Download Regulation of Thyroid Hormone Receptor Isoforms in Physiological

Regulation of Thyroid Hormone Receptor Isoforms in
Physiological and Pathological Cardiac Hypertrophy
Koichiro Kinugawa, Katsunori Yonekura, Ralff C.J. Ribeiro, Yoko Eto, Teruhiko Aoyagi,
John D. Baxter, S. Albert Camacho, Michael R. Bristow, Carlin S. Long, Paul C. Simpson
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
Abstract—Physiological and pathological cardiac hypertrophy have directionally opposite changes in transcription of
thyroid hormone (TH)-responsive genes, including ␣- and ␤-myosin heavy chain (MyHC) and sarcoplasmic reticulum
Ca2⫹-ATPase (SERCA), and TH treatment can reverse molecular and functional abnormalities in pathological
hypertrophy, such as pressure overload. These findings suggest relative hypothyroidism in pathological hypertrophy, but
serum levels of TH are usually normal. We studied the regulation of TH receptors (TRs) ␤1, ␣1, and ␣2 in pathological
and physiological rat cardiac hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH target
genes, ␣- and ␤-MyHC and SERCA. All 3 TR subtypes in myocytes were downregulated in 2 hypertrophy models with
a hypothyroid-like mRNA phenotype, phenylephrine in culture and pressure overload in vivo. Myocyte TR␤1 was
upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine, T3), in culture and exercise in vivo.
In myocyte culture, TR overexpression, or excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs
and promoters. In addition, TR cotransfection and treatment with the TR␤1-selective agonist GC-1 suggested different
functional coupling of the TR isoforms, TR␤1 to transcription of ␤-MyHC, SERCA, and TR␤1, and TR␣1 to ␣-MyHC
transcription and increased myocyte size. We conclude that TR isoforms have distinct regulation and function in rat
cardiac myocytes. Changes in myocyte TR levels can explain in part the characteristic molecular phenotypes in
physiological and pathological cardiac hypertrophy. (Circ Res. 2001;89:591-598.)
Key Words: thyroid hormone receptor 䡲 physiological and pathological hypertrophy 䡲 ␣1-adrenergic receptor
䡲 cardiac myocyte 䡲 rat
C
also called the fetal program.9 The fact that TH treatment can
reverse these genetic changes in some models of pathological
hypertrophy is additional evidence for a hypothyroid state,
but TH blood levels are usually normal.3 Conversely, physiological hypertrophy caused by exercise is characterized by
hyperthyroid-like changes in TH target genes, yet again blood
TH levels are normal.10 Only in cardiac hypertrophy during
normal postnatal development are changes in TH-responsive
genes attributable clearly to altered TH levels.11,12
Transcription of TH target genes is mediated by nuclear
TH receptors (TRs), with 2 genes, ␣ and ␤, encoding at least
4 major TRs, ␤1, ␤2, ␣1, and ␣2,13,14 as well as minor
variants, including TR␤3.15 TRs ␤1, ␣1, and ␣2 are expressed
widely. TR␤2 is present mainly in pituitary cells but might be
a small fraction of T3-binding TRs in heart, as found in one
study.16 TR␣2 binds thyroid response elements (TREs) on
DNA but does not bind 3,3⬘,5-triiodo-L-thyronine (T3), the
ardiac hypertrophy is sometimes considered a single
process that leads invariably to myocardial dysfunction
(pathological hypertrophy). However, physiological hypertrophy exists in which cardiac function is maintained or
enhanced, including normal cardiac development, exercise
training, and thyroid hormone (TH) treatment. Exercise and
TH can reverse molecular and functional abnormalities in
pathological hypertrophy without decreasing ventricular
mass, indicating that physiological and pathological hypertrophy are qualitatively distinct processes.1– 6
TH-responsive genes in cardiac muscle include ␣-myosin
heavy chain (MyHC) and sarcoplasmic reticulum Ca2⫹ATPase (SERCA), which are induced by TH, and ␤-MyHC,
which is repressed.7,8 An intriguing observation is that pathological hypertrophy is characterized by hypothyroid-like
changes in these target genes, with decreases in ␣-MyHC and
SERCA and increases in ␤-MyHC, a molecular phenotype
Original received June 19, 2001; revision received July 26, 2001; accepted July 27, 2001.
From the Division of Cardiology (K.K., M.R.B.), University of Colorado Health Sciences Center, Denver, Colo; Department of Cardiovascular
Medicine (K.Y., Y.E., T.A.), Graduate School of Medicine, University of Tokyo, Japan; Department of Pharmaceutical Sciences (R.C.J.R.), University
of Brasília, Brazil; Metabolic Research Unit, Diabetes Center, and Department of Medicine (J.D.B.), University of California, San Francisco, Calif;
Department of Medicine (Cardiology) (S.A.C.), San Francisco General Hospital, San Francisco, Calif; Cardiology Section (C.S.L.), Denver Health
Medical Center, Denver, Colo; and Veterans Affairs Medical Center and Cardiovascular Research Institute and Department of Medicine (P.C.S.),
University of California, San Francisco, Calif.
Presented in part at the 71st and 73rd Scientific Sessions of the American Heart Association, Dallas, Tex, November 8 –11, 1998, and New Orleans,
La, November 12–15, 2000, and published in abstract form (Circulation. 1998;98[suppl I]:I-837 and 2000;102[suppl II]:II-72).
Correspondence to Paul C. Simpson, MD, VA Medical Center (111-C-8), 4150 Clement St, San Francisco, CA 94121. E-mail [email protected]
© 2001 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
591
592
Circulation Research
September 28, 2001
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
active form of TH, and can function as a dominant negative.13,17 TRs ␤1 and ␣1 bind T3 with similar affinity but
exhibit subtle differences in binding to TREs and cofactors
and in forming homodimers and heterodimers with retinoid X
receptors.13,18 –20 Consistent with these biochemical differences, mouse TR knockouts reveal nonredundant TR functions in many tissues.19 –21 In the heart, however, TR knockouts suggest that the transcription effects of TH are mediated
largely by TR␣1.17,22 TR functions have not been evaluated
systematically in isolated myocytes.
