Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders

Molecular and Cellular Endocrinology xxx (2015) 1e8

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Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders Caroline A. Lino, Ivson Bezerra da Silva, Caroline E.R. Shibata, Priscilla de S. Monteiro, Maria Luiza M. Barreto-Chaves* Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 July 2015 Received in revised form 10 August 2015 Accepted 10 August 2015 Available online xxx

Suboptimal intrauterine conditions as changed hormone levels during critical periods of the development are considered an insult and implicate in physiological adaptations which may result in pathological outcomes in later life. This study evaluated the effect of maternal hyperthyroidism (hyper) on cardiac function in adult offspring and the possible involvement of cardiac Renin-Angiotensin System (RAS) in this process. Wistar dams received orally thyroxin (12 mg/L) from gestational day 9 (GD9) until GD18. Adult offspring at postnatal day 90 (PND90) from hyper dams presented increased SBP evaluated by plethysmography and worse recovery after ischemia-reperfusion (I/R), as evidenced by decreased LVDP, þdP/dT and edP/dT at 25 min of reperfusion and by increased infarct size. Increased cardiac Angiotensin I/II levels and AT1R in hyper offspring were verified. Herein, we provide evidences that maternal hyperthyroidism leads to altered expression of RAS components in adult offspring, which may be correlated with worse recovery of the cardiac performance after ischemic insults and hypertension. © 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Gestational hyperthyroidism Cardiac renin-angiotensin system Fetal programming

1. Introduction The fetal programming hypothesis suggests that individuals submitted to suboptimal conditions in utero present an increased risk for pathological outcomes in later life. This concept raised over the last 20 years and is strongly supported by epidemiological and experimental evidences in which the offspring exposed to intrauterine disturbs is generally accompanied by low birth weight and increased susceptibility to cardiovascular, renal and metabolic disorders (Barker and Bagby, 2005; Barker, 2007). The Barker hypothesis proposes that fetuses respond to these conditions through alterations on its own growth and metabolism, ensuring immediate survival (Gluckman and Hanson, 2006). The maternal hormonal status has a marked influence on perinatal development and provides a long-term impact on energy balance that might predispose the offspring to cardiovascular diseases (Sullivan et al., 2011). Thyroid hormones (TH) play important roles in fetal normal development and, in many instances, in postnatal organ systems

* Corresponding author. Laboratory of Cellular Biology and Functional Anatomy, Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes 2415, Sao Paulo, SP, 05508-900, Brazil. E-mail address: [email protected] (M.L.M. Barreto-Chaves).

maturation, primarily related to metabolism (Bruno et al., 2005; Fowden et al., 2005). Nonetheless, the knowledge regarding thyroid gland or TH bioavailability is restricted (Fowden et al., 2005). Thyroid gland does not become functional until the middevelopmental period and remains relatively inactive until birth (Fisher et al.,1977). However, as a diffusible lipophilic compound, TH reach the fetuses through maternal uterus. A fine regulation on the thyrotropin/free thyroxine ratio is fundamental during perinatal development since fetal exposure to higher levels of maternal TH may impair its physiological maturation (Fisher et al., 2000). The most frequent thyroid diseases during pregnancy are hyperand hypothyroidism, including their variants. Unlike congenital hypothyroidism, which is usually permanent, gestational hyperthyroidism is commonly transient and ranges prevalence between 0.05% and 0.2% of pregnant women (Fern
andez-Soto et al., 1998). Recently, a study from our group demonstrated that maternal hyperthyroidism is associated with alterations in fetal development (Lino et al., 2014). Specifically, it is accompanied by altered pattern of expression of Renin-Angiotensin System (RAS) components evidenced on gestational day 18 (GD18) and GD20. These results may represent an important predisposal factor for cardiovascular diseases in adulthood. However, little is known about the association between those endocrine systems during critical periods of heart development.

http://dx.doi.org/10.1016/j.mce.2015.08.015 0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Lino, C.A., et al., Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders, Molecular and Cellular Endocrinology (2015), http://dx.doi.org/10.1016/j.mce.2015.08.015

