Perinatal growth restriction (GR) is associated with heightened sympathetic tone and hypertension. We have previously shown that naturally occurring neonatal GR programmes hypertension in male but not female mice. We therefore hypothesized that intact ovarian function or post-ovariectomy (OVX) oestrogen administration protects GR female mice from hypertension. Utilizing a non-interventional model that categorizes mice with weanling weights below the tenth percentile as GR, control and GR adult mice were studied at three distinct time points: baseline, post-OVX and post-OVX with oral oestrogen replacement. OVX elicited hypertension in GR mice that was significantly exacerbated by psychomotor arousal (systolic blood pressure at light to dark transition: control 122±2; GR 119±2; control-OVX 116±3; GR-OVX 126±3 mmHg). Oestrogen partially normalized the rising blood pressure surge seen in GR-OVX mice (23±7% reduction). GR mice had left ventricular hypertrophy, and GR-OVX mice in particular had exaggerated bradycardic responses to sympathetic blockade. For GR mice, a baseline increase in baroreceptor reflex sensitivity and high frequency spectral power support a vagal compensatory mechanism, and that compensation was lost following OVX. For GR mice, the OVX-induced parasympathetic withdrawal was partially restored by oestrogen (40±25% increase in high frequency spectral power, P<0.05). In conclusion, GR alters cardiac morphology and cardiovascular regulation. The haemodynamic consequences of GR are attenuated in ovarian-sufficient or oestrogen-replete females. Further investigations are needed to define the role of hormone replacement therapy targeted towards young women with oestrogen deficiency and additional cardiovascular risk factors, including perinatal GR, cardiac hypertrophy and morning surge hypertension.

CLINICAL PERSPECTIVES

  • Sexual dimorphism has been observed in the developmental origins of cardiovascular disease, with young adult women relatively protected from perinatal GR-induced hypertension.

  • Utilizing a translational model, we identified heightened baroreceptor sensitivity and heart rate variability in young adult mice with a history of neonatal GR. Following OVX, those markers of cardiac vagal tone decreased, and growth-restricted mice developed morning surge hypertension in the absence of oral oestrogen replacement.

  • Our findings provide insight into the cardioprotective mechanisms empowered in young women and identify a patient population, those with ovarian insufficiency and GR-related hypertension, which may uniquely benefit from oestrogen replacement therapy.

INTRODUCTION

Perinatal growth-restricted infants face a constellation of challenges and must compensate to minimize the risk of life-long morbidity [1]. In addition to the reprogramming of metabolism to optimize the incorporation of limited nutritional resources, the circulatory system must adapt to altered caloric demands and body dimensions. Whether a primary consequence of growth restriction (GR) or a compensatory response, former GR infants have increases in sympathetic tone and are at a greater risk of hypertension than the general population [2,3].

Premature infants face many of the same challenges encountered by intrauterine growth-restricted term infants. Both have global impairment in growth and development and marked reductions in nutritional stores. By the time premature infants reach 36 weeks postmenstrual age, nearly 90% have developed ex utero GR and up to 70% have elevated arterial pressures throughout infancy [4,5]. As adults, former premature infants have restricted cardiac dimensions and increased arterial pressures, with the greatest alterations seen in men [68].

Investigations in rats have shown late gestation uterine artery ligation and maternal malnutrition elicit intrauterine GR with long-term phenotypic changes in adult offspring, including increased sympathetic tone and hypertension [911]. Female offspring in these studies are partially protected from the damaging effects of intrauterine GR, but this protection is lost with ovariectomy (OVX) or age-related ovarian failure [9,12]. Considering the inter-species differences in developmental trajectory, the critical third trimester of human neurodevelopment and cardiac growth may be modelled in neonatal mice. Utilizing a non-interventional model, we have previously shown neonatal GR programmes hypertension in male but not female mice [13].

In the context of our neonatal GR model, we sought to define the oestrogen-dependent haemodynamic adaptations that occur in young adult female mice that partially protect them from programmed hypertension. We hypothesized that neonatal GR increases sympathetic activation, but ovarian function or oestrogen replacement therapy facilitates physiological compensation through enhanced baroreceptor reflex control to suppress the development of adult hypertension.

MATERIALS AND METHODS

Ethical approval

All procedures were approved by the University of Iowa Animal Care and Use Committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals: Eighth Edition, published by the National Research Council: National Academies Press, Washington, DC.

