The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues using modern system biology approaches. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics, metabolomics, protein structure, post-translational modifications, minimally invasive metabolic rate information 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) Our previous work on the regulation of oxidative phosphorylation has concentrated on isolated mitochondria that we have extrapolated to in vivo conditions. Based on our realization that isolated mitochondria are structurally compromised when compared to the normal cellular structure, we have now moved our non-invasive optical studies of the chromophores of oxidative phosphorylation to evaluate the enzymatic activity of mitochondrial energy conversion to the study of the isolated working perfused heart. We have established a working rabbit heart preparation in the laboratory where we can simultaneously monitor heart work, metabolic rate and optically follow the redox state of all of the Complexes of oxidative phosphorylation, non-invasively. A white light LED tipped catheter has been developed to perform transmural optical spectroscopy studies on the perfused heart while perflurocarbon (PFC) perfusions have been shown to attempt to mimic oxygen delivery by blood hemoglobin. Our initial studies have revealed that the saline perfused heart is partially anoxic with up to 20% of the heart not being adequately oxygenated at the level of the mitochondria. The heart has adapted to these conditions by reducing cardiac performance in concert with the reduced oxygen delivery. The addition of the PFC to the perfusion media that increases oxygen content, roughly 3 fold, improved cardiac performance and we find no evidence of tissue hypoxia mimicking conditions in vivo. The isolated perfused heart is a major tool in the understanding of cardiac metabolism and function. These technical developments of a transmission optical spectroscopy approach together with a optically neutral PFC to provide oxygenation should greatly improve the utility of this preparation in analyzing cardiac metabolism in the intact heart. 2) 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(PD). The goal of these studies is to unravel acute energy conversion regulation in this bacterium and then look for similar mechanisms in mammalian mitochondria. With the growing interest in the microbiome, these studies should also provide new insight into the acute regulation of bacterial energy metabolism that has not been extensively studied. This might provide new tools in generating novel antibiotics. Over the last year we have begun a systems biology approach to studying the metabolic function of PD including the quantitation of the oxidative phosphorylation Complexes along with the absorption spectrum of the chromophores associated with these Complexes, protein and metabolite content (so called proteomics and metabolomics), ion transport, oxidative phosphorylation Complex redox state, oxygen consumption, fixed acid production, membrane potential and the NADH redox state. The acute work function of the bacteria we are exploring is volume regulation, that is the work associated with the bacterium adjusting to environmental salt content. These changes routinely occur in the soil or clinically in the gut and skin. We have found a remarkable greater than 4 fold increase in respiratory and fixed acid production response to alterations in ion concentration is not due to the energetic requirements of ion transport, but the direct activation of metabolism and alteration of the oxidative phosphorylation complexes. We are currently exploring where this increase in energy conversion is utilized in the osmotic response of the bacterium as well as the nature of the regulation of glucose transport and cytochrome oxidase by potassium. 3) One hypothesis concerning the coordinated regulation of oxidative phosphorylation complexes is the formation of super complexes of these enzymes. In a super complex all of the complexes associated with generating the mitochondrial membrane potential, a primary energy source for the formation of ATP, are all co-localized in a large single unit that could modulate the function of all simultaneously via structural alterations of the Supercomplex. This is an attractive concept since simple modifications could result in alterations in several enzyme systems simultaneously. However, it is controversial whether these complexes actually exist in functioning mitochondria or whether they are an artifact of the detergents used to isolate the membrane bound proteins. To address this problem we have begun experiments with Dr. Heck in the Netherlands to examine whether the Complexes are structurally close to each other in intact mitochondria by using lipid permeable crosslinking agents in intact mitochondria to make close protein associations permanent for mass spectroscopy analysis. Initial studies support the notion of the formation of multi Complex Super-Complexes with an extensive interaction of the Complexes of oxidative phosphorylation including Complex V usually ignored in these analysis. Many new proteins protein interactions were also discovered by this screening approach that will need to be confirmed with other methods and approaches. Clearly, the protein-protein interactome is extensive in the mitochondria. This approach also allowed us to separate the different compartments of the organelle including the matrix, interspace and different sides of the individual membranes. This should provide a novel insight into the compartmentation and interaction of the mitochondrial proteome. Current studies are focusing on this approach in intact cells to get the interactions as close as possible to the in vivo condition. Hopefully these studies will reveal the role of protein protein interactions in the regulation of mitochondria metabolism in the heart.
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