Thyroid Hormone Induces Cardiac Myocyte Hypertrophy in a Thyroid Hormone Receptor
1-Specific Manner that Requires TAK1 and p38 Mitogen-Activated Protein Kinase
Koichiro Kinugawa,
Mark Y. Jeong,
Michael R. Bristow and
Carlin S. Long
Division of Cardiology (M.R.B., C.S.L.), University of Colorado Health Sciences Center, Cardiology Section (C.S.L.), Denver Health Medical Center, Denver, Colorado 80204
Address all correspondence and requests for reprints to: Carlin S. Long, M.D., 777 Bannock Street, Box 0960, Denver, Colorado 80204. E-mail: clong{at}dhha.org.
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ABSTRACT
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Alterations in TR [thyroid hormone (TH) receptor]1 isoform expression have been reported in models of both physiologic and pathologic cardiac hypertrophy as well as in patients with heart failure. In this report, we demonstrate that TH induces hypertrophy as a direct result of binding to the TR
1 isoform and, moreover, that overexpression of TR
1 alone is also associated with a hypertrophic phenotype, even in the absence of ligand. The mechanism of TH and TR
1-specific hypertrophy is novel for a nuclear hormone receptor and involves the transforming growth factor ß-activated kinase (TAK1) and p38. Mitigating TR
1 effects, both TR
2 and TRß1 attenuate TR
1-induced myocardial growth and gene expression by diminishing TAK1 and p38 activities, respectively. These findings refine our previous observations on TR expression in the hypertrophied and failing heart and suggest that manipulation of thyroid hormone signaling in an isoform-specific manner may be a relevant therapeutic target for altering the pathologic myocardial program.
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INTRODUCTION
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IT IS WELL accepted that alterations in thyroid function occur in patients with heart failure (1, 2, 3, 4). Although previously felt to represent the euthyroid-sick syndrome rather than frank hypothyroidism, recent data suggest that a primary change in the myocardial response to thyroid hormone might underlie some of the alterations in myocardial form and function seen in the failing heart. In fact, the use of the thyroid hormone (TH) supplementation as a means of increasing cardiac function for patients with heart failure has met with limited success (5, 6, 7). This tactic is considered by many to be suboptimal, however, because thyroid supplementation may be associated with potential adverse effects on heart rate and myocardial oxygen consumption. With increased use of ß-blockade in heart failure patients, these side effects may well be controlled, and interest in TH therapy for these patients has been renewed. Furthermore, several TH analogs with limited effects on heart rate have also been developed and, in preliminary clinical trials, have been associated with improved myocardial function (8).
In response to our observation that myocardial TH receptor (TR) isoform expression is decreased in patients with heart failure (9), it is possible that these changes may be responsible, at least in part, for certain aspects of the failure phenotype. In the work described here, we have found that TR isoforms have differential effects on the cardiac myocyte phenotype. Specifically, TR
appears to be linked to robust changes in cardiac myocyte growth that are dependent upon the p38MAPK cascade. In contrast, TRß does not induce a growth program, limits p38 activation, and stimulates the classic thyroid-responsive cardiac myocyte genes [namely
MHC (myosin heavy chain) and SERCA (sarcoplasmic reticulum Ca2+-ATPase)].
These data support our hypothesis that changes in the expression of TR isoforms and their signaling partners are likely to play a direct role in myocardial growth and gene expression in heart failure. It is tempting to speculate from these findings that manipulation of the TH:TR axis in an isoform-specific manner may represent a new therapeutic approach to CHF that may complement treatment profiles already in use for this devastating syndrome.
