ONTOGENY AND HORMONAL CONTROL OF CARDIAC NADH SHUTTLES

University Of Iowa, Iowa City IA

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Abstract

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.