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337 Threonine Thyroid Hormone Receptor ß Mutant Derived from the S Family1
Department of Medicine, Division of Endocrinology and Metabolism, University of California-San Diego, La Jolla, California 92093-0618
Address all correspondence and requests for reprints to: Wolfgang H. Dillmann, M.D., Department of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0618. E-mail: wdillman{at}ucsd.edu
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Abstract |
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337T) derived
from the S kindred. The mutant receptor was originally identified in a
patient with generalized resistance to thyroid hormone. Cardiac
expression of the mutant receptor was achieved by a transgenic approach
in mice. As the genes for myosin heavy chains (MHC
and MHCß) and
the cardiac sarcoplasmic reticulum Ca2+ adenosine
triphosphatase (SERCA2) are known to be regulated by T3,
their cardiac expression was analyzed. The messenger RNA levels for
MHC and SERCA2 were markedly down-regulated, MHCß messenger RNA
was up-regulated. Although T3 levels were normal in these
animals, this pattern of cardiac gene expression mimics a hypothyroid
phenotype. Cardiac muscle contraction was significantly prolonged in
papillary muscles from transgenic mice. The electrocardiogram of
transgenic mice showed a substantial prolongation of the QRS interval.
Changes in cardiac gene expression, cardiac muscle contractility, and
electrocardiogram are compatible with a hypothyroid cardiac phenotype
despite normal T3 levels, indicating a dominant negative
effect of the T3Rß mutant. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß
receptors, resulting in a dominant negative action. In one kindred with
RTH, designated the S family, resulting from a deletion of threonine at
position 337, a single patient was found to be homozygous for the
T3Rß mutant (10). This child showed profound resistance
to thyroid hormone and developed a hyperactive syndrome accompanied by
marked tachycardia contributing to the child’s early demise. To
explore in further detail the influence that the expression of a mutant
T3Rß in cardiac myocytes has on cardiac gene expression
and the cardiac phenotype, we generated transgenic mice with the mutant
complementary DNA (cDNA). These were bred for homozygosity and used for
all experiments throughout our study. In this mouse model, thyroid
hormone levels were normal. However, the expression of myosin heavy
chain (MHC) isoforms and the gene coding for the Ca2+
adenosine triphosphatase (ATPase) of the sarcoplasmic reticulum as well
as cardiac muscle contractile changes and prolongation of the QRS
interval in the electrocardiogram (ECG), resembled a significantly
hypothyroid cardiac phenotype. The T3Rß mutant induced
changes are described in further detail in this report. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. For the generation of
transgenic animals, a large amount of plasmid was cleaved with
SalI and HindIII, and the resulting fragment was
purified. The protocol to generate transgenic mice was described by
Hogan (12). Briefly, the female pronuclei of eggs from superovulated
C57BL/6 x BALB/c mice were injected with 1–2 pl of the purified
DNA fragment at a concentration of 2 µg/ml. The injected eggs were
then transferred into the oviduct of pseudopregnant BALB/c mice.
Litters were delivered after approximately 20 days gestation.
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). If
the transgene was inserted into the mouse genome, ApaI and
PstI digestion would generate a 1579-bp fragment that
specifically hybridized to the probe. To determine the number of copies of the transgene integrated into the genome of the transgenic mice, 0.5–2.0 µg genomic DNA were used, previously isolated from the tails of wild-type and homozygous transgenic mice. DNA was ribonuclease treated and bound to a nylon membrane with a slot blot apparatus. The blot was probed with a 220-bp 5'-fragment corresponding to the first 70 amino acids of the hT3Rß, and the slope of a line, plotting photographic density on the y-axis and DNA concentration on the x-axis, showed that there was one copy of the transgene integrated into one chromosome of the transgenic mice.
Blood from wild-type and transgenic mice was collected to determine the content of circulating total T3 and T4 hormones. Serum analysis was performed at the University of California-San Diego Medical Center/Clinical Chemistry Laboratory. In addition, TSH levels were determined by Dr. Forrest’s laboratory (Mt. Sinai Hospital, New York, NY) (13).
RNA isolation and Northern blot analysis
Isolation of tissue RNA was performed as described by
Chomczynski and Sacchi (14). For electrophoresis, Northern transfer,
and hybridization, the protocol was as described previously (15). RNA
was visualized and photographed before transfer to Magna Graph
membranes (Micron Separations Inc., Westboro, MA) overnight in 10
x SSC (standard saline citrate). Membranes were subsequently baked at
80 C in a vacuum oven for 1–2 h. The probes used to study gene
transcription are specified as follows. To characterize the
hT3Rß transgene expression, a full-length rat
T3R cDNA was used. Restriction fragments of cDNAs for
SERCA2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were
isolated and used to characterize the expression of these genes in the
heart of wild-type and transgenic animals. Highly specific
oligonucleotides complementary to the MHC or MHCß messenger RNA
(mRNA) served to detect their respective mRNA on Northern blots.
