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产生细胞兴奋-收缩（E-C）偶联缺陷。这些缺陷是造成收缩异常的原因，这种收缩异常是维持大面积心肌梗死或患有终末期扩张型和缺血性心肌病的心源性休克的典型特征。我们有三个具体目标：1)我们将研究成人心室肌细胞中心脏E-C偶联增益和亚细胞Ca2+释放事件的代谢调节。主要目的是确定E-C偶联是否优先依赖于糖酵解和氧化代谢产生的ATP；2)我们将确定单Ca2+通道特性是如何通过糖酵解和氧化代谢调节的。我们还将确定Ca2+电流和ryanodine受体在代谢抑制期间Ca2+火花概率变化中的相对作用；3)我们将研究代谢抑制产生的总跨膜Ca2+通量的变化，并确定在这些条件下Ca2+电流激活钠钙交换的程度。我们的一般方法是研究亚细胞Ca2+运动和跨膜Ca2+通量对代谢抑制剂的反应，在装载荧光Ca2+指标的大鼠和兔的膜片夹离体心室心肌细胞中。代谢抑制剂可选择阻断糖酵解代谢、氧化代谢，或同时阻断糖酵解和氧化代谢。我们将使用新的共聚焦成像策略记录亚细胞Ca2+运动在代谢应激异常高的空间和时间分辨率。我们将首次评估代谢抑制对大鼠和兔心室肌细胞细胞贴附斑块中l型Ca2+通道单通道特性的影响。我们将使用一种新的表观荧光方法来梳理代谢抑制对l型Ca2+通道和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.