Cardiovascular Research

Cardiovascular Research 1997 36(2):236-245; doi:10.1016/S0008-6363(97)00186-7
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Changes in collagen phenotypes during progression and regression of cardiac hypertrophy

Chun Mu Yang, Vijaya Kandaswamy, David Young and Subha Sen^*

Department of Molecular Cardiology (FF4-09), Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

^* Corresponding author. Tel. (+1-216) 4442056; Fax. (+1-216) 4449263; E-mail sens@ccsmtp.ccf.org

Received 30 August 1996; accepted 26 June 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Excessive deposition of collagen has been implied to be responsible for abnormal stiffness and altered cardiac function during hypertrophy and heart failure. In the present paper we studied the changes in collagen and their phenotypes during development of cardiac hypertrophy in spontaneously hypertensive rats (SHR) compared to age- and sex-matched Wistar Kyoto (WKY). We also studied the changes in collagen after regression of hypertrophy, with antihypertensive therapy with ACE inhibitors, captopril (C) and lisinopril (L). Method: Collagen was extracted from the heart tissue by cyanogen bromide (CNBr) digestion. Collagen phenotypes were separated and quantified by SDS–polyacrylamide gel electrophoresis. The transcript levels (mRNA) of collagen phenotypes were determined by Northern analysis. Results: Our studies showed that the ventricular collagen and their phenotypes did not alter in SHR during the first 6 months of progression of hypertrophy when compared to WKY. After 40 weeks, however, in SHR there was an unexpected rise in collagen content and the distribution of collagen phenotype differs compared to WKY, especially during the chronic phase of hypertrophy (65 weeks of age). In WKY during the aging process there was a gradual increase in type III collagen, whereas in SHR it plateaus after 40 weeks of age. Treatment with antihypertensive drugs captopril and lisinopril showed a similar degree of reduction in blood pressure (p<0.001), regressed hypertrophy (p<0.001), and reduced collagen, whereas decrease in type I to III ratio was found with captopril only, but not with lisinopril. This decrease in type I to III ratio due to captopril treatment is primarily due to an increase in type III collagen (both protein and transcript level) in SHR. Conclusion: Our data showed, for the first time, that during the chronic phase of hypertrophy in SHR there is a gradual reduction in type I to III ratio, primarily due to a lack of increase in type III collagen during chronic phase of hypertrophy. This suggests that quality of collagen is an important factor in determining the degree of cardiac stiffness. Our data also showed that not all ACE inhibitors have similar actions on collagen phenotype production. This suggests that perhaps the mechanism of action of ACE inhibitors on collagen are independent of its effect on angiotensin II formation.

KEYWORDS Collagen phenotypes; Captopril; Lisinopril; Angiotensin II; Regression of Hypertrophy; Rat

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Myocardial hypertrophy has been described as an increase in myocardial mass and myocyte size associated with an increase in interstitial collagen within the extracellular matrix. This has been shown in both humans and rats. The capability of the heart's function is believed to be predicted by the amount of collagen present in the ventricle. Recently, it has been shown that quality rather than the quantity of the collagen present in the heart is perhaps more important in determining cardiac function [1]. It is important to understand the alterations that occur in the collagens during development of hypertrophy as the structural remodeling that takes place in the heart is an important factor for the transition of hypertrophy to heart failure [2, 3]. Sen and Bumpus [4] previously showed that hypertrophy can be regressed by using various antihypertensive drugs. Captopril, an ACE inhibitor, is the only antihypertensive drug that not only regressed cardiac hypertrophy, but also reduces collagen. The purpose of the present study is to elucidate the molecular changes that occur during development of hypertrophy, especially the alteration in the formation of collagen, both at transcriptional and protein levels, and the effect of converting enzyme inhibitors on collagen formation. Mukherjee and Sen [5] reported that, although both captopril and hydrazine can lower blood pressure in spontaneously hypertensive rats (SHR), a genetically hypertensive rat model, only captopril resulted in the regression of hypertrophy and reduction in collagen content of the heart, whereas hydralazine, despite normalization of the blood pressure, did not alter collagen or cardiac hypertrophy. Furthermore, captopril also resulted in redistribution of collagen phenotypes Type I and Type III, of cardiac tissue both in rats [5] and humans [6]. These data suggested that alteration in collagen phenotypes in SHR is independent of blood pressure control and myocardial mass.