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RESULTS
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Cellular Distribution of Endogenous and Overexpressed TR Isoforms
To better understand the role of individual TR isoforms in the heart, a series of adenoviral vector constructs containing each of the TR isoforms found in the heart (TR
1, TR
2, and TRß1) (9) was developed. As indicated by immunostaining (Fig. 1A
), EMSA (Fig. 1B
), and Western blot (Fig. 1C
, upper panel), all three TRs can be successfully overexpressed in cardiac myocytes, with over 90% of myocytes successfully infected at multiplicity of infection (MOI) of approximately 15. Importantly, radioligand binding assays confirm that the adenoviral overexpression system increases cellular TRs by only approximately 2- to 4-fold when compared with control cells (Fig. 1C
, lower panel; basal binding is
0.5 fmol/106 cells, which increases to
1.0 fmol/106 at 5 MOI and approximately 2 fmol/106 at 50 MOI). Unexpectedly, distribution of expressed human (h) TRs appears to show some isoform specificity. Specifically, unless overexpressed to very high levels (>200 MOI), hTRß1 is localized in the nucleus. In contrast, both hTR
1 and hTR
2 are found in both cytosolic and nuclear fractions. As shown by EMSA, both nuclear and cytosolic TRs are fully competent for binding to a consensus thyroid-responsive element (TRE). Notably, T3 (1100 nM) did not change the localization pattern of overexpressed TRs (data not shown), as seen by others in different systems (10, 11).

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Fig. 1. Cardiac Myocyte Expression of Human TRs
A, Immunostaining. Neonatal rat cardiac myocytes (MCs) were exposed to adenovirus at 100 MOI for 72 h. Upper panels are immunofluoresence pictures of cells infected with the indicated AdTRs incubated with the C1 antibody that only recognizes human TR isoforms ( and ß). Bottom panels were the same cells coincubated with antibody to sarcomeric -actin which identifies cardiac myocytes. Less than 5% of cells were sarcomeric actin negative (nonmyocytes, NMCs). A myocyte without expression of human TRß1 ( ) is identified. Note the restriction of hTRß1 expression to the nucleus of these cells, whereas both hTR 1 and hTR 2 appear to be distributed in both nuclear and cytoplasmic compartments. B, EMSA for the DR4 (direct repeat-4) TRE. Cells were exposed to adenovirus at 50 MOI for 48 h. For supershift assays, the same human TR-specific antibodies used in Fig. 1C were used, and are denoted as + isoform-specific Ab. B1 and B2 consists of heterodimers of retinoid X receptor (RXR , ß, or ) and TR (1 molecule of each), and homodimers of TRs (two TR molecules), respectively. No monomer binding was observed. Competitor lanes were with unlabeled oligonucleotide. The 200 MOI lane for hTRß was included because this was the only condition where cytosolic hTRß was found. C, Quantification and subcellular location of human TR overexpression in neonatal rat cardiac myocytes. Myocytes were infected with the individual AdTRs at the indicated MOIs for 48 h. Fractionated cell extracts were prepared and subjected to Western blotting with human-specific TR antibodies in the upper panels (hence no rat TR is detected in uninfected lanes). In the binding experiments, cell extracts from equal numbers of cells were subjected to [125I]T3-binding assay as described previously (44 ). Notably, expression of TR 1 was readily found in both nuclear and cytoplasmic fractions, whereas AdTRß1 expression was generally limited to the nucleus.
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Overexpression of TR
1 Induces Myocyte Hypertrophy Independent of Ligand
Consistent with reports from our lab and others, T3 stimulates cardiac myocyte hypertrophy in culture (
75% increase in synthesized protein, P < 0.05, n = 5) with an EC50 of approximately 0.3 nM (12, 13, 14). As shown in Fig. 2A
, even in the absence of exogenous hormone, AdTR
1 (but not TRß1 or TR
2) also increased protein synthesis and cell surface area (1.88 ± 0.13-fold over control surface area at an MOI of 50, P < 0.05). AdTR
1-induced hypertrophy was enhanced by addition of T3, but not with the TRß-selective agonist GC-1 (15) (Fig. 2B
). Notably, hypertrophy induced by both T3 and AdTR
1 was inhibited by both AdTR
2 and AdTRß1 (Fig. 2
, B and C).

