Thyroid hormone and cardiac function in mice deficient in thyroid hormone receptor-alpha or -beta : an echocardiograph study

Roy E. Weiss1, Claudia Korcarz1, Olivier Chassande3, Kevin Cua1, Peter M. Sadow2, Eugene Koo1, Jacques Samarut3, and Roberto Lang1

Departments of 1 Medicine and 2 Pathology, University of Chicago, Chicago, Illinois 60637; and 3 Laboratoire de Biologie Moleculaire et Cellulaire de l'Ecole Normale Supérieure de Lyon, 69364 Lyon, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the effect of thyroid hormone (TH) receptor (TR)alpha and -beta isoforms in TH action in the heart. Noninvasive echocardiographic measurements were made in mice homozygous for disruption of TRalpha (TRalpha 0/0) or TRbeta (TRbeta -/-). Mice were studied at baseline, 4 wk after TH deprivation (using a low-iodine diet containing propylthiouracil), and after 4-wk treatment with TH. Baseline heart rates (HR) were similar in wild-type (WT) and TRalpha 0/0 mice but were greater in TRbeta -/- mice. With TH deprivation, HR decreased 49% in WT and 37% in TRbeta -/- mice and decreased only 5% in TRalpha 0/0 mice from baseline, whereas HR increased in all genotypes with TH treatment. Cardiac output (CO) and cardiac index (CI) in WT mice decreased (-31 and -32%, respectively) with TH deprivation and increased (+69 and +35%, respectively) with TH treatment. The effects of CO and CI were blunted with TH withdrawal in both TRalpha 0/0 (+8 and -2%, respectively) and TRbeta -/- mice (-17 and -18%, respectively). Treatment with TH resulted in a 64% increase in LV mass in WT and a 44% increase in TRalpha 0/0 mice but only a 6% increase in TRbeta -/- mice (ANOVA P < 0.05). Taken together, these data suggest that TRalpha and TRbeta play different roles in the physiology of TH action on the heart.

left ventricular mass; thyrotropin; cardiac index; cardiac output; shortening fraction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THYROID HORMONE (TH) exerts a profound effect on the chronotropic and ionotropic function of the heart (25, 28). TH deficiency results in systolic and diastolic dysfunction with reduction in heart rate and force of contraction and an increase in cardiac relaxation (18, 23). The molecular basis for the negative ionotropic consequences of hypothyroidism can be linked to decreased expression of the sarcoplasmic reticulum Ca2+ ATPase gene (42) and myosin heavy chain (MHC)alpha and increase in MHCbeta (27, 42). The latter results in decreased speed of systolic contraction due to the increased expression of myosin V3, which has a lower ATPase activity (2, 44). The molecular basis for the ionotropic effect seen in hypothyroidism, namely the bradycardia, is related to decreased expression of the hyperpolarization-activated, cyclic nucleotide-gated genes 2 or 4, which are specific targets of the TH receptor (TR)alpha gene (19).

TH action is mediated by its interaction with specific nuclear TRs functioning as ligand-dependent transcription factors that modulate the expression of target genes (30, 33). The two TR genes TRalpha and TRbeta have substantial structural and sequence similarities. Each generates multiple TR proteins by alternative splicing (alpha 1 and alpha 2; beta 1, beta 2, and beta 3) or alternative start sites (Delta alpha 1, Delta alpha 2, revErb). TRalpha 2 binds to DNA, but, due to a sequence difference at the ligand-binding site, it does not bind TH and thus does not function as a TR proper (35). The relative expression of TR genes and the distribution of their products vary among tissues and during different stages of development (20, 31, 46). Furthermore, an internal promoter, located within intron 7 of the TRalpha gene, is responsible for the expression, in mice, of truncated isoforms of TRalpha 1 and TRalpha 2, (TRDelta alpha 1 and TRDelta alpha 2) containing the carboxy-terminal segment of the molecule. These additional products of the TRalpha gene may play a role in downregulation of transcriptional activity (5, 17, 40). TRalpha 1 and TRalpha 2 are the major isoforms of TR expressed in the heart (24). Although TRbeta 1 is expressed at low levels (10), TRbeta 2 is also expressed (43). The use of mice with disruption of either the TRbeta or TRalpha gene has led to the conclusion that the effect of L-triiodothyronine (L-T3) on the heart is mediated predominantly by TRalpha (19, 21, 22, 38, 48). These studies have primarily examined the effect of TH deprivation and have not examined the effect of long-term TH excess.

The purpose of the present study was to examine the contribution that both TRbeta and TRalpha make in the presence of TH withdrawal and TH treatment on the chronotropic and ionotropic effect on the heart in vivo as determined by echocardiographic parameters. Echocardiography uniquely allows the study of sequential changes in TH action over time in the same animals. We have confirmed that chronotropic effects of TH on the heart are primarily TRalpha dependent. Furthermore, we have demonstrated that in an intact animal the action of TH on the heart is both TRbeta and TRalpha dependent.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mice. Mice were weaned on the 4th wk after birth and fed a rodent diet (no. 5053; Lab Diet, Brentwood, MO) containing 0.53 ppm iodine and were given tap water ad libitum. They were housed three to five mice per cage in an environment of controlled 19°C temperature and 12-h alternating dark-artificial light cycles. All animal experiments were performed according to approved protocols at the University of Chicago by the Institutional Animal Care and Use Committee.

