Abstract
This study aimed to investigate whether nitric oxide participates in the cardiovascular function and haemodynamic adaptation to acute haemorrhage in animals with thyroid disorders. Sprague-Dawley rats aged 2months old treated with T3 (hyper, 20μg/100g body weight) or 0.02% methimazole (hypo, w/v) during 28days were pre-treated with NG nitro-l-arginine methyl ester (L-NAME) and submitted to 20% blood loss. Heart function was evaluated by echocardiography. Measurements of arterial blood pressure, heart rate, nitric oxide synthase activity and protein levels were performed. We found that hypo decreased fractional shortening and ejection fraction and increased left ventricle internal diameter. Hyper decreased ventricle diameter and no changes in cardiac contractility. Haemorrhage elicited a hypotension of similar magnitude within 10min. Then, this parameter was stabilized at about 30–40min and maintained until finalized, 120min. L-NAME rats showed that the immediate hypotension would be independent of nitric oxide. Nitric oxide synthase inhibition blunted the changes of heart rate induced by blood loss. Hyper and hypo had lower atrial enzyme activity associated with a decreased enzyme isoform in hypo. In ventricle, hyper and hypo had a higher enzyme activity, which was not correlated with changes in protein levels. Haemorrhage induced an increased heart nitric oxide production. We concluded that thyroid disorders were associated with hypertrophic remodelling which impacted differently on cardiac function and its adaptation to a hypovolemia. Hypovolemia triggered a nitric oxide synthase activation modulating the heart function to maintain haemodynamic homeostasis. This involvement depends on a specific enzyme isoform, cardiac chamber and thyroid state.
Introduction
Hypovolemia, secondary to major blood loss, frequently precedes multiple organ dysfunctions (De Santis & Singer 2015). Activation of several neurohormonal factors (nitric oxide (NO), catecholamines, endothelins, vasopressin, renin–angiotensin system) is involved in the restoration of vascular volume and blood pressure following bleeding (Fujisawa et al. 1999, Paczwa & Ganten 1999). This adaptive response to the decrease in the total blood volume implies a peripheral vasoconstriction which induces a redistribution of blood flow to the vital organs within which highlights the heart (Schadt & Hasser 2004). Previously, we demonstrated that hypovolemic state induced by acute haemorrhage provoked a heterogeneous and dynamic NO synthase (NOS) activation modulating the cardiovascular response in young rats. Increased cardiac endothelial NOS expression is an early molecular response to regulate cardiac function after blood loss. Inducible NOS becomes a major source of cardiac NO production in later stages, which could be determinant of heart dysfunction after 120min of sustained haemorrhagic shock (Balaszczuk et al. 2006).
On the other hand, it is well known that cardiovascular function is also influenced by the autonomic nervous system and numerous endocrine hormones in which thyroid hormones have relevance. Thyroid hormone deficiencies, as well as excesses, result in profound changes in cardiac function regulation and cardiovascular haemodynamia mediated by genomic and non-genomic effects (Vargas-Uricoechea et al. 2014). A functional relation involving thyroid hormones, endothelial cells and NO has been extensively described in the past several years. There are several studies that showed that thyroid hormones and NO are involved in many different signalling pathways related to normal post-natal cardiac development, maturation and function (Lepic et al. 2006). We have previously demonstrated that thyroid hormones are able to regulate intrinsic heart rate (HR) in a heart without autonomic regulation. According to our results, NO pathway would be involved in this mechanism. Thyroid hormones modulate NO steady-state level which may act as a messenger to modulate the mitochondrial bioenergetic function, resulting in an NO-mediated regulation of the heart pacemaker activity (Fellet et al. 2004, 2006, 2008). Additionally, we demonstrated that hypothyroidism contributes in a differential way to ageing-induced changes in the myocardium and aorta tissues. Low thyroid hormones levels would enhance the ageing effect on the heart related to cardiac NO production (Sarati et al. 2012). We also revealed that the heart of male and female rats undergoes distinct adaptive responses to hyperthyroidism that confer to the latter, a relatively stronger adaptation profile that appears to be related to the ability to regulate NO production (Rodríguez et al. 2015).