Recently, more evidence has been presented defining a role of angiotensin II (AII), a potent vasoconstrictor, in stimulation of cultured myocyte growth and smooth muscle cell hyperplasia [7–9]. There is some evidence as well for the existence of independent intracardiac renin angiotensin systems [10]. Taken together, these data suggest that angiotensin II may play an important role in cardiac collagen gene expression and may be responsible for myocardial fibrosis and thereby stiffness [11, 12]. Although increasing evidence indicates that collagen production may occur both at the translational and transcriptional levels, most data presented to date are in acute in vitro models using cell culture systems. These models, however, cannot precisely reflect the in vivo situation in hypertensive hypertrophy, which is a long-term progressive process. In the present work, we studied effects of the converting enzyme inhibitors captopril and lisinopril on collagen production and distribution of collagen phenotypes I and III, both at the protein and mRNA levels. The purpose of this study is two-fold: to understand the mechanism of action and uniqueness of captopril compared with an equally potent converting enzyme inhibitor (lisinopril) and to evaluate the effect of prevention of AII formation on collagen production, including their phenotypes Type I and Type III formation. The present study describes the changes in collagen of the ventricular tissue during development of hypertrophy, and also the effect of ACE inhibitors on cardiac tissue collagen after regression of hypertrophy.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
All male SHR and age- and sex-matched normotensive Wistar–Kyoto (WKY) rats used in this study were obtained from Taconic Farm (Germantown, NY). All chemicals and solvents used in this study were ACS certified analytical reagents. The following materials were from the sources indicated: human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probe (Clontech), mouse collagen cDNA plasmid a2 (I) probe, pAZ1002, and mouse collagen cDNA plasmid a1 (III) probe, pMCS1.7 (from Dr. Crombrugghe, Department of Molecular Genetics, University of Texas), random primed DNA labeling kit (Boehringer Mannheim), Genescreen nylon hybridization membrane and a-32P-dCTP (Dupont).

2.1 Study groups
Male SHR and age- and sex-matched WKY rats from 8 weeks to 65 weeks of age were chosen to study progression of hypertrophy and its transition to heart failure. For the regression study, 32 week old SHR were used. Each study group consisted of at least 10 rats.

All animals were kept under the same conditions and fed Purina Rat Chow. For antihypertensive treatment the rats were treated with captopril (50 mg/kg/day) or lisinopril (10 mg/kg/day) each, given in drinking water, for 13 weeks. Arterial blood pressure was measured from the last three weeks of treatment using a tail cuff method as described by Sen et al. [13]. At the end of the treatment period, rats were killed by decapitation. Whole hearts were excised and were freed of blood. The ventricles were blotted and weighed. The left ventricles were separated and frozen in liquid nitrogen and then transferred to a –70°C freezer until use.

2.2 Determination of hydroxyproline
The ventricular collagen was quantified from the hydroxyproline content. The amount of hydroxyproline in the left ventricles was determined by using a modified method of Bergman and Loxley [14]. All tissue samples (approximate 10 mg) were taken from the same area of left ventricular wall.

The myocardial tissue was homogenized and hydrolyzed with 6 normal HCl at 110°C for 24 hours under vacuum. Hydrolysis samples were dried with a flash evaporator. 0.5 ml of oxidant (chloramine T) was added, vortexed and allowed to stand for 4 minutes. To this was added 3.25 ml Ehrlich's reagent (3 ml of Ehrlich's + 16 ml of isopropanol). The tubes were kept at 25°C for 18 hours, and the intensity of the rate of coloration was measured at 558 nm using a Beckman DU spectrophotometer (Beckman Instruments, Inc, Fullerton, CA). A conversion factor of 8.2 was used to convert hydroxyproline to collagen [14].

2.3 Phenotyping of collagen
The purification, digestion of collagen with cyanogen bromide and separation of Type I and Type III collagen by SDS–PAGE, and their quantification by gel scanning was performed following the procedure described by Mukherjee and Sen [5].

