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Circulation. 1997;96:4002-4010

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(Circulation. 1997;96:4002-4010.)
© 1997 American Heart Association, Inc.

Articles

Effect of Angiotensin-Converting Enzyme Inhibition on Myocardial Fibrosis and Function in Hypertrophied and Failing Myocardium From the Spontaneously Hypertensive Rat

Wesley W. Brooks, DSc; Oscar H. L. Bing, MD; Kathleen G. Robinson, BS; Mara T. Slawsky, MD, PhD; David M. Chaletsky, MPH; ; Chester H. Conrad, MD, PhD

From the Department of Veterans Affairs Medical Center, Boston, Mass, and the Department of Medicine, Boston University School of Medicine, Boston.

Correspondence to Wesley W. Brooks, DSc, Research Service(151), Boston VA Medical Center, 150 South Huntington Ave, Boston, Mass 02130. E-mail conrad.chester{at}boston.va.gov


*    Abstract
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 *Abstract
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 down arrowResults
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Background After a period of stable hypertrophy, male spontaneously hypertensive rats (SHR) develop heart failure between 18 to 24 months of age, with depression of active myocardial function and increased passive stiffness. We tested the hypothesis that chronic ACE inhibition by captopril would prevent and possibly reverse impairment of myocardial function.

Methods and Results Male SHR and normotensive Wistar-Kyoto rats (WKY) were assigned to no treatment or captopril treatment (2 g/L in drinking water) begun at ages 12, 18, and 21 months; animals were studied at 24 months of age, or earlier when evidence of heart failure was found in SHR (mean age, 19±2 months). In an additional group, captopril treatment was begun when SHR developed heart failure; surviving animals were studied at 24 months of age. In untreated SHR, relative to WKY, isometric stress development at Lmax, maximum rate of stress development, and shortening velocity were depressed, whereas passive stiffness was increased, in association with the development of myocardial fibrosis. In the SHR treated before cardiac dysfunction, captopril administration attenuated hypertrophy and prevented contractile dysfunction, fibrosis, and increased passive stiffness. Captopril treatment begun after cardiac function was impaired reduced left ventricular hypertrophy but did not restore intrinsic contractile function or reduce fibrosis or passive stiffness.

Conclusions In the male SHR, early treatment with captopril was associated with the most marked attenuation of dysfunction relative to the untreated SHR. Treatment initiated after the onset of heart failure improved clinical signs of heart failure and decreased left ventricular hypertrophy in surviving animals but did not reverse the fibrosis and contractile dysfunction associated with heart failure.


Key Words: angiotensin-converting enzyme inhibition • myocardial fibrosis • myocardial hypertrophy • heart failure


*    Introduction
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 *Introduction
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The SHR is a laboratory model of naturally developing hypertension that appears to be similar in a number of respects to essential hypertension in humans1–3. In the SHR model, persistent hypertension begins when the rats are {approx}2 months of age4,5 and is followed by a relatively long period of stable, compensated hypertrophy. At {approx}18 months of age in the male SHR, the first indications of left ventricular pump6,7 and muscle dysfunction8,9 appear. Evidence of dysfunction is also observed in the female SHR although events appear to be delayed by a few months.10 These findings are followed by pathophysiological manifestations of overt heart failure (mean age of failure is {approx}20 months in the male SHR, with evidence of failure in most animals by 24 months of age).3,9,11 At 18 to 24 months, with the advent of the heart failure state, the male SHR demonstrates a marked upregulation of genes encoding extracellular matrix components12 associated with an increase in myocardial fibrosis, passive stiffness, and impaired contractile function relative to age-matched nonfailing SHR and normotensive WKY rats.3,9

In the clinical setting, patients with heart failure of varying origin are well recognized to benefit from ACE inhibitor therapy.13 Pfeffer et al10 have shown that long-term captopril administration prevents impairment of hemodynamic function in the female SHR. However, the relation between the time treatment is initiated in the course of the disease and the prevention or reversal of intrinsic myocardial dysfunction and hemodynamic impairment has not been elucidated.

