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Thyroid Division (J.P., R.P., H.T., S.D.C., P.R.L.), Brigham and Women’s Hospital, Harvard Institute of Medicine, Boston, Massachusetts 02115; Department of Internal Medicine and Endocrinology (J.P.), University Medical School of Warsaw, 02–097 Warsaw, Poland; Nuclear Magnetic Resonance Laboratory for Physiological Chemistry (J.H., J.S.I.), Brigham and Women’s Hospital, Boston, Massachusetts; Thyroid Unit (H.K., E.D.A., F.E.W.), Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: P. Reed Larsen, M.D., FACP, FRCP, Chief, Thyroid Division, Brigham and Women’s Hospital, 560 Harvard Institute of Medicine, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115. E-mail: larsen{at}rascal.med.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-MHC promoter. Despite high
myocardial D2 activity, myocardial T3 was, at
most, minimally increased in TG myocardium. Although, plasma
T3 and T4, growth rate as well as the heart
weight was not affected by TG expression, there was a significant
increase in heart rate of the isolated perfused hearts, from 284 ±12
to 350 ± 7 beats/min. This was accompanied by an increase in
pacemaker channel (HCN2) but not -MHC or SERCA II messenger RNA
levels. Biochemical studies and 31P-NMR spectroscopy showed
significantly lower levels of phosphocreatine and creatine in TG
hearts. These results suggest that even mild chronic myocardial
thyrotoxicosis, such as may occur in human hyperthyroidism, can cause
tachycardia and associated changes in high energy phosphate compounds
independent of an increase in SERCA II and -MHC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thyroxine (T4) is a tetra-iodinated iodothyronine prohormone, which must be mono-deiodinated in the outer ring to T3 to be activated (1, 8). In mammals, there are two isoenzymes that can catalyze this conversion, the types 1 (D1) and 2 (D2) 5' iodothyronine deiodinases (8). In rat tissues expressing D2, a substantial portion of the nuclear receptor-bound T3 is provided by the intracellular conversion of T4 to T3 by this isoenzyme (9). This enzyme is a critical component of the homeostatic mechanism for maintaining the tissue T3 under a variety of stresses because it can increase the efficiency of T4 activation when T4 production is reduced as in iodine deficiency (10).
The coding sequence and 3' untranslated region of the human type 2 deiodinase have been recently identified (11, 12, 13). The messenger RNA (mRNA) is highly expressed not only in the human brain and pituitary, as it is in the rat and mouse, but also in myocardium and skeletal muscle, which is not the case in the rodent (12, 13, 14). The expression of D2 in the myocardium raises the possibility that, in humans, this tissue can respond not only to changes in plasma T3, but also to those in T4. Thus, the human heart may resemble the pituitary and brain with respect to sources of intracellular T3. This could contribute to the sensitivity of pulse rate to minimal increases in circulating T4. The contribution of the T3 generated by the action of D2 to total myocardial T3 in the human heart under normal or pathological conditions remains to be determined. However, it seems likely that with respect to the potential for the intracellular T4 to contribute to intracellular T3 in this organ, rodents are not a faithful model of the human situation.
Studies of the effects of thyroid hormone on the myocardium of
experimental animals are often performed by giving exogenous
T4 or T3 to hypothyroid
rats, generally in large excess, for relatively short time periods.
Although this has allowed the identification of number of
T3-responsive genes, including - and ß-MHC,
SERCA II, HCN2 as well as inducing acute changes in the cardiac
physiology of the hyperthyroid animal, it does not faithfully replicate
the pathophysiology of human hyperthyroidism. Clinical hyperthyroidism
is typically present for at least 6 months in a progressively more
symptomatic form before coming to medical attention (1).
Biochemically, e.g. in terms of suppression of serum TSH, it
has likely to have been present for an even longer period. Usually,
there is only a 2- to 3-fold increase in serum T4
(1). For these reasons, the animal experiments do not
accurately replicate human hyperthyroidism.