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In adults, the crosstalk between TH and RAS on cardiac function has been previously described by our group and others (Kobori et al., 1999; Hu et al., 2003; Ibrahim et al., 2006; Carneiro-Ramos et al., 2007; Diniz et al., 2007; Barreto-Chaves et al., 2010; Carneiro-Ramos et al., 2010). Besides the systemic RAS, a cardiac RAS consisting of locally synthesized Renin, Angiotensinogen, Angiotensin-Converting Enzyme (ACE) and Type 1 and 2 Angiotensin II receptors (AT1 and AT2, respectively) acts under different conditions and independently from systemic RAS to control the cardiovascular function (Bader, 2002). Angiotensin II (Ang II) generated locally has other non-hemodynamic effects involved with cardiac hypertrophy in adult myocardium, as cardiomyocyte growth and fibrosis (Fischer and Hilfiker-Kleiner, 2007; Morgan and Baker, 1991), and then the local generation of this peptide may represent highest clinical relevance. In fact, there is accumulating evidence that this peptide has important paracrine/autocrine functions (Danser, 1996; Dzau, 1988). These actions are primarily mediated by binding to AT1R, which has been shown to be responsible for the main physiological and pathophysiological effects of Ang II (Higuchi et al., 2007). In the present study, we tested the hypothesis that maternal hyperthyroidism induces alterations on cardiovascular function of adult offspring and those alterations are associated with cardiac RAS activation.

administration in the drinking water (12 mg/L) from gestational day 9 (GD9), whereas the control group received vehicle solution, as previously described (Araujo et al., 2011). GD9 was chosen as the onset of the treatment due the importance of maternal TH at the first half of pregnancy, before the endogenous TH production by fetuses (Karakawa et al., 1989). Considering that higher levels of TH may cause abnormalities in maternal behavior and impairment in lactation (Rosato et al., 1992), maternal treatment was maintained until GD 18, ensuring the offspring survival. Neonatal offspring group was obtained at the first day of postnatal life (PND1) and the adult offspring group was obtained at PND90. The litters were maintained with mothers until weaning (PND21), and then the male offspring from control and hyper dams were separated (2 pups/litter were used for adult offspring group). 2.3. Serum thyroid hormone levels Trunk blood samples were centrifuged (3000 rpm for 15 min at 4  C) and the supernatants were stored at 80  C freezer. Serum TH levels were determined using a radioimmunoassay kit for total T3 and total T4 (Coat-A-Count kit, Siemens Healthcare Diagnostics, Los Angeles, CA, USA) according to the manufacturer specifications. 2.4. Hemodynamics

2. Materials and methods 2.1. Animals All surgical procedures and protocols were performed in accordance with the Ethical Principles in Animal Research set forth by the Brazilian College of Animal Experimentation and approved by the Animal Research Ethics Committee of the University of Sao Paulo, Institute of Biomedical Sciences, Brazil. Sexually mature male and female Wistar rats (90 days old) were obtained from the Institute animal facilities, housed under controlled temperature and light (24  C, 12/12 h light/dark cycle) and given free access to standard rat chow and water. 2.2. Experimental procedures Female rats were caged overnight with males (2 females/male) and the vaginal smear was analyzed at the microscope to confirm the copulation. The day of sperm detection was considered the gestational day 0 (GD0) (Grazeliene et al., 2006). All dams were housed individually and randomized into control and hyperthyroid (hyper) groups (7 dams/group), according to Fig. 1. Maternal hyperthyroidism was induced by thyroxine (T4)

Systolic blood pressure (SBP) and heart rate (HR) were determined in conscious rats using a non-invasive tail-cuff plethysmography method (Kent Scientific, Lietchfield, CT, USA). Rats were acclimatized to the apparatus by daily sessions for two weeks before the final measurements. Adult offspring was preheated for 5 min and three stable consecutive measurements were averaged as described previously (Carneiro-Ramos et al., 2010). 2.5. Ischemia-reperfusion Isolated heart experiments were also performed. After euthanasia by decapitation, hearts from the adult offspring were quickly excised and retrograde perfused via aorta by a Langendorff apparatus using a modified Krebs-Henseleit gassed (95% O2 and 5% CO2) buffer, as previously described (Tavares et al., 2013). Hearts were perfused under constant flow rate (10 ml/min) during 30 min (stabilization period) for baseline recording and then submitted to a global ischemia (zero flow) during 20 min. The flow was restarted and the hearts were reperfused for 25 min (reperfusion period). The ventricular function was assessed using a pressure transducer connected to a saline-filled latex balloon inserted into the left ventricle (LV). The balloon volume was adjusted to permit an end

Fig. 1. Scheme of experimental protocol used in the study.

