Control Of Cellular Energy Metabolism

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

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 imaging 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)We have expanded our transmission optical spectroscopy investigation of the functioning of mitochondria in the intact beating heart to focus on ischemic reperfusion in the mouse heart model working with the MRC group at Cambridge University. We have also validated the use of the chromophores of Complex III of oxidative phosphorylation to provide a non-invasive determination of mitochondrial membrane potential in the beating heart. Using this approach we have established the dynamics of the mitochondrial membrane potential and overall redox poise of the mitochondria during ischemia, contraction and reperfusion under a variety of conditions. Initial findings suggest a rapid recovery of the mitochondria membrane potential at reperfusion that could contribute to both reactive oxygen species generation as well as the accumulation of Ca in the mitochondria matrix. Both of these events could contribute to mitochondria damage during the reperfusion phase and methods to differentiate these complications are being evaluated (see Below). We are continuing to develop a new model to perform these optical studies on the heart, in vivo. 2) We have completed a full 3D high resolution electron microscopy screen on hearts exposed to different degrees of ischemia before the reperfusion event. These studies demonstrate highly heterogenous morphological responses while a degradation of the mitochondria reticulum, previously described by our lab as critical in energy distribution in the heart. These data may suggest that the degradation of the mitochondria reticulum may be one of the early damage events in reperfusion injury. 3) Working with Dr. Murphy, we have developed a system using RHOD2 as a fluorescent indicator of mitochondrial free calcium in the intact perfused heart that permits the monitoring of the fluorescence from RHOD2 while tracking the primary and secondary filtering of light by the heart wall. This is done by alternating a white light transmission study with a 532nm laser fluorescence study in the same heart over time. This permits the correction of primary and secondary optical filtering of the fluorescent data that has been a major problem in intact tissue studies in the past. Using this approach we have made the surprising observation that mitochondrial calcium does not increase when one of the putative major calcium transports, MCU, is knocked out but the metabolic and functional response to hormonal stimulation is unchanged. This result demonstrates that changes in intramitochondrial calcium are not required for modest increases in workload in the heart. This finding suggests that one of the current models of cardiac energy regulation by mitochondria matrix calcium. We are currently expanding this approach to monitor reactive oxygen species generation in the heart under reperfusion conditions. 4) 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 recently completed our initial 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. We demonstrated in this period that the previously described metabolic homeostasis described in the mammalian heart, that is constant free energy available in ATP as well as the mitochondria proton motive during increases is workload, exists in PD. That is PD can increase it metabolic rate by over 8 fold and maintain constant proton motive force as well as increase ATP content. Currently we are specifically looking for the enzymatic pathways in substrate oxidation that are regulated by work in the bacterium. It is hoped that this simple system will provide insight into the molecular mechanisms involved in the regulation of oxidative phosphorylation in mammalian mitochondria. 4) Another classical approach to studying physiological process like the control of energy metabolism, is to look at extreme cases. In mammals, the shrew represents one of the highest resting metabolic rates with a resting heart rate approaching 1000 beats/min. We are studying the shrew with hopes of understanding how this animal has adapted to these extreme metabolic rates will provide insight into the human condition. We have successfully established one of the two Cryptotis parva shrew colonies in the world with the help of Dr. Nissar Darmani at Western University of Health Sciences. We have completed the genome of this animal, a proteomic screen of its organ systems as well as a 3D FIBSEM examination of the mitochondria /tissue structure in heart, liver, kidney and skeletal muscle. These studies reveal the most extensive mitochondria reticulum system yet observed in the aerobic skeletal muscle while the heart structure seems relatively conserved even in comparison to larger animals. The most dramatic effect was in the liver where the mitochondria content is essentially equivalent to the heart, suggesting a major metabolic capacity in this small animals liver not present in larger species. This has led to a new hypothesis with regard to the distribution of mitochondria in mammalian tissues. Where the mitochondria and energy utilizing systems can be co-located such as the ER in liver and basolateral membrane of the kidney, the mitochondria are compartmentalized to meet the local needs. In tissues that have highly diffuse energy needs such as striated muscle, a mitochondria reticulum is necessary to deliver energy over nearly the entire cell. This hypothesis may explain the variance in mitochondria morphology in different tissues. 5) Together with numerous programs at the NIH and collaboration with Siemens Medical Systems we have developed a low field 0.55T MRI system for monitoring physiological and structural function in humans. In addition to its low cost, good image quality and contrast as well as the ability to tolerate the use of interventional devices without associated radiofrequency heating, we have exploited this magnetic field to evaluate gaseous oxygen as a MRI contrast agent permitting the evaluation of lung ventilation, delivery of oxygen to the heart and potentially the relative oxygen consumption by the heart when combined with blood flow measures.