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Fig. 2. Overexpression of TR 1 Induces Myocyte Hypertrophy
A, MOI-dependent effects on protein synthesis by AdTRs. Cells were infected with AdßGal, AdTR 1, AdTR 2, or AdTRß1 for 48 h at the designated MOI. Radiolabeled protein content (RLP) was normalized to AdßGal at identical MOIs and at the 0.3 MOI level for the subsequent increases in AdßGal itself. For comparison, the RLP seen with 100 nM T3 alone is shown. B, Effects of T3 or GC-1 on protein synthesis. Cells were infected with AdßGal, AdTR 1, AdTR 2, or AdTRß1 for 48 h at 10MOI with various concentrations of T3 or GC-1. Values were normalized to vehicle + AdßGal at 10MOI. C, Effects of AdTR 2 or AdTRß1 on AdTR 1-induced hypertrophy. Cells were treated with AdTR 1 at 10MOI ( ) with the addition of AdTR 2 or AdTRß1 at the indicated MOIs for 48 h.
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TH and TR
1 Activate the p38 Signaling Cascade and Hypertrophy Is p38 Dependent
Unexpectedly, hypertrophy induced by either T3 or AdTR
1 was inhibited by preincubation with the p38 inhibitor SB201290 (SB, Fig. 3A
). The IC50 of SB201290 was approximately 30 nM, consistent with specific inhibition of p38 (16). Specificity for the p38 family was confirmed using infection with dominant-negative (DN) adenoviral vectors for MKK3 or p38
. The failure of either the MEK1/2 inhibitors U0126 and PD98059 (PD98059 not shown), or infection with AdJNK1DN to inhibit hypertrophy provide additional support for a p38-specific pathway (Fig. 3B
). Although members of the nuclear hormone receptor family have not previously been thought to directly activate the stress kinase [p38 and c-Jun N-terminal kinase (JNK)] family of signaling intermediates, our results with p38 inhibitors suggest that TH/TR
1 induced hypertrophy requires this arm of the MAPK signaling cascade. Supporting a direct effect for p38, both T3 and TR
1 stimulated a rapid increase in the phosphorylated form of p38 and subsequently its kinase activity (Fig. 3C
). The TRß1-specific agonist GC-1 had no effect (data not shown). As shown in the upper panel of Fig. 3C
, adenoviral overexpression of both TRß1 and the DN TR
2 inhibited p38 activation by T3 (4-fold induction in AdbGal cells vs. a 1.2-fold increase in AdTR
2 and 1.5-fold increase in AdTRß1 cells). Because these effects on myocyte growth pointed to an involvement upstream of p38 itself, we focused on a possible interaction between TR
1 and the proximate MAPK kinases (MAPKKs, MKKs) and MAPK kinase kinases (MAPKKKs). As indicated in Fig. 4
, A and B, both AdTR
1 and T3 stimulated MKK3/6 phosphorylation and the kinase activity of the MAPKKK, TAK1. Notably, although TR
2 inhibited T3-induced TAK1 kinase activity, AdTRß1 had no apparent effect. Specificity for the p38 arm of the MAPK family was also shown by the inability of T3 or adenoviral overexpression of any TR to activate either the ERK or JNK cascades in cardiac myocytes (Fig. 4C
).

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Fig. 3. TH and TR 1 Hypertrophy Is p38 Dependent
A, Dose-dependent effects of SB202190 on T3 and AdTR 1-induced myocyte growth. Cells were pretreated with the indicated dose of SB202190 (SB) or null SB202474 (Null) for 30 min, followed by the addition of AdßGal (50MOI, not shown), AdßGal+T3 (100 nM) or AdTR 1 for 48 h. Values were normalized to that of AdßGal + vehicle. B, Cells were pretreated with vehicle (DMSO) or U0126 (1 µM) for 30 min or AdJNK1DN, AdMKK3DN, or Adp38 DN for 24 h. Cells were subsequently infected with AdßGal (50 MOI, not shown), and treated with T3 (100 nM, AdßGal+ T3), or AdTR 1 for 48 h. Values were normalized to that of AdßGal + vehicle. C, T3/TR stimulation of p38MAPK. Cells were treated with T3 (100 nM) or AdTR 1 (50 MOI) for the designated times (left and middle panels) or infected with AdßGal or AdTRs (50 MOI) for 24 h and T3 (100 nM) added for an additional 15 min (right panel). Phospho-p38 was then determined by Western blotting. Both T3 and AdTR 1 activate p38 (bottom panel). AdßGal cells were treated with T3 (15 min) or AdTR 1 (24 h) at indicated doses. In vitro p38 activity was measured by immune complex kinase assay with GST-ATF2.