The purpose of this study, therefore, was to examine the relation between age of initiation of ACE inhibitor treatment, including its administration before or subsequent to the development of heart failure, and the prevention and/or reversal of fibrosis and intrinsic myocardial dysfunction in the male SHR.


*    Methods
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 *Methods
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Materials and Preparation
Animal Model
One hundred eight male SHR and 12 WKY rats were purchased from Taconic (Germantown, NY). All rats were housed two per container and fed regular rat chow and water. Groups of SHR were treated by addition of captopril to drinking water (2 g/L of drinking water). This concentration has been shown to be effective in preventing hemodynamic impairment associated with chronic hypertrophy in the SHR.10 Groups of animals had treatment initiated at 12, 18, and 21 months of age (SHRRx12, SHRRx18, SHRRx21, respectively). An additional group of SHR was treated with captopril when evidence of impaired cardiac function was detected clinically and documented by echocardiographic studies.14 Fig 1Down is a schematic representation of the times captopril treatment was initiated to the various SHR groups used in this experimental protocol. Control groups included untreated SHR and age-matched WKY. Animals were monitored with measurements of body weight and blood pressure. Animals were also observed several times a week for the presence of tachypnea and labored respiration; when these findings were clearly evident, animals were either killed and studied or treated with captopril. Animals not demonstrating respiratory difficulties were studied at 24 months of age.



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  		Figure 1. Study protocol. Captopril treatment was initiated in four groups of SHRs beginning at 12, 18, and 21 months of age (SHRRx12, SHRRx18, SHRRx21). Captopril was also administered to an additional group of SHR at the onset of heart failure (SHR-FRx). All captopril-treated animals were studied at 24 months of age; untreated animals were studied at 24 months or at the onset of failure (if before 24 months).

Echocardiographic Methods
Two dimensional echocardiographic examination was performed on conscious, nonfailing SHR (SHR-NF) and failing SHR (SHR-F) as previously described.14 All studies were performed with a 7.5-MHz short focus transducer with a 60-Hz frame acquisition rate (model 77020A, Hewlett-Packard). Images were obtained in the parasternal long-axis and short-axis and apical two-chamber and four-chamber views. Gain and contrast settings were adjusted by the operator (M.T.S.) to achieve a well-balanced gray-scale appearance on the video display. Real-time images were stored on VHS videotape for subsequent off-line analysis. Ventricular volume (V) was calculated assuming the ventricle to be a prolate spheroid, formed by rotation of an ellipse about its major axis: V=(4/3) · {pi} · (L/2) · (W/2)2, where L is the major axis (length) and W the minor axis (width). The relationship between the long-axis cross-sectional area (A) and the dimensions is A={pi} · (L/2) · (W/2), or A={pi}/4 · L · W, so that W can be expressed as W=4 · A/({pi}L), and volume can be expressed as V=([8/3]/{pi}) · A2/L. End-diastolic volume (EDV) can therefore be represented as EDV=([8/3]/{pi}) · LVAd2/LVLd, where LVAd is end-diastolic area and LVLd is end-diastolic length; end-systolic volume (ESV) as ESV=([8/3]/{pi}) · LVAs2/LVLs, where LVAs is end-systolic area and LVLs is end-systolic length. Ejection fraction ([EDV-ESV]/EDV) was calculated as an index of left ventricular systolic function. The high-frequency transducer used in this study allowed us to obtain good-quality transthoracic images of the beating heart. With a small sector size and shallow imaging depth, sampling rate for two-dimensional imaging was {approx}60 Hz. Given heart rates of 200 to 300 bpm, {approx}8 to 16 frames were recorded per cardiac cycle.

Mechanical Studies
Experimental Preparation
After the animal was killed, hearts were quickly removed and placed in oxygenated Krebs-Henseleit solution at 28°C. The left ventricular posterior papillary muscle was dissected free, mounted between two spring clips, and placed vertically in a 100-mL acrylic chamber containing Krebs-Henseleit solution of the following composition (mmol/L): NaCl 120, KCl 5.9, NaHCO3 25, NaH2PO4 1.2, MgCl2 1.2, CaCl2 1.0, and dextrose 11.5. The solution was bubbled with a gas mixture containing 95% O2 and 5% CO2 and equilibrated to pH 7.4 at 28°C. The muscle preparation was placed between parallel platinum plate electrodes and field stimulation was carried out at a rate of 0.2 Hz, using square-wave pulses 5 ms in duration. The voltage was set 10% above threshold. The spring clip on the upper end of the muscle was attached to a low-inertia DC pen motor (G100-PD, General Scanning) and the lower clip to a semiconductor strain gauge force transducer (DSC-3, Kistler-Morse). A digital computer with an analog/digital interface allowed control of either force or length of the preparation. Force and length data were sampled at a rate of 1 kHz and stored on disk for later analysis.