To provide a model that might better reflect events in the hyperthyroid
human myocardium with respect to the sources of
T3, we have prepared transgenic (TG) mice in
which human D2 is driven by the mouse -MHC promoter and,
therefore, expressed at high levels in the myocardium. The mice are
systemically euthyroid but have some, but not all, physiological,
biochemical, and molecular changes in the heart consistent with
thyrotoxicosis. Perfused hearts were tachycardic, had an increase in
rate pressure product and a decrease in phosphocreatine (PCr) without
any changes in creatine kinase activity. The HCN2 mRNA was modestly
increased, but no significant changes were found in the expression of
-MHC or SERCA II genes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation and screening of transgenic animals
The coding region of the human D2 complementary DNA (cDNA)
(Genethon clone supplied by Drs. St. Germain and Galton, Dartmouth
Medical Center, Lebanon, NH) and the selenocysteine insertion sequence
(SECIS) of rat selenoprotein P (SelP), as a 1.9-kb fragment with the
potential poly A site, were subcloned between the mouse -MHC 5'
flanking region and the additional polyadenylation sequence of human GH
(vector provided by Dr. Jeffrey Robbins, Division of Molecular
Cardiovascular Biology, University of Cincinnati Medical Center,
Cincinnati, OH) to form plasmid pHT1402 (Fig. 1
). The entire 8.4-kb transgene was
released from the plasmid pHT1402 by BamHI digestion.
Approximately 4 ng of the gel purified transgene were microinjected
into each male pronuclei of 1-day-old mouse zygotes of the inbred
strain FVB/C57, and these were reimplanted into the uteri of
pseudopregnant foster mice at The Beth Israel Transgenic Facility.
Litters were obtained after 21 days. Between age 14 and 18 days,
preweaned mice were identified by gender, marked by earlobe punching,
and approximately 10 mm of tail tip was removed for genotyping. Genomic
DNA was generated by overnight digestion with proteinase K and SDS.
After high salt precipitation in the presence of SDS, the supernatant
was phenol/chloroform extracted and DNA precipitated by ethanol.
Genomic DNA (10 µg) was digested with XbaI (20 U/µg of
DNA) subjected to electrophoresis through the 1% agarose gel and
transferred to GeneScreen Plus nylon membranes (NEN Life Science Products, Boston, MA). Hybridization was performed using a 0.3
kb XbaI, AccI fragment of rat D2 cDNA, which
is virtually identical in sequence to the mouse and human D2 genes
(13, 15). The rat D2 cDNA was kindly provided by Drs.
St. Germain and Galton (Dartmouth Medical Center, Lebanon, NH). Two
transgenic lines were identified and expanded. Littermates served as
controls unless indicated.
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Determination of myocardial T3 concentration
Animals were anesthetized by carbon dioxide and decapitated.
After wide opening of chest cavity, hearts (including both atria and
ventricle) were rapidly dissected from the great vessels, divided in
half, rinsed in ice-cold PBS, and frozen in liquid nitrogen. Half of
each heart, about 50 mg, was used for measurement of
T3 content. Each tissue was homogenized in 1 ml
of methanol using a Brinkmann Instruments, Inc. (Westbury,
NY) homogenizer. For protein measurement, a 50-µl aliquot of each
homogenate was solubilized by adding 10 µl of 1 M NaOH.
The solubilized protein was diluted in water and concentration of
protein was measured using Bio-Rad Laboratories, Inc.
