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Am J Physiol Cell Physiol 294: C1227-C1233, 2008. First published March 5, 2008; doi:10.1152/ajpcell.00328.2007
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CELLULAR AND MITOCHONDRIAL METABOLISM

H9c2 cardiomyoblasts produce thyroid hormone

Christof Meischl,1,10 Henk P. Buermans,2,10 Thierry Hazes,1 Marian J. Zuidwijk,2 René J. P. Musters,2,10 Christa Boer,2 Arthur van Lingen,3 Warner S. Simonides,2,10 Marinus A. Blankenstein,4 Corrine Dupuy,5 Walter J. Paulus,2,10 C. Erik Hack,6 Carrie Ris-Stalpers,7 Dirk Roos,8 and Hans W. M. Niessen1,8,9

1Department of Pathology, 2Department of Physiology, 3Department of Nuclear Medicine, and 4Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands; 5Unité 486 Institut National de la Santé et de la Recherche Médicale, Université Paris 11, Châtenay-Malabry Cedex, France; 6Department of Immunopathology, Sanquin Research, Amsterdam, The Netherlands; 7Laboratory of Pediatric Endocrinology, Academic Medical Center, Amsterdam, The Netherlands; 8Department of Blood-Cell Research, Sanquin Research, Amsterdam, The Netherlands; 9Department of Cardiac Surgery, VU University Medical Center, Amsterdam, The Netherlands; and 10IcaR-VU, VU University Medical Center, Amsterdam, The Netherlands

Submitted 27 July 2007 ; accepted in final form 3 March 2008


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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Thyroid hormone acts on a wide range of tissues. In the cardiovascular system, thyroid hormone is an important regulator of cardiac function and cardiovascular hemodynamics. Although some early reports in the literature suggested an unknown extrathyroidal source of thyroid hormone, it is currently thought to be produced exclusively in the thyroid gland, a highly specialized organ with the sole function of generating, storing, and secreting thyroid hormone. Whereas most of the proteins necessary for thyroid hormone synthesis are thought to be expressed exclusively in the thyroid gland, we now have found evidence that all of these proteins, i.e., thyroglobulin, DUOX1, DUOX2, the sodium-iodide symporter, pendrin, thyroid peroxidase, and thyroid-stimulating hormone receptor, are also expressed in cardiomyocytes. Furthermore, we found thyroglobulin to be transiently upregulated in an in vitro model of ischemia. When performing these experiments in the presence of 125I, we found that 125I was integrated into thyroglobulin and that under ischemia-like conditions the radioactive signal in thyroglobulin was reduced. Concomitantly we observed an increase of intracellularly produced, 125I-labeled thyroid hormone. In conclusion, our findings demonstrate for the first time that cardiomyocytes produce thyroid hormone in a manner adapted to the cell's environment.

DUOX; cardiomyocyte; ischemia; heart failure

THYROID HORMONE influences the basal metabolic rate of a wide range of tissues and is in the cardiovascular system an important regulator of cardiac function and cardiovascular hemodynamics (7). Thyroxine (T4) is the main product of thyroid secretion and is deiodinated in peripheral tissues to triiodothyronine (T3), the biologically active thyroid hormone (3). T3 either regulates gene expression by binding to high-affinity nuclear receptors that, in turn, recognize specific response-element sequences in the promoters of T3-target genes (11) or exerts different nonnuclear effects by as yet less well characterized mechanisms.

In the heart T3 acts directly on the cardiomyocytes where it stimulates heart rate and contractile performance, the later mediated, to a large extent, by increased expression of the calcium adenosine triphosphatase of the sarcoplasmic reticulum and a decreased expression of phospholamban.

Pathophysiologically, T3 has been shown to be important for the induction of different forms of cardiac hypertrophy (19) and for the response of the heart to ischemic stress (17). The exact mechanisms are still under investigation.

Traditionally, the thyroid gland has been considered the sole source of thyroid hormone. However, several studies by the group of Taurog (9, 22) and others (16) suggested the possibility of a low-level generation of thyroid hormone in organs/tissues other than the thyroid gland. Taurog et al. (9) demonstrated in thyroidectomized rats that in all indexes of T4 action examined, namely growth, metabolic rate, heart rate, and pituitary, adrenal and reproductive function, the restorative or maintenance activities of large and repeatedly administrated quantities of iodide were identical with those of minute quantities of T4. In a later study of comparable experimental design they showed by scans of paper chromatograms the presence of T4 in the plasma of the thyroidectomized rats (22). Obregon et al. (16) later showed by specific RIAs that in rats that had received a single dose of 131I after thyroidectomy, T4 and T3 could be detected in the liver, kidney, brain, and heart long after these hormones had disappeared from the circulation. However, to our knowledge these studies were never followed up to elucidate the extrathyroidal source of these hormones. Now, based on these studies, the apparent need of cardiomyocytes for thyroid hormone under (patho)physiological conditions and on the increasingly recognized endocrine function of the heart (14), we speculated that cardiomyocytes under certain conditions might be able to generate at least low levels of thyroid hormone (s).