Male mice were 50-90 days old at the time of initial blood sampling. Three hundred microliters of blood were obtained by retroorbital vein puncture under light methoxyflurane (Pitman Moore, Mundelein, IL) anesthesia. Bleeding was generally done between 0900 and 1200. Serum was separated by centrifugation and stored at -20°C until analyzed.

The TRbeta knockout mice were produced by insertion of the LacZ-NeoR cassette downstream of the splice site in exon 4, eliminating the expression of the DNA and ligand-binding domains of TRbeta 1 and TRbeta 2 (TRbeta -/-) (16). The TRalpha knockout mouse was produced by insertion of the LacZ-NeoR cassette downstream of exon 3 and replacing exons 5-7, thus effectively abolishing not only the generation of full TRalpha 1 and TRalpha 2 transcripts but also that of TRDelta alpha 1 or TRDelta alpha 2 (TRalpha 0/0) by removal of the transcription start point at intron 7 (17). The gene sequence for rev-erbA-alpha protein encoded by the opposite strands for the TRalpha (45) remains intact. In both sets of mice, the recombinant ES cells were derived from 129sv mice and were implanted into C57BL/6 recipient blastocysts. C57BL/6 mice were mated to each chimeric mouse and then back-crossed three to four times into the same strain, thereby diluting the 129sv background. The TRalpha 0/0 and TRbeta -/- mice were crossed for greater than five generations to select wild-type mice that had similar backgrounds.

Induction of hypothyroidism and treatment with TH. TH deficiency was induced in 10 male mice of each type (wild type, TRalpha 0/0, and TRbeta -/-) with a low-iodine diet containing 0.15% 5-propyl-2-thiouracil (PTU) for 4 wk. Blood samples were obtained from the retroorbital vein after recording of echocardiograms as described in Echocardiograms. The same mice were then placed for 4 wk on L-thyroxine (L-T4) contained in the drinking water (2 µg/ml). Mice drank ~5 ml/day, resulting in a total dose of 10 µg L-T4 · day-1 · mouse-1.

Hormone measurements. Serum total T4 was measured by coated-tube RIAs (DPC; Diagnostic Products, Los Angeles, CA) with 25 µl of serum. The sensitivity of this assay is 0.2 µg T4/dl (2.6 nmol/l). The interassay coefficients of variation were 5.4, 4.2, and 3.6% at 3.8, 9.4, and 13.7 µg/dl T4, respectively.

Serum TSH was measured in 50 µl of serum by use of a sensitive, heterologous, disequilibrium, double-antibody precipitation RIA, as previously described (41). The sensitivity of this assay was 5-10 mU/l. The intra-assay and interassay coefficients of variation were, respectively, 16 and 27% at 20 mU/l, 6.3 and 8.2% at 200 mU/l, 5.4 and 9.8% at 850 mU/l, and 10 and 24% at 2,000 mU/l. Samples containing >1,000 and >10,000 mU thyroid-stimulating hormone (TSH)/l were diluted 10- to 100-fold, respectively, with zero TSH mouse serum obtained from wild-type mice treated with a suppressive dose of T4 (41).

Ventricular weight. At the end of the 4 wk of L-T4 treatment and after completion of the final echocardiographic recording, mice were killed and their hearts excised. The left ventricles were separated and carefully isolated by trimming the atria and the valves, blotted of excess fluid, and were weighed on a Mettler Balance (model no. AG245) with an accuracy of ± 0.01 mg.

Echocardiograms. For animal preparation, 30 mice were studied, including 10 wild-type, 10 TRbeta -/-, and 10 TRalpha 0/0 mice, as described.

Before acquisition of cardiac ultrasound recordings, anesthesia was induced by administering chloral hydrate (500 mg/5 ml; Pennex Pharmaceutical) intraperitoneally in a 4% solution in PBS at a dose of 0.4 mg/g mouse. Animals were then secured to a custom-made waterbed in a shallow left lateral decubitus position to facilitate imaging. The bed was connected to a circulating water bath set at 40°C to prevent hypothermia. The actual bed temperature was maintained at 38°C throughout the experiment, and every effort was made to maintain constant temperature, but the application of gel for echocardiography was an unavoidable variable. Transthoracic echocardiography was performed three times during the experiment. Baseline recordings were obtained at 8 wk of age, after 4 wk with PTU treatment (as described, at 12 wk of age), and after 4 wk of L-T4 treatment (as described, at 16 wk of age).