(Hercules, CA) protein assay kit. To assess the recovery of
T3, approximately 500 cpm of the high specific
activity [125I]-T3
(NEN Life Science Products, Boston, MA) were added to the
rest of each homogenate and counted. Homogenates were then spun for 20
min at 5,000 x g and the supernatant mixed with 2 ml
of chloroform. Thyroid hormone was extracted into aqueous solution by
two successive 0.5 ml aliquots of 0.4 M
NH4OH. The supernatants were pooled after
centrifugation for 20 min at 5,000 x g. Any possible
traces of chloroform in pooled supernatants were removed by adding 1 ml
of ethyl ether and gravity separation. Samples were then evaporated in
a lyophilizer (Freezemobile 12 SL, The Virtis Co., Gardiner, NY) and
redissolved in 400 µl of 0.01 M NaOH. Each
sample was again counted to determine the T3
recovery which ranged from 60–75%. Duplicate samples of solubilized
T3 were assayed in sodium salicylate/0.2
M glycine acetate buffer, pH 8.6, using specific
and sensitive rabbit polyclonal T3 antibodies
(17, 18). Standards were prepared in 0.01
M NaOH and ranged from 0.5–16 pg of
T3/tube. Additionally, the same aliquot of each
sample and standard was used to determine nonspecific binding of
[125I] T3 in the absence
of T3 antibodies. This did not differ between
standards and samples (18). Linearity of measurement was
confirmed by assay of four serial 2-fold dilutions of
T3 extracts from rat heart and liver.
Perfusion protocol
Mice of both genders from both lines were heparinized (5000 U/kg
BW, administered ip) 10–15 min before cervical dislocation. Their
hearts were excised and immediately arrested by placing in ice-cold
perfusion buffer. After cannulation of the aorta, each heart was
perfused by the Langendorff method at constant pressure of 80 mmHg and
at 37.5 C with modified Krebs Henseleit bicarbonate buffer (118
mM NaCl, 4.7 mM KCl, 1.2 mM
MgSO4·7H2O, 2.5
mM
CaCl2·2H2O, 0.5
mM Na2EDTA, 25 mM
NaHCO3, 10 mM glucose, and 0.5
mM pyruvate). All buffers were gassed with 95%
O2/5% CO2 to give a pH of
7.4 at 37 C.
Cardiac function was recorded as the rate pressure product (RPP), the product of heart rate and left ventricular developed pressure (LVDP), using a water-filled polyethylene balloon in the left ventricle. The size of the balloon was carefully matched to the size of the ventricle (19). The balloon was connected via a water-filled tube to a pressure transducer (Stratham P23Db, Gould, Oxnard, CA) attached to a MacLab (ADInstruments, Milford, MA) analog digital converter, sampling at 200 samples/sec. The balloon was inflated to give an end diastolic pressure (EDP) of approximately 8 mmHg. Intraventricular pressure development was prevented by inserting a short piece of polyethylene (PE10) tubing through the apex of the left ventricle.
Hearts were placed in a 10 mm NMR tube, and the effluent from the heart was suctioned from above. The effluent flow rate was measured in a volumetric container, which allowed the coronary flow rates to be calculated. The perfusion system was then placed into the magnet at the correct height to give 80 mmHg pressure at the level of the heart. The temperature of the system was maintained at 37.5 C by external heating of the NMR tube with warm air and by keeping all perfusion buffers in water jacketed containers (20). Each group of hearts was subjected to a stabilization period of 30 min, during which the probes were tuned and the magnet shimmed. Following that, two 8 min spectra were acquired. At the end of each experiment the hearts were blotted and weighed, and stored at -80 C.
31P NMR spectroscopy and data analysis
Spectra were acquired using a GE-400 wide bore Omega NMR
spectrometer (GE, Fremont, CA) operating at the
31P resonance frequency of 161.94 MHz. A 10-mm
glass NMR tube (Wilmad, Buena, NJ) containing the isolated heart
preparation was inserted into a custom built
1H/31P double-tuned probe
(Morris Instruments, Ontario Canada) situated in the center of a 9.4 T
superconducting magnet. The spectra were acquired as described
previously (20, 21). Quantification of ATP, PCr and Pi
concentrations from spectral peak areas was achieved using
biochemically determined ATP concentration in a separate group of
hearts which were freeze-clamped after the same period of perfusion.
