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*LIOTHYRONINE
Endocrinology Vol. 145, No. 6 2767-2774
Copyright © 2004 by The Endocrine Society

The Metabolic and Cardiovascular Effects of Hyperthyroidism Are Largely Independent of ß-Adrenergic Stimulation

Eric S. Bachman, Thomas G. Hampton, Harveen Dhillon, Ivo Amende, JuFeng Wang, James P. Morgan and Anthony N. Hollenberg

Department of Medicine (E.S.B., H.D., A.N.H.), Division of Endocrinology; and Department of Medicine (T.G.H., I.A., J.W., J.P.M.), Division of Cardiology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Eric S. Bachman, Beth Israel Deaconess Medical Center, Division of Endocrinology, Room 316, RN 99 Brookline Avenue, Boston, Massachusetts 02215. E-mail: ebachman{at}caregroup.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hyperthyroidism and states of adrenergic hyperactivity have many common clinical features, suggesting similar pathogenic mechanisms of action. The widespread use of ß-adrenergic receptor (ßAR) antagonists (ß-blockers) to treat hyperthyroidism has led to the belief that the physiological consequences of thyroid hormone (TH) excess are mediated in part via catecholamine signaling through ßARs. To test this hypothesis, we compared the response to TH excess in mice lacking the three known ßARs (ß-less) vs. wild-type (WT) mice. Although ß-less mice had a lower heart rate at baseline in comparison to WT mice, the metabolic and cardiovascular responses to hyperthyroidism were equivalent in both WT and ß-less mice. These data indicate that the metabolic and cardiovascular effects of TH excess are largely independent of ßARs. These findings suggest that the efficacy of clinical treatment of hyperthyroidism with ß-blockers is due to antagonism of sympathetic signaling, and that this process functions independently of TH action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE AND the sympathetic nervous system (SNS) regulate numerous, diverse physiological processes. These two systems share many functions that suggest common mechanisms of action, although they are anatomically and molecularly distinct. For example, thyroid hormone (TH) and catecholamines, the endogenous ligands for the SNS, are necessary for normal thermogenesis and cold resistance (1, 2). In the heart, thyroid hormone causes cardio-stimulatory effects that are similar to catecholamine-mediated sympathetic stimulation of ß-adrenergic receptors (ßARs) (3, 4). Clinical states of thyroid excess (e.g. hyperthyroidism) resemble states of catecholamine excess (e.g. pheochromocytoma), suggesting synergistic mechanisms of action (5). Finally, effective treatment of hyperthyroidism includes the use of pharmacologic blockade of ßARs, suggesting that ßARs play an important role in the pathogenesis of hyperthyroidism (6).

It has not been conclusively determined whether the physiologic effects of TH and ßARs during hyperthyroidism are additive or synergistic. THs are synthesized as T4 and, to a lesser degree, as T3 in the thyroid gland. T3, the more active congener, is primarily the result of peripheral conversion of T4 in tissues. To mediate its effects on gene transcription, T3 enters the cell and binds to three nuclear thyroid receptors (TRs, the two TRß isoforms and TR{alpha}1), which bind to both positive and negative thyroid response elements in the regulatory regions of target genes. This leads to the recruitment of transcriptional coregulators and enables transcriptional regulation (7). In addition, nongenomic actions of TH have been postulated via the action of T4 at the plasma membrane (4, 8, 9). TH excess results in increased cardiac stimulation including tachycardia, augmented contractility, and a rise in metabolic demand (9). The chronic effects of T3 excess can cause cardiomyopathy and death (10). These effects are associated with altered expression of cardiac-specific structural and regulatory genes, which include increased transcription of cardiac stimulatory ßARs (11, 12). The relative contributions of these individual cardiac genes in hyperthyroid-induced cardiac stimulation are not known.

