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Mcc- fa cp Rats
J Mol Cell Cardiol 31, 1527–1537 (1999) Article No. jmcc.1999.0985, available online at http://www.idealibrary.com on
Effect of Ovariectomy and Estrogen Replacement on Cardiovascular Disease in Heart Failure-Prone SHHF/Mcc-fa cp Rats Leslie C. Sharkey1, Bethany J. Holycross2, Sonhee Park3, Laura J. Shiry1, Toni M. Hoepf1, Sylvia A. McCune3 and M. Judith Radin1 1 3
Department of Veterinary Biosciences, 2Department of Medical Biochemistry, Department of Food Science and Technology, The Ohio State University, Columbus, OH 43210, USA
(Received 9 November 1998, accepted in revised form 13 May 1999) L. C. S, B. J. H, S. P, L. J. S, T. M. H, S. A. MC M. J. R. Effect of Ovariectomy and Estrogen Replacement on Cardiovascular Disease in Heart Failure-Prone SHHF/Mcc-facp Rats. Journal of Molecular and Cellular Cardiology (1999) 31, 1527–1537. The importance of endogenous and exogenous estrogen levels to the development of cardiovascular disease in women in controversial. The purpose of our study was to examine the effect of estrogen on the development of hypertension, cardiac hypertrophy, ventricular function, and gene expression for atrial natriuretic peptide (ANP) and components of the renin angiotensin system in spontaneously hypertensive heart failure rats (SHHF/Mcc-facp). Development of hypertension was prevented in 3-month-old ovariectomized rats receiving subcutaneous 17b-estradiol implants (EST) compared to ovariectomized (OVX) and controls (CON). EST had the least left ventricular hypertrophy, CON were intermediate, and OVX had the most (P<0.05), correlating well with systolic blood pressure. OVX had significantly lower percentage V1 myosin isoform compared to EST and CON, indicating reversion to a more immature phenotype associated with hypertrophy. Similarly, OVX had decreased percentage left ventricular shortening fraction by echocardiography compared to EST and CON. These changes were not accompanied by alterations in plasma ANP, or in expression of mRNA for left ventricular ANP, renal renin, or hepatic angiotensinogen. Serum angiotensin converting enzyme activity was lower in EST compared to CON or OVX. When 17b-estradiol was given to 17-month-old rats that had naturally ceased estrous cycling, there was no effect on hypertension, progression of cardiac functional decline, or survival. In conclusion, estradiol treatment given prior to the development of hypertension in SHHF prevented left ventricular hypertrophy and hypertension. Development of congestive heart failure was not delayed if 17b-estradiol was begun in the post-menopausal period. Effectiveness of estrogen therapy may depend on age or whether hypertension is already established at the time treatment is 1999 Academic Press begun. K W: Hypertension; Myosin isozyme; Atrial natriuretic peptide; Renin angiotensin system; Left ventricular hypertrophy; Left ventricular fractional shortening; Estradiol; Angiotensin converting enzyme.
Introduction Despite clear epidemiological evidence that the incidence of heart disease in women increases as estrogen levels fall in the post-menopausal years, the benefits of estrogen replacement therapy are still
debated. Studies of the administration of exogenous estrogen have shown increases, decreases, or no effect on blood pressure, and improvement or no effect on cardiac contractility in women (Pines et al., 1992; Snabes et al., 1997). The inconsistency in data likely arises from the use of a wide variety
Please address correspondence to: M. Judith Radin, Department of Veterinary Biosciences, 1925 Coffey Road, Columbus, OH 43210, USA. E-mail: [email protected]
0022–2828/99/081527+11 $30.00/0
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Table 1 Body weight, organ weights, and plasma estrogen levels for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats OVX (n=6) Body weight (g) Heart weight (g) Left ventricle+septum (g) Left ventricle/heart weight (%) Atria (g) Right ventricle (g) Heart: brain weight Left ventricle: brain Uterus (g) Brain (g) Heart: body weight Left ventricle: body weight Plasma 17b-estradiol at 13 weeks of treatment (pg/ml)
EST (n=6)
294.6±8.1∗ 1.25±0.01∗ 0.88±0.03∗ 70.6±2.1∗ 0.18±0.02∗† 0.16±0.03∗ 0.69±0.01∗ 0.49±0.01∗ 0.17±0.03∗ 1.80±0.02∗ 0.426±0.010∗ 0.294±0.011∗ 27.70±2.8∗
176.3±4.7† 0.87±0.03† 0.53±0.01† 61.9±2.2† 0.13±0.02∗ 0.09±0.01∗ 0.51±0.01† 0.32±0.01† 0.44±0.03† 1.68±0.03† 0.462±0.032∗ 0.302±0.007∗ 182±7.15†
CON (n=5) 236.8±6.6‡ 1.06±0.04‡ 0.73±003‡ 68.8±1.1∗† 0.21±0.01† 0.11±0.01∗ 0.61±0.02‡ 0.42±0.01∗† 0.53±0.05† 1.75±0.04∗† 0.446±0.011∗ 0.310±0.004∗ 63.75±9.25‡
Within rows, data with different symbols are significantly different (P<0.05), data that share a symbol within the same row are not statistically different.
of estrogen preparations, and the inability to control other important factors like diet, exercise, and smoking that affect cardiovascular health. Estrogen treatment of spontaneously hypertensive rats (SHR) was associated with a significant reduction in the degree of hypertension, but this treatment had no effect on blood pressure in normotensive Wistar–Kyoto rats (Hoeg et al., 1977). A study of Wistar rats showed that estrogen replacement reversed deleterious effects of ovariectomy on percent V1 isozymes and heart function (Scheuer et al., 1987). Our study is designed to examine the effect of ovariectomy and ovariectomy with estrogen supplementation on cardiac hypertrophy and function in the lean female SHHF/Mcc-facp (abbreviated SHHF) rat. We will simultaneously evaluate the expression of the atrial natriuretic peptide (ANP) gene in the left ventricle, renal renin expression, and hepatic angiotensinogen expression and angiotensin converting enzyme activity to determine if changes in these neurohumoral systems correlate with estrogen treatment and any associated differences in the cardiovascular system. Additionally, we will determine if estradiol supplementation beginning when the rats naturally cease estrus cycling delays onset of congestive heart failure. All SHHF rats spontaneously develop hypertension, cardiac hypertrophy, activation of neurohumoral systems, and terminal congestive heart failure as animals age in the absence of dietary or surgical interventions, making them a superior model for human heart disease (McCune et al., 1995; Holycross et al., 1997). As in humans, SHHF females progress to heart failure later than SHHF males. Females typically
show decline in ventricular function and signs of congestive heart failure after they are no longer demonstrating estrous cycles (Sharkey et al., 1998). This strain provides a unique model to study the effects of ovarian hormone loss in a “high-risk” population. We hypothesize that the development of hypertension, left ventricular hypertrophy, and impaired left ventricular systolic function will be accelerated in the ovariectomized animals compared to control rats, and that estrogen supplementation will prevent these changes. Estrogen status may also alter the transcription of genes that contribute to the regulation of cardiovascular function such as ANP, renin, and angiotensinogen.
Materials and Methods Animals Lean female spontaneously hypertensive heart failure (SHHF/Mcc-facp) rats were obtained from Dr Sylvia McCune’s breeding colony at The Ohio State University. These rats were housed two or three to a cage in shoe-box-style polycarbonate cages on corn cob bedding in a temperature controlled room with a 12-h light–dark cycle. Rats were fed ad libitum Prolab Rat/Mouse/Hamster diet (Agway, Syracuse, NY, USA). Water was provided free choice in water bottles. The animal facility is AAALACaccredited and the animal protocol defining experimental procedures was approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.
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Duograph (Model ICT-2H, Middleton, WI, USA). Three acceptable tracings were obtained for each animal, and the values were averaged.
Blood collection and analysis
Figure 1 Fasted body weight in grams for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05, all three groups are significantly different.
Experiment 1 At the beginning of the study, 3-month-old rats had baseline systolic blood pressure measurements taken by the tail-cuff method, and baseline samples of serum and plasma were collected after a 24-h fast. Additionally, all rats were documented to be having normal estrous cycles by vaginal cytology. The rats were grouped by threes according to initial blood pressure measurements, and then randomly assigned to ovariectomy (OVX, n=6), ovariectomy with estrogen replacement therapy (EST, n=6), or control groups (CON, n=5). EST rats received a subcutaneous 60-day release hormone implant pellet containing 1.5 mg of 17-b estradiol (Innovative Research of America, Sarasota, FL, USA) placed in the interscapular region at the time they were ovariectomized. OVX and CON rats received 1.5mg placebo pellets. Pellets were replaced every 60 days for the duration of the study. Approximately every 4 weeks, blood pressure measurements were performed in the morning before placing the rats in metabolic cages. Rats were bled from the tail for fasted levels of atrial natriuretic peptide. M-mode echocardiograms were performed at 22 weeks after ovariectomy. Animals were killed after 24 weeks of treatment (9 months of age) by intraperitoneal injection of 100 mg/kg pentobarbital. At the end of the study, organs were weighed, and tissue samples collected, and terminal blood samples were collected by cardiac puncture.
Blood pressure measurement Conscious resting tail-cuff blood pressures were taken every 4 weeks for 24 weeks using a Gilson
Blood samples were collected following a 24-h fast. Tubes for plasma collection contained lithium heparin and aprotinin. Plasma ANP concentrations were determined at 0, 4, 12, and 24 weeks using a RIA kit (Peninsula Laboratories, Inc., Belmont, CA, USA) modified by the method of Radin et al. (1992). Serum 17b-estradiol levels were measured after 13 weeks, corresponding to 30 days after the most recent pellet implantation. 17b-estradiol was determined using a RIA kit (Coat-A-Count Estradiol-6, Diagnostic Products Corporation, Los Angeles, CA, USA). Serum angiotensin converting enzyme (ACE) activity was measured at 8 and 24 weeks by detecting generation of hippuric acid from hippuryl--histidyl--leucine (Lieberman, 1975).