After removal of the papillary muscles, the left and right ventricles were carefully separated. Tissues were gently blotted and weighed. Samples of left and right ventricle were dried at 60°C for 24 hours and reweighed to calculate wet/dry ratios. LV/BW and RV/BW were used as indices of ventricular hypertrophy.

After muscles were mounted, they were equilibrated by isotonic contraction at a light load (on the order of 0.4 g/mm2). After this period, muscles were gradually stretched to the peak of the active stress versus length relationship (Lmax, defined as the muscle length resulting in peak active stress). The overall muscle equilibration time period before study was 90 to 120 minutes. Isometric contraction parameters from five twitches were averaged; these were resting stress ({varsigma}resting, g/mm2); active stress ({varsigma}active, g/mm2), defined as peak isometric stress minus resting stress; peak rate of isometric stress development (peak+d{varsigma}/dt, g/mm2/s); electromechanical delay (EMD, ms), defined as the time from stimulation to the onset of stress development; time to peak stress (TPS, ms), defined as the time from the onset of stress development to the time of peak stress; and time from peak stress to 50% relaxation (RT1/2, ms). Stress-velocity relations (shortening velocity versus load) were determined from muscle shortening velocity measurements after "quick releases" 100 ms after stimulation to loads ranging from 0.5 g to peak isometric stress. Values were subsequently normalized for muscle length and cross-sectional area, as described previously.3,8

Stress-Strain Analysis
Analysis of passive central segment stiffness was done as previously described.9,15 Briefly, two silk markers, {approx}1 to 2 mm apart, were applied to the muscle preparations. Application of the markers had no significant effect on active stress development. The preparation is scanned longitudinally by a laser beam at a rate of 1 kHz, and the resulting decrease in reflected light is detected by a photodiode. The time between marker detection events is converted to a central segment length signal (Lcs), which is sampled by the computer along with whole muscle length and stress. Central segment dimensions were measured at three levels in both the frontal and lateral projections with the use of a cathetometer-telescope combination (Gaertner Scientific Corp) and cross-sectional area calculated assuming an elliptical configuration. Passive stress/length relations were determined by applying length ramps to the whole papillary muscle at a rate of 1.0 mm/s, corresponding to a normalized rate of length change on the order of 0.1 muscle lengths/s, with a range of stress on the order of 0.1 to 1.25 g/mm2. Stress and central segment length were sampled and central segment stress-strain relations were derived from these measurements as previously described.9 It should be noted that stiffness was determined over a relatively large range of stress, the upper limit of which was above the usual physiological range.

Central segment stress ({varsigma}cs) was defined as tension normalized by instantaneous cross-sectional area, calculated from the measured cross-sectional area assuming incompressibility: CSAcs(inst)=CSAcs(ref)[Lcs(ref)/Lcs(inst)], where CSAcs(inst) is instantaneous central segment cross-sectional area, CSAcs(ref) is the cross-sectional area at the reference length, Lcs(inst) is instantaneous central segment length, and Lcs(ref) is the reference central segment length. Natural strain ({epsilon}) is defined as {epsilon}=ln (L/Lo), where L is length and L0 is length at zero stress (or "slack length"). Because of the exponential nature of the stress-strain relation, and therefore the shallow slope at low loads, the determination of true slack length (as used in the traditional definition of strain) is subject to considerable experimental error. Therefore, a modified natural strain definition was used in the present study: {epsilon}cs=ln (Lcs/L0.1), where Lcs is instantaneous central segment length, and L0.1 is central segment length at a load of 0.1 kdyne/mm2. Using this definition, {epsilon}=0 at a near-slack "reference length" at which {varsigma}=0.1 kdyne/mm2.