Animal model

Adult C57Bl/6J mice (Jackson Laboratory) delivered naturally and were maintained on standard rodent chow (7013; Harlan Teklad) throughout pregnancy and lactation. Neonatal GR was identified by a day 21 weight either less than 80% of the mean weight of the littermate controls or less than the tenth percentile for the colony [13,14]. Beginning at 3 months, female mice were utilized for baseline radiotelemetry or echocardiography. To assess the potential protective effect of ovarian function, bilateral OVX was completed at 4 months (control 17.7±2.2 weeks; GR 17.6±2.3 weeks). Post-OVX data were collected an additional 4–6 weeks after the ovariectomies were performed [15]. Analgesia during OVX included flunixin meglumine (2.5 mg/kg, subcutaneously once or twice daily for 48 h with the first dose given at the time of isoflurane induction) and 0.5% bupivacaine applied to the wound margin. To test the protective effect of oestrogen replacement on GR mice, six GR and four control mice underwent OVX followed 4–6 weeks later by post-OVX recordings, then repeat recordings 3 weeks after beginning daily oral oestrogen at a dose shown to provide physiological replacement (1.12 μg of 17β-oestradiol in 60 mg of hazelnut cream) [16].

Radiotelemetry

Carotid radiotelemetry catheters (PA-C10; Data Sciences International) were implanted during isoflurane-induced general anaesthesia [17]. Flunixin meglumine (2.5 mg/kg) was administered subcutaneously at the time of anaesthetic induction and 0.5% bupivacaine was applied to the wound margin. After a 7 day recovery period, arterial pressures, heart rate and relative locomotor activity were recorded for 10 s every 5 min for 60 h (encompassing three dark cycles and two light cycles). Immediately thereafter, arterial waveforms were recorded at 2000 Hz to calculate spontaneous baroreflex activity by the sequence technique and spectral analysis by fast Fourier transformation. This sampling occurred at the end of the light cycle, during a 10 min phase of inactivity. Baroreflex events were defined as reciprocal changes in blood pressure and heart rate occurring over the course of four cardiac cycles, and murine heart rate-specific values were utilized to calculate the absolute power of the low frequency (0.1–1.5 Hz) and high frequency (1.5–4.0 Hz) spectral components, both of which are influenced predominately by parasympathetic alteration [17,18]. After these observational recordings, mice received a single intraperitoneal injection of the α1-adrenergic receptor antagonist prazosin (1 mg/kg), followed in 24 h by the nicotinic receptor antagonist chlorisondamine (2.5 mg/kg). Haemodynamic responses were assessed 30–60 min after the injections, and the transmitters were then magnetically deactivated and OVX was performed. After 4–6 weeks of recovery, the transmitters were reactivated and the telemetry protocol was repeated in its entirety. For the final cohort of mice, post-OVX recordings were followed by daily oestrogen therapy and a final set of recordings 3 weeks thereafter. No mouse underwent all three sets of recordings. At the end of the protocol, the mice were killed by bilateral thoracotomy while under isoflurane anaesthesia.

Echocardiography

Echocardiograms of the left ventricle were obtained using the VisualSonics Vevo 2100 High Resolution Imaging System (VisualSonics Inc.) by an investigator blinded to group assignment. Isoflurane was titrated to maintain heart rate between 450 and 600 beats/min. M-mode recordings were obtained of the left ventricles in the parasternal short axis view at the level of the left ventricular papillary muscles. Measurements were made of the left ventricular internal dimension in diastole and systole (LVIDd and LVIDs respectively) and left ventricular posterior wall thickness in diastole and systole (LVPWd and LVPWs respectively). These measurements were then used to calculate the left ventricular ejection fraction (EF) and fraction shortening (FS), and left ventricular volumes, as previously described [19].

Oestradiol levels

Eight adult mice underwent bilateral OVX. Fourteen days later, plasma was collected for oestradiol determination. The oral oestradiol replacement regimen was then initiated, and both 3 and 5 weeks into oestrogen replacement, 4 h after the daily dose of oestradiol was administered, plasma was again collected. To further define the upper range of physiological oestradiol levels, plasma was collected from three dams in the third trimester of pregnancy (two were at E19 and delivered within 24 h and the third was at E14 and delivered 5 days later). All samples were collected at the end of the light cycle (to match the timing of the heart rate variability assessments), and all samples were immediately frozen at −80°C until being thawed a single time for determination of oestradiol concentration by ELISA (Calbiotech, ES180S-100, standard range: 3–300 pg/ml).

