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Institute of Clinical Pharmacology, University Hospital of Mannheim, Faculty for Clinical Medicine Mannheim, University of Heidelberg (B.M.W.S., N.M., A.C.G., H.-C.T., M.F., M.C., M.W.), 68135 Mannheim, Germany; and Medical Department 4/Nephrology, University of Erlangen-Nürnberg (B.M.W.S.), 90471 Nürnberg, Germany
Address all correspondence and requests for reprints to: Martin Wehling, M.D., Institute of Clinical Pharmacology, Faculty of Clinical Medicine, Ruprecht Karls University of Heidelberg, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany. E-mail: . martin.wehling{at}kpha.ma.uni-heidelberg.de
Abstract
T3 has been shown to exert cardiovascular effects. These effects have not yet been defined with regard to the mode of action (nongenomic vs. genomic) and with regard to an interaction with the adrenergic system in humans. To address these issues we conducted a randomized, double blind, 6-fold cross-over trial in 18 healthy male volunteers. After pretreatment with the ß-agonist dobutamine, the ß-blocking agent esmolol, or placebo (0.9% NaCl), 100 µg T3 or placebo were injected. Primary target variables were systemic vascular resistance (SVR) and cardiac output (CO) within 45 min after injection of T3 vs. placebo after placebo pretreatment. Sympatho-vagal balance was assessed by measurement of heart rate variability.
T3 caused a lower SVR and a higher CO than placebo (P < 0.001) after pretreatment with placebo. An increased low frequency (LF)/high frequency (HF) ratio (power in LF/power in HF band) after T3 compared with placebo (P = 0.004) suggests an increase in sympathetic tone. After pretreatment with dobutamine, the effects of T3 on SVR and CO were abolished, and the effect on LF/HF ratio was reversed. After pretreatment with esmolol, the effects on SVR and LF/HF ratio were reversed. Our data show, for the first time, nongenomic cardiovascular effects of T3 in humans.
IN HYPERTHYROIDISM, leading cardiovascular features are increased cardiac output (CO; caused by increased heart rate and enhanced myocardial contractility) and decreased systemic vascular resistance (SVR) (1). In hypothyroidism, the opposite effects occur. Furthermore, thyroid hormone causes increased sympathetic tone (2). Recently, it has been shown that administration of T3 has beneficial effects after open heart surgery in adults (3) and children (4) and lowers systemic vascular resistance (SVR) and increases cardiac output (CO) in cardiac failure (5). These clinical characteristics of thyroid hormone have not yet been characterized with regard to the mode of action, genomic vs. nongenomic transmission.
At the molecular level, both genomic and nongenomic cardiovascular effects of thyroid hormone have been observed. Nongenomic actions of T3 trigger changes in intracellular calcium levels in the myocardium via activated reverse mode Na+/Ca2+ exchange (6), increased sarcoplasmatic reticulum calcium-adenosine triphosphatase activity (7), and coupled glucose/Ca2+ uptake (8, 9). Furthermore, T3 has been shown to rapidly shorten action potential duration in rat ventricular myocytes by a nongenomic mechanism (10). Rapid vasodilatory effects of T3 are postulated to be mediated by both smooth muscle cells and endothelium (11, 12).
Experimentally, genomic and nongenomic actions can be differentiated in various ways. Genomic responses require de novo synthesis of proteins and thus are sensitive to inhibition of transcription and translation, e.g. by actinomycin D and cycloheximide. They are unlikely to occur within less than 1 h. Nongenomic effects may occur within seconds or minutes. They are insensitive to inhibition of transcription and translation (13). The clinical studies mentioned above did not measure cardiac function within 2 h of administration of T3. Therefore, either early genomic responses or persisting rapid nongenomic effects may contribute to the cardiovascular changes observed in response to T3.
Given this, the numerous observations of clearly nongenomic T3 effects in cell cultures and animal models still lack in vivo confirmation in man. Therefore, we investigated the acute effects of 100 µg T3 on CO and SVR as primary target parameters in healthy euthyroid male volunteers in a randomized, placebo-controlled, double blind study. We also assessed sympatho-vagal balance to characterize a possible relation between nongenomic T3 effects and the autonomic nervous system in vivo.
As shown for aldosterone, nongenomic cardiovascular effects of steroid hormones may depend on an interplay with the autonomic nervous system (14). Therefore, we simulated different ß-adrenergic situations by infusion of a ß-agonist (dobutamine) and a ß-blocking agent (esmolol) before injecting T3. Cardiovascular parameters were measured by impedance cardiography (ICG), heart rate variability (HRV) by digital electrocardiography (ECG) recording.