Assuming that passive myocardial stress({varsigma})-strain({epsilon}) relations are exponential in nature, the relation can be expressed as {varsigma}=ce(k{epsilon}). Using a log transformation, log({varsigma})=log(c)+k{epsilon}. The central segment stiffness constant, kcs, was derived from the slope of the log({varsigma}) versus {epsilon} relation.

Histological Analysis
Samples of left ventricular free wall were obtained at the time the animals were killed, placed in Karnovsky's fixative, and prepared for microscopy and histological analysis as previously described.8,9 At the conclusion of the mechanical studies, the central segment of the left ventricular papillary muscle was fixed with a preload equivalent to the resting stress at Lmax. Histological sections were stained with Masson's trichrome and with picrosirius red. Picrosirius red sections were examined under polarized light.16 Areas of connective tissue network and myocytes were quantified by a semiautomated computer-based video analysis system with the ColorImage image analysis software package.17 Images were acquired with a binocular microscope (Leitz Ortholux) with a video camera (Javelin model JE3462 RGB). The software has the capability of identifying the area in an image corresponding to a specified spectral range. For each field of a given slide, a region of interest was identified and the computer was used to discriminate all areas within that region meeting the specified spectral criteria. The relative area occupied by connective tissue and myocytes (representative of volume fraction, assuming a random three-dimensional distribution) was quantified in at least 5 fields from each slide, and the results were averaged. Slides from 5 to 13 left ventricles were examined from each group.

Statistical Analysis
A two-way ANOVA with replication was used to examine group and treatment effects. The Newman-Keuls multiple-sample comparison test18 was used to localize differences where appropriate. Data are expressed as mean±SD.


*    Results
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 up arrowMethods
 *Results
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Development of Heart Failure and Survival Data
One hundred eight SHR and 12 WKY rats were entered into the study. SHR were studied when failure was suspected, or at 24 months if failure did not occur by that time. Clinical findings suggesting heart failure were tachypnea and labored respiration. Animals were only considered to have heart failure, however, when documented by pathological findings as described previously,3,9,11 which included pleural and/or pericardial effusions, left atrial thrombi, and right ventricular hypertrophy. Forty-five SHR were treated before heart failure (SHRRx12, n=14; SHRRx18, n=16; and SHRRx21, n=15) and 12 SHR were treated after heart failure was documented (SHR-FRx). Fifty-one SHR were never treated (SHRNo Rx). Of the group of 63 initially untreated SHR (51 untreated and 12 treated only after the onset of failure), 54 of 63 (85%) died or developed heart failure by 24 months of age; 17 SHR died of unknown cause (no obvious cardiac pathology) or had to be killed for other reasons (stroke, debilitation, or tumor); 5 SHR died with autopsy evidence of heart failure (the pathological criteria for failure were the same as that used for study animals); and 20 were studied when they developed heart failure. Twelve SHR developed evidence of heart failure and were treated (SHR-FRx group). Overall, 37 of 63 (59%) of untreated SHR (this includes the SHR-FRx group, which developed failure and were then treated) developed heart failure by 24 months of age. None of the SHR treated before heart failure (n=45) developed pathological findings consistent with heart failure.

Physiological Parameters
Peak systolic blood pressure, body weight, cardiac chamber weights, and chamber weights normalized for body weight are presented in Table 1Down. Treatment of SHR with captopril initiated at 12, 18, and 21 months of age (SHRRx12, SHRRx18, SHRRx21) was associated with a significant decrease in the left ventricular weight/body weight and right ventricular weight/body weight in all treated SHR (P<.01) compared with untreated SHR. Both LV/BW and RV/BW, however, were significantly lower in WKY relative to SHR with treatment initiated at 12 months of age (SHRRx12 versus WKY; P<.01). In a subgroup of 12 SHR-F, captopril treatment was begun at the time of heart failure (SHR-FRx). In addition to tachypnea and labored respiration, the presence of depressed function was documented in this subgroup of animals by echocardiogram (Table 2Down). Six of 12 SHR-FRx (50%) treated with captopril for 2 to 4 months survived to 24 months of age and were then studied. At the time of study, 1 of 6 had effusions, 5 of 6 had atrial thrombi, and all had right ventricular hypertrophy. When SHR-F were treated after heart failure (SHR-FRx), LV/BW was significantly reduced (P<.01, Table 2Down) relative to the untreated SHR-F group.