   METHODS
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 METHODS
RESULTS
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Rats. We purchased male Wistar rats from the Harlan Laboratories. Rat care and experimental procedures were performed according to national guidelines and with the approval of the VU University Medical Center, Amsterdam, and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).

H9c2 cells and metabolic inhibition. We purchased H9c2 cardiomyoblasts from American Type Culture Collection and cultured them in Dulbecco's modified Eagle's medium (DMEM; Cambrex) containing 10% (vol/vol) heat-inactivated fetal calf serum (Cambrex), 100 IU penicillin per milliliter (Yamanouchi), 100 µg streptomycin per milliliter (Radiopharma-Fisiopharma), and 2 mmol/l L-glutamine (Invitrogen), under 5% CO2 at 37°C. As an experimental in vitro model for ischemia we used metabolic inhibition as described elsewhere (15). Briefly, we exposed the cells to metabolic inhibition buffer (in mmol/l: 5 NaCN, 20 2-deoxyglucose, 0.9 CaCl2, 106 NaCl, 38 NaHCO3, 4.4 KCl, and 1 MgCl2; pH 6.6) for the indicated periods under 5% CO2 at 37°C. For reperfusion we cultured the cells for 2 h in DMEM following metabolic inhibition.

Isolation of mRNA and generation of cDNA. We isolated total RNA from H9c2 cells with the RNeasy Fibrous Tissue Kit (QIAGEN) and generated cDNA using Superscript III reverse transcriptase (Invitrogen) with Oligo (dT)15 primers (Promega).

PCR and cycle sequencing. We amplified fragments of DUOX1, DUOX2, and thyroglobulin cDNA derived from rat heart tissue by PCR on the Rapid Cycler (Idaho Technology) with 50 cycles at 95°C for 10 s, denaturing temperature for 30 s, and 72°C for 15 s and slope S9. The denaturing temperatures for DUOX1, DUOX2, and thyroglobulin were 62°C, 66°C, and 56°C, respectively. The primer sequences were 5'-TCAAGGGGAGTGGATTTGGCTTCGG-3' and 5'-CATCCACGACTCGGATCTGTCCAGG-3' for DUOX1, 5'-CCTGTTACTGTGATTGACTACTTTGAGG-3' and 5'-CTGTCTGGAAGCAGCTGGACAGTG-3' for DUOX2, and 5'-GCAGAACAACCACCATCACTGGAGC-3' and 5'-TGGCACTGGGGACTCTGGACTTGAC-3' for thyroglobulin. The amplification of DUOX 2 necessitated a second, seminested PCR step with primers 5'-CCTGTTACTGTGATTGACTACTTTGAGG-3' and GAGCCACCACCCAAGCAATAATCAGAC. All primer combinations were intron spanning to preclude contamination by genomic DNA. The reaction volume of 15 µl contained 2 units of Taq DNA polymerase (Promega), 2 units of TaqStart antibody (Clontech Laboratories), 50 ng of each primer, 3 nmol of each of the dNTPs (Promega) and reaction buffer (50 mmol/l KCl, 1.5 mmol/l MgCl2, 0.1% Triton X-100, 10 mmol/l Tris, and 8% DMSO, pH 9 at 25°C). The PCR reaction took place in 10-µl glass capillaries (Idaho Technology). We cycle sequenced the purified templates with the Big Dye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer) and ran them on an ABI 377 XL Automated DNA Sequencer (PerkinElmer). We performed sequence analysis with the Sequence Analysis, Sequence Navigator, and Auto Assembler software (all from PerkinElmer).

Western blot analysis. We harvested H9c2 cells or total rat heart tissue into modified Laemmli sample buffer and, after SDS-PAGE and blotting, we analyzed them for thyroglobulin or DUOX expression with monoclonal antibodies 8–575/14 (human and rat thyroglobulin; from C. Ris-Stalpers), an antibody directed against the EF hand-containing domain of DUOX (from C. Dupuy) or an antibody raised against the first intracellular part of human DUOX1 (6) (from F. Miot). We visualized the blots by enhanced chemiluminescence (Amersham) and the LAS-3000 imaging system (Fuji Photo Film,) and analyzed them with Advanced Image Data Analyzer software, version 3.28.001 (Raytest). FRTL-5-cell lysates were from the laboratory of C. Ris-Stalpers.