The adrenergic receptors (ARs) are essential for cardiovascular modulation (13). ARs are seven-pass transmembrane proteins that include six {alpha}ARs and three ßARs, all of which are receptors for the endogenous catecholamines norepinephrine and epinephrine (14). Stimulation of ßARs by catecholamines activates adenylate cyclase and results in an increase in intracellular cAMP. The three known ßARs are expressed in cardiac myocytes and regulate heart rate (HR), contractility, and vasoactivity (15, 16). Gene disruption experiments have suggested that the ß1AR primarily mediates cardiac chronotropy and inotropy, whereas the ß2 and ß3ARs regulate vascular tone and metabolic rate (13, 17, 18). Previous studies indicate that there is redundancy in the function of ßARs, and compensation when one or more of the ßARs are deleted (13, 19). Similar to T3 excess, overstimulation of ßARs in catecholamine-secreting tumors leads to cardiac hypertrophy, heart failure, and death (20). A direct, mechanistic relationship between T3 and ßARs in hyperthyroidism, however, has not been established.

The similarities between the effects of T3 excess and catecholamine excess on cardiovascular physiology are well known (10). Evidence that ßARs may contribute to hyperthyroidism includes increased expression of ßARs in thyrotoxicosis (10), significant improvement in cardiovascular symptoms with ß-blockade (21), and efficacy of ß-blockers in normalizing metabolic demand (22). The downstream effects of ßAR stimulation includes genes that are also regulated positively by T3, such as L-type calcium channels and phospholamban (4, 16). Nonetheless, the cardiovascular effects in hyperthyroidism may be the consequence of unique actions of T3 and ßARs (23, 24). For example, hyperthyroidism is associated with normal levels of catecholamines (10). Also, ß-blockade fails to completely reverse the physiology and symptoms of hyperthyroidism (25), and overall adrenergic responsiveness remains unchanged during hyperthyroidism (12). The hypothesis that TH and ßARs share common mechanisms of action in states of TH excess, therefore, has not been definitively addressed.

We directly tested the hypothesis that TH and ßARs operate independently in states of TH excess by inducing hyperthyroidism in mice lacking the known ßARs, and comparing these to wild-type (WT) mice. We found that the metabolic and cardiovascular effects of hyperthyroidism are preserved in mice lacking ßARs, suggesting that these effects are independent of ß-adrenergic stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
This study was performed with approval by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center. Handling and care of mice were consistent with federal and institutional guidelines. Twelve- to 14-wk-old male WT mice and mice lacking the three known ßARs (ß-less) mice were available in our laboratory as described in a prior study (26). These outbred mice derived from the same founder parents, and have similar body weights and TH levels at 12–14 wk of age (26). Pooled data from two semi-independent lines of both WT and ß-less mice were analyzed. Age- and weight-matched WT and ß-less mice were genotyped by Southern blot to confirm lack of the three known ßARs (17, 18, 19).

Animals were housed at room temperature (22–24 C) on a 14- to 10-h light and dark cycle with food and water provided ad libitum. WT and ß-less mice are euthyroid (26).

Oxygen consumption was measured in a Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments, Columbus, OH) over 3 d after acclimation. Heat production was calculated from 24-h oxygen consumption according to the Weir equation within the CLAMS software (Columbus Instruments, Columbus, OH).

Electrocardiogram (ECG) recording
ECGs were recorded in conscious mice as described previously (27). Briefly, mice were carefully positioned on custom-manufactured disposable platforms embedded with three electrodes (Bio-DETEK, Narragansett, RI). The size and arrangement of the electrodes are configured to advance contact with three of the animals’ paws to provide an ECG signal that is equivalent to Einthoven lead II. The electrodes were connected to a custom-designed amplifier and analog-to-digital converter. The signals were digitized at a sampling rate of 2 kHz. Because even modest handling of these mice induces significant alterations in HR (28), each mouse was permitted to acclimate for 10 min before collection of baseline data. Only data from continuous recordings of 20–30 ECG signals were used in the analyses.

ECG analyses
Analyses of the ECGs were performed as described previously (27).

