The role of thyroid hormone in the pathophysiology of heart failure: clinical evidence

Thyroid hormone (TH) has a fundamental role in cardiovascular homeostasis in both physiological and pathological conditions, influencing cardiac contractility, heart rate (HR), diastolic function and systemic vascular resistance (SVR) through genomic and non-genomic mediated effects. In heart failure (HF) the main alteration of thyroid function is referred to as “low-triiodothyronine (T3) syndrome” (LT3S) characterized by decreased total serum T3 and free T3 (fT3) with normal levels of thyroxine (T4) and thyrotropin (TSH). Even if commonly interpreted as an adaptive factor, LT3S may have potential negative effects, contributing to the progressive deterioration of cardiac function and myocardial remodeling in HF and representing a powerful predictor of mortality in HF patients. All these observations, together with the early evidence of the benefits of T3 administration in HF patients indicate that placebo-controlled prospective studies are now needed to better define the safety and prognostic effects of chronic treatment with synthetic TH in HF.     

Differences in nuclear thyroid hormone receptors among species

Hepatic nuclear thyroid hormone receptors from rat, dog, chicken, and rainbow trout were compared. Receptor affinities for 3,5,3'-triiodo-L-thyronine (T3) were similar in preparations from rat, dog, and chicken, using isolated nuclei and nuclear extracts. Rainbow trout nuclear receptor showed a lower affinity for T3. Almost half of the receptors were released into the medium with rat and chicken nuclei, and 79.7 +/- 1.1% of the receptors were released with rainbow trout nuclei, when isolated nuclei were incubated with T3 at 22 degrees for 2 hr. The affinity constant of rat liver receptor for calf thymus DNA-cellulose at 0.17 M KCl, pH 7.4, was 3.98 +/- 1.47 x 10(5) M-1, when determined using DNA-cellulose columns. The number of salt bridges involved in DNA binding of the rat receptor was 5.73 +/- 0.38. When receptor-DNA interactions were compared among species, significant differences were found, but the receptors from dog and rainbow trout liver were similar. Sephacryl S-200 column chromatography showed that chicken receptor had a Stokes radius significantly smaller than that of rat receptor. Partial proteolysis of T3-receptor complex using trypsin alpha-chymotrypsin, elastase, and papain produced distinct T3-binding fragments in different species. Our data provide evidence that nuclear thyroid hormone receptors from different species have significant structural dissimilarities.     

Characterization of nuclear thyroid hormone receptors of cultured skin fibroblasts from patients with resistance to thyroid hormone

Nuclear thyroid hormone receptors of patients with the syndrome of resistance to thyroid hormone were investigated in cell lines from seven patients in four affected families and compared to results from six normals. Fibroblasts cultured from skin biopsies were used. When binding affinity and capacity for L-triiodothyronine (T3) were examined by incubating whole cells or isolated nuclei, no significant differences were found. The amount of receptor released during the incubation of nuclei (9.3% to 19.0% of total nuclear receptors) was also within the normal range in these patients. When T3 binding assays were performed on 0.3 mol/L KCl extracted receptor, a significant decrease in binding capacity (MBC) without a difference in binding affinity (Ka) was observed in four patients and a lower Ka with normal MBC was found in two patients. Recovery of receptors in saline extracts, from patients' fibroblasts showing a low MBC, was low in comparison to normals. Lability of salt extracted receptors at 38 degrees C was normal and salt extractability of T3 occupied receptors, examined by incubation of [125I]-T3 labeled nuclei with various concentrations of KCl, was only slightly decreased. This lower salt extractability of receptors was insufficient to account for the low MBC obtained by Scatchard analysis of T3 binding to nuclear extracts. Gel filtration and density gradient sedimentation of salt-extracted receptors showed Stokes radius of 34 A, and sedimentation coefficient of 3.4 S in all patients and normals. From these values, molecular weight of 49,000 and total frictional ratio (f/fo) of 1.4 were calculated for nuclear receptors from patients and normals, suggesting a somewhat asymmetrical shape of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of thyroid hormone in blood pressure homeostasis: evidence from short-term hypothyroidism in humans