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Fig. 4. TH and TR 1 Activate MKK3/6 and TAK1, But Not ERK or JNK
A, Activated (phosphorylated) MKK3 (upper band) and MKK6 (lower band) expression increase in T3 and AdTR 1 treated cells. B, In vitro TAK1 activity was measured by immune complex kinase assay using MalMKK3 in cells treated with T3 alone (15 min) or in the presence of the indicated AdTRs (48 h infection). C, Phosphorylated and total ERK1/2 and JNK1/2 expression were also examined in similarly treated T3 and TR infected cells. As a positive control, cells were treated with 20% of fetal bovine serum (FBS) for 30 min.
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TR
1 and TR
2 Interact with TAK1 in Cytosol
TAK1 is a member of the MAPKKK family activated by various cytokines including the transforming growth factor ß ligands (17). In general, TAK1 forms a complex with other adapter proteins and kinases, ultimately resulting in its own activation and stimulation of downstream kinases including p38. Although multiple partners have been identified for TAK1, an interaction with the nuclear hormone receptor family has not been previously appreciated. When overexpressed in cardiac myocytes, both TR
1 and TR
2 isoforms were found to colocalize with endogenous TAK1 (Fig. 5A
), a finding that also extended to endogenous rat TR
1 (Fig. 5B
). As reported by others (18), TAK1 was found only in cytosolic fraction (Fig. 5C
). The interaction appears to be specific for the TR
isoforms because overexpressed TRß1 was never found in complex with TAK1 even under circumstances of nuclear overflow with MOIs of > 200 for 48 h (data not shown). TRß1 did, however, interact with p38
, a finding that did not extend to either TR
1 or TR
2 (Fig. 5D
). Furthermore, in a cell-free system, TRß1 reduced both autophosphorylation of p38 and phosphorylation of its substrate ATF2 but did not appear to affect MKK6 phosphorylation of p38 (Fig. 5E
).

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Fig. 5. Cytosolic TR 1 Interacts with TAK1
A, TR 1 and TR 2, (but not TRß1) interact with TAK1. Lanes 13: Human-specific TR antibody was validated for Western blotting with control human TRs synthesized in rabbit reticulocyte lysate [TR 1 ( 48 kDa), TR 2 ( 58 kDa), and TRß1 ( 52 kDa)]. Doublets represent lysate-specific in vitro processing and are not seen in AdTR-infected cells. Lanes 46: Myocytes were infected with AdTRs at 50 MOI for 24 h followed by immunoprecipitation of endogenous TAK1. This was subjected to Western for TR. Lanes 79: Expression of human TRs in each sample was confirmed using the same antibody. B, Whole cell extract from uninfected cells was immunoprecipitated with rabbit IgG or rat-specific TR 1 antibody, and subjected to Western blotting for TAK1. C, Western blotting and immunofluorescence microscopy for endogenous cardiac myocyte TAK1 expression. D and E, TRß1 (but not TR 1 and TR 2) interacts with p38 and diminishes its kinase activity. Cells were infected with AdTRs and Adp38 WT for 24 h. Total p38 was immunoprecipitated, and subjected to Western for humanTR (C1). E, In vitro-synthesized human TRß1 or control rabbit reticulocyte lysate was mixed with active MKK6 or active p38 ( 68 kDa), and their activities measured on unactive recombinant GST-p38 ( 64 kDa) or GST-ATF2 ( 40 kDa), respectively. SB202190 was used at 10 nM.