The ATP resonance area (average of and 
phosphate areas) divided
by the wet weight (mgww) of each heart was used to convert the
resonance areas of the other phosphorus containing metabolites using
their saturation factors previously determined in our laboratory for
PCr (1.2) and inorganic phosphate, Pi (1.15), relative to ATP.
Myocardial pH was estimated using the chemical shifts of the Pi peak
relative to the PCr peak using titration curves determined previously
in our laboratory (pH = ppm·0.724 + 3.5455 were ppm reflects the
chemical shift between Pi and PCr). Cytosolic free ADP concentration
was calculated using the equilibrium constant of the CK reaction
(Keqm = 1.99 x 109
M-1) and using
metabolite values obtained by NMR spectroscopy and biochemical analysis
(22, 23).
Biochemical analysis
A separate group of hearts was freeze-clamped after the same
period of perfusion. These were stored at -80 C, and used for
biochemical determination of ATP, PCr, Creatine (Cr),
glucose-6-phosphate (G6P). ATP, PCr, Cr, glycogen, and G6P were
extracted in 6% perchloric acid and assayed using spectrophotometric
techniques as described (24). ATP, PCr, Cr, and G6P
results were calculated in mmol/mg of protein and expressed in
mM concentration using the conversion factor 0.17
(protein/wet weight ratio) and factor 0.48 (water/wet weight ratio).
For glycogen measurements the wet/dry weight ratio of each heart was
determined, and results expressed as µmol/g dry wt (µmol glucosyl
units/g dry wt for glycogen). Creatine kinase activity (CK
Vmax) and the amount of this activity
attributable to each isoenzyme of CK as well as adenosine kinase (AK)
activity were measured using methods previously described
(25). The cardiac tissue was homogenized for 10 sec at 4 C
in potassium phosphate buffer containing 1 mmol/liter EDTA and 1
mmol/liter ß-mercaptoethanol, pH 7.4 (final concentration of 5 mg
tissue/ml). Triton X-100 was then added to the homogenate at a final
concentration of 0.1%. The CK activity was measured in tissue
homogenates at 30 C (25). CK activities were measured in
units of IU per mg protein and converted to mM/sec using
the measured concentrations of cardiac protein, assayed in the samples
before the addition of Triton X-100 using the Lowry method
(26). All values are expressed as mM/sec at 37
C (the results were multiplied by the factor 1.8 to convert from 30–37
C). The percent of total CK activity attributable to each isoenzyme was
measured using a Helena Cardio-Rep CK isoenzyme analyzer (Beaumont, TX)
(25).
Deiodinase assays
Tissues were homogenized on ice in buffer containing 1 x
PE (0.1 M potassium phosphate and 1 mM EDTA),
250 mM sucrose and 10 mM DTT (pH 6.9). D2
assays were performed in the presence of 1 nM
T4 with or without 1 mM PTU and/or
100 nM T3, as described earlier
(27).
Isolation and analysis of RNA
RNA was extracted from the tissue using TriZol reagent
(Life Technologies, Inc., Rockville, MD) according to the
manufacturer’s protocol and RNA concentration was estimated from the
A260 value. Northern analysis was performed using of 10 µg total RNA
by standard methods as described earlier (7). A mouse HCN2
cDNA fragment (
0.5 kb) was made by RT-PCR from euthyroid mouse
cortex RNA (7). Specific -MHC oligonucleotide was a
gift of Dr. W. H. Dillmann (Department of Medicine, Division of
Endocrinology and Metabolism, University of California San Diego, La
Jolla, CA) and ß-MHC oligonucleotide was obtained from Life Technologies, Inc. (Rockville, MD). Both oligonucleotides were
designed from the nonhomologous 3' regions of the mouse myosin heavy
chain cDNAs and have been described earlier (28). Mouse
ß-actin cDNA was a gift of Dr. B. M. Spiegelman (Dana Farber
Cancer Institute, Boston, MA). Rat cyclophilin cDNA was a gift of Dr.
