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Increased Expression of Thyroid Hormone Receptor Isoforms in End-Stage Human Congestive Heart Failure¹

Dipartimento di Medicina Sperimentale e Patologia (G.d.’A., C.R.T.d.G., D.M., D.P., L.P.-P., P.G., F.S.), and Istituto di Chirurgia del Cuore e Grossi Vasi (F.M.), Università degli studi di Roma "La Sapienza," Rome 00161, Italy

Address all correspondence and requests for reprints to: Francesco Saverio Celi, M.D., I Cattedra di Endocrinologia, Dipartimento di Medicina Sperimentale e Patologia, Policlinico Umberto I, Viale Regina Elena, 324, 00161 Roma, Italy. E-mail: francescosaverio.celi{at}uniroma1.it

	Abstract

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Thyroid hormone plays an important role on myocardial development and function. The local effects of thyroid hormone are mediated by the receptor isoforms ultimately driving the expression of cardiac-specific genes. Although overt and subclinical thyroid dysfunction causes well-known changes in the cardiovascular system, little is known about local thyroid hormone action in normal and failing human myocardium. With a newly developed multiplex competitive RT-PCR method, we evaluated the expression of thyroid hormone receptor (TR) isoforms -1, -2, and ß-1 in normal human hearts and in end-stage congestive heart failure. A statistically significant difference in the expression of all three TR isoforms was observed among samples from normal subjects, ischemic heart disease (IHD), and dilated cardiomyopathy (DCM). In DCM, compared with normal, the studied TR isoforms were significantly increased. In IHD, the increased expression was found significant only for -1 and -2 isoforms. No differences were observed between the pathologic groups. In conclusion, a coordinated increment in the expression of the TR isoforms was observed in both DCM and IHD by multiplex competitive RT-PCR. The observed changes could represent a compensatory mechanism to myocardial failure or to locally altered thyroid hormone action.

	Introduction

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THYROID HORMONE ACTION is regulated by a redundant multistep system (1) ultimately mediated by the interaction of hormone bound to its nuclear receptor (TR) and thyroid hormone–responsive elements, thus inducing or inhibiting the target genes’ transcription. At present four TR isoforms, derived from the alternative splicing of two genes (2), have been characterized. Among them, the ß-2 isoform is preferentially localized in the central nervous system with little expression in the other tissues (3). TRs belong to the superfamily of nuclear receptors characterized by the presence of a DNA-binding domain, a hinge section; and, with the exception of the -2 isoform, a hormone-binding domain. Experimental evidence demonstrates that the latter inhibits the hormonal action (4).

It is well known that thyroid hormone plays an important role in myocardium development and function through the regulation of cardiac-specific genes such as and ß myosin heavy chain isoforms, sarcoplasmic reticulum Ca²⁺ ATPase, voltage-gated potassium channels, and others (5, 6, 7, 8). Although overt and subclinical thyroid dysfunction causes distinct changes in the cardiovascular system (9, 10), little is known about local thyroid hormone action in cardiac disease. Subjects with advanced congestive heart failure (CHF) often show abnormal local thyroid hormone metabolism (11). Some studies suggest that thyroid hormone supplements are useful adjuncts both in the treatment of CHF (12, 13) and in the postoperative period after complex cardiac surgical procedures (14). These findings have been obtained mostly from experimental models of dysfunction, and little information is available from humans. At the present, data concerning TR regulation in failing human myocardium are scarce.

The aim of this study was to evaluate the expression of TR isoforms in normal human hearts and in CHF of different etiologies by multiplex competitive RT-PCR.

	Materials and Methods

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Study population

Human heart specimens from subjects with end-stage CHF were obtained from 14 patients who underwent orthotopic heart transplant for either ischemic heart disease (IHD, n = 8) or dilated cardiomyopathy (DCM, n = 6) at our institution. Sixteen left ventricular biopsies from unaffected myocardium obtained during elective coronary bypass surgery for single-vessel disease were used as controls. The local ethical committee approved the protocol, and all patients gave informed consent to the procedure. Exclusion criteria for both study groups and controls were abnormal thyroid hormone values; therapy with amiodarone, propanolol, thyroid hormone, and derivatives; or use of dopamine during a period of 4 weeks preceding heart surgery. For the control group, additional exclusion criteria were New York Heart Association class > II and ejection fraction < 50%. Relevant clinical data are reported in Table 1.

Tissues from both diseased (n = 14) and control (n = 5) hearts were snap frozen in liquid nitrogen and stored at -80 C. Myocardial samples from diseased hearts were obtained from areas of the left ventricle devoid of scarring. Samples were homogenized in Trizol (Life Technologies, Milano, Italy), and RNA extraction was performed according to the manufacturer’s instructions. Total RNA was resuspended in DEPC-treated water. Spectrophotometry was performed in duplicate with two different sample dilutions within the linear reading range of the apparatus (Ultrospec 2000, Amersham Pharmacia Biotech, Milano, Italy).