Ovariectomy procedure and hormone implants Seven mg of ketamine (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA, USA) and 0.7 mg xylazine (Rompun, Bayer Corporation, Shawnee Mission, KS, USA) per 100 g body weight were administered intraperitoneally. For the ovariectomy groups, the ovaries were exteriorized, ligated, and removed via bilateral paralumbar incisions, which were closed with wound clips. Additionally, a small area of the dorsal interscapular region was clipped and disinfected with alcohol. A 1-cm incision was made with scissors, and a hemostat was used to undermine a short distance under the skin and place a subcutaneous hormone pellet in the EST rats or placebo pellet in the OVX and CON. The wounds were closed with wound clips. Vaginal cytology, plasma estrogen levels, and examination of the ovaries and uterus at necropsy verified successful ovariectomy and estrogen treatment.
Echocardiograms Echocardiograms were performed on a Sonos echocardiograph machine using a 7.5 megahertz pediatric transducer (Hewlett-Packard, Inc, Waltham, WA, USA). M-mode measurements were taken of systolic and diastolic left ventricular internal dimensions and R–R interval. Restraint was provided by intraperitoneal injection of 5.0 mg ketamine and 0.5 mg xylazine per 100 g body weight. The left ventricular shortening fraction and heart rate in
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RNA isolation and Northern blot analysis
Figure 2 Systolic blood pressure by the tail cuff method in mmHg for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05 for EST different from OVX and CON, #P<0.05 for differences between all three groups.
beats/min were calculated from measured parameters.
Tissue collection Heart, kidneys, liver, uterus, and the brain were weighed. Tissue samples were quick frozen on dry ice and stored at −70°C. Once the whole heart weight was measured, the atria were separated by a single cut with a razor blade and weighed together. The right ventricle was removed by cutting along its attachment to the left ventricle and septum and weighed separately. The left ventricle and septum were weighed as a single unit. A single cross-section of the intact left ventricle and septum was made at approximately the level of the left coronary artery, and saved in 10% neutral buffered formalin. The remainder of the heart was frozen immediately on dry ice. All animals were examined for the presence or absence of ovaries. The same investigator (LCS) did all prosections. Formalin fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin and Masson’s trichrome strains.
Myosin isozymes Isolation and separation of myosin isozymes from the left ventricle were done following the method of Hoh et al. (1978). Myosin extracts were prepared and stored at −70°C until analysis by 3.5% polyacrylamide gel electrophoresis using non-dissociating conditions. Gels were stained with Coomasie blue and quantitated by a laser densitometer (LKB Gelscan XL, LKB Probkter AB, Bromma, Sweden).
Total RNA was extracted from quick-frozen left ventricle, kidney, and liver samples by the method of Chirgwin et al. (1979) and stored at −70°C. Northern blot analysis was done to identify and quantitate mRNA for renal renin, hepatic angiotensinogen, and left ventricular proANP as previously described (Sharkey et al., 1998). Full-length complementary DNA (cDNA) probes for rat renin (1.4 kb) and rat angiotensinogen (1.7 kb) were generously provided by Dr Kevin Lynch, University of Virginia, Charlottesville, VA, USA (Burnham et al., 1987). Dr Christopher Glembotski, San Diego State University, San Diego, CA, USA provided the full length cDNA probe for ANP (Glembotski et al., 1987). Expression of ANP mRNA in the left ventricle was normalized to a constitutively expressed mRNA, glyceraldehyde dehydrogenase (GAPDH). A 905 bp cDNA probe for GAPDH was acquired from Ambion, Incorporated (Austin, TX, USA) and is reported to have a high degree of binding for rat mRNA (Sabath et al., 1990). Dried blots were wrapped in plastic wrap and placed on a Molecular Dynamic’s Phosphor ImagerTM, and ImageQuantTM Software was used for densitometric analysis. After initial analysis, blots were stripped by boiling for 10–15 min in 0.1× SSPE. Successful stripping was confirmed by placing the blots on a Phosphor imager for 24-h, resulting in no image on scanning. The blots were then considered ready for constitutive mRNA probing.
Experiment 2 Sixteen 13-month-old lean female SHHF rats were initially documented to be having normal estrous cycles by vaginal cytology and were then monitored until cycling ceased naturally at 17 months of age. Previous studies have shown that this is the age at which endogenous serum estrogen declines to levels comparable to that observed with ovariectomy (Sharkey et al., 1998). At that time, baseline systolic blood pressure and left ventricular shortening fractions were determined as described above. Rats were paired by left ventricular fractional shortening and randomly assigned to either estradiol supplementation therapy (HRT, n=8) or control menopausal (MENO, n=8) groups. HRT rats were subcutaneously implanted with a 90-day release pellet containing 1.5 mg of 17b-estradiol (Innovative Research of American, Sarasota, FL, USA), while MENO received 1.5 mg placebo pellets. Pellets were replaced every 90 days for the duration of the
Ovariectomy and Estrogen Replacement in SHHF
Figure 3 V1% myosin isozyme in ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05 for OVX v. EST and CON.
experiment. Systolic blood pressure measurements and echocardiography were repeated every 45 days until the rats developed congestive heart failure, and either died spontaneously or were euthanized due to distress. Serum ACE activity was measured at 18.5 and 23 months of age. Organ weights were measured at the end of the study.
Statistical analysis Data are expressed as the mean±standard error of the mean; unless otherwise specified, all comparisons were done using ANOVA. Differences were reported as significant if the value of P<0.05 and the Tukey–Kramer Multiple Comparisons post-test was used for mean separation. The Kaplan–Meier curves were constructed and the log rank test was used to compare the survival of the HRT and MENO rats that were in terminal congestive heart failure at the time of death. Organ weights in Experiment 2 were compared using the Student’s t-test.
Results Experiment 1 As determined by vaginal cytology, all CON rats were cycling normally, while OVX rats were persistently in diestrus or proestrus, and EST rats were in continuous estrus. The success of the ovariectomy procedure was further confirmed by examination of the reproductive tract at necropsy. OVX rats lacked ovaries and had grossly atrophic uteri that weighed significantly less than CON and EST rats (Table 1). All OVX rats showed histologic
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Figure 4 Left ventricular fractional shortening percentage by M-mode echocardiography for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05 for OVX v. EST and CON.
evidence of uterine atrophy compared to EST and CON. There was no evidence of uterine neoplasia in any of the animals. 17b-Estradiol levels were determined to confirm the efficacy of treatment. Levels were measured at 13 weeks, corresponding to 30 days after the implantation of a 60-day pellet (Table 1). At this time, OVX had the lowest 17bestradiol levels, CON were significantly higher than OVX, and EST were higher than the other two groups. Effective estrogen replacement at the end of the study was demonstrated by the normalization of uterine weights in the 17b-estradiol treated ovariectomized rats, with EST and CON rats having similar uterine weights that were significantly greater than OVX. All three groups had similar fasted body weights at the beginning of the study, but by 4 weeks and, until the end of the study, the weights were significantly different between groups. OVX were the heaviest, CON were intermediate, and EST weighed the least (Fig. 1). The heart weights were significantly different between all three groups when compared as gross weight or relative to brain weights, with the OVX having the largest hearts and the EST the smallest (Table 1). The weights of the left ventricles and septa showed a similar pattern. The left ventricle and septum also made up proportionately more of the total heart weight in OVX compared to EST. The atria in the EST were significantly lighter than CON, but were not different from OVX. In contrast, the right ventricles were similar in all groups. Histologic evaluation of the left ventricle and septum showed no differences in myocardial fibrosis, which was minimal in all animals. Treatment also affected cardiovascular functional parameters. By 1 month after treatment, EST had
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expressed as a ratio to constitutive GAPDH expression (Fig. 5). EST significantly lowered serum ACE activity by 28 and 22% compared to CON at 8 and 24 weeks, respectively (Table 2). Conversely, OVX significantly increased serum ACE activity by 23 and 15% compared to CON at those same times (Table 2). There were no differences in hepatic angiotensinogen mRNA expression or renal renin mRNA expression relative to GAPDH at the end of 24 weeks (Table 2).