In this study, we tested the hypothesis that hypothyroidand hyperthyroid-like transcription changes in pathological
and physiological hypertrophy are caused by altered TR
levels in heart and myocytes. Recent studies in the aging rat
and in human end-stage heart failure reveal changes in
myocardial TR levels that correlate with changes in TH target
gene transcription.23,24 However, possible changes in TR
levels in hypertrophy have not been examined, and TR
functions on target promoters in myocytes have not been
compared. We measured TRs in rat hypertrophy models in
culture and in vivo and tested TR functions in culture using
transfection and treatment with T3 or a TR␤1-selective
agonist.
Materials and Methods
Cell Culture, Hypertrophy, and Transfection
Ventricular myocytes from 1-day-old rats were plated at low density
in MEM with 5% calf serum and studied in serum-free MEM exactly
as in Deng et al.25 Cardiac nonmyocytes in preplates were expanded
to near confluence in MEM with 5% serum.26 Cultures were treated
with phenylephrine (PE) (Sigma No. P6126), T3 (Sigma No. T5516),
GC-1 (gift from G. Chiellini and T.S. Scanlan, Department of
Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, San Francisco, Calif),18 or their
vehicles (ascorbic acid for PE, NaOH for T3, and DMSO for GC-1).
Myocyte hypertrophy was quantified by content of radiolabeled
protein (RLP) after continuous incubation with 14C- or 3Hphenylalanine. Myocytes were doubly transfected using calcium
phosphate coprecipitation27 with expression plasmids for the human
TRs under control of the cytomegalovirus promoter (CMV)28 and
chloramphenicol acetyl transferase (CAT) reporter plasmids containing the rat promoters for ␣-MyHC (2900 bp), ␤-MyHC (3300 bp), or
SERCA (3500 bp).29
Hypertrophy In Vivo
Hypertrophy was induced in adult male Wistar rats by voluntary
wheel running for 10 weeks (age 7 to 17 weeks) or by ascending
aortic constriction (pressure gradient ⬇75 mm Hg) for 4 weeks (age
10 to 14 weeks).30 Serum levels of total 3,3⬘,5,5⬘-tetraiodo-Lthyronine (T4) and total T3 were measured by ELISA (ICN
Pharmaceuticals).
RNase Protection Assay (RPA) for TH-Responsive
mRNAs
Total RNA was extracted from cells or ventricles with TRIZol
(GIBCO) and used in RPA with a probe for ␣- and ␤-MyHC and a
probe for SERCA2a plus GAPDH as an internal control (see
expanded Materials and Methods, available in the online data
supplement at http://www.circresaha.org).25
TH Receptors
For TR mRNAs, RPA was used with probes specific for the rat TR
isoforms, and GAPDH was used as an internal control (see the
expanded Materials and Methods section).24 For TR proteins, cul-
tured myocyte nuclear and cytoplasmic fractions27 were used in
Western blot with monoclonal antibodies from Santa Cruz (J51/SC737 specific for human TR␤1; J52/SC-738 reactive with rat and
human TR␤1).31 Human and rat TR␤1 proteins for standards were
synthesized using rabbit reticulocyte lysate (TNT T7 Quick Coupled
Transcription/Translation System, Promega).28,32
Statistics
Data shown are mean⫾SE and were compared by one-way ANOVA
and the Newman-Keuls test.
An expanded Materials and Methods section can be found in the
online data supplement available at http://www.circresaha.org.
Results
TH-Responsive mRNAs in Hypertrophy Models in
Culture and In Vivo
We first confirmed regulation of TH target mRNAs in cardiac
hypertrophy models in culture and in vivo (Table 1). In
cultured neonatal rat myocytes, TH (T3 100 nmol/L) caused
hypertrophy, increased ␣-MyHC and SERCA mRNA levels,
and repressed ␤-MyHC to 5% of control. The ␣1-adrenergic
receptor (AR) hypertrophic agonist PE (20 ␮mol/L) had
opposite effects on these mRNAs, increasing ␤-MyHC and
decreasing SERCA and ␣-MyHC. Prazosin (2 ␮mol/L) inhibited all PE effects, confirming ␣1-AR dependence (n⫽3
cultures, data not shown).
Under our basal, serum-free culture conditions, medium T3
levels averaged about 0.1 nmol/L over the 3-day time course
of these experiments, presumably reflecting myocyte release
of stored T3.33–35 Interestingly, when 1000-fold excess T3
(100 nmol/L) was added to PE-treated myocytes, all PE
effects on TH-responsive transcripts were reversed, with
significant increases in ␣-MyHC and SERCA and decreases
in ␤-MyHC (Table 1). The antagonistic relationship between
␣1-AR and T3 signaling was not explained by a lesser degree
of hypertrophy, because hypertrophy was even greater with
PE plus T3 (Table 1).
In intact rat hypertrophy models, voluntary running exercise stimulated hypertrophy with an mRNA profile similar to
T3 in culture (Table 1). Conversely, pressure overload by
ascending aortic constriction caused hypertrophy with
changes in TH target mRNAs opposite to exercise and similar
to PE in culture (Table 1). Thus, in agreement with previous
studies (discussed in the Introduction), 2 hypertrophy models
had signature mRNA changes of heightened thyroid signaling, T3 in culture and exercise in vivo. In contrast, 2 other
models had a directionally opposite pattern of hypothyroidlike signaling, PE in culture and pressure overload in vivo. T3
and T4 blood levels in the intact rat models were similar
among all 4 groups, with total T4 ⬇40 nmol/L (3 to 3.2
␮g/dL) and total T3 ⬇1 nmol/L (52 to 55 ng/dL) (n⫽6 to 13,
P⫽NS), as found by others.3,10 Thus, mRNA changes were
not explained by changes in circulating TH concentration.