Data acquisition. Cardiac ultrasound imaging was performed using a high-frequency 15-MHz linear transducer (Sonos 5500; Agilent, Andover, MA) at a maximum frame rate of 120 frames/s. Parasternal long- and short-axis views were obtained after adjusting gain settings for optimal epicardial and endocardial wall visualization. From the short-axis view, left ventricular (LV) M-mode tracings were obtained. From an unconventional, more superior parasternal long-axis view, ascending aortic two-dimensionally targeted M-modes were recorded. Ascending aortic pulse wave Doppler velocities were obtained from the suprasternal window by means of a pediatric short focal length, 12-MHz phased array transducer (Sonos 5500; Agilent Technologies). To improve image quality, an acoustic coupling gel standoff was mounted to the probe. This resulted in a 1- to 1.5-cm standoff between the transducer and the chest wall, enabling the transducer to work at its ideal focal length. To further improve quality by decreasing artifacts, the gel (Aquasonic 100; Parker, Orange, NJ) was centrifuged at 2,000 g to remove air bubbles.

Echocardiographic loops of >= 20 cardiac cycles containing the two-dimensional data, M-mode tracings, and Doppler velocity panels were stored digitally on magneto-optical disk for off-line analysis.

Measurements. From the short-axis view, epicardial and endocardial LV areas were measured offline at end systole and end diastole. Images were considered adequate for measurement when >75% of the epicardial and endocardial contour could be adequately visualized. In accordance with American Society of Echocardiography recommendations, the short-axis endocardial border was traced on the innermost endocardial edge, whereas the epicardial border was traced along the first bright pixel immediately adjacent to the darker myocardium (Fig. 1B). The LV length, defined as the distance between the apex and the midmitral annulus, was obtained from the parasternal long-axis views in which the mitral annular plane and the apex were well defined (Fig. 1A). LV measurements were made from at least three cardiac cycles, at both end systole and end diastole.


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Fig. 1.   Representative echocardiograms used for measurement of left ventricular (LV) mass by use of the area-length method. A: in the parasternal long-axis view, the ventricular length (L) is measured from the endocardial apex to the mitral valve annulus (MVA). B: in the short-axis view, the LV area is traced at the level of the papillary muscles (arrow). The tip of the papillary muscles was used as a landmark reference.

LV mass was calculated using the formula
2-D area-length method LV mass 

=[1.05 (5/6 A<SUB>1</SUB>(L + t) − 5/6 A<SUB>2</SUB>L]
where 1.05 is the specific gravity of muscle, A1 and A2 are the epicardial and endocardial parasternal short-axis area, respectively, L is the parasternal long-axis length, and t is the wall thickness calculated from A1 and A2 (Fig. 1).

Two-dimensionally targeted M-mode echocardiographic images were obtained at the level of the papillary muscles from the parasternal short-axis view and recorded at a speed of 150 cm/s (Fig. 2). LV internal diameters and wall thickness (leading edge to trailing edge) were obtained at end systole and end diastole from cross-sectional short-axis views. Heart rate was measured, and shortening fraction (SF), the echocardiographic equivalent of ejection fraction, was calculated from these tracings by using the formula
%SF = [(LVedd − LVeds)/LVedd)] × 100
where LVedd and LVeds are the LV internal diameter at end diastole and end systole, respectively.


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Fig. 2.   Representative echocardiogram used for calculation of shortening fraction (%SF). This is an LV 2-dimensionally targeted M-mode tracing at midcavity level, where LV end-diastolic (LVedd) and end-systolic (LVesd) dimensions are measured and used for calculating %SF.

For both two-dimensional and M-mode calculations, LV end-diastolic measurements were obtained at the peak of the R wave, whereas end-systolic measurements were obtained at the time of minimal chamber area.

Aortic stroke volume (SVao) was calculated from pulse wave aortic Doppler recordings and measurements of the proximal ascending aortic diameter (Fig. 3). Because of the pulsatile nature of the cardiovascular system, velocities are not constant throughout the cardiac cycle, thereby requiring temporal integration of the Doppler velocities known as the time velocity integral (TVI). The cross-sectional area of the aorta (CSAao) was calculated assuming a constant circular orifice throughout the cardiac systole by use of the formula
CSA<SUB>ao</SUB> = (AO diameter/2)<SUP>2</SUP> × &pgr;

SV<SUB>ao</SUB> = CSA<SUB>ao</SUB> × TVI<SUB>ao</SUB>

CO<SUB>ao</SUB> = SV<SUB>ao</SUB> × HR

CI = CO<SUB>ao</SUB>/body wt
where CO is cardiac output, CI is cardiac index, and HR is heart rate.


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Fig. 3.   Two-dimensionally targeted M-mode tracing of proximal ascending aorta (A) and aortic Doppler velocity profile (B). AO, aorta; LA, left atrium.