Each ECG signal was analyzed using e-MOUSE (Mouse Specifics, Inc., Boston, MA), an Internet-based physiologic waveform analysis platform. The software uses peak detection to calculate HR. HR variability (HRV) was calculated as the mean of the differences between sequential HRs for the recorded ECG signals. Calculation of first and second derivatives and algebraic "if-thens" search the ECG signals for probable P-wave peaks and onset and termination of QRS complexes. The end of the T-wave of each signal was defined as the point where the signal intersected the isoelectric line (the mean voltage between the preceding P-wave and QRS interval). We included the inverted and biphasic portions of the T-wave in our calculations of the QT (interval between start of Q-wave and end of T-wave in ECG) and rate-corrected QT (QTc) intervals (29). The software plots its interpretation of P, Q, R, S, and T for each beat so that unfiltered noise or motion artifacts are rejected. e-MOUSE then calculates the mean of the ECG time intervals for each set of waveforms.

Serum chemistries
Serum was obtained by cardiac puncture after euthanasia, and T4 was measured in duplicate (Linco Inc., St. Charles, MO).

ßAR responsiveness
To confirm that ß-less mice lack ßAR signaling in the heart pharmacologically, we administered the nonspecific ß-agonist isoproterenol, which has been shown to induce tachycardia, ischemia, and cardiac hypertrophy in normal mice (27, 30). Acute responses to isoproterenol are mediated by the ß1AR (17). Isoproterenol was administered to WT and ß-less mice twice daily for 3 d to confirm that ß-less mice lack acute and chronic responsiveness to ß-agonists.

Induction of hyperthyroidism
Experimental hyperthyroidism was induced in age and weight-matched WT and ß-less male mice (31) by a single, daily ip injection of T3 (Sigma Aldrich Chemicals, St. Louis, MO) at a dose designed to induce modest hyperthyroidism (4 µg/d = 150 ng/g·d) for 14 d (32). Animals were given either T3 or an equal volume of saline. The injections were performed at the same time of day throughout the study. Animals were studied at least 8 h after the last saline or T3 injection. To test whether ßARs are required for increased metabolic rate in response to hyperthyroidism, saline, and T3-treated WT and ß-less mice were placed in metabolic chambers (CLAMS) and heat production was calculated based on measured oxygen consumption.

Hemodynamics
Hemodynamic measurements were performed in vivo as described previously (33). Briefly, a carotid artery of each mouse was isolated and cannulated with a 1.4-F high-fidelity microtip catheter (Millar Instruments, Houston, TX), connected to an analog-to-digital recorder (PowerLab ML820, AD Instruments, Boulder, CO). After blood pressure (BP) was recorded, the transducer was advanced into the left ventricle (LV). LV peak systolic pressure (LVSP) and end-diastolic pressures (LVEDP), HR, and maximum rates of pressure rise (dP/dtmax) and fall (–dP/dtmin) were recorded at a sampling rate of 1kHz. Rate-pressure product (RPP) was calculated as LVSP multiplied by HR.

After measurements, mice were euthanized by ip administration of pentobarbital (150 mg/kg), consistent with the American Veterinary Medical Association Panel on Euthanasia guidelines. Excised hearts, excluding atria and blotted dry, were then weighed.

Gene expression analysis
Hearts were dissected from freshly euthanized mice and quick-frozen in liquid nitrogen. Total RNA was extracted from whole heart tissue using RNA STAT60 (Tel-Test, Friendswood, TX). Two micrograms of RNA were used to synthesize cDNA using the Superscript in vitro transcription kit (Invitrogen, La Jolla CA) according to the manufacturer’s instructions. Quantitative PCR (QPCR) assays were used to measure relative expression of, HCN2 (hyperpolarization-activated cyclic nucleotide-gated channel 2), sarcoplasmic reticulum calcium ATPase (SERCAII), phospholamban, {alpha}-myosin heavy chain (MHC), and ß-MHC. QPCR assays for the above mentioned genes were purchased from Applied Biosystems (Foster City, CA). 18S RNA was used for normalization in multiplexed reactions when assaying for the genes of interest to minimize variability. QPCR was performed using the MX4000 real-time PCR machine (Stratagene, La Jolla, CA).