TR Levels in Hypertrophy Models in Culture
To test if some change in thyroid signaling besides TH
concentration might explain changes in transcription of THresponsive genes in hypertrophy, we first measured TR levels
in cultured myocytes. In cultured myocytes under basal,
serum-free conditions, RPAs detected the mRNAs for TRs
Kinugawa et al
TABLE 1.
Thyroid Hormone Receptors in Hypertrophy
593
TH-Responsive mRNAs in Hypertrophy Models in Culture and in Vivo
In culture
Vehicle
Cultures, n
5
RLP, %
T3 100 nmol/L
5
PE 20 ␮mol/L
5
194⫾16*
PE⫹T3
5 (RLP 3)
100
148⫾2*
244⫾15†
␣-MyHC
100
256⫾8*
82⫾8
155⫾5†
␤-MyHC
100
5⫾1*
298⫾30*
127⫾8†
SERCA
100
142⫾13*
56⫾10*
103⫾7†
Sham
mRNAs, %
In vivo
Sedentary
Ex
AC
Rats, n
13
29
14
Body, g
467⫾18
459⫾12
484⫾11
494⫾14
Heart, g
1.00⫾0.04
1.12⫾0.02*
1.49⫾0.06†
1.04⫾0.07
Left ventricle, g
0.77⫾0.03
0.88⫾0.01*
1.16⫾0.05†
0.83⫾0.06
LV/body, g/kg
1.66⫾0.03
1.94⫾0.04*
2.40⫾0.08†
1.68⫾0.04
100
117
145
101
Hypertrophy, %
8
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
mRNAs, %
␣-MyHC
100
118⫾1*
43⫾6†
102⫾5
␤-MyHC
100
29⫾2*
262⫾8†
105⫾4
SERCA
100
120⫾2*
50⫾6†
98⫾4
In culture, neonatal rat cardiac myocytes were treated with vehicle, T3, or PE for 72 hours; in the
PE⫹T3 group, T3 was also present for the final 24 hours. RLP was an index of hypertrophy, and MyHC
and SERCA mRNAs were quantified by RNase protection in 3 ␮g total RNA. In adult male rats,
hypertrophy was induced by voluntary running exercise (Ex) for 10 weeks or ascending aortic
constriction (AC) for 4 weeks, and left ventricular (LV) mRNAs were quantified as in culture. The mRNA
levels are normalized to the vehicle or sedentary groups. ␤-MyHC as fraction of total MyHC mRNA
was 44% in vehicle cultures and 18% in sedentary hearts. Values are mean⫾SE.
*P⬍0.05 vs vehicle or sedentary; †P⬍0.05 vs PE or sham.
␣2, ␣1, and ␤1, with a molar ratio of ⬇7:3:1. TR␤2 mRNA
was not found, using a probe validated in rat pituitary GC
cells (not shown), and TR␤3 was not tested. Treatment with
T3 under conditions that stimulated hypertrophy (Table 1)
caused an increase in TR␤1 mRNA, with a maximum
⬇2-fold and an EC50 ⬇3 nmol/L, but had no effect on TR␣2
or ␣1 (Figure 1A). T3 induction of TR␤1 in myocytes was
significant as early as 12 hours and maximum at 48 hours
(data not shown). Thus TR␤1 itself was a TH-responsive
gene in myocytes and was increased in T3-stimulated
hypertrophy.
PE-induced hypertrophy in cultured myocytes had an
effect different from T3 and reduced the levels of all 3 TR
isoform mRNAs to 50% to 75% of control (Figure 1B). The
EC50 for TR repression was about 3 ␮mol/L (Figure 1B),
similar to the EC50 for PE-induced hypertrophy, and the PE
effect was detected as early as 6 hours (data not shown). PE
is an ␣1-AR agonist with weak ␤-AR activity, and specific
␣1-AR repression of TRs was confirmed in separate experiments with norepinephrine, isoproterenol, prazosin, and timolol (all 2 ␮mol/L, n⫽3 to 4 cultures for each, data not shown).
Interestingly, and similar to the other TH target mRNAs
(Table 1), addition of T3 reversed PE repression of TR␤1, but
TRs ␣1 and ␣2 were not changed (Figure 1B).
To test if TR mRNA levels reflected TR protein, we
measured endogenous TRs in cultured myocytes by Western
blot. None of the commercial antibodies from Santa Cruz or
Affinity BioReagent gave specific signals for TRs ␣1 and ␣2
(not shown), but TR␤1 protein was detected in nuclear
extracts (Figure 1). PE reduced TR␤1 protein significantly,
whereas T3 increased TR␤1 and reversed TR␤1 repression
caused by PE (Figures 1A and 1B). Thus TR␤1 mRNA and
protein changes were concordant.
Cultured cardiac nonmyocytes also expressed mRNAs for
all 3 of the TRs found in myocytes, ␣1, ␣2, and ␤1, but levels
were not altered by PE 20 ␮mol/L or T3 100 nmol/L (n⫽3
cultures, data not shown). No response to PE can be explained by the absence of ␣1-ARs in nonmyocytes.36 No
response to T3 is compatible with a requirement for cellspecific factors such as MEF-2 in myocyte TR signaling.37,38
TR mRNA Levels in Hypertrophy Models In Vivo
Control adult male rat hearts contained mRNAs for TRs ␣2,
␣1, and ␤1 at equal molar ratios (⬇1:1:1). Physiological
hypertrophy stimulated by voluntary running exercise mimicked T3 in culture, with an increase in TR␤1 mRNA and no
change in TRs ␣1 and ␣2 (Figure 2). Pathological hypertrophy stimulated by ascending aortic constriction was similar to
PE in culture, with decreases in all 3 TR isoform mRNAs
(Figure 2), detectable as early as 1 week after banding (not
shown). Thus, in distinct hypertrophy models in vivo as in
culture, TR isoform levels and TH-responsive transcript
levels were related directly (Figure 2 and Table 1).
Cotransfection of TR Isoforms and
TH-Responsive Promoters
To test directly whether changes in TR levels could alter
TH-responsive transcription, we cotransfected cultured myo-
594
Circulation Research
September 28, 2001
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
Figure 1. Regulation of TR isoforms with hypertrophy in culture.
