The overall objective of this proposal is to further define those factors in the normal and post-ischemic heart which integrate and regulate the production of high-energy phosphate compounds, namely ATP and phosphocreatine. The integration of these metabolites is viewed to be the function of the creatine kinase reaction. The net production of ATP is the consequence of mitochondrial oxidative phosphorylation. Both of these processes will be investigated in this proposal. The model is the isolated, perfused, isovolumic working rabbit heart. The analytical methodology will extensively involve the use of 31-phosphorus nuclear magnetic resonance techniques. Both kinetic and thermodynamic approaches will be employed. To this end, three specific aims have been outlined: 1. Saturation transfer and 2-D NMR studies. These biophysical techniques will be used to measure the flux rates of high-energy phosphate mediated by creatine kinase and oxidative phosphorylation in the normal and post-ischemic heart. The goal will be to determine the degree to which ischemic induced cell damage results in fundamental changes in energy production and integration. 2. In vivo respiratory control studies. Recent studies have suggested that the adenine nucleotide control of oxidative phosphorylation relates to the availability of ADP at the mitochondrial adenine nucleotide translocase. This hypothesis will be tested in vivo using pace induced changes in function to stress the myocardium in a graded manner. By assuming that the cytoplasmic form of creatine kinase is in """"""""near-equilibrium"""""""", one can use the equilibrium constant for this reaction to calculate the free ADP content of the heart. This will then be correlated to changes in the rates of oxygen consumption, to estimate the apparent Km for ADP induced respiration. 3. Energy supply/demand balance: The [PCr]/[Pi] ratio. This ratio is now thought to be an indication of the balance between metabolic supply and energetic demand. This will be explored in the perfused heart using the pace-jump protocol to perturb the steady state. The degree to which this balance is altered by ischemic induced cell damage will also be assessed. Overall, these fundamental studies will provide new insights into the dynamic and responsive bioenergetic status of both the normal and ischemic myocardium.