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  		Table 1. Blood Pressure, Body Weight, Cardiac Chamber Weight, and Ratios


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  		Table 2. Age, Body Weight, Cardiac Chamber Weights, Ratios, and Echocardiographic Data

Data from SHR treated at the onset of failure (SHR-FRx) and SHR-F are summarized in Table 2Up. The mean age at study of SHR-F (19±2 months) was slightly but significantly lower than the other two groups (P<.01) because the SHR-F were studied when failure developed. In echocardiographic studies of unanesthetized rats, SHR-F demonstrated significantly enlarged end-diastolic and end-systolic left ventricular volumes relative to SHR-NF. The left ventricular ejection fraction was 45±7% in the SHR-F and 73±2% in the untreated SHR-NF group (P<.01). Captopril treatment (SHR-FRx), initiated at the time of heart failure and continued for 2 to 4 months did not significantly improve these indices despite a reduction in LV/BW ratio. The RV/BW ratio was not significantly reduced with captopril in SHR-FRx relative to SHR-F group.

Isometric Contraction Parameters and Shortening Velocity
Papillary muscle data for the untreated SHR versus captopril-treated SHR groups are presented in Table 3Down. Papillary muscle cross-sectional area was not significantly different among SHR groups. Active isometric stress ({varsigma}active, normalized for muscle cross-sectional area) and maximum rate of stress development (+d{varsigma}/dt) were increased with captopril treatment (SHRRx12, SHRRx18, SHRRx21) compared with untreated SHR (SHRNo Rx) (treatment effect P<.001 by ANOVA; see Table 3Down for subgroup comparisons). There was no significant difference in active stress or maximum rate of stress development in SHR treated with captopril for 12 months versus untreated WKY (WKY versus SHRRx12; Table 3Down). Captopril treatment with the onset of failure resulted in no significant improvement in either of these contractile indices (SHR-FRx compared with SHR-F; Table 3Down).


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  		Table 3. Isolated Muscle Parameters

In animals treated with captopril at 12, 18, and 21 months of age to 24 months (SHRRx12, SHRRx18, SHRRx21 groups), electromechanical delay time (EMD) was significantly decreased versus untreated SHR (SHRNo Rx; Table 3Up). Time to peak stress (TPS) of SHR treated at 12 and 18 months was significantly abbreviated compared with untreated SHR (P<.01). The relaxation time index (RT1/2) was significantly abbreviated in untreated SHR relative to SHR treated at 12, 18, and 21 months of age (P<.01). There was no significant effect of treatment of failing SHR (SHR-FRx) versus untreated SHR-F (SHR-F) on any of the measured physiological parameters (Table 3Up).

Quick-release stress-velocity relationships are presented in Fig 2Down. At all loads examined, velocity of shortening was less in papillary muscles from SHR-F than WKY and SHR-NF (left panel), although to a lesser degree at higher loads. Captopril treatment (SHRRx12, SHRRx18, SHRRx21 groups; right panel) before heart failure was associated with increased V0.5 in all groups relative to untreated SHR (SHRRx12, SHRRx18, SHRRx21 groups combined versus all untreated SHR; treatment effect P<.01, Table 3Up). However, captopril treatment of the failing SHR did not significantly improve V0.5 (SHR-FRx versus untreated SHR-F (SHR-F; Table 3Up) or stress-velocity relationships (Fig 2Down).



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  		Figure 2. Effect of captopril on quick release (100 ms after stimulation) stress-velocity relationships. Data from untreated spontaneously hypertensive rats with failure (SHR-F) and without failure (SHR-NF), and normotensive WKY rats are presented in the left graph; the effects of captopril treatment of SHR are presented in the right graph. As noted in Table 3Up, shortening velocity was significantly reduced (P<.01) in SHR-F in comparison to WKY rats. The shortening velocity in all captopril-treated SHR groups (SHRRx12, SHRRx18, SHRRx21) was significantly greater (P<.01) than that in untreated SHR. Data are mean±SD; 6 to 14 papillary muscles per group.

