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. 2004 Oct;85(5):287–294. doi: 10.1111/j.0959-9673.2004.00399.x

The effect of enalapril on the cardiac remodelling in ovariectomized spontaneously hypertensive rats

Wellington V Santos 1, Leila MM Pereira 1, Carlos A Mandarim-de-Lacerda 1
PMCID: PMC2517526  PMID: 15379961

Abstract

Angiotensin-converting enzyme inhibitors reduce the blood pressure (BP) and inhibit the generation of the angiotensin II from the inactive angiotensin I. Ten 28-week-old spontaneously hypertensive rats (SHRs) had their ovaries bilaterally removed and five rats were left intact and studied for 7 additional weeks: intact group, ovariectomized group (ovx SHRs) and ovariectomized + enalapril group (ovx + en). BP was higher in ovx SHRs and lower in treated ovx SHRs. Left ventricular (LV) mass index was greater in untreated ovx SHRs and smaller in ovx + en group. The LV cardiomyocyte (cmy) mean cross-sectional area, measured by stereology, was greater in ovx SHRs and smaller in both intact and ovx + en SHRs. Ovx significantly decreased the density of intramyocardial blood vessels (ive), but administration of enalapril was able to restore the density of the ive to that seen in intact group. The worst ive:cmy ratio was found in untreated ovx SHRs, the intact group showed a 90% greater ratio, and the treated ovx group showed a 150% greater ratio than the untreated ovx group. In conclusion, ovariectomy, in SHRs, causes cardiac hypertrophy and an unfavourable myocardial remodelling. Of the spectrum of changes seen, the major effect of enalapril appears to be mediated via an increase in the density of ive.

Keywords: cardiac hypertrophy, enalapril, hypertension, ovariectomy, spontaneously hypertensive rats, stereology

It is well known that the incidence of cardiovascular events in premenopausal women is lower than that in men of the same age, and that after the menopause, cardiovascular morbidity and mortality in women become similar, if not higher, than that in men, indicating that female sex hormones play an important protective role upon the maintenance of the vasculature (Rappelli 2002). The postmenopausal metabolic syndrome consists of obesity, insulin resistance and hyperinsulinaemia, conveying increased sodium reabsorption, stimulation of the sympathetic nervous system and smooth muscle growth resulting in hypertension (Fisman et al. 2002).

The precise mechanism through which menopause favours the development of hypertension is still a matter of debate (Beaufils 2000; Rappelli 2002). Similarly, the influence of menopause on the ventricular function and remodelling remains undefined (Mercier et al. 2002).

The class of antihypertensives known as angiotensin- converting enzyme (ACE) inhibitors reduces the blood pressure (BP) and inhibits the generation of the haemodynamically active octapeptide angiotensin II from the inactive decapeptide angiotensin I. The use of ACE inhibitors can lead to a partial or complete reversion of the hypertension and associated target-organ lesions (Brilla 2000a, 2000b). The development of hypertension in spontaneously hypertensive rats (SHRs), regardless of sex steroids, is mediated by the renin–angiotensin system (RAS) (Reckelhoff et al. 2000). However, gender differences in RAS components seem to exist and may play a central role in the BP control. In normotensive populations, for example, the plasma renin activity is significantly higher in men than that in women, and it is higher in postmenopausal vs. premenopausal women (Fisman et al. 2002). Oestrogen is protective against hypertension, possibly by amplifying the vasodilator contributions of Ang (I–VII), while it reduces the formation and vasoconstrictor actions of Ang II (Brosnihan et al. 1997).

Ovariectomy enhances RAS in SHRs, simulating RAS in male SHRs (Bachmann et al. 1993). The use of inhibitors of the ACE attenuates hypertension and the consequent cardiac remodelling in SHRs (Mandarim-de-Lacerda et al. 2002; Zorzi et al. 2002; Der Sarkissian et al. 2003). Therefore, this study was proposed to investigate the blood pressure alteration and the cardiac remodelling of untreated ovariectomized SHRs and also ovariectomized SHRs treated with the drug enalapril, an inhibitor of the ACE.