Data analysis

All values are presented as means ± S.E.M. GR compared with control body weights were analysed by Student's t test, and GR compared with control litter size was analysed by the Mann–Whitney Rank Sum Test. Dark cycle and light cycle tabulated data were compared by three-way ANOVA factoring for lighting cycle, GR and OVX status. Hourly blood pressure data were compared within the three cohorts (baseline, OVX and OVX-oestrogen) by two-way repeated measures ANOVA factoring for time of day and GR. All other comparisons were by two-way ANOVA, factoring for GR and OVX/oestrogen status. Post-hoc analysis (Holm–Sidak method) was performed if statistically significant differences were detected (P<0.05). All analyses were performed using SigmaStat 3.0 (SPSS Inc.).

RESULTS

Animal model

The initial 76 litters spontaneously ranged in size from 2 to 12 pups, and all 486 pups (including 255 females) were fostered by their birth dam. There was an inverse correlation between litter size and weanling weight (Figure 1A, R=−0.36, P<0.001). To minimize the number of unused pups, we applied these data to our breeding strategy and increased the likelihood of identifying a balanced ratio of control compared with GR mice by cross-fostering <1-day-old mice into litters of six compared with 11 or 12 pups. For the 324 female mice subsequently fostered by this complementary approach, weanling weight again inversely correlated with litter size (Figure 1B, R=−0.66, P<0.001). Overall, GR, defined by a weanling weight less than the historically determined tenth percentile threshold of 6.8 g [20], developed in 11% of mice from litters containing up to ten pups and 32% of mice from litters containing 11 or 12 pups. Ultimately, the control group consisted of 30 mice from 19 litters and the GR group consisted of 29 mice from 17 litters. Although GR mice tended to come from larger litters than controls (median litter size: 11 compared with six), this did not reach statistical significance (P=0.054), and nine litters provided both GR and control mice. Birth weights of the mice that went on to develop neonatal GR did not significantly differ from control values (control: 1.71±0.06 g, GR: 1.85±0.13 g). By definition, neonatal GR mice weighed less than controls at weaning (6.3±0.4 g compared with 9.0±0.2 g, P<0.001), but partially caught up as adults (23.7±0.9 g compared with 25.7±0.9 g, P=0.11).

Impact of litter size on neonatal growth

Figure 1
Impact of litter size on neonatal growth

Female C57BL/6 mice were alternatively kept with their birth dam (A, n=255) or cross-fostered into litters of six compared with 11 or 12 mice (B, n=324). In both cases, weaning weight was inversely proportional to litter size (R=−0.36 and −0.66 respectively; both P<0.001). Neonatal GR, defined by a weanling weight below 6.8 g, was more likely to occur when mice were fostered in litters of 11 or 12 pups, but it also occurred in 11% of the pups fostered in smaller litters.

Figure 1
Impact of litter size on neonatal growth

Female C57BL/6 mice were alternatively kept with their birth dam (A, n=255) or cross-fostered into litters of six compared with 11 or 12 mice (B, n=324). In both cases, weaning weight was inversely proportional to litter size (R=−0.36 and −0.66 respectively; both P<0.001). Neonatal GR, defined by a weanling weight below 6.8 g, was more likely to occur when mice were fostered in litters of 11 or 12 pups, but it also occurred in 11% of the pups fostered in smaller litters.

Radiotelemetry

For both GR and control mice, time of day (dark cycle compared with light cycle) significantly influenced arterial pressures, heart rate and locomotor activity (all P<0.01) (Table 1). Independently of GR or oestrogen therapy, OVX mice had lower heart rates than ovarian-sufficient mice (571±4 compared with 601±5 beats/min, P<0.001). Baseline blood pressure was not significantly altered by GR, but following OVX, increased systolic blood pressures were seen in GR compared with control mice (Table 1; GR-OVX 124±1, control-OVX 121±1 mmHg, P<0.05). To better define the blood pressure regulatory pattern of GR and control mice, hourly data were analysed. This ambulatory blood pressure assessment confirmed normal arterial pressures in pre-OVX GR mice (Figure 2A). In contrast, GR-OVX mice had significantly higher systolic pressures immediately prior to and following the removal of ambient lighting (Figure 2B: light cycle to dark cycle transition occurs at 1800). As shown in Figure 2C, this increase in GR-OVX compared with control-OVX arterial pressure at the time of awakening correlated with an increase in GR-OVX compared with GR-baseline blood pressure suggesting OVX unmasked a predisposition of GR mice towards ‘rising surge hypertension’. Applying the conventional equation for surge hypertension (rising blood pressure minus blood pressure nadir at rest), GR-OVX mice had significantly higher surges (GR-OVX: 14±4; control-OVX: 7±2 mmHg, P<0.05) and were more likely than control-OVX mice to have a rising surge in excess of 15 mmHg (46% compared with 0%, P<0.05 by Fisher exact test). Oestrogen replacement effectively normalized the arterial blood pressure of GR mice (Table 1 and Figure 2D) and reduced the rising blood pressure surge of GR-OVX mice to 77±7% of pre-oestrogen replacement values (P=0.07). Taken together, the haemodynamic data suggest that the OVX-induced hypertension seen in GR mice may be influenced by oestrogen-modulated alterations in arousal-evoked sympathetic activation.