Arterial hypertension is known to be frequently associated with thyroid dysfunction, with a particularly high prevalence in chronic hypothyroidism. However, to our knowledge no comprehensive study addressed causal mechanisms possibly involved in this association. We here report the physiological relationships between blood pressure and neuro-humoral modifications induced by acute hypothyroidism in normotensive subjects. Twelve normotensive patients with previous total thyroidectomy were studied. Ambulatory 24-h blood pressure monitoring was performed, and free T(3), free T(4), TSH, PRA, aldosterone, cortisol, adrenaline, and noradrenaline were assayed 6 wk after oral L-T(4) withdrawal (phase 1) and 2 months after resumption of treatment (phase 2). During the hypothyroid state (TSH, 68.1 +/- 27.7 microIU/ml; mean +/- SD), daytime arterial systolic levels slightly, but significantly, increased (125.5 +/- 9.7 vs. 120.4 +/- 10.8 mm Hg; P < 0.05), and daytime diastolic levels (84.6 +/- 7.9 vs. 76.4 +/- 6.8 mm Hg; P < 0.001), noradrenaline (2954 +/- 1578 vs. 1574 +/- 962 pmol/liter; P < 0.001), and adrenaline (228.4 +/- 160 vs. 111.3 +/- 46.1 pmol/liter; P < 0.05) also increased. PRA remained unchanged (0.49 +/- 0.37 vs. 0.35 +/- 0.21 ng/ml.h; P = NS), whereas both aldosterone (310.3 +/- 151 vs. 156.9 +/- 67.5 pmol/liter; P < 0.005) and cortisol (409.2 +/- 239 vs. 250.9 +/- 113 pmol/liter; P < 0.02) significantly increased. By using univariate logistic regression daytime arterial diastolic values, noradrenaline and aldosterone were found to be significantly related to the hypothyroid state (P < 0.02, P < 0.036, and P < 0.024, respectively). In conclusion, our data show that thyroid hormones participate in the control of systemic arterial blood pressure homeostasis in normotensive subjects. The observed sympathetic and adrenal activation in hypothyroidism, which is reversible with thyroid hormone treatment, may also contribute to the development of arterial hypertension in human hypothyroidism.     

Thyroid hormone resistance in the heart: role of the thyroid hormone receptor beta isoform

Several cardiac genes possess thyroid hormone (TH) response elements regulated by TH receptors. Mutation in TR-beta gene causes the human syndrome of resistance to TH, which is characterized by elevated serum concentration of T(4) and T(3) and variable degrees of insensitivity to TH. It is unclear, however, whether a mutant TR-beta could function as a dominant negative in the heart when expressed from the endogenous locus. A well-described resistance to TH (Delta337T) was either introduced into germline of mice (KI-mut) or expressed as a transgene in the heart using a cardiac-specific promoter (KS-mut). Mice were studied at baseline, after 5-propyl-2-thiouracil (PTU) or after PTU and T(3) treatment (PTU + T(3)). PTU + T(3) treatment significantly increased left ventricular mass in all groups compared with baseline measurements, although the increase in left ventricular mass was significantly less in KI-mut animals. Baseline heart rates (HRs) were similar in wild-type (WT) and KI-mut but were lower in KS-mut animals. After TH deprivation (PTU), HR decreased in WT and KI-mut animals; similarly, HR increased in WT and KI-mut after PTU + T(3). In contrast, HR in KS-mut animals did not change after either treatment. Except for cardiac hypertrophy, the presence of a germline TR-beta mutation had surprisingly little effect on cardiac function.     

Solubilized nuclear thyroid hormone receptors in circulating human mononuclear cells