Materials and methods

Sample and procedures

Fifteen 28-week-old SHRs were selected from a colony maintained in the Laboratory of Morphometry & Cardiovascular Morphology (http://www.2.uerj.br/~lmmc) and used for this study. Animals were individually housed in a temperature (20 ± 2 °C), humidity (60 ± 10%) and 12-h light/dark cycle (artificial lights, 7–19 h) controlled room and had free access to a standard rat chow (Nuvilab®, Parana, Brazil) (Reeves et al. 1993) and fresh water. Rats were acclimated for 5 days in this environment, using a randomized complete block design; they were divided into three groups of five rats each. SHRs of two groups were bilaterally ovariectomized and SHRs of one group were left intact. The animal experimentation ethics committee of the State University of Rio de Janeiro approved the protocols. Animals care was according to the ‘Guide for the Care and Use of Laboratory Animals’ published by the US National Institutes of Health (NIH publication n°85-23, revised 1996).

SHRs were anaesthetized (intraperitonial sodium pentobarbital 10 mg/kg to each rat) before the surgical procedure, the ovaries were bilaterally clamped and removed through a ventral abdominal midline small incision, the uterine tubes were ligatured and the uterus left intact, then the abdominal wall was sutured. After the surgery, rats were maintained in good conditions, and physiological oestrous was periodically examined by microscopic evaluation of vaginal smears. Fifteen days from the date of surgery, blood samples were collected and a significant low level of oestradiol was confirmed (enzyme immunoassay; 582251 oestradiol EIA kit, Cayman Chemical Co., Denver, CO, USA) before the initiation of the dosing part of the experiment.

The SHRs were studied for seven additional weeks: (a) intact group (not operated SHRs), (b) ovariectomized group (ovx SHRs), and (c) ovariectomized plus enalapril group (ovx + en, ovariectomized SHRs received enalapril maleate, Sigma Chemical Co., St Louis, MO, USA, Lot 38H0500, 20 mg/kg/day in the drinking water).

The systolic blood pressure (BP) and body mass were measured weekly during the study; BP was verified in conscious rats using the non-invasive method of the tail-cuff plethysmography (Letica LE 5100, Panlab®, Barcelona, Spain).

On the day of euthanasia (50th day), rats were deeply anaesthetized (intraperitonial sodium pentobarbital 15 mg/kg) and heart was stopped in diastole by the left ventricular (LV) injection of 3.0 ml KCl at 10%. The heart was then removed, the LV was separated, including the interventricular septum, and measured (water displacement weighting Scherle's methods) (Scherle 1970). LV mass index was determined as LV mass-to-body mass ratio.

Tissue processing

LV was cut at random according to the orientator method (Mattfeldt et al. 1990). The fragments were randomly embedded without a reference to the position of the specimen in the first cut. LV fragments were kept for 48 h at room temperature in fixative (freshly prepared 4% w/v formaldehyde in 0.1 m phosphate buffer pH 7.2) (Carson et al. 1973) and embedded in Paraplast plus® (Sigma Chemical Co.). Several (up to 10) 3-µm thick sections were systematically and uniformly random sampled and stained with Masson trichrome or picro-sirius red. LV myocardium was analysed considering the cardiomyocytes and the cardiac interstitium (that was subdivided into intramyocardial vessels and connective tissue with nerves).

