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Hypertension. 1997;30:1029-1034

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(Hypertension. 1997;30:1029-1034.)
© 1997 American Heart Association, Inc.


Articles

Cardiomyocyte Apoptosis and Cardiac Angiotensin-Converting Enzyme in Spontaneously Hypertensive Rats

Javier Díez; Angel Panizo; Marta Hernández; Francisco Vega; Iosu Sola; María Antonia Fortuño; Javier Pardo

From the Vascular Pathophysiology Unit, School of Medicine (J.D., M.H., M.A.F.), and the Department of Pathology, University Clinic, School of Medicine (A.P., F.V., I.S., J.P.), University of Navarra, Pamplona, and the Department of Medicine, School of Medicine, University of Zaragoza (J.D.), Spain.


*    Abstract
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Abstract Increased apoptosis has been reported in the heart of rats with spontaneous hypertension and cardiac hypertrophy. This study was designed to investigate the relationship between apoptosis and hypertrophy in cardiomyocytes from the left ventricle of spontaneously hypertensive rats (SHR). In addition, we evaluated whether the development of cardiomyocyte apoptosis is related to blood pressure or to the activity of the local angiotensin-converting enzyme (ACE) in SHR. The study was performed in 16-week-old SHR, 30-week-old untreated SHR, and 30-week-old SHR treated with quinapril (10 mg · kg-1 · d-1) during 14 weeks before they were killed. Cardiomyocyte apoptosis was assessed by direct immunoperoxidase detection of digoxigenin-labeled 3'-hydroxyl ends of DNA. Nuclear polyploidization measured by DNA flow cytometry was used to assess cardiomyocyte hypertrophy. Compared with 16-week-old normotensive Wistar-Kyoto rats, 16-week-old SHR exhibited increased blood pressure (P<.001), increased rate of tetraploidy (P<.05), and similar levels of ACE activity and apoptosis. Compared with 30-week-old Wistar-Kyoto rats, 30-week-old SHR showed increased blood pressure (P<.001), increased ACE activity (P<.05), increased rate of tetraploidy (P<.01), and increased apoptosis (P<.01). Untreated 30-week-old SHR exhibited similar values of blood pressure and tetraploidy and higher ACE activity (P<.05) and apoptosis (P<.001) than 16-week-old SHR. A direct correlation (P<.01) was found between ACE activity and the apoptotic index in untreated 30-week-old SHR. The long-term administration of quinapril was associated with the normalization of ACE activity and apoptosis in treated SHR. These results suggest that the timing and mechanisms responsible for apoptosis and hypertrophy of cardiomyocytes are different in SHR. Whereas hypertrophy seems to be an earlier alteration that develops in parallel with hypertension, apoptosis develops later in association with overactivity of the local ACE. Our data suggest that cell death dysregulation may be a novel target for antihypertensive agents that interfere with the renin-angiotensin system in hypertension.


Key Words: angiotensin-converting enzyme • apoptosis • cardiomyocytes • hypertrophy • rats, inbred SHR


*    Introduction
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Apoptosis is an active, tightly regulated, energy-requiring process in which cell death follows a programmed sequence of events.1 Fragmentation of chromosomal DNA is the biological hallmark of apoptosis.2 This process of internucleosomal fragmentation of DNA appears to be a genetic event that requires gene transcription and translation3 and may be stimulated or inhibited by a variety of regulatory factors including growth factors and cytokines.4 Apoptotic cells undergo extracellular degeneration or phagocytosis by macrophages and neighboring cells.5 Thus, apoptosis plays a role in the regulation of cell mass and architecture in many tissues.6

Terminally differentiated cells such as cardiomyocytes are not believed to undergo apoptosis under normal conditions. However, recent evidence suggests that apoptosis can be induced in cardiomyocytes by a variety of insults, which include mechanical stretch,7 pressure overload,8 ischemia reperfusion,9 10 and cytokine induction of inducible NO synthase.11

Increased apoptosis has been demonstrated recently in whole sections of the hypertrophied LV of SHR.12 13 In addition, in spontaneously hypertensive mice, enhanced apoptosis was identified in the cardiomyocytes of the LV compared with the normotensive control heart.12

At the present time, the mechanisms that are responsible for increased cardiomyocyte apoptosis in rodents with genetic hypertension remain unknown. In addition, it is unclear whether cardiomyocyte apoptosis is a prerequisite for cardiac remodeling or whether hypertrophy represents a failure of compensatory apoptosis. Therefore, the present study was designed to determine the relationship that exists in the development of apoptosis and hypertrophy in the LV of rats at different ages. In addition, we evaluated whether the development of cardiomyocyte apoptosis is related to hemodynamic (ie, high BP) or to nonhemodynamic (ie, overactivity of the local ACE) factors in SHR.