Subjects and Methods
Study volunteers
Eighteen healthy male volunteers were included in the study. All subjects gave written informed consent to participate in the study. They were subjected to a medical examination within 2 wk before inclusion in the study. The examination included medical history, a physical examination, a 12-lead ECG, and clinical laboratory parameters, including free T3 (fT3), free T4, and TSH to assure a euthyroid state.
The study was performed subsequent to a study of rapid nongenomic aldosterone effects, which used a very similar design (14). No volunteer participated in both studies.
Study procedures
The study was designed as a randomized, placebo-controlled, 6-fold cross-over trial, blinded with respect to the ß-adrenergic modulators for the subjects and double blinded with respect to the T3/placebo treatment. It was conducted according to the guidelines for good clinical practice and the Declaration of Helsinki after approval by the institutional review board of the Faculty for Clinical Medicine Mannheim, University of Heidelberg (Heidelberg, Germany). All 18 enrolled volunteers were randomly subjected to 6 test periods, with a minimum wash-out interval of 7 d.
Volunteers were in house at 0730 h on each study day. After a standard breakfast, study procedures were started by bringing volunteers into the supine position and inserting three indwelling catheters into forearm veins: one for the continuous infusion of the modulator of the adrenergic system (dobutamine or esmolol or placebo), one for T3 or placebo injection, and one for blood sampling (on the arm not receiving T3). After a 30-min resting period, baseline data from ICG and digital ECG were obtained over 12 min. These results will be referred to as baseline 1. Then continuous infusion of the ß-adrenergic modulator was started. The initial doses were 3.75 µg/kg BW/min for dobutamine and 0.1 mg/kg BW·min after a bolus injection of 0.2 mg/kg BW for esmolol. After 10 min cardiovascular parameters were reassessed, and the changes in SVR were evaluated. These infusions were intended to cause a shift in SVR reflecting 30% of the maximal effect of the particular drug (14). If the target change in SVR (Table 2
) had not been reached, the dose of the continuous infusion was increased or decreased by 2.5 µg/kg BW·min for dobutamine and 0.035 mg/kg BW·min for esmolol, and the procedure was repeated without delay. After SVR was measured to be within the target range, the dose was kept constant again for 10 min. In case the target range was not reachable after three dose adjustments, study procedures were continued without reaching the SVR target level (Table 2). Subsequently, baseline data from ICG and digital ECG were obtained over 12 min. Baseline values were calculated as the mean of four consecutive measurements performed at 3-min intervals. These results will be referred to as baseline 2. Then T3 (100 µg) or placebo was injected into one of the catheters over 1 min, and cardiovascular parameters were measured for 45 min. During the test volunteers remained in the supine position. Vital parameters were permanently monitored throughout the test. Blood pressure, heart rate (Omnicare CS24, Hewlett-Packard Co., Boblingen, Germany), and ICG parameters (Cardioscreen, Medis GmbH, Ilmenau, Germany) were measured at 3, 6, 9, 12, 15, 20, 25, 30, 35, 40, and 45 min. Parameters of HRV were assessed at 5-min intervals.
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ICG was performed using standard methods as described previously (15). Stroke volume was computed by Bernstein’s formula (16). CO was calculated as stroke volume x heart rate, and SVR as 80 x (mean arterial pressure - 3)/CO.
Time domain parameters of HRV, i.e. pNN50 (percentage of differences between adjacent normal RR intervals longer than 50 msec) and rMSSD (square root of the mean of squared differences between adjacent normal RR intervals), were calculated at 5-min intervals. Power spectral density for the low frequency (LF) and high frequency (HF) bands of HRV was calculated by fast Fourier transformation at 256-sec intervals. Sympatho-vagal balance was assessed by the LF/HF ratio (17).
The fT3 and T3 levels in plasma were measured by a commercial ELISA (Roche Molecular Biochemicals, Mannheim, Germany). As fT3 levels after 3 min in almost all cases exceeded the upper limit of reliable quantification of the test (61.4 pmol/liter), these data are not shown. Levels higher than 61.4 were measured twice. For measurement of total T3 levels, sera were adequately diluted.