Table 1
Arterial blood pressure (BP), heart rate and relative locomotor activity were recorded every 5 min for 60 h, allowing for comparison of dark cycle and light cycle values

The effects of neonatal GR, bilateral OVX and OVX plus oral oestradiol administration (OVX-E2) were analysed by three-way ANOVA factoring for time, growth and ovarian status. *P< 0.05 compared with control; #P< 0.05 compared with baseline; n=11 control baseline, 7 GR baseline, 9 control OVX, 11 GR OVX, 3 control OVX-E2, 6 GR OVX-E2; AU: arbitrary units.

BaselineOVXOVX-E2
ControlGRControlGRControlGR
Dark cycle 
 Systolic BP (mmHg) 128±1 127±1 125±3 128±2* 130±2 126±2 
 Mean BP (mmHg) 113±1 114±2 110±2 112±2 113±3 110±2 
 Diastolic BP (mmHg) 97±1 99±2 95±2 97±3 96±4 93±2 
 Heart rate (beats/min) 618±11 613±7 591±7# 588±7# 569±18# 570±24# 
 Activity (AU) 14±3 12±2 10±2 9±1 13±3 13±3 
Light cycle 
 Systolic BP (mmHg) 117±1 116±2 114±2 119±2* 118±1 116±2 
 Mean BP (mmHg) 103±1 103±2 100±1 104±2 102±2 100±1 
 Diastolic BP (mmHg) 87±1 89±2 86±1 90±3 87±3 84±2 
 Heart rate (beats/min) 588±10 586±9 560±10# 570±9# 552±22# 545±8# 
 Activity (AU) 5±1 5±1 4±1 4±1 4±1 5±1 
BaselineOVXOVX-E2
ControlGRControlGRControlGR
Dark cycle 
 Systolic BP (mmHg) 128±1 127±1 125±3 128±2* 130±2 126±2 
 Mean BP (mmHg) 113±1 114±2 110±2 112±2 113±3 110±2 
 Diastolic BP (mmHg) 97±1 99±2 95±2 97±3 96±4 93±2 
 Heart rate (beats/min) 618±11 613±7 591±7# 588±7# 569±18# 570±24# 
 Activity (AU) 14±3 12±2 10±2 9±1 13±3 13±3 
Light cycle 
 Systolic BP (mmHg) 117±1 116±2 114±2 119±2* 118±1 116±2 
 Mean BP (mmHg) 103±1 103±2 100±1 104±2 102±2 100±1 
 Diastolic BP (mmHg) 87±1 89±2 86±1 90±3 87±3 84±2 
 Heart rate (beats/min) 588±10 586±9 560±10# 570±9# 552±22# 545±8# 
 Activity (AU) 5±1 5±1 4±1 4±1 4±1 5±1 

Ambulatory blood pressures

Figure 2
Ambulatory blood pressures

Systolic blood pressure was measured by radiotelemetry in adult female control mice (closed symbols) and neonatal growth restricted mice (GR, open symbols) at baseline (A, triangles), after bilateral OVX (B and C, circles) and 3 weeks after post-OVX oestradiol therapy (D, squares). Compared with control-OVX mice (B) and GR-baseline mice (C), GR-OVX mice had increased arterial pressures in the absence of oestrogen replacement. *P<0.05 compared with control-OVX and P<0.05 compared with GR-baseline.

Figure 2
Ambulatory blood pressures

Systolic blood pressure was measured by radiotelemetry in adult female control mice (closed symbols) and neonatal growth restricted mice (GR, open symbols) at baseline (A, triangles), after bilateral OVX (B and C, circles) and 3 weeks after post-OVX oestradiol therapy (D, squares). Compared with control-OVX mice (B) and GR-baseline mice (C), GR-OVX mice had increased arterial pressures in the absence of oestrogen replacement. *P<0.05 compared with control-OVX and P<0.05 compared with GR-baseline.

