Effects of thyroid hormone on cardiac beta-adrenergic responsiveness in conscious baboons

BACKGROUND: Many of the cardiovascular manifestations of thyroid hormone excess resemble those produced by sympathoadrenal stimulation. The objective of this study was to determine the effects of thyroid hormone excess on myocardial beta-adrenergic expression and responsiveness to infused agonists in the primate heart. METHODS AND RESULTS: The responses of left ventricular isovolumic contraction (dP/dt(max)) and relaxation (tau) during graded dobutamine infusion were studied both before and after 4 weeks of thyroid hormone administration in 8 chronically instrumented baboons. At matched (atrially paced) heart rates, thyroid hormone significantly increased resting dP/dt(max) (3073+/-1034 versus 2318+/-829 mm Hg/s, P<.05) and decreased tau (24.0+/-5.5 versus 28.2+/-5.4 ms, P<.05). The change from baseline for dP/dt(max) and tau in response to beta1-adrenergic stimulation was significant at each dobutamine dose (2.5 to 10 microg x kg(-1) x min(-1)), but when expressed as a percent change, it was similar before versus after thyroid hormone. Similar changes were found when beta2-adrenergic stimulation was produced by terbutaline infusion in three additional baboons. beta-Adrenergic receptor (betaAR) expression was higher in five thyroxine-treated than in five control baboons (37.4+/-1.2 versus 15.7+/-3.2 fmol/mg, P<.001), and this was due to a greater increase in the beta2AR (5.9+/-1.5 to 20.6+/-1.2 fmol/mg, P<.001) than the beta1AR (9.7+/-1.7 to 16.8+/-0.1 fmol/mg, P<.01) subtype. CONCLUSIONS: In the primate heart, thyroid hormone produces positive inotropic and lusitropic effects in the resting state and upregulates both beta1AR and beta2AR, with the beta2AR increase predominating. At equivalent rates, however, thyroid hormone excess does not appear to enhance the sensitivity of left ventricular contractility and relaxation to either beta1- or beta2-adrenergic stimulation.     

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5'-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides -870 to -650) and TRE2 (nucleotides -272 to -40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter-luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides -716 to -731) represents TRE1 and that the sequence GGGTCC (nucleotides -117 to -112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.     

Determination of a specific 5-HT2a antagonist (M100907) in rat and dog plasma at the femtomole/milliliter level by gas chromatography-mass spectrometry

Hypothyroidism was induced in a group of male Fischer 344 rats by administration of 0.05% propylthiouracil (PTU) in the drinking water for 12 weeks. Control rats were not treated. Plasma levels of thyroid hormones indicated that PTU treatment had produced severe thyroid hormone deficiency. In PTU-treated rats compared to control rats, levels of total T3 and total T4 were reduced 54.5% and 53.7%; while levels of free T3 and free T4 were reduced 87.1% and 96.5%. Functional hypothyroidism was demonstrated by: (i) a 49.1% decrease in hepatic plasma membrane alpha1-adrenergic receptor binding, and (ii) a 11.2-fold increase in hepatic gamma-glutamyltranspeptidase activity; relative to the expression of these parameters in control rats. Membranes were isolated from hippocampi of control, PTU-induced hypothyroid and thyroxine-replaced rats and specific adrenergic receptor binding determined by radioligand binding techniques. Hypothyroidism resulted in a shift in the balance of alpha1 and beta2 adrenergic receptor binding by evoking: an increase in alpha1-adrenergic receptor binding to 1.57-fold of control levels; and, a decrease in beta2-adrenergic receptor binding to 64% of control levels. Thyroid hormone replacement carried out in PTU-treated hypothyroid rats at 30 microg/kg s.c. per day for the last 3 days of the 12 week PTU-treatment protocol, which reversed physical and functional hypothyroidism, reversed the observed changes in hippocampal adrenergic receptor binding, indicating them to be thyroid hormone, and not PTU, -dependent. This receptor shift evoked by hypothyroidism may, in part, explain the protective effect of hypothyroidism on ischemia-induced hippocampal damage by favoring inhibitory input and limiting excitotoxic input by catecholamines.     