G. Adler and W. Chin (Brigham and Women’s Hospital, Boston, MA).
Labeling of - and ß-MHC probes was performed by 5' end-labeling
method using T4-polynucleotide kinase from
New England Biolabs, Inc. (Beverly, MA) and
[32P] ATP from NEN Life Science Products (Boston, MA). The remaining probes were radiolabeled
using standard random nanomer method and
[32P] dCTP.
Statistical analysis
All results are expressed as means ± SEM.
Statistical analysis was done using SPSS, Inc. program
version 8 (Chicago, IL). WT and TG mice were compared using ANOVA or
Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Southern
blotting identified a 3-kb XbaI fragment of the mouse type 2
deiodinase (dio2) gene as well as a 1.35-kb band of the
human D2 transgene both containing sequences homologous to the
fragment of the second exon of the rat dio2 gene used as
a probe (Fig. 1). Two founders, one female and one male, were selected
and bred. Based on inheritance and the signal intensity, line 1 had
about 50 copies of transgene in the same chromosome and line 2, about
50 copies of transgene in at least two different chromosomes. Neither
line exhibited any phenotypic developmental abnormalities nor showed
any increased mortality. The growth rate, body weight (BW) and heart to
body weight ratio (H/BW) were not statistically different between WT
and TG mice (estimated marginal means for 14 week old mice were:
24.3 g vs. 24.0 g, P = 0.87 for BW
and 4.32 mg/g vs. 4.14 mg/g, P = 0.59 for
H/BW ratio). Both WT and TG males had slightly larger body weight then
age-matched females (estimated marginal means for 14-week-old mice were
26.8 for males and 21.7 for females, P = 0.16, WT plus
TG combined).
Northern blot analysis in both lines confirmed a high level of
transgene expression in the heart and lungs, the organs where the
-MHC gene is normally expressed (29). The D2
activity in the TG heart was almost 100 and 1000 times higher,
respectively, than in the pituitary gland or in the cortex where
D2, but not the -MHC gene, is normally expressed. The kinetic
studies of outer ring T4 deiodination showed,
typical for D2, Km of about 1 nM, and lack of PTU
inhibition. There were no age (tested by the regression analysis,
R = 0.27, P = 0.32) or gender-dependent
significant differences in transgene expression (males 58.7 ± 8,
and females 81 ± 8 pmol of I released/mg·h, P =
0.09, n = 11 for males and n = 5 for females). Based on a
2 h assay with 300 µg of myocardial protein, wild-type mouse
heart did not contain D2 activity and no mD2 mRNA was found
by Northern analysis.
Thyroid hormone concentrations in myocardium and blood
The myocardial T3 concentration was
12.8 ± 0.9 ng of T3/g of protein in TG
(n = 17) and 11.2 ± 0.8 ng of T3/g of
protein in WT mice, P = 0.2 (n = 15). There was a
tendency for the myocardial T3 to be higher in TG
than in WT males, but this was not statistically significant (14.8
vs. 11.4 ng of T3/g of protein,
P = 0.09). There were no significant differences in
myocardial T3 content between lines 1 and 2 (11.3
and 13.6 ng of T3/g of protein, P
= 0.31). Serum T3 and T4
concentrations were not different between TG and littermate WT mice
indicating that there was no significant increase in systemic
T3 production (0.48 vs. 0.53 ng/ml,
P = 0.15, n = 48 and 27.7 vs. 27.7
ng/ml, P = 0.96, n = 49 for
T3 and T4, respectively).
There were no gender differences in serum T3 and
T4 concentrations (0.50 vs. 0.51
ng/ml, P = 0.63 and 28.3 vs. 27.1 ng/ml,
P = 0.91, males vs. females,
respectively).
Myocardial function and high energy phosphate compounds
Myocardial performance parameters are shown in Fig. 2A. The perfused TG and WT hearts (8
littermate and 4 inbred FVB/C57 wild-type mice matched for age and
gender) were the same size. Transgenic hearts exhibited greater basal
cardiac function as reflected in a higher basal heart rate and rate
pressure product, although 2/3 of this effect was due to the increase
in heart rate (Fig. 2A). Therefore contractile function (expressed as
RPP/g) in the TG hearts was increased about 25% compared with
wild-type hearts (2.87 ± 0.23 x 105
mmHg/min/gww vs. 2.18 ± 0.12 x
105 mmHg/min/gww; P = 0.014).