Briefly, the heart myocardial tissue was homogenized in a glass homogenizer (10 ml vol) with PBS at 4°C. The homogenate was centrifuged at 4000xg for 10 minutes. The residue was resuspended in 2% SDS and rehomogenized at room temperature. The homogenate was centrifuged again and rehomogenized in 2% SDS. The procedure was repeated four times. The remaining residue was further extracted three times with PBS to remove the excess SDS. The residue was then rehomogenized in acetone and centrifuged at 4000xg for 10 minutes. The supernatant was discarded, the step was repeated, and the pellet was dried under vacuum.

The acetone dry powder was homogenized in a glass homogenizer with 0.6 ml of 70% formic acid (volume per volume) per 100 mg of original tissue. The homogenate was then transferred to a 15 ml graduated polyethylene centrifuge tube and any remaining residue was washed from the homogenizer with 0.6 ml formic acid. The homogenate was made up to a volume of 1.5 ml with 70% formic acid per 100 mg original tissue. Cyanogen bromide crystals were added to this volume to produce a concentration of 20 mg/ml. Nitrogen gas was bubbled through the mixture, the tubes were sealed, and the reaction was allowed to proceed for 18 hours at 25°C. To facilitate mixing, the tubes were positioned at an angle of 30°C. At the completion of the reaction, the digest was centrifuged at 5000xg for 20 minutes. 0.3 ml of the supernatant was removed and dried under a vacuum. The material was then dissolved in a sample buffer in preparation for polyacrylamide gel electrophoresis. The amount to be loaded was determined by hydroxyproline estimation on the lyophilized powder obtained from 0.5 ml of the supernatant. Collagen phenotyping was done by polyacrylamide gel electrophoresis. It was performed on a vertical gel (protein N2, BioRad Laboratories, Richmond, CA) by stacking and separating gel concentrations of 4% and 12%, respectively. Samples of a 10 µl volume are loaded into each well and stacking was allowed to proceed at a current of 25 mA. Once the samples entered the separating gel, the current was increased to 35 mA, and electrophoresis continued until the dye marker reached a level two thirds from the top. When electrophoresis was completed, the gel was removed and stained for one hour by gentle shaking in 250 ml of aqueous solution containing 0.1 to 5% Coomassie Blue-250, 30% methanol and 7.5% acetic acid and was destained for 24 to 48 hours by continuous shaking with several changes of acetic acid. All staining and destaining reactions, as well as gel buffers, were filtered to minimize contamination for subsequent scanning. Gels were scanned for quantification using Helena Laboratories, quick scan (Beaumont, TX) [1]. Data are expressed as percent distribution/mg collagen.

2.4 RNA extraction and RNA–DNA hybridization
Isolation of total RNA from the rat left ventricles was performed as in the procedure described by Chirgwin et al. [15] with minor modification [16]. The ventricle was homogenized in 4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol solution at room temperature. Sequentially, 0.1 volume of 2 M sodium acetate, pH 4.0, 1.0 volume phenol (water saturated) and 0.2 volume of chloroform were added to the homogenate. The mixture was vortexed and put on ice for 15 minutes and centrifuged in a Sorvall centrifuge at 10 000xg at 4°C for 20 minutes. The aqueous phase containing the RNA was transferred to a Corex glass centrifuge tube and purified via two rounds of isopropanol precipitation and one of ethanol precipitation. The purified RNA sample was dissolved in diethyl pyrocarbonate (DEPC) treated water. RNA concentration was determined by its absorbance at 260 nm. Integrity of the RNA was ascertained by the appearance of the 18s and 28s RNA bands on ethidium bromide stained agarose gel.

