ONTOGENY AND HORMONAL CONTROL OF CARDIAC NADH SHUTTLES

心脏 NADH 穿梭的个体发育和激素控制

• 批准号: 2465747
• 负责人: THOMAS D SCHOLZ
• 金额: $ 10万
• 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 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2465747
• 关键词: NAD(P)H oxidoreductase; antisense nucleic acid; embryo /fetus cell /tissue; enzyme activity; enzyme mechanism; genetic transcription; genetic translation; gestational age; glycerol 3 phosphate dehydrogenase; heart cell; heart metabolism; hormone regulation /control mechanism; in situ hybridization; insulin; liver metabolism; malate dehydrogenase; membrane transport proteins; mitochondrial membrane; myocardium; myogenesis; newborn animals; nucleic acid probes; sheep; thyroid hormones; western blottings

Substrates for myocardial adenosine triphosphate (ATP) production shift from glucose and lactate to fatty acids in the developing heart. Cytosolic reduced nicotinamide adenine dinucleotide (NADH) produced during the oxidation of glucose and lactate must be re-oxidize for glycolytic ATP production to continue. The malate/aspartate (mal/asp) and alpha- glycerophosphate (alpha-GP) shuttles, critical pathways for oxidation of cytosolic NADH in the neonatal heart, decline significantly after birth. The goals of the current proposal are to identify the enzymatic steps which regulate ma1/asp and alpha-GP shuttle activities. The specific aims are to examine the ontogeny of activity and gene expression of the proteins involved in these pathways as well as to determine the enzymatic steps specifically regulated by thyroid hormone and insulin. The initial aim of the current proposal will be to define the age- dependent changes in the mal/asp and alpha-GP shuttles in fetal and newborn sheep myocardium. The twin pregnant ewe model will then be used to determine the effect of insulin and altered thyroid function on shuttle capacity in the fetal heart. Twin fetal sheet will receive saline infusion (control twin) or infusion of either insulin or triiodothyronine or thyroidectomy (test twin). Shuttle capacities will be measured using isolated cardiac mitochondria from these animals. Myocardium will also be obtained to measure transcription and translation products of the genes which encode the mitochondrial proteins of the mal/asp and alpha-GP shuttles. Quantitative Northern blots and immunoblots will be performed using probes and antibody to the aspartate/glutamate carrier (AGC), the oxoglutarate/malate carrier (OMC), and the mitochondrial isoforms of malate dehydrogenase, aspartate aminotransferase, and alpha-GP dehydrogenase (malpha-GPDH). To begin to explore the importance of maximal NADH shuttle activity on the developing heart, the temporal and spatial distribution of these genes within the embryonic heart will also be explored. In situ hybridization of the antisense RNA probes to the AGC, OMC, and malpha-GPDH genes will be performed using embryonic sheep. It is clear that the glycolytically active fetal heart must oxidize cytosolic NADH via the mal/asp and alpha-GP shuttles in order to allow maximal flux through the reactions of glycolysis. Identifying regulatory sites and hormonal regulators of the mal/asp and alpha-GP shuttles will serve as the foundation of the long-term objective of these studies which is to establish the relationship between normal myocardial energetics and cardiac morphogenesis and growth.

心肌三磷酸腺苷 (ATP) 生产转移的底物 在发育中的心脏中从葡萄糖和乳酸到脂肪酸。胞质 还原过程中产生的烟酰胺腺嘌呤二核苷酸（NADH） 葡萄糖和乳酸的氧化必须重新氧化为糖酵解 ATP 生产继续进行。苹果酸/天冬氨酸 (mal/asp) 和 α- 甘油磷酸（α-GP）穿梭，氧化的关键途径 新生儿心脏中的胞质NADH在出生后显着下降。 当前提案的目标是确定酶促步骤 调节 ma1/asp 和 alpha-GP 穿梭活动。具体目标 是为了检查活性的个体发育和基因表达 参与这些途径的蛋白质以及确定酶促 具体由甲状腺激素和胰岛素调节的步骤。 当前提案的最初目标是定义年龄 胎儿和胎儿中 mal/asp 和 alpha-GP 穿梭的依赖性变化 新生羊心肌。然后，双胞胎怀孕母羊模型将被用于 确定胰岛素和甲状腺功能改变对航天飞机的影响 胎儿心脏的容量。双胎床单将接受生理盐水输注 （对照双胞胎）或输注胰岛素或三碘甲状腺原氨酸或 甲状腺切除术（测试双胞胎）。 航天飞机容量将使用以下方式测量 从这些动物中分离出心脏线粒体。心肌也会 获得用于测量基因的转录和翻译产物 编码 mal/asp 和 alpha-GP 的线粒体蛋白 班车。将进行定量 Northern 印迹和免疫印迹 使用天冬氨酸/谷氨酸载体 (AGC) 的探针和抗体， 氧化戊二酸/苹果酸载体 (OMC) 和线粒体亚型 苹果酸脱氢酶、天冬氨酸转氨酶和 α-GP 脱氢酶（malpha-GPDH）。开始探索最大的重要性 NADH 在发育中的心脏、时间和空间上的穿梭活动 这些基因在胚胎心脏内的分布也将是 探索过。反义RNA探针与AGC的原位杂交， OMC 和 malpha-GPDH 基因将使用胚胎羊进行。 很明显，具有糖酵解活性的胎儿心脏必须氧化 胞质 NADH 通过 mal/asp 和 alpha-GP 穿梭机 通过糖酵解反应的最大通量。识别监管 mal/asp 和 alpha-GP 穿梭机的位点和激素调节剂将 作为这些研究长期目标的基础 是为了建立正常心肌能量学与 心脏形态发生和生长。