There was no difference in +dP/dt between groups (TG mice: 4222 ±
381 mmHg/sec vs. WT mice: 3843 ± 121 mmHg/sec;
P = 0.08). Similarly, there was no increase in the
relaxation rate in the transgenic hearts at baseline (TG mice:
-2327 ± 228 mmHg/sec vs. WT mice: -2550 ± 95
mmHg/sec; P = 0.18).
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, B and
2C). Biochemical measurements confirmed lower PCr as well as showing
depressed total creatine concentration in TG hearts (Table 1). The levels of ATP, Pi and free energy
of hydrolysis of ATP were unchanged in the TG hearts. The calculated
intracellular pH was also unchanged. There was no difference in the
myocardial levels of glycogen or glucose-6-phosphate (G-6-P). CK
activity in the transgenic heart was unchanged (51.0 ± 7.0
mM/sec in WT and 48.9 ± 3.0 mM/sec in TG
mice, P = 0.79, n = 4 for each group) and there
were no differences in the activity of CK isoenzymes (Table 1).
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-MHC, ß-MHC, SERCA II, and HCN2 in the
ventricular myocardium by Northern analysis (Fig. 3). The mRNA for the HCN2 gene has
recently been shown to increase in response to thyroid hormone in rats
(7). We also found this was increased in TG mice
(P < 0.05, n = 12, Fig. 3). Surprisingly, -MHC
and SERCA II mRNAs were not altered (Fig. 3). The ß-MHC mRNA was
significantly increased in TG hearts, although in the euthyroid state
both WT and TG myocardium expressed almost exclusively -MHC mRNA
(Fig. 3).
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-MHC-D2
transgene-MHC promoter,
which is positively regulated by thyroid hormone (30).
However, because the endogenous -MHC mRNA was not affected by the
rather minimal increase in myocardial T3, it was
not clear if endogenous T3 contributed to the
high D2 expression in TG myocardium. Furthermore, it might be
expected that increased myocardial T4 to
T3 conversion by hD2 might protect the heart
against hypothyroidism. To explore these issues, three groups of TG and
WT animals were kept on a low-iodine, PTU containing diet for 4, 8, and
12 days, respectively. This time was chosen based on preliminary
experiments in which D2 activity fell to undetectable levels after
3 weeks on this regimen. D2 activity in the myocardium fell in
parallel with the serum T4 concentration with the
lowest level found at 12 days (Fig. 4).
These results confirmed the positive feedback of the D2 transgene
by the endogenous thyroid hormone. At the same time, there was a
time-dependent decrease in -MHC and an increase in ß-MHC mRNA
levels in both TG and WT myocardium (Fig. 4). There was no
statistically significant difference between WT and TG mice in the
-MHC, ß-MHC, or SERCA mRNA level (by ANOVA, P >
0.25 for the effect of transgene on the expression of each mRNA) during
induction of hypothyroidism suggesting that there was no protection of
myocardial thyroidal status by myocardial hD2 (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MHC promoter. D2 activity was also present in lung
because the -MHC promoter also directs expression in the intimal
walls of the veins and venules in that organ (29). The
most surprising result of the experiment was the absence of a major
effect on the myocardial T3 concentration
considering the level of D2 expression. In the cerebral cortex,
for example, much lower D2 activity results in sufficient local
T3 production to saturate approximately 80% of
the thyroid hormone receptors (9). The failure of tissue
T3 concentrations to rise to higher levels could
have a number of explanations. D2 activity is expressed only in
myocytes, which make up only about 50% of the myocardial cells and
account for about 70% of the total myocardial proteins
(3). Because the T3 concentration
was measured in whole heart homogenate and denominated by total
myocardial protein, the difference between WT and TG is slightly
underestimated. Nevertheless, even if corrected for the above factor,
the increase in myocardial T3 concentration is