Myocardial Stiffness
Fig 3Down demonstrates the effects of captopril treatment on myocardial stiffness. Captopril treatment before failure was associated with significantly reduced myocardial stiffness, kcs, in SHR captopril-treated groups (SHRRx12, SHRRx18, SHRRx21 groups) versus untreated SHR (SHRNo Rx) (Table 3Up). In contrast, in the SHR-F group, treatment did not significantly reduce myocardial stiffness (SHR-F versus SHR-FRx; Table 3Up).



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  		Figure 3. Effects of captopril on myocardial stiffness of left ventricular papillary muscles from captopril-treated and untreated SHR and normotensive WKY rats. Treatment was initiated in SHR at 12, 18, and 21 months of age (SHRRx12, SHRRx18, SHRRx21) and animals were studied at 24 months. In the SHR-F group, treatment for 2 to 4 months was begun at the time of failure and data were compared with untreated SHR with heart failure. A significant effect (P<.01) of captopril treatment in the SHR was noted when captopril was administered prior to the onset of failure. Data are mean±SD; 6 to 12 papillary muscles per group.

The relationship between myocardial stiffness and shortening velocity is presented in Fig 4Down. There is an overall negative correlation between shortening velocity and stiffness of the left ventricular papillary muscle (r=-.96).



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  		Figure 4. Effect of captopril on the relationship between shortening velocity and myocardial stiffness of left ventricular papillary muscles from captopril-treated and untreated SHR and normotensive WKY rats. Treatment was initiated in SHR at 12, 18, and 21 months of age (SHRRx12, SHRRx18, SHRRx21) and animals were studied at 24 months. In the SHR-F group, treatment for 2 to 4 months was begun at the time of heart failure. Data are mean±SD; 6 to 12 papillary muscles per group.

Histology
Representative histological sections of left ventricular free wall from the WKY, SHR-NF and SHR-F and 12-month captopril-treated SHR are presented in Fig 5Down, and a quantitative histological analysis of left ventricular papillary muscles in Table 4Down. Fig 6Down is a cross-section of the left ventricular free wall from normotensive WKY and failing SHR stained with picrosirius red, viewed under polarized light. Myocardial fibrosis was increased and myocyte area was decreased in failing SHR (SHR-F) relative to WKY (P<.05). Fibrosis and myocyte area in captopril-treated SHR (SHRRx12, SHRRx18) was comparable to that in WKY (Table 4Down). Fibrosis was not significantly reduced by captopril when treatment was begun at 21 months of age (SHRRx21). Furthermore, fibrosis and reduced myocyte area were not altered when captopril administration was initiated at the time of failure (SHR-FRx versus WKY; Table 4Down).



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  		Figure 5. Light micrographs of left ventricular free wall sections from the normotensive WKY rat (top left), the untreated SHR-NF (top right), the SHR with heart failure (bottom left), and the SHR treated with captopril for 12 months (bottom right). All sections were stained with Masson's trichrome; x40 objective.


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  		Table 4. Histological Analysis (Left Ventricular Papillary Muscles)



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  		Figure 6. Collagen network of left ventricular myocardium from normotensive WKY (A) and SHR with heart failure (B) using picrosirius red, viewed under polarized light. Note the increase in interstitial fibrosis and the extensive collagen network (as indicated by the bright birefringent fibers) in the left ventricle of SHR-F.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study demonstrates that captopril administered prior to the onset of heart failure prevents impairment of myocardial function in the SHR. Treatment decreases hypertrophy and prevents the development of structural changes (fibrosis and cell loss) and mechanical dysfunction associated with the development of heart failure. The degree to which captopril prevents contractile dysfunction appears to be related to the age at which captopril treatment was initiated and the duration of captopril administration (ie, a greater effect with earlier and more prolonged treatment). In contrast, captopril treatment after the onset of failure results in a reduction of left ventricular hypertrophy, but contractile dysfunction and abnormal structure are not reversed. It is likely that there are irreversible events with heart failure (eg, myocyte loss) and other changes that may be irreversible or only slowly reversible (eg, fibrosis). From these studies, one cannot exclude the possibility that improvement in myocardial function in these animals with heart failure might be demonstrable if captopril treatment were to be continued for a longer period of time. On the other hand, there may be little or no improvement in intrinsic myocardial function, and the functional improvement with treatment seen in animal models and in the clinical setting may reflect stabilization of disease progression, with improved cardiac function secondary to alterations in loading conditions. It should be noted that the observed reduction in systolic function in the SHR (as measured in vivo by ejection fraction, Table 2Up) may be due in part to differences in loading conditions (ie, different levels of end-systolic stress).