Experiment 2
Figure 5 Plasma atrial natriuretic peptide (ANP) in pg/ ml in ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. No differences were detected between the groups at any time. +P<0.05 for 24 week v. baseline (0 week) values for all groups. The table shows left ventricular ANP mRNA expression normalized to GAPDH. No differences were detected between groups.
significantly lower systolic blood pressure than OVX or CON, and this difference persisted throughout the study (Fig. 2). OVX and CON had similar pressures except for week 20, when CON blood pressure was significantly lower than OVX, but significantly higher than EST. Therefore, estrogen treatment prevented the increase in systolic blood pressure seen in the CON and OVX groups over the study period. OVX had significantly lower V1 percentage isozyme compared to EST and CON (Fig. 3), lower left ventricular shortening fraction (Fig. 4). CON and EST had similar V1 percentage isozyme and left ventricular shortening fractions. The heart weight correlated positively with systolic blood pressure with a correlation coefficient of 0.868 (P<0.0001, 95% confidence interval 0.654 to 0.954), but did not correlate with the left ventricular fractional shortening (P=0.146) by linear regression. There were no differences in plasma ANP concentrations between OVX, EST or CON at any time point (Fig. 5). Plasma ANP concentration increased over time, as is characteristic for this strain of rat (McCune et al., 1995), and all three groups had significantly greater plasma ANP levels on week 24 when compared to their respective baseline value (week 0). As expected based on the plasma levels, there were no differences between the groups in left ventricular expression of ANP mRNA when
Estrogen supplementation was determined to be effective in that HRT rats maintained larger uteri (0.65±0.09 g) compared to MENO (0.45±0.05 g), tended to have a smaller body weights (Fig. 6), and had vaginal cytology compatible with estrogen supplementation. Both groups of rats were hypertensive at the start of the experiment [Fig. 7(a)]. Unlike the previous experiment, there was no effect of estrogen supplementation on systolic blood pressure or left ventricular shortening fraction (Fig. 7). Both groups showed a similar decline in left ventricular shortening fraction with time, and there was no significant difference between groups at the time of terminal CHF (33±2% for HRT, n=5 v. 30±6% for MENO, n=5). Whilst there was a trend for ACE activity to be lower in HRT, this did not reach significance at either 18.5 (15.05±0.92 nmol/ml/min for HRT v 16.74±0.39 nmol/ml/min for MENO, P=0.097) or 23 (15.48±1.16 nmol/ml/min for HRT v 19.78±1.51 nmol/ml/min for MENO, P=0.064) months of age. MENO rats showed a progressive increase in ACE activity over time, eventually resembling values observed in OVX rats from Experiment 1. Three MENO and two HRT rats developed tumors and died prior to the development of congestive heart failure. These were considered non-events and were not used for the following analysis. The mean survival time for HRT was 23.27 months (range 18.3–26.5, confidence interval 21.04– 25.49 months) and for MENO was 22.60 months (range 19.1–24.5, confidence interval 20.85– 24.38 months). The difference was not significant by log rank analysis, indicating that estrogen supplementation of rats that were already hypertensive and had undergone spontaneous cessation of cycling did not significantly prolong survival. There was no significant difference between groups for heart weight (2.13±0.15 g for HRT v. 2.63±0.32 g for MENO).
Ovariectomy and Estrogen Replacement in SHHF
Figure 6 Body weight in g for lean female SHHF rats supplemented with estrogen beginning at 17 months of age (HRT) and menopausal controls (MENO). There were no significant differences between the groups at any time.
Discussion Interpretation of the data on the cardiovascular effects of estrogen in people is still debated. Despite the well-documented differences between men and women in the incidence of heart disease, there is still not a consensus that estrogen is responsible for improved cardiovascular health in women during the premenopausal years. Studies of the relationship of blood pressure and natural fluctuations in estrogen levels show decreased blood pressure in the mid-trimester of pregnancy coincident with a massive increase in total estrogen (Swartz et al., 1995) and an increase in blood pressure during menopause when estrogen levels are falling (Bittner, 1995). These patterns would suggest that estrogen might influence the regulation of blood pressure. It is extremely difficult to evaluate the effects of exogenous estrogen in women due to the variety of preparations available, patterns of use, and the presence of numerous other factors such as obesity, smoking, diet, and exercise that also have significant impact on cardiovascular health but are difficult to control. Despite these difficulties, several studies have shown that post-menopausal estrogen
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treatment decreases blood pressure (Utian et al., 1978; Lind et al., 1979). Another author observed the effect of particular preparations of replacement on blood pressure, and found that equine estrogens and artificial estrogens administered with progestins increase pressure, while other estrogens do not (Jesperson, 1997). In order to clarify the effects of estrogen on blood pressure in a more controlled setting than is possible with human subjects, the effects of estrogen treatment on hypertension have been investigated in several hypertensive strains of rats. As in our study with the SHHF rat, treatment of intact female SHRSP with 17b-estradiol or of female SHR with mestranol resulted in a significant reduction in the degree of hypertension attained compared to placebo-treated animals (Hoeg et al., 1977; Eiff et al., 1985). The pressures of normotensive female Wistar–Kyoto rats were not affected by the same therapy, suggesting that estrogen may have a more pronounced effect on blood pressure in hypertensive animals. The improved prognosis for women with congestive heart failure compared to men suggests that female hormones might support cardiac function; however, studies on the effect of estrogen replacement therapy on heart function in healthy postmenopausal women had conflicting results. One found that Doppler derived parameters of aortic flow were improved after 1 year of therapy (Pines et al., 1992), while another failed to find significant improvements in M-mode, two-dimensional, and Doppler echocardiographic analysis of heart function after 12 weeks of treatment (Snabes et al., 1997). The capacity of estrogen to support heart function in people is unclear at this time. 17b-estradiol treatment appeared to have a beneficial effect on the heart in female SHHF rats, documenting the potential for estrogen to significantly alter heart function and cardiac hypertrophy in individuals genetically predisposed to the development of cardiovascular disease. Similar to our
Table 2 Serum ACE activity after 8 and 24 weeks of treatment and expression of renal renin and hepatic angiotensinogen mRNA relative to GAPDH expression at 24 weeks of treatment for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats from Experiment 1.
Group
ACE activity 8 weeks (nmol/ml/min)
ACE activity 24 weeks (nmol/ml/min)
Renin: GAPDH 24 weeks
Angiotensinogen: GAPDH 24 weeks
OVX EST CON
20.90±0.94∗ 12.28±0.97† 16.96±1.20‡
19.67±0.50∗ 13.22±0.28† 17.02±0.95‡
0.341±0.044∗ 0.324±0.030∗ 0.353±0.039∗
3.179±0.281∗ 3.050±0.193∗ 2.300±0.110∗
Within columns, data with different symbols are significantly different (P<0.05) data that share a symbol within the same column are not statistically different.
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Figure 7 (a) Systolic blood pressure by tail method in mmHg; and (b) left ventricular fractional shortening (%) for lean female SHHF rats supplemented with estrogen beginning at 17 months of age (HRT) and menopausal controls (MENO). There were no significant differences between the groups at any time.
study, Schaible et al. (1984) found that ovariectomy of Wistar rats resulted in a reduction in V1 myosin isozyme and calcium-myosin ATPase activity. Decreased V1 percentage and increased V3 percentage myosin isozymes are associated with pathologic left ventricular hypertrophy (Buttrick et al., 1994). In contrast, exercise-associated physiologic hypertrophy in female rats is associated with increase in V1 percentage (Geenen et al., 1996). Several studies have documented depression of cardiac function in isolated hearts from ovariectomized rats (Schaible et al., 1984; Scheuer et al., 1987) and prevention of the myosin shift and cardiac dysfunction by estrogen administration (Scheuer et al., 1987). We were able to confirm the in vivo significance of these estrogen-associated cardiac functional differences in failure prone SHHF rats by the use of echocardiography. Estrogen could exert effects on the cardiovascular system by a number of mechanisms, including modulating the activity of the renin angiotensin system (RAS). SHHF rats show a progressive activation of the RAS, which peaks as the rats develop terminal congestive heart failure (Holycross et al., 1997). In this study, OVX, EST, and CON rats had comparable hepatic angiotensinogen mRNA and renal renin mRNA expression despite significantly lower blood pressures in EST compared to OVX and CON. A similar lack of effect on renal renin and hepatic angiotensinogen gene expression was also seen when SHHF were ovariectomized after onset of hypertension (Sharkey et al., 1998). Women taking oral estrogens have a consistent, marked increase in plasma angiotensinogen; however, this increase is not observed with transdermal preparations (Adashi et al., 1996). Concentration of
oral estrogens in the liver as the result of a substantial first pass effect, as well as the presence of an estrogen responsive sequence in the 5′-flanking region of the promoter inducing angiotensinogen transcription most likely contribute to this effect (Feldmer et al., 1991). Because our treatment was administered subcutaneously, we did not anticipate a marked estrogenic effort on hepatic angiotensinogen expression. Likewise, we did not observe differences between the groups in renal renin expression. Estrogen has the potential to influence the transcription of renin through regulatory sequences analogous to estrogen-receptor binding sites in the 5′-flanking region of the gene (Fukamizu et al., 1988). Ovariectomy and estrogen supplementation has been shown to have variable effects on gene expression in other rat strains. Ovariectomy was not associated with changes in renal renin or hepatic angiotensinogen gene expression in stroke-prone SHR (Bachmann et al., 1993); however, both were decreased in ovariectomized Okamoto SHR (Chen et al., 1992). Estrogens have been shown to decrease ACE activity by approximately 20% in post-menopausal women (Proudler et al., 1995) and in monkeys (Brosnihan et al., 1997). Estradiol administration to ovariectomized SHHF in experiment 1 resulted in a similar 22–28% decrease in ACE activity compared to controls. Spontaneous cessation of cycling in older SHHF resulted in a gradual increase in ACE activity, eventually resulting in values comparable to those obtained by SHHF that were ovariectomized at a young age (OVX, experiment 1). Administration of estradiol to menopausal rats resulted in maintenance of premenopausal levels of ACE activity, but did not lower ACE activity or modify already
