excess thyroid hormone is associated with changes in cardiovascular system , including increase in heart rate , blood volume , cardiac contractility , and cardiac output [ 1 , 2 ] and decrease in systemic vascular resistance , whereas thyroid hormone deficiency has been associated with the opposite effects . it has been postulated that these changes are the result of both a direct regulation of cardiac - specific genes by triiodothyronine ( t3 ) [ 4 , 5 ] and indirect changes in hemodynamic function . atrial natriuretic peptide ( anp ) is a hormone synthesized by atrial cardiomyocytes [ 7 , 8 ] and is involved in blood pressure and electrolyte homeostasis . human studies have shown that plasma anp secretion is affected by thyroid hormones , being higher in hyperthyroid patients compared to euthyroid controls [ 1012 ] and returning to normal levels after appropriate treatment . there is a strong positive relationship between circulating thyroid hormones and both atrial and ventricular anp mrna contents [ 14 , 15 ] , supporting the hypothesis that anp synthesis and/or secretion from atrial cardiomyocytes remain under thyroid hormone control . the exact mechanism(s ) by which thyroid hormones regulate cardiac anp secretion have not been fully elucidated . in vitro studies , in rat atrial myocytes , have shown a direct , dose - dependent stimulation of anp synthesis and secretion by thyroid hormones , but in vivo studies are missing . on the other hand , atrial stretch and/or pressure within the atria is considered the primary mechanism that regulates anp secretion from cardiomyocytes [ 17 , 18 ] . therefore , in chronic hyperthyroidism , the increased anp secretion could be the result of both the direct action of thyroid hormones on the myocardium and/or the indirect action through the alterations in peripheral circulation that may drive the change in cardiac work [ 1921 ] . the aim of this study was to examine whether in vivo thyroid hormone regulation of anp secretion is the result of a direct action on the myocardium and/or of an indirect action through the alterations they pose in the peripheral circulation . we examined the cardiac anp secretion ( baseline and volume expansion induced ) in healthy subjects before and after per os t3 administration for 3 days to establish serum t3 levels compatible with hyperthyroidism . fourteen healthy male volunteers were recruited in the study ( mean age 29  4.3 years ) . none of them was using any medication known to interfere with thyroid hormones metabolism or cardiovascular system function . 50  g t3 was administered per os twice daily for three days . to exclude serum t3 level variation ( due to its t1/2 of ~8 hours ) , before t3 initiation and in the morning of the fourth day , the participants reported in the testing room between 8:00  am and 8:30  am after an overnight fasting . a heparinized indwelling intravenous ( iv ) line was placed in a forearm vein , and the subject remained in the supine position until the end of the study . blood samples for measurement of anp , pra , aldo , t4 , t3 , tsh , serum creatinine , sodium , and potassium levels were obtained after 30  minutes of being in the supine position before ( i.e. , baseline ) and after a 1500  ml normal saline ( nsal ) infusion ( iv , rate 25  ml / min ) . new blood samples were thereafter obtained for anp , pra , and aldo measurement at 30 , 60 , 75 , 90 , and 120 minutes from the completion of nsal - induced volume expansion ( 60 minutes from initiation of nsal ) . all blood samples were immediately centrifuged , and plasma or serum was separated and frozen at 70c until assayed . for anp determinations , venous blood was drawn directly into ice - chilled disposable glass tubes containing edta ( 1  mg / ml ) and 500  iu / ml aprotinin . plasma was immediately separated by centrifugation for 15 minutes at 4c and stored frozen at 70c until the assay . plasma anp levels were measured by ria as previously described   using an antiserum to human anp ( 128 ) ( nichols institute diagnostics ltd . , uk ) . pra was measured using solid - phase ria from diasorin ( saluggia , italy ) . for the statistical analysis , all pra levels below the sensitivity value ( 0,2  ng / ml / hr ) serum aldo , t3 , t4 , and tsh concentrations were measured using commercial ria kits . additionally , two urine samples were collected for two- and- a- half - hour periods , at baseline and after nsal infusion for urine aldo , electrolytes , and creatinine level measurement . baseline and 60- and 120-minute blood pressure ( bp ) and heart rate ( hr ) were recorded . mean arterial blood pressure ( map ) was calculated as follows : 
								 ( 1)map = diastolic  bp+(13pulse  pressure ) . map was used for the determination of the systemic vascular resistance ( svr ) according to the formula 