Data analysis. Values are reported as means ± SD. P values were calculated by two-way ANOVA when mice of different genotypes and treatment were compared and by the Student's t-test when comparisons were made within the same genotype with the Statview 5.0 program (SAS Institute, Cary, NC).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thyroid function tests and body weights. The thyroid function tests before and after treatment with PTU and L-T4 are shown in Table 1. Determination of the effect of TH on heart physiology could not be done with baseline measurements alone, because the mice had different serum concentrations of T4 and TSH. Therefore, mice of all genotypes were made deficient in TH by PTU treatment for 4 wk. This treatment normalized the serum T4 levels to <0.02 µg/dl in all groups, whereas the TSH concentration increased 340-, 48-, and 149-fold in the wild-type, TRalpha 0/0, and TRbeta -/- mice, respectively. L-T4 treatment suppressed the TSH in all genotypes to <20 mU/l, whereas serum T4 levels were similar in wild-type and TRalpha 0/0 mice, TRbeta -/- mice had slightly higher concentrations.

                              
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Table 1.   Thyroid function tests and body weights

At baseline, TRbeta -/- mice had markedly elevated serum T4 and TSH, consistent with their state of resistance to TH. Four-week treatment with PTU resulted in a decrease of serum T4 levels below the limit of detection with a concomitant increase in TSH. In the TRalpha 0/0 mice, the serum TSH did not reach the level attained in the wild-type or TRbeta -/- mice. Treatment with L-T4 suppressed the TSH in all mice, and although the serum T4 concentration attained in the TRbeta -/- mice was not different from that in wild-type mice, it was slightly higher in the TRalpha 0/0 mice (P < 0.05). There were no significant differences in body weight at baseline for any of the three genotypes. Mice treated with PTU for 4 wk did not gain weight during this time. However, after 4 wk of L-T4 treatment, body weight increased by 26, 13, and 32% in wild-type, TRalpha 0/0, and TRbeta -/- mice, respectively, compared with baseline weights.

Effect of TH on heart rate. Heart rate in these mice determined during the echocardiograph analysis demonstrated resting tachycardia in untreated TRbeta -/- mice (415 ± 10 beats/min) relative to the untreated wild-type mice (372 ± 10 beats/min) and TRalpha 0/0 mice (407 ± 10), likely reflective of the higher baseline T4 levels in these mice. The difference in basal heart rate between wild-type and TRalpha 0/0 mice was not significant. During TH deprivation, heart rates significantly decreased in both wild-type (49 ± 6%) and TRbeta -/- (39 ± 6%) mice and changed only a small amount in the TRalpha 0/0 mice (5 ± 6%). Although TH treatment resulted in an increase in the wild-type mice (43 ± 6%), there was only minimal change in the TRbeta -/- (5 ± 2%), yet a moderate increase in the TRalpha 0/0 (21 ± 6%) compared with wild-type mice (Fig. 4).


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Fig. 4.   Heart rate (HR) in wild-type mice, mice homozygous for disruption of thyroid hormone receptor-beta (TRbeta -/-) and mice homozygous for disruption of TRalpha (TR0/0) at baseline (filled bar), with TH deprivation [5-propyl-2-thiouracil (PTU); gray bar], and with L-thyroxine (L-T4) treatment (open bar). A: HR; B: %difference in HR. Statistical significance, P <0.05: * difference between baseline values, same genotype; Delta difference from wild-type, same treatment; nabla difference from PTU treatment, same genotype. BPM, beats/min.

Echocardiographic analyses. The SF, equivalent to the ejection fraction, was significantly reduced in the hypothyroid wild-type mice (25.6 ± 2.3%) compared with pretreatment baseline (38.4 ± 2.3). Although there was a trend toward having decreased SF in TRbeta -/- and TRalpha 0/0 mice, it was not significantly different from that of wild-type mice (Fig. 5, A-C). There were no significant changes in SF with L-T4 treatment in the different groups compared with baseline or compared with the hypothyroid state. Preload CO (Fig. 5, D-F) and CI (Fig. 5, G-I) decreased by 31 ± 8% and 33 ± 10%, respectively, in hypothyroid mice and increased by 69 ± 10% and 35 ± 8%, respectively, in hyperthyroid wild-type mice. Neither the TRbeta -/- nor the TRalpha 0/0 mice had a response in CO or CI to TH deprivation, and although the TRalpha 0/0 mice had a slight increase in CI and CO in response to TH treatment, it was not as robust as that seen in the wild-type mice (Fig. 5).


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Fig. 5.   SF (A-C), cardiac output (CO; D-F), and cardiac index (CI; G-I) in wild-type, TRbeta -/-, and TRalpha 0/0 mice at baseline (filled bar), with TH deprivation (PTU; gray bar), and with L-T4 treatment (open bar). Top (A, D, G): absolute values; middle (B, E, H): %difference from untreated; bottom (C, F, I): %difference between hypothyroid and hyperthyroid. Statistical significance, P < 0.05: * difference between baseline values, same genotype; Delta difference from wild type, same treatment; nabla difference from PTU treatment, same genotype.