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TRs Exhibit Isoform-Specific Changes in Myocyte Gene Program
Given the reported physiologic effects of T3 on myocyte gene expression (12, 13, 19, 20), we found that AdTR
1 initiated a gene profile more consistent with a pathologic/fetal myocyte program [increasing ßMHC, skeletal
-actin (skACT), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP), while decreasing
MHC and SERCA2 expression (Fig. 6
)]. AdTR
1 also caused a down-regulation of the myocyte expression of endogenous TR
1 and TRß1. The fetal gene program induced by AdTR
1 was abrogated somewhat, however, by cotreatment with T3 and coinfection with AdTR
2 (data not shown). At relatively low MOIs (1050), AdTR
2 also inhibited T3-induced increases in skACT, ANP, or BNP expression; however, MOIs of more than 200 were required for attenuation of T3-induced increased expression of
MHC and SERCA. T3-induced inhibition of ßMHC expression was not affected in AdTR
2-treated cells, at any MOI. Notably, overexpression of TRß1 induced a gene program that was sharp contrast to TR
1, reflecting a marked TR isoform-specific gene program. The gene expression profile seen with AdTRß1 was, in fact, quite similar to that observed with T3 [increases in
MHC, SERCA2, and endogenous TRß1, and repression of ßMHC mRNA to nearly undetectable levels (Fig. 6
)]. Addition of T3 to AdTRß1 further enhanced the change in
MHC, SERCA, and endogenous TRß1 expression. Expression of skACT, ANP, and BNP in AdTRß1-infected cells differed from T3 treatment, exhibiting a substantial inhibition in AdTRß1 infected cells (Fig. 6
).

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Fig. 6. TR Isoform-Specific Changes in the Cardiac Myocyte Gene Program
Cells were treated with AdßGal at 50 MOI with or without T3 (100 nM) for 72 h and compared with cells infected with AdTR 1 or AdTR 2, or AdTRß1 at 50 MOI. Values of the corresponding AdßGal group were set at 100%, and data are presented as percentage of change from 100%, n = 34. As such, a value of 0% equals no change from AdßGal infected cells and 100% represents a doubling of signal. All signals were corrected for RNA loading using an internal GAPDH signal.
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DISCUSSION
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Recent work from both our investigative group and others has renewed the interest in a possible therapeutic role for the TH:TR axis in patients with heart failure. In this regard, we previously reported isoform-specific alterations in TR expression in both human and experimental hypertrophy/failure (9, 12). Changes in TR isoforms were believed to play a role in the development and/or maintenance of the pathologic cardiac myocyte gene program and, as such, represented possible therapeutic targets. In the present study, we extend these findings and report previously unappreciated isoform-specific, nongenomic activities for TR isoforms in cardiac myocytes. The major conclusions from these investigations are: 1) T3-induced cardiac myocyte hypertrophy is p38 dependent and requires TR
1 and activation of the TGFß-activated kinase, TAK1 and 2) The ultimate effect of TH on myocardial growth/gene expression is the result of the combinatorial effects (both complementary and antagonistic) of the three TR isoforms found in heart, TR
1, TR
2, and TRß1.
T3 and TR
1 Activate TAK1 and the p38 Cascade
Several lines of evidence indicate that the effects of TH on the myocyte gene program are characterized by an adult/physiologic phenotype. The work presented here, however, shows that T3 stimulates p38, the arm of the MAPK family most frequently associated with pathologic hypertrophy (21). In these investigations, we examine this seemingly contradictory phenomenon. Our studies indicate that T3-induced p38 activity and myocyte growth is exclusively due to the action of the TR
1 isoform on the upstream kinase, TAK1. The sequential activation of TAK1 and p38 by T3/TR
1 is capable of being modulated at two points in the cascade. First, TR
2 can compete with TR
1 for binding to TAK1, and second, TRß1 can associate with, and inhibit, the downstream target p38. This TRß1-p38 interaction is similar to the interaction of TRß1 with ERK2 (20, 22, 23) and likely occurs in the nucleus where the majority of activated p38 is found (24). Thus, although the TR
1 isoform facilitates T3-induced P38 activity, the P38 activity (and program of pathological hypertrophy) can be altered by both TR
2 and TRß1. Given that both TR
1 and TR
2 interact with TAK1, the interaction domain must reside within the common 5'-half of the TR proteins, a domain known to interact with other transcription factors or signaling molecules such as MEF2 or ERK (22, 25, 26, 27). Activation of TAK1 does not appear to result from any inherent kinase activity of TR
1 itself, but rather likely results from an adapter function of TR
1, possibly similar to that described for the TAK1-binding protein (TAB1) (28). TR
1, however, was not found to interact with TAB1 (data not shown). Although somewhat unexpected by us, the finding that TRs may have differential subcellular locations and shuttle between nuclear and cytoplasmic compartments has been reported by a number of investigative groups (10, 11, 29, 30, 31). The mechanisms of nucleo-cytoplasmic shuttling have not been identified with certainty, but may involve protein partners (10) and possibly posttranslational modification of TRs [i.e. phosphorylation (22)]. Notably, this has not reliably been altered by ligand (10, 30, 31). The exact mechanism for either the differential localization or movement from one compartment to the other in the cardiac myocyte context has not been identified in the present work and is certainly worthy of further investigation.