Please cite this article in press as: Lino, C.A., et al., Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders, Molecular and Cellular Endocrinology (2015), http://dx.doi.org/10.1016/j.mce.2015.08.015

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Table 1 Neonatal (PND1) and adult offspring (PND90) variables.

Neonatal offspring

Adult offspring

Variable

Control

Body Weight (g) Heart Weight (mg) Heart Weight/Body Weight (mg/g) Kidney Weight (mg) Kidney Weight/Body Weight (mg/g) Total T4 (mg/dL) Total T3 (ng/dL) Water intake (mL/day) Food intake (g/day) Heart Weight (g) Heart Weight/Tibia Length (g/mm)

5.94 32.26 5.44 26.70 4.50 5.20 53.82 45.97 21.94 0.944 0.025

± ± ± ± ± ± ± ± ± ± ±

Hyper 0.058 0.52 0.081 0.44 0.068 0.16 4.10 3.98 0.54 0.02 0.008

5.38 30.82 5.73 26.78 4.88 5.48 43.37 40.55 24.53 1.075 0.028

± ± ± ± ± ± ± ± ± ± ±

0.045* 0.47 0.080* 0.67 0.096* 0.04 0.79 3.37 0.33* 0.05 0.015

Data are expressed as mean ± SEM. Neonatal offspring (n ¼ 72 control and n ¼ 47 hyper); Adult offspring (n ¼ 5 control and n ¼ 4 hyper). *P < 0.05 vs. control.

diastolic pressure (EDP) of 8e10 mmHg in both groups. The functional parameters, LVDP, HR, þdP/dT, dP/dT and left ventricular end-diastolic pressure (LVEDP) were continuously monitored by a recording system (PowerLab Chart 7-Lab, AD Instruments, Australia). 2.6. Myocardial infarct size The extent of irreversible myocardial ischemic damage was measured as described previously with modifications (Budas et al.,

Blood Pressure (mmHg)

A 200 180

*

160 140 120 100

Control

Hyper

B Heart Rate (bpm)

500 400 300

2010). At the end of the reperfusion period (25 min), transversal slices of hearts were cut, incubated with 1% 2,3,5triphenyltetrazolium chloride solution (TTC; SigmaeAldrich, St. Louis, MO) for 15 min in water bath at 37  C in the dark and then immersed into a 10% formalin overnight at 4  C. The slices were photographed for quantification of unstained area; pale area corresponds to the infarcted area, which was expressed as percentage of the whole section. 2.7. Immunoblotting Western blot analysis was performed as previously described (Lino et al., 2014). Heart samples were harvested using an extraction buffer on a tissue homogenizer. Homogenates were centrifuged and the supernatants stored at 80  C freezer. Protein concentration of samples was determined according to Bradford method (Bradford, 1976) and 30ug of total protein was electrophoresed in a 10% sodium dodecyl sulfate polyacrylamide gel for 1 h and then blotted onto a nitrocellulose membrane by semidry electroblotting (Bio-Rad Laboratories, Hercules, CA). The blots were incubated overnight at 4  C with the primary antibody raised against Angiotensin precursor expression (recognizes angiotensin I and angiotensin II), Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) (Santa Cruz Biotechnology, CA, USA) and Angiotensin II type 1 and 2 Receptors (AT1R and AT2R, respectively) (Alomone Labs, Jerusalem, Israel) using 1:1000 dilution in a 0.1% Tween 20 Tris-buffered saline (TTBS). The nitrocellulose membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature in 1:10,000 dilution in TTBS and washed again. Signal detection was visualized using chemiluminescence detection reagents (ECL, Amersham Biosciences) according to the manufacturer's instructions and exposure to X-ray film. The specific protein bands were quantified by densitometry using Image J software. GAPDH was used as internal control and the quantitative analysis was calculated as percentage in relation to control.

200

2.8. Statistical analysis

100

Data are presented as mean ± standard error of the mean (SEM). Control and hyper groups were compared using unpaired Student's t-test. Values of p < 0.05 were considered statistically significant.

0

Control

Hyper

Fig. 2. Analysis of systolic blood pressure (A) and heart rate (B) in conscious adult offspring through tail-cuff plethysmography. Data are expressed as mean ± SEM and represent the mean of at least 7 animals/group; control group (open bars) and hyper group (closed bars). *P < 0.05 vs. control.