Multiplex competitive RT-PCR

We recently described a rapid PCR-based method to synthesize polyA-tailed RNA internal standards for multiplex competitive PCR (15). Briefly, three polyA-tailed RNA internal standards, sharing the same primers and most of the sequence for 1, 2, and ß1 human TR cDNAs, were generated by in vitro transcription (Promega Corp., Milano, Italy) (Table 2). The standards were measured by spectrophotometry, serially diluted with DEPC-treated H₂O to 10⁷–10³ molecules/µL and stored at -80 C. Aliquots of 100 ng of human myocardial total RNA obtained from individual control or study subjects were reverse transcribed in 50 mM Tris HCl (pH 8.3 at 25 C), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 200 µM each dNTP, 10 µM of oligo-dT primer, 20 U RNAsin (Promega Corp.), and 200u M-MLV RT (Promega Corp.) in a final volume of 25 µL in the presence of 10⁷–10³ molecules of internal standards for 120 min at 37 C. The reaction was terminated by heating at 70 C for 15 min. Five microliters of the reaction product were then amplified using the "hot start" technique in a final volume of 50 µL in 1x PCR buffer, 2.5 mM MgCl₂, 200 µM each dNTP, and 10 µM of each primer. After an initial denaturation of 2 min at 94 C, 5 µL of enzyme solution containing 1U of Taq polymerase (Promega Corp.) in 1x PCR buffer, was added to the reaction and 35 cycles of 54 C 1 min, 72 C 1 min, 94 C 1 min were performed followed by a final step of 72 C for 10 min. The samples were run on a 3% agarose gel, and the amount of RNA was estimated as the equivalence between each target and internal standard band intensity. Each experiment was performed in triplicate; data are expressed as number of molecules/100 ng total RNA ± SE.

Immunohistochemistry was performed on myocardial samples from all explanted hearts. Tissue was obtained from the left ventricle adjacent to the areas of sampling for RT-PCR within 30 min of explant, snap frozen in liquid nitrogen and stored at -80 C. Four-micrometer-thick cryostat sections were treated with 3% hydrogen peroxide for 20 min and then incubated 1 h at room temperature with a polyclonal antibody raised against an amino acid sequence from the D domain common to TRs -1 and -2 (16) (dilution 1:200). The antibody was a generous gift from Dr. L. J. DeGroot (University of Chicago, Chicago, IL). The avidin-biotin peroxydase complex was used to label the primary antibody. The reaction product was detected using 3,3-diaminobenzidine. Control experiments were accomplished by omitting the primary antibody.

Proteins for Western blotting were extracted from frozen explanted myocardium and pooled (n = 11) control specimens. Samples were snap frozen in liquid nitrogen and stored at -80 C. Each sample was homogenized on ice with 1 mL of lysis buffer containing 2% SDS, 10% glycerol, 5% mercaptoethanol, and 6.25 mmol/L Tris HCl buffer, pH 6.8. Protein concentrations were quantified using a protein assay kit (Bio-Rad Laboratories, Inc. Segrate, Italy) with bovine serum protein as reference protein. For each sample, 30 µg of protein were separated by SDS-PAGE (10%) and electroblotted onto polyvinylidene fluoride sheets. Blots were blocked with 5% nonfat dry milk (Carnation, Glendale, CA) in 1x TBST (Tris 100 mM, NaCl 150 mM, Tween 20 0.05%, pH 7.4) and incubated with two monoclonal antibodies (dilution 1:200) recognizing: 1) an epitope common to TRß-1 and TR-1 in the hormone-binding domain (Affinity BioReagents, Inc., Golden, CO); 2) an epitope specific for the ß-1 isoform (Affinity BioReagents, Inc.). A monoclonal antibody against a-sarcomeric actin (Sigma, Milano, Italy) was used to confirm equal protein loading. The blots were developed by chemiluminescence (ECL, Amersham Pharmacia Biotech).

Statistical analysis

Overall differences in TR isoform expression among groups were determined by the Kruskal-Wallis test (17, 18), which is equivalent to an ANOVA of the ranks. When the test indicated a significant difference, individual study groups were compared with one another and the controls using the Mann-Whitney rank sum test. For this test, a value of P < 0.06 was considered statistically significant.

	Results

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Evaluation of thyroid hormone receptor isoform expression

A statistically significant difference was observed in the expression of -1 TR among normal subjects, IHD, and DCM (6,400 ± 2,205 vs. 65,625 ± 16,780 vs. 158,333 ± 68,882, P = 0.008) using the Kruskal-Wallis test. When comparing with the Mann-Whitney rank sum test normal vs. IHD and vs. DCM, a statistically significant increase of -1 TR expression was observed in the pathological samples (P < 0.06); no significant difference was observed between IHD and DCM. Similarly, the expression of -2 TR was significantly different among the three groups (2,700 ± 1,820 vs. 30,000 ± 7,559 vs. 30,000 ± 8,944, P = 0.008). A statistically significant increase of -2 TR expression was observed when comparing normal vs. IHD (P < 0.06) and normal vs. DCM (P < 0.06). No significant difference was observed between IHD and DCM. A significant overall difference in ß-1 expression was also observed comparing normal, IHD, and DCM (2,800 ± 1,800 vs. 33,937 ± 12,310 vs. 53,330 ± 16,470, P = 0.008). However, a significant pairwise increase was observed only when comparing normal vs. DCM (P < 0.06) (Fig. 1).

