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）：胞质氧化/还原状态与心脏功能之间的重要关系越来越多地认识到。在新生儿和肥大心脏中的葡萄糖和乳酸，典型的燃料的利用会产生烟酰胺腺苷二核苷酸（NADH），必须被氧化以维持胞质氧化还原状态。苹果酸/天冬氨酸班车在维持氧化还原比的维持中起着至关重要的作用，这是通过在细胞质中氧化的NADH并赋予线粒体内电子传输链的减少等效物。对心肌中苹果酸/天冬氨酸班车的调节和重要性的完全理解，由于缺乏天冬氨酸 - 谷氨酸载体载体（AGC）的序列而受到阻碍。最近，已经建议两种与成人发作的瓜氨酸血症和阿拉尔1相关的蛋白质，以作为AGC蛋白。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）在大脑中以其在突触谷氨酸摄取中的作用而表征，也可能在心脏线粒体中提供AGC功能。我们已经开发了一系列试剂，使我们能够研究这些潜在的AGC蛋白，并进一步探索心肌肌细胞中苹果酸/天冬氨酸班车的代谢重要性。该提案的具体目的是：1）确认EAAT-1是心脏和脑线粒体中的AGC； 2）证明苹果酸/天冬氨酸穿梭的内部线粒体膜载体调节线粒体中的班车通量； 3）表明，通过限制苹果酸/天冬氨酸班车通量，功能在正常和肥大的新生儿和成年肌细胞中受到损害。 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苹果酸/天冬氨酸班车。使用培养的新生儿和成年肌细胞，将改变线粒体膜蛋白的水平，并将探索对苹果酸/天冬氨酸班车活动和肌细胞功能的影响。在细胞质和线粒体之间的界面上，苹果酸/天冬氨酸穿梭的位置可直接影响线粒体对肌原纤维和能量产生的收缩功能。对这一途径的完全理解对于完全定义这两个隔室之间的相互作用至关重要。