Immunohistochemistry. We fixed frozen sections (4 µm) in cold acetone, blocked the slides at room temperature with normal swine serum (Dakopatts), and incubated the slides with a DUOX-specific antibody (diluted 1:100) for 60 min. After incubation of the slides first with an horseradish peroxidase-conjugated secondary antibody and then with 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich), we counterstained them with hematoxylin.

Immunofluorescence microscopy. Two days before metabolic inhibition, we passaged H9c2 cells onto sterile Lab-Tek II chamber CC2 glass slides (Nalge Nunc International) to reach about 80–90% confluency at the day of the experiment.

After incubation with metabolic-inhibition buffer or culture medium, paraformaldehyde fixation, and permeabilization, we incubated the samples with the primary antibody 8-575/14 (diluted of 1:100). Subsequently, we stained the samples with FITC-conjugated secondary antibody (Dakopatts) and with 4',6 diamidino-2-phenylindole-containing mounting medium (H1200, Vectashield; Vector Laboratories).

We qualitatively analyzed sections with a 3I Marianas digital-imaging microscopy workstation (Zeiss Axiovert 200M inverted microscope; Carl Zeiss). We recorded and stored exposures, objective, montage, and pixel binning automatically with each image.

Monocrotaline-induced right ventricular hypertrophy and congestive heart failure. All animals received a single subcutaneous injection with either saline (control group), 30 mg monocrotaline (Sigma-Aldrich)/kg body wt (hypertrophy group), or 80 mg monocrotaline/kg body wt (congestive heart failure group).

Gene-expression analysis of proteins necessary for thyroid hormone synthesis. We used spotted oligonucleotide microarrays to analyze the cardiac mRNA expression of components necessary for thyroid hormone generation in normal, hypertrophic, and failing ventricular tissue (4). We induced right ventricular hypertrophy and congestive heart failure by administration of monocrotaline as described above. We assigned a total of 27 animals randomly to three groups and at days 10, 19, and 25 after treatment, and we randomly picked 2 animals from each group. We rinsed the hearts by perfusion and separated the left ventricle, right ventricle, and interventricular septum. We weighed all tissues and snap froze the samples in liquid nitrogen. We isolated total RNA from the right ventricle with TRIzol (Invitrogen) and quantified the RNA concentrations by A260 measurement. We constructed a common reference RNA pool by pooling RNA from interventricular septum samples of animals euthanized at days 19 and 25. We generated cDNA strands from 30 µg total RNA. All microarray procedures were performed as previously described (2, 4). We hybridized pairs of labeled reference pool (Cy3) and ventricular sample (Cy5) per microarray slide. For all 4,803 features on the array, we corrected mean Cy3 and Cy5 intensities for background and averaged the duplicate signals for each feature on the array and the GAPDH replicates (52x). Spot signals had to be at least three times higher than the local background to allow further processing. In addition, we excluded genes with an average spot intensity below 256, taken over all 54 channels of the 27 arrays, from further processing. Furthermore, we did not allow more than 3 of the 54 channel intensities per gene with a signal intensity below 100. Only genes without flags in any of the 27 arrays were selected for further analysis. For the remaining 3,148 genes, we log2 transformed, mean centered, and standardized Cy3 and Cy5 mean channel intensities. We also calculated, mean centered, and standardized ratios (Cy5 to Cy3). For the purpose of the present study, we selected and analyzed thyroid-related genes with two-factor ANOVA followed by Bonferroni's multiple-comparison test (P < 0.05). The selected genes were thyroglobulin (GenBank: AB035201), sodium-iodide symporter (GenBank: U60282), pendrin (GenBank: NM_019214), thyroid peroxidase (GenBank: L20319), and thyroid-stimulating hormone receptor (GenBank: NM_012888).

Analysis of incorporation of 125I into proteins and modified RIA. We cultured H9c2 cells for 88 h in the presence of 50 µCi 125I/ml and subsequently exposed the cells to metabolic inhibition in the presence of the same activity of 125I per milliliter. Thereafter, we lysed the cells and determined the protein concentrations of the samples with the Bio-Rad Protein Assay. For analysis of which proteins contained 125I, we performed SDS-PAGE with 70 µg protein/sample. We assessed organification of proteins by overnight autoradiography with a Storage Phosphor Screen and subsequent analysis on a STORM 820 Gel and Blot Imaging System (both Molecular Dynamics). Subsequently, we assessed the identity of the organified protein band by Western blot analysis with antibody 8-575/14 as described above.