Steady-state levels of mRNA were determined by Northern and dot blot analysis. Total RNA was denatured in the solution containing 17.5% formaldehyde and 10xSSC at 65°C for 15 minutes and transferred to ice for making dots. Each lane contained 20 µg RNA. The RNA were applied on the Genescreen nylon membrane and UV crosslinked with Stratalinker UV crosslinker according to the manufacturer's instructions and baked at 80°C for 2 hours in a vacuum oven. Blots were prehybridized overnight at 42°C in 5xSSC, 0.12% SDS, 10% dextran sulfate, 50% formamide, 1x Denhardt's solution, 200 mg/ml ssDNA, 20 mM Tris-HCl, pH 7.4. pAZ1002 contains the cDNA fragment of mouse collagen a1(I) and pMCS-1.7 contains the cDNA fragment of mouse collagen a1(III). The 850 bp fragment of mouse collagen a2(I) cDNA was released by restriction enzyme XhoI, and the 1700-bp fragment of mouse collagen a1(III) cDNA was released by EcoRI and the cDNAs were separated by agarose gel and purified with a Gene Clean Kit according to manufacturer's instruction (Bio 101 Inc). The cDNA probes were labeled with a-32P-dCTP using the random primer extension labeling system. The probe was added to the prehybridization mixture at the concentration of 1x10⁶ cpm/ml. The hybridization was performed at the same temperature for 24 hours. After hybridization, the blots were washed in 2xSSC for 30 minutes at room temperature and 2xSSC and 0.1% SDS at 42°C for 30 minutes and 50°C for 15 minutes. The blots were exposed on X-ray films with intensifying screen for 1–2 days at –70°C. Relative amounts of each mRNA were determined by video densitometry (Image Analyzer, Fotodyne Inc, New Berlin, Wis.) in the linear response range of the X-ray films. The densitometric residues of specific mRNAs were ‘normalized’ by that of GAPDH mRNA, which was used as an internal control [34].

2.5 Statistical analysis
Results are expressed as mean±SEM. Data were analyzed by Student's t-tests and by ANOVA as appropriate, followed by Tukey's test and F-tests. The SAS system was used for all analyses (SAS Institute Inc, SAS/STAT User's Guide, Version 6, 4th edition, vol 2, SAS Institute Inc, Cary, NC, 1990). Data were considered significant when P<0.05.

The investigator conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Blood pressure during development of hypertrophy in SHR and WKY
The changes in blood pressure in SHR and normal Wistar–Kyoto rats during progression of hypertrophy are summarized in Table 1. In the SHR blood pressure was elevated as early as 8 weeks of age and remained elevated throughout the rest of the experimental period (155 mmHg vs. 113 mmHg at 8 weeks of age P<0.001 and 195±5 mmHg vs. 115±5 mmHg in WKY, P<0.001 at 65 weeks of age). The heart weight/body weight ratio during progression of hypertrophy in SHR and WKY is shown in Table 1. SHR had hypertrophied hearts from as early as 8 weeks (3.3±0.05 vs. 2.9±0.06 mg/g) and remained hypertrophied until 65 weeks of age (3.6±0.06 vs. 2.0±0.02 mg/g, P<0.01).

View this table: [in this window] [in a new window]   Table 1 Blood pressure, ventricular weight during progression of hypertrophy in SHR and WKY

 
3.2 Collagen concentration
Changes in collagen concentration during development of hypertrophy are summarized in Fig. 1. From 8 weeks to 24 weeks of age, the collagen concentration (mg/g wet tissue) did not differ between SHR and WKY rats, despite persistence of hypertension and hypertrophy. However, at 40 weeks of age there was an unexpected increase in collagen concentration (10±0.5 mg/g wet tissue in SHR vs. 7±1 in WKY, P<0.05) which was even further increased at the age of 65 weeks (23.7±0.5 vs. 14.4±0.8 mg/g wet tissue, P<0.001) (Fig. 1). These data showed that despite persistence of high blood pressure and cardiac hypertrophy, during the initial phase of hypertrophy, the collagen component of the heart was not altered. However, during the chronic phase of hypertrophy (to 65 weeks of age), the collagen concentration was increased in the SHR compared to WKY and remained elevated.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Changes in ventricular collagen concentration (mg/g wet tissue) during progression of hypertrophy to heart failure. Each time point represents n = 10. Note: During the initial phase of hypertrophy in SHR, despite the presence of hypertension, the collagen concentration was similar to normal. In SHR, an unexpected increase was observed only during the later phase.

