Control of Cardiac Metabolism: NADH Shuttles/Hypertrophy

心脏代谢的控制：NADH 穿梭/肥大

• 批准号: 7019127
• 负责人: THOMAS D SCHOLZ
• 金额: $ 36.01万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-15 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7019127
• 关键词: age difference; aspartate; bioenergetics; cardiac myocytes; cellular respiration; excitatory aminoacid; heart metabolism; hypertrophy; laboratory rat; malates; membrane transport proteins; mitochondrial membrane; nicotinamide adenine dinucleotide; oxidation reduction reaction; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): The important relationship between the cytosolic oxidation/reduction state and cardiac function is becoming increasingly recognized. Utilization of glucose and lactate, typical fuels in the neonatal and hypertrophied hearts, generates reduced nicotinamide adenine dinucleotide (NADH) that must be oxidized in order to maintain the cytosolic redox state. The malate/aspartate shuttle plays a pivotal role in the maintenance of the redox ratio by oxidizing NADH in the cytosol and delivering reducing equivalents to the electron transport chain within the mitochondria. A complete understanding of the regulation and importance of the malate/aspartate shuttle in myocardium has been hampered by lack of sequence for the aspartate-glutamate carrier (AGC), the electrogenically-driven inner mitochondrial membrane carrier of the malate/aspartate shuttle. Recently, two proteins associated with adult-onset citrullinemia, citrin and aralar 1, have been suggested to serve as AGC proteins. Based on our preliminary data, we hypothesize that the excitatory amino acid transporter-1 (EAAT-1), characterized in brain for its role in synaptic glutamate uptake, may also provide AGC function in cardiac mitochondria. We have developed a set of reagents that will allow us to study these potential AGC proteins and to further explore the metabolic importance of the malate/aspartate shuttle within the cardiac myocyte. The specific aims of this proposal are to: 1) confirm that EAAT-1 serves as an AGC in cardiac and brain mitochondria; 2) demonstrate that the inner mitochondrial membrane carriers of the malate/aspartate shuttle regulate shuttle flux in the mitochondria; and 3) show that by limiting malate/aspartate shuttle flux, function is compromised in normal and hypertrophied neonatal and adult myocytes. Microscopic immunolabeling investigations and kinetic studies using purified protein in proteoliposomes will be performed to confirm Aim 1. To approach the other aims, we have developed sense and antisense adenoviral and expression vector constructs containing the genes encoding EAAT-1, citrin, aralar 1 and the oxoglutarate-malate carrier, the latter being the other inner mitochondrial membrane protein of the malate/aspartate shuttle. Using cultured neonatal and adult myocytes, levels of the inner mitochondrial membrane proteins will be altered and the effect on malate/aspartate shuttle activity and myocyte function will be explored. At the interface between the cytosol and the mitochondria, the malate/aspartate shuttle is positioned to directly impact upon contractile function of the myofibrils and energy production by the mitochondria. A complete understanding of this pathway is essential to fully define the interaction between these two compartments.

描述（由申请人提供）：细胞质氧化/还原状态与心脏功能之间的重要关系正变得越来越被认识到。葡萄糖和乳酸（新生儿和肥大心脏中的典型燃料）的利用会产生还原型烟酰胺腺嘌呤二核苷酸（NADH），必须将其氧化才能维持细胞质氧化还原状态。苹果酸/天冬氨酸穿梭机通过氧化细胞质中的 NADH 并将还原当量传递到线粒体内的电子传递链，在维持氧化还原比方面发挥着关键作用。由于天冬氨酸-谷氨酸载体（AGC）（苹果酸/天冬氨酸穿梭的电驱动的线粒体内膜载体）的序列缺乏，阻碍了对心肌中苹果酸/天冬氨酸穿梭的调节和重要性的全面理解。最近，两种与成人发病的瓜氨酸血症相关的蛋白质，citrin 和 aralar 1，被建议作为 AGC 蛋白质。根据我们的初步数据，我们假设兴奋性氨基酸转运蛋白 1 (EAAT-1) 在大脑中的特征在于其在突触谷氨酸摄取中的作用，也可能在心脏线粒体中提供 AGC 功能。我们开发了一套试剂，使我们能够研究这些潜在的 AGC 蛋白，并进一步探索心肌细胞内苹果酸/天冬氨酸穿梭的代谢重要性。该提案的具体目的是：1）确认EAAT-1在心脏和脑线粒体中充当AGC； 2)证明苹果酸/天冬氨酸穿梭的线粒体内膜载体调节线粒体中的穿梭通量； 3)表明，通过限制苹果酸/天冬氨酸穿梭通量，正常和肥大的新生儿和成人肌细胞的功能会受到损害。将使用蛋白脂质体中的纯化蛋白进行显微免疫标记研究和动力学研究以确认目标 1。为了实现其他目标，我们开发了正义和反义腺病毒和表达载体构建体，其中包含编码 EAAT-1、citrin、aralar 1 和氧化戊二酸-苹果酸载体，后者是苹果酸/天冬氨酸穿梭的另一种线粒体内膜蛋白。使用培养的新生儿和成人肌细胞，线粒体内膜蛋白的水平将发生改变，并将探讨其对苹果酸/天冬氨酸穿梭活性和肌细胞功能的影响。在细胞质和线粒体之间的界面，苹果酸/天冬氨酸穿梭的位置直接影响肌原纤维的收缩功能和线粒体的能量产生。完整理解该途径对于完全定义这两个区室之间的相互作用至关重要。