*    Methods
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Animals and Design
All procedures were in accordance with institutional guidelines for animal research. The rats were provided by Iffa-Credo (L'Abresle, France). The SHR and their normotensive genetic controls, WKY, were studied in the following manner: (1) 16-week-old SHR (n=10) and WKY (n=10), observed in our colony from 14 weeks of age and showing stable BP over a 2-week period, were killed (groups SHR16, WKY16); (2) untreated 16-week-old SHR (n=10) and WKY (n=10) were observed for an additional 14 weeks and killed at 30 weeks of age (groups SHR30 and WKY30); and (3) 16-week-old SHR (n=10) were treated with quinapril (10 mg · kg-1 · d-1) for 14 weeks and killed at 30 weeks (group SHR-Q30). The drug was dissolved in drinking water, and the concentration was adjusted for the daily water intake and body weight to obtain an average daily dose of 10 mg/kg body wt. All rats were housed in individual cages and were fed a standard rat chow and tap water ad libitum. They were maintained in a quiet room at constant temperature (20°C to 22°C) and humidity (50% to 60%). All the manipulations were carried out in accordance with institutional guidelines.

Before the animals were killed, they were anesthetized with methohexital (50 mg/kg IP). Once the animals had been killed by decapitation, the heart was removed en bloc, and the cardiac dimensions were measured. In each animal, the cardiac index was calculated by dividing the heart weight by the body weight. After the equator of the heart was selected as representative of the entire LV, ventricular wall thickness was measured across the free wall. The LV was dissected and washed thoroughly with normal saline to remove the contaminating blood and then immediately processed for biochemical and morphological studies. For histopathological and immunohistochemical investigations, the hearts were fixed in 10% buffered formalin and embedded in paraffin.

Measurement of BP and LV ACE Activity
Systolic BP was measured every 2 weeks in all animals by the standard tail-cuff method with an LE 5007 Pressure Computer (Letica Scientific Instruments).14

ACE activity was determined in homogenized LV by measurement of the generation of the dipeptide histidyl-leucin from the synthetic substrate N-hippuryl-L-histidyl-L-leucine.15 The dipeptide histidyl-leucin was determined after the fluorometric assay, which was previously developed in our laboratory.16

Morphological Studies
Coronal sections of the LV obtained from its equator were prepared for light microscopy as reported previously by Doering et al.17 A complete cross section of the heart was used for the morphometric analysis. Each block was cut serially at 5 µm, and sections were stained with hematoxylin-eosin and Masson's trichrome.

Cardiomyocyte diameter and collagen volume fraction were determined with an automatic image analyzer (Microm IP 1.6; Microm). The diameter of representative LV cardiomyocytes was measured according to a methodology described previously18 19 20 and as modified in our laboratory.21 Areas that contained properly oriented cross sections of cardiomyocytes were selected. The distance across the cell at its narrowest plane across the nucleus was measured in 75 cells from each LV (25 from the epicardium, 25 from the mesocardium, and 25 from the endocardium), and the average diameter was calculated. The collagen volume fraction was calculated as the sum of the surfaces of the connective tissue of the coronal section divided by the total surface of the section as previously described.21 22

In Situ Detection of Apoptosis
Sections from the LV were deparaffinized, transferred to xylene, and rehydrated in descending concentrations of alcohol. After rehydration, the slides were incubated with 20 µg of proteinase K per milliliter in phosphate-buffered saline. Endogenous peroxidase was inactivated by 3% hydrogen peroxide. Tissue sections were stained with an ApopTag system (Oncor) that identifies cells with internucleosomal fragmentation of DNA. The procedure was performed according to the manufacturer's instructions. The method is based on the preferential binding of terminal deoxynucleotidyl transferase (TdT) to the 3'-hydroxyl ends of DNA.23 Briefly, residues of biotinylated dUTP were catalytically added to the ends of DNA fragments with the enzyme TdT. For negative controls, deionized water was used instead of TdT. After end-labeling, the sections were incubated with avidin–horseradish peroxidase, stained with diaminobenzidine, and counterstained with ethyl green to detect the biotin-labeled nuclei. Apoptotic bodies stained brown. ApopTag staining was biologically validated in a tissue known to exhibit high rates of programmed cell death (ie, human infarcted myocardium).24 As shown in Fig 1Down, cardiomyocyte nuclei with labeled oligonucleosomal DNA were clearly visible in this tissue because of their intense brown staining.