Considering that thyroid status alterations are one of the major endocrine diseases in adulthood and its association with a significant increase in cardiovascular risk in the middle-aged, the aim of the present work was to analyze whether changes in NO signalling participate in the cardiovascular manifestations of thyroid disorders and whether these changes are involved in haemodynamic adaptation to acute haemorrhage in animals with thyroid disorders.
Materials and methods
Animals
Male Sprague-Dawley rats 2months old from the breeding laboratories of the ‘Facultad de Farmacia y Bioquímica’ (Universidad de Buenos Aires, Argentina) were used throughout the study. Rats were housed in humidity- and temperature-controlled environment with an automatic 12h light:12h darkness cycle. Rats were fed standard rat chow from Ganave (Buenos Aires, Argentina) and received tap water ad libitum up to the day of the experiments. All procedures were reviewed and approved by the National Food, Drug and Medical Technology Administration (ANMAT) and National Department of Health and Environment, Argentina (No. 6344/96).
Rats were randomly assigned to one of the three groups:
Control rats (Eut, n=15): euthyroid animals who received s.c. injections of 0.9% NaCl (0.1mL/100g body weight (BW) every 2nd day during 28days.
T3-treated rats (Hyper, n=15): animals received s.c. injections of T3 (Sigma, 20μg/100g body weight) every 2nd day during 28days (Heron & Rakusan 1996).
Methimazole-treated rats (hypo, n=15): animals were rendered hypothyroid after 28days of treatment with 0.02% methimazole (w/v) in the drinking water (Franco et al. 2006).
Determination of treatment efficacy
In order to confirm the hypo- and hyperthyroid states, serum thyroid-stimulating hormone (TSH), total triiodothyronine (T3) and thyroxin (T4) (TSH kit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA) were measured by radioimmunoassay at the beginning and the end of the experimental period. Intra- and inter-assay coefficients of variation for TSH were 8.7% and 13.4%, respectively (Greeley et al. 1982). T3 inter- and intra-assay coefficients of variation varied from 4.2 to 6.0 and 5 to 6.5%, respectively; T4 inter- and intra-assay coefficients of variation varied from 7.1 to 7.4 and 2.9 to 5.1%, respectively).
Echocardiographic measurements
After 28days of treatment, or control period, rats were anesthetized with urethane (1.0g/kg, i.p.), their chests were shaved under aseptic conditions and echocardiographic measurements were performed in the left lateral decubitus position. Two-dimensional directed M-mode images were obtained using a Sonoscape (A6 Vet) system with a 9–4MHz transducer. Measurements were taken in the right parasternal short axis plane at the level of the mitral valve leaflets. LV internal diameter (LVID), LV posterior wall thickness (PWT) and anterior wall thickness (AWT) were measured in both systole (s) and diastole (d). Ejection fraction (EF), fractional shortening (FS) and systolic volume were measured from ventricular internal diameters by the echocardiography system. All determinations were made according to the guidelines of the American Society of Echocardiography. Each rat was then instrumented with catheters. Animals were kept under anaesthesia by additional small doses of urethane throughout the experiment. Body temperature was monitored with a rectal probe and maintained at 37.0±0.5°C with heating lamps to avoid the influence of temperature on cardiovascular parameters during the experiment.
Cardiovascular assessments in thyroid disorders
After 28days of treatment, the animals were anesthetized with urethane (1.0g/kg i.p.). To ensure an open airway, a tracheotomy was performed using polyethylene tubing (3.5 or 4mmID, Portex). Mean arterial pressure (MAP) was measured through a cannula inserted into the right femoral artery and connected to a pressure transducer (Statham P23 ID, Gould Inst, Cleveland, OH, USA); measurements were recorded with a polygraph (Physiograph E & M, Houston, TX, USA) during the whole experiment. Heart rate (HR) was determined from the pulsatile pressure signal by beat-to-beat conversion with a tachograph amplifier (model S77-26 tachometer, Coulbourn Instruments, Allentown, PA, USA). The Labtech Notebook program (Laboratory Technology, Wilmington, MD, USA) was used for data acquisition.
Experimental protocol
Eut, hyper and hypo animals were sub-divided into two experimental groups:
The hypovolemic state in H and H+LNAME groups was induced through an acute haemorrhage using a cannula inserted into the left femoral artery (Riviero, PR10). The bleeding was done by a loss of 20% of the blood volume during 2min, at constant flux. The volume was calculated individually for every animal, from the total blood volume (7% of the body mass).
