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. ? ?
| Ralphe, J Carter; Bedell, Kurt; Segar, Jeffrey L et al. (2005) Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism. Am J Physiol Heart Circ Physiol 288:H2521-6 |
| Ralphe, J Carter; Segar, Jeffrey L; Schutte, Brian C et al. (2004) Localization and function of the brain excitatory amino acid transporter type 1 in cardiac mitochondria. J Mol Cell Cardiol 37:33-41 |
| van Natta, Timothy L; Ralphe, J Carter; Mascio, Christopher E et al. (2004) Ontogeny of vascular growth factors in perinatal sheep myocardium. J Soc Gynecol Investig 11:503-10 |