Cultured neonatal rat cardiac myocytes were treated with T3 (A)
or PE (B) for 72 hours, and TR isoform mRNAs were assayed in
10 ␮g total RNA by RNase protection (left and middle). Alternatively, cells were treated for 120 hours, and TR␤1 protein in
equal numbers of myocyte nuclei was quantified by Western
blot (right). In the PE⫹T3 groups, cells treated with PE 20
␮mol/L were also treated with T3 100 nmol/L for the final 24
hours (mRNA) or 72 hours (protein). Representative gels are
shown; mean data are from 5 cultures for mRNA and 3 cultures
for protein.
cytes with expression plasmids for the human TR isoforms
and reporter plasmids for the TH-responsive genes (Figure 3).
The Western blot in Figure 3A shows that human TR␤1 was
detected in nuclear extracts from TR␤1-transfected myocytes,
with overflow of transfected TR␤1 into the cytosolic fraction,
suggesting saturating levels.31 Similarly, an antibody recognizing both human and rat TR␤1 indicated that total TR␤1
nuclear levels in transfected cultures were about 2-fold over
endogenous (Figure 3A). With ⬇5% of myocytes transfected,39 this result implied about 40-fold overexpression of
TR␤1.
We used this system to test the effects of increasing TR
isoform levels on ␣-MyHC, ␤-MyHC, and SERCA promoters. In vehicle-treated control myocytes, overexpression of
TR␣1 activated the ␣-MyHC promoter, and TR␤1 repressed
␤-MyHC (Figure 3B). TR␣2 had no effect. Induction of
␣-MyHC indicated that TR␣1 was expressed, even though
Western blots were unsatisfactory for TR␣ protein levels.
In PE-treated myocytes transfected with empty vector, the
␤-MyHC promoter was activated, and ␣-MyHC and SERCA
promoters were repressed (Figure 3C), similar to the PE
effects on endogenous mRNAs (Table 1). Increasing the
levels of TR␣1 by transfection reversed PE repression of
␣-MyHC, whereas TR␤1 overexpression significantly op-
Figure 2. Regulation of TR isoforms with hypertrophy in vivo.
Hypertrophy was induced in adult male rats by voluntary running exercise (Ex) for 10 weeks or ascending aortic constriction
(AC) for 4 weeks, and left ventricular TR isoform mRNAs were
assayed as in Figure 1. Top, Representative gel is shown. Bottom, Mean values from the indicated numbers of rats, normalized to the sedentary control group.
posed the effects of PE on both ␤-MyHC and SERCA.
Interestingly, forced expression of TR␣2, the non-T3-binding
TR, slightly but significantly enhanced PE repression of
␣-MyHC and SERCA (Figure 3C).
In T3-treated myocytes transfected with empty vector, the
␤-MyHC promoter was repressed and ␣-MyHC and SERCA
were activated (Figure 3D), as expected from T3-induced
mRNA changes (Table 1). Overexpression of TR␤1 in
T3-treated myocytes significantly enhanced induction of the
SERCA promoter, but TRs ␣1 and ␣2 had no effects over T3
alone (Figure 3D).
Taken together, these cotransfection results showed that
increasing the levels of T3-binding TRs altered THresponsive transcription and antagonized the effects of PE on
TH-responsive genes. In addition, the results suggested different functions of TRs in myocytes, regulation of ␣-MyHC
by TR␣1, and regulation of ␤-MyHC and SERCA by TR␤1.
The repressive effect of TR␣2 only in PE-treated cells
Kinugawa et al
Thyroid Hormone Receptors in Hypertrophy
595
TABLE 2. Potencies of T3 and GC-1, a TR␤1-Selective
Agonist, in Cultured Myocyte Transcription and Hypertrophy
EC50, nmol/L
Assay
mRNA level and promoter activity
␤-MyHC
T3
GC-1
0.06–0.09
0.05–0.08
SERCA
0.6–0.8
0.7–1
TR␤1
3
2
␣-MyHC
RLP
0.1–0.2
1
0.1
2
EC50s were estimated from the dose-response curves in Figures 1A and 4A
through 4C. The curve for TR␤1 mRNA induction by GC-1 is not shown, and
TR␤1 promoter activity was not tested. GC-1 is at least 10-fold less potent than
T3 in stimulation of ␣-MyHC and RLP; the true EC50s for GC-1 induction of
␣-MyHC and RLP might be even higher, because the responses are not quite
saturated at the highest doses studied (Figures 4B and 4C).
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
␣-MyHC and RLP. These data were consistent with the
cotransfection experiments (Figure 3) and suggested preferential coupling of TR␤1 to ␤-MyHC and SERCA, whereas
TR␣1 regulated ␣-MyHC and total protein content. In addition, GC-1 increased TR␤1 mRNA by 203⫾28% at 100
nmol/L with an EC50 of 2 nmol/L (P⬍0.05 versus vehicle,
n⫽5), almost identical to the effect of T3 (Figure 1 and Table
2), consistent with the notion that TR␤1 activated its own
transcription.
Figure 3. TR transfection in cultured myocytes. Cultured myocytes were cotransfected with CAT reporter plasmids (5 ␮g) and
CMV-driven human TR expression plasmids or equal amounts
of empty CMV vector (2 to 5 ␮g). SV40-driven secreted alkaline
phosphatase (1 ␮g, Clontech) was included to control for transfection efficiency. A, Western blots used TR␤1-specific antibodies from Santa Cruz (human-specific J51/SC-737 and rat- and
human-reactive J52/SC-738) with myocyte nuclear and cytosolic
extracts after 72 hours of transfection (left and middle). Rat and
human TR␤1 proteins from in vitro transcription/translation were
used as standards (⬇55-kD) (right). B through D, Myocytes were
treated for 72 hours after transfection with vehicle (B); PE 20
␮mol/L (C); or T3 10 nmol/L (D). Values are from 5 cultures.