Statistics
Data are presented as the means ± SE. Comparisons between saline-treated and ß-less mice were performed using Student’s t test for unpaired observations. Differences were considered significant with P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Baseline characteristics
Baseline body weight and TH levels were not significantly different between WT and ß-less mice (Table 1Go). Baseline metabolic rate, as measured by oxygen consumption, is 12–14% lower in ß-less compared with WT mice (26). As shown in Table 1Go, HR in conscious, unrestrained ß-less mice is significantly lower compared with WT mice. HRV is not significantly between WT and ß-less mice. The PR interval (the time from the onset of ventricular depolarization to the onset of ventricular depolarization) and QTc interval [the time of ventricular depolarization and repolarization, corrected for HR (29)] are significantly prolonged in ß-less compared with WT mice.


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  		TABLE 1. Baseline characteristics of 12-wk-old, conscious WT and ß-less mice

 
ß-Agonist stimulation is absent in ß-less mice
To confirm that ß-less mice lack responsiveness to ß-agonists, WT and ß-less mice were treated with saline or the nonspecific ß-agonist isoproterenol for 3 d. This treatment is known to cause cardiac hypertrophy via stimulation of ßARs in normal hearts (27, 30). Heart weight (HW) of saline-treated WT and ß-less mice is not significantly different (Fig. 1AGo). In response to isoproterenol, however, HW increases 14% in WT mice but does not change in ß-less mice (Fig. 1AGo). Isoproterenol treatment increases HR and causes ST segment depression in WT mice, but not in ß-less mice (Fig. 1BGo). These data, which are the first detailed cardiac studies in ß-less mice, confirm that the cardiovascular effects of ß-agonists are absent in ß-less mice. In addition to prior metabolic data, these data also support the view that there are only three functional ßARs in mice (26).



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  		FIG. 1. Cardiac responses to ß-agonist stimulation are absent in ß-less mice. Twelve-week-old WT and ß-less mice (n = 5 in each group) were treated with saline or isoproterenol (ISO) twice daily for 3 d. A, HW in saline-treated (open bars) and ISO-treated (filled bars) in WT and ß-less mice (*, P < 0.05 compared with saline-treated mice). B, ECGs in response to saline (upper panel) and ISO (lower panel) in WT and ß-less (right) mice.

 
Induction of hyperthyroidism
Saline or T3 treatment has no effect on body weight (BW) (Fig. 2AGo). Levels of T4 in saline-treated groups are normal in both WT and ß-less mice, and are consistent with a euthyroid state. Administration of T3 for 2 wk results in hyperthyroidism, as shown by lower T4 levels expected from T3-induced feedback on hypothalamic TRH and pituitary TSH production, and subsequently decreased T4 production in the thyroid gland (Fig. 2BGo). Oxygen consumption and metabolic rate (kilocalories/minute) in saline-treated ß-less mice are significantly lower than in WT mice, as reported previously (26). Administration of T3, however, causes a comparable approximately 14% increase of in metabolic rate in both groups (Fig. 2CGo). There were no differences in physical activity in any group tested (data not shown).



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  		FIG. 2. Induction of hyperthyroidism by T3. Twelve-week-old male WT and ß-less mice were treated with saline (open bars) or T3 (filled bars). A, Body weight was unchanged with T3. B, Serum T4 levels were comparably normal in saline-treated WT and ß-less mice. T3 treatment suppressed T4 levels less than assay (<) in WT and ß-less mice. C, Oxygen consumption, expressed as calculated heat production per mouse, was slightly lower in saline-treated ß-less (filled bars) vs. WT (open bars) mice. Both groups demonstrated comparable increases in oxygen consumption in response to T3 administration (n = 6 each group; *, P < 0.05 compared with saline-treated from the same group).

