Metabolic Regulation of Cardiac E-C Coupling

描述(由申请人提供)：本提案的长期目标是确定缺血和充血性心力衰竭常见的代谢异常如何导致细胞兴奋-收缩(E-C)偶联的缺陷。这些缺陷是导致收缩异常的原因，在持续大型心肌梗死或患有终末期扩张性和缺血性心肌病的患者中，这些异常是典型的心源性休克。我们的目标有三个：1)我们将研究成人心肌细胞E-C偶联增益和亚细胞内钙释放事件的代谢调节。一个主要的目标是确定E-C偶联是否优先依赖于糖酵解而不是氧化代谢产生的ATP；2)我们将确定单个钙通道的性质是如何由糖酵解相对于氧化代谢来调节的。我们还将确定在代谢抑制过程中钙电流和兰尼定受体在钙触发概率变化中的相对作用；3)我们将研究代谢抑制产生的跨膜总钙流量的变化，并确定在这些条件下钙电流激活钠钙交换的程度。我们的一般方法是研究膜片钳分离的大鼠和兔心肌细胞内钙离子运动和跨膜钙通量对代谢抑制剂的反应。代谢抑制剂将被选择来阻断糖酵解代谢、氧化代谢，或者同时阻断糖酵解代谢和氧化代谢。我们将使用新的共聚焦成像策略，以异常高的空间和时间分辨率记录代谢应激期间的亚细胞钙运动。我们将首次评估代谢抑制对大鼠和兔心室肌细胞膜上L钙通道单通道特性的影响。我们将使用一种新的荧光方法来筛选代谢抑制对L类钙通道与钠钙交换器之间复杂相互作用的影响。更好地了解这些问题将有助于开发新的治疗方法来恢复心力衰竭患者的收缩功能。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine how metabolic abnormalities common to ischemia and congestive heart failure produce defects in cellular excitation-contraction (E-C) coupling. These defects are responsible for the contractile abnormalities that typify cardiogenic shock in patients sustaining a large myocardial infarction or suffering from end-stage dilated and ischemic cardiomyopathies. We have three specific aims: 1) We will investigate the metabolic regulation of cardiac E-C coupling gain and subcellular Ca2+ release events in adult ventricular myocytes. A major goal is to determine whether E-C coupling is preferentially dependent upon ATP derived from glycolysis versus oxidative metabolism; 2) We will determine how single Ca2+ channel properties are regulated by glycolytic versus oxidative metabolism. We will also determine the relative roles of the Ca2+ current, and the ryanodine receptor, on changes in Ca2+ spark probability during metabolic inhibition; 3) We will study alterations in total transmembranous Ca2+ flux produced by metabolic inhibition, and determine the extent to which Ca2+ current activates sodium-calcium exchange under these conditions. Our general approach is to study the response of subcellular Ca2+ movements and transmembranous Ca2+ fluxes to metabolic inhibitors, in patch clamped isolated ventricular cardiac myocytes from rats and rabbits loaded with fluorescent Ca2+ indicators. Metabolic inhibitors will be chosen to block, alternatively, glycolytic metabolism, oxidative metabolism, or both glycolytic and oxidative metabolism simultaneously. We will use novel confocal imaging strategies to record subcellular Ca2+ movements during metabolic stress with unusually high spatial and temporal resolution. We will, for the first time, assess the effects of metabolic inhibition on the single channel properties of L-type Ca2+ channels in cell-attached patches on rat and rabbit ventricular myocytes. We will use a novel epifluorescence approach to sort out the effects of metabolic inhibition on the complex interaction between L-type Ca2+ channels and the sodium-calcium exchanger. A better understanding of these issues will assist in the development of new therapies to restore contractile function in patients with cardiac failure.