(Figure 3C) was consistent with a weak dominant-negative
effect of this non–T3-binding TR (see the Introduction) that
could be detected only when levels of the T3-binding TRs
were reduced by PE (Figure 1B).
Effects of a TR␤1-Selective Agonist on Myocyte
Transcription and Hypertrophy
To test different functional roles for TR isoforms by a second
approach not involving transfection, we treated myocytes
with GC-1, which has ⬇10-fold selectivity for TR␤1 versus
TR␣1 but has not been tested with TRs ␤2 and ␤3, and
compared the responses to T3, which binds with similar
affinity to both TRs ␤1 and ␣1.18,35 We assayed both
TH-responsive promoter activities and endogenous mRNAs
(Figures 4A and 4B) and also RLP, an index of total protein
content or overall myocyte size (Figure 4C). As shown in
Figures 4A through 4C and summarized in Table 2, the
EC50s for GC-1 induction of SERCA and repression of
␤-MyHC were identical to the EC50s for T3, whereas GC-1
was at least 10-fold less potent than T3 in induction of
Discussion
The main new finding in this study was the direct relationship
between TR levels and transcription of TH-responsive genes
in different types of hypertrophy. All TRs were downregulated in pressure overload, a model of pathological hypertrophy with a hypothyroid-like molecular phenotype. Conversely, TR␤1 was upregulated in the exercise model of
physiological hypertrophy, where TH-responsive transcription was enhanced. These results, together with the TR
overexpression experiments in cultured myocytes, suggest
that the distinct molecular signatures in different types of
hypertrophy can be caused in part by altered TR levels. Thus
the fetal program in pathological hypertrophy can be viewed
as the result of cellular hypothyroidism, caused by reduced
TR levels, whereas physiological hypertrophy can be seen as
a state of relative cellular hyperthyroidism, attributable to
increased TR␤1. The results also indicate that TR isoforms
have different transcription functions in rat myocytes and
raise the possibility of additional alteration of TH signaling in
hypertrophy.
We detected 3 TRs in rat myocytes, ␤1, ␣1, and ␣2, and all
were reduced by pressure overload in vivo and ␣1-AR
signaling in culture (Figures 1 and 2), the models with
hypothyroid-like transcription changes (Table 1). Reversal of
hypothyroid signaling when TRs ␤1 and ␣1 were overexpressed in the PE culture model supported the causal significance of TR downregulation (Figure 3C). Downregulation of
dominant-negative TR␣2 in both models would have been
expected to partly counteract the hypothyroid effects of
decreased TRs ␤1 and ␣1, because overexpressed TR␣2 had
hypothyroid effects when TRs ␤1 and ␣1 were reduced by PE
(Figure 3C). Nevertheless, global TR downregulation in both
596
Circulation Research
September 28, 2001
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
Figure 4. Effects of GC-1, a TR␤1 agonist, in cultured myocytes. Cultured myocytes were treated for 72 hours with the
indicated doses of T3 or GC-1. A, Myocytes were transfected with 5 ␮g of each
CAT reporter plasmid. B, Endogenous
mRNAs were assayed in 3 ␮g total RNA
by RNase protection. C, Cells were
labeled continuously with 14Cphenylalanine to quantify RLP. Values
are from 3 to 4 cultures.
models of pathological hypertrophy was associated with
reduced transcription of TH target genes.
Abnormal TR levels are also seen at a terminal stage of
pathological hypertrophy, severe human heart failure. In
human heart failure, TR␣1 is decreased and TR␣2 is increased, probably because of altered splicing of the TR␣
gene, and the increase in TR␣2 correlates with reduced
␣-MyHC transcription.24 In our rat models of PE treatment
and pressure overload, the global decrease in all TR mRNAs
could be explained by reduced TR gene transcription or
increased TR mRNA degradation. Thus the specific mechanism for altered TR levels might differ among species or
models, but the human and rat studies both suggest that TR
downregulation is a mechanism for hypothyroid-like signaling in pathological hypertrophy. This relationship was additionally strengthened by our converse results in physiological
models.
TR upregulation was seen in exercise, a model of physiological hypertrophy with normal or enhanced contractile
function.30 Voluntary running exercise caused an increase in
TR␤1 but no change in TR␣ (Figure 2). T3 in culture also
caused selective induction of TR␤1 (Figure 1) and similarly
induces a physiological hypertrophy in vivo.5 Our finding that
the TR␤1 gene is TH-responsive in myocytes but the TR␣
gene is not agrees with some studies in the intact rat,40,41 but
not with others,42 and can be explained by positive TREs in
the TR␤1 promoter that are absent in TR␣.43,44 Furthermore,
the TR␤1 gene seemed to be activated by TR␤1 itself,
because T3 and the TR␤1-selective agonist GC-1 induced
TR␤1 with similar potency (Table 2). Thus, exercise-induced
increases in TR␤1 levels should be partly self-sustaining, and
the increase in TR␤1 would augment transcription of downstream TH target genes on the basis of our transfection studies
(Figure 3).
TR␤1 in myocytes differed from TR␣1 in induction by T3
and also in function. In this study, we made the first
systematic comparison of TR function in isolated myocytes
and found by 2 complimentary approaches that TR␤1 induced SERCA and TR␤1 itself and repressed ␤-MyHC,
whereas TR␣1 increased ␣-MyHC and protein content (Figures 3 and 4 and Table 2). Notably, the same pattern of TR
function was seen with TR cotransfection and with GC-1,
which has ⬇10-fold selectivity for TR␤1.18 Distinct TR
isoform effects on different promoters might be explained by
isoform-specific interactions with cofactors or differing affinities for TREs (see the Introduction). Interestingly, target
gene–specific differences were evident even for a single TR
isoform in that TR␤1 more potently repressed ␤-MyHC than
it activated SERCA and activated SERCA more potently than
TR␤1 itself (Table 2). Also intriguing was the correlation of
TR␣1 signaling with increased total protein content, an index
of overall hypertrophy (Figure 4C and Table 2). Increased
Kinugawa et al
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
protein content requires increased translation as well as
transcription, thus raising the question of nongenomic effects
of T3 via TR␣1.