To analyze the putative generation of T4 and T3, we added 120 µg protein/sample into each of two Coat-A-Count tubes (Diagnostic Products) specific for free T4 and free T3. After overnight incubation at 4°C, we washed the tubes two times with H2O and measured the bound activities with a 1282 Compugamma counter (Wallac). Empty Coat-A-Count tubes were measured as background control samples. To evaluate whether the observed differences were significant, we performed a one-way ANOVA analysis followed by Bonferroni's multiple-comparison test. We considered a P value (two-sided) of <0.05 to represent a significant difference.

Image editing. In Figs. 1A and 2, A and B, we cropped intervening lanes with the Adobe Photoshop 5.5 software (Adobe Systems) to remove samples unrelated to this paper. We did not perform any further editing.


Figure 1
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  		Fig. 1. Expression of thyroglobulin (TG). A: mRNA isolated from total rat heart tissue as template to amplify a fragment corresponding to TG (expected fragment length: 302 bp) by RT-PCR. Subsequently, we confirmed the sequence identity of the PCR fragment by cycle sequencing. M, 100-bp DNA marker; W, water control; S, cDNA sample. B: expression of TG in H9c2 and FRTL-5 cells (a rat thyrocyte cell line). Note, we applied an arbitrary amount of FRTL-5-cell lysate as positive control; the blot shown is representative of three identical experiments. C: negative control (right) and staining of H9c2 cells for TG (left) with antibody 8-575/14 specific for rat TG (white), demonstrating the perinuclear localization of TG (nucleus: grey) (magnification, x640); the picture shown is representative of three identical experiments. D: expression of TG after the indicated periods of metabolic inhibition, followed or not by 2 h of reperfusion. We loaded 70 µg of protein per lane; the blot shown is representative of three identical experiments.

 

Figure 2
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  		Fig. 2. Microarray analysis of cardiac mRNA expression of functional components necessary for thyroid hormone synthesis in the thyroid gland. We analyzed by microarray the right ventricular mRNA expression levels of TG (A), the sodium-iodide symporter (NIS) (B), pendrin (C), thyroid peroxidase (TPO) (D), and thyroid-stimulating hormone receptor (Tshr) (E) 25 days after subcutaneous injection of saline (CON), 30 mg monocrotaline/kg body wt (hypertrophy group, HYP), or of 80 mg monocrotaline/kg body wt (congestive heart failure group, CHF), respectively. The graphs indicate the expression levels relative to that found in the control animals. *Significant difference in mRNA expression between the control and the chronic heart failure sample (P < 0.05).

 

   RESULTS
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 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Expression of thyroglobulin in H9c2 cells. To test our hypothesis that cardiomyocytes can generate thyroid hormone in response to ischemia, we first analyzed the expression of thyroglobulin, the matrix protein essential for thyroid hormone synthesis, in H9c2 cells, a rat cardiomyoblast cell line. Control H9c2 cells expressed thyroglobulin at the mRNA and protein level (Fig. 1, A and B), and digital imaging demonstrated an intense perinuclear localization (Fig. 1C) that remained unchanged under metabolic inhibition (data not shown). Western blot analysis showed that the expression of thyroglobulin was transiently increased under metabolic inhibition, with maximal expression after 30 min of metabolic inhibition and a subsequent decline of expression in time (Fig. 1D). The transient upregulation of thyroglobulin was accompanied by a downward shift in mass of the thyroglobulin-specific band with a return to its original mass upon subsequent reperfusion (Fig. 1D).

Increased expression of thyroglobulin in a rat model of induced right ventricular hypertrophy. In an effort to assess whether the upregulation of thyroglobulin was limited to ischemia, we analyzed cardiomyocyte hypertrophy, another context where an important role for thyroid hormone has been described (19). In a rat model of induced hypertrophy, namely monocrotaline-induced right ventricular hypertrophy, we also found by Western blot analysis an increased expression of thyroglobulin in hypertrophic hearts compared with control hearts (data not shown). These findings correlated well with microarray analyses that showed expression of thyroglobulin in the rat heart that was increased at day 25 of right ventricular hypertrophy induced by low doses of monocrotaline and significantly so at day 25 of right ventricular congestive heart failure induced by high doses of monocrotaline (P < 0.05; Fig. 2A).