View larger version (43K): [in this window] [in a new window]   Figure 1. A, Multiplex competitive RT-PCR. Aliquots of 100 ng of total human myocardial RNA were reverse transcribed in the presence of 107–103 molecules of polyA-tailed RNA internal standards for human thyroid hormone receptor -1, -2, and ß-1 isoforms. In this myocardial sample, we found that the amount of -1, -2, and ß-1 was 105, 104, and 104 molecules/100 ng total RNA, respectively. B, Comparison among study groups of TR isoforms expression. Data are expressed as molecules/100 ng total RNA ± SE.

Immunoblotting experiments performed with antibodies to both TRß-1 and TR-1 and to a specific epitope for the ß-1 isoform displayed a single band of 48 kDa, corresponding to the molecular weight of TR -1. The abundance of the immunoreactive band was higher in diseased myocardium from IHD and DCM, compared with pooled normal samples (Fig. 2, A and B).

View larger version (85K): [in this window] [in a new window]   Figure 2. A, Western blot analysis of TR isoforms. The monoclonal antibody against an epitope common to TRs ß-1 and -1 recognizes a specific band at 48 Kd, corresponding to the molecular weight of -1 TR. B, Immunoblotting with antiactin antibody is used as reference. C, Immunohistochemistry with a polyclonal antibody against an amino acid sequence common to TRs -1 and -2. All myocyte nuclei show a strong immunostaining. Only a few interstitial fibroblasts are stained (original magnification 25x).

	Discussion

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Our results demonstrate a coordinated increase in the expression of TR isoforms in human failing myocardium, irrespective of the etiology. The immunoblotting experiments suggest that these changes correlate with TRs translation.

The data currently available on TRs expression in CHF are scarce and conflicting. A report from Sylvén et al. (19) described a decrease in -1 transcript and no difference in -2 and ß-1 isoforms in failing human myocardium. On the other hand, recent work performed on myocardium from dogs with overt CHF (20) showed an increase in ß-1 and ß-2 TR isoforms, while no difference was observed in -2 expression, and -1 was not evaluated. A possible explanation for these discrepancies could result from differences in the species involved and the experimental methods (i.e. semiquantitative PCR and Rnase protection assay vs. a multiplex competitive RT-PCR). Moreover, no information was available on thyroid status or patient use of medication that might affect thyroid homeostasis. It is also worth noticing that in our study the use of multiplex competitive RT-PCR, by comparing RNA molecules with internal standards, has allowed an accurate quantitation of the transcript independently from the effects of possible changes in the local thyroid hormone homeostasis that could alter the transcription rate of commonly used reference genes (21, 22).

In our study, the greatest increase in expression was observed in mRNA for the -1 receptor. Smaller but significant increments were also observed in ß-1 and -2 isoforms. It is worth noting that the -1 receptor appears to play a major role in heart rate regulation and ventricular repolarization (23). The increase observed in -2 mRNA is somewhat surprising because this receptor has been reported to exert a dominant inhibitory action on transcription. On the other hand, the increase in this TR isoform is proportionally smaller relative to the other hormone binding isoforms. Because -2 derives from an alternative splicing of the gene, one could speculate that, in this case, its expression is nonspecifically driven at a lower rate by the -1 transcription.

The pattern of gene expression in failing myocardium (24) is strikingly similar to the one observed in experimental hypothyroidism (25). The increase in TR isoforms observed in our study could thus be interpreted as a compensatory mechanism to a cardiac hypothyroidism (5, 6, 7, 8, 9) generated by altered local thyroid hormone metabolism. Such a mechanism, if confirmed, could represent a molecular basis for the treatment of CHF with thyroid hormone supplements. On the other hand, one could hypothesize that the increase in the TRs could result in a higher degree of unbound receptor, with negative transcription activity, thus protecting the failing myocardium from thyroid hormone action.

In conclusion, our results indicate that TR up-regulation in CHF is a reactive, secondary phenomenon in an attempt of the myocardium to cope with the increased hemodynamic stress. Further studies, both in experimental models and in humans, will be needed to evaluate the role of thyroid hormone supplement in the management of CHF.

	Acknowledgments

 
We thank Dr. Leslie J. DeGroot and Dr. Emmanuele A. Jannini for critical review of the manuscript and Ms. Loreta Petricca for excellent technical assistance.

	Footnotes

 
¹ This work was supported by Telethon Italy Grant E.763, Ministero Università e Ricerca Scientifica e Tecnologica (MURST 1999).

Received September 22, 2000.

Revised December 28, 2000.

Accepted January 30, 2001.

	References

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