Previous studies in the SHR19 have demonstrated that in addition to left ventricular hypertrophy there is also an increase in interstitial collagen. Structural remodeling of the extracellular matrix has been implicated in alterations in myocardial stiffness,16,19 which may contribute to both systolic and diastolic dysfunction in hypertrophied hearts. In previous studies, we have observed a marked increase in myocardial fibrosis and stiffness in the male SHR during the transition to failure at 18 to 24 months of age,9 a time that corresponds to the first appearance of hemodynamic and functional impairment.3,6–12,14 Consistent with these findings, histological analysis of the myocardium in the present study demonstrates increased fibrosis and decreased fractional myocyte area in the SHR with heart failure.

In studies of the myocardial infarction model, hypertrophied noninfarcted papillary muscles from rats with large infarctions were found to have a marked increase in hydroxyproline content and increased myocardial stiffness 6 weeks after coronary artery ligation20; treatment with captopril for 3 weeks starting 3 weeks after infarction did not prevent the increase in myocardial hydroxyproline content and passive stiffness. Smits et al,21 on the other hand, demonstrated that captopril treatment early after myocardial infarction not only reduced left ventricular hypertrophy but also completely inhibited increased DNA synthesis and collagen deposition. Thus the effects of early treatment of animals with myocardial infarction may be analogous to those in the SHR treated before the onset of contractile dysfunction, fibrosis, and heart failure. In the present study the increased myocardial stiffness in the aging SHR was associated with decreased shortening velocity (Fig 4Up). A similar relationship between stiffness and shortening velocity with myocardial hypertrophy has been noted by others.22 Treatment with captopril ameliorated the depression of shortening velocity seen in untreated animals. It is not possible, however, from the present studies, to determine if there is a direct relationship between stiffness and shortening or whether both result from other structural and functional changes.

The protection afforded by captopril may be attributed in part to a reduction in blood pressure and reduced left ventricular loading caused by inhibition of the renin-angiotensin system.13 In the present study, peak systolic pressure was reduced on average from 180 to 145 mm Hg over a 3- to 12-month period of treatment. However, the relation between blood pressure reduction and protection by angiotensin converting enzyme inhibition is not entirely straightforward. Laboratory data have demonstrated regression of left ventricular hypertrophy by ACE inhibitors at doses that do not lower blood pressure23,24 as well as in the presence of a fixed pressure overload as the result of aortic constriction.25–27 In the male SHR-F, blood pressure was reduced to {approx}140 mm Hg in comparison to nonfailing SHR, and captopril treatment of SHR-F did not further decrease peak systolic pressure although hypertrophy was reduced. Captopril has also been shown to significantly reduce LV/BW ratio of normotensive female rats yet did not lower the blood pressure.10 Thus although the increase in myocardial hypertrophy and fibrosis seems to be partially related to elevated blood pressure in male and female animals, other factors may also be involved.

The transition from hypertrophy to failure has been observed to be associated with elevations of transforming growth factor-ß1, fibronectin, and pro-{alpha}1(I) and pro-{alpha}1(III) collagen mRNA.12 Transforming growth factor (TGF-ß1), which increases before increases in collagen mRNA in the heart, has been shown to induce expression of mRNAs coding for extracellular matrix proteins28 including collagen type I and III29 and to inhibit collagenase activity.30 Angiotensin II has been shown to increase fibroblast proliferation31,32 and augments, up to threefold, the expression and secretion of latent TGF-ß1 in neonatal cardiac fibroblasts.33 While there may be differences between neonatal and adult fibroblasts, angiotensin II–induced activation of TGF-ß1 gene expression may be an important mechanism by which collagen genes are stimulated in fibroblasts, resulting in increased extracellular matrix production.

