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1Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion 57069; and 2South Dakota Health Research Foundation, Cardiovascular Research Institute, Sioux Falls, South Dakota 57105
Submitted 9 June 2003 ; accepted in final form 6 August 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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hypertension; sick euthryoid syndrome; hyperthyroidism
An additional stressor on the cardiopulmonary system is thyroid hormone dysfunction. For example, hyperthyroidism increases the metabolic demands of the body and stresses the cardiovascular and respiratory systems (22, 29). In contrast, patients with heart failure can actually exhibit a "sick euthryoid" syndrome, characterized by decreased or normal levels of triiodothyronine (T3) and increased levels of reverse T3 (9, 23, 35). The possibility of using thyroid hormone replacement as a therapeutic measure has been approached in patients with heart failure (13, 23). Thus, increasing thyroid levels in SHHF rats may actually improve their cardiopulmonary status.
In this study, we investigated the effects of experimentally induced hyperthyroidism in male and female SHHF rats on control of breathing and cardiac function. Gender-matched Wistar-Kyoto (WKY) rats were used as normotensive, euthryoid controls. We hypothesized that the combined effects of hyperthyroidism and hypertension would have a more deleterious effect on the cardiopulmonary system of male than female SHHF rats, because hypertension is not tolerated as well in male as in female rats, and this should be further exacerbated by the superimposition of hyperthyroidism, leading to heart failure. An alternative hypothesis may be that if SHHF rats actually exhibit a sick euthryoid syndrome, administration of thyroid hormones may be beneficial.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Rats were housed three to four per cage according to genotype and gender. Light cycles consisted of 12 h on (6 AM) and 12 h off (6 PM). SHHF rats of each gender and genotype were divided randomly into two groups: 1) those that received water and powdered Purina rat chow ad libitum (controls) and 2) those that received water and powdered Purina rat chow to which 0.6% desiccated thyroid gland (DTG; Sigma Chemical, St. Louis, MO) was added for 8 wk (treated). WKY rats received water and powdered Purina rat chow ad libitum. SHHF rats consisted of seven control and nine DTG-treated males and seven control and eight DTG-treated females, and the WKY rats consisted of eight males and eight females. The method used to induce hyperthyroidism has been used previously by our group (11).
Measurement of ventilation and O2 consumption. To evaluate ventilation in air and in response to a brief hypercapnic challenge (7% CO2 in O2), conscious rats were placed in a 19-cm-long x 9.5-cm-diameter Plexiglas cylindrical chamber. One end of the chamber contained ports to allow air or the test gases to enter. At this end, chamber temperature was monitored using a Digetec thermometer. Pressure changes associated with ventilation were measured using a low-pressure transducer (Statham, Hato Rey, Puerto Rico) coupled to a Grass polygraph recording system. With the use of Boyle's law, the pressure changes were calibrated with a glass syringe attached to the chamber by injection of a known volume of gas into the chamber. This barometric method to evaluate ventilation has been used previously in our laboratory (33, 34). Ventilatory parameters included tidal volume, frequency of breathing, and minute ventilation.
A port was connected to a rotameter on the other side of the chamber to determine the flow rate through the chamber. A Beckman OM-14 O2 analyzer and a Vacumed CO2 analyzer were used to measure the fractional content of O2 and CO2 entering and leaving the chamber. Measurements of flow rate and the fractional contents of gases entering and exiting the chamber were used to determine O2 consumption using the flow through system. O2 consumption, minute ventilation, and tidal volume were corrected by body weight (BW) as follows: tidal volume ·100–1·BW–1, minute ventilation · 100–1·BW–1, and O2 consumption/BW. Another parameter that was calculated from the data was the ventilatory equivalent (ventilation ÷ O2 consumption), which was used to determine how well ventilation and O2 consumption were matched.