								 ( 2)svr = mapcardiac  output . all participants underwent a comprehensive 2d and tissue doppler imaging transthoracic echocardiography at baseline ( i.e. , before treatment with t3 ) and after 3 days of treatment , performed by a single experienced echocardiographer with the ultrasound apparatus atl - ultramark 9 ( bothell , seattle , usa ) and a 2.5  mhz transducer . complete m mode , two dimensional , and spectral and color doppler recordings were made with subjects in the left lateral decubitus position using conventional parasternal and apical views , according to the standardization of the american society of echocardiography . three consecutive sinus beats were measured , and the averaged doppler analysis of the transmitral early ( e ) and late ( a ) inflow velocity was performed in the apical 4-chamber view . systolic function of the left ventricle was also evaluated . the study was conducted in accordance with the 1964 declaration of helsinki and was approved by the ethics committee at the haf and va general hospital of athens . the hormone responses were calculated either as the peak minus baseline ( or  response ) or as the integrated area under the curve ( auc ) values on the baseline ( 0 minute ) corrected response , using the trapezoidal model . one - way anova and paired t - test were used for statistical comparisons in each group . nonpaired t - test and mann whitney , for non - parametric data , were used for comparisons between groups . t3 administration resulted in a significant increase in serum t3 , a significant decrease in serum tsh , and a small but significant decrease in serum t4 levels ( table 1 ) . similarly , the induced hyperthyroid serum t3 levels resulted in a nonsignificant increase in plasma anp , aldo , and pra ( table 1 ) . t3 administration was followed by a significant increase in shortening fraction , velocity of circumferential fiber shortening , ejection fraction , stroke volume , stroke index , and cardiac output and a significant decrease in left ventricular ( lv ) end - systolic diameter , lv end - systolic volume , lv ejection time , peak e velocity , acceleration velocity , and systemic vascular resistance ( table 2 ) . on the contrary , peak a velocity and left atrial diameter were not significantly affected ( table 2 ) . the nsal - induced plasma volume expansion was followed by a significant decrease in pra and aldo levels without significantly affecting map and heart rate over each study period ( i.e. , before and after t3 administration ; table 3 ) . on the contrary , plasma anp levels increased significantly compared with baseline values in both study periods ( table 3 , figure 1 ) . however , hormone responses to nsal - induced volume expansion , measured as the integrated area under the curve values corrected on the baseline (  auc ) , were comparable before and after t3 administration for anp , pra , and aldo ( table 3 , figure 1 ) . plasma volume expansion did not alter urine aldo excretion but affected the urine electrolyte excretion . urine sodium and potassium levels increased significantly after nsal - induced volume expansion compared to levels before nsal infusion ( table 4 ) and to comparable degree in both study periods that is , the nsal - induced significant changes (  responses ) were comparable before and after t3 administration . this has been shown in isolated cardiac preparations [ 23 , 24 ] and in intact animals with volume expansion or direct atrial distention [ 2426 ] . however , in vitro studies in rat atrial myocyte cultures have shown that anp synthesis and secretion are also stimulated directly by thyroid hormones in a dose - dependent manner . there are no data in vivo , indicating direct or indirect thyroid hormone stimulatory effect on myocardial anp secretion . our study is the first to show that a short - term increase in t3 levels , induced by an exogenous administration of t3 , despite reaching t3 values comparable to that seen in hyperthyroidism , did not have any significant impact on plasma anp levels . we administered t3 for 3 days , as the 72-hour length has been reported to be an adequate time frame for a t3-induced anp - mrna synthesis and subsequent significant protein secretion . have demonstrated that treatment of neonatal rat cardiomyocytes ( primary cultures ) with t3 results in a significant increase in pronatriodilantin ( the precursor for anp ) mrna , whereas matsubara et al . have shown a significant increase in secreted anp levels following treatment with t3 in primary cultures . the effect in the former study was already apparent at 12 hours and reached its maximal effect at 48 hours , whilst in the later became apparent at 48 hours . the stimulatory effect of t3 was near maximal at a concentration of 5  10  m , which correlates very well with the affinity of t3 for its nuclear receptor . our study is in accordance with previous studies showing a significant increase in lv contractility following t3 administration , resulting in a profound increase in stroke volume , cardiac output , and cardiac index [ 1 , 2 ] . however , the non - significant influence of short - term hyperthyroidism in left atrial diameter and in peak a velocity , which reflect atrial contraction , indicate that the resulting alterations in cardiac preloading conditions   were not sufficient enough to significantly affect atrial wall stretch . on the other hand , the significant decrease in peak e velocity , which represents an early active relaxation of the left ventricle , in conjunction with the unchanged peak a velocity and left atrial diameter , may indicate a more active relaxation of the left ventricle and rapid filling phase [ 2931 ] without significant change in atrial function . nevertheless , if the action of thyroid hormones were to be direct on cardiac myocytes , one should expect a significant increase in plasma anp levels following t3 administration . the above - mentioned alterations in cardiovascular system do not seem enough to increase atrial stretch and hence anp secretion . in healthy men , blood volume expansion has previously been shown to increase plasma anp levels , and this increase closely correlates with the relative intravascular expansion . intravascular volume expansion following nsal infusion in our subjects is validated by the significant decrease of pra and aldo levels . the comparable nsal - induced decrease in pra and serum aldo levels before and after t3 administration and the non - significant change in the map and heart rate indicate comparable plasma volume expansions , which have been proved to induce increased cardiac preload [ 33 , 34 ] , velocity of the diastolic rapid filling phase [ 29 , 30 ] , cardiac output and stroke volume [ 3436 ] . in agreement this , a comparable amount of nsal - induced natriuresis was observed in our study participants before and after t3 administration . therefore , thyroid hormone action in our population was exerted predominantly through indirect modification of the peripheral cardiovascular system rather than a directed effect . our observation that the short - term hyperthyroid t3 levels in normal men do not affect baseline plasma anp levels is in contrast to what is seen in chronic hyperthyroid patients , where anp levels are significantly increased [ 1013 ] . the possibility that this finding is attributed to different serum t4 levels should be excluded taking into account that the stimulating effect of t4 on cellular anp content and anp - mrna levels in rat atria is entirely induced after its conversion to t3 by type i 5-deiodinase . based on our observations , we can speculate that the increased plasma anp levels in long - lasting hyperthyroidism are mainly the result of hemodynamic changes on the heart and peripheral circulation that move the cardiovascular system to a new functional equilibrium accompanied by increased atrial stretch . this is supported by the fact that hyperthyroid patients with atrial fibrillation have significantly higher plasma anp levels compared to patients without atrial fibrillation . additionally , artificial pacing in intact animals   as well as in humans   increases the secretion of anp through the elevation of atrial pressure , despite the stable normal thyroid hormone levels . the nondifferent baseline and post nsal - infusion plasma anp levels in our study may indicate that short - term hyperthyroid serum t3 levels are not able to induce hemodynamic changes sufficient enough to increase atrial stretch . in conclusion , our data indicate that thyroid hormone - induced myocardial anp secretion in healthy subjects is not the result of a direct action on the myocardium , rather , it is mainly the result of an indirect modification in cardiovascular hemodynamics that lead to increased atrial stretch .
background . 
 excess circulating thyroid hormones are associated with increased cardiac atrial natriuretic peptide ( anp ) secretion but the exact mechanisms involved have not been fully elucidated in vivo . methods . to examine whether thyroid hormone regulation of anp secretion is the result of a direct action on the myocardium and/or of an indirect action through alterations in the peripheral circulation , plasma anp levels ( baseline and volume expansion - induced ) 
 were evaluated in 14 healthy men , before and after triiodothyronine ( t3 ) administration . 
 results . 
 t3 administration was followed by a significant increase in serum t3 levels and a significant decrease in serum tsh levels , without significantly affecting anp levels . 
 systemic vascular resistance , plasma rennin activity ( pra ) , and aldosterone ( aldo ) levels , as well as indices of left atrial function , were not significantly altered , despite a significant increase in cardiac output . 
 plasma volume expansion , induced by a 1500  ml normal saline ( nsal ) infusion , both before and after t3 administration , was followed by a significant decrease in pra and aldo and a significant increase in plasma anp levels , without significantly affecting the mean blood pressure ( bp ) and heart rate ( hr ) in each study period . 
 the nsal - induced response , measured as the integrated area under the curve corrected for baseline values ( -auc ) , was not different after t3 administration for anp , aldo , pra , hr , and mean bp . conclusion . in vivo 
 thyroid hormone - induced myocardial anp secretion is the result of an indirect action mainly through hemodynamic changes that increase atrial stretch .