SummaryThe Myocardial Response to TH Results from the Combinatorial Effects of Individual TR Isoforms (Fig. 7
)
Our investigations indicate that T3-induced gene expression in neonatal cardiac myocytes is the result of two parallel signaling cascades. One is the classical, direct (or genomic) pathway in which TH interacts with nuclear TRs, likely bound to characteristic TREs on target genes. The second pathway, novel for a nuclear hormone receptor, is a true cascade in which T3 activates TAK1 through the action of cytoplasmic TR
1. This activation ultimately results in the stimulation of a series of p38-dependent processes that include myocyte protein synthesis (hypertrophy) and the induction of a set of genes whose expression characterizes the pathologic growth program (skACT, ANP, and BNP). This action of the TR
1 isoform is tempered somewhat by opposing effects of both the TR
2 and TRß1 receptor isoforms which prevent the tonic activation of the p38 arm at two points in the cascade, while maintaining expression of TRE-dependent genes. This latter effect may be largely due to the T3-dependent up-regulation of the TRß1 isoform, which appears to be a potent stimulus for
MHC and SERCA expression. As such, T3-responsive genes can be divided into three categories. The first group consists of TRE-containing genes that are regulated by both TR
1 and TRß1, a category that includes
MHC and SERCA. Observations on TR
1-deficient mice (32, 33) and data from our transfection study (12) also suggest significant roles of TR
1 on these genes. The second group is made up of TRE-containing genes that are mostly regulated by TRß1. This category consists of ßMHC and endogenous TRß1 and is supported by our previous promoter assays suggesting a TRß1-specific role in the regulation of the latter (12). For both groups, TRE-mediated effects appear to be dominant in the final response to TH. The third category includes skACT, ANP, BNP, and endogenous TR
1, genes that do not contain TREs in their promoter regions and are only modestly responsive to TH. For these genes, it appears that the TR
1 stimulation of the p38 pathway is dominant in the final response to TH. Consistent with these findings, IL-1ß, which is a strong p38 activator (34, 35), has synergistic effects with T3 for increases in ANP and BNP expression as well as myocyte protein synthesis (our unpublished observations). We have reported that the failing human heart exhibits fetal gene expression (36, 37, 38). Because human heart failure is often associated with enhanced p38 activity (39, 40, 41), a TRß-specific agonist could result in a decrease in p38 activation and an alteration in the fetal gene program, and possibly the growth program. In contrast, thyroid hormone supplementation could theoretically lead to excessive p38 activation and additional myocyte injury, particularly if there were an imbalance in TR expression toward TR
1 as seen in some cases of pathologic hypertrophy (12). Although there is a currently available TRß agonist, GC-1, it is probably not as highly TRß specific as would be necessary (15), and its bioavailability has been questioned (42). Additionally, relative to TR
1, TRß1 gene expression in the human left ventricle is quite low (9). Therefore, the utility of a TRß1 agonist in the treatment of heart failure will need to await the development of a more highly selective TRß1 ligand.

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Fig. 7. Proposed Schema of T3/TR Isoform-Specific Action on Cardiac Myocyte MAPK Signaling and Gene Program
See text for details.