Procedures used to evaluate ventilation and O2 consumption. Each rat was weighed and then placed in the chamber and exposed to air for 30 min of acclimatization. Ventilation and O2 consumption were determined. Then the rat was exposed to the hypercapnic challenge for 5 min, and its ventilation was evaluated. Subsequently, the rat was removed from the chamber, and its rectal temperature was measured using a thermometer-thermocouple system (Sensortek, Clifton, NJ). The ventilatory responses to hypercapnia were determined by subtracting the air value preceding the exposure value and dividing by the air value.
Measurement of cardiovascular parameters. Hemodynamic measurements were conducted shortly after the ventilatory studies were completed. Rats were anesthetized with ketamine hydrochloride (30 mg/kg im) plus xylazine (5 mg/kg im) and placed on a 37°C heating pad (Braintree Scientific, Braintree, MA), and an intratracheal tube was inserted. Subsequently, an ultraminiature catheter containing a pressure transducer (model SPR-407, Millar, Houston, TX) was inserted into the left ventricle via the right common carotid artery. Right ventricular hemodynamic parameters were measured using a specially designed curved ultraminiature catheter (model SPR-407, curved, Millar) inserted through the right jugular vein. The catheters were connected to a Crystal Biotech DataFlow System (Hopkinton, MA) to evaluate left and right ventricular pressures (LVP and RVP, respectively), left and right end-diastolic pressures, and heart rate (HR). At the end of the experiment, the rat was euthanized with an overdose of thiopental sodium (Pentothal), and hematocrit and heart weight (HW) were evaluated.
Serum thyroid hormone (T3 and thyroxine) levels. At the end of the experiments, blood was collected by cardiac puncture. The blood was spun down at 3,000 rpm, and the serum was decanted and frozen at –80°C until analyzed for thyroid hormone levels. Total thyroid hormone plasma levels were determined using RIA kits (Diagnostic Systems, Webster, TX). The procedure followed the basic principle of RIA, where there is competition between a radioactive and a nonradioactive antigen for a fixed number of antibody-binding sites. The amount of 125I-labeled T3 or thyroxine (T4) bound to the antibody is inversely proportional to the concentration of unlabeled T3 or T4.
All reagents and samples were allowed to reach room temperature (
25°C) and then thoroughly mixed by gentle inversion. Unbound materials were removed by decanting and washing the coated tubes before counting in a gamma counter (COMPAC 120). The detection limits for the assays were 4.3 ng/dl for T3 and 0.4 µg/dl for T4. Samples of standards, controls, and unknowns were assayed in duplicate. The intra-assay coefficient of variation was 4%.
Data analysis. O2 consumption, ventilatory parameters, thyroid hormone levels, HW-to-BW ratio (HW/BW), ventilatory responses to hypercapnia, and the cardiovascular parameters were compared for effects of gender and treatment using a two-factor ANOVA. Another two-way ANOVA was used to compare genotype and gender in the untreated controls. If the ANOVA was significant (P < 0.05), a post hoc unpaired Student's t-test with Bonferroni's correction was used. Values are means ± SE.
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RESULTS |
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In the SHHF rats, there was a borderline trend toward an interaction (P = 0.056), but gender [F(1,7) = 12.94, P < 0.001] and treatment [F(1,27) = 8.96, P < 0.006] were significant for serum T3 levels (Table 1). DTG treatment increased T3 levels in male, but not female, SHHF rats. There was no difference in baseline T3 levels between untreated WKY and SHHF male rats.
BW, body temperature, and O2 consumption. SHHF rats were significantly heavier than WKY rats (Table 2). BW was significantly less (P < 0.01) in DTG-treated male SHHF than in control rats. Administration of DTG had no significant effect on BW in SHHF female rats. Body temperature was significantly higher in male and female SHHF than in gender-matched WKY rats (P < 0.05 for each). Body temperatures were elevated in male DTG-treated SHHF rats relative to genotype control rats body (P = 0.015) but not DTG-treated SHHF females relative to controls. There were genotype differences in weight-corrected O2 consumption in male [F(1,27) = 29.4, P < 0.00003] and female [F(1,27) = 14.6, P = 0.00817] rats. In general, weight-corrected O2 consumption was higher in WKY than in SHHF rats. DTG treatment increased O2 consumption in male and female SHHF rats (P < 0.001 for each). Thus treatment with DTG had different effects on BW in male and female SHHF rats but similar effects on O2 consumption in both genders.