In order to assess iodothyronine receptor interactions in man, we have developed a receptor assay for T3 and T4 in solubilized nuclear extracts from circulating mononuclear cells. This assay utilizes the technique of salt solubilization to isolate nuclear receptors and employs standard saturation analysis for T3 and T4 to determine maximal binding capacity (MBC) and equilibrium dissociation constants (Kd). We have determined that 11 normal subjects had a MBC for T3 of 1.20 +/- 0.20 pmol/mg DNA (+/- SE) and a Kd of 3.4 +/- 0.2 X 10(-10) M; the T4 MBC was 8.44 +/- 1.22 pmol/mg DNA and the Kd was 2.7 +/- 0.3 X 10(-10) M. Hypothyroid patients had a mean T3 MBC of 7.32 +/- 2.28 pmol/mg DNA and a mean T4 MBC of 40.04 +/- 21.36 pmol/mg DNA (P less than 0.05 compared to normal). Obese subjects (n = 12) had a basal fed MBC that was 0.66 +/- 0.13 pmol/mg DNA for T3 (P less than 0.05 compared to normal) and was 3.58 +/- 0.56 pmol/mg DNA for T4 (P less than 0.01 compared to normal). During fasting, the average T3 MBC increased to 1.43 +/- 0.31 pmol/mg DNA and the average T4 MBC increased to 9.63 +/- 2.46 pmol/mg DNA, values that are both significantly higher than those in the fed period; the dissociation constants were unaltered in obese subjects (compared to normals) in fed and fasting states. Gel filtration with 0.5 M agarose was employed to ascertain if the physicochemical properties of the solubilized mononuclear human cell receptor were similar to those previously observed in rat and human liver and kidney receptors. The elution profile obtained was similar to that reported earlier. The major binding activity has an estimated Stokes radius of 35 A and a molecular weight ratio of approximately 50,000 daltons. These studies indicate that: 1) high affinity T3 and T4 receptors exist in human mononuclear cells and have properties similar to those for T3 and T4 described previously in rat liver; 2) T3 and T4 receptor number tends to increase in hypothyroid subjects and tend to be lower in obese patients than in normal weight control subjects; 3) fasting is associated with an increase in both T3 and T4 MBC; and 4) despite their apparent physicochemical similarity, T3 receptors in rat liver and human mononuclear cells may be regulated differently, at least during fasting since hepatic T3 receptors decrease in the fasted rat. Collectively, these observations support the concept that human white cell T3 nuclear receptor binding is capable of rapid fluctuations, suggesting a mechanism for homeostatic regulation of T3 action.     

Purification and characterization of rat liver nuclear thyroid hormone receptors

Nuclear thyroid hormone receptor was purified to 904 pmol of L-3,5,3'-triiodothyronine (T3) binding capacity per mg of protein with 2.5-5.2% recovery by sequentially using hydroxylapatite column chromatography, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column chromatography, DEAE-Sephadex column chromatography, and heparin-Sepharose column chromatography. Assuming that one T3 molecule binds to the 49,000-Da unit of the receptor, we reproducibly obtained 6.4-14.7 micrograms of receptor protein with 4.2-4.9% purity from 4-5 kg of rat liver. Elution of receptor from the heparin-Sepharose column was performed using 10 mM pyridoxal 5'-phosphate, which was observed to diminish binding of receptor to heparin-Sepharose or DNA-cellulose. This effect was specific for pyridoxal 5'-phosphate, since related compounds were not effective. Purified receptor bound T3 with high affinity (6.0 X 10(9) liter/mol), and the order of affinity of iodothyronine analogues to purified receptor was identical to that observed with crude receptor preparations [3,5,3'-triiodothyroacetic acid greater than L-T3 greater than D-3,5,3'-triiodothyronine (D-T3) greater than L-thyroxine greater than D-thyroxine]. Purified receptor had a sedimentation coefficient of 3.4 S, Stokes radius of 34 A, and calculated molecular mass of 49,000. Among several bands identified by silver staining after electrophoresis in NaDodSO4/polyacrylamide gels, one 49,000-Da protein showed photoaffinity labeling with [125I]thyroxine that was displaceable with excess unlabeled T3. The tryptic fragment and endogenous proteinase-digested fragment of the affinity-labeled receptor showed saturable binding in 27,000-Da and 36,000-Da peptides, respectively. These molecular masses are in agreement with estimates from gel filtration and gradient sedimentation, indicating that affinity labeling occurred at the hormone binding domain of nuclear thyroid hormone receptor. This procedure reproducibly provides classical native rat liver T3 nuclear receptor in useful quantity and purity and of the highest specific activity so far reported.     

Existence of nuclear thyroid hormone receptors in the porcine thyroid gland and the rat thyroid cell line FRTL-5

We have investigated whether nuclear T3 receptors exist in the thyroid cell. Nuclear proteins extracted from porcine thyroid nuclei with 0.4 mol/l KCl were incubated with [125I]T3. The mixture was then analysed by sucrose density gradient ultracentrifugation which revealed that the T3-binding proteins migrated at the same position of 3.6 S as rat liver nuclear T3 receptors. Fractionation by high performance liquid chromatography using a size exclusion column and an ion exchanger column also demonstrated elution patterns of T3-binding similar to those of the rat liver receptor. Scatchard plots of crude nuclear extracts from porcine thyroid represented a curvilinear pattern. However, when the nuclear proteins partially purified by a DEAE column chromatography were analysed, a single binding component was found; the association constant was 4.1 x 10(10) l/mol and the maximal binding capacity was 602 fmolT3/mg protein. Displacement study with several T3 analogues showed a highly selective affinity for L-T3. Cultured rat thyroid cells of the FRTL-5 line also contained a single class of saturable, high affinity T3-binding site. Subconfluent cells in 100-mm dishes were incubated with increasing amounts of [125I]T3 at 37 degrees C for 3 h and radioactive T3 in isolated nuclei was counted. Scatchard analysis of data showed that the association constant and the maximal binding capacity were 3.44 +/- 0.63 x 10(10) l/mol and 63.7 +/- 17.8 fmolT3/mg protein, respectively. These results strongly suggest that there are nuclear T3 receptors, indistinguishable from the hepatic T3 receptors, in the porcine thyroid and rat FRTL-5 cells.     