Effect of TH on LV mass. LV mass was determined by area- to length-based estimates via transthoracic echocardiographic measurements in diastole (ALd) and systole (ALs). Although there was no significant difference in LV mass between baseline and hypothyroid mice in any genotype, L-T4 treatment resulted in an increase in the ALd of wild-type (0.079 ± 0.001 to 0.126 ± 0.016 g, P < 0.05) and TRalpha 0/0 mice (0.079 ± 0.012 to 0.114 ± 0.021 g, P < 0.05) but less so in the TRbeta -/- mice (0.085 ± 0.011 to 0.089 ± 0.009 g, P > 0.05) (Fig. 6). Similar changes were seen in ALs and in ALs index and ALd index when corrected for body weight. At autopsy, upon completion of the L-T4 treatment arm of the experiment the LV weight (mg) per gram of mouse correlated with the echocardiographic measurements (3.9 vs. 4.2 mg/g, respectively, for the wild-type mice; 3.4 vs. 3.5 mg/g, respectively, for the TRbeta -/- mice; and 4.0 vs. 4.0 mg/g, respectively, for the TRalpha 0/0 mice.)


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Fig. 6.   LV mass measured by echocardiography. A: absolute mass (g) during diastole (ALd). * P < 0.05 compared with baseline in same genotype. B: %difference from baseline. * P < 0.05 compared with difference from wild type; Delta , %difference between TRbeta -/- and TRalpha 0/0.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of TH on the heart is both intrinsic and extrinsic. Studies demonstrating effects of TH on isolated cardiac muscle (19, 34) or perfused intact hearts (37) give insight into the intrinsic effects of TH on cardiac function, but noninvasive assessment of cardiovascular function has not been well reported in vivo. Technological advances, such as smaller probes and the creation of high-frequency linear transducers, have allowed accurate echocardiographic analysis of small mice and enabled one to obtain in vivo measurements of cardiac function (8, 11, 13-15, 36). Echocardiographic measurements can accurately determine dynamic changes in LV mass (8). Furthermore, studies using mice with deletion of either of the TRs allows one to delineate their contribution to TH action. This study demonstrates the role that TRbeta and TRalpha play in TH action in the heart. Whereas TRalpha 1 and TRalpha 2 are the major TR isoforms expressed in the heart, it is not surprising that wild-type and TRbeta -/- mice had a decreased heart rate in response to TH deprivation, whereas the TRalpha 0/0 mice were unresponsive (32, 48). Whereas all three genotypes demonstrated an increase in heart rate in response to TH treatment, there was a blunted response in both the TRalpha 0/0 and TRbeta -/- mice, indicating that TRbeta also plays a role in the increase in heart rate associated with TH treatment. This study does not indicate whether the TRbeta responsiveness is a direct effect on the heart or an indirect effect on sympathomimetic factors, which, in turn, increase heart rate.

Contrary to the heart rate data presented in this paper, two previous reports, including one from our laboratory (19, 32), have demonstrated baseline bradycardia in the TRalpha 0/0 mice. Variables that may account for this discrepancy include 1) the method of anesthesia, 2) the number of mice analyzed, and 3) the body temperature of the animal at the time of analyses. Although Gloss et al. (19) used a ketamine-xylazine cocktail, Macchia et al. (32) used chloral hydrate as we did in the present report. Although Gloss et al. analyzed six animals and reported a difference in heart rate, Macchia et al. required 43 wild-type mice to show significant differences, due to the variability in heart rate. Furthermore, our measurement of heart rate was done during simultaneous echocardiograph analysis, and although every effort was attempted to maintain euthermia in the mice, there may have been differences in body temperature to account for the differences in heart rate at baseline.

TRalpha 1 knockout mice had an average heart rate 20% lower than that of wild-type mice, with prolonged QRS and QT durations at baseline and with TH treatment, suggesting that TRbeta can also affect heart rate (49). Transgenic mice with myocardium-specific expression of a mutant TRbeta (Delta 377T) demonstrated heart failure in vitro but not in vivo, suggesting that diminished performance in these mice may be compensated for by other mechanisms in vivo (38).

Change in %SF was limited to the wild-type mice. Although there was a trend toward the TRbeta -/- mice having decreased %SF with TH deprivation and an increase with TH treatment, this did not reach significance. Furthermore, CO and CI were increased in response to TH treatment and were decreased in response to TH withdrawal in the wild-type mice, and these changes were blunted in the both the TRbeta -/- and TRalpha 0/0 mice. Therefore, whereas TRalpha seems to be more important than TRbeta for the chronotropic effects of TH on cardiac function, both TRalpha and TRbeta are necessary for the ionotropic effects. In humans, hyperthyroidism results in an increase in echocardiograhic indexes of myocardial contractility (12, 39). Whereas normal CO in humans is 4.0-6.0 l/min, it is >7.0 and <4.0 l/min in hyperthyroid and hypothyroid humans, respectively.

Hyperthyroidism has been reported to result in increased cardiac mass in both humans (7) and mice (4, 26). The mechanism of increased cardiac mass may be related to the local activation of the renin-angiotensin system in the heart in response to TH that can be reversed with angiotensin-converting enzyme inhibitors (1, 3, 29). In addition, the TH-induced increase in heart size may be due, in part, to coordinated capillary and myocardial growth that is TH dependent (6, 9, 47). The absence of TRbeta prevented the TH-mediated increase in cardiac mass, whereas the absence of TRalpha did not. TRbeta may be the important TR for the generation of tissue angiotensin-converting enzyme. In our mice, the absolute T4 concentrations were higher in the TRbeta -/- mice compared with the TRalpha 0/0 mice only. This difference may partially account for the increase in LV mass seen in TRbeta -/- mice but cannot explain the difference compared with wild-type mice.