The L-NAME, an unspecific inhibitor of the NOS, was administrated as a continual infusion through a cannula inserted into the right femoral vein (Riviero PR 10, 0.5mg/kg/h IV=100μL/h).
At the end of each experimental protocol, rats were killed by pneumothorax and heart was removed. Western blot analysis for NOS was performed in this tissue, and NOS activity was measured according to the method of the conversion of [14C (U)]-l-arginine to [14C (U)]-l-citrulline.
Nitric oxide synthase activity
Capacity for cardiac NO formation was assessed determining NOS activity in right atria and left ventricle from Eut, hyper and hypo animals by measuring the conversion of [14C (U)]-l-arginine to [14C (U)]-l-citrulline. Tissue homogenates (approximately 50µg protein) were incubated in Tris–HCl buffer (pH 7.4) containing 1µg/mL l-arginine, [14C (U)]-l-arginine (346µCi/mL), l-valine (67mM), NADPH (1mM), calmodulin (30nM), tetrahydrobiopterin (5µM) and CaCL2 (2mM) for 60min at room temperature. At the end of the incubation period, the NOS reaction was arrested by the addition of a buffer solution containing 20mM HEPES buffer and 20mM EDTA, pH5.5. Reaction mixtures were loaded onto cation exchange columns (Dowex AG 50W-X8, Na+ form; Bio-Rad) and [14C (U)]-l-citrulline was eluted from columns with 0–50mL ddH2O. The amount of [14C (U)]-l-citrulline eluted was quantified using a liquid scintillation counter (Wallac 1414 WinSpectral; EG&G Company, Turku, Finland) as described previously (Sarati et al. 2012). All compounds, except [14C (U)]-l-arginine monohydrochloride (346mCi/mmol, Amersham Life Science), were purchased from Sigma Chemic. Protein determination was made using the Lowry method, with bovine serum albumin as a standard.
Calcium dependence
In order to determine calcium dependence, atria and ventricle NOS activity was determined using [14C (U)] arginine as substrate as described above. Tissue slices (2–3mm thick) from Eut, hypo and hyper animals were obtained. Some slices from haemorrhaged groups were pre-incubated (15min) with calmidazolium (Cz, 1µM) (Elesgaray et al. 2008) before incubation with [14C (U)] l-arginine during 30min at 37°C. The amount of [14C] l-citrulline obtained was determined with a liquid scintillation counter. Nitric oxide production (measured as pmol of [14C] citrulline) was expressed in pmol/g wet weight min.
Western blot analysis
The right atria and left ventricle samples were homogenized on ice with a Tissue Tearor (Biospec Products) in homogenization buffer (50mmol/L Tris, 0.1mmol/L EDTA, 0.1mmol/L EGTA, 1% Triton, 1mmol/L PMSF, 1μmol/L pepstatin, 2μmol/L leupeptin, 1× protease inhibitor cocktail from Roche Diagnostics). Protein concentration in the Triton-soluble supernatant was determined using the Lowry assay. Equal amounts of protein (100μg protein/lane) were separated by electrophoresis in 7.5% SDS-polyacrylamide gels (Bio-Rad), transferred to a nitrocellulose membrane (Bio-Rad) and then incubated with rabbit polyclonal anti-NOS antibodies, diluted at 1:500. The primary antibodies were: polyclonal rabbit anti-inducible NOS (iNOS) (epitope at the carboxy terminus), anti-endothelial NOS (eNOS) (epitope at the amino terminus) and anti-neuronal NOS (nNOS) (epitope at the amino terminus). Finally, a secondary immunoreaction with a goat anti-rabbit antibody conjugated with horseradish peroxidase (dilution 1:5000) was performed. Samples were revealed by chemiluminescence using Kalium reagent for 2–4min. Density of the respective bands was quantified by densitometric scanning of Western blots using a Hewlett-Packard scanner and Totallab analyzer software (Biodynamics, Seattle, WA, USA), and protein amounts were calculated by comparing with the densitometric values of the corresponding standard. Protein levels were expressed as a ratio of the optical densities of NOS isoforms and β-actin band (using anti-beta actin, clone EP1123Y, rabbit monoclonal antibody) to check for any inaccuracies in protein loading.