Esmolol and dobutamine are commercially available drugs [brand names Brevibloc (Baxter Deutschland GmbH, Unterschleissheim, Germany) and Dobutamin Fresenius (Fresenius AG, Bad Homburg, Germany)]. Placebo was isotonic (0.9%) NaCl solution. For sake of readability these drugs will be referred to as pretreatment, although they were continuously administered until the end of the study procedure. T3 was the commercially available drug Thyrotardin inject N (Henning Berlin GmbH \|[amp ]\| Co., Berlin, Germany).
Statistical methods
The statistical analysis was performed using SPSS software version 8.0 (SPSS, Inc., Chicago, IL). All individual data of ICG, HRV, and safety measures were analyzed descriptively by computation of means, SEM, and confidence intervals. In the tables and figures, the mean and SEM are shown.
For statistical inference the percent change from baseline 2 was calculated for each variable and each measurement time point. The percent differences from baseline 2 of CO and SVR in the placebo/placebo vs. placebo/T3 periods were the primary target variables. These were tested using multifactorial ANOVA with the factors treatment, time point, and treatment-time point interaction. In the case of statistically significant differences in ANOVA, post-hoc analysis of single time points was performed using t tests.
Regarding primary target parameters, the level of significance was set at 0.025 for ANOVA to adjust for the number of two target variables. The comparison of T3 and placebo after pretreatment with dobutamine or esmolol, testing of parameters of HRV and post-hoc analyses were exploratory, the level of significance was set at 0.05. In the figures, SEs are given that were estimated as pooled SEs based on the sampling error adjusted for the above model. The pharmacokinetics of T3 and fT3 are presented as the time-concentration profile.
Results
Demographics
Important clinical characteristics and demographic features are shown in Table 1.
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Table 2
shows the primary cardiovascular parameters SVR and CO before administration of dobutamine, esmolol, and placebo (baseline 1) and after titration as described above (baseline 2). All values are means of four measurements performed during the 12-min baseline periods (at 3, 6, 9, and 12 min). The shift in SVR between baseline 1 and baseline 2 was in the intended range.
Changes in the parameters of HRV reflect the intended modulation of the sympatho-vagal balance. After dobutamine pretreatment measures of vagal tone such as pNN50 and rMSSD decreased, and the LF/HF ratio increased, whereas pNN50 and rMSSD increased slightly after esmolol. Only the LF/HF ratio after esmolol pretreatment unexpectedly increased.
Effect of T3 on SVR and CO after placebo pretreatment
Figure 1 shows the time course of SVR after placebo pretreatment with injection of T3 vs. placebo. SVR was lower after T3 compared with placebo injection. The difference between both treatment periods is statistically significant (P < 0.001, by ANOVA). Evaluation of single time points shows that the effect is already statistically significant 3 min after T3/placebo injection (P = 0.025, t test) and again after 20 (P = 0.032, by t test), 35 (P = 0.023, by t test), and 40 (P = 0.013, by t test) min.
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shows the time course of CO after placebo pretreatment with infusion of T3 vs. placebo. CO was higher with T3 than with placebo infusion. The difference between treatment periods is statistically significant (P < 0.001, by ANOVA). Evaluation of single time points shows that the difference is of borderline statistical significance 3 min after the start of T3 infusion (P = 0.051, by t test) and after 20 min (P = 0.050, by t test). At 35 and 40 min statistically significant differences occur (P = 0.008 and P = 0.011, respectively). This effect is due to an higher stroke volume after T3 than after placebo (P = 0.005) and a slightly higher heart rate (P = 0.07; Table 4). This suggests that a positive inotropic and a positive chronotropic effect contribute to the increase in CO.
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After pretreatment with dobutamine no significant differences of SVR and CO between placebo and T3 were found (P = 0.753 and P = 0.812, respectively; Table 3). Further analysis shows that after dobutamine pretreatment heart rate is lower with T3 than with placebo treatment, compensating the effect of the higher stroke volume (Table 4).
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). However, analyzing the single time points, only a significant effect on CO could be demonstrated with esmolol pretreatment after 30 min (P = 0.031, by t test). This effect is due to a lower heart rate with T3 compared with placebo and an identical stroke volume (Table 4). HRV
After placebo pretreatment, T3 significantly increases the LF/HF ratio compared with placebo, reflecting an increased sympathetic and/or reduced parasympathetic activity (P = 0.004, by ANOVA; Fig. 3). After pretreatment with dobutamine and esmolol, opposite changes in sympatho-vagal balance occur (P < 0.001 and P = 0.008, respectively; Table 3). Regarding the time domain parameters pNN50 and rMSSD, no statistically significant differences were found (data not shown).