3. Results 3.1. Maternal hyperthyroidism In order to confirm the maternal hyperthyroid status, serum levels of TH were determined by radioimmunoassay at PND1. As

Please cite this article in press as: Lino, C.A., et al., Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders, Molecular and Cellular Endocrinology (2015), http://dx.doi.org/10.1016/j.mce.2015.08.015

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C.A. Lino et al. / Molecular and Cellular Endocrinology xxx (2015) 1e8 Table 2 Cardiovascular parameters of adult offspring at the stabilization time.

Stabilization time

Variable

Control

LVDP (mmHg) LVEDP (mmHg) þdP/dT (mmHg/s) -dP/dT (mmHg/s) HR (bpm)

67.47 5.19 2937.16 1968.57 351

Hyper ± ± ± ± ±

8.52 1.46 435.33 401.69 44

69.10 10.51 3476.12 2461.82 326

± ± ± ± ±

7.95 5.47 450.52 500.02 57

LVDP e left ventricular developed pressure; LVEDP e left ventricular end-diastolic pressure; þdP/dT and edP/dT e maximal positive and negative first derivative of left ventricular pressure, respectively; HR e heart rate. Data represent the mean of the last 5 min of the stabilization time. Data are expressed as mean ± SEM; n ¼ 6 in both groups.

A

B

C

LVDP

+dP/dt

125

* ...........................

75 50 25 0

Control

125

75 50 25 0

Hyper

D

.......................... *

100

Control

HR

Control

Hyper

% to stabilization time

% to stabilization time

0

50 25 0

Control

Hyper

PP 200

400

50

.......................... *

75

LVEDP

150

.............................

100

F

E

100

125

Hyper

300 200 100 0

...........................

Control

Hyper

% to stabilization time

100

150

% to stabilization time

150

% to stabilization time

% to stabilization time

150

-dP/dt

150 100

............................

50 0

Control Hyper

Fig. 3. Cardiac functional response to I/R of adult offspring. Data are expressed as mean ± SEM and represent the mean of the 25 min of reperfusion in relation to stabilization time (100%). (A) LVDP e left ventricular developed pressure; (B) þdP/dt e positive first derivative of left ventricular pressure; (C) edP/dt e negative first derivative of left ventricular pressure; (D) HR e heart rate; (E) LVEDP e left ventricular end-diastolic pressure; (F) PP e perfusion pressure. Control group (open bars) and hyper group (closed bars); n ¼ 6; *p < 0.05 vs. control.

expected, in response to T4 administration in the drinking water, total T3 and total T4 fractions were significantly augmented in hyper group of dams (Total T3: 178.06 ± 9.1 vs. 72.25 ± 4.3 ng/dL in control; Total T4: 16.71 ± 4.06 vs. 2.43 ± 0.59 ug/dL in control, p < 0.05). In addition, the water (60.50 ± 0.97 vs. 45.59 ± 6.68 mL/ day in control, p < 0.05) and food (27.92 ± 2.47 vs. 22.89 ± 2.21 g/ day in control, p < 0.05) intakes were significantly increased in hyper dams when compared to control. Besides that, maternal body weight was not different at PND1 (254.02 ± 16.41 vs. 258.71 ± 14.45 g in control, p < 0.05).

Hyperthyroidism also caused a significant increase in the heart weight/tibia length ratio (0.024 ± 0.001 vs. 0.019 ± 0.001 g/mm in control, p < 0.05), indicating the development of cardiac hypertrophy. No difference was observed regarding the litter size (9 ± 1 vs. 10 ± 2 in control). 3.2. Maternal hyperthyroidism impairs fetal growth Fetal exposure to maternal excess of TH during gestation presented an intrauterine growth restriction as evidenced by lower