 
3.3 Collagen phenotypes during progression of hypertrophy
The % distribution of Type I and III collagen during progression of hypertrophy in SHR and WKY is shown in Fig. 2. During the aging process in SHR and WKY, the percent distribution of Type I and Type III per mg collagen did not change until 40 weeks of age. However, at 40 weeks of age the Type I collagen increased compared to previous age groups in WKY (p<0.05), and this increase in Type I collagen persisted until 65 weeks of age. The distribution of Type I collagen in SHR shown during progression of hypertrophy is shown in Fig. 1. Similar to WKY, up to 40 weeks of age there was no change in collagen despite the presence of hypertrophy and hypertension. Although there was a trend, but there was no significant increase in percent distribution of Type I collagen in SHR during aging process. However, when compared to SHR and WKY, a slight increase was noted in WKY compared to SHR.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 The distribution of Type I collagen during progression of hypertrophy in spontaneously hypertensive and normotensive rats. Each time point represents n = 12, P<0.05. Note: The percent distribution of Type I/per mg of collagen did not alter significantly during progression of hypertrophy in SHR, but increased slightly in WKY at 40 and 65 weeks of age.

 
The percent distribution of Type III collagen is summarized in Fig. 3. Distribution of Type III collagen in WKY remained unaltered until 40 weeks of age where there was a significant increase compared to previous age groups and remained elevated up to 65 weeks of age. In SHR, on the other hand, the percent distribution of Type III collagen remained unaltered throughout the progression of hypertrophy. This resulted in a significant increase in percent distribution of Type III collagen in WKY compared to SHR. This data showed that the ratio of Type III/Type I collagen is significantly different between WKY and SHR. In older WKY, myocardial collagen has more Type III compared to SHR.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 The percent distribution of ventricular collagen Type III during progression of hypertrophy. Each time point represents n = 12, P<0.05. Note: In WKY the percent distribution of Type III/mg collagen gradually increased from 24 weeks to 65 weeks of age. In SHR the Type III collagen did not alter up to 65 weeks of age.

 
3.4 Alteration of mRNA of collagen Type I and III during progression of hypertrophy in hypertension
Northern blot analysis of total RNA from SHR and WKY rats has resolved to a major band of 4.5 Kb and a minor species of 4.8 Kb, whereas only a single transcript of 5.3 Kb was obtained for Type III mRNA (Fig. 4). Density scans of the autoradiograph showed that during progression of aging, the transcript level of Type I collagen in SHR and WKY rats decreases slowly from 8 weeks to 65 weeks of age from a density of 0.8 unit to 0.45 (P<0.05) for SHR, and from 1.2 to 0.6 in WKY (Fig. 5). Type III transcript level, on the other hand, also decreases significantly in SHR rats during this period from 1.1 to 0.2 density unit, P<0.001 (Fig. 5). Compared to WKY, the Type III mRNA levels in SHR were significantly reduced from 40 to 65 weeks of age.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Total RNA was isolated from ventricle, and Northern blot analyses were performed. Total RNA (10 µg) was loaded for each lane. Representative Northern blot for collagen Type I is shown in Lane 1 and for collagen Type III in Lane 2. The message for type I showed two transcript (4.5 Kb and 4.8 Kb) and type III showed one transcript of 5.3 Kb.

 

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Ventricular collagen transcript levels (mRNA) for Type I and Type III collagen in SHR and WKY rats during progression of hypertrophy. The data were expressed as arbitrary densitometric units. Each time point represents n = 12, P<0.01. Note: (Top Panel) Type I mRNA. The mRNA level of Type I collagen gradually decreased during aging process. The Type III mRNA level in SHR and WKY in lower panel. Type III mRNA level did not alter significantly in WKY during growth and development. In SHR, the mRNA level gradually decreased from 1.1 unit to 0.2 at 65 weeks of age.

 
3.5 Effect of antihypertensive treatment with lisinopril and captopril on collagen
The effect of ACE inhibitors lisinopril and captopril on blood pressure is shown in Table 2. Both lisinopril and captopril effectively control blood pressure from 189 mmHg to 126 mmHg (C) and 116 mmHg (L) and resulted in regression of hypertrophy by normalizing the ventricular weight/body weight ratio (Table 2) (3.5±0.13 untreated vs. 3.05±0.12, P<0.01, captopril and 2.9±0.1, P<0.01, lisinopril treated). The total collagen content is also significantly reduced due to treatment with both ACE inhibitors (Table 2) (13±0.4 mg/g vs. 8.8±0.5 captopril and 10.2±0.4 lisinopril treated).