TRalpha is the predominant isoform in the heart. We have shown that TRalpha is required for the changes in heart rate seen with TH deprivation or treatment. However, the TH-induced increase in cardiac mass is TRbeta dependent. Taken together, these data suggest that TRalpha and TRbeta play different roles in the physiology of TH action on the heart. Furthermore, TRbeta may play a more important role in indirect effect of TH on the heart.


    ACKNOWLEDGEMENTS

This study was supported in part by Grant DK-58258 (to R. E. Weiss) from the National Institute of Diabetes and Digestive and Kidney Diseases and by the Seymour J. Abrams Thyroid Research Fund and the Ministry of Research ACI 283 (to J. Samarut).


    FOOTNOTES

Address for reprint requests and other correspondence: R. E. Weiss, Dept. of Medicine, MC 3090, Univ. of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: rweiss{at}medicine.bsd.uchicago.edu).

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

10.1152/ajpendo.00019.2002

Received 18 January 2002; accepted in final form 16 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Asahi, T, Shimabukuro M, Oshiro Y, Hisashi Y, and Takasu N. Cilazapril prevents cardiac hypertrophy and postischemic myocardial dysfunction in hyperthyroid rats. Thyroid 11: 1009-1015, 2001[ISI][Medline].

2.   Barany, M. ATPase activity of myosin correlated with speed of muscle shortening. J Gen Physiol 50, Suppl6: 197-218, 1967[Abstract/Free Full Text].

3.   Basset, A, Blanc J, Messas E, Hagège A, and Elghozi J-L. Renin-angiotensin system contribution to cardiac hypertrophy in experimental hyperthyroidism: an echocardiographic study. J Cardiovasc Pharmacol 37: 163-172, 2001[ISI][Medline].

4.   Bedotto, JB, Gay RG, Graham SD, Morkin E, and Goldman S. Cardiac hypertrophy induced by thyroid hormone is independent of loading conditions and beta adrenoceptor blockade. J Pharmacol Exp Ther 248: 632-636, 1989[Abstract].

5.   Chassande, O, Fraichard A, Gauthier K, Flamant F, Legrand C, Savatier P, Laudet V, and Samarut J. Identification of transcripts initiated from an internal promoter in the c-erb-Aalpha locus that encode inhibitors of retinoic acid receptor-alpha and triiodothyronine receptor activities. Mol Endocrinol 11: 1278-1290, 1997[Abstract/Free Full Text].

6.   Chilian, WM, Wangler RD, Peters KG, Tomanek RJ, and Marcus ML. Thyroxine-induced left ventricular hypertrophy in the rat. Anatomical and physiological evidence for angiogenesis. Circ Res 57: 591-598, 1985[Abstract].

7.   Ching, GW, Franklyn JA, Stallard TJ, Daykin J, Sheppard MC, and Gammage MD. Cardiac hypertrophy as a result of long-term thyroxine therapy and thyrotoxicosis. Heart 75: 363-368, 1996[Abstract].

8.   Collins, KA, Korcarz CE, Shroff SG, Bednarz JE, Fentzke RC, Lin H, Leiden JM, and Lang RM. Accuracy of echocardiographic estimates of left ventricular mass in mice. Am J Physiol Heart Circ Physiol 280: H1954-H1962, 2001[Abstract/Free Full Text].

9.   Craft-Cormney, C, and Hansen JT. Early ultrastructural changes in the myocardium following thyroxine-induced hypertrophy. Virchows Arch B Cell Pathol Incl Mol Pathol 33: 267-273, 1980[ISI][Medline].

10.   Falcone, M, Miyamoto T, Fierro-Renoy F, Nacchia E, and DeGroot LJ. Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform. Endocrinology 131: 2419-2429, 1992[Abstract].

11.   Feldman, MD, Erikson JM, Mao Y, Korcarz CE, Lang RM, and Freeman GL. Validation of a mouse conductance system to determine LV volume: comparison to echocardiography and crystals. Am J Physiol Heart Circ Physiol 279: H1698-H1707, 2000[Abstract/Free Full Text].

12.   Feldman, T, Borow KM, Sarne DH, Neumann A, and Lang RM. Myocardial mechanics in hyperthyroidism: importance of left ventricular loading conditions, heart rate and contractile state. J Am Coll Cardiol 7: 967-974, 1986[ISI][Medline].

13.   Fentzke, RC, Korcarz CE, Lang RM, Lin H, and Leiden JM. Dilated cardiomyopathy in transgenic mice expressing a dominant- negative CREB transcription factor in the heart. J Clin Invest 101: 2415-2426, 1998[Abstract/Free Full Text].

