ATP DEPENDENCE OF ION GRADIANTS IN NORMOXIC HEARTS

该建议的假设是稳态心肌Ca 2+和 Na+离子梯度由肌浆平衡状态设定， 肌网Ca~（2+）ATP酶和肌膜Na~+/K~+ ATP酶 因此，Ca2+和Na+的梯度取决于反应， ATP水解的自由能Δ GATP。 SPECIFIC AIM 1将开发两种具有降低的deltaGATP的模型系统， 充氧灌注的大鼠心脏。 提供ATP的底物流量 合成定义了这两个模型：模型1 ATP合成将是 糖酵解;模型ATP合成将是氧化的。 模型1约束 三羧酸循环可用的乙酰辅酶A通量， 代谢抑制剂 因此，模型1中的能量需求和deltaGATP为 由糖酵解的底物水平磷酸化决定。 Model 2将 消耗心脏的糖原和底物氧化将受到限制， 非糖酵解底物的可用性。 能源需求和 模型2中的deltaGATP由氧化磷酸化设定， 其由衬底可用性控制。 在两种型号中，deltaGATP 将因工作需求的增加而进一步减少。31p核磁共振光谱法 将测量计算所需的磷酸化代谢物 deltaGATP。 此外，氧消耗、底物氧化和 将测定乳酸盐产生。 具体目标2使用这些模型 以确定deltaGATP和[Ca2 +] i之间的关系。 这将是 使用装载水母发光蛋白的心脏来测量Ca2+瞬变， 峰值收缩[Ca2 +] i和舒张[Ca2 +] i作为deltaGATP， 减少，调节Ca~（2+）内流和外流。具体目标3 使用这些模型来定义deltaGATP和 SL Na+梯度。 这将使用23Na NMR光谱进行， 测量[Na +] i，39K NMR光谱测量[K +] i和87Rb NMR 光谱法以测量模型1和2中的Na +/K + ATP酶活性。 Ca2+和Na+梯度的改变是由于 心肌缺血 这些变化构成了 缺血时的损伤。 这些调查将 模拟缺血的能量后果， 复杂的影响。理解对人类社会的积极贡献 正常心脏中离子稳态的控制可导致改善的 缺血性综合征的治疗。

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.