Interaction of amiodarone and desethylamiodarone with solubilized nuclear thyroid hormone receptors

The mechanisms of action of the potent antiarrhythmic drug amiodarone are unknown. However, amiodarone and its abundant metabolite, desethylamiodarone, bear a striking structural resemblance to thyroid hormones. In addition, certain cardiac electrophysiologic effects of amiodarone treatment are similar to those of hypothyroidism. These facts suggest that amiodarone or desethylamiodarone could be acting, in part, by blocking thyroid hormone action. Because thyroid hormones are known to act through nuclear receptor proteins, the binding of amiodarone and desethylamiodarone was measured to nuclear extracts derived from human lymphocytes, bovine atrium and ventricle and rat liver. The capacity of increasing concentrations of amiodarone and desethylamiodarone nuclear extracts to block receptor binding of radiolabeled triiodothyronine (T3) in a standard in vitro competition assay was tested. Nuclear extracts demonstrated only minimal binding to amiodarone. However, all receptor preparations had substantial affinities (KD) for the desethyl analog: lymphocyte, 8.6 microM; atrium, 35.0 microM; ventricle, 26.9 microM and liver, 8.6 microM. Desethylamiodarone accumulates in very large quantities in parenchymatous organs during long-term amiodarone treatment. Taking its usual therapeutic serum level (about 4 microM or 2.7 micrograms/ml) as an estimate of intranuclear concentration, desethylamiodarone would partially saturate nuclear thyroid hormone receptors in several different tissues, including the heart. Thus, amiodarone treatment may exert some of its electrophysiologic effects by metabolic conversion to desethylamiodarone. This metabolite may then exclude thyroid hormone from nuclear receptor sites within the myocardium.     

Nuclear receptors for thyroid hormone: evidence for nonrandom distribution within chromatin.

Chromatin receptor proteins appear to mediate some actions of thyroid hormone. In this study, sheared mammalian chromatin containing [125I]triiodothyronine (T3) bound by these receptors was separated using sucrose gradient velocity sedimentation. T3-receptor complexes were distributed throughout the chromatin fractions, but were enriched in the slowly sedimenting fractions. The latter contain most of the template capacity for RNA synthesis and most of the endogenous RNA polymerase activity but a minor portion of the total DNA. Formaldehyde treatment of chromatin containing receptor-bound [125 I ]T3 resulted in fixation of radioactivity, as evidenced by its migration with chromatin after equilibrium density gradient sedimentation in both cesium chloride and Conray. This fixation implies that the T3 receptor protein is closely associated with chromatin. These results suggest that proteins involved in the regulation of gene function may be nonrandomly distributed within chromatin subfractions, and are consistent with a direct role for thyroid hormone in regulating genetic expression.     

Putative nuclear triiodothyronine receptors in tadpole erythrocytes: regulation of receptor number by thyroid hormone