14.   Fentzke, RC, Korcarz CE, Shroff SG, Lin H, Leiden JM, and Lang RM. The left ventricular stress-velocity relation in transgenic mice expressing a dominant negative CREB transgene in the heart. J Am Soc Echocardiogr 14: 209-218, 2001[ISI][Medline].

15.   Fentzke, RC, Korcarz CE, Shroff SG, Lin H, Sandelski J, Leiden JM, and Lang RM. Evaluation of ventricular and arterial hemodynamics in anesthetized closed-chest mice. J Am Soc Echocardiogr 10: 915-925, 1997[ISI][Medline].

16.   Gauthier, K, Chassande O, Platerotti M, Roux JP, Legrand C, Rousset B, Weiss R, Trouillas J, and Samarut J. Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development. EMBO J 18: 623-621, 1999[Abstract/Free Full Text].

17.   Gauthier, K, Plateroti M, Harvey CB, Williams GR, Weiss RE, Refetoff S, Willott JF, Sundin V, Roux JP, Malaval L, Hara M, Samarut J, and Chassande O. Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha  locus. Mol Cell Biol 21: 4748-4760, 2001[Abstract/Free Full Text].

18.   Gay, RG, Raya TE, Lancaster LD, Lee RW, Morkin E, and Goldman S. Effects of thyroid state on venous compliance and left ventricular performance in rats. Am J Physiol Heart Circ Physiol 254: H81-H88, 1988[Abstract/Free Full Text].

19.   Gloss, B, Trost SU, Gluhm WF, Swanson EA, Clark R, Winkfein R, Janzen KM, Giles W, Chassande O, Samarut J, and Dillmann WH. Cardiac ion channel expression and contractile function in mice with deletion of thyroid hormone receptor alpha or beta. Endocrinology 142: 544-550, 2001[Abstract/Free Full Text].

20.   Hodin, RA, Lazar MA, and Chin WW. Differential and tissue-specific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone. J Clin Invest 85: 101-105, 1990[ISI][Medline].

21.   Johansson, C, Gothe S, Forrest D, Vennstrom B, and Thoren P. Cardiovascular phenotype and temperature control in mice lacking thyroid hormone receptor-beta or both -alpha 1 and -beta . Am J Physiol Heart Circ Physiol 276: H2006-H2012, 1999[Abstract/Free Full Text].

22.   Johansson, C, Lannergeren J, Lunde PK, Vennstrom B, Thoren P, and Westerblad H. Isometric force and endurance in soleus muscle of thyroid hormone receptor-alpha 1 or -beta -deficient mice. Am J Physiol Regul Integr Comp Physiol 278: R598-R603, 2000[Abstract/Free Full Text].

23.   Kahaly, G, Mohr-Kahaly S, Beyer J, and Meyer J. Left ventricular function analyzed by Doppler and echocardiographic methods in short term hypothyroidism. Am J Cardiol 75: 645-648, 1995[ISI][Medline].

24.   Kinugawa, K, Yonekura K, Ribeiro RCJ, Eto Y, Aoyagi T, Baxter JD, Camacho SA, Bristow MR, Long CS, and Simpson PC. Regulation of thyroid hormone receptor isoforms in physiological and pathological cardiac hypertrophy. Circ Res 89: 591-598, 2001[Abstract/Free Full Text].

25.   Klein, I. Thyroid hormone and the cardiovascular system. Am J Med 88: 631-637, 1990[ISI][Medline].

26.   Klein, I. Thyroxine-induced cardiac hypertrophy: time course of development and inhibition by propranolol. Endocrinology 123: 203-210, 1988[Abstract].

27.   Klein, I, and Hong C. Effects of thyroid hormone on cardiac size and myosin content of the heterotopically transplanted rat heart. J Clin Invest 77: 1694-1698, 1986[ISI][Medline].

28.   Klein, I, and Ojamaa K. Thyroid hormone and the cardiovascular system. N Engl J Med 344: 501-509, 2001[Free Full Text].

29.   Kobori, H, Ichihara A, Miyashita Y, Hayashi M, and Saruta T. Local renin-angiotensin system contributes to hyperthyroidism-induced cardiac hypertrophy. J Endocrinol 160: 43-47, 1999[Abstract/Free Full Text].

30.   Lazar, MA. Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14: 184-193, 1993[ISI][Medline].

31.   Macchia, E, Nakai A, Janiga A, Sakurai A, Fisfalen ME, Gardner P, Soltani K, and DeGroot LJ. Characterization of site-specific polyclonal antibodies to c-erbA peptides recognizing human thyroid hormone receptors alpha 1, alpha 2, and beta  and native 3,5,3'-triiodothyronine receptor, and study of tissue distribution of the antigen. Endocrinology 126: 3232-3239, 1990[Abstract].

32.   Macchia, PE, Takeuchi Y, Kawai T, Cua K, Gauthier K, Chassande O, Seo H, Hayashi Y, Samarut J, Murata Y, Weiss RE, and Refetoff S. Increased sensitivity to thyroid hormone in mice deficient in thyroid hormone receptor alpha. Proc Natl Acad Sci USA 98: 394-354, 2001.