 
Cardiovascular responses to hyperthyroidism are preserved in ß-less mice
In conscious WT and ß-less mice, administration of T3 resulted in similar, significant increases in HR of approximately 15% [from 719 beats per minute (bpm) to 820 bpm in WT; 553 to 636 bpm in ß-less mice, respectively], indicating that ßARS are not required for a chronotropic response to TH. To examine cardiac dynamics in more detail, we next studied anesthetized mice. Similar to what is observed in conscious animals, HR is significantly lower in anesthetized ß-less mice compared with anesthetized WT mice (Table 2Go). Mean BP is comparable in WT and ß-less mice. In response to T3, HR increases significantly in WT mice (+26%) and ß-less mice (+66%) (Fig. 3AGo). The HR in anesthetized, T3-treated ß-less and WT mice is comparable [481 bpm ± 52 vs. 545 ± 38, bpm, not significant (NS)], suggesting that ßARs are not required for maximal HR in response to TH excess. T3 treatment does not result in changes in BP in WT mice (Fig. 3BGo). ß-less mice, however, show small but significant increases in mean blood pressure after treatment with T3 (Fig. 3BGo). LVSP is modestly higher in saline-treated ß-less compared with saline-treated WT mice. We noted that the placement of the pressure transducer was slightly more difficult in the carotid artery of ß-less mice, indicating vasoconstriction an possibly increased peripheral vascular resistance. Treatment with T3 increased LVSP increases modestly in WT mice in response to T3 administration but does not increase further in ß-less mice (Fig. 3CGo). The RPP, an index of cardiac metabolic demand, is comparable at baseline in both groups of mice (Table 2Go). The RPP increases significantly in both WT (+42%) and ß-less mice (+81%) in response to T3 administration (Fig. 3DGo).


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  		TABLE 2. Baseline cardiovascular parameters in 12-wk-old anesthetized mice

 


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  		FIG. 3. Cardiovascular responses to T3 treatment in anesthetized, instrumented mice. Twelve-week-old WT and ß-less mice (n = 6 each group) were treated with ip saline (open bars, control) or T3 (filled bars) for 14 d. A, HR. B, Mean BP. C, LVSP. D, RPP. E, HW to BW ratio. Error bars represent SEM for each group; *, P < 0.05 compared with saline-treated from the same group; #, P < 0.001 compared with WT treated with T3.

 
Chronic stimulation with either TH or ßARs results in cardiac hypertrophy (32, 34). To test whether ßARs are required for hyperthyroidism-induced cardiac hypertrophy, we compared the hypertrophic responses to hyperthyroidism in WT and ß-less mice. Hypertrophy was assessed by the HW normalized to BW (HW/BW) in WT and ß-less mice in response to T3, although these groups had similar BW so that HW or HW/BW could be used for comparisons. T3 increases HW/BW significantly in WT mice (+46%) and ß-less mice (+12%, P < 0.05 compared with saline-treated), although the cardiac hypertrophy is significantly greater in WT than ß-less mice (Fig. 3EGo). Consistent with an increase in heart size relative to body size, we observed increased myofibrillar width in both WT and ß-less groups of mice in response to T3 administration (data not shown).

Cardiac contractility is another cardiac index that is stimulated by both TH and ß-agonists. We next performed hemodynamic measurements to determine the role of ßARs in mediating TH excess-mediated alterations in cardiac contractility. Baseline contractility (dP/dtmax) in ß-less mice is slightly increased compared with WT mice, although this did not reach statistical significance (9385 ± 1977 in ß-less mice vs. 6256 ± 1157 mm Hg/sec in WT mice, NS; Fig. 4AGo). Baseline relaxation (dP/dtmin) is not significantly different in ß-less mice compared with WT (6930 ± 1318 vs. 4332 ± 403, NS). The hemodynamic responses to T3 are comparable in WT and ß-less mice (Fig. 4Go, A and B). The inotropic response to T3 administration is equivalent in WT and ß-less mice, as reflected by comparable, significant increases in dP/dtmax (+78% and +77%). A significant lusitropic response to T3 administration is also seen in both in WT and ß-less mice (+145% and +63% increase, respectively, Fig. 4Go, A and B). Thus, ßARs are not required for the increases in cardiac contractility and relaxation that occur as a result of hyperthyroidism.



