Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart. Am J Physiol Heart Circ Physiol 314:H704-H715
Liu, Fan; Lössl, Philip; Rabbitts, Beverley M et al. (2018) The interactome of intact mitochondria by cross-linking mass spectrometry provides evidence for coexisting respiratory supercomplexes. Mol Cell Proteomics 17:216-232
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Chen, Zhe; Qi, Yun; French, Stephanie et al. (2015) Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function. Mol Biol Cell 26:674-84
Kooragayala, Keshav; Gotoh, Norimoto; Cogliati, Tiziana et al. (2015) Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria. Invest Ophthalmol Vis Sci 56:8428-36

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