Materials
The antibodies against the three isoforms of NOS (iNOS (610333), eNOS (610298) and nNOS (610311) were supplied by BD Biosciences and anti β-actin by Millipore (04-1116). Secondary antibody (170-6515) was by Bio-Rad laboratories. The Western Blot Detection System was supplied by Amersham Pharmacia Biotech. Biochemicals were supplied by Sigma.
Ethical approval for animal experimentation
Animals were cared for according to regulation 6344/96 of Argentina’s National Food, Drug and Medical Technology Administration (ANMAT). Experiments with animals had been performed in accordance with UK legal requirements. Experimental procedures were approved by the ethics committee of the Facultad de Farmacia y Bioquímica (CICUAL; EXP UBA N° 0054570), Universidad de Buenos Aires, Argentina.
Statistical analysis
Data in tables and figures are mean values±s.e.m. Data were evaluated with univariate and multivariate approaches for a completely randomized design, with a structure of two factors (haemorrhage and thyroid hormones). For each variable, ANOVA or MANOVA analysis was performed when appropriate. The Levene’s and Shapiro–Wilk’s tests were used to evaluate homogeneity of variances and normality of data, respectively.
When normality and homogeneity of variances assumptions were satisfied, the Bonferroni multiple comparison test was run. In the case of non-homogenous variances, a multiple comparison test, such as Tamhane, was run. To detect association among variables, a correlation analysis was performed and the Pearson coefficient was calculated. All statistical procedures were performed using the SPSS statistical software package version 22.0; statistical significance was set at P<.05.
Results
Treatment efficacy
Treatment with methimazole and T3 was effective in establishing a hypothyroid and hyperthyroid state, respectively. TSH plasmatic levels were higher and lower in hypo and hyper rats, respectively, than Eut animals. T3 and T4 levels decreased in hypo rats, while T4 increased in hyper animals. Body weights were similar in the three groups of animals. Basal MAP values were similar in Eut, hypo and hyper rats. However, basal HR values were lower and higher in hypo and hyper rats compared with Eut animals, respectively (Table 1).
Biological variables.
| Group of animals | Eut | Hypo | Hyper |
|---|---|---|---|
| TSH (ng/mL) | 14.75±0.83 | 35.57±4.35* | 5.57±0.03* |
| T3 (ng/dL) | 1.131±0.123 | 0.750±0.036* | 1.034±0.036 |
| T4 (ug/mL) | 2.475±0.031 | 1.034±0.036* | 3.775±0.270* |
| BW (g) | 337±12 | 338±12 | 298±12 |
| HR (b.p.m.) | 352±15 | 214±13* | 424±15* |
| MAP (mmHg) | 80±4 | 70±4 | 76±2 |
| LVIDd (mm) | 5.40±0.18 | 5.99±0.19* | 4.7±0.10* |
| LVIDs (mm) | 2.80±0.06 | 3.01±0.10* | 2.21±0.07* |
| AWTd (mm) | 1.70±0.02 | 1.37±0.01* | 2.01±0.10 |
| AWTs (mm) | 2.87±0.03 | 2.10±0.02* | 2.8±0.10 |
| PWTd (mm) | 2.10±0.17 | 1.50±0.10* | 2.03±0.15 |
| PWTs (mm) | 2.90±0.08 | 2.43±0.12* | 3.23±0.08 |
| EF (%) | 86±3 | 83±1* | 88±3 |
| FS (%) | 56±2 | 46±2* | 51±2 |
AWTd, anterior wall thickness in diastole; AWTs, anterior wall thickness in systole; BW, body weight; EF, ejection fraction; Eut, euthyroid rats; FS, fractional shortening; HR, heart rate; Hyper, hyperthyroid rats; Hypo, hypothyroid rats; LVIDd, LV internal diameter in diastole; LVIDs, LV internal diameter in systole; MAP, mean arterial pressure; PWTd, posterior wall thickness in diastole; PWTs, posterior wall thickness in systole; T3, triiodothyronine; T4, total thyroxine; TSH, thyroid-stimulating hormone. Data are mean±s.e.m.; n=15; *P<0.05 vs Eut rats.