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T3 serum levels did not change during titration. Pretreatment did not influence T3 levels after the injection of T3 or placebo (Fig. 4). The fT3 and T3 levels reached supraphysiological levels and remained in this range during the study period. As fT3 levels 3 min after the injection of T3 exceeded the upper limit of the test (61.4 pmol/liter) in most cases, exact values cannot be calculated at this time point. If compared with periods in which fT3 levels were measurable at both this time point and 15 min after injection, it is fair to assume that these values were below 100 pmol/liter.
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There were no adverse events related to the T3 injection.
Discussion
In this study nongenomic cardiovascular effects of T3 are shown in vivo in humans for the first time. Rapid, nongenomic effects of T3 have been shown in various in vitro systems (18). Most known effects involve calcium membrane transport or intracellular calcium levels (19). Such effects have been found in rat liver cells (20), red blood cells (lacking a nucleus and thus a genomic effector) (21), and rat myocytes (22). The T3-induced decrease in systemic vascular resistance in rats may be explained by direct modulation of endothelium-independent and endothelium-dependent vasoregulation (11). In isolated cardiac myocytes, T3 has been shown to alter sodium currents in a manner consistent with inotropic changes (23). This includes changes in sodium-calcium exchange to produce increased contractility (24). The rapid response of certain voltage-gated potassium channels to thyroid hormone may explain the observations that T3 treatment lowered the prevalence of atrial fibrillation in patients after coronary artery bypass surgery (25). Furthermore, it has been shown recently that T3 by a nongenomic mechanism shortens action potential duration in rat ventricular myocytes (10). The relevance of nongenomic T3 effects in cardiovascular physiology is further supported by cycloheximide-insensitive, early modulation of ß-adrenoceptor density by T3 in cultured embryonic cardiac myocytes (2), whereas late up-regulation of ß-adrenoceptors was cycloheximide sensitive. This direct interaction may explain T3-dependent sensitization of the ß-adrenergic increase in ventricular contractility (26).
The T3 effects shown in this study are clearly nongenomic, as on SVR they were observed within 3 min after the injection of T3. This short time frame clearly excludes a genomic effect (13). For other cardiovascular parameters statistically significant effects occurred at the earliest 30 min after T3 injection. As the study was not powered to detect changes at single time points, their evaluation was explorative only, and visible effects were detected within 10 min. Furthermore, even an effect of T3 30 min after injection is not likely to be compatible with genomic effects.
Evaluating T3 effects after placebo pretreatment, the in vivo data presented in this study are in line with in vitro data (6, 7, 8, 9, 10, 11, 12); we observed an increase in CO that reflects positive inotropism and a decrease in SVR that reflects vasodilatation. In addition, the increase in the LF/HF ratio is compatible with an increase in sympathetic activity. These data are also compatible with the results of the studies by Klemperer et al. (3) and Hamilton et al. (5) in patients after coronary artery bypass surgery or suffering from severe heart failure. In these studies an increase in CO and a decrease in SVR have also been described after the administration of T3. However, cardiovascular parameters were measured 2 h after the start of T3 infusion. Therefore, early genomic effects cannot be ruled out. Notably, these studies were conducted in patients suffering from a disease (advanced congestive heart failure) or undergoing an intervention (cardiopulmonary bypass) known to decrease T3 levels and thus causing the low T3 syndrome. The study presented here was conducted in healthy euthyroid male volunteers. This may explain why the effects seen in the patient studies were larger than those in the study presented here. Even the small effects seen here are of potential physiological and definite pharmacological importance if extrapolated to patients with hypothyroidism. This is documented by cardiac rhythm disorders in response to rapid infusion of thyroid hormone in hypothyroid patients (27).
The potency of thyroidal hormones to provoke nongenomic effects may depend on the system studied. For safety reasons, we felt that only T3 can be used in humans in our experimental setting given its rather short half-life. However, after having shown rapid, nongenomic T3 effects in this setting and knowing the excellent tolerability of the T3 injection, it is of interest to look for nongenomic effects especially of L-T4, which, according to some preclinical models, may provoke nongenomic effects at lower (physiological) doses.