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established hypertension. Ultimately, development of congestive heart failure was not significantly delayed in those SHHF who began estradiol treatment in the perimenopausal period. Similarly, in a previous study, decline of cardiovascular function and death from congestive heart failure was not hastened when SHHF were ovariectomized after the development of hypertension but prior to menopause (Sharkey et al., 1998). The role of ACE in maintenance of hypertension and development of congestive heart failure remains uncertain. It has been suggested that hypertension may be more important than ACE activity in promoting cardiac hypertrophy (Yoshida et al., 1988). Our studies suggest that estradiol treatment of young SHHF may prevent development of hypertension in association with lowering of ACE activity; however, a cause and effect relationship is not certain and requires further study. Once hypertension has become established, estradiol therapy alone may be inadequate to lower blood pressure. However, possible benefits of estrogen therapy in conjunction with other antihypertensive measures remain to be explored. Estrogen could modify the effects of the RAS by several other mechanisms that were not evaluated in this experiment. Estrogens modify the number of angiotensin subtype 1 receptors in a tissuespecific manner that may effect fluid balance and blood pressure (Seltzer et al., 1992). Changes in sensitivity to components of the renin angiotensin system may also occur. For example, chronic estrogen treatment significantly decreases angiotensin II-induced contractile responsiveness of rat aortic rings (Cheng and Gruelter, 1992). In addition to the RAS, the natriuretic peptides make up another neuroendocrine system that can impact cardiovascular homeostasis. ANP is of interest in the study of hypertension and heart disease because of its potential to antagonize the RAS, and its effects on plasma volume and blood pressure via natriuretic, diuretic, and vasorelaxant properties. There are consistent increases in the plasma levels of ANP in people with left ventricular dysfunction and in SHHF rats as they progress through cardiac hypertrophy to failure (Francis et al., 1990; McCune et al., 1995; Arai et al., 1988). Augmented expression of ANP mRNA in the left ventricles of SHHF rats accompanies the increase in plasma concentrations associated with hypertension and cardiac hypertrophy (McCune et al., 1995). Ovariectomy and estrogen treatment failed to cause changes in plasma ANP concentration or ANP mRNA expression in the left ventricle relative to control animals, despite significant differences in
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blood pressure and cardiac hypertrophy. Data demonstrating increases in plasma ANP in women taking oral contraceptives or who are pregnant suggest that estrogen, or increases in blood volume induced by estrogen, could be associated with changes in plasma ANP (Davidson et al., 1988; Sala et al., 1995). In summary, estradiol status had a significant impact on cardiac hypertrophy and cardiovascular function when examined in lean female SHHF rats prior to the development of hypertension. Our data show that ovariectomy of prehypertensive heart failure-prone SHHF results in greater cardiac hypertrophy and impaired cardiac function. The administration of subcutaneous 17b-estradiol prevented ovariectomy-associated decreases in left ventricular shortening fraction and V1 percentage myosin isozyme, but the data do not indicate that exogenous estrogen improves left ventricular function compared to normally cycling, failure-prone rats. Heart function and myosin isozymes of EST remain comparable to CON. Interestingly, 17b-estradiol treatment prevented the rise in blood pressure normally observed in this hypertensive rat strain, resulting in significantly lower pressures in EST compared to OVX and even CON animals. This suggests that hormone supplementation may improve blood pressure control beyond merely counteracting the effects of the removal of the ovaries. The changes in heart size and function associated with estrogen status were not associated with significant differences in renal renin, hepatic angiotensinogen, or left ventricular ANP mRNA expression, but were associated with decreases in ACE activity. The mechanisms by which estrogen improved cardiovascular parameters in ovariectomized SHHF may relate to improved blood pressure control. If estradiol administration was started in the perimenopausal period, after hypertension was established, cardiac hypertrophy, decline in cardiovascular function, and onset of congestive heart failure was not delayed. Further work is needed to clarify the mechanisms by which estrogen works in SHHF rats.
Acknowledgements This work was supported in part by US Public Health Service Grant HL48835. Salaries and research support was also provided in part by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, Ohio State University.
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References A EY, 1996. Transdermal estrogen replacement. In: Adashi EY, Rock JA, Rosenwaks Z (eds). Reproductive Endocrinology, Surgery and Technology. Philadelphia: Lippincott Raven Publishers. 1784–1795. A H, N K, S Y, M N, S A, Y Y, I H, S S, M M, O H, N S, I H, 1988. Augmented expression of atrial natriuretic peptide in ventricles of spontaneously hypertensive rats (SHR) and SHR-stroke prone. Circ Res 62: 926–930. B J, W J, H C, W A, C A, G D, 1993. Modulation of blood pressure and the renin-angiotensin system in transgenic and spontaneously hypertensive rats after ovariectomy. J Hypertens 11(Suppl. 5): S226– S227. B V, 1995. Hypertension in Women. Curr Op. Neph Hypertens 92: 3431–3435. B KB, W D, A MS, H C, L P, F CM, 1997. Effects of chronic hormone replacement on the renin-angiotensin system in cynomolgus monkeys. J Hypertension 15: 719–726. B C, H-J C, F B, L K, 1987. Molecular cloning of rat renin cDNA and its gene. Proc Natl Acad Sci USA 84: 602–609. B PM, K M, L LA, S J, 1994. Alterations in gene expression in the rat heart after chronic pathological and physiological loads. J Mol Cell Cardiol 26: 61–67. C Y, N A, O S, 1992. Androgen-dependent angiotensinogen and renin messenger RNA expression in hypertensive rat. Hypertension 19: 456– 463. C DY, G CA, 1992. Chronic estrogen alters contractile responsiveness to AII and norepinephrine in the female rat aorta. Eur J Pharm 215: 171–176. C J, P A, MD R, R W, 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299. D B, R CD, V GJ, 1988. Atrial natriuretic peptide, plasma renin activity, and aldosterone in women on estrogen therapy and with premenstrual syndrome. Fertility and Sterility 50: 743–746. E AW, L HM, K R, 1985. The protective effect of estrogen on high blood pressure. Basic Res Cardiol 80: 191–201. F M, K M, T S, M JJ, G D, 1991. Glucocorticoid and estrogen responsive elements in the 5′ flanking region of the rat angiotensinogen gene. J Hypertens 9: 1005–1012. F GS, B C, J DE, K PC, N H, L C, K SH, R-T E, Y S, 1990. Comparison of neuroendocrine activation in patients with left ventricular dysfunction with and without congestive heart failure. Circulation 82: 1724– 1729. F A, N K, C T, S M, N K, O H, N S, M K, 1988. Structure of the rat renin gene. J Mol Biol 201: 443–450. G DL, M A, B PM, 1996. Angiotensin receptor 1 blockade does not prevent physiological cardiac hypertrophy in the adult rat. J Appl Phys 81: 816–821.
G C, O M, L X, S P, H P, K R, G R, 1987. The characterization of atrial natruiuretic peptide expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe. Endocrinol Biol 121: 843–853. H JM, W LR, W MH, 1977. Estrogen attenuation of the development of hypertension in spontaneously hypertensive rats. Am J Physiol 233: H369–H373. H J, MG P, H P, 1978. Electrophoretic analysis of multiple forms of rat cardiac myosins; effects of hypophysectomy and thyroxin replacement. J Mol Cell Cardiol 11: 1053–1078. H B, S B, D R, MC S, 1997. Plasma renin activity in heart failure prone (SHHF/ Mcc-facp) rats. Am J Physiol 273: H228–H233. J CM, 1997. Hormone replacement therapy and high blood pressure. Am J Hypertens 10: 366–367. L J, 1975, Elevation of serum angiotensin-converting-enzyme (ACE) level in sarcoidosis. Am J Med 59: 365–372. L T, C EC, H WM, L C, M PG, O Q, G JA, 1979. Prospective controlled trial of six forms of hormone replacement therapy given to postmenopausal women. Br J Obset Gyn 86: 1–29. MC S, P S, R M, J R, 1995. SHHF/Mccfacp rat model: a genetic model of congestive heart failure. In: Singal P, Dixon I, Beamish R, Dhalla R (eds). Mechanisms of Heart Failure. Kluwer Academic Publishers, 91–106. P A, F EZ, A D, D Y, A M, L Y, 1992. Long-term effects of hormone replacement therapy on Doppler derived parameters of aortic flow in postmenopausal women. Chest 102: 1496–1498. P AJ, H-A AI, C D, F I, R JM, S JC, 1995. Hormone replacement therapy and ACE activity in postmenopausal women. Lancet 346: 89–90. R MJ, J J, MC SA, H R, 1992. Effects of enalapril and clonidine on glomerular structure, function, and atrial natriuretic peptide receptors in SHHF/Mcc-facp rats. J Cardiovasc Pharmacol 19: 464– 472. S D, B H, P M, 1990. Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned Thelper lymphocyte. Gene 91: 185–191. S C, C M, A G, M T, Z A, M L, 1995. Atrial Natriuretic peptide and hemodynamic changes during normal human pregnancy. Hypertension 25 (part 1): 631–636. S TF, M A, C C, S J, 1984. The effects of gonadectomy on left ventricular function and cardiac contractile proteins in male and female rats. Circ Res 54: 38–49. S J, M A, S TF, C J, 1987. Effects of gonadectomy and hormonal replacement on rat hearts. Circ Res 61: 12–19. S A, P JEP, V PN, C FMA, L C, S T, S MK, S JM, 1992. Estrogens regulate ACE and angiotensin receptors in female rat anterior pituitary. Neuroendocrinology 55: 460–467.
Ovariectomy and Estrogen Replacement in SHHF S LC, H BJ, P S, MC SA, H R, R MJ, 1998. Effect of ovariectomy in heart failure-prone SHHF/Mcc-facp rats. Am J Physiol 275: R1968–R1976. S MC, P JP, K HA, D JK, Y R, Z WA, 1997. Physiologic estradiol replacement therapy and cardiac structure and function in normal postmenopausal women: a randomized double blind, placebo controlled, crossover trial. Obstet Gyn 89: 332– 339. S, J, F R, F W 1995. Clinical
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pharmacology of estrogens: cardiovascular actions and cardioprotective benefits of replacement therapy on postmenopausal women. J Clin Pharmacol 35: 1–16. U WH, 1978. Effect of postmenopausal estrogen therapy on diastolic blood pressure and body weight. Maturitas 34: 3–8. Y K, K M, C DJ, J CI, 1998. Angiotensin-converting enzyme inhibition and salt in experiment myocardial infarction. J Cardiovasc Pharmacol 32: 357–365.