Echocardiographic measurements
Table 1 also shows echocardiographic data for all groups. LV systolic and diastolic chamber diameters increased in hypo rats; meanwhile, these diameters decreased in hyper animals. LV anterior and posterior wall thickness in both systole and diastole remained unchanged between Eut and hyper animals; however, a reduction in these parameters was observed in hypo group. LV ejection fraction and fractional shortening did not change between Eut and hyper animals, but they both were reduced in hypothyroid state. Figure 1 shows representative images of M-mode echocardiographic tracing.
Left ventricular representative images of M-mode echocardiographic tracing from euthyroid (panel A), hypothyroid (panel B) and hyperthyroid rats (panel C).
Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0203
Changes in systemic haemodynamic parameters during and after haemorrhagic state
Figure 2 illustrates the time course of MAP and HR during 120min after bleeding. Baseline MAP (mm Hg) measurements were not different among the three groups of rats. Haemorrhage induced a marked decrease in MAP in Eut group, which reached a value of 44±4mm Hg at 10min following the bleeding period (P<0.001 vs basal values), with subsequent stabilization at about 55±4mm Hg at 35min (P<0.01 vs basal values). The pressure response to bleeding was similar in the three experimental groups. However, the magnitude of immediate hypotension after bleeding was greater in animals with thyroid alterations. The pressure remained low during the entire experimental time presenting lower values of animals with thyroid disorders (panel A). L-NAME treatment has not altered basal MAP in the three experimental groups. Blood pressure in L-NAME Eut-treated rats group was 37mm Hg (P<0.01 vs basal values) at 10min haemorrhage, rising to 82±4 (P=ns), 100±4 (P<0.01) and 105±4mm Hg (P<0.01) during 35, 60 and 120min, respectively, following the bleeding. The inhibition of NO system induced a recovery of the MAP after bleeding, registering a stabilization of this parameter at 60min after haemorrhage, at values higher than hypo and hyper animals. In relation to the hypothyroid rats, the L-NAME treatment induced a greater hypotensive response after bleeding. However, in the later stages, MAP was gradually increased stabilizing in 53mmHg at about 65min. The hyperthyroid animals presented a lower hypotensive response to the euthyroid, but managed to reach MAP values close to the baseline at about 100min (panel B).
Changes in mean arterial pressure (MAP) and heart rate (HR) during and after haemorrhage in euthyroid (Eut), hypothyroid (hypo) and hyperthyroid rats (Hyper) (panels A and C). Changes in MAP and HR during and after haemorrhage in L-NAME-treated Eut, hypo and hyper rats (panels B and D). ♦ Eut rats; ■ hypo rats; ▲ hyper rats. Points are mean values (error bars represent s.e.m.); n=15/group; *P<0.05 vs basal values.
Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0203
Panel C illustrates the time course of the HR in Eut, hypo and hyper animals after bleeding. Basal HR of Eut animals was around 352±15b.p.m., while that of the hypo and hyper rats was 214±15b.p.m. and 424±13b.p.m., respectively. The haemorrhage induced a bradycardia of short duration followed by a gradual increase in this parameter in all the groups. Eut animals have attained the stabilization of this parameter at about 30min after haemorrhage, whereas this time was between 10 and 15min to achieve the stabilization in hypo and hyper rats (panel C). Treatment with L-NAME annulled the changes of HR to haemorrhage in Eut and hypo rats. L-NAME hyper-treated animals showed a similar HR response after withdrawal (panel D).
Nitric oxide synthase activity and Western blot
Figure 3 (panel A) shows that hypo and hyper animals exhibited a decreased atrial NOS activity compared with Eut rats. Bleeding increased NOS activity in all groups of animals. However, the magnitude of change was greater in Eut and hyper rats compared with hypo animals. Endothelial NOS isoform decreased in hypo rats (panel B). There were no differences between Eut and hyper groups of animals. Haemorrhage increased eNOS protein levels in Eut and hypo rats. However, hyper animals showed decreased eNOS protein levels after withdrawal. Inducible and neuronal NOS were lower in hypo rats compared with Eut group. Inducible and neuronal NOS proteins levels did not change in Eut and hyper rats (panels C and D). Bleeding increased iNOS proteins levels in all groups of animals; meanwhile, it did not change nNOS protein levels in experimental groups (panel C and D).