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were expressed as a percentage of baseline values (during stabilization) in each group (Fig. 3). The offspring hearts from hyper dams presented a significant decrease in LVDP (29.86%), þdP/dT (24.57%) and dP/dT (29.85%) at 25 min of reperfusion (p < 0.05) (Fig. 3). No change was observed in parameters as HR, LVEDP and PP. 3.4. Determination of infarct size At the end of I/R, perpendicular slices from the long axis of hearts were cut and stained with triphenyltetrazolium chloride (TTC) to differentiate necrotic (white) and viable (red) tissue. The offspring hearts from hyper dams presented an infarct area higher (68.77%) than from control dams (25.28 ± 2.28 vs. 42.67 ± 5.92 in control, p < 0.05) (Fig. 4). 3.5. Maternal hyperthyroidism upregulates cardiac angiotensin peptides and AT1R in adult offspring The expression of AngI/AngII on cardiac tissue of adult offspring from hyper group were significantly higher (approximately 50%, p < 0.05) compared to control (Fig. 5A). In addition, the expression of AT1R was also increased in hyper group (25% vs. control, p < 0.05; Fig. 5B), while no difference was observed in AT2R protein expression between groups (Fig. 5C). 4. Discussion

Fig. 4. Analysis of the extension of infarcted area measured by TTC staining in adult offspring submitted to ischemia-reperfusion. (A) Quantification of the uncolored area in relation to total area (%); (B) representative image of a transversal slice of heart stained with TTC from control and hyper adult offspring. Data are expressed as mean ± SEM, n ¼ 6; control group (open bars) and hyper group (closed bars). *P < 0.05 vs. control.

birth weight of neonatal offspring (p < 0.05) assessed at PND1 (Table 1). No significant difference was observed for neonatal heart weight or kidney weight (Table 1).

3.3. Effect of maternal hyperthyroidism in adult offspring The effect of maternal hyperthyroidism in different variables of adult offspring was also evaluated (Table 1). No difference was detected on TH levels between groups. Maternal hyperthyroidism induced a small (approximately 11%) but significant increase in food intake of hyper adult offspring (p < 0.05) and no difference was observed in the water intake. No difference was also observed in parameters as heart weight or heart weight/tibia length ratio (Table 1). Cardiac hemodynamic parameters were evaluated by using indirect method (tail-cuff plethysmography). The data showed a significant increase in SBP (11%) in offspring of TH-treated dams (155.83 ± 3.89 vs. 143.2 ± 0.39 mmHg in control, p < 0.05), indicating that maternal hyperthyroidism during pregnancy may lead to hypertension in the adult life (Fig. 2A) whereas no difference related to HR was detected (388 ± 20 vs. 373 ± 36 bpm in control, p ¼ 0.72) (Fig. 2B). In order to evaluate the cardiac function in offspring of THtreated dams when submitted to an ischemic insult, the functional response to I/R was also evaluated. Baseline cardiac function was assessed by measurement of four different parameters: LVDP (mmHg), LVEDP (mmHg), þdP/dT, dP/dT (mmHg/s) and HR (bpm) which were evaluated as the mean of the last 5 min of the stabilization period prior the I/R event (Table 2). No difference was observed during the stabilization time. Functional recovery values

Programming by adverse conditions during fetal development is considered the main determinant of offspring life-course health (Santos et al., 2015). There is growing evidence that the offspring of women submitted to adverse outcomes during pregnancy presents an increased risk of diseases in the adulthood, including cardiovascular diseases as hypertension. It is well known that pregnancy induces many physiological changes in maternal thyroid function which are essential since the prenatal exposure to TH is necessary for normal postnatal development of heart, lung and brain (Bale, 2015; Morreale et al., 2000). On the other hand, gestational hyperthyroidism reaches prevalence from 0.05% to 0.2% of pregnant women (Fern
andez-Soto et al., 1998) and may be associated with different repercussions. In a recent study, using the same experimental approach, we demonstrated that maternal hyperthyroidism is associated with increased cardiac weight in pups at GD18 and GD20 and also altered pattern of expression in RAS components (Lino et al., 2014). In the present study, we tested the hypothesis that maternal hyperthyroidism leads to subsequent changes on cardiovascular function of adult offspring through RAS programming mechanisms. The existence of a crosstalk between thyroid hormones and renin angiotensin system in the control of cardiac function in adults has been described in previous studies (Kobori et al., 1997; Hu et al., 2003; Carneiro-Ramos et al., 2007). However, to our knowledge, this is the first study that correlates maternal hyperthyroidism with higher susceptibility to cardiac disorders in the adult offspring, which were accompanied by changed pattern of RAS expression. Our findings show that fetal exposure to maternal TH excess leads to intrauterine fetal growth restriction (IUGR) as evidenced by low birth weight (LBW) of neonatal offspring. In fact, other authors has previously described that maternal hyperthyroidism is associated with increased risk of IUGR (Bahn et al., 2011) and decreased head circumference in children born to hyperthyroid mothers (Ohrling et al., 2014). Although there is no consensus for the lower birth weight this might occur due to the presence of thyroid autoantibodies in the fetus, which are prevalent in up to 10% of the population (Hollowell et al., 2002). Nevertheless, IUGR may permanently influence the endocrine system by affecting its