To further assess the potential for autonomic dysregulation, haemodynamics were recorded during pharmacological challenge. Intraperitoneal injection of prazosin lowered arterial pressures and eliminated the increase in blood pressure seen in GR-OVX mice (Figure 3). Despite the reduction in arterial pressure, prazosin also significantly lowered the heart rate of GR-OVX mice (Figure 3), again suggesting an increase in cardiac sympathetic tone. To further assess autonomic tone, the ganglionic blocker chlorisondamine was administered. Unlike prazosin, chlorisondamine minimally attenuated the systolic blood pressure elevation seen in GR-OVX compared with control-OVX mice (92±5 compared with 82±5 mmHg; P=0.08; Figure 4). Furthermore, independently of OVX or oestrogen therapy, post-chlorisondamine heart rates were consistently higher in GR compared with control mice (481±14 compared with 434±18 beats/min; P<0.05; Figure 4). Taken together the pharmacological data suggest GR-OVX mice have heightened cardiac sympathetic tone independently of post-ganglionic neurotransmission.

Haemodynamic effects of sympathetic blockade

Figure 3
Haemodynamic effects of sympathetic blockade

Control (grey bars) and GR mice (white bars) were injected with the α1-adrenergic receptor antagonist prazosin (1 mg/kg intraperitoneally). Systolic blood pressure (left column) and heart rate (right column) were recorded over the subsequent 60 min. *P<0.05 compared with corresponding control and #P<0.05 compared with GR-baseline.

Figure 3
Haemodynamic effects of sympathetic blockade

Control (grey bars) and GR mice (white bars) were injected with the α1-adrenergic receptor antagonist prazosin (1 mg/kg intraperitoneally). Systolic blood pressure (left column) and heart rate (right column) were recorded over the subsequent 60 min. *P<0.05 compared with corresponding control and #P<0.05 compared with GR-baseline.

Haemodynamic effects of pharmacological denervation

Figure 4
Haemodynamic effects of pharmacological denervation

Control (grey bars) and GR mice (white bars) were injected with the nicotinic receptor antagonist chlorisondamine (2.5 mg/kg intraperitoneally) to induce ganglionic blockade (GB). Systolic blood pressure (A and C) and heart rate (B and D) were recorded over the subsequent 60 min. *P<0.05 compared with corresponding control.

Figure 4
Haemodynamic effects of pharmacological denervation

Control (grey bars) and GR mice (white bars) were injected with the nicotinic receptor antagonist chlorisondamine (2.5 mg/kg intraperitoneally) to induce ganglionic blockade (GB). Systolic blood pressure (A and C) and heart rate (B and D) were recorded over the subsequent 60 min. *P<0.05 compared with corresponding control.

To more specifically analyse resting cardiac autonomic tone, we next interrogated spontaneous baroreceptor reflex activity while mice rested in the midst of the diurnal light cycle. Consistent with the OVX-induced reduction in baseline heart rate (Table 1), both GR and control mice had fewer baroreceptor events following OVX, with or without oestrogen therapy (Figure 5A). Independently of OVX or oestrogen, GR mice had decreased baroreceptor event frequency (GR overall: 5.7±0.9; control overall: 10.3±1.1 events per 1000 beats; Figure 5A, P<0.01). For individual events, baroreceptor reflex sensitivity was increased in GR mice at baseline, but this was lost following OVX, and then significantly restored by oestradiol therapy (Figure 5B). Subsequent spectral analysis revealed a significant increase in baseline low-frequency and high-frequency spectral power in GR compared with control mice (Figure 6). The increase in GR spectral power was lost following OVX regardless of oestrogen therapy, providing converging evidence that the hypertension seen in GR-OVX mice is partially mediated by a loss of compensatory baroreceptor or vagal tone. To screen for the possibility of GR-induced cardiac structural or functional impairment as a contributing factor in the observed cardiovascular dysregulation, echocardiography was performed.

Effect of neonatal GR on baroreceptor reflex activity and sensitivity

Figure 5
Effect of neonatal GR on baroreceptor reflex activity and sensitivity

During the light cycle that followed the radiotelemetry recordings, spontaneous baroreceptor reflex events (A) and sensitivity (B) were recorded in adult female control (grey bars) and GR mice (white bars) both before and after bilateral OVX with or without oestrogen therapy (E2). *P<0.05 compared with corresponding control and #P<0.05 compared with baseline.

Figure 5
Effect of neonatal GR on baroreceptor reflex activity and sensitivity

During the light cycle that followed the radiotelemetry recordings, spontaneous baroreceptor reflex events (A) and sensitivity (B) were recorded in adult female control (grey bars) and GR mice (white bars) both before and after bilateral OVX with or without oestrogen therapy (E2). *P<0.05 compared with corresponding control and #P<0.05 compared with baseline.