Previous results from rat and human models are consistent
with the distinct transcription functions of TR isoforms we
show in this study. In the aging rat, increased ␤-MyHC
transcription correlates with decreased TR␤1 levels and
reduced ␣-MyHC correlates with reduced TR␣1.23 A correlation between levels of TR␣1 and ␣-MyHC is also seen in
the failing human heart.24 A preferential effect of GC-1 on
myocardial SERCA and ␤-MyHC is not seen in the rat in
vivo, but this might be because of lower bioavailability of
GC-1 in cardiac tissue.35 On the other hand, the mouse might
be different from rat and human, because knockout studies
suggest that most cardiac effects of T3 can be accounted for
by TR␣1.17,22 Whether both TRs are required for full TH
effects in heart is one key consideration in potential therapeutic uses of TH signaling,8 and additional study is needed.
Finally, the question arises of whether other mechanisms
alter TH signaling in hypertrophy in addition to changes in
TR levels. Pertinent is the effect of T3 in the culture model,
where a 1000-fold increase in medium T3 (from 0.1 to 100
nmol/L) reversed PE antagonism of all TH-responsive genes,
including the MyHCs, SERCA, and TR␤1, despite PE reduction of TR levels (Table 1 and Figure 1). Thus, excess T3 in
culture could counteract the hypothyroid effects of reduced
TRs. In vivo as well, high doses of T3 reverse hypothyroid
transcription in pressure overload models3,5 despite the reduced TR levels we show in this study (Figure 2). The
reversal effect of excess T3 can be explained partly by
induction of TR␤1 and downstream changes in other THresponsive genes, but the T3 effect also suggests that cellular
T3 levels might be limiting in some cases. Thus it is possible
that myocyte T3 metabolism33–35 is changed in hypertrophy,
with increases in cellular T3 with exercise and decreases with
pressure overload and PE, and this requires additional study.
In summary, changes in TR levels can explain at least in
part the characteristic hypothyroid- and hyperthyroid-like
transcription in different types of hypertrophy. TH signaling
is impaired in pathological hypertrophy and enhanced in
physiological hypertrophy.
Acknowledgments
This work was supported in part by grants from the National
Institutes of Health to P.C.S. and C.S.L. J.D.B. is consultant and
Deputy Director for Karo Bio AB, which has commercial interests in
this area of research. We thank Marietta Paninbatan, Heather Lucas,
Anna Casulo, Yuren Wei, Gail Morris, and Mary Atz for excellent
technical assistance.
References
1. Scheuer J, Malhotra A, Hirsch C, Capasso J, Schaible TF. Physiologic
cardiac hypertrophy corrects contractile protein abnormalities associated
with pathologic hypertrophy in rats. J Clin Invest. 1982;70:1300 –1305.
2. Schaible TF, Ciambrone GJ, Capasso JM, Scheuer J. Cardiac conditioning ameliorates cardiac dysfunction associated with renal hypertension in rats. J Clin Invest. 1984;73:1086 –1094.
3. Izumo S, Lompre AM, Matsuoka R, Koren G, Schwartz K, Nadal-Ginard
B, Mahdavi V. Myosin heavy chain messenger RNA and protein isoform
transitions during cardiac hypertrophy: interaction between hemodynamic
and thyroid hormone-induced signals. J Clin Invest. 1987;79:970 –977.
Thyroid Hormone Receptors in Hypertrophy
597
4. Orenstein TL, Parker TG, Butany JW, Goodman JM, Dawood F, Wen
WH, Wee L, Martino T, McLaughlin PR, Liu PP. Favorable left ventricular remodeling following large myocardial infarction by exercise
training: effect on ventricular morphology and gene expression. J Clin
Invest. 1995;96:858 – 866.
5. Chang KC, Figueredo VM, Schreur JHM, Kariya K, Weiner MW,
Simpson PC, Camacho SA. Thyroid hormone improves function and Ca2⫹
handing in pressure overload hypertrophy: association with increased
sarcoplasmic reticulum Ca2⫹-ATPase and ␣-myosin heavy chain in rat
hearts. J Clin Invest. 1997;100:1742–1749.
6. Gomberg-Maitland M, Frishman WH. Thyroid hormone and cardiovascular disease. Am Heart J. 1998;135:187–196.
7. Dillmann WH. Biochemical basis of thyroid hormone action in the heart.
Am J Med. 1990;88:626 – 630.
8. Klein I, Ojamaa K. Thyroid hormone and the cardiovascular system.
N Engl J Med. 2001;344:501–509.
9. Simpson PC, Long CS, Waspe LE, Henrich CJ, Ordahl CP. Transcription
of early developmental isogenes in cardiac myocyte hypertrophy. J Mol
Cell Cardiol. 1989;21(suppl V):79 – 89.
10. Rupp H, Wahl R. Influence of thyroid hormones and catecholamines on
myosin of swim-exercised rats. J Appl Physiol. 1990;68:973–978.
11. Chizzonite RA, Zak R. Regulation of myosin isoenzyme composition in
fetal and neonatal rat ventricle by endogenous thyroid hormones. J Biol
Chem. 1984;259:12628 –12632.
12. Lompre AM, Nadal-Ginard B, Mahdavi V. Expression of the cardiac
ventricular ␣- and ␤-myosin heavy chain genes is developmentally and
hormonally regulated. J Biol Chem. 1984;259:6437– 6446.
13. Lazar MA. Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev. 1993;14:184 –193.
14. Ribeiro RC, Apriletti JW, Wagner RL, West BL, Feng W, Huber R,
Kushner PJ, Nilsson S, Scanlan T, Fletterick RJ, Schaufele F, Baxter JD.
Mechanisms of thyroid hormone action: insights from x-ray crystallographic and functional studies. Recent Prog Horm Res. 1998;53:351–392.
15. Williams GR. Cloning and characterization of two novel thyroid hormone
receptor ␤ isoforms. Mol Cell Biol. 2000;20:8329 – 8342.