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Figure 1. Standardization of fragmented DNA detection by in situ DNA end-labeling with an ApopTag kit in infarcted myocardium from humans. Apoptotic cardiomyocytes in the infarct center are identified by their brown nuclei (magnification x480).

Cardiomyocytes, which were well-shaped, elongated, and striated cells, were easily distinguished morphologically, under a light microscope at high magnification, from other rare nonmyocytes. Tissue sections from each myocardial specimen were examined microscopically at x40 magnification, and at least 200 cardiomyocytes were counted in a minimum of five high-power fields separately in the subepicardial, midmyocardial, and subendocardial layers. The percentage of apoptotic cells was determined with an apoptotic index; the apoptotic index was calculated by dividing the number of positive-staining cardiomyocyte nuclei by the total number of cardiomyocyte nuclei and multiplying that value by 100. The pathologist who analyzed the specimens was unaware of the experimental group for all the rats examined.

Determination of DNA Ploidy
A number of experimental and clinical studies support the concept that an association exists between an increase in the number of polyploid cardiomyocytes and the process of cardiac hypertrophy.25 26 27 28 29 30 31 In this study, nuclear polyploidization was determined by DNA flow cytometry. For flow cytometric study, one to four 1.5x1.5x1.5-mm cubes were taken from each LV and immediately frozen in liquid nitrogen and stored at -80° C until analysis. At the time of analysis, nuclear suspensions were obtained according to the method described by Vindelov et al.32 Briefly, samples were placed in tissue culture medium (RPMI-I640) supplemented with 10% fetal calf serum and processed into single-cell suspensions with scissors. After centrifugation for 5 minutes at 2000 rpm, the pellet was resuspended in 2.5 mL of citrate buffer. After another centrifugation, the following were added successively: 1800 µL of solution containing trypsin, 1500 µL of solution containing trypsin inhibitor and ribonuclease A, and 1500 µL of solution containing propidium iodide. Before the analysis, the cell sample was filtered through a 50-µmol/L nylon mesh.

A minimum of 2x104 cardiomyocytes per sample was analyzed on an Arhens-FCS II flow cytometer as reported previously.31 The cells were chosen randomly from each myocardial layer. The flow cytometric parameters evaluated included DNA ploidy and the percentage of cells in each area by use of the software Arhens-Acas 5.0 that was included in the system.

Statistical Analysis
Results are presented as mean±SEM computed from the average measurements obtained from each group of rats. Differences among the five groups of rats were tested by a two-way ANOVA. Subsequent analysis for significant differences between two groups was performed with a multiple comparison test (Scheffé's method). The correlation between continuously distributed variables was tested by univariate regression analysis. The significance level was assumed at a value of P<.05.


*    Results
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BP and ACE Activity
Systolic BP was increased significantly in SHR16 compared with WKY16 (Table 1Down). LV ACE activity was similar in SHR16 and WKY16 (Table 1Down).


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Table 1. Blood Pressure and Cardiac ACE Activity in Normotensive and Hypertensive Rats: Effects of Chronic Treatment With Quinapril

Untreated SHR30 exhibited significantly higher values of systolic BP than WKY16 and WKY30 (Table 1Up). LV ACE activity was increased significantly in SHR30 compared with WKY16, WKY30, and SHR16 (Table 1Up). Treatment with quinapril was associated with normalization of BP and ACE activity in SHR-Q30 (Table 1Up).

LV Hypertrophy and Fibrosis
Cardiac index, LV wall thickness, cardiomyocyte diameter, and the rate of tetraploid cardiomyocytes were significantly higher in SHR16 than in WKY16 (Table 2Down). The collagen volume fraction was also increased in SHR16 compared with WKY16 (Table 2Down). Therefore, hypertrophy and fibrosis were present in the LV in SHR16.


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Table 2. Parameters Related to LV Hypertrophy and Fibrosis in Normotensive and Hypertensive Rats: Effects of Chronic Treatment With Quinapril

These two pathological changes were observed also in the LV in SHR30, as assessed by the abnormally high values of cardiac index, LV wall thickness, cardiomyocyte diameter, number of tetraploid nuclei, and collagen volume fraction observed in these rats (Table 2Up). Treatment with quinapril was associated with the normalization of all the above morphological parameters in SHR-Q30 (Table 2Up). It therefore appears that quinapril was able to reverse hypertrophy and fibrosis present in the LV in SHR-Q30.