Impact of OVX of heart rate variability

Figure 6
Impact of OVX of heart rate variability

Spectral power was calculated from the same high-fidelity radiotelemetry recordings utilized for baroreceptor reflex analysis. Heart rate variability was analysed at both low frequency (LF HRV, A) and high frequency (HF HRV, B) before and after bilateral OVX with or without oestrogen therapy (E2). *P<0.05 compared with control, #P<0.05 compared with GR-baseline and P<0.05 compared with GR-OVX.

Figure 6
Impact of OVX of heart rate variability

Spectral power was calculated from the same high-fidelity radiotelemetry recordings utilized for baroreceptor reflex analysis. Heart rate variability was analysed at both low frequency (LF HRV, A) and high frequency (HF HRV, B) before and after bilateral OVX with or without oestrogen therapy (E2). *P<0.05 compared with control, #P<0.05 compared with GR-baseline and P<0.05 compared with GR-OVX.

Echocardiography

Independently of ovarian or oestrogen status, GR mice had significantly decreased left ventricular volumes in diastole (GR overall: 59±2 μl; control overall: 67±2 μl; P<0.05) (Table 2). As estimated by left ventricular FS, intrinsic cardiac function was not altered by either GR or OVX. Related to the decrease in diastolic volumes with preserved FS, GR mice had independent reductions in left ventricular stroke volumes (GR overall: 34±1 μl; control overall: 38±2 μl; P<0.05). When indexed to current body weight, there were no longer significant differences in left ventricular volumes, but adult left ventricular posterior wall thickness (LVPW) was significantly increased by neonatal GR, regardless of ovarian or oestrogen status (GR overall: 36±1; control overall: 31±2 mm/kg; P<0.05).

Table 2
Left ventricular echocardiography was performed on control and growth-restricted mice (GR), including those that underwent bilateral OVX alone or OVX with oral oestrogen replacement (OVX-E2)

*P< 0.05 for GR compared with control by two-way ANOVA; n=15 control baseline, 16 GR baseline, 11 control OVX, 13 GR OVX, 4 control OVX-E2, 6 GR OVX-E2; left ventricular volumes were excluded for three mice as statistical outliers; these values in μl were 21 (control mouse at baseline), 107 (GR mouse at baseline) and 19 (GR mouse after OVX-E2).

BaselineOVXOVX-E2
ControlGRControlGRControlGR
LVPW, diastole (mm) 0.80±0.04 0.77±0.03 0.82±0.06 0.80±0.06 0.67±0.05 0.72±0.06 
 (mm/kg) 30±1 33±2* 34±3 38±3* 30±3 36±4* 
LV volume, diastole (μl) 66±2 61±3* 64±4 60±4* 70±3 57±2* 
 (μl/g) 2.5±0.1 2.6±0.1 2.7±0.2 2.8±0.2 3.1±0.2 2.9±0.1 
Stroke volume (μl) 39±2 34±2* 38±3 35±2* 37±2 33±2* 
 (μl/g) 1.5±0.1 1.4±0.1 1.6±0.1 1.6±0.1 1.7±0.1 1.7±0.1 
FS (%) 32±2 29±2 33±3 32±3 27±1 29±1 
BaselineOVXOVX-E2
ControlGRControlGRControlGR
LVPW, diastole (mm) 0.80±0.04 0.77±0.03 0.82±0.06 0.80±0.06 0.67±0.05 0.72±0.06 
 (mm/kg) 30±1 33±2* 34±3 38±3* 30±3 36±4* 
LV volume, diastole (μl) 66±2 61±3* 64±4 60±4* 70±3 57±2* 
 (μl/g) 2.5±0.1 2.6±0.1 2.7±0.2 2.8±0.2 3.1±0.2 2.9±0.1 
Stroke volume (μl) 39±2 34±2* 38±3 35±2* 37±2 33±2* 
 (μl/g) 1.5±0.1 1.4±0.1 1.6±0.1 1.6±0.1 1.7±0.1 1.7±0.1 
FS (%) 32±2 29±2 33±3 32±3 27±1 29±1 

Oestradiol levels

To determine whether the oral oestradiol treatment regimen matched the physiological levels previously reported [16], plasma oestradiol concentrations were determined (Figure 7A). Prior to treatment, OVX mice had a mean plasma oestradiol level of 5.6±0.8 pg/ml with a range of 2.8–8.2 pg/ml. The two levels below the lowest 3.0 pg/ml standard (2.8 and 2.9 pg/ml) were excluded from further analysis. During oral oestradiol therapy, the same OVX mice had stable plasma oestradiol levels of 38±19 pg/ml after 3 weeks of treatment and 40±15 pg/ml after 5 weeks of treatment, with a final range of 6.5–102 pg/ml (P=0.07 compared with OVX). In every case, plasma oestradiol levels were increased by oral oestradiol administration (Figure 7B). In comparison, third trimester maternal plasma oestradiol levels were 220±8.9 pg/ml with a range of 205–236 pg/ml, values that were significantly higher than those seen in OVX mice, even after oestradiol therapy (Figure 7C).