The oligonucleotides were labeled using terminal deoxynucleotide
transferase and [-32P]deoxy-CTP. SERCA2, rat
T3R, and GAPDH fragments were labeled with
[-32P]deoxy-CTP by a random hexamer priming protocol
(16) to a specific activity of about 5 x 108 cpm/µg
and hybridized to Northern filters as described previously in a 50%
formamide-containing solution at 42 C overnight. Filters were
subsequently washed to a stringency of 0.5 x SSC (1 x
SSC = 0.15 M NaCl and 0.015 M sodium
citrate, pH 7.0)-0.1% SDS at 55 C.
Isolated papillary muscle experiments
Contractile parameters of papillary muscles were measured as
previously described (17). Briefly, left ventricular papillary muscles
from the hearts of six mutant T3Rß mice and eight
wild-type mice were excised under oxygenated Tyrode solution (136
mM NaCl, 5.4 mM KCl, 1 mM
MgCl2, 0.33 mM NaH2PO4,
10 mM HEPES, and 10 mM glucose, pH 7.40)
containing 30 mM 2,3-butanedione monoxime and 2.5
mM CaCl2. They were inserted into 
-shaped
clamps made from strips of platinum foil, tied with 6.0 braided silk
suture, and mounted on hooks of platinum wire in a 0.5-ml muscle
chamber.
Muscles were perfused with 2.5 mM Ca2+ Tyrode solution at 37 C and stimulated at 2 and 6 Hz through the platinum clamps (5 V, 0.25-msec duration). Force was measured with an isometric force transducer (OPT1L, Scientific Instruments, Heidelberg, Germany) and recorded on a strip chart recorder. Muscles were stretched over 30–60 min to the length at which active force development was maximal (Lmax). Forces [in millinewtons (mN)] were normalized by the muscle cross-sectional areas to yield stresses (in millinewtons per mm2). The cross-sectional area was calculated for each muscle as the ratio of muscle volume (determined by weighing) and muscle length at Lmax.
The time to peak tension was determined as the time from 10% of tension development to the peak of contraction. Relaxation times RT50 and RT90 were determined as the time from the peak of contraction to 50% or 10%, respectively, of the maximum developed stress during relaxation. Data are expressed as the mean ± SEM. Statistical comparisons were made using unpaired Student’s t tests.
Electrophysiological measurements in mouse heart
For ECG measurements, 6 transgene positive mice, 10 hypothyroid
mice, and 5 age-matched normal mice were analyzed. Hypothyroidism was
established in the mice by feeding the animals iodine-deficient, 0.15%
5-propyl-2-thiouracil-containing food pellets (Harlan Teklad, Madison,
WI) for 4 weeks. The described procedures were performed in
accordance with the guidelines established by the committee on animal
research at the University of California-San Diego. When mice were
sedated after ip injection of a ketamine-xylazine cocktail, they were
kept in a supine position by four limb restrainers. Four needles were
placed sc on each limb close to the trunk, and an ECG with the leads I,
II, III, AVR, AVL, and AVF was obtained. The ECG was acquired with an
analog to digital conversion at 2000 Hz on an IBM-compatible 486
computer using Windaq software (Data Instruments, Akron, OH). The
following ECG parameters were measured: 1) duration of atrial
depolarization (P), 2) duration of excitation progression from the
atrium to the ventricle (PQ), 3) duration of depolarization of the
ventricles (QRS), and 4) duration of excitation and repolarization of
the ventricles (QT). Parameters were measured in each lead and
averaged.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was used to generate transgenic mice. Southern
blot analysis of tail DNA digested and hybridized with a
hT3Rß probe indicated that we had generated one founder
line. It was of interest to determine how many copies of the mutant
T3Rß cDNA were integrated in the genome of the transgenic
line. Genomic DNA from tails of wild-type and homozygous transgenic
animals was prepared and used in a comparative slot blot using a probe
from the 5'-end of the cDNA that preferentially hybridized with the
T3Rß gene. Figure 2 shows
the autoradiograph of the slot blot. Densitometric quantification of
this slot blot revealed that one copy of the transgene had been
integrated into one chromosome.
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and T3Rß genes, mRNA
was prepared from the hearts of transgenic animals and control animals
and analyzed by Northern blot. Figure 3
clearly shows that in comparison with transgene-negative mice (lanes 1
and 2) homozygous T3Rß mutant transgenic animals contain
a substantial amount of mutant hT3Rß mRNA in their hearts
(lanes 3 and 4). The levels of wild-type T3R1,
T3R2, and T3Rß1 mRNA were not
significantly different in the hearts of wild-type and
T3Rß mutant mice.