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  		FIG. 4. Effects of TH on LV contracility, relaxation, and gene expression. In all panels, open bars denote saline-treated and filled bars denote T3-treated. A, Maximal rate of pressure rise (dP/dtmax). B, Maximal rate of pressure fall (dP/dtmin); saline-treated (open bars), T3-treated (filled bars); C, QPCR for HCN2, SERCA2A and phospholamban gene expression; (n = 4 in each group). *, P < 0.05 compared with saline-treated of same genotype; #, P < 0.001 compared with WT treated with T3.

 
Cardiac function is mediated by numerous genes that regulate HR, contractility, and hypertrophy. We asked whether the changes in cardiovascular dynamics in response to T3 were accompanied by similar changes in gene expression in WT and ß-less mice. We compared relative gene expression of a subset of genes in WT and ß-less mice by QPCR analysis. In rodents, the hyperthyroidism-induced increase in HR is associated with increased expression of the pacemaker gene responsible for the hyperpolarization-activated current (HCN2) (32, 35, 36). The expression of HCN2, normalized to 18 S RNA, increases in both WT (6-fold increase) and in ß-less mice (2.5-fold increase) in response to T3 administration (upper panel, Fig. 4CGo). These results are consistent with induction of hyperthyroidism in both T3-treated WT and ß-less mice, and demonstrate that ßARs are not required for increased HCN2 expression in response to T3. Cardiac function is also modulated by the SERCAII, which is regulated positively by T3 (32, 37). The expression of SERCAII increased approximately 2-fold in WT mice, but ß-less mice showed no significant increase (lower panel, Fig. 4CGo). Expression of phospholamban, which is a reversible inhibitor of SERCAII and is regulated by ß-adrenergic stimulation, did not change in either group in response to T3 administration (Fig. 4CGo). The ratio of phospholamban/SERCAII, which has been described as an index of cardiac contractility (38), was not significantly different in WT compared with ß-less mice (data not shown). We also compared the expression of {alpha}-MHC and ß-MHC in response to T3 because these genes are regulated by hemodynamic load and direct transcriptional activation by T3 (4, 39). We observed a significant induction in {alpha}-MHC levels in T3-treated WT mice (7-fold increase), whereas ß-less mice showed elevated baseline levels of {alpha}-MHC (7-fold compared with WT mice) and no increase in response to T3 administration (data not shown). There were no significant baseline differences in ß-MHC levels between WT vs. ß-less mice, and neither group showed a significant change in response to T3 administration (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Significant changes in metabolism and cardiovascular function occur in mice devoid of all three known ßARs in response to TH excess. These findings demonstrate that ßARs are not required for the metabolic and cardiovascular effects of hyperthyroidism in mice. Although ß-adrenergic antagonists are often prescribed for the treatment of hyperthyroidism, our data suggest that the mechanisms of T3 and ßAR signaling in hyperthyroidism are independent.

The ß-less mouse model we used in these studies offers several advantages for the study of hyperthyroidism. First, mice and humans share all of the major ligands and receptors of the thyroid and the SNS. Secondly, THs and the SNS have similar general effects on cardiac physiology, although species differences have been reported in the regulation of ßARs in hyperthyroid hearts (24). Thirdly, ß-less mice are euthyroid, have serum levels of T4 and T3 comparable with those in WT mice, and do not have significant differences in body temperature (26). The use of ß-less mice, which are genetically null for all three ßARs, avoids the possibility that data are obfuscated by incomplete ßAR blockade, indirect effects mediated by ßARs in other tissues, or potential compensation by other ßARs (19). Therefore, ß-less mice enabled us to effectively query the link between hyperthyroidism and ßAR signaling.