Please cite this article in press as: Lino, C.A., et al., Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders, Molecular and Cellular Endocrinology (2015), http://dx.doi.org/10.1016/j.mce.2015.08.015

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C.A. Lino et al. / Molecular and Cellular Endocrinology xxx (2015) 1e8

Fig. 5. Analysis of RAS components in adult offspring. Immunocontent evaluated by Western Blot of Angiotensin I/Angiotensin II (A), Type 1 Angiotensin II receptor e AT1R (B) and Type 2 Angiotensin II receptor e AT2R (C). GAPDH was used as an internal control. Data are expressed as mean ± SEM and represent the mean of at least 4 animals/group; control group (open bars) and hyper group (closed bars); *p < 0.05 vs. control.

programming during development (Radetti et al., 2004). In addition, some studies have suggested an association between (LBW) and increased SBP in later life (Gamborg et al., 2007; Lawlor et al., 2007). The potential mechanisms underlying this association in adulthood remain unclear. Interestingly, elastin synthesis within the wall of large elastic arteries is thought to be impaired, leading to the concept that altered artery structure in IUGR infants might be a possible mechanism for the elevated SBP in later life (Martyn and Greenwald, 1997). However, although low birth weight is associated with altered hemodynamic in adulthood, the associations are predominantly driven by factors such as adult body size (Miles et al., 2011) and the SBP that rises throughout life (Franklin et al., 1997). Therefore, it is possible that individuals with (LBW) may be predisposed to greater age-related changes in SBP,

which may be associated with cardiovascular risk in later life. Despite the consistent epidemiological results, there are few studies regarding the underlying pathophysiological mechanisms for an increased cardiovascular susceptibility among (LBW). Thus, considering the clinical relevance and consistence with Barker hypothesis (Barker, 1995) we used different approaches to evaluate the effect of maternal hyperthyroidism in hemodynamic parameters of adult offspring. Non-invasive blood pressure measurements using tail-cuff plethysmography assays were done to evaluate SBP and HR in conscious rats. A subtle and statistically significant augment of SBP was observed on hyper group. Ibrahim et al. (2006) demonstrated that tail-cuff and direct arterial pressures can be measured equivalently in both normotensive and hypertensive rats. In that study the authors point out that blood pressure (BP) is

