To define biochemical function in hearts cells, we have developed an isolated cardiomyocyte model which can be used in conjunction with NMR spectroscopy to continuously probe the interaction between real time metabolism and ion transport. We have done initial studies using P NMR to monitor energetic metabolites in conjunction with Na NMR to monitor sodium transport. Preliminary data suggest that discrete source and/or location (membrane vs cytoplasm) of cellular energy are critical to the maintenance of myocyte Na- gradients and that there are differences in this function in different models of disease (spontaneous hypertension (SH), diabetes mellitus (DM) and chronic hyperlipidemia (HPL) compared to controls. We proposed to further explore these mechanism by using inhibitors of Na transport (Na, K-ATPase inhibitors, such as ouabain), and specific inhibitors of energetics (2-deoxyglucose or iodoacetate to block glycolysis, oligomycin to block oxidative phosphorylation, and dinitrophenol to uncouple oxidative phosphorylation) alone and combined. Specific questions are: 1) What proportion of Na transport is supported by glycolytic versus oxidative processes, during normoxia or ischemia? 2) How are Na transport and bioenergetics correlates of these altered by disease states? 3) Are altered Na transporter processes the basis of specific pathophysiologic events? 4) Can Na ad P NMR be used to define these processes? To further use this model to investigate clinically relevant problems, additional studies will be done to evaluate the role of the myocyte (as opposed to endothelial, smooth muscle, and white blood cells) in preconditioning protection against the effect of prolonged ischemia. It is hypothesized that prolonged maintenance of Na,K, transport function is intrinsically involved in the preconditioning protection. Smaller increases in Nai during prolonged ischemia stabilized the membrane potential and decrease Na+ and Ca2+ exchange, thereby decreasing Ca2+ overload. Further, it is hypothesized that the protective effect is also related to maintenance of glycolytic function during and after prolonged ischemia. Both of these processes will be monitored with combined Na and P NMR. The goal of this project are two: 1) to demonstrate that specific abnormalities in Na transport are important determinants of cardiac pathophysiology; 2) to explore the use of NMR techniques as a diagnostic tool to evaluate pathophysiological processes with an """"""""eye"""""""" to adapt these techniques for clinical use. These studies will allow delineation of the mechanisms and energetics of Na transport which will allow characterization of disease processes. Myocytes from controls rats and animal models of cardiovascular disease (DM, SH, HPL) will be studied under baseline conditions and during inhibition of specific transport and metabolic processes.
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