Effect of Ovariectomy and Estrogen Replacement on Cardiovascular Disease in Heart Failure-Prone SHHF/Mcc-fa cp Rats Leslie C. Sharkey1, Bethany J. Holycross2, Sonhee Park3, Laura J. Shiry1, Toni M. Hoepf1, Sylvia A. McCune3 and M. Judith Radin1 1 3
Department of Veterinary Biosciences, 2Department of Medical Biochemistry, Department of Food Science and Technology, The Ohio State University, Columbus, OH 43210, USA
(Received 9 November 1998, accepted in revised form 13 May 1999) L. C. S, B. J. H, S. P, L. J. S, T. M. H, S. A. MC M. J. R. Effect of Ovariectomy and Estrogen Replacement on Cardiovascular Disease in Heart Failure-Prone SHHF/Mcc-facp Rats. Journal of Molecular and Cellular Cardiology (1999) 31, 1527–1537. The importance of endogenous and exogenous estrogen levels to the development of cardiovascular disease in women in controversial. The purpose of our study was to examine the effect of estrogen on the development of hypertension, cardiac hypertrophy, ventricular function, and gene expression for atrial natriuretic peptide (ANP) and components of the renin angiotensin system in spontaneously hypertensive heart failure rats (SHHF/Mcc-facp). Development of hypertension was prevented in 3-month-old ovariectomized rats receiving subcutaneous 17b-estradiol implants (EST) compared to ovariectomized (OVX) and controls (CON). EST had the least left ventricular hypertrophy, CON were intermediate, and OVX had the most (P<0.05), correlating well with systolic blood pressure. OVX had significantly lower percentage V1 myosin isoform compared to EST and CON, indicating reversion to a more immature phenotype associated with hypertrophy. Similarly, OVX had decreased percentage left ventricular shortening fraction by echocardiography compared to EST and CON. These changes were not accompanied by alterations in plasma ANP, or in expression of mRNA for left ventricular ANP, renal renin, or hepatic angiotensinogen. Serum angiotensin converting enzyme activity was lower in EST compared to CON or OVX. When 17b-estradiol was given to 17-month-old rats that had naturally ceased estrous cycling, there was no effect on hypertension, progression of cardiac functional decline, or survival. In conclusion, estradiol treatment given prior to the development of hypertension in SHHF prevented left ventricular hypertrophy and hypertension. Development of congestive heart failure was not delayed if 17b-estradiol was begun in the post-menopausal period. Effectiveness of estrogen therapy may depend on age or whether hypertension is already established at the time treatment is 1999 Academic Press begun. K W: Hypertension; Myosin isozyme; Atrial natriuretic peptide; Renin angiotensin system; Left ventricular hypertrophy; Left ventricular fractional shortening; Estradiol; Angiotensin converting enzyme.
Introduction Despite clear epidemiological evidence that the incidence of heart disease in women increases as estrogen levels fall in the post-menopausal years, the benefits of estrogen replacement therapy are still
debated. Studies of the administration of exogenous estrogen have shown increases, decreases, or no effect on blood pressure, and improvement or no effect on cardiac contractility in women (Pines et al., 1992; Snabes et al., 1997). The inconsistency in data likely arises from the use of a wide variety
Please address correspondence to: M. Judith Radin, Department of Veterinary Biosciences, 1925 Coffey Road, Columbus, OH 43210, USA. E-mail: [email protected]
0022–2828/99/081527+11 $30.00/0
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Table 1 Body weight, organ weights, and plasma estrogen levels for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats OVX (n=6) Body weight (g) Heart weight (g) Left ventricle+septum (g) Left ventricle/heart weight (%) Atria (g) Right ventricle (g) Heart: brain weight Left ventricle: brain Uterus (g) Brain (g) Heart: body weight Left ventricle: body weight Plasma 17b-estradiol at 13 weeks of treatment (pg/ml)
EST (n=6)
294.6±8.1∗ 1.25±0.01∗ 0.88±0.03∗ 70.6±2.1∗ 0.18±0.02∗† 0.16±0.03∗ 0.69±0.01∗ 0.49±0.01∗ 0.17±0.03∗ 1.80±0.02∗ 0.426±0.010∗ 0.294±0.011∗ 27.70±2.8∗
176.3±4.7† 0.87±0.03† 0.53±0.01† 61.9±2.2† 0.13±0.02∗ 0.09±0.01∗ 0.51±0.01† 0.32±0.01† 0.44±0.03† 1.68±0.03† 0.462±0.032∗ 0.302±0.007∗ 182±7.15†
CON (n=5) 236.8±6.6‡ 1.06±0.04‡ 0.73±003‡ 68.8±1.1∗† 0.21±0.01† 0.11±0.01∗ 0.61±0.02‡ 0.42±0.01∗† 0.53±0.05† 1.75±0.04∗† 0.446±0.011∗ 0.310±0.004∗ 63.75±9.25‡
Within rows, data with different symbols are significantly different (P<0.05), data that share a symbol within the same row are not statistically different.
of estrogen preparations, and the inability to control other important factors like diet, exercise, and smoking that affect cardiovascular health. Estrogen treatment of spontaneously hypertensive rats (SHR) was associated with a significant reduction in the degree of hypertension, but this treatment had no effect on blood pressure in normotensive Wistar–Kyoto rats (Hoeg et al., 1977). A study of Wistar rats showed that estrogen replacement reversed deleterious effects of ovariectomy on percent V1 isozymes and heart function (Scheuer et al., 1987). Our study is designed to examine the effect of ovariectomy and ovariectomy with estrogen supplementation on cardiac hypertrophy and function in the lean female SHHF/Mcc-facp (abbreviated SHHF) rat. We will simultaneously evaluate the expression of the atrial natriuretic peptide (ANP) gene in the left ventricle, renal renin expression, and hepatic angiotensinogen expression and angiotensin converting enzyme activity to determine if changes in these neurohumoral systems correlate with estrogen treatment and any associated differences in the cardiovascular system. Additionally, we will determine if estradiol supplementation beginning when the rats naturally cease estrus cycling delays onset of congestive heart failure. All SHHF rats spontaneously develop hypertension, cardiac hypertrophy, activation of neurohumoral systems, and terminal congestive heart failure as animals age in the absence of dietary or surgical interventions, making them a superior model for human heart disease (McCune et al., 1995; Holycross et al., 1997). As in humans, SHHF females progress to heart failure later than SHHF males. Females typically
show decline in ventricular function and signs of congestive heart failure after they are no longer demonstrating estrous cycles (Sharkey et al., 1998). This strain provides a unique model to study the effects of ovarian hormone loss in a “high-risk” population. We hypothesize that the development of hypertension, left ventricular hypertrophy, and impaired left ventricular systolic function will be accelerated in the ovariectomized animals compared to control rats, and that estrogen supplementation will prevent these changes. Estrogen status may also alter the transcription of genes that contribute to the regulation of cardiovascular function such as ANP, renin, and angiotensinogen.
Materials and Methods Animals Lean female spontaneously hypertensive heart failure (SHHF/Mcc-facp) rats were obtained from Dr Sylvia McCune’s breeding colony at The Ohio State University. These rats were housed two or three to a cage in shoe-box-style polycarbonate cages on corn cob bedding in a temperature controlled room with a 12-h light–dark cycle. Rats were fed ad libitum Prolab Rat/Mouse/Hamster diet (Agway, Syracuse, NY, USA). Water was provided free choice in water bottles. The animal facility is AAALACaccredited and the animal protocol defining experimental procedures was approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee.
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Duograph (Model ICT-2H, Middleton, WI, USA). Three acceptable tracings were obtained for each animal, and the values were averaged.
Blood collection and analysis
Figure 1 Fasted body weight in grams for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05, all three groups are significantly different.
Experiment 1 At the beginning of the study, 3-month-old rats had baseline systolic blood pressure measurements taken by the tail-cuff method, and baseline samples of serum and plasma were collected after a 24-h fast. Additionally, all rats were documented to be having normal estrous cycles by vaginal cytology. The rats were grouped by threes according to initial blood pressure measurements, and then randomly assigned to ovariectomy (OVX, n=6), ovariectomy with estrogen replacement therapy (EST, n=6), or control groups (CON, n=5). EST rats received a subcutaneous 60-day release hormone implant pellet containing 1.5 mg of 17-b estradiol (Innovative Research of America, Sarasota, FL, USA) placed in the interscapular region at the time they were ovariectomized. OVX and CON rats received 1.5mg placebo pellets. Pellets were replaced every 60 days for the duration of the study. Approximately every 4 weeks, blood pressure measurements were performed in the morning before placing the rats in metabolic cages. Rats were bled from the tail for fasted levels of atrial natriuretic peptide. M-mode echocardiograms were performed at 22 weeks after ovariectomy. Animals were killed after 24 weeks of treatment (9 months of age) by intraperitoneal injection of 100 mg/kg pentobarbital. At the end of the study, organs were weighed, and tissue samples collected, and terminal blood samples were collected by cardiac puncture.
Blood pressure measurement Conscious resting tail-cuff blood pressures were taken every 4 weeks for 24 weeks using a Gilson
Blood samples were collected following a 24-h fast. Tubes for plasma collection contained lithium heparin and aprotinin. Plasma ANP concentrations were determined at 0, 4, 12, and 24 weeks using a RIA kit (Peninsula Laboratories, Inc., Belmont, CA, USA) modified by the method of Radin et al. (1992). Serum 17b-estradiol levels were measured after 13 weeks, corresponding to 30 days after the most recent pellet implantation. 17b-estradiol was determined using a RIA kit (Coat-A-Count Estradiol-6, Diagnostic Products Corporation, Los Angeles, CA, USA). Serum angiotensin converting enzyme (ACE) activity was measured at 8 and 24 weeks by detecting generation of hippuric acid from hippuryl--histidyl--leucine (Lieberman, 1975).