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.
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, MHCß, and SERCA2 mRNAs. Figure 4A shows the results of a blot that had
successively been hybridized with four different probes. The last
hybridization was carried out with a probe for GAPDH to confirm equal
loading of RNA in each lane. Expression of the MHCß gene was markedly
increased in transgenic mice compared with that in wild-type mice.
Wild-type mice had an undetectable amount of MHCß mRNA in a Northern
blot and MHCß protein in a Coomassie-stained gel (Fig. 4B). The
ventricular homogenates of wild-type and transgenic mice were prepared
according to a protocol from J. Robbins and run on a low percentage,
glycerol-containing SDS gel followed by Coomassie blue staining (21).
For the MHC gene, the Northern blot showed a 34% reduction in
expression of this gene in the heart of T3Rß mutant mice.
This number was determined by densitometry of the autoradiograph shown
in Fig. 4A.
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). It should be noted that heterozygous transgenic animals
showed a lesser degree of down-regulation of SERCA2 mRNA than the
homozygous animals (data not shown). Upon densitometry of the Northern
blot shown in Fig. 4A and another blot (not shown), digital values
could be assigned for the intensity of each band. We then performed a
statistical analysis on these values from a total of eight transgenic
animals and eight wild-type control animals. The MHC values,
normalized to the GAPDH values, were set at 1 for controls and were
determined to be 0.6575 for transgenics, which was calculated to be a
34% reduction with a P value of 0.022. The SERCA values,
also normalized to the GAPDH values, were set at 1 for controls and
were determined to be 0.678 for transgenics, which was calculated to be
a 32% reduction with a P value of 0.013. As both the SERCA2
down-regulation and the MHC isoform shift could prolong cardiac
contraction, papillary muscles were isolated from normal and transgenic
mice to perform contractile studies.
Contractile behavior of isolated papillary muscle from
T3Rß mutant mice
To characterize the cardiac contractile phenotype of transgenic
mice, we determined force development in isolated left ventricular
papillary muscles from transgenic and wild-type mice (Fig. 5). Muscles from mutant thyroid hormone
receptor transgenic mice displayed a slight prolongation of time to
peak tension, amounting to 12% at 2 Hz stimulation frequency
(P < 0.01) and 10% at 6 Hz stimulation frequency.
Relaxation was more markedly impaired. RT50 increased by
20% at 2 Hz (P < 0.001) and 21% at 6 Hz
(P < 0.01). The delay of the late phase of relaxation
was even more pronounced, as RT90 was increased by 26% at
2 Hz and 25% at 6 Hz (P < 0.001).
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ECG changes in the heart of mutant T3Rß
transgenic mice
We compared electrocardiograms obtained from the mutant thyroid
hormone receptor transgenic mice with hypothyroid and normal mice. The
QRS intervals in transgenic (14.2 ± 1.5 msec) and hypothyroid
(12.9 ± 1.6 msec) animals were markedly prolonged compared with
that in control animals (10.6 ± 0.4 msec). In addition, the P
wave and the PQ interval were markedly prolonged in hypothyroid mice,
but not in the mutant T3Rß transgenic mice. These results
are summarized in Table 2. The changes in
electrophysiological parameters induced by the expression of a mutant
thyroid hormone receptor resemble in part the changes in hypothyroidism
(24).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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337T mutant T3Rß isoform derived from the S family in
the heart of transgenic mice exerts a strong dominant negative effect,
leading to a hypothyroid pattern of gene expression, contractile
function, and ECG changes. These changes occur despite normal
T3, T4, and TSH levels. A relatively
hypothyroid cardiac status and a normal set-point of the
pituitary-thyroid feedback loop most likely result from significant
expression of the T3Rß mutant in cardiac myocytes, as
demonstrated in Northern blots. Expression of the T3Rß
mutant occurs most likely at insignificant levels in the pituitary,
where it would otherwise impair negative T3 feedback by
lowering TSH production (25). Strong expression of the mutant
T3Rß in cardiac myocytes is in line with the expression
pattern of the cytomegalovirus enhancer/chicken ß-actin promoter,
which leads to a strong expression in myocytes, also observed for other
transgenes (17).
Analysis of the expression pattern of the MHC and MHCß genes
revealed a pattern compatible with a hypothyroid status. The expression
of the MHCß gene was significantly increased in the hearts of
transgenic T3Rß mutant mice. This gene has been shown to
be markedly thyroid hormone responsive. The marked increase in its
expression in the hypothyroid heart may be in part mediated by
increased transcription of the MHCß gene (26).