quite modest. This finding cannot be explained by the artifactual
degradation of T3 during tissue processing
because there was no significant degradation of
T3 in tissue homogenate when incubated at 4 C for
6 h (data not shown). Nor can it be explained by intracellular
T4 to T3 conversion in WT
myocardium because this is not detectable. There are other potential factors that could lead to lower T3 production than one might anticipate based on the results of in vitro D2 assays. An as yet unidentified thiol-containing cellular cofactor is required for iodothyronine deiodination. Because the mouse myocyte does not normally convert T4 to T3, the level of this cofactor may be much lower than in brain, pituitary gland or brown fat. Alternatively, there may be limited T4 uptake by the rodent myocyte. The molecular mechanism for T4 and T3 transport is only now being unraveled. One report indicates that T4 transport into the heart is not temperature-dependent as is that of T3, suggesting that it may not be an active process (31). Furthermore, when T3 is produced, it may well diffuse rapidly from myocytes into the circulation due to the high myocardial blood flow. Although this is an attractive hypothesis, the similar concentrations of serum T4 and T3 in the sera of the TG and WT animals imply that the rate of total body conversion of T4 to T3 is not significantly increased by the expression of D2 in the myocardium. In all species examined to date, the fraction of T4 converted to T3 per 24 h in the whole animal is less than 50% (32, 33). If that fraction were to increase significantly, one would expect a downward adjustment of T4 production by the hypothalamic-pituitary feedback system such that the serum T4 might well be reduced with no change or perhaps a slight increase in serum T3. The opposite change in serum T4 has been documented in the C3H mouse in which a genetic decrease in D1 expression causes a 2-fold increase in circulating T4 but no change in serum T3 (34, 35). The intact mouse is a closed system so that thyroid status must remain constant despite any changes in the relative fraction of T4 to T3 conversion.
Thyroid hormone response elements have been identified in the murine
-myosin heavy chain promoter, which was used to induce
myocardial-specific D2 expression (29, 30). Thus, one
would anticipate that a feed-forward mechanism might be present in the
TG myocardium to increase D2 expression. The sensitivity of
D2 expression to thyroid hormone is apparent from the results in
Fig. 4. We took advantage of the sensitivity of the endogenous - and
ß-MHC promoters to thyroid status to determine whether the expression
of D2 would protect the myocardium against the effects of
hypothyroidism. Although the thyroid hormone-dependence of D2
expression makes this experiment more problematic than it would be if
D2 expression remained constant, we found no evidence that the
transgene affects the onset of hypothyroidism-induced changes in
myocardial gene expression (Fig. 4).
There was remarkably little effect of the transgene expression on the
mouse. Growth rates were not different between TG and littermate
controls and there was no alteration in the ratio of heart to body
weight, which is a parameter commonly increased by excess thyroid
hormone (36). This is consistent with the concept that the
increased protein synthesis and hypertrophy of the hyperthyroid heart
requires the increased myocardial work normally associated with
systemic thyrotoxicosis (36). Unloading the heart by
heterotopic cardiac transplantation has been shown to decrease overall
protein synthesis and heart weight (37). In the same
model, thyroid hormone excess induced increases in -MHC mRNA is seen
although unloading of the heart by itself leads to similar gene
expression rearrangement as occurs in hypothyroidism (5).
In euthyroid hearts, for example, there are minimal, if any, changes in
-MHC mRNA levels induced by treatment with excess thyroid hormone
(7, 38).