Stereology and data analysis

The analysis used a videomicroscope (Leica model DMRBE microscope, Kappa CF 15/5 video-camera, Sony triniton monitor) and a test-system with 36 test-points and a frame of known area (Mandarim-de-Lacerda 2003). The reference volume was estimated by point counting using the points hitting the global myocardium (PT). The number of points (Pp) hitting the cardiomyocytes (cmy), the intramyocardial vessels (ive) and the interstitial connective tissue (ct) were counted to estimate the volume densities of these structures (Vv[structure] : = Pp[structure]/PT) (: = is used to indicate that it is an estimate). The number of cardiomyocytes and intramyocardial vessels was counted in the test-area (QA), because they did not cross the frame forbidden line or its extensions (a two-dimensional 2800 µm2 test-frame area calibrated with a Leitz micrometer 1 mm/100) (Gundersen 1977). The cardiomyocyte mean cross-sectional area was estimated as A[cmy] : = Vv[cmy]/2QA[cmy]µm2. The intramyocardial vessel-to-cardiomyocyte ratio was determined as Vv[ive]/Vv[cmy], and the intramyocardial vessels length density (Lv[ive] : = 2QA[ive]) was also estimated (Mandarim-de-Lacerda 2003).

The biometric parameter differences were tested by the analysis of variance and the post hoc test of Newman–Keuls. Stereology differences were tested by both the non-parametric Kruskal–Wallis analysis of variance and the Mann–Whitney test. In all cases, P < 0.05 was considered statistically significant (Zar 1999). All analyses were performed using GraphPad Prism® version 4.0 for Windows® (GraphPad Software, San Diego, CA, USA).

Results

Biometrical results are shown in Figures 1–4, and stereological results are shown in Figures 5–8.

Figure 1.

Figure 1

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Blood oestradiol levels in spontaneously hypertensive rats after 15 days of ovariectomy (mean and SE).

Figure 4.

Figure 4

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Left ventricular (LV) mass index in spontaneously hypertensive rats with 7 weeks of treatment (mean and SE) (ovx = ovariectomized SHRs, en = enalapril). Mann–Whitney test: in signalled cases, when compared, P < 0.05; if: [a] when compared with intact group, [b] with ovx group.

Figure 5.

Figure 5

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Volume densities of the intramyocardial vessels [ive] and interstitial connective tissue [ct] in spontaneously hypertensive rats after 7 weeks of treatment (mean and SE) (ovx = ovariectomized SHRs, en = enalapril). Mann–Whitney test: in signalled cases, when compared, P < 0.05; if: [a] when compared with intact group, [b] with ovx group.

Figure 8.

Figure 8

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The intramyocardial vessel [ive]-to-cardiomyocyte [cmy] ratio in spontaneously hypertensive rats after 7 weeks of treatment (mean and SE) (ovx = ovariectomized SHRs, en = enalapril). Mann–Whitney test: in signalled cases, when compared, P < 0.05; if: [a] when compared with intact group, [b] with ovx group.

Blood oestradiol

A significant reduction in blood oestradiol levels occurred 15 days after the ovariectomy (Figure 1). Therefore, ovx SHRs began the experiment with 40% of the blood oestradiol levels shown by the intact SHRs. Both body mass and blood pressure analyses also showed significant differences among the groups.

Body mass

Intact SHRs stabilized their body mass after the second week of the experiment (Figure 2). They presented a lower body mass than the ovx groups; in the fourth week, the ovx SHRs body mass was nearly 20% greater than the intact SHRs. This difference was maintained throughout the study, but the enalapril-treated ovx SHR group showed a small body mass reduction in the last 3 weeks of the experiment (Figure 2), although this was still significantly greater than that in the intact SHRs.

Figure 2.

Figure 2

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Body mass in spontaneously hypertensive rats during 7 weeks after ovariectomy (mean and SE). After the second week of experiment, all groups were different from the intact group (ovx = ovariectomized SHRs, en = enalapril). ANOVA and Newman–Keuls test: in signalled cases, when compared, P < 0.05; if: [a] when compared with intact group.

Blood pressure

BP showed marked variation among the groups during the experiment (Figure 3). The differences began to be significant in the second week and became more accentuated after that. At the second week of experiment, the untreated ovx SHRs showed a BP 15% higher than that seen in intact SHRs (which showed a steadily increasing BP throughout the experiment). In treated ovx SHRs, BP reduction also began at the second week of experiment, reaching normal levels (mean ± SD of 122 ± 7 mmHg) at the end of the experiment. BP was more than 45% lower in ovx + en SHRs than in ovx SHRs, and more than 35% lower than intact SHRs.