View this table: [in this window] [in a new window]   Table 2 Effect of antihypertensive treatment on blood pressure, ventricular weight and collagen in SHR and WKY

 
3.6 Effect of captopril and lisinopril on collagen phenotypes in SHR and WKY rats
The effect of lisinopril and captopril on collagen phenotypes is summarized in Fig. 6. Treatment with captopril caused a significant decrease in the ratio of Type I/Type III collagen compared to untreated control (1.2±0.16 in untreated vs. 0.56±0.04 captopril treated P<0.01) (Fig. 6); on the other hand, treatment with lisinopril caused a slight but not significant decrease in the ratio of Type I/Type III collagen phenotype pattern (1.2±0.16 untreated vs. 0.83±0.04 lisinopril, P = NS). These data showed a subtle difference in the effect of captopril and lisinopril on collagen phenotypes. This reduction in ratio of Type I/Type III is due to an increase in Type III collagen by captopril treatment. Captopril treatment resulted in an increase in Type III collagen from 2.6 mg/g to 6.4 mg/g (P<0.05). Lisinopril treatment, on the other hand, did not alter Type III collagen.

View larger version (49K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of converting enzyme inhibitors lisinopril and captopril on collagen phenotypes in spontaneously hypertensive rat hearts. In captopril-treated SHR, there was a significant decrease in Type I/Type III ratio (P<0.05), whereas with lisinopril the Type I/Type III ratio did not change compared to untreated SHR. Each group represents n = 10, P<0.05.

 
3.7 Effect of captopril and lisinopril on transcript levels of collagen phenotypes
To study the effects of two angiotensin II converting enzyme inhibitors, captopril and lisinopril, on the cardiac procollagen Type I and Type III transcript levels, we measured the abundance of mRNA for proa2(I) and proa1(III) collagen from left ventricular tissue. The results are summarized in Fig. 7. Dot blot hybridization was performed to localize both Type I and Type III collagen mRNA species. The data showed that the probes hybridized were specific to collagen mRNA and the molecular weight is similar to what has been reported earlier. In SHR captopril treatment increased the proa2(I) from 0.70±0.02 units to 0.87±0.04 units (P<0.01), whereas lisinopril treatment did not alter the transcript level of proa2(I) (0.70±0.09 vs. 0.72±0.04 units, P = NS). The transcript levels of Type III remained unaltered due to ACE therapy.

View larger version (49K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Effect of antihypertensive treatment on collagen Type I transcript level in spontaneously hypertensive rats treated with captopril and lisinopril. Each group represents n = 10, P<0.05. Top pane: Captopril treatment caused an increase in the transcript level of Type I collagen, whereas lisinopril did not result in any change. Bottom panel: The changes in Type III collagen mRNA level in SHR. There was no change in transcript level of Type III collagen treatment with either captopril or lisinopril.

 
In WKY rats, there was no significant difference among three groups for both Type I and Type III collagen mRNA. Antihypertensive treatment (Type I collagen transcript level: 0.88±0.09 untreated WKY, 1.07±0.06 captopril treated and 0.86±0.04 lisinopril treated, P = 0.1; and Type III mRNA 1.00±0.08 untreated; 1.11±0.08 captopril treated; 0.86±0.07 lisinopril treated, (P = 0.2).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
This study demonstrates that alteration in collagen levels and phenotypes occurs during progression of hypertrophy as well as after its regression by two selective antihypertensive ACE inhibitors. This study also showed that the two inhibitors do not have similar effects on the collagen phenotypes. (Figs. 5–7).

Our data showed that during progression of hypertrophy, myocardial collagen levels remain unaltered during the initial phase, and increase unexpectedly during the chronic phase. We have also shown that this alteration of collagen phenotypes occurs differently in SHR compared to WKY rats. By relative quantification, we found that the percent distribution of Type I collagen increased gradually in WKY compared to SHR during aging. On the other hand, the concentration of Type III collagen in SHR remained unaltered during the chronic phase of hypertrophy (Fig. 5), compared to WKY. This is the first documented evidence of a major difference in collagen III phenotype distribution in SHR compared to normotensive control. These data suggest that during the chronic phase of hypertrophy, the SHR heart collagen phenotypic distribution is different compared to WKY; especially WKY has more Type III collagen. The alteration of the collagen phenotype at the transcriptional level also showed that the transcript level for Type III collagen remains unaltered in WKY throughout the period of progression of hypertrophy, whereas it gradually decreased in SHR rats. These increases in collagen in older SHR and changes in their phenotypes appear to occur independent of longstanding hypertension and hypertrophy (Table 1), as hypertrophy and hypertension both were present in the SHR up to 6 months, when the collagen level was not different from age-matched WKY.