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Control of breathing. Weight-corrected minute ventilation (Table 3) exhibited a genotype difference [F(1,27) = 5.42, P = 0.029]. There were no differences between untreated SHHF and WKY female rats in this parameter, but weight-corrected ventilation was greater in WKY male than in SHHF male rats (P = 0.01). A significant treatment effect was noted in weight-corrected minute ventilation [F(1,27) = 148.9, P = 0.0093], with an increase noted in DTG-treated SHHF male (P = 0.019), but not female, rats.
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The increase in minute ventilation in DTG-treated SHHF male rats was due to a significant increase in weight-corrected tidal volume (P = 0.05; Table 3). This parameter was not significantly elevated in DTG-treated SHHF female rats. In contrast, frequency of breathing was increased significantly by DTG treatment in SHHF female (P < 0 0.01), but not SHHF male, rats. Thus the increase in weight-corrected minute ventilation in DTG-treated SHHF male rats was due to small significant increases of tidal volume, whereas in SHHF female rats the major response to DTG treatment was an increase in breathing frequency.
To determine whether ventilation and O2 consumption were affected in a parallel manner by DTG treatment, the ventilatory equivalent (ventilation ÷ O2 consumption) was evaluated (Fig. 1). When untreated SHHF and WKY rats are compared, a genotype [F(1,27) = 9.97, P = 0.0043] and gender difference [F(1,27) = 4.59, P = 0.043] become apparent. SHHF rats have higher ventilatory equivalents than WKY rats, and this parameter is higher in male than in female rats (Fig. 1). In male and female SHHF rats, the ventilatory equivalent was significantly decreased by DTG treatment [F(1,23) = 6.07, P = 0.02] relative to the untreated gender-matched controls. DTG treatment caused the ventilatory equivalents of SHHF rats to reach levels noted in WKY male rats and eliminated the gender difference noted in the untreated SHHF rats.
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The ventilatory responses to hypercapnia (Fig. 2) in SHHF rats exhibited a significant gender difference [F(1,27) = 12.28, P = 0.002] and a treatment effect [F(1,27) = 4.78, P = 0.04]. In SHHF male and female rats individually, the ventilatory response to hypercapnia was maintained with DTG administration. The ventilatory response to hypercapnia was greater in untreated WKY than in SHHF rats (P < 0.04).
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Cardiovascular parameters. HW/BW (Table 4) exhibited a significant gender and genotype interaction in the untreated groups [F(1,27) = 5.76, P = 0.0245]. HW/BW was higher in untreated SHHF rats of both genders than in WKY rats (P < 0.001 for each) and higher in female than in male rats. DTG treatment increased HW/BW further in SHHF rats (P < 0.00001).
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There was no significant genotype difference in HR (Table 4). Male and female SHHF rats exhibited a treatment effect [F(1,15) = 52.6, P < 0.001], with a marked elevation of HR after DTG.
DTG-treated SHHF animals showed an interaction between gender and treatment [F(1,15) = 11.07, P = 0.0034], with an increase in LVP in female, but not male, rats (Fig. 3A). LVP was higher in untreated male and female SHHF than in untreated WKY rats (P < 0.001 for both). Thus DTG did not increase LVP further in SHHF male rats but did increase LVP in female rats. To evaluate left ventricular contractility, the first derivative of LVP (dP/dt) was determined (Fig. 3B). In SHHF rats, DTG had no significant effect. Untreated WKY male and female rats exhibited a higher dP/dt than gender-matched SHHF rats (P < 0.004 for each gender).