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MATERIALS AND METHODS
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Cell Culture
Ventricular myocytes from 1-d-old rats were cultured as described (9). Vehicle for T3 (Sigma, St. Louis, MO) was NaOH. GC-1 (a gift from G. Chiellini and T. S. Scanlan, University of California San Francisco) and U0126 (Cell Signaling, Beverly, MA) were dissolved in DMSO. Effects of SB202190 (Calbiochem, San Diego, CA) were always compared with those of inactive SB202474. All animal experimentation described was approved by the University of Colorado Institutional Animal Care and Use Committee and was conducted in accord with accepted standards of humane animal care as outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. As described previously, cells are kept in 5% serum containing medium (which has not been previously stripped of TH) for 24 h, followed by washing and change to serum-free medium whose residual T3 content has been measured at approximately 0.1 nM (12).
Adenoviral Constructs
Adenoviral (Ad) constructs for ß-galactosidase (AdßGal), HA-tagged, constitutively activated MAPK kinase (MKK-6, AdMKK6CA) or DN MKK-3 (AdMKK3DN), DN JNK (AdJNK1DN) and Flag-tagged Adp38
DN or wild-type p38
(Adp38
WT) were provided by J. Han (Scripps Institute), L. Heasley (University of Colorado), and K. A. Heidenreich (Denver Veterans Affairs Medical Center, Denver, CO), respectively. By X-Gal staining, approximately 95% of cells expressed ß-Gal protein after exposure to AdßGal for 72 h at a MOI of 1 or more. Expression of other constructs was confirmed by Western blot with epitope-specific antibodies (Roche Molecular Biochemicals, Indianapolis, IN).
Preparation of Adenoviral Constructs for TRs and Overexpression in Myocytes
cDNAs for human TR/c-erbA isoforms (
1,
2, and ß1) were provided by R. C. J. Ribeiro (University of Brasilia, Brasilia, Brazil) and J. D. Baxter (University of California, San Francisco, San Francisco, CA). The full-length cDNAs were subcloned into adenovirus shuttle vector (pAC-CMV), and transfected together with adenoviral arm in 293 cells (43). Plaques negative for X-Gal staining were selected, and ones positive for TR cDNA by PCR were purified.
Western Blot Analysis and T3-Binding Assay
Total or fractionated cell extracts from equal number of cells were subjected to Western blot analysis (12) or [125I]T3-binding assay (44). Antibodies used included those for phopho-p38
/ß, p38
, phospho-MKK3/6, phospho-ERK1/2, ERK1/2, phospho-JNK1/2 from Cell Signaling (all polyclonal, used at 1:1000 dilution); for MKK3 (I-20), p38
/ß(A-12), TAK1 (C-9, M-579), JNK2/1 (D-2), TRß1 (J51), TR
/ß (C-1) from Santa Cruz Biotechnology (Santa Cruz, CA) (used at 0.22 µg/ml); and for TR
1 (PA1211A) from Affinity BioReagents (Golden, CO) (1:200 dilution). Recombinant TR proteins were synthesized using expression vectors for human TRs (gifts from R. C. J. Ribeiro and J. D. Baxter) and rabbit reticulocyte lysate (TNT T7 Quick Coupled System; Promega, Madison, WI).
Immune Complex Kinase Assay
Total cell extract was immunoprecipitated with p38
/ß (A-12) or TAK1 (C-9) antibody (4 µg/ml). The immune complexes were used for in vitro kinase assay (45) with 32P-
ATP and 2 µg of recombinant glutathione-S-transferase (GST)-ATF2 (Santa Cruz Biotechnology) or inactive MalE-MKK6 (Upstate Biotechnology, Waltham, MA) as substrates at 30 C for 20 min. Phosphorylated products were analyzed by SDS-PAGE. Aliquots of immune complexes were also blotted for total p38
(Cell Signaling) or TAK1 (M-579).