Apoptosis of Cardiomyocytes
In situ end-labeling–detected apoptosis was confined predominantly to the cardiomyocytes of the LV in the animals studied. Apoptosis was rare in the interstitial or endothelial cells and smooth muscle cells of the intramyocardial arteries and arterioles.

Apoptosis of cardiomyocytes was identified in myocardial specimens from approximately half of the animals from the WKY16 and SHR16 groups. Apoptosis was predominant in the subendocardium. Apoptotic cardiomyocytes were seen in most of the rats in the WKY30, SHR30, and SHR-Q30 groups; again, apoptotic cells were seen predominantly in the subendocardial layers. Whereas in WKY30 apoptosis was identified in isolated cells, in SHR30 and SHR-Q30 apoptosis appeared to occur in small groups of noncontiguous cells (Fig 2Down).



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Figure 2. Photomicrographs show apoptotic cardiomyocytes identified by their brown nuclei in the LV from 30-week-old normotensive WKY (top, magnification x240), in untreated 30-week-old SHR (middle, magnification x480), and in quinapril-treated 30-week-old SHR (bottom, original magnification x240).

No significant differences were found in the apoptotic index between WKY16 and SHR16 animals (0.29±0.01 versus 0.30±0.01) (Fig 3Down). The apoptotic index was significantly higher (P<.001) in SHR30 (2.12±0.20) than in WKY16, SHR16, and WKY30 (0.22±0.02) (Fig 3Down). The value of this parameter was normal in SHR-Q30 (0.26±0.02) (Fig 3Down).



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Figure 3. Values of the apoptotic index calculated in each experimental group. The bars represent the mean±SEM of 10 animals in each group. Scheffé's two-way ANOVA test was applied to assess the statistical significance of differences among groups. WKY16 indicates 16-week-old normotensive WKY; WKY30, 30-week-old WKY; SHR16, 16-week-old SHR; SHR30, 30-week-old SHR; and SHR-Q30, quinapril-treated 30-week-old SHR. *P<.001 vs WKY16, SHR16, WKY30, and SHR-Q30.

As shown in Fig 4Down, a direct correlation was found between LV ACE activity and the apoptotic index (y=-0.270+0.003x, r=.847, P<.01) in untreated SHR30. No correlations were found between these two parameters in the remaining groups of rats.



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Figure 4. Scatterplot shows direct correlation (y=-0.270+ 0.003x) between LV ACE activity and the apoptotic index in 10 untreated 30-week-old SHR. Correlation determined by univariate regression analysis.


*    Discussion
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*Discussion
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The main finding of the present study is that stimulation of cardiomyocyte apoptosis in the LV of SHR is related in a temporal way to local ACE activity and not to BP.

The mechanisms of increased cardiomyocyte apoptosis in rats with genetic hypertension are not well known. In a recent work, Teiger et al8 showed that apoptosis is induced selectively in cardiomyocytes after acute hemodynamic stimulation secondary to aortic stenosis in the rat. Overstretching secondary to acute mechanical loading of the myocardial muscle has been shown to be coupled with apoptosis of cardiomyocytes.7 Overstretching may also increase the expression of the cell surface molecule Fas,33 34 , which has been found to be involved in apoptotic cardiomyocyte death.9 Therefore, it is possible that the physical forces may induce cardiomyocyte apoptosis in conditions of experimentally induced pressure overload of the heart. Although no association was found in this study between hypertension and apoptosis in 16-week-old SHR, a role for long-term hemodynamic overload cannot be excluded in enhanced cardiac apoptosis in 30-week-old SHR.

An alternative explanation may arise from several observations. (1) Previous studies have shown that the LV ACE is induced35 and that angiotensin II is stimulated36 in the hearts of rats subjected to experimental aortic stenosis. (2) Booz and Baker37 reported recently that angiotensin II exerts an antigrowth effect in rat ventricular cardiomyocytes that can be blocked by AT2 antagonists. Interestingly, angiotensin II may activate apoptosis directly via the AT2 receptor.38 (3) The NO-releasing drug C87-3754 is capable of preventing apoptosis in cardiomyocytes exposed to overstretching.7 Therefore, it can be hypothesized that an imbalance between increased angiotensin II and decreased NO secondary to the enhanced activity of the cardiac ACE may be involved in apoptosis of cardiomyocytes in conditions of pressure overload. The association found in this study between enhanced apoptosis and exaggerated ACE activity in untreated SHR and the effect of the ACE inhibition on apoptosis in quinapril-treated SHR supports this possibility.