Plasma oestradiol levels

Figure 7
Plasma oestradiol levels

Oestradiol levels were determined by ELISA with linear values obtained utilizing standards ranging from 3 to 300 pg/ml (A, R2=0.97). Plasma was obtained 2 weeks after bilateral OVX, 3 weeks after starting oestradiol therapy and again 5 weeks after starting oestradiol therapy (B and C; n=6). Comparative plasma was obtained from third trimester pregnant dams that were not receiving exogenous oestrogen (C, n=3). *P<0.05 compared with OVX and OVX-E2.

Figure 7
Plasma oestradiol levels

Oestradiol levels were determined by ELISA with linear values obtained utilizing standards ranging from 3 to 300 pg/ml (A, R2=0.97). Plasma was obtained 2 weeks after bilateral OVX, 3 weeks after starting oestradiol therapy and again 5 weeks after starting oestradiol therapy (B and C; n=6). Comparative plasma was obtained from third trimester pregnant dams that were not receiving exogenous oestrogen (C, n=3). *P<0.05 compared with OVX and OVX-E2.

DISCUSSION

Research into the developmental origins of cardiovascular disease has identified a strong sexual dimorphism with heightened susceptibility towards hypertension seen in males. Utilizing our translational neonatal GR model, we identified key phenotypic changes in GR-OVX mice that are analogous to those reported in low birth weight or premature infants [2,58], including (i) hypertension, (ii) autonomic dysregulation, and (iii) abnormalities in cardiac morphology. We further demonstrated the therapeutic utility of daily oral oestrogen replacement therapy to restore the cardiac vagal tone of GR-OVX mice, and dampen the emergence of morning surge hypertension in a target population at increased risk of cardiovascular disease.

The light–dark transition surge hypertension we uncovered in GR-OVX mice is a novel finding. Previous studies have demonstrated that postmenopausal status strongly predicts non-dipping nocturnal and morning surge hypertension [21,22]. Of greatest concern, surge hypertension is strongly associated with cardiovascular mortality [2224], perhaps through an association with increased sympathetic tone and reduced baroreceptor sensitivity [2527], and women are exquisitely sensitive to non-dipping-associated end organ damage [23].

The data we obtained during ambulatory blood pressure monitoring and pharmacological autonomic blockade highlight the complexity of cardiovascular regulation and the ways that ovarian status influences the predisposition of GR mice to cardiac dysregulation. At baseline, ovarian sufficient GR mice appeared to have a well-balanced increase in cardiac parasympathetic and sympathetic tone. Following OVX, the significant reduction in cardiac vagal tone, as detected by baroreceptor reflex and spectral analysis, appeared to unmask an underlying increase in sympathetic activation, and this was most dramatically manifested as morning surge hypertension. The presence of a GR-induced increase in sympathetic tone was further supported by the results of autonomic blockade. During prazosin administration, systemic vascular resistance and blood pressure consistently decrease with variable effects on heart rate [28]. Typically, prazosin-induced hypotension leads to baroreceptor reflex activation and β-adrenergic receptor mediated tachycardia, but that reflex tachycardia is partially opposed by the bradycardia that occurs as a direct result of prazosin-induced cardiac α-adrenergic receptor blockade [28]. For ovarian sufficient GR mice, the heightened baroreceptor reflex sensitivity may have increased reflex tachycardia, but that exaggerated response may have been attenuated to control levels by an increase in prazosin-induced bradycardia. For GR mice, OVX reduced baroreceptor reflex sensitivity to control levels, but might not have attenuated persistent hypersensitivity to direct α-adrenergic receptor antagonist-mediated bradycardia, potentially contributing to the observed reduction in post-prazosin tachycardia seen in GR-OVX mice. This indirect evidence of heightened cardiac sympathetic tone is consistent with the increase in post-ganglionic blockade heart rate and the decrease in baroreceptor event frequency demonstrated in GR mice, independently of ovarian or oestrogen status. Overall, the relative cardioprotection of GR female compared with male mice appears to emanate from an ovarian-dependent enhancement in cardiac vagal tone as a critical buffer for the heightened sympathetic responses that may otherwise be observed [29]. This interpretation of our data is consistent with clinical data showing that OVX interferes with vagally mediated heart rate regulation in young women, in a manner that is normalized by oestrogen replacement [30,31].