16. Schwartz HL, Lazar MA, Oppenheimer JH. Widespread distribution of
immunoreactive thyroid hormone ␤2 receptor (TR ␤2) in the nuclei of
extrapituitary rat tissues. J Biol Chem. 1994;269:24777–24782.
17. Macchia PE, Takeuchi Y, Kawai T, Cua K, Gauthier K, Chassande O,
Seo H, Hayashi Y, Samarut J, Murata Y, Weiss RE, Refetoff S. Increased
sensitivity to thyroid hormone in mice with complete deficiency of
thyroid hormone receptor ␣. Proc Natl Acad Sci U S A. 2001;98:
349 –354.
18. Chiellini G, Apriletti JW, al Yoshihara H, Baxter JD, Ribeiro RC, Scanlan
TS. A high-affinity subtype-selective agonist ligand for the thyroid
hormone receptor. Chem Biol. 1998;5:299 –306.
19. Hsu J-H, Brent GA. Thyroid hormone receptor gene knockouts. Trends
Endocrinol Metab. 1998;9:103–112.
20. Zhang J, Lazar MA. The mechanism of action of thyroid hormones. Annu
Rev Physiol. 2000;62:439 – 466.
21. Forrest D, Vennstrom B. Functions of thyroid hormone receptors in mice.
Thyroid. 2000;10:41–52.
22. Gloss B, Trost SU, Bluhm WF, Swanson EA, Clark R, Winkfein R,
Janzen KM, Giles W, Chassande O, Samarut J, Dillmann WH. Cardiac
ion channel expression and contractile function in mice with deletion of
thyroid hormone receptor ␣ or ␤. Endocrinology. 2001;142:544 –550.
23. Long X, Boluyt MO, O’Neill L, Zheng JS, Wu G, Nitta YK, Crow MT,
Lakatta EG. Myocardial retinoid X receptor, thyroid hormone receptor,
and myosin heavy chain gene expression in the rat during adult aging. J
Gerontol A Biol Sci Med Sci. 1999;54:B23–B27.
24. Kinugawa K, Minobe WA, Wood WM, Ridgway EC, Baxter JD, Ribeiro
RC, Tawadrous MF, Lowes BA, Long CS, Bristow MR. Signaling
pathways responsible for fetal gene expression in the failing human heart:
evidence for altered thyroid hormone receptor gene expression. Circulation. 2001;103:1089 –1094.
25. Deng X-F, Rokosh DG, Simpson PC. Autonomous and growth factorinduced hypertrophy in cultured neonatal mouse cardiac myocytes: comparison with rat. Circ Res. 2000;87:781–788.
26. Palmer JP, Hartogensis WE, Patten M, Fortuin FD, Long C.
Interleukin-1␤ induces cardiac myocyte growth but inhibits cardiac
fibroblast proliferation in culture. J Clin Invest. 1995;95:2555–2564.
27. Kariya K, Karns LR, Simpson PC. An enhancer core element mediates
stimulation of the rat ␤-myosin heavy chain promoter by an ␣1-adrenergic
agonist and activated ␤-protein kinase C in hypertrophy of cardiac
myocytes. J Biol Chem. 1994;269:3775–3782.
598
Circulation Research
September 28, 2001
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
28. Umesono K, Evans RM. Determinants of target gene specificity for
steroid/thyroid hormone receptors. Cell. 1989;57:1139 –1146.
29. Patten M, Hartogensis WE, Long CS. Interleukin-1␤ is a negative transcriptional regulator of ␣1-adrenergic induced gene expression in cultured
cardiac myocytes. J Biol Chem. 1996;271:21134 –21141.
30. Eto Y, Yonekura K, Sonoda M, Arai N, Sata M, Sugiura S, Takenaka K,
Gualberto A, Hixon ML, Wagner MW, Aoyagi T. Calcineurin is activated
in rat hearts with physiological left ventricular hypertrophy induced by
voluntary exercise training. Circulation. 2000;101:2134 –2137.
31. Lin KH, Willingham MC, Liang CM, Cheng SY. Intracellular distribution
of the endogenous and transfected beta form of thyroid hormone nuclear
receptor visualized by the use of domain-specific monoclonal antibodies.
Endocrinology. 1991;128:2601–2609.
32. Ribeiro RC, Kushner PJ, Apriletti JW, West BL, Baxter JD. Thyroid
hormone alters in vitro DNA binding of monomers and dimers of thyroid
hormone receptors. Mol Endocrinol. 1992;6:1142–1152.
33. Everts ME, Verhoeven FA, Bezstarosti K, Moerings EP, Hennemann G,
Visser TJ, Lamers JM. Uptake of thyroid hormones in neonatal rat cardiac
myocytes. Endocrinology. 1996;137:4235– 4242.
34. Ribeiro RC, Cavalieri RR, Lomri N, Rahmaoui CM, Baxter JD, Scharschmidt BF. Thyroid hormone export regulates cellular hormone content
and response. J Biol Chem. 1996;271:17147–17151.
35. Trost SU, Swanson E, Gloss B, Wang-Iverson DB, Zhang H, Volodarsky
T, Grover GJ, Baxter JD, Chiellini G, Scanlan TS, Dillmann WH. The
thyroid hormone receptor–selective agonist GC-1 differentially affects
plasma lipids and cardiac activity. Endocrinology. 2000;141:3057–3064.
36. Stewart AFR, Rokosh DG, Bailey BA, Karns LR, Chang KC, Long CS,
Kariya K, Simpson PC. Cloning of the rat ␣1C-adrenergic receptor from
37.
38.
39.
40.
41.
42.
43.
44.
cardiac myocytes: ␣1C, ␣1B, and ␣1D mRNAs are present in cardiac
myocytes, but not in cardiac fibroblasts. Circ Res. 1994;75:796 – 802.
Lee Y, Nadal-Ginard B, Mahdavi V, Izumo S. Myocyte-specific enhancer
factor 2 and thyroid hormone receptor associate and synergistically activate the ␣-cardiac myosin heavy-chain gene. Mol Cell Biol. 1997;17:
2745–2755.