Ovariectomy procedure and hormone implants Seven mg of ketamine (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA, USA) and 0.7 mg xylazine (Rompun, Bayer Corporation, Shawnee Mission, KS, USA) per 100 g body weight were administered intraperitoneally. For the ovariectomy groups, the ovaries were exteriorized, ligated, and removed via bilateral paralumbar incisions, which were closed with wound clips. Additionally, a small area of the dorsal interscapular region was clipped and disinfected with alcohol. A 1-cm incision was made with scissors, and a hemostat was used to undermine a short distance under the skin and place a subcutaneous hormone pellet in the EST rats or placebo pellet in the OVX and CON. The wounds were closed with wound clips. Vaginal cytology, plasma estrogen levels, and examination of the ovaries and uterus at necropsy verified successful ovariectomy and estrogen treatment.
Echocardiograms Echocardiograms were performed on a Sonos echocardiograph machine using a 7.5 megahertz pediatric transducer (Hewlett-Packard, Inc, Waltham, WA, USA). M-mode measurements were taken of systolic and diastolic left ventricular internal dimensions and R–R interval. Restraint was provided by intraperitoneal injection of 5.0 mg ketamine and 0.5 mg xylazine per 100 g body weight. The left ventricular shortening fraction and heart rate in
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RNA isolation and Northern blot analysis
Figure 2 Systolic blood pressure by the tail cuff method in mmHg for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05 for EST different from OVX and CON, #P<0.05 for differences between all three groups.
beats/min were calculated from measured parameters.
Tissue collection Heart, kidneys, liver, uterus, and the brain were weighed. Tissue samples were quick frozen on dry ice and stored at −70°C. Once the whole heart weight was measured, the atria were separated by a single cut with a razor blade and weighed together. The right ventricle was removed by cutting along its attachment to the left ventricle and septum and weighed separately. The left ventricle and septum were weighed as a single unit. A single cross-section of the intact left ventricle and septum was made at approximately the level of the left coronary artery, and saved in 10% neutral buffered formalin. The remainder of the heart was frozen immediately on dry ice. All animals were examined for the presence or absence of ovaries. The same investigator (LCS) did all prosections. Formalin fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin and Masson’s trichrome strains.
Myosin isozymes Isolation and separation of myosin isozymes from the left ventricle were done following the method of Hoh et al. (1978). Myosin extracts were prepared and stored at −70°C until analysis by 3.5% polyacrylamide gel electrophoresis using non-dissociating conditions. Gels were stained with Coomasie blue and quantitated by a laser densitometer (LKB Gelscan XL, LKB Probkter AB, Bromma, Sweden).
Total RNA was extracted from quick-frozen left ventricle, kidney, and liver samples by the method of Chirgwin et al. (1979) and stored at −70°C. Northern blot analysis was done to identify and quantitate mRNA for renal renin, hepatic angiotensinogen, and left ventricular proANP as previously described (Sharkey et al., 1998). Full-length complementary DNA (cDNA) probes for rat renin (1.4 kb) and rat angiotensinogen (1.7 kb) were generously provided by Dr Kevin Lynch, University of Virginia, Charlottesville, VA, USA (Burnham et al., 1987). Dr Christopher Glembotski, San Diego State University, San Diego, CA, USA provided the full length cDNA probe for ANP (Glembotski et al., 1987). Expression of ANP mRNA in the left ventricle was normalized to a constitutively expressed mRNA, glyceraldehyde dehydrogenase (GAPDH). A 905 bp cDNA probe for GAPDH was acquired from Ambion, Incorporated (Austin, TX, USA) and is reported to have a high degree of binding for rat mRNA (Sabath et al., 1990). Dried blots were wrapped in plastic wrap and placed on a Molecular Dynamic’s Phosphor ImagerTM, and ImageQuantTM Software was used for densitometric analysis. After initial analysis, blots were stripped by boiling for 10–15 min in 0.1× SSPE. Successful stripping was confirmed by placing the blots on a Phosphor imager for 24-h, resulting in no image on scanning. The blots were then considered ready for constitutive mRNA probing.
Experiment 2 Sixteen 13-month-old lean female SHHF rats were initially documented to be having normal estrous cycles by vaginal cytology and were then monitored until cycling ceased naturally at 17 months of age. Previous studies have shown that this is the age at which endogenous serum estrogen declines to levels comparable to that observed with ovariectomy (Sharkey et al., 1998). At that time, baseline systolic blood pressure and left ventricular shortening fractions were determined as described above. Rats were paired by left ventricular fractional shortening and randomly assigned to either estradiol supplementation therapy (HRT, n=8) or control menopausal (MENO, n=8) groups. HRT rats were subcutaneously implanted with a 90-day release pellet containing 1.5 mg of 17b-estradiol (Innovative Research of American, Sarasota, FL, USA), while MENO received 1.5 mg placebo pellets. Pellets were replaced every 90 days for the duration of the
Ovariectomy and Estrogen Replacement in SHHF
Figure 3 V1% myosin isozyme in ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05 for OVX v. EST and CON.
experiment. Systolic blood pressure measurements and echocardiography were repeated every 45 days until the rats developed congestive heart failure, and either died spontaneously or were euthanized due to distress. Serum ACE activity was measured at 18.5 and 23 months of age. Organ weights were measured at the end of the study.
Statistical analysis Data are expressed as the mean±standard error of the mean; unless otherwise specified, all comparisons were done using ANOVA. Differences were reported as significant if the value of P<0.05 and the Tukey–Kramer Multiple Comparisons post-test was used for mean separation. The Kaplan–Meier curves were constructed and the log rank test was used to compare the survival of the HRT and MENO rats that were in terminal congestive heart failure at the time of death. Organ weights in Experiment 2 were compared using the Student’s t-test.
Results Experiment 1 As determined by vaginal cytology, all CON rats were cycling normally, while OVX rats were persistently in diestrus or proestrus, and EST rats were in continuous estrus. The success of the ovariectomy procedure was further confirmed by examination of the reproductive tract at necropsy. OVX rats lacked ovaries and had grossly atrophic uteri that weighed significantly less than CON and EST rats (Table 1). All OVX rats showed histologic
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Figure 4 Left ventricular fractional shortening percentage by M-mode echocardiography for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. ∗P<0.05 for OVX v. EST and CON.
evidence of uterine atrophy compared to EST and CON. There was no evidence of uterine neoplasia in any of the animals. 17b-Estradiol levels were determined to confirm the efficacy of treatment. Levels were measured at 13 weeks, corresponding to 30 days after the implantation of a 60-day pellet (Table 1). At this time, OVX had the lowest 17bestradiol levels, CON were significantly higher than OVX, and EST were higher than the other two groups. Effective estrogen replacement at the end of the study was demonstrated by the normalization of uterine weights in the 17b-estradiol treated ovariectomized rats, with EST and CON rats having similar uterine weights that were significantly greater than OVX. All three groups had similar fasted body weights at the beginning of the study, but by 4 weeks and, until the end of the study, the weights were significantly different between groups. OVX were the heaviest, CON were intermediate, and EST weighed the least (Fig. 1). The heart weights were significantly different between all three groups when compared as gross weight or relative to brain weights, with the OVX having the largest hearts and the EST the smallest (Table 1). The weights of the left ventricles and septa showed a similar pattern. The left ventricle and septum also made up proportionately more of the total heart weight in OVX compared to EST. The atria in the EST were significantly lighter than CON, but were not different from OVX. In contrast, the right ventricles were similar in all groups. Histologic evaluation of the left ventricle and septum showed no differences in myocardial fibrosis, which was minimal in all animals. Treatment also affected cardiovascular functional parameters. By 1 month after treatment, EST had
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expressed as a ratio to constitutive GAPDH expression (Fig. 5). EST significantly lowered serum ACE activity by 28 and 22% compared to CON at 8 and 24 weeks, respectively (Table 2). Conversely, OVX significantly increased serum ACE activity by 23 and 15% compared to CON at those same times (Table 2). There were no differences in hepatic angiotensinogen mRNA expression or renal renin mRNA expression relative to GAPDH at the end of 24 weeks (Table 2).
Experiment 2
Figure 5 Plasma atrial natriuretic peptide (ANP) in pg/ ml in ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats. No differences were detected between the groups at any time. +P<0.05 for 24 week v. baseline (0 week) values for all groups. The table shows left ventricular ANP mRNA expression normalized to GAPDH. No differences were detected between groups.