Expression of DUOX1 and DUOX2 in H9c2 cells. We recently found NOX2 expression in cardiomyocytes (12). As the NOX homologues DUOX1 and DUOX2 have been shown to be involved in thyroid hormone synthesis, we subsequently analyzed the expression of these proteins. H9c2 cells indeed expressed DUOX1 and DUOX2 at the mRNA level (Fig. 3, A and B) and at the protein level (Fig. 3C). Notably, the antisera directed against DUOX do not permit distinction between DUOX1 and DUOX2.


Figure 3
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  		Fig. 3. Expression of DUOX1 and DUOX2. A and B: we used mRNA isolated from total rat heart tissue as a template to amplify by RT-PCR fragments corresponding to DUOX1 (expected fragment length: 254 bp) (A) and DUOX2 (expected fragment length: 174 bp) (B). We subsequently confirmed the sequence identity of the PCR fragments by cycle sequencing. M, 50-bp DNA markers; W, water control (bands at 60 bp: primer dimers); S, cDNA samples. C: Western blot analysis of DUOX expression in H9c2 and FRTL-5 cells. Note, we applied an arbitrary amount of FRTL-5-cell lysate as positive control.

 
mRNA expression in rat hearts of components necessary for thyroid hormone generation. To further test our hypothesis that the expression of thyroglobulin and DUOX in cardiomyocytes may be related to cardiac thyroid hormone synthesis, we analyzed the cardiac expression by microarray at the mRNA level of the components necessary for the synthesis of thyroid hormone in the thyroid gland. We performed the analysis with mRNA obtained from right ventricular myocardium of control rats and of rats with monocrotaline-induced right ventricular hypertrophy or congestive heart failure (4). We found low but significant expression of all the remaining components required for thyroid hormone synthesis, namely, the sodium-iodide symporter, pendrin, thyroid peroxidase, and the receptor for thyroid-stimulating hormone (Fig. 2). We observed a significant increase in mRNA expression for pendrin in the congestive heart failure samples compared with the mRNA expression in control animals (P < 0.05).

Incorporation of 125I into thyroglobulin and synthesis of thyroid hormone by H9c2 cells. To address the possibility that the expression in cardiomyocytes of proteins related to thyroid hormone biosynthesis indeed serves the function of hormonogenesis, we analyzed the metabolism of 125I in H9c2 cells. We incubated H9c2 cells for 88 h in the presence of 50 µCi 125I/ml and subsequently induced ischemia for 30 min in the presence of the same concentration of 125I. Analysis by SDS-PAGE revealed that 125I was incorporated exclusively into one band that, upon immunostaining, corresponded to thyroglobulin (Fig. 4A). Whereas the thyroglobulin protein expression showed an increase under metabolic inhibition, 125I incorporation into thyroglobulin was clearly decreased under metabolic inhibition (Fig. 4A). When analyzing these cardiomyocyte lysates with a modified RIA specific for free T4 and free T3, we found a low but significant generation of both hormones (P < 0.05) that was clearly increased under metabolic inhibition in case of T3 (P < 0.05; Fig. 4B).


Figure 4
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  		Fig. 4. Incorporation of 125I into TG and generation of thyroid hormones by H9c2 cells. We cultured H9c2 cells for 88 h in the presence of 50 µCi 125I/ml. Thereafter, we induced ischemia for 30 min in the presence of the same concentration of 125I and subsequently lysed and analyzed the cells. A: SDS-PAGE separation and subsequent autoradiography of the samples. We loaded 70 µg of protein per lane. The corresponding bar graph visualizes the band intensities relative to the control band. Blot shown is representative of three identical experiments. B: we added cell lysates, normalized to contain 120 µg of protein, into RIA tubes that were coated with antibodies specific for free T4 (left) and free T3 (right), respectively. After overnight incubation at 4°C, we washed the samples two times and analyzed them in a gamma counter. Bar graphs show the retained radioactivity after the subtraction of the averaged activity of the empty background control tubes (n = 5). *Significant difference between sample and background control (P < 0.05); {dagger}significant difference between ischemia and control (P < 0.05).

 
In summary, our findings support the concept of cardiomyocyte-specific thyroid hormone synthesis at a low level. Assumedly, hormone synthesis takes place in the perinuclear thyroglobulin matrix, and the hormone is liberated from this matrix during ischemia at an increased rate.