Putative thyroid hormone (TH) receptors have been detected in the nuclei of red blood cells (RBCs) from Rana catesbeiana tadpoles, and their binding characteristics have been examined. Nuclear T3 saturation analyses were carried out in vitro in intact RBCs suspended in phosphate-buffered amphibian Ringer. After incubation with T3, intact nuclei were obtained by centrifugation after lysing the cells in a sucrose-Tris-HCl buffer containing 0.2% saponin and then adding Triton X-100 (final concentration 0.125%) to the lysed mixture to reduce nonspecific binding to less than 10% of total binding. Scatchard analysis of equilibrium binding data revealed that RBCs from premetamorphic (first year) tadpoles contained 502 +/- 39 (SE) T3 binding sites per nucleus; in prometamorphic (second year) tadpoles the number had increased to 844 +/- 39. Development was also accompanied by some increase in the affinity of these sites; dissociation constant (Kd) = 1.8 +/- 0.39 X 10(-11) M and 0.95 +/- 0.108 X 10(-11) M in pre- and prometamorphic tadpoles, respectively. The number of sites per nucleus in both pre- and prometamorphic tadpole RBCs was greatly increased by pretreatment in vivo with either T3 or T4 for 6-10 days; a comparable number of sites per nucleus (2225 +/- 65) was observed after maximal stimulation by either hormone at both stages of development. Significant increases in receptor number were observed 10 days after injection of 0.03 nmol T3 or 0.06 nmol T4 into tadpoles weighing 13-15 g; maximal effects were obtained with 0.1 nmol T3 or 1.0 nmol T4. On the basis of these observations and the evidence that more TH is present in pro- than in premetamorphic tadpoles, it is suggested that the spontaneous increase in receptor number in RBC nuclei associated with progression from the pre- to prometamorphic phase is due, at least in part, to increased levels of endogenous TH.     

Functional activation of cerebral metabolism in mice with mutated thyroid hormone nuclear receptors

Neonatal hypothyroidism impairs structural maturation in the brain and results in diminished electrical activities and energy metabolism. We recently found that glucose utilization (CMR(glc)) is markedly depressed throughout the brain in mice with targeted mutations in thyroid hormone receptor alpha1 (TR alpha 1), but not TR beta. Previous studies had shown that CMR(glc) increases linearly with spike frequency in the afferent pathways to synapse-rich regions in neuropil, but not in neuronal cell bodies. To determine whether the decreased CMR(glc) in mutant TR alpha 1(PV/+) mice reflected lesser synaptic density or reduced functional activity in existing synapses, we stimulated vibrissae unilaterally and measured CMR(glc) bilaterally in four stations of the whisker-to-barrel cortex pathway. Baseline CMR(glc) (unstimulated side) was markedly lower in all four stations in the TR alpha 1(PV/+) mutants than in wild-type controls, even though Northern blot and immunohistochemical examinations showed normal Na(+),K(+)-adenosine triphosphatase expression and neuronal differentiation. Despite the lower baseline CMR(glc), however, vibrissal stimulation evoked percent increases in CMR(glc) in the TR alpha 1(PV/+) mutants that were as great as those in wild-type mice. These results indicate that in the TR alpha 1(PV/+) mutants there it is a reduction in synaptic density that is responsible for the decrease in CMR(glc), but functionality of existing synapses is retained.     

Lymphocyte thyroid hormone receptors in obesity

Triiodothyronine (T3)-receptor characteristics of isolated circulating human mononuclear cells have been studied in a group of obese patients who claimed to be unable to lose weight on conventional 4.2 MJ (1000 kcal) diets. The cells of the obese patients exhibited a lower receptor capacity than those of a control group of non-obese subjects but the difference was not significant. There was a significant fall (P less than 0.01) in receptor capacity in the obese patients after 12 weeks in a 1.34 MJ (320 kcal) per d formula diet and this provides a further mechanism whereby a fall in metabolic rate takes place in response to severe dietary restriction. Some patients who also received T3 (60 micrograms/d) in addition to the formula diet showed a further fall in the receptor numbers. These findings may partly account for the previously reported resistance to thyroid hormones in obesity.     

Possible role of histones in the organization of rat liver thyroid hormone receptors in chromatin

The effects of histone subfractions on rat liver thyroid hormone receptor-DNA interaction were examined using an in-vitro DNA-cellulose binding assay. H1 histones bound to DNA showed reversible and potent inhibition of receptor-DNA binding without affecting receptor hormone binding. Poly-lysine, bovine serum albumin, ovalbumin and cytochrome c did not alter receptor-DNA binding. H1 histone subfractions (calf thymus lysine-rich histone (CTL)-1, CTL-2 and CTL-3) showed potent inhibition of receptor-DNA binding indistinguishable from each other. The quantity of H1 histone subfractions bound to DNA was the same. Although each subfraction has different functional properties, inhibition of receptor-DNA binding was a common feature of all the H1 histone subfractions, which is important for the non-random distribution of the receptor in chromatin. Binding of the receptor to core histones was investigated; it was found to bind to core histones more potently than to other proteins (H1 histone, ovalbumin and cytochrome c). Among core histone subfractions, H4 histone bound to the receptor most potently and is the candidate to be one of the acceptor sites of the receptor in chromatin.