The most striking evidence for myocardial thyrotoxicity of the TG
animals was detected in the performance of the isolated heart. There
was an approximately 20% increase in heart rate and an 30% increase
in the rate pressure product (Fig. 2a). This result is consistent with
earlier studies in isolated perfused rat hearts where alterations in
thyroid status cause parallel alterations in basal heart rate
(39, 40). It is also consistent with the current
interpretation that the increase in heart rate induced by thyroid
hormone is, at least partially, intrinsic to the muscle and does not
require either changes in the autonomic nervous system or circulating
catecholamines. A potential explanation for the increase in the
intrinsic heart rate is the increased HCN2 expression found in TG
animals. We have recently observed that HCN2 is thyroid
hormone-responsive in rats although the major change in this mRNA, like
that for -MHC, occurs during the hypothyroid to euthyroid transition
(7). In acutely thyrotoxic rats, there was a doubling of
HCN2 mRNA from the hypothyroid to euthyroid state but only a 15%
further increase during transition from euthyroidism to
hyperthyroidism. Little is known of the factors regulating the mouse
HCN2 gene. A recent communication suggests that it too may be thyroid
hormone responsive. HCN2 mRNA levels were reduced about 50% in
hypothyroid mice and were twice normal in hyperthyroid mice
(41). In addition, the level of HCN2 mRNA was shown to be
primarily regulated by rather than by ß thyroid hormone receptors
(42). This could account for the fact that there was an
increase in the mRNA for this gene but not that of -MHC or SERCA II
in the TG hearts. Such a species difference would also raise the
possibility that in humans the HCN2 gene might also be positively
regulated between the euthyroid and hyperthyroid state. This could
account for the common observation of tachycardia as one of the
earliest physical manifestations of thyrotoxicosis in humans. Although
an increase in spontaneous heart rate correlated with the increase in
HCN2 gene expression in the transgenic ventricles, it is well known
that thyroid hormone action may be chamber specific and further studies
analyzing HCN2 expression in atrial pacemaking cells will be needed to
determine whether similar effects occur (43).
In association with the increased intrinsic heart rate, the
31P NMR as well as biochemical measurements
demonstrated a significant reduction in phosphocreatine in the
transgenic hearts (Fig. 2, B and C, and Table 1). This was associated
with a decrease in creatine level in the TG mice (Table 1). Both of
those findings have been reported in the myocardium of hyperthyroid
rats (20, 44). The decreased PCr may make the TG mouse
heart more susceptible to ischemic challenge with a more rapid decrease
in ATP and a greater increase in Pi than occurs under normal
circumstances. Such effects could then lead to decreases in myocardial
pH and reduced cardiac function (25). Testing to determine
the validity of such predictions is currently in progress.
The changes, such in myocardial performance and biochemistry, induced by chronic D2 overexpression are unexpected. Some of the more striking alterations in gene expression expected on the basis of earlier short-term, high dose of exogenous thyroid hormones did not occur. In humans, only modest increases in serum T3 and T4 (within the normal range) are required to cause suppression of TSH. There is considerable controversy about whether or not such subclinical hyperthyroidism, manifested only by a suppressed TSH, is physiologically significant (45). Because the changes demonstrated in these mice occur with minimal increases in myocardial T3 together with the fact that the human myocardium also expresses D2 mRNA, modest increases in circulating T4 and T3 would have similar effects on the human myocardium. Supporting this is a recent report that in a group of patients with normal thyroid hormone levels but suppressed TSH, 24-h Holter monitoring showed an increase in heart rate from 71 to 82 beats per minute compared with age-matched controls (45). Thus, mice expressing a D2 transgene may provide a model for evaluation of the consequences of mild chronic thyrotoxicosis on myocardial function which is hard to generate by any other technique.
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Footnotes |
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2 Supported by a Student’s fellowship from Dr. Saal van
Zwanenbergstichting, Stichting Bekker-la Bastide-Fonds and Stichting
Dr. Hendrik Muller’s Vaderlandsch Fonds from Netherlands.
Received July 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MHC403/+ mouse model of familial hypertrophic
cardiomyopathy. J Clin Invest 101:1775–1783[Medline]
337 threonine thyroid hormone receptor
beta mutant derived from the S family. Endocrinology 140:897–902This article has been cited by other articles:
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