Conserved residues amino-terminal of cytoplasmic tyrosines contribute to the SHP-1-mediated inhibitory function of killer cell Ig-like receptors

The heart has been recognized as a major target of thyroid hormone action. Our study investigates both the regulation of cardiac-specific genes and contractile behavior of the heart in the presence of a mutant thyroid hormone receptor beta1 (T3Rbeta1-delta337T) derived from the S kindred. The mutant receptor was originally identified in a patient with generalized resistance to thyroid hormone. Cardiac expression of the mutant receptor was achieved by a transgenic approach in mice. As the genes for myosin heavy chains (MHC alpha and MHC beta) and the cardiac sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2) are known to be regulated by T3, their cardiac expression was analyzed. The messenger RNA levels for MHC alpha and SERCA2 were markedly down-regulated, MHC beta messenger RNA was up-regulated. Although T3 levels were normal in these animals, this pattern of cardiac gene expression mimics a hypothyroid phenotype. Cardiac muscle contraction was significantly prolonged in papillary muscles from transgenic mice. The electrocardiogram of transgenic mice showed a substantial prolongation of the QRS interval. Changes in cardiac gene expression, cardiac muscle contractility, and electrocardiogram are compatible with a hypothyroid cardiac phenotype despite normal T3 levels, indicating a dominant negative effect of the T3Rbeta mutant.     

Two separate NCoR (nuclear receptor corepressor) interaction domains mediate corepressor action on thyroid hormone response elements

The nuclear corepressor (NCoR) binds to the thyroid hormone receptor (TR) in the absence of ligand. NCoR-TR interactions are mediated by two interaction domains in the C-terminal portion of NCoR. Binding of NCoR to TR results in ligand-independent repression on positive thyroid hormone response elements. The interactions between NCoR interaction domains and TR on DNA response elements, however, have not been well characterized. We have found that both interaction domains are capable of binding TR on thyroid hormone response elements. In addition, the NCoR interaction domains interact much more strongly with the TR than those present in the silencing mediator of retinoic acid and TRs (SMRT). Furthermore, deletion of either NCoR interaction domain does not significantly impair ligand-independent effects on positive or negative thyroid hormone response elements. Finally, both NCoR interaction domains appear to preferentially bind TR homodimer over TR-retinoid X receptor heterodimer in electrophoretic mobility shift assays. These data suggest that either NCoR interaction domain is capable of mediating the ligand-independent effects of TR on positive and negative thyroid hormone response elements.     

Cell type-dependent modulation of the dominant negative action of human mutant thyroid hormone beta 1 receptors

BACKGROUND: Mutations in the ligand-binding domain of the thyroid hormone receptor beta (TR beta) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TR alpha and the non-mutated TR beta alleles by the TR beta 1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants. MATERIALS AND METHODS: Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TR beta 1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TR beta 1 expression in these two cell types. In order to determine how TR beta 1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed. RESULTS: Transfection studies using WT h-TR beta 1 in HeLa and NIH3T3 cells, showed that the 3,3',5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TR beta 1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif. CONCLUSIONS: The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.     

Reduced beta 1 receptor messenger RNA abundance in the failing human heart

Heart failure in humans is characterized by alterations in myocardial adrenergic signal transduction, the most prominent of which is down-regulation of beta 1-adrenergic receptors. We tested the hypothesis that down-regulation of beta 1-adrenergic receptors in the failing human heart is related to decreased steady-state levels of beta 1 receptor mRNA. Due to the extremely low abundance of beta 1 receptor mRNA, measurements were possible only by quantitative polymerase chain reaction (QPCR) or by RNase protection methods. Because the beta 1 receptor gene is intronless and beta 1 receptor mRNA abundance is low, QPCR yielded genomic amplification in total RNA, and mRNA measurements had to be performed in poly (A)(+)-enriched RNA. By QPCR the concentration of beta 1 receptor mRNA varied from 0.34 to 7.8 x 10(7) molecules/microgram poly(A)(+)-enriched RNA, and the assay was sensitive to 16.7 zeptomol. Using 100-mg aliquots of left ventricular myocardium obtained from organ donors (nonfailing ventricles, n = 12) or heart transplant recipients (failing ventricles, n = 13), the respective beta 1 mRNA levels measured by QPCR were 4.2 +/- 0.7 x 10(7)/micrograms vs. 2.10 +/- 0.3 x 10(7)/micrograms (P = 0.006). In these same nonfailing and failing left ventricles the respective beta 1-adrenergic receptor densities were 67.9 +/- 6.9 fmol/mg vs. 29.6 +/- 3.5 fmol/mg (P = 0.0001). Decreased mRNA abundance in the failing ventricles was confirmed by RNase protection assays in total RNA, which also demonstrated a 50% reduction in beta 1 message abundance. We conclude that down-regulation of beta 1 receptor mRNA contributes to down-regulation of beta 1 adrenergic receptors in the failing human heart.