Total NOS activity in right atria from euthyroid (Eut), hypothyroid (hypo) and hyperthyroid (hyper) rats (panel A). The values are mean±s.e.m.; n=15/group; *P<0.05 vs Eut rats; †P<0.05 vs rats without haemorrhage. Representative Western Blots of eNOS (panel B), iNOS (panel C), nNOS (panel D) carried out on proteins from Eut, hypo and hyper right atria. Histograms illustrate the mean NOS protein values for each group. All experiments were performed in triplicate. Each blot was normalized with the expression of the β-actin from the same gels. Data are mean±s.e.m.; n=7/group; *P<0.05 vs Eut rats; †P<0.05 vs rats without haemorrhage. EutH, euthyroid haemorrhaged rats; hyperH, hyperthyroid haemorrhaged rats; hypoH, hypothyroid haemorrhaged rats.
Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0203
Figure 4 (panel A) shows that hypo and hyper rats showed increased left ventricle NOS activity compared with Eut animals. Haemorrhage increased NOS activity in all groups of animals. eNOS, iNOS and nNOS protein levels did not change with thyroid status. Bleeding increased eNOS protein levels in Eut and hypo animals (panel B) and iNOS proteins levels in Eut rats (panel C). nNOS did not change with haemorrhage (panel D).
Total NOS activity in left ventricle from euthyroid (Eut), hypothyroid (hypo) and hyperthyroid (hyper) rats (panel A). The values are mean±s.e.m .; n=15/group; *P<0.05 vs Eut rats; †P<0.05 vs rats without haemorrhage. Representative Western blots of eNOS (panel B), iNOS (panel C), nNOS (panel D) carried out on proteins from Eut, hypo and hyper left ventricle. Histograms illustrate the mean NOS protein values for each group. All experiments were performed in triplicate. Each blot was normalized with the expression of the β-actin from the same gels. Data are mean±s.e.m.; n=7/group; *P<0.05 vs Eut rats; †P<0.05 vs rats without haemorrhage. EutH, euthyroid haemorrhaged rats; hyperH, hyperthyroid haemorrhaged rats; hypoH, hypothyroid haemorrhaged rats.
Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0203
Figure 5 (panel A) showed that when NOS activity was evaluated on atria slices, we obtained similar results to those obtained using homogenates. Hypo and hyper animals exhibited a decreased atrial NOS activity compared with Eut rats and bleeding increased NOS activity in all groups of animals. Calmidazolium treatment attenuated NOS activity in Eut and hypo animals without modification in hyper group of animals. In ventricle, hypo and hyper rats showed increased left ventricle NOS activity compared with Eut animals, and haemorrhage increased NOS activity in all groups of animals. Pre-treatment with calmidazolium attenuated and blunted the increase in NOS activity induced by haemorrhage in Eut and hypo animals, respectively. The NOS activity increase after bleeding was not altered by calmidazolium in hyper animals.
Total NOS activity in right atria (panel A) and left ventricle (panel B) slices from euthyroid (Eut), hypothyroid (hypo) and hyperthyroid (Hyper) rats. CZ, calmidazolium; EutH, euthyroid haemorrhaged rats; EutHCZ, euthyroid haemorrhaged rats pre-treated with CZ; hyperH, hyperthyroid haemorrhaged rats; hyperHCZ, hyperthyroid haemorrhaged rats pre-treated with CZ; hypoH, hypothyroid haemorrhaged rats; hypoHCZ, hypothyroid haemorrhaged rats pre-treated with CZ. Data are mean±s.e.m.; n=7/group; *P<0.05 vs Eut rats; †P<0.05 vs rats without haemorrhage.
Citation: Journal of Endocrinology 230, 2; 10.1530/JOE-16-0203
Discussion
This study provides new evidences that changes in cardiac function associated with thyroid disorders and hypovolemia not only involve effects on sympathetic nervous system, but may also involve changes in the response of the myocytes to NO bioavailibity. This study investigated the role of NO in the cardiovascular adaptation following acute haemorrhage in rat with thyroid disorders. TSH measurements showed that T3 and methimazol treatment were effective to establish hyper- and hypothyroid state, respectively. In our experimental condition, basal MAP values were similar in the three experimental groups. These findings are surprising. It would be expected that MAP decreases and increases in hypo- and hyperthyroid state, respectively. However, our results showed that MAP did not change compared with euthyroid control rats. The maintainence of arterial pressure values in thyroid disorders may be due to changes induced on diastolic pressure similar in magnitude to the changes induced on systolic pressure despite having very different HR values. It is important to consider that this discrepancy with others researchers might be due to the different duration and degree of hypo- and hyperthyroidism developed in our experimental conditions. Additionally, we have shown that L-NAME infusion did not alter basal blood pressure values in experimental groups. It is probable that in this condition, the inhibition of the constitutive NOS activity is only partial, being the quantity of NO that exceeds sufficient to maintain MAP within the basal range.