Moriscot AS, Sayen MR, Hartong R, Wu P, Dillmann WH. Transcription
of the rat sarcoplasmic reticulum Ca2⫹ adenosine triphosphatase gene is
increased by 3,5,3⬘-triiodothyronine receptor isoform-specific interactions with the myocyte-specific enhancer factor-2a. Endocrinology.
1997;138:26 –32.
Kariya K, Karns LR, Simpson PC. Expression of a constitutively-activated mutant of the ␤-isozyme of protein kinase C in cardiac myocytes
stimulates the promoter of the ␤-myosin heavy chain isogene. J Biol
Chem. 1991;266:10023–10026.
Bishop CM, McCabe CJ, Gittoes NJ, Butler PJ, Franklyn JA. Tissuespecific regulation of thyroid hormone receptor mRNA isoforms and
target gene proteins in domestic ducks. J Endocrinol. 2000;165:607– 615.
Haddad F, Qin AX, McCue SA, Baldwin KM. Thyroid receptor plasticity
in striated muscle types: effects of altered thyroid state. Am J Physiol.
1998;274:E1018 –E1026.
Balkman C, Ojamaa K, Klein I. Time course of the in vivo effects of
thyroid hormone on cardiac gene expression. Endocrinology. 1992;130:
2001–2006.
Ishida T, Yamauchi K, Ishikawa K, Yamamoto T. Molecular cloning and
characterization of the promoter region of the human c-erbA ␣ gene.
Biochem Biophys Res Commun. 1993;191:831– 839.
Sakurai A, Miyamoto T, DeGroot LJ. Cloning and characterization of the
human thyroid hormone receptor ␤1 gene promoter. Biochem Biophys Res
Commun. 1992;185:78 – 84.
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
Regulation of Thyroid Hormone Receptor Isoforms in Physiological and Pathological
Cardiac Hypertrophy
Koichiro Kinugawa, Katsunori Yonekura, Ralff C.J. Ribeiro, Yoko Eto, Teruhiko Aoyagi, John
D. Baxter, S. Albert Camacho, Michael R. Bristow, Carlin S. Long and Paul C. Simpson
Circ Res. 2001;89:591-598; originally published online September 13, 2001;
doi: 10.1161/hh1901.096706
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/89/7/591
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2001/09/11/hh1901.096706.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
2901/R1
online methods
Expanded Methods: Thyroid Hormone Receptors in Hypertrophy
Expanded Methods. Thyroid Hormone Receptors in Hypertrophy, Kinugawa et al.
RNase protection assays for TRs, MyHCs, and SERCA.
Probes for TR RNase protection assays
A. TRα probe
PF 538 bp
Probe
TR α1 mRNA
Pst I
DBD
Xba I
LBD
TRα1 protein
Probe
TR α2 mRNA
PF 159 bp
Pst I
DBD
TRα2 protein
B. TRβ1 probe
PF 329 bp
Probe
TRβ1 mRNA
Xba I
DBD
LBD
TRβ1 protein
C. TRβ2 probe
PF 521 bp
Probe
TRβ2 mRNA
Xba I
LBD
DBD
TRβ2 protein
The Figure depicts the riboprobes used in RNase protection assay for TR mRNAs.
Similar shading within TR α or β isoforms indicates identical mRNA sequence. PF is the mRNA
1
Expanded Methods: Thyroid Hormone Receptors in Hypertrophy
fragment protected by the probe. The corresponding TR proteins are shown, with the DNAbinding domain (DBD) and ligand-binding domain (LBD) indicated. The TRα probe (Panel A)
contained a PstI-XbaI fragment of rat TRα1 cDNA and protected a fragment of 538-bp for TRα1
(1279-1816 nucleotides of GenBank Accession number M18028) and 159-bp for TRα2 (14711629 nucleotides of M31174). The TRβ1-specific probe (Panel B) contained the fragment from
the 5’-untranslated region to an XbaI site and protected 323-bp (227-555 nucleotides of J03933).
The TRβ2-specific probe (Panel C) contained the fragment from the 5’-untranslated region to an
XbaI site and protected 521-bp (1-521 nucleotides of M25071). All plasmids for riboprobes were
sequenced, and linealized at the 3’-end of the antisense strand with appropriate restriction
enzymes. TR probes were made with a specific activity of ~9x108 cpm/µg, and hybridized with
10 µg total RNA. After incubation at 42°C overnight, the RNA-probe mix was digested with
RNase A/T1 (1:100, RPAII, Ambion) at 37°C for 30 min, and protected fragments were
separated on a 6% denaturing polyacrylamide gel. In each gel lane, all TR isoforms, and
GAPDH as an internal control, were quantified by densitometry and corrected for background.
TR signals were normalized to GAPDH.
The StyI-digested rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe (pTRIGAPDH-Rat, Ambion) protected a 134-bp fragment (complimentary to nucleotides 369-503 of
X02231). The GAPDH probe (specific activity ~2x108 cpm/µg for experiments with MyHC and
SERCA, ~0.5x108 cpm/µg for experiments with TRs) was mixed in each sample as an internal
control.
The probe specific for the rat SERCA2 gene protected a 333-bp fragment of SERCA2a
(3014-3347 nucleotides of X15635). The probe for rat MyHC was complimentary to the 3’-end
of β-MyHC mRNA; it protected a 300-bp fragment (5626-5925 nucleotides of X15939), but also
hybridized to α-MyHC, and protected a 175-bp fragment (5656-5830 nucleotides of X15938).
Riboprobes were labeled with [α-32P]UTP using Maxiscript kit (Ambion). The MyHC and
SERCA probes had a specific activity of ~0.5x108 and ~1.0x108 cpm/µg, respectively, and were
2
Expanded Methods: Thyroid Hormone Receptors in Hypertrophy
used with 3 µg total RNA. All probe mixes were run on the same gel with RNA from the in vivo
experiments.
3