Among the potential proximal causes of autonomic imbalance, there is experimental support for GR-associated over-activation of the renin–angiotensin system as well as dysregulated central leptin signalling [9,12]. To this list, we now add an association between reduced left heart volumes and downstream sympathetic activation. We have identified a strikingly similar small left heart syndrome in adult mice exposed to the SSRI sertraline as neonates [19]. In these mice, a restriction in neonatal cardiac development led to programmed increases in adult heart rate, metabolic rate and urinary catecholamine excretion. Our investigations have consistently focused on the neonatal window of susceptibility because that is the critical final phase of murine cardiomyocyte proliferation, a developmental stage analogous to the third trimester of human gestation [32,33]. Of concern, former premature or low birth weight infants have now been shown to have a long-term reduction in left ventricular volumes [6,34].

We previously reported that maternal carbenoxolone administration leads to neonatal GR, stress hypertension, heightened superoxide production and impaired baroreceptor sensitivity in male mice, probably a consequence of increased transplacental glucocorticoid exposure [17]. Although the physiological effects of OVX extend beyond oestrogen deficiency, oestradiol is known to attenuate oxidative stress, possibly by reducing angiotensin receptor signalling [35,36]. Notably, oestrogen replacement, renin–angiotensin system blockade and renal denervation are each capable of normalizing the blood pressure of ovariectomized rats [9,10]. Clinical studies have shown combined progestin plus oestrogen replacement exerts greater effects than placebo or oestrogen alone in reducing the morning blood pressure surge seen in postmenopausal women despite unfavourable effects on intra-office blood pressure readings [3739]. Further investigations are needed to determine the relative importance of perinatal glucocorticoid exposure, oxidative stress and enhanced adrenergic signalling in the development of GR-associated cardiovascular dysregulation and left ventricular hypertrophy. Clinical investigations have already identified morning surge hypertension as an independent predictor of left ventricular hypertrophy in elderly populations [40,41].

Therapeutically, the oestradiol concentrations we measured are consistent with prior studies in mice [16]. The observed variability in these investigations has likewise been seen during clinical therapy in both young women with primary ovarian failure and older women with surgically induced or age-related menopause [42,43]. The pharmacokinetic variability emphasizes the importance of clinical dosing to achieve efficacy, followed by concentration monitoring to minimize the risk of adverse effects. For our investigations, after we documented cardiovascular improvement during oestradiol treatment, we obtained levels to confirm treatment was not eliciting concentrations above the physiological range. Although our studies were designed to be translational, extensive monitoring for adverse effects was beyond the scope of these investigations. Meta-analysis in heterogeneous populations has shown cardioprotection when hormone therapy is initiated within 10 years of menopause, but concerns remain regarding thromboembolic risk [44]. Assessment of the relative risk compared with benefit of oestrogen therapy for high-risk subpopulations within existing clinical studies would inform the therapeutic potential of individualized hormone replacement, perhaps targeted towards young adults with a personal history of neonatal GR and subsequent early-onset menopause [45].

In conclusion, neonatal GR leads to reduced adult left ventricular volumes with relative cardiac hypertrophy and markers of increased sympathetic tone. In the presence of intact ovarian function, arterial pressures are not elevated. Following OVX, there is vagal withdrawal, the bradycardic response to hypertension is attenuated, and a surge hypertension phenotype is observed. The results provide insight into the role of oestrogen in programmed hypertension and help to identify a patient population that may benefit from preservation of ovarian function. Further investigations into the links between the early environmental perturbations and later physiological alterations are needed to identify interventions to prevent or better palliate an important subset of cardiovascular disease.

AUTHOR CONTRIBUTION

Sarah Haskell, Veronica Peotta, Benjamin Reinking, Catherine Zhang and Robert Roghair were involved in the conception and design of the experiments. Sarah Haskell, Veronica Peotta, Benjamin Reinking, Catherine Zhang, Vivian Zhu, Elizabeth Kenkel and Robert Roghair were involved in the collection, analysis and interpretation of data. Sarah Haskell and Robert Roghair drafted the article or revised it critically for important intellectual content.

FUNDING

This work was supported by the National Institutes of Health [grant numbers HD050359 and HL007485]; and the American Heart Association (Undergraduate Student Research Fellowship) [grant numbers 10UFEL4140149 and 13UFEL17240085].

Abbreviations

     
  • FS

    fractional shortening

  •  
  • GR

    growth restriction

  •  
  • LVPW

    left ventricular posterior wall thickness

  •  
  • OVX

    ovariectomy

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