We confirmed that hyperthyroidism was induced in T3-treated mice by measuring metabolic rate and demonstrating suppressed T4 levels. ß-Less mice have lower whole body oxygen consumption than WT mice before T3 treatment, although the primary tissue(s) responsible for this phenotype is not known (26). Hypermetabolism induced by hyperthyroidism may be due to the effects of TH on numerous tissues, including adipose, nerve, muscle, and visceral organs (40). In some tissues, such as brown adipose tissue, TH, and ßARs act synergistically via independent signaling pathways that result in cooperative regulation of metabolically active genes (41). The significant increase in metabolic rate in T3-treated ß-less and WT mice is consistent with the hyperthyroidism that has been observed with comparable administration of T3 (31, 35). Also, the increase in metabolic rate in mice treated with T3 is similar to that observed in patients with hyperthyroidism (40). The proportionate increases in metabolic rate in WT and ß-less mice after T3 treatment confirm prior reports that there may be independent thermogenic mechanisms that are mediated by TH and ßARs (42, 43). The metabolic effects of T3 excess are, therefore, most likely additive to the effects of ßAR signaling in cardiac tissue.

HR is dependent on many factors, including body size, temperature, age, circulating volume, the autonomic nervous system, and thyroid status. Telemetric studies demonstrated comparable HR between WT mice and mice deficient in ß1ARs, ß2ARs, or both ß1 and ß2ARs (13, 17). We found a significant reduction in HR in mice lacking all three of the known ßARs compared with WT mice. This could be due to the additional absence of the ß3AR, or to higher HR in the outbred WT mice in the present experiments. In response to T3, HR increased comparably and significantly in ß-less mice and WT mice. This is a similar finding to prior studies that employed a combination of ß-blockade and T3 treatment (44). This demonstrates that ßARs are not required for the chronotropic responses to hyperthyroidism. We observed a significant increase in HCN2 expression in both WT and ß-less mice, which may provide one possible mechanistic basis for the observed tachycardic response to T3. However, ßARs may be necessary to achieve maximal HR, because HR in awake, T3-treated ß-less mice remained significantly lower than in T3-treated WT mice. This may partly explain the efficacy of ß-blockade in patients with hyperthyroidism because peak HR can be reduced in hyperthyroidism by preventing ßAR signaling (24).

We found that cardiac hypertrophy in response to hyperthyroidism developed in both WT and ß-less mice. Cardiac hypertrophy can result from numerous physiological and molecular mechanisms, many of which are not fully understood. Hypertrophy as a compensatory response to increased cardiac load, however, might be expected to occur similarly in T3-treated WT and ß-less mice because both groups of mice had comparably elevated cardiovascular indices. Why, then, was cardiac hypertrophy significantly greater in WT than ß-less mice? ß-Agonist (isoproterenol)-induced cardiac hypertrophy, for one, is absent in ß-less mice. Thus, cardiac hypertrophy in ß-less mice in response to T3 cannot have occurred via direct stimulation of ßARs. Prior studies have indeed shown that ß blockade during hyperthyroidism prevents cardiac hypertrophy in rats (45). We observed differences in the levels of expression of {alpha}MHC between WT and ß-less mice (4), but these do not explain T3-induced cardiac hypertrophy in ß-less mice, because {alpha}MHC was not induced in ß-less mice and basal levels were much higher. Noting that catecholamine-induced cardiac hypertrophy is believed to result from independent {alpha}AR-induced and ßAR-induced pathways (46), our results may be due to unopposed stimulation of cardiac {alpha}ARs during hyperthyroidism. Signaling via the {alpha}1AR has been shown, for example, to be important in promoting cardiac hypertrophy (15). On the other hand, overexpression of the {alpha}1A-AR results in increased contractility but not hypertrophy (47). Unopposed {alpha}AR stimulation could also explain the lack of SERCa induction in ß-less mice, given that stimulation of {alpha}1ARs has been shown to block thyroid-induced transcription of this gene (48). In addition, the ßAR-independent effects of T3 on intracellular signaling (49) could have resulted in variable responses to T3 in WT and ß-less mice in the presence and absence of catecholamine stimulation, respectively. Finally, gene expression studies do not always accurately reflect protein levels, as has been observed for SERCa expression (50). Gene expression or proteomic profiling from hearts of T3-treated WT and ß-less mice may demonstrate which genes mediate cardiac hypertrophy directly by TH signaling (i.e. T3) (48, 51, 52) compared with ßAR signaling. If preventing ßAR signaling during hyperthyroidism leads to reduced cardiac hypertrophy but normal cardiac function, this finding would have important implications for the treatment of human hyperthyroidism, in which cardiovascular compromise is a major contributor to mortality and morbidity (3). We conclude that ßARs are not required for cardiac hypertrophy in response to hyperthyroidism.

