The hypothesis of this proposal is that steady state myocardial Ca2+ and Na+ ion gradients are set by an equilibrium state of the sarcoplasmic reticulum (SR) Ca2+ ATPase and the sarcolemmal (SL) Na+/K+ ATPase reactions, respectively Thus, the Ca2+ and Na+ gradients depend on the free energy of ATP hydrolysis, deltaGATP.
SPECIFIC AIM 1 will develop two model systems with reduced deltaGATP in the oxygenated perfused rat heart. The substrate flux that provides ATP synthesis defines these two models: MODEL 1 ATP synthesis will be glycolytic; MODEL ATP synthesis will be oxidative. MODEL 1 restrains the flux of acetyl-CoA available to the tricarboxylic acid cycle using metabolic inhibitors. Hence, energy demand and deltaGATP in MODEL 1 is set by substrate level phosphorylation of glycolysis. MODEL 2 will deplete hearts of glycogen and substrate oxidation will be limited by the availability of non-glycolytic substrates. Hence, energy demand and deltaGATP in MODEL 2 is set by oxidative phosphorylation, the rate of which is controlled by substrate availability. In both MODELS deltaGATP will be further reduced by increased work demand. 31P NMR spectroscopy will measure the phosphorylated metabolites necessary to calculate deltaGATP. In addition, oxygen consumption, substrate oxidation, and lactate production will be determined.
SPECIFIC Aim 2 uses these MODELS to define the relationship between deltaGATP and [Ca2+]i. This will be done using aequorin-loaded hearts to measure the Ca2+ transient, the peak systolic [Ca2+]i and the diastolic [Ca2+]i as deltaGATP is decreased and the influx and efflux of Ca2+ modulated.
SPECIFIC Aim 3 uses these MODELS to define the relationship between deltaGATP and the SL Na+ gradient. This will be done using 23Na NMR spectroscopy to measure [Na+]i, 39K NMR spectroscopy to measure [K+]i and 87Rb NMR spectroscopy to measure Na+/K+ ATPase activity in MODELS 1 and 2. Alterations in the Ca2+ and Na+ gradients occur as a result of myocardial ischemia. These alterations underlie a significant portion of the damage that occurs during ischemia. These investigations will mimic the energetic consequence of ischemia without some of its complicating effects. Understanding the energetic contribution to the control of ion homeostasis in normal hearts may lead to improved therapies for ischemic syndromes.
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| Liu, Sihao; Hatano, Ben; Zhao, Minghui et al. (2011) Role of peroxisome proliferator-activated receptor {delta}/{beta} in hepatic metabolic regulation. J Biol Chem 286:1237-47 |