When we evaluated cardiac function associated with thyroid status, echocardiographic data confirmed that hypothyroid animals have FS and EF decreased and increased left ventricle internal diameter. This would indicate that myocardial contractility would be altered and the ventricle would not be filling properly especially during diastole in these animals. It is important to note that FS depends primarily on afterload. The most common causes of decreased FE in hypothyroidism would be blockages in the coronary arteries, increased blood pressure, heart rhythm disturbances or weakening of the heart muscle. In this context, hypothyroidism could be associated with increased prevalence of cardiac heart failure associated with thyroid hormones deficiencies (Biondi 2012). On the other hand, although hyperthyroid animals showed a decrease in the diameter of the ventricle, no change was observed in cardiac contractility in our experimental model. Taken together, these results suggest that hypo and hyper rats would exhibit different thyroid disorder-induced remodelling adaptation. Hypothyroidism would exhibit a greater eccentric hypertrophic response, while hyperthyroid animals would exhibit concentric hypertrophic response. These findings agreed with other authors who described the development of the eccentric hypertrophy in hypothyroidism (Wang et al. 2010, Sarati et al. 2012) and concentric left ventricle hypertrophy associated with thyrotoxicosis (Abergel et al. 1995). The mechanisms of the animal model of thyroid disturbances-induced cardiac hypertrophy are multifactorial. It is not clear whether thyroid hormone status-induced cardiac hypertrophy results from a direct effect on the heart, alterations of the adrenergic nervous system signalling or altered cardiac loading conditions. Taking into account the latter, we evaluate cardiovascular haemodynamic changes to haemorrhage in animals with thyroid disorders. It is well known that cardiovascular adaptation to this hypovolemic state is under dynamic control of the sympathetic and parasympathetic divisions, the magnitude of haemorrhage, the rate of bleeding, and the species examined (Schadt & Ludbrook 1991). Different circulating endocrine and local paracrine factors such as NO have been postulated to modulate the cardiovascular response to hypovolemia (Goldstein et al. 1999). In this study, we showed that haemorrhage elicited a significant decrease between 50 and 59% of arterial blood pressure within 10min after bleeding from basal values in all experimental groups. The magnitude of this immediate hypotension would seem similar in all rats. After this time, this parameter increased stabilizing its values at about 30–40min and was maintained until finalized, 120min. Pre-treatment with L-NAME before bleeding induced a similar immediate decrease (at about 60%) in all experimental groups. This immediate hypotension was followed by a faster recovery of blood pressure to basal values in euthyroid and hyperthyroid haemorrhaged rats. This parameter did not reach basal values in hypo rats. Taken together, these findings suggest that the immediate hypotension would be independent of NO system; however, after this time (10min), NO would modulate systemic vascular response probably due a direct vasodilatory action on vascular smooth muscle especially in euthyroid and hyperthyroid animals. This effect of NO seems to be lower in hypothyroidism in which basal values of MAP were not reached. However, we cannot throw away the effects of NO on the integrated mechanisms which become activated in response to haemorrhage as well as the release of several neurohormonal vasoconstrictor factors (catecholamines, endothelins, vasopressin, renin–angiotensin system) (Fujisawa et al. 1999, Moreno et al. 2002).