We were surprised to observe that SERCAII expression did not increase with T3 administration in the ß-less mice, despite increases in contractility ex vivo. The effects of hyperthyroidism on SERCAII expression have been reported to be small and regional (atrial) (53), however, allowing for the possibility that our analysis of whole hearts was not sensitive enough to detect localized changes in SERCAII expression. With regard to cardiac contractility, there are numerous potential mechanisms that could have contributed to increased cardiac contractility in ß-less mice independently of SERCAII, including other calcium receptors (ryanodine receptor RyR2) (54), {alpha} adrenergic stimulation (47), phosphatidylinositol 3-kinase (55), posttranslational modifications (56), and altered calcium dynamics in response to increased HR (57).

ß-Adrenergic antagonists are used early and often in the treatment of hyperthyroidism, although their mechanism of action and effectiveness in treating hyperthyroidism had not previously been defined. Our findings show that the mechanisms of T3 and ßAR signaling in hyperthyroidism are independent because the effects of TH excess on cardiac function persist even in the absence of ßARs. These observations support the concurrent use of antithyroidal medication and ß-adrenergic blockade in the clinical setting. Our data showing T3-induced cardiac hypertrophy is ameliorated by the absence of ßARs further underscores the importance of reducing ßAR signaling via ß-blockade in the treatment of hyperthyroidism. In summary, ßARs are not required for the metabolic and cardiovascular effects of hyperthyroidism in mice, suggesting that the thyroid and SNS axes act independently in this pathologic state. These results may have considerable clinical relevance for treating hyperthyroidism in humans.


   Footnotes
 
These studies were funded by the National Institutes of Health [1 K08 DK60664-01 (to E.S.B.); DK56123 (to A.H.)]. I.A. was generously supported by Förderkreis zur Verbesserung des Gesundheitswesens e.V. and August Eilert GmbH, Alfeld, Germany.

E.S.B. and T.G.H. contributed equally to this work.

T.G.H. is owner of Mouse Specifics, Inc., a company organized to commercialize the ECG technology described in Materials and Methods. The other authors declare no financial conflicts of interest.

Abbreviations: AR, Adrenergic receptor; ßAR, ß-AR; ß-less, ßAR null; BP, blood pressure; bpm, beats per minute; BW, body weight; ECG, electrocardiogram; HCN2, hyperpolarization cyclic nucleotide gated current gene 2; HR, heart rate; HRV, HR variability; HW, heart weight; LV, left ventricle; LVSP, left ventricular systolic pressure; MHC, myosin heavy chain; NS, not significant; QPCR, quantitative PCR; QRS, ECG complex interval between onset of Q-wave and end of S-wave; QT, interval between start of Q-wave and end of T-wave in ECG; QTc, rate-corrected QT; RPP, rate-pressure product; SERCAII, sarcoplasmic reticulum calcium ATPase; SNS, sympathetic nervous system; WT, wild-type.

Received December 9, 2003.

Accepted for publication March 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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