The results of the study periods in which modulators of the adrenergic system were administered are surprising. We expected that ß-adrenergic action and thyroid hormone effects would be additive or even superadditive. However, dobutamine blunts the thyroid hormone effects. The most likely explanation for this result may be a strong adrenergic stimulation by dobutamine, which may not allow for additional measurable T3 effects. However, this does not explain the reverse effect of T3 on LF/HF ratio, an effect that suggests a sympatholytic or vagotonic action of T3 on a stimulated adrenergic system, which is also suggested by the decreased heart rate. With regard to these results, one should notice that LF/HF ratio decreased slightly compared with that at baseline 2, but was still higher than that before dobutamine infusion. Furthermore, T3 did not reverse the dobutamine and esmolol effects, but augmented them. Given this, T3 augments the esmolol effect, but, the other way around esmolol pretreatment reversed the T3 effect seen after placebo pretreatment (increase in CO and decrease in SVR). In principle, the same is true with dobutamine, but the effect is not statistically significant: the small effect of T3 with dobutamine pretreatment also augments the dobutamine effect, but, again the other way around the effects of T3 after placebo pretreatment (increase in CO, decrease in SVR) were diminished or almost blunted by dobutamine pretreatment. Therefore, the data presented here are not in conflict with the results of the study by Martin et al. (28), in which increased cardiac effects of isoproterenol have been shown after treatment with T3 for 14 d. Our results are further in line with the results of an experimental study showing that T3 increases cell shortening of isolated cardiac myocytes within 5 min, but is ineffective in producing the same effect after isoproterenol pretreatment (29).
We analyzed for thyroid hormone effect the parameters from which SVR and CO are derived, i.e. heart rate, mean arterial pressure, and stroke volume. This analysis showed that a positive inotropic effect of T3 occurs with placebo and dobutamine pretreatment, but not with esmolol pretreatment. This suggests that the positive inotropic effect of T3 depends on ß-adrenoceptors in the myocardium, which is in line with data showing that T3 rapidly increases ß-adrenoceptor density in cultured embryonic cardiomyocytes (2). Whereas no changes in mean arterial pressure could be observed, heart rate was differentially influenced by T3 depending upon pretreatment: with placebo pretreatment, a statistically insignificant positive chronotropic effect occurred, but after dobutamine and esmolol pretreatment, T3 caused a negative chronotropic effect. This is not explained by direct cardiac effects of T3, but, rather, by an effect on the autonomic nervous system, because the changes in heart rate parallel those observed in the LF/HF ratio. The mechanism of this interaction with the autonomic nervous system remains the subject of further examinations.
The results of this study are comparable to those from two recent studies by our group showing modulation of nongenomic aldosterone effects by the adrenergic system (14, 30). In the latter study aldosterone elevated mean arterial pressure during ß-antagonism and decreased mean arterial pressure during ß-agonism. Nongenomic aldosterone effects reversed the effect of the adrenergic modulators esmolol and dobutamine. Here, the T3 effects on SVR and CO augment the effects of the adrenergic modulator. Modulation by the adrenergic system seems to be a common feature of nongenomic aldosterone and T3 effects despite the fact that the changes are in different directions.
The serum levels of fT3 and T3 reached in this study are in the pharmacological range, this may limit their relevance from a physiological point of view. However, the effects occur at serum levels that are still far below concentrations eliciting nonspecific nongenomic steroid actions (
10 µmol/liter) (13). The almost immediate action of T3 is of clinical relevance for the clinician administering T3, e.g. in hypothyroid coma. Furthermore, in certain clinical situations, such as prolonged or difficult weaning from cardiopulmonary bypass, T3 might have an immediate effect that could be used therapeutically.
This study for the first time gives evidence of rapid nongenomic T3 effects in humans. The effects are measurable even in euthyroid subjects and may be clinically relevant, e.g. in patients with hypothyroidism or low T3 syndrome. More studies are needed to clarify the mechanisms of interaction between the autonomic nervous system and rapid nongenomic T3 effects and to define the possible clinical implications of these effects.
Acknowledgments
Footnotes
This work was supported by the German Bundesministerium für Bildung, Wissenschaft, Forschung, und Technologie (01EC9407).
Abbreviations: CO, Cardiac output; ECG, electrocardiography; fT3, free T3; HF, high frequency; HRV, heart rate variability; ICG, impedance cardiography; LF, low frequency; pNN50, percentage of differences between adjacent normal RR intervals longer than 50 msec; rMSSD, square root of the mean of squared differences between adjacent normal RR intervals; SVR, systemic vascular resistance.
Received June 29, 2001.
Accepted January 10, 2002.
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