Focusing on chronotropic response, our results showed an increase and decrease of basal pacemaker activity in hyper- and hypothyroid rats compared with the respective euthyroid animal. These changes would confirm the tachycardic action of T3 (Sun et al. 2001). NOS inhibition was not modified at basal values of HR in the three groups of animals, but blunted the changes induced by blood loss. Hypovolemic state provoked after the expected immediate reflex-induced tachycardia, a bradycardic stage followed by a gradual increase in HR during 120min (Balaszczuk et al. 2006). It is known that blood pressure is maintained in the early stage of haemorrhage by reflex increase in HR, vascular resistance and peripheral sympathetic nerve activity. The inhibition of NO system would affect the immediate baroreflex response in our experimental model. The bradycardia, observed in the early stages, may result from alterations in the activation of unmyelinated vagal afferents (C fibers) from the left ventricle induced by the loss of 20% in the blood volume. A decrease in HR may seem unreasonable during haemorrhage, but it could be a part of a complex reflex in order to reduce an ongoing blood loss by reducing blood pressure by means of peripheral vasodilatation and, at the same time, maintain organ blood flow. The absence of the bradycardia suggests that NO could be involved in the cholinergic modulation of HR in the early stage of hypovolemic state. However, the relationship between the NO system and the absence of the later increase in HR was not well understood. It was reported that systemic inhibition of NOS in vivo in humans, by L-NMMA, significantly reduced renal plasma flow in the absence of alterations in glomerular filtration rate, blood pressure or pulse rate (Wolzt et al. 1997). By contrast, Schmetterer et al. (1999) showed a significant decrease in heart rate after infusion of L-NMMA. These findings suggest that nitric oxide, present in the sinoatrial and atrioventricular nodes, seems to play an important role in pacemaker activity control. The action of L-NAME on the later tachycardia may be a primary action due to inhibition of NO pathway or a secondary effect resulting from the absence of the maintained hypotension after the haemorrhage.
Considering NO system in the heart, our results showed that animals with thyroid alterations have a lower atrial NOS activity than Eut rats. This lower activity of the enzyme is associated with a decrease in the protein levels of the three NOS isoforms in hypo rats. Meanwhile, hyperthyroid animals showed no changes in protein levels of the three isoforms of the enzyme. These findings allow us to think that negative modulators of the enzyme, like caveolins, are probably increased in hyperthyroidism.
In addition, a contradictory result was found in the left ventricle. Animals with thyroid disorders had a higher enzyme activity than euthyroid and this rise was not correlated with changes in NOS protein levels. Positive modulators of NOS activity would be exacerbated in thyroid disorder.
Focusing on the physiological involvement of NO during hypovolemic state, we observed that acute haemorrhage results in an excessive production of NO in right atria as well as in left ventricle at 120min after blood loss in all experimental groups. In the atrium, increased NOS activity induced by bleeding could be due to increased endothelial and inducible isoform of the enzyme in euthyroid and hypothyroid animals and only iNOS protein levels in hyperthyroidism. Experiments with calmidazolium confirmed these findings. Conversely, withdrawal induced a decrease in protein levels of eNOS in hyperthyroidism.
On the other hand, the left ventricle increased NOS activity induced by haemorrhage could be due to a rise in eNOS protein levels in euthyroid and hypothyroid animals. Additionally, hypovolemia increased iNOS protein levels in euthyroid rats. Our results also suggested that the NOS activity changes induced by hypovolemic state could involve alteration of positive modulators of the enzyme in hyperthyroidism. Experiments with calmidazolium confirmed these findings. Calmodulin antagonist attenuated NOS activity increase in Eut animals as well as blunted the rise in hypo rats and did not modify it in hyper group.
In summary, the key findings of this study are that thyroid hormones deficiencies, as well as excesses result in alterations of cardiac function regulation and cardiovascular haemodynamia. Although hypothyroidism and hyperthyroidism are associated with cardiac remodelling, they affect cardiac function and haemodynamic parameters in a different way. Our results demonstrated that both thyroid disorders were associated with hypertrophic remodelling which was impacted differently on cardiac function and consequently its adaptation to a hypovolemic state. Additionally, although the effect of the thyroid disorders on NO production depends on the studied cardiac chamber, the impact of bleeding is similar in both chambers. Hypovolemia induced by acute haemorrhage triggered a NOS activation modulating the heart function to maintain haemodynamic homeostasis. The involvement of NO pathway depended on a specific NOS isoform, cardiac chamber and thyroid state.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the University of Buenos Aires Grants 20020110100020.
Acknowledgements
The authors wish to thank Dr Carlos Libertum from IBYME, Institute-CONICET for the TSH plasma determinations, Gabriela Pidal from Facultad de Ciencias Veterinarias, Universidad de Buenos Aires for echocardiographic measurements and Ana Borthwick for proofreading and language assistance.
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