Captopril has been shown to prevent the increase in DNA and collagen synthesis in noninfarcted cardiac muscle after coronary artery ligation.34 Enalapril treatment has also been shown to prevent the increase in the fraction of myocardium occupied by fibrosis and the decrease in myocellular cross-sectional area in male SHR when treatment was begun at 3 months of age and continued up to 14 months of age.35 It has been shown, however, that cardiac angiotensin II formation is not blocked by ACE inhibition (suggesting that other proteases are involved36). Thus the effect of captopril may result, at least in part, from effects other than ACE inhibition. In particular, it is possible that the captopril may act by inhibition of degradation of the vasodepressor peptide bradykinin.37 In a study of the noninfarcted region of the rat myocardial infarction model, however, Smits et al21 have shown that the angiotensin II receptor blocker losartan prevents fibrosis at sites remote from the infarction, suggesting that angiotensin II mediated events may be more important than the bradykinin pathway with respect to the development of myocardial fibrosis.

Gene expression of components of the renin-angiotensin system have been demonstrated in myocardium.38–40 Increased ACE activity in right and left myocardium after myocardial infarction has been observed while plasma renin and serum ACE remain at normal levels.41 The colocalization of ACE and angiotensin II receptors further supports the concept that angiotensin II is produced locally,42 particularly in the SHR, in which the tissue renin-angiotensin system has been reported to be abnormally activated.43 In chronic pressure overload, both ACE mRNA and ACE activity have been found to be increased in rat heart as was the fractional conversion of angiotensin I to II.44 In the aging SHR, left ventricular ACE mRNA expression is also upregulated in the SHR-NF and to a greater extent in the SHR-F (unpublished observations). Cardiac ACE45 and AT1 receptor binding density46 have been studied in the myocardial infarction model in the rat. AT1 receptor binding increased by day 3 after infarction and was followed by increased ACE binding at 1 week. ACE binding density subsequently increased progressively from 2 to 8 weeks in fibroblasts and fibroblast-like cells and was associated with collagen I gene expression and fibrillar collagen accumulation.46 These data demonstrate a sequence of local events in stressed myocardium resulting in a connective tissue response.

In the present study, treatment with captopril after the onset of heart failure did not reverse structural or functional abnormalities. Captopril administered to 12 animals with heart failure (6 of which survived until study) for 2 to 4 months was effective in reducing left ventricular hypertrophy but not fibrosis. In a study of SHR with established hypertension and cardiac and vascular hypertrophy, 16 weeks of high-dose ACE inhibitors reduced blood pressure and cardiac hypertrophy but did not reverse vascular fibrosis.47 Although it is possible that with more prolonged treatment, improvement in active and passive mechanical properties might be demonstrated in myocardium from male SHR-F, these findings appear to be generally consistent with the concept that captopril prevents fibroblast proliferation and collagen synthesis, but is ineffective in reversing these effects once fibrosis is well established.

In conclusion, the present study demonstrates that long-term captopril administration can prevent impaired contractile function and heart failure in the aging male SHR. A point of clinical relevance suggested by the data is that the earlier captopril is initiated, the greater the degree of prevention of pathological changes to the myocardium and myocardial dysfunction. Treatment initiated at the time of failure (SHR-FRx) reduces left ventricular hypertrophy but does not significantly ameliorate fibrosis, increased passive myocardial stiffness, or contractile dysfunction.


*    Selected Abbreviations and Acronyms
 
LV/BW and RV/BW = left and right ventricular wet weight normalized to body weight
SHR = spontaneously hypertensive rat
WKY = Wistar-Kyoto (rat)


*    Acknowledgments
 
This study was supported by Medical Research Funds from the Department of Veterans Affairs. This work was done during the tenure of a Clinician-Scientist Award from the American Heart Association (C.H.C.).

Received May 23, 1997; revision received August 4, 1997; accepted August 22, 1997.


*    References
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