EMSA
Both nuclear and cytosolic fractions were resuspended in equivalent volumes for comparison of relative TR expression in EMSA (45). For supershift assay, antibodies for TR
1 (PA1211A, 1:10 dilution), TRß1 (J51), or retinoid X receptor (
: D-20, ß: C-20,
: Y-20, Santa Cruz Biotechnology) (used at 20 µg/ml) were added to samples for 30 min at 4 C before incubation with 32P-labeled direct repeat 4 (DR4) oligonucleotide (Santa Cruz Biotechnology). Unlabeled DR4 was used as competitor at 100-fold molar excess.
Immunostaining
Myocyte expression of exogenous human TR or endogenous TAK1 was visualized by immunostaining (35) using TR (C1) or TAK1 (C-9) antibody (4 µg/ml) and fluorescein isothiocyanate-labeled anti-IgG1 antibody (Santa Cruz Biotechnology). Cardiac myocytes were identified with sarcomeric
-actin antibody (1:50 dilution, 5C5, Sigma) and rhodamine-conjugated anti-IgM antibody (Santa Cruz Biotechnology).
Immunoprecipitation
Total cell extract immunoprecipitated by TAK1 (C-9), p38
/ß (A-12), or TR
1 antibody (FL-408, Santa Cruz Biotechnology) was subjected to Western blot analysis for TR (C1) or TAK1 (C-9). Aliquots of immune complexes were also blotted for total p38
(Cell Signaling) or TAK1 (M-579).
Kinase Assay
Human TR protein (
10 ng) synthesized in rabbit reticulocyte lysate was mixed with 10 ng of active MalE-MKK6 (
81 kDa, Upstate Biotechnology) or active GST-p38
(
68 kDa, Calbiochem), and incubated with 2 µg of inactive GST-p38
(
64 kDa, Upstate Biotechnology) or GST-ATF2 as substrates, respectively. Autophosphorylation of p38
was also assessed with 1µg of the active GST-p38
. Kinase reaction was carried out at 30 C for 20 min and products analyzed by SDS-PAGE (45).
Growth Assay
After treatment, myocytes were incubated in fresh media containing 14C-phenylalanine for 24 h. The incorporated [14C]-phenylalanine into synthesized protein allowed for quantification of hypertrophy at the steady state by radiolabeled protein assay (12).
Ribonuclease (RNase) Protection Assay
Total RNA (5 µg) was used in RNase protection assay (9, 12) with rat probes for
/ß-MHC, SERCA2, ANP, skACT, TR
1/TR
2, TRß1, and GAPDH as an internal control.
Data Analyses and Statistics
Specific signals obtained from Western, kinase assay, or RNase protection assay were quantified by densitometry. All values were normalized to the appropriate controls. Data were compared by one-way ANOVA and the Newman-Keuls test. Mean ± SE is shown.
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ACKNOWLEDGMENTS
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We thank Mary Atz and Albina Nesterova for excellent technical help; Ralff C. J. Ribeiro and John D. Baxter for TR plasmids; J. Han, K. Heidenreich, and L. Heasley for adenoviral constructs; and G. Chiellini and T. S. Scanlan for GC-1.
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FOOTNOTES
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This work was supported by grants from the National Institutes of Health (to M.R.B. and C.S.L.). M.J. was supported by the Sarnoff Endowment for Cardiovascular Science.
Present address for M.Y.J.: Boston University School of Medicine, Department of Internal Medicine, 715 Albany Street, E-113, Boston, Massachusetts 02118.
Present address for K.K.: Department of Cardiovascular Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
First Published Online April 14, 2005
Abbreviations: Ad, Adenoviral; ANP, atrial natriuretic peptide; ßGal, ß-galactosidase; BNP, brain natriuretic peptide; DN, dominant negative; GST, glutathione-S-transferase; hTR, human TR; JNK, c-Jun N-terminal kinase; MHC, myosin heavy chain; MKK, MAPK kinase; MOI, multiplicity of infection; RNase, ribonuclease; SERCA, sarcoplasmic reticulum Ca2+-ATPase; skACT, skeletal
-actin; TAK, TGFß-activated kinase; TH, thyroid hormone; TR, thyroid hormone receptor; TRE, thyroid-responsive element; WT, wild type.
Received for publication December 13, 2004.
Accepted for publication April 5, 2005.
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