Figure 3.

Figure 3

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Systolic blood pressure alteration in spontaneously hypertensive rats during 7 weeks after ovariectomy (mean and SE). After the second week, all groups showed different blood pressure levels (ovx = ovariectomized SHRs, en = enalapril). ANOVA and Newman–Keuls test: in signalled cases, when compared, P < 0.05; if: [a] when compared with intact group, [b] with ovx group.

LV mass index

In comparison with intact SHRs, LV mass index was 15% greater in untreated ovx SHRs and 14% smaller in ovx + en group (Figure 4).

Volume density

The intramyocardial vessel and interstitial connective tissue volume densities were significantly different among the groups (Figure 5). The ovx SHRs showed the smallest Vv[ive] and the greatest Vv[ct], while enalapril-treated ovx SHRs showed the greatest Vv[ive] (20% greater than that seen in the intact SHRs and 150% greater than that seen in the untreated ovx SHRs) and the smallest Vv[ct] (50% smaller than that found in the intact SHRs and 70% smaller than that found in the untreated ovx SHRs).

Cardiomyocyte mean cross-sectional area

The A[cmy] grew up in untreated ovx SHRs reaching a value that was 35% greater than that observed in both the intact SHRs and the ovx + en SHRs (Figure 6). No difference was found comparing these two last groups.

Figure 6.

Figure 6

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Cardiomyocyte mean cross-sectional area in spontaneously hypertensive rats after 7 weeks of treatment (mean and SE) (ovx = ovariectomized SHRs, en = enalapril). Mann–Whitney test: in signalled cases, when compared, P < 0.05; if: [a] when compared with intact group, [b] with ovx group.

Length density of intramyocardial vessels

Lv[ive] showed a significant reduction in untreated ovx SHRs (Figure 7). Treatment of ovx SHRs with enalapril partially reversed the decreased Lv[ive] levels shown in untreated ovx SHRs. Lv[ive] levels in ovx + en SHRs were 75% of those seen in intact SHRs.

Figure 7.

Figure 7

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Length density of intramyocardial vessels in spontaneously hypertensive rats after 7 weeks of treatment (mean and SE) (ovx = ovariectomized SHRs, en = enalapril). Mann–Whitney test: in signalled cases, when compared, P < 0.05; if: [a] when compared with intact group, [b] with ovx group.

Intramyocardial vessel-to-cardiomyocyte ratio (ive:cmy)

The ive:cmy ratio summarizes the quantitative myocardial alterations due to ovariectomy and the treatment with enalapril (Figure 8). Ovx resulted in a ratio of ive:cmy that was <50% of that seen in intact SHRs. Treatment of ovx SHRs with enalapril fully reversed this trend such that the resulting ratio was approximately 125% of that seen in intact SHRs.

Discussion

Cardiovascular system alterations in the genetically hypertensive rats with concomitant ovariectomy and hypertension treatment is the focus of this study. Comparison was made between mature female SHRs left intact and ovariectomized SHRs. Furthermore, some of the ovariectomized SHRs were treated with enalapril. The study analysed the BP alteration and the cardiac remodelling using a quantitative approach. Ovariectomized SHRs showed considerable changes in all measured parameters when compared with intact SHRs. Treatment with enalapril generally acted to normalize these changes by reducing BP and cardiac hypertrophy and, best of all, by enhancing the intramyocardial vessel-to-cardiomyocyte ratio.