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RVP (Fig. 4A) was elevated by DTG treatment in SHHF rats [F(1,15) = 36.2, P < 0.0001]. RVP was higher in untreated male WKY male than in untreated SHHF male rats (P = 0.02), whereas no differences were noted in RVP between untreated female WKY and SHHF rats. Right ventricular contractility (Fig. 4B) was increased by DTG treatment in SHHF rats [F(1,15) = 21.93, P = 0.00014]. Right heart contractility in untreated groups was not different from that in male or female rats of either genotype, and there were no significant differences between genotypes.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Regardless of gender, DTG-treated SHHF rats exhibited increased HW/BW, HR, weight-corrected O2 consumption, RVP, and right ventricular contractility relative to controls. In contrast, LVP was elevated in DTG-treated SHHF male, but not female, rats. Potential mechanisms for these findings are discussed below.
BW and DTG treatment. SHHF female rats maintained BW relative to untreated controls, although BW was lower in WKY BW than in SHHF rats (28, 40). In contrast, DTG-treated SHHF male rats lost weight. These results suggest an interaction between gender and thyroid hormone effects in rats (32, 40). Maintenance of BW necessitates that the amount of food intake balances use of food for energy, such as activities, growth, and maintenance of body temperature. In DTG-treated SHHF female rats, body temperature was similar to that in control female SHHF rats, but O2 consumption was increased. Thus DTG-treated SHHF female rats needed to eat more and/or lose less heat to maintain BW than did male rats (25).
A potential limitation in incorporating DTG in the food is that the amount consumed per animal is unknown and is linked to the amount an animal eats. A potential benefit of incorporating DTG into food is that it is administered without injections, pumps, or pellets, methods that increase stress on the animals.
Hyperthyroidism and the cardiovascular system. Hyperthyroidism induced experimentally by administration of DTG, T4, or T3 has profound effects on cardiovascular function (10, 15, 28, 25, 37). Gay and coworkers (10) showed increases in HR, RVP, LVP, ventricular contractility, and mean circulatory filling pressure in male Sprague-Dawley (SD) rats treated for 8–10 days with T4. Total peripheral resistance and LVP and venous compliance were decreased relative to controls. Effects on HR, left ventricular dP/dt, and LVP were similar in female SD rats treated with T3 for 14 days. Increased RVP and pulmonary arterial pressure in hyperthyroid female SD rats occurred concomitantly with ventricular hypertrophy, a positive chronotropic effect, and an increased cardiac output. Many of these cardiovascular effects were also noted in the present study in SHHF rats of both genders.
Few studies have investigated the effects of hyperthyroidism in rat models of hypertension, such as the SHR. When Heckmann and Zimmer (15) administered T3 to female SHR, 30% of the animals died, whereas there was no mortality in SD female rats. In the SHR that survived T3 treatment, HR and HW/BW were markedly increased, but LVP, right ventricular weight, left ventricular dP/dt, cardiac output, and total peripheral resistance were not affected. These results are in contrast to those in the present study in SHHF male rats, in that their response to thyroid hormone treatment is similar to that of SD female rats. In comparison, DTG-treated SHHF female rats showed no increase in LVP. In the present study, DTG was administered for 8 wk, rather than 1–2 wk, before cardiovascular measurements. The attenuated cardiovascular responses of SHHF rats to DTG may be the result of a euthryoid sick syndrome. Potential mechanisms for development of this disorder in SHHF rats may include elevations of reverse T3, cytokines such as tumor necrosis factor-
, and stress hormones such as glucocorticoids (9, 37), all of which inhibit to various extent deiiodinonases, which convert T4 to T3. In addition, the lack of significant increases in T4 levels with treatment suggests that absorption of this hormone is not normal in SHHF rats. Increases in O2 consumption in male and female SHHF rats suggest that our treatment was increasing thyroid hormone levels. Further evidence for this possibility was noted in a preliminary study using WKY rats treated with the same or lower levels of DTG, in which we found marked increases in T4 and T3 serum levels in this genotype (unpublished observations).
Effects of hyperthyroidism on ventilation. Hyperthyroidism can affect control of breathing through various mechanisms. These include increased metabolic demand and body temperature (37), elevated LVP (7), increased blood pressure (11), remodeling of the respiratory muscles and lungs (1, 20), increased hematocrit (25), and alteration of neurotransmitter levels in brain regions involved in control of breathing (16, 21, 38).
