title: Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism.
abstract: In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P < 0.05) and decreased in PTU-treated (2.0 +/- 0. 3 vs. 5.2 +/- 1; P < 0.05) rats. Aralar1 mRNA levels were unchanged in T3-treated and somewhat decreased in PTU-treated (7.1 +/- 1.0 vs. 9.3 +/- 0.1, P < 0.05) rats. Citrin mRNA levels were decreased in T3-treated and unchanged in PTU-treated rats. EAAT1 protein levels (arbitrary units) in T3-treated cardiac mitochondria were increased compared with controls (8.9 +/- 0.4 vs. 5.9 +/- 0.6; P < 0.005) and unchanged in PTU-treated mitochondria. No difference in malate/aspartate shuttle capacity was found between PTU-treated and control cardiac mitochondria. Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.
authors.affiliation: Department of Pediatrics, Roy and Lucille Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA. carter.ralphe@chp.edu
authors.forename: J Carter
authors.initials: JC
authors.lastname: Ralphe
authors.forename: Kurt
authors.initials: K
authors.lastname: Bedell
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Segar
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Scholz
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D006332:Cardiomegaly / Q000150:complications / Q000188:drug therapy / Q000378:metabolism*; D027341:Excitatory Amino Acid Transporter 1; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D050491:Glutamate Plasma Membrane Transport Proteins; D006980:Hyperthyroidism / Q000139:chemically induced / Q000150:complications / Q000378:metabolism*; D007037:Hypothyroidism / Q000150:complications / Q000188:drug therapy / Q000378:metabolism*; D015151:Immunoblotting; D008293:Malates / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014284:Triiodothyronine / Q000494:pharmacology
chemical_list: D027322:Amino Acid Transport System X-AG; D027341:Excitatory Amino Acid Transporter 1; D050491:Glutamate Plasma Membrane Transport Proteins; D008293:Malates; D012333:RNA, Messenger; C493392:Slc1a3 protein, rat; D027981:Symporters; D014284:Triiodothyronine; D001224:Aspartic Acid; C030298:malic acid