The incidences of overweight and obesity are increased in postmenopausal women when compared with those in the general population. The complex mechanism of obesity-related hypertension can play a relevant role in explaining the high prevalence of hypertension after the menopause (Rappelli 2002). The positive correlation between leptin and body fat mass has caused some investigators to speculate that leptin resistance contributes to obesity. Ovariectomy-induced weight gain is believed to be caused by the early drop in leptin level, probably originated from a reduced sensitivity in leptin signal (Chu et al. 1999). However, the loss of ovarian function in human and rat is associated with increased fat mass gain and increased circulating leptin levels, but the loss of ovarian function in rats is not associated with a change in leptin sensitivity (Chen & Heiman 2001). In the present study, untreated and treated ovariectomized SHRs showed a body mass gain in relation to intact SHRs. We know that SHRs were highly sensitive to oestrogen deficiency, ovariectomy-induced earlier and greater cancellous bone loss in the SHRs than in the WKY, with greater increases in bone turnover rate, eroded surface, activation frequency and a decrease in the ratio of labelled to eroded perimeter in proximal tibial metaphysis at 2 weeks after surgery (Liang et al. 1997), but a recent study has demonstrated that postmenopausal hypertensive rats (blood oestradiol levels approximately 50 pg/ml) are more suitable models for the postmenopausal hypertension study and its consequences than ovariectomized rats (Fortepiani et al. 2003).

Oestrogen replacement significantly attenuates the pressure response to Ang II in rats, significantly reduces plasma ACE activity in association with a reduction in circulating levels of Ang II and also reduces tissue levels (kidney and aorta) of ACE (Brosnihan et al. 1997). Oestrogen replacement therapy reduces the risk of atherosclerotic change associated with the decrease of aorta ACE activity (Tanaka et al. 1997). Thus, inhibitors of the ACE seem to be a logical antihypertensive treatment to reduce postmenopausal hypertension. Different inhibitors of the ACE are efficient at reducing cardiac hypertrophy and hypertension in SHRs (Amrani et al. 1994; Zorzi et al. 2002). In this investigation, enalapril was found to be highly efficient at reducing hypertension and cardiac hypertrophy of ovariectomized SHRs.

Although sex hormones are important for maintaining normal heart weights and myosin isoenzyme balance in rats, they do not appear to be important in the adaptation shown in the heart when exposed to increased physiological or pathological loads (Malhotra et al. 1990). Myosin isoenzyme distribution in the adult heart is unaltered by ovariectomy, suggesting that oestrogen loses its ability to regulate expression of this protein in the mature heart (Morris et al. 1998). Present results suggest that sex hormones are not directly involved in the cardiac capability to respond to pressure overload, and that ovariectomized SHRs became more hypertensive and developed extensive cardiac hypertrophy that was effectively reversed by antihypertensive therapy.

The present study demonstrated an association of ovariectomy with worst intramyocardial vessels stereology in SHRs. The vascular system is associated with hypertension and menopause in different ways, the endothelin system, for example, is related to many forms of cardiovascular disease in experimental animals and humans (Schiffrin 1998), endothelin-1 represents an integral mechanism involved in the increase of mean arterial pressure following ovariectomy in rats (Mercier et al. 2002). Oestrogen seems to preserve the nitric oxide-mediated portion of flow/shear stress-induced dilation in female SHRs, resulting in a lower maintained wall shear stress in female than in male SHRs; the lower wall shear stress may contribute to the mechanisms by which oestrogen lowers BP and the incidence of cardiovascular diseases in women (Huang et al. 1998). Otherwise, a cellular mechanism of oestrogen-induced vascular relaxation involving inhibition of Ca2+ entry into vascular smooth muscle is gender dependent (Crews & Khalil 1999). Oestrogen has been shown not to affect arterial muscle relaxation (Packer et al. 2001), but a combination of oestrogen and progesterone may have a small attenuating effect on cardiovascular reactivity (Eikelis & Van Den Buuse 2000).

In summary, BP is improved in genetic hypertensive rats a few days after bilateral ovariectomy causing cardiac hypertrophy and worst myocardial remodelling. This condition is efficiently treated with enalapril, suggesting the RAS involvement in this hypertension. The more important aspect of the treatment is an enhancement of the density of the intramyocardial vessels.

Acknowledgments

The authors thank Tathiany de Souza Marinho and Ana Claudia Viana Soares for their technical assistance. The authors are grateful to referee for excellent reviewing the manuscript and contributing valuable comments. The Brazilian agencies CNPq and Faperj support the Laboratory of Morphometry & Cardiovascular Morphology.

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