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highly variable with some evidence that accuracy of the tail-cuff method is proportional to the level of BP and dependent on genetic background that also may modulate stress responses and/or biological variability (Ibrahim et al., 2006). In order to clarify the possible cardiovascular damage caused by maternal hyperthyroidism we evaluated the post-ischemic recovery of cardiac function in isolated hearts from adult offspring and analyzed subsequently the infarcted area by using TTC staining. The Langendorff heart perfusion system is a classic model to estimate cardiac functional alterations in the absence of a neurohumoral influence (Langendorff, 1895) and commonly used to study the effect of different experimental procedures on I/R injury. Our findings on cardiac function confirm and expand those observed by tail-cuff plethysmography, demonstrating that hyper group presents impairment on cardiac performance when submitted to an insult, as evidenced by a decreased left ventricular developed pressure (LVDP), þdP/dT and edP/dT observed at 25 min of reperfusion. It is interesting to note that while in the present study we found that maternal hyperthyroidism increased the vulnerability to adult offspring ischemic injury, in a previous study conducted in adult hyperthyroid rats isolated hearts subjected to I/R presented an improved recovery of cardiac function in relation to euthyroid hearts (Tavares et al., 2013). In addition, some previous studies based on experimental evidences have demonstrated that TH induce cardioprotection, which has been shown to be very similar to that observed during preconditioning (Pantos et al., 2002, 2005). Therefore, our results are consistent with the idea about developmental programming since a specific challenge as elevated TH levels during a critical developmental time window may result in effects completely different of those observed in adult life. Besides the damage on cardiac performance after insult observed in adult offspring from hyperthyroid dams, our results indicate a greater and significant infarct area in these animals. Interestingly, the thyroid status of hyper offspring was not influenced by maternal TH excessive levels since in the adult offspring the TH levels were similar to control. In this sense, according to a recent review, TH action is regulated by a multiplicity of mechanisms and their circulating levels not always are predictive for the cellular availability and activity (Colicchia et al., 2014). Finally, an important finding in this present study in relation to its potential as a possible mechanism to try understand the cardiovascular consequences for maternal hyperthyroidism is the observation that cardiac RAS components were changed in the adult offspring. Modulation of RAS components has been pointed as one of the important pathways related to fetal programming (Ojeda et al., 2008). Particularly in our model, because TH modulates the activity of cardiac RAS components in adult life (Hu et al., 2003; Carneiro-Ramos et al., 2007) and exerts critical importance in coordinating morphogenic processes, it is possible that RAS may contribute to outcomes observed in adulthood offspring. In addition to structural changes that occur in the fetus, enduring gene expression patterns that are established in the uterus are believed to result from epigenetic processes such as DNA methylation, histone modification, and/or ribonucleic acid interactions within the promoter region of regulatory genes (Hochiberg et al., 2011; Thornburg, 2011). Such epigenetic mechanisms may underlie many of the deleterious actions in different organs during fetal period. In this study we showed, for the first time, that maternal hyperthyroidism increases AngI-Ang II levels and upregulates AT1R in the hearts from adult offspring, while no change was observed in the cardiac AT2R expression. Although the best experimental approach to assess the cardiac Ang II levels is still discussed in the literature, their levels in addition to elevated cardiac AT1R correspond to especially important factors of cardiac susceptibility to

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cardiovascular diseases. These changes might be involved with the impairment on cardiac performance as observed when hearts were submitted to an ischemia insult. Other studies have linked environmental insults, leading to epigenetic alterations of AT1R and AT2R expression in adrenal gland and kidney during fetal life, with consequent alteration in their pattern of expression in adult life, which can ultimately lead to hypertension (Bogdarina et al., 2007, 2010). Although some studies have found a critical role for the RAS in developmental programming of the kidney and other organs (Woods et al., 2001; Grigore et al., 2007), to our knowledge this is the first time that maternal hyperthyroidism has been suggested as a potential stimulus that lead to altered angiotensin II levels and AT1R expression in adulthood. In conclusion, by using an original model of fetal programming (Lino et al., 2014) we provide evidences that maternal hyperthyroidism leads to altered pattern of expression of RAS components in adult offspring and that may be correlated to the worse recovery of the cardiac performance observed after ischemic insult. We expect that future studies using similar approach will provide new and important findings that will help understanding the link between fetal programming induced by maternal hyperthyroidism and the pathogenesis of some forms of hypertension. Funding This work was supported by grants from the Fundaç~ ao de  Pesquisa do Estado de Sa ~o Paulo (FAPESP; grant 2008Amparo a 01489-5). Disclosures No conflicts of interest, financial or otherwise are declared by the author(s). Author contributions Author contributions: C.A.L., I.B.S., C.E.R.S. and P.S.M. performed experiments; C.A.L. and I.B.S. prepared figures; M.L.M.BeC. conception and design of research; C.A.L., I.B.S. and M.L.M.BeC. edited and revised manuscript; C.A.L., I.B.S. and M.L.M.BeC. interpreted results of experiments; M.L.M.BeC. approved final version of manuscript. Acknowledgments We are grateful to Dr. Joel Heimann and Dr. Miriam Dolnikoff for invaluable discussions related to fetal programming and perinatal environment, as well as to Marina Fevereiro for the technical assistance provided. C.A. Lino and I.B. Silva are recipients of a ~o de Amparo a  Pesquisa do Estado de scholarship from the Fundaça ~o Paulo (FAPESP, Foundation for the Support of Research in the Sa State of Sao Paulo). References Araujo, A.S., Diniz, G.P., Seibel, F.E., Branchini, G., Ribeiro, M.F., Brum, I.S., Khaper, N.,
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Please cite this article in press as: Lino, C.A., et al., Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders, Molecular and Cellular Endocrinology (2015), http://dx.doi.org/10.1016/j.mce.2015.08.015