   DISCUSSION
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 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The goal of the present study was to test the hypothesis that cardiomyocytes are capable of generating low amounts of thyroid hormone in an intracrine fashion. This hypothesis is supported by our findings that 1) rat hearts express all components essential for thyroid hormonogenesis, 2) 125I is integrated specifically into thyroglobulin in H9c2 cells, 3) in cell lysates low but significant radioactive levels of both forms of thyroid hormone T4 and T3 were detected, and 4) these levels were increased significantly under metabolic inhibition in case of T3.

While there have been sporadic (1, 8, 13, 20) and sometimes contradictory reports (5) of extrathyroidal expression of the components necessary for thyroid hormone synthesis, this is, to the best of our knowledge, the first study addressing the possibility that nonthyroidal cells may express the complete set of the thyroid hormone-generating protein machinery. The low levels both of protein expression and of thyroid hormone generation probably explain why these findings have escaped detection until now.

One argument that can be raised against our hypothesis of cardiomyocyte-generated thyroid hormone is that the heart does not possess the follicular structure that is important for the function of the thyroid gland. However, we propose that the cardiomyocyte-generated hormone serves exclusively intracrine purposes and does not affect the systemic hormone levels that are regulated by the thyroid gland. This hypothesis is also supported by the early reports of extrathyroidal thyroid hormone generation that demonstrated the necessity of high doses for iodide supplementation to be effective (9). Apparently cardiomyocytes under physiological conditions import only minimal amounts of iodide.

One of the primary reactions of cardiomyocytes to both hypertrophic and ischemic stimuli, however, seems to be an increased expression of thyroglobulin. However, because there was a concomitant release of the hormone from thyroglobulin as assessed by RIA, we found a reduced radioactive signal in the thyroglobulin-specific band under metabolic inhibition. Release of thyroid hormone from thyroglobulin in the thyroid gland is mediated primarily by cathepsins K and L, two cysteine proteinases (10). Of these, at least cathepsin L is ubiquitously expressed, including the myocardium (21). Conversion of T4 into the bioactive T3 is catalyzed by iodothyronine 5'-monodeiodinases, types 1 and 2, both of which are expressed in human and rat myocardium (18). The increased bioavailability of active thyroid hormone in cardiomyocytes in ischemia and, possibly, hypertrophy seems to be regulated, therefore, by a concomitantly increased synthesis, as evidenced by an increased expression of thyroglobulin, and an increased release of the hormone from thyroglobulin that theoretically could be mediated by cathepsins L and/or K.

Physiologically, it is advantageous for cells to be able to generate thyroid hormone in circumstances where due to the blocked blood flow not only the supply of oxygen is compromised but also that of blood-borne thyroid hormone. This relative independence from systemic thyroid hormone allows cardiomyocytes to initiate cell-protective mechanisms even before local circulation is reestablished.

Although our finding that H9c2 cells seem to be capable of thyroid hormone generation needs to confirmed in primary cells, our results suggest that cardiomyocytes indeed can generate low levels of thyroid hormone in an intracrine fashion. The precise regulatory mechanisms that control the bioavailability of cardiomyocyte-generated thyroid hormone need yet to be elucidated. Given the central importance of thyroid hormone in cardiac (patho)physiology, understanding the regulation and role of cardiomyocyte-generated hormone will be relevant for understanding and treating heart disease. It will be of relevance, for example, to learn whether the autoantibodies in Graves' disease that bind and stimulate the thyroid-stimulating hormone receptor also induce an increased thyroid hormone generation in cardiomyocytes and in doing so contribute to the cardiac symptoms found in these patients.


   GRANTS
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 ABSTRACT
 METHODS
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 GRANTS
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C. Meischl was supported by a grant from the Netherlands Heart Foundation (2002B010).


   DISCLOSURES
 
The authors declare to not have any competing financial interests.

Present address for H. P. Buermans: Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Present address for C. Boer: Abbott B.V., Siriusdreef 51, 2132 WT Hoofddorp, The Netherlands.

Present address for C. Dupuy: Unité mixte de recherché 8125, Université Paris-Sud XI, Villejuif Cedex, France.

Present address for C. E. Hack: Crucell Holland, Archimedesweg 4, 2333 CN Leiden, The Netherlands.


   ACKNOWLEDGMENTS
 
We thank F. Miot for providing the DUOX-specific antiserum and M. M. Wiegman and T. Weijer for technical advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Meischl, VU Univ. Medical Center, Dept. of Pathology, Rm. nr. 1B116, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands (e-mail: c.meischl{at}vumc.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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