An increase in O2 demand is a powerful stimulus to increase ventilation. One measure of the efficacy of the respiratory system to respond to changes in metabolism is the ventilatory equivalent (ventilation ÷ O2 consumption). In the present study, ventilatory equivalent was significantly lower in DTG-treated SHHF male and female rats than in untreated controls. These data suggest that DTG-treated SHHF rats did not increase ventilation to match O2 consumption. Although there was a clear gender difference in WKY ventilatory equivalent, DTG did not alter this relation or its magnitude. In support for the latter findings, Hillbom and Poso (17) rendered male rats hyperthyroid by administering T3 for 6 days and found no effect on blood gases. This indicates that ventilation and metabolism were matched in these animals. Ianuzzo and coworkers (19) induced hyperthyroidism in male SD rats by subcutaneous injection of T4 for 6 wk and noted no effect on tidal volume or frequency, although diaphragmatic fiber types were markedly altered and fiber diameters were smaller. These ventilatory results contrast with our findings in SHHF, but not WKY, rats and suggest that the type and length of thyroid hormone treatment and underlying factors such as strain influence control of breathing.
In hyperthyroid human subjects, ventilation is in excess of metabolic demands (20, 29). This is especially apparent when the subject exercises. However, the level of exercise that can be attained by a hyperthyroid subject is generally lower than that attained by matched euthyroid subjects (20). Ventilatory responses to hypoxic and hypercapnic gas challenges are also greater in hyperthyroid subjects (29). In the present study, the ventilatory response to hypercapnia was not altered by DTG treatment. However, untreated WKY rats exhibited higher responses to hypercapnia than did untreated SHHF rats. This was especially true in WKY compared with SHHF male rats.
Other factors that may modulate ventilation are reflexes associated with the cardiovascular system. For example, Crisp and colleagues (7) reported that elevating LVP in anesthetized dogs decreased phrenic nerve activity, an index of ventilatory drive to the diaphragm. In a separate study, Crisp and coworkers (6) noted that elevating RVP had no effect on ventilation. Thus the depressed ventilatory equivalent in DTG-treated SHHF male and female rats may be related to their elevated LVP. However, LVP is similar in untreated and DTG-treated SHHF male rats and higher than in untreated SHHF female rats, but ventilatory equivalent is higher in untreated SHHF male rats than in DTG-treated male or untreated female rats. These results suggest that not only LVP, but also gender, in SHHF rats affects control of ventilation.
Studies that investigate the interaction between hypertension, gender, and control of breathing focus on their roles in sleep apnea syndrome (cessation of airflow during sleep) (2). Male gender and hypertension are significant risk factors in humans for developing sleep apnea (26). Clinically, sleep apnea exacerbates underlying cardiovascular disease or may contribute to its etiology and may increase morbidity and mortality. Treatment with antihypertensive drugs or assisted ventilation may be helpful in some patients (29).
Animal models of hypertension have been useful to study hypertension and abnormal control of breathing. Carley and coworkers (3) reported that male SHR exhibited increased postsigh apneas (a measure of control of breathing) during sleep relative to normotensive controls. Administration of hydralazine, which normalized the blood pressure in SHR, markedly decreased sleep apnea and the number of postsigh apneas (4).
These and other studies in SHR and in hypertensive humans indicate a significant relation between hypertension and abnormalities of control of breathing (26). Except in the epidemiological studies and the present study, the role of gender has not been specifically investigated in modulating hypertensive factors that also influence control of breathing.
In summary, this study investigated how the interaction between hypertension, hyperthyroidism, and gender affects control of cardiopulmonary function. These results suggest a host of further studies cited above to understand how thyroid hormone therapy affects remodeling of the heart, lungs, and central nervous system in SHHF male and female rats. Our original hypothesis that hyperthyroidism would have a more deleterious effect on the cardiopulmonary system of SHHF male rats was not supported, but, rather, DTG actually may have a beneficial effect in this genotype. SHHF rats may serve as a good model to investigate a beneficial role of thyroid hormone abnormalities associated with heart failure.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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