significantly lower systolic blood pressure than OVX or CON, and this difference persisted throughout the study (Fig. 2). OVX and CON had similar pressures except for week 20, when CON blood pressure was significantly lower than OVX, but significantly higher than EST. Therefore, estrogen treatment prevented the increase in systolic blood pressure seen in the CON and OVX groups over the study period. OVX had significantly lower V1 percentage isozyme compared to EST and CON (Fig. 3), lower left ventricular shortening fraction (Fig. 4). CON and EST had similar V1 percentage isozyme and left ventricular shortening fractions. The heart weight correlated positively with systolic blood pressure with a correlation coefficient of 0.868 (P<0.0001, 95% confidence interval 0.654 to 0.954), but did not correlate with the left ventricular fractional shortening (P=0.146) by linear regression. There were no differences in plasma ANP concentrations between OVX, EST or CON at any time point (Fig. 5). Plasma ANP concentration increased over time, as is characteristic for this strain of rat (McCune et al., 1995), and all three groups had significantly greater plasma ANP levels on week 24 when compared to their respective baseline value (week 0). As expected based on the plasma levels, there were no differences between the groups in left ventricular expression of ANP mRNA when
Estrogen supplementation was determined to be effective in that HRT rats maintained larger uteri (0.65±0.09 g) compared to MENO (0.45±0.05 g), tended to have a smaller body weights (Fig. 6), and had vaginal cytology compatible with estrogen supplementation. Both groups of rats were hypertensive at the start of the experiment [Fig. 7(a)]. Unlike the previous experiment, there was no effect of estrogen supplementation on systolic blood pressure or left ventricular shortening fraction (Fig. 7). Both groups showed a similar decline in left ventricular shortening fraction with time, and there was no significant difference between groups at the time of terminal CHF (33±2% for HRT, n=5 v. 30±6% for MENO, n=5). Whilst there was a trend for ACE activity to be lower in HRT, this did not reach significance at either 18.5 (15.05±0.92 nmol/ml/min for HRT v 16.74±0.39 nmol/ml/min for MENO, P=0.097) or 23 (15.48±1.16 nmol/ml/min for HRT v 19.78±1.51 nmol/ml/min for MENO, P=0.064) months of age. MENO rats showed a progressive increase in ACE activity over time, eventually resembling values observed in OVX rats from Experiment 1. Three MENO and two HRT rats developed tumors and died prior to the development of congestive heart failure. These were considered non-events and were not used for the following analysis. The mean survival time for HRT was 23.27 months (range 18.3–26.5, confidence interval 21.04– 25.49 months) and for MENO was 22.60 months (range 19.1–24.5, confidence interval 20.85– 24.38 months). The difference was not significant by log rank analysis, indicating that estrogen supplementation of rats that were already hypertensive and had undergone spontaneous cessation of cycling did not significantly prolong survival. There was no significant difference between groups for heart weight (2.13±0.15 g for HRT v. 2.63±0.32 g for MENO).
Ovariectomy and Estrogen Replacement in SHHF
Figure 6 Body weight in g for lean female SHHF rats supplemented with estrogen beginning at 17 months of age (HRT) and menopausal controls (MENO). There were no significant differences between the groups at any time.
Discussion Interpretation of the data on the cardiovascular effects of estrogen in people is still debated. Despite the well-documented differences between men and women in the incidence of heart disease, there is still not a consensus that estrogen is responsible for improved cardiovascular health in women during the premenopausal years. Studies of the relationship of blood pressure and natural fluctuations in estrogen levels show decreased blood pressure in the mid-trimester of pregnancy coincident with a massive increase in total estrogen (Swartz et al., 1995) and an increase in blood pressure during menopause when estrogen levels are falling (Bittner, 1995). These patterns would suggest that estrogen might influence the regulation of blood pressure. It is extremely difficult to evaluate the effects of exogenous estrogen in women due to the variety of preparations available, patterns of use, and the presence of numerous other factors such as obesity, smoking, diet, and exercise that also have significant impact on cardiovascular health but are difficult to control. Despite these difficulties, several studies have shown that post-menopausal estrogen
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treatment decreases blood pressure (Utian et al., 1978; Lind et al., 1979). Another author observed the effect of particular preparations of replacement on blood pressure, and found that equine estrogens and artificial estrogens administered with progestins increase pressure, while other estrogens do not (Jesperson, 1997). In order to clarify the effects of estrogen on blood pressure in a more controlled setting than is possible with human subjects, the effects of estrogen treatment on hypertension have been investigated in several hypertensive strains of rats. As in our study with the SHHF rat, treatment of intact female SHRSP with 17b-estradiol or of female SHR with mestranol resulted in a significant reduction in the degree of hypertension attained compared to placebo-treated animals (Hoeg et al., 1977; Eiff et al., 1985). The pressures of normotensive female Wistar–Kyoto rats were not affected by the same therapy, suggesting that estrogen may have a more pronounced effect on blood pressure in hypertensive animals. The improved prognosis for women with congestive heart failure compared to men suggests that female hormones might support cardiac function; however, studies on the effect of estrogen replacement therapy on heart function in healthy postmenopausal women had conflicting results. One found that Doppler derived parameters of aortic flow were improved after 1 year of therapy (Pines et al., 1992), while another failed to find significant improvements in M-mode, two-dimensional, and Doppler echocardiographic analysis of heart function after 12 weeks of treatment (Snabes et al., 1997). The capacity of estrogen to support heart function in people is unclear at this time. 17b-estradiol treatment appeared to have a beneficial effect on the heart in female SHHF rats, documenting the potential for estrogen to significantly alter heart function and cardiac hypertrophy in individuals genetically predisposed to the development of cardiovascular disease. Similar to our
Table 2 Serum ACE activity after 8 and 24 weeks of treatment and expression of renal renin and hepatic angiotensinogen mRNA relative to GAPDH expression at 24 weeks of treatment for ovariectomized (OVX), estrogen-treated ovariectomized (EST), and control (CON) lean female SHHF rats from Experiment 1.
Group
ACE activity 8 weeks (nmol/ml/min)
ACE activity 24 weeks (nmol/ml/min)
Renin: GAPDH 24 weeks
Angiotensinogen: GAPDH 24 weeks
OVX EST CON
20.90±0.94∗ 12.28±0.97† 16.96±1.20‡
19.67±0.50∗ 13.22±0.28† 17.02±0.95‡
0.341±0.044∗ 0.324±0.030∗ 0.353±0.039∗
3.179±0.281∗ 3.050±0.193∗ 2.300±0.110∗
Within columns, data with different symbols are significantly different (P<0.05) data that share a symbol within the same column are not statistically different.
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Figure 7 (a) Systolic blood pressure by tail method in mmHg; and (b) left ventricular fractional shortening (%) for lean female SHHF rats supplemented with estrogen beginning at 17 months of age (HRT) and menopausal controls (MENO). There were no significant differences between the groups at any time.
study, Schaible et al. (1984) found that ovariectomy of Wistar rats resulted in a reduction in V1 myosin isozyme and calcium-myosin ATPase activity. Decreased V1 percentage and increased V3 percentage myosin isozymes are associated with pathologic left ventricular hypertrophy (Buttrick et al., 1994). In contrast, exercise-associated physiologic hypertrophy in female rats is associated with increase in V1 percentage (Geenen et al., 1996). Several studies have documented depression of cardiac function in isolated hearts from ovariectomized rats (Schaible et al., 1984; Scheuer et al., 1987) and prevention of the myosin shift and cardiac dysfunction by estrogen administration (Scheuer et al., 1987). We were able to confirm the in vivo significance of these estrogen-associated cardiac functional differences in failure prone SHHF rats by the use of echocardiography. Estrogen could exert effects on the cardiovascular system by a number of mechanisms, including modulating the activity of the renin angiotensin system (RAS). SHHF rats show a progressive activation of the RAS, which peaks as the rats develop terminal congestive heart failure (Holycross et al., 1997). In this study, OVX, EST, and CON rats had comparable hepatic angiotensinogen mRNA and renal renin mRNA expression despite significantly lower blood pressures in EST compared to OVX and CON. A similar lack of effect on renal renin and hepatic angiotensinogen gene expression was also seen when SHHF were ovariectomized after onset of hypertension (Sharkey et al., 1998). Women taking oral estrogens have a consistent, marked increase in plasma angiotensinogen; however, this increase is not observed with transdermal preparations (Adashi et al., 1996). Concentration of
oral estrogens in the liver as the result of a substantial first pass effect, as well as the presence of an estrogen responsive sequence in the 5′-flanking region of the promoter inducing angiotensinogen transcription most likely contribute to this effect (Feldmer et al., 1991). Because our treatment was administered subcutaneously, we did not anticipate a marked estrogenic effort on hepatic angiotensinogen expression. Likewise, we did not observe differences between the groups in renal renin expression. Estrogen has the potential to influence the transcription of renin through regulatory sequences analogous to estrogen-receptor binding sites in the 5′-flanking region of the gene (Fukamizu et al., 1988). Ovariectomy and estrogen supplementation has been shown to have variable effects on gene expression in other rat strains. Ovariectomy was not associated with changes in renal renin or hepatic angiotensinogen gene expression in stroke-prone SHR (Bachmann et al., 1993); however, both were decreased in ovariectomized Okamoto SHR (Chen et al., 1992). Estrogens have been shown to decrease ACE activity by approximately 20% in post-menopausal women (Proudler et al., 1995) and in monkeys (Brosnihan et al., 1997). Estradiol administration to ovariectomized SHHF in experiment 1 resulted in a similar 22–28% decrease in ACE activity compared to controls. Spontaneous cessation of cycling in older SHHF resulted in a gradual increase in ACE activity, eventually resulting in values comparable to those obtained by SHHF that were ovariectomized at a young age (OVX, experiment 1). Administration of estradiol to menopausal rats resulted in maintenance of premenopausal levels of ACE activity, but did not lower ACE activity or modify already