The above hypothesis deserves some comments. It has been proposed that growth factors such as angiotensin II, which has been shown to result in immediate early gene expression and hypertrophy of neonatal cardiomyocytes,39 may induce apoptosis in the aging and chronically overloaded heart.40 41 On the other hand, although recent evidence suggests that NO is capable of triggering apoptosis in cardiomyocytes,11 42 it also has been shown that NO may protect against apoptotic cell death induced by the superoxide anion.43 44 45 This can be important in conditions of enhanced afterload of the myocardium that increase oxygen consumption and lead to the generation of superoxide anions, which may activate the apoptosis program of cardiomyocytes.46

The pathophysiological meaning of increased cardiomyocyte apoptosis in SHR also remains a matter of discussion. Teiger et al8 showed that apoptosis occurs in the initial phase after the aortic stenosis and is of short duration, clearly preceding and then fading at the time of cardiac hypertrophy. Thus, these authors suggested that apoptosis can trigger cardiac remodeling. Clearly, this cannot be applied to the SHR we studied because apoptosis of cardiomyocytes appears later than the two lesions defining hypertensive cardiac remodeling (ie, cardiomyocyte hypertrophy and interstitial fibrosis). Nevertheless, because only two time points were examined, we cannot rule out undetected apoptosis at an earlier time in the development of hypertension.

In SHR, it is known that aging or persistent elevations in afterload are associated with the occurrence of heart failure.47 Bing48 has hypothesized that apoptosis may be a mechanism for loss of viable cardiomyocytes, myocardial dysfunction, and the transition to heart failure associated with long-term pressure overload. This is supported by the observation that cardiomyocyte apoptosis is significantly higher in the failing heart of SHR compared with the nonfailing heart of SHR.13 In this setting, it should be noted that although the absolute number of apoptotic cardiomyocytes found in the LV of the SHR we studied was small, the loss of cells, nevertheless, can be significant because the time course for apoptotic cell death is only a few hours. For example, Colucci49 proposed that assuming that a cell undergoing apoptosis is detectable for a maximum of 24 hours,50 it could be estimated that the loss of only 0.2% of cells per day, if sustained for 1 year, might result in the loss of >50% of the total pool of cardiomyocytes.

If apoptosis plays a role in the loss of cardiomyocytes in arterial hypertension, then interruption of the signals that stimulate apoptosis or the pathways that couple those signals to cell death may be of value in preventing or reversing the process leading to heart failure in this disease.48 49 The present study provides the first data that suggest that long-term antihypertensive treatment with an ACE inhibitor blocks apoptosis of cardiomyocytes in adult rats with genetic hypertension. Additional studies are necessary to establish whether the beneficial effects of ACE inhibitors in patients with heart failure51 are due in part to the ability of these drugs to suppress the apoptotic loss of cardiomyocytes by diminishing angiotensin II and/or increasing bradykinin and, in turn, NO.52

In conclusion, our findings suggest that cardiomyocyte apoptosis may be related to exaggerated ACE activity in the LV of adult SHR. Nevertheless, the long-term impact of arterial hypertension cannot be excluded as an additional facilitating factor in the development of cardiomyocyte programmed cell death. Because apoptosis of cardiomyocytes appears late in the process of hypertensive heart disease in SHR, our data favor the hypothesis that this alteration may be associated with a transition to reduced cardiomyocyte function via a loss of viable cardiomyocytes. Finally, our observations support the contention of Hamet et al12 that pharmacological interventions in hypertension should be applied not only to normal cell growth but also to apoptosis in target organs.


*    Selected Abbreviations and Acronyms
 
ACE = angiotensin-converting enzyme
BP = blood pressure
LV = left ventricle
NO = nitric oxide
SHR = spontaneously hypertensive rat(s)
WKY = Wistar-Kyoto rat(s)


*    Footnotes
 
Reprint requests to Javier Díez, MD, PhD, Unidad de Fisiopatología Vascular, Facultad de Medicina, C/Irunlarrea s/n, 31080 Pamplona, Spain.

Received January 9, 1997; first decision February 5, 1997; accepted April 15, 1997.


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*References
 
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