The presence of a strong negative thyroid hormone response element
(TRE) in the promoter of this gene has been postulated (27), which
could explain our findings. The mechanism by which the mutant receptor
would up-regulate the MHCß promoter is by competition for DNA binding
of the wild-type receptor. The wild-type receptor binding to a negative
TRE would be associated with hormone and, in contrast to a positive
TRE, with a powerful corepressor that is able to silence transcription
in an as yet not fully explained fashion. The mRNA levels of the MHC
and SERCA2 were reduced in the transgenic animals by 34% and 32%,
respectively. These findings can be explained by the presence of
positive-acting TREs in the promoter of both the SERCA2 and the MHC
gene, which has been reported previously (26, 28, 29, 30). Analogous to the
action on the negative TRE, the mutant receptor would compete with the
wild-type receptor to occupy the positive TREs. In this case, however,
the mutant receptor cannot associate with the coactivators necessary to
stimulate transcription, because of its lack of hormone-binding
capacity (31). Whether the mutant receptor, binding to a positive TRE,
is associated with a corepressor that cannot be released because the
mutant receptor has impaired hormone binding is not yet clear.
The combined decrease in SERCA2 mRNA and MHC RNA levels and the
significant increase in MHCß mRNA levels should lead to the cardiac
contractile abnormalities that we indeed observed. Cardiac relaxation
is influenced by the calcium pump of the sarcoplasmic reticulum (SR),
which, during diastole, transports calcium from the cytoplasm into the
vesicular structure of the SR, lowering free cytosolic calcium levels.
The lower calcium levels result in diminished interaction of the
globular head of myosin with actin in the thin filament, accelerating
diastolic relaxation. The most marked change in contractile parameters
that we observed was a significant delay in cardiac muscle relaxation
that is in line with diminished SERCA2 expression. In addition, cardiac
contraction was changed, as indicated by the delayed time to peak
tension. This parameter is influenced by the speed and magnitude of the
calcium increase during the systolic contraction phase, but also by the
activity of the myosin ATPase of the globular head of myosin. The
higher the myosin ATPase activity, the faster the globular head of
myosin moves along the actin thin filament. MHC that forms myosin V1
has a higher myosin ATPase activity than myosin V3 that is composed of
two MHCß molecules. In addition, a decrease in SERCA activity will
lead to decreased filling of the SR, resulting in diminished calcium
release during systole that will further contribute to a prolongation
of the time needed to reach peak tension. In contrast to the delay in
force development during contraction and the prolongation in force
decay indicated by delayed relaxation, the maximally developed force
was not altered. Therefore, neither alteration in MHCß isoform
predominance nor the 32% decrease in SERCA2 mRNA levels influenced
this parameter.
The ECGs obtained in the T3Rß mutant mice indicated that some electrophysiological parameters of the heart are also compatible with a hypothyroid phenotype similar to changes in contractile behavior. Despite normal thyroid hormone values, the QRS complex in the transgenic animals is markedly prolonged. The detailed molecular mechanisms underlying this change are currently unclear. Thyroid hormone-induced changes in specific ion channels that may contribute to a prolongation of the action potential have been explored to only a very limited extent. For example, the outward potassium channel, K1T, that is composed of genes coding for the outward rectifying potassium channels, KV 4.2 and KV 4.3, is markedly thyroid hormone responsive (32). In addition, it has been shown that the mRNA coding for the KV 1.5 ion channel, which contributes to a transient outward and delayed rectifying current, is also markedly decreased by hypothyroidism (33).
It has been reported that some effects of thyroid hormone on ion channels, such as effects on sodium channels, occur very rapidly and may be mediated by extranuclear effects that are not mediated by T3 binding to thyroid hormone receptors. Our results are of specific interest related to such a potential extranuclear mechanism. We found marked changes in the expression of T3-responsive genes such as MHC isoforms and SERCA2. In addition to hypothyroid contractile and ECG changes in transgenic animals, the T3Rß mutant mice have normal thyroid hormone levels, which strongly argues that the observed changes are mediated by a T3 receptor-based mechanisms of hormone action. Additional studies in the T3Rß mutant transgenic mice and in T3 receptor isoform knockout mice that are now becoming available will allow us to explore the nuclear vs. extranuclear effects of T3 in greater detail.
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Acknowledgments |
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Footnotes |
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2 Clinical Associate Professor, Texas Tech University (Amarillo,
TX).
Received June 19, 1998.
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References |
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- and ß-myosin heavy chain mRNA levels in
rabbit heart. Biochemistry 23:1596–1599[CrossRef][Medline]
-myosin heavy
chain gene promoter. Transgenic Res 4:397–405[CrossRef][Medline]
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