The purpose of these studies is to establish a better understanding of the energy metabolism in tissues, in vivo. Towards this goal, the laboratory concentrates on the use of non-invasive and non-destructive techniques to evaluate the biochemical and physiological function of the heart and skeletal muscle with regard to energy metabolism. The following major findings were made over the last year: 1) It was demonstrated that inorganic phosphate (Pi) can play a complex role in the regulation of oxidative phosphorylation. Using a variety of approaches it was demonstrated that Pi can modulate both the generation of NADH at the level of the dehydrogenases, the flow of reducing equivalents down the cytochrome chain, and finally Pi serves as a substrate for the final phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). Pi was shown in increase the net generation of NADH using NADH enzyme dependent fluorescence recovery after photobleaching (ED-FRAP) on intact mitochondria. These studies revealed that Pi can increase the rate of mitochondrial generation of NADH by greater than 70%. This was associated with an increase in reducing equivalent flow from cytochrome b to cytochrome c based on optical absorption spectroscopy using an optical integration sphere technology developed in the laboratory. These two new activation sites were identified in addition to the conventional control site as a substrate for the phosphorylation of ADP to ATP. The mechanisms of action of phosphate on these processes is being investigated and include the role of Pi in the regulation of matrix volume and pH as well as direct effects of Pi on these enzymatic processes. The balanced activation of oxidative phosphorylation by phosphate occurs over a physiological concentration (K1/2 <1 mM) and is likely to play a key role in the maintaining the energy homeostasis of the heart at high work load. 2) The energy metabolism of the intact heart has been suggested to be highly heterogeneous based on the distribution of radioactive microspheres determined from surgical extraction procedures. Using MRI and optical fluorescence microscopy as non-invasive imaging tools, the distribution of iron or fluorescently labeled microspheres was compared to extracellular MRI contrast agents and deuterium oxide first pass kinetics in the canine heart, in vivo. These studies revealed that the heterogeneity of blood flow, reported in the earlier studies, was due to the geometry of the vessels and not blood flow alone. These data suggest that the blood flow and energy metabolism of the heart in vivo is very homogeneous not varying by more than 20% over the majority of the left ventricle. 3) The temporal characteristics of mitochondrial NADH fluorescence in intact cardiac myocytes were evaluated in confocal microscopy. Analysis of this data demonstrated that the temporal fluctuations are simply related to the signal to noise of the confocal instrument and not to cellular metabolic processes. These data suggest that rapid fluctuations (0.1 to 1sec) in mitochondrial membrane potential or NADH redox state are not occurring under resting conditions. 4) The use of GD-DTPA as an agent to image metabolically compromised cardiac tissue using MRI was evaluated. This contrast agent was found by others to concentrate in damaged myocardium 10 to 20 minutes after bolus infusion. Using equilibrium binding techniques, we demonstrated that this trapping of GD-DTPA was not due to specific binding to damaged heart cells, but more likely due to restricted diffusion in the damaged myocardium.
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