Ovariectomy and Estrogen Replacement in SHHF
established hypertension. Ultimately, development of congestive heart failure was not significantly delayed in those SHHF who began estradiol treatment in the perimenopausal period. Similarly, in a previous study, decline of cardiovascular function and death from congestive heart failure was not hastened when SHHF were ovariectomized after the development of hypertension but prior to menopause (Sharkey et al., 1998). The role of ACE in maintenance of hypertension and development of congestive heart failure remains uncertain. It has been suggested that hypertension may be more important than ACE activity in promoting cardiac hypertrophy (Yoshida et al., 1988). Our studies suggest that estradiol treatment of young SHHF may prevent development of hypertension in association with lowering of ACE activity; however, a cause and effect relationship is not certain and requires further study. Once hypertension has become established, estradiol therapy alone may be inadequate to lower blood pressure. However, possible benefits of estrogen therapy in conjunction with other antihypertensive measures remain to be explored. Estrogen could modify the effects of the RAS by several other mechanisms that were not evaluated in this experiment. Estrogens modify the number of angiotensin subtype 1 receptors in a tissuespecific manner that may effect fluid balance and blood pressure (Seltzer et al., 1992). Changes in sensitivity to components of the renin angiotensin system may also occur. For example, chronic estrogen treatment significantly decreases angiotensin II-induced contractile responsiveness of rat aortic rings (Cheng and Gruelter, 1992). In addition to the RAS, the natriuretic peptides make up another neuroendocrine system that can impact cardiovascular homeostasis. ANP is of interest in the study of hypertension and heart disease because of its potential to antagonize the RAS, and its effects on plasma volume and blood pressure via natriuretic, diuretic, and vasorelaxant properties. There are consistent increases in the plasma levels of ANP in people with left ventricular dysfunction and in SHHF rats as they progress through cardiac hypertrophy to failure (Francis et al., 1990; McCune et al., 1995; Arai et al., 1988). Augmented expression of ANP mRNA in the left ventricles of SHHF rats accompanies the increase in plasma concentrations associated with hypertension and cardiac hypertrophy (McCune et al., 1995). Ovariectomy and estrogen treatment failed to cause changes in plasma ANP concentration or ANP mRNA expression in the left ventricle relative to control animals, despite significant differences in
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blood pressure and cardiac hypertrophy. Data demonstrating increases in plasma ANP in women taking oral contraceptives or who are pregnant suggest that estrogen, or increases in blood volume induced by estrogen, could be associated with changes in plasma ANP (Davidson et al., 1988; Sala et al., 1995). In summary, estradiol status had a significant impact on cardiac hypertrophy and cardiovascular function when examined in lean female SHHF rats prior to the development of hypertension. Our data show that ovariectomy of prehypertensive heart failure-prone SHHF results in greater cardiac hypertrophy and impaired cardiac function. The administration of subcutaneous 17b-estradiol prevented ovariectomy-associated decreases in left ventricular shortening fraction and V1 percentage myosin isozyme, but the data do not indicate that exogenous estrogen improves left ventricular function compared to normally cycling, failure-prone rats. Heart function and myosin isozymes of EST remain comparable to CON. Interestingly, 17b-estradiol treatment prevented the rise in blood pressure normally observed in this hypertensive rat strain, resulting in significantly lower pressures in EST compared to OVX and even CON animals. This suggests that hormone supplementation may improve blood pressure control beyond merely counteracting the effects of the removal of the ovaries. The changes in heart size and function associated with estrogen status were not associated with significant differences in renal renin, hepatic angiotensinogen, or left ventricular ANP mRNA expression, but were associated with decreases in ACE activity. The mechanisms by which estrogen improved cardiovascular parameters in ovariectomized SHHF may relate to improved blood pressure control. If estradiol administration was started in the perimenopausal period, after hypertension was established, cardiac hypertrophy, decline in cardiovascular function, and onset of congestive heart failure was not delayed. Further work is needed to clarify the mechanisms by which estrogen works in SHHF rats.
Acknowledgements This work was supported in part by US Public Health Service Grant HL48835. Salaries and research support was also provided in part by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, Ohio State University.
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References A EY, 1996. Transdermal estrogen replacement. In: Adashi EY, Rock JA, Rosenwaks Z (eds). Reproductive Endocrinology, Surgery and Technology. Philadelphia: Lippincott Raven Publishers. 1784–1795. A H, N K, S Y, M N, S A, Y Y, I H, S S, M M, O H, N S, I H, 1988. Augmented expression of atrial natriuretic peptide in ventricles of spontaneously hypertensive rats (SHR) and SHR-stroke prone. Circ Res 62: 926–930. B J, W J, H C, W A, C A, G D, 1993. Modulation of blood pressure and the renin-angiotensin system in transgenic and spontaneously hypertensive rats after ovariectomy. J Hypertens 11(Suppl. 5): S226– S227. B V, 1995. Hypertension in Women. Curr Op. Neph Hypertens 92: 3431–3435. B KB, W D, A MS, H C, L P, F CM, 1997. Effects of chronic hormone replacement on the renin-angiotensin system in cynomolgus monkeys. J Hypertension 15: 719–726. B C, H-J C, F B, L K, 1987. Molecular cloning of rat renin cDNA and its gene. Proc Natl Acad Sci USA 84: 602–609. B PM, K M, L LA, S J, 1994. Alterations in gene expression in the rat heart after chronic pathological and physiological loads. J Mol Cell Cardiol 26: 61–67. C Y, N A, O S, 1992. Androgen-dependent angiotensinogen and renin messenger RNA expression in hypertensive rat. Hypertension 19: 456– 463. C DY, G CA, 1992. Chronic estrogen alters contractile responsiveness to AII and norepinephrine in the female rat aorta. Eur J Pharm 215: 171–176. C J, P A, MD R, R W, 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299. D B, R CD, V GJ, 1988. Atrial natriuretic peptide, plasma renin activity, and aldosterone in women on estrogen therapy and with premenstrual syndrome. Fertility and Sterility 50: 743–746. E AW, L HM, K R, 1985. The protective effect of estrogen on high blood pressure. Basic Res Cardiol 80: 191–201. F M, K M, T S, M JJ, G D, 1991. Glucocorticoid and estrogen responsive elements in the 5′ flanking region of the rat angiotensinogen gene. J Hypertens 9: 1005–1012. F GS, B C, J DE, K PC, N H, L C, K SH, R-T E, Y S, 1990. Comparison of neuroendocrine activation in patients with left ventricular dysfunction with and without congestive heart failure. Circulation 82: 1724– 1729. F A, N K, C T, S M, N K, O H, N S, M K, 1988. Structure of the rat renin gene. J Mol Biol 201: 443–450. G DL, M A, B PM, 1996. Angiotensin receptor 1 blockade does not prevent physiological cardiac hypertrophy in the adult rat. J Appl Phys 81: 816–821.
G C, O M, L X, S P, H P, K R, G R, 1987. The characterization of atrial natruiuretic peptide expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe. Endocrinol Biol 121: 843–853. H JM, W LR, W MH, 1977. Estrogen attenuation of the development of hypertension in spontaneously hypertensive rats. Am J Physiol 233: H369–H373. H J, MG P, H P, 1978. Electrophoretic analysis of multiple forms of rat cardiac myosins; effects of hypophysectomy and thyroxin replacement. J Mol Cell Cardiol 11: 1053–1078. H B, S B, D R, MC S, 1997. Plasma renin activity in heart failure prone (SHHF/ Mcc-facp) rats. Am J Physiol 273: H228–H233. J CM, 1997. Hormone replacement therapy and high blood pressure. Am J Hypertens 10: 366–367. L J, 1975, Elevation of serum angiotensin-converting-enzyme (ACE) level in sarcoidosis. Am J Med 59: 365–372. L T, C EC, H WM, L C, M PG, O Q, G JA, 1979. Prospective controlled trial of six forms of hormone replacement therapy given to postmenopausal women. Br J Obset Gyn 86: 1–29. MC S, P S, R M, J R, 1995. SHHF/Mccfacp rat model: a genetic model of congestive heart failure. In: Singal P, Dixon I, Beamish R, Dhalla R (eds). Mechanisms of Heart Failure. Kluwer Academic Publishers, 91–106. P A, F EZ, A D, D Y, A M, L Y, 1992. Long-term effects of hormone replacement therapy on Doppler derived parameters of aortic flow in postmenopausal women. Chest 102: 1496–1498. P AJ, H-A AI, C D, F I, R JM, S JC, 1995. Hormone replacement therapy and ACE activity in postmenopausal women. Lancet 346: 89–90. R MJ, J J, MC SA, H R, 1992. Effects of enalapril and clonidine on glomerular structure, function, and atrial natriuretic peptide receptors in SHHF/Mcc-facp rats. J Cardiovasc Pharmacol 19: 464– 472. S D, B H, P M, 1990. Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin 2-induced transcript in a cloned Thelper lymphocyte. Gene 91: 185–191. S C, C M, A G, M T, Z A, M L, 1995. Atrial Natriuretic peptide and hemodynamic changes during normal human pregnancy. Hypertension 25 (part 1): 631–636. S TF, M A, C C, S J, 1984. The effects of gonadectomy on left ventricular function and cardiac contractile proteins in male and female rats. Circ Res 54: 38–49. S J, M A, S TF, C J, 1987. Effects of gonadectomy and hormonal replacement on rat hearts. Circ Res 61: 12–19. S A, P JEP, V PN, C FMA, L C, S T, S MK, S JM, 1992. Estrogens regulate ACE and angiotensin receptors in female rat anterior pituitary. Neuroendocrinology 55: 460–467.
Ovariectomy and Estrogen Replacement in SHHF S LC, H BJ, P S, MC SA, H R, R MJ, 1998. Effect of ovariectomy in heart failure-prone SHHF/Mcc-facp rats. Am J Physiol 275: R1968–R1976. S MC, P JP, K HA, D JK, Y R, Z WA, 1997. Physiologic estradiol replacement therapy and cardiac structure and function in normal postmenopausal women: a randomized double blind, placebo controlled, crossover trial. Obstet Gyn 89: 332– 339. S, J, F R, F W 1995. Clinical
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pharmacology of estrogens: cardiovascular actions and cardioprotective benefits of replacement therapy on postmenopausal women. J Clin Pharmacol 35: 1–16. U WH, 1978. Effect of postmenopausal estrogen therapy on diastolic blood pressure and body weight. Maturitas 34: 3–8. Y K, K M, C DJ, J CI, 1998. Angiotensin-converting enzyme inhibition and salt in experiment myocardial infarction. J Cardiovasc Pharmacol 32: 357–365.