33.   Mangelsdorf, DL, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, and Evans RM. The nuclear receptor superfamily: the second decade. Cell 83: 835-839, 1995[ISI][Medline].

34.   McDonough, KH, Chen V, and Spitzer JJ. Effect of altered thyroid status on in vitro cardiac performance in rats. Am J Physiol Heart Circ Physiol 252: H788-H795, 1987[Abstract/Free Full Text].

35.   Mitsuhashi, T, Tennyson GE, and Nikodem VM. Alternative splicing generates messages encoding rat c-erbA proteins that do not bind thyroid hormones. Proc Natl Acad Sci USA 85: 5804-5805, 1988[Abstract].

36.   Mor-Avi, V, Korcarz C, Fentzke RC, Lin H, Leiden JM, and Lang RM. Quantitative evaluation of left ventricular function in a t transgenic mouse model of dilated cardiomyopathy with 2-dimensional contrast echocardiography. J Am Soc Echocardiogr 12: 209-214, 1999[ISI][Medline].

37.   Pachucki, J, Hopkins J, Peeters R, Tu H, Carvalho SD, Kaulbach H, Abel ED, Wondisford FE, Ingwall JS, and Larsen PR. Type 2 iodothyronine deiodinase transgene expression in the mouse heart causes cardiac-specific thyrotoxicosis. Endocrinology 142: 13-20, 2001[Abstract/Free Full Text].

38.   Pazos-Maoura, C, Abel ED, Boers ME, Moura E, Hampton TG, Wang J, Morgan JP, and Wondisford FE. Cardiac dysfunction caused by myocardium-specific expression of a mutant thyroid hormone receptor. Circ Res 86: 700-706, 2000[Abstract/Free Full Text].

39.   Petretta, M, Bonaduce D, Spinelli L, Vicario MLE, Nuzzo V, Marciano F, Camuso P, DeSanctis V, and Lupoli G. Cardiovascular haemodynamics and cardiac autonomic control in patients with subclinical and overt hyperthyroidism. Eur J Endocrinol 145: 691-696, 2001[ISI][Medline].

40.   Plateroti, M, Gauthier K, Domon-Dell C, Freund JN, Samarut J, and Chassande O. Functional interference between thyroid hormone receptor alpha  (TRalpha ) and natural truncated TRDelta alpha isoforms in the control of intestine development. Mol Cell Biol 21: 4761-4772, 2001[Abstract/Free Full Text].

41.   Pohlenz, J, Muqueem A, Cua K, Weiss RE, Van Sande J, and Refetoff S. Improved radioimmunoassay for measurement of mouse TSH in serum: strain differences in TSH concentration and thyrotropin sensitivity to thyroid hormone. Thyroid 9: 1265-1271, 1999[ISI][Medline].

42.   Rohrer, D, and Dillmann WH. Thyroid hormone markedly increase the mRNA coding for sacroplasmic reticulum Ca2+ ATPase in the heart. J Biol Chem 263: 6941-6944, 1988[Abstract/Free Full Text].

43.   Schwartz, H, Lazar M, and Oppenheimer J. Widespread distribution of immunoreactive thyroid hormone beta 2 receptor (TRbeta 2) in the nuclei of extrapituitary rat tissues. J Biol Chem 269: 24777-24782, 1994[Abstract/Free Full Text].

44.   Schwartz, K, Lecarpentier Y, Martin JL, Lompre AM, Mercadier JJ, and Swyngohedauw B. Myosin isoenzyme distribution correlates with speed of myocardial contraction. J Mol Cell Cardiol 13: 1071-1075, 1981[ISI][Medline].

45.   Spanjaard, R, Nguyen V, and Chin W. Rat rev-erbA alpha, an orphan receptor related to thyroid hormone receptor, binds to specific thyroid hormone response elements. Mol Endocrinol 8: 286-295, 1994[Abstract].

46.   Strait, KA, Schwartz HL, Perez-Castillo A, and Oppenheimer JH. Relationship of c-erbA mRNA content to tissue triiodothyronine nuclear binding capacity and function in developing and adult rats. J Biol Chem 265: 10514-10521, 1990[Abstract/Free Full Text].

47.   Tomanek, RJ, and Busch TL. Coordinated capillary and myocardial growth in response to thyroxine treatment. Anat Rec 251: 44-49, 1998[ISI][Medline].

48.   Weiss, RE, Murata Y, Cua K, Hayashi Y, Forrest D, Seo H, and Refetoff S. Thyroid hormone action on liver, heart and energy expenditure in thyroid hormone receptor beta  deficient mice. Endocrinology 139: 4945-4952, 1998[Abstract/Free Full Text].

49.   Wikström, L, Johansson C, Salto C, Barlow C, Campos Barros A, Baas F, Forrest D, Thorén P, and Vennström B. Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor alpha 1. EMBO J 17: 455-461, 1998[Abstract/Free Full Text].


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