The most intriguing observation in this study is that, although both converting enzyme inhibitors, lisinopril and captopril, lowered blood pressure, regressed hypertrophy, and reduced myocardial collagen, treatment with captopril resulted in an increased Type III collagen protein, but lisinopril did not. This is the first evidence documenting the difference between the mechanism of action of these two ACE inhibitors. This change in Type III collagen distribution occurs in SHR independent of blood pressure control and myocardial mass and, more importantly, likely to be independent of levels of angiotensin II. As both drugs controlled blood pressure to the same degree, regressed hypertrophy to the same level and prevented formation of angiotensin II in vivo, it appears that the mechanism of collagen phenotype changes is not exclusively due to the prevention of angiotensin II formation.

The arrangement and content of the extracellular matrix of the heart is intimately associated with cardiac function during development and disease state [17, 18]. Although several distinct types of collagen exist in the extracellular matrix, the cardiac interstitium is primarily composed of Type I and Type III collagen. Previous structural and biochemical studies have shown an alteration in collagen types in the interstitial matrix of the heart [3, 18, 19].

Remodelling of the collagen matrix with abnormal diastolic and systolic stiffness of the myocardium has been suggested to be characteristic of changes in pressure-overload myocardial hypertrophy [20–23]. It has also been suggested that interstitial fibrosis represents a determinant of pathologic hypertrophy in hypertension [22]. The mechanism of this impaired diastolic function in patients with pressure-overload hypertrophy is unknown. Collagen is likely to play an important role in the stiffness of the myocardium, depending on the matrix formation [24–27]. Therefore, it is likely that an increased accumulation of collagen, as well as alteration of the distribution of the phenotype, may compromise cardiac function. Changes in the organization and accumulation of this interstitial collagen can be correlated with concurrent changes in myocardial compliance [28]. Therefore, it is important to understand what causes alteration in collagen phenotypes during progression of hypertrophy, both at the message and protein levels, and also to discern how the accumulation of collagen can be prevented by appropriate antihypertensive drugs and thereby to understand the mechanism by which these antihypertensive treatments can prevent increased biosynthesis of collagen.

In animal models various antihypertensive drugs have been used to study the effect of pressure overload on cardiac function. Rossi and Peres [29] have shown that the growth of the cellular constituents of the myocardial parenchyma and stoma seem to have different regulatory mechanisms. This finding raises the intriguing possibility that the growth and regression of cellular constituents in cardiac hypertrophy are distinctly different biologic phenomena. Their data showed [29] that although long-term treatment with captopril reduced collagen volume fraction in the aortic banded model of hypertension and hypertrophy, during the prevention treatment in which the animals were treated from the time of banding, the collagen volume fraction remained unchanged. These results indicate that in the aortic constriction model, the development of myocardial cell hypertrophy will depend, at least in part, on cardiac workload. However, these studies do not rule out the involvement of local growth factor at the cardiac myocyte level [29]. Many drugs have been used to treat hypertension and hypertrophy, but converting enzyme inhibitors have been found to be the most effective class of drug, not only in regressing hypertrophy but also reducing collagen. Sen and Bumpus have shown [3] that administration of -methyldopa resulted in an increase in myocardial collagen and that administration of vasodilators showed no change in collagen, despite normalization of the blood pressure, but captopril treatment led to both regression of hypertrophy and reduction of myocardial collagen. An important question is what is the unique property of captopril that reduces collagen, whereas the other classes of antihypertensive drug have failed to do so.

One of the known mechanisms of action of the ACE inhibitor captopril is prevention of AII formation. Also, it has been suggested that AII may play a role in myocyte growth, as well as increase in myocardial collagen in the rodent model. Our study demonstrated that administration of ACE inhibitor showed very little correlation between myocyte growth and formation of AII in the plasma [30] and in the tissue (Sen et al., unpublished observation). Our data showed that although captopril was one of the drugs found to reduce collagen in hypertensive hypertrophy, the mechanism appeared not to be through AII formation only, the known mechanism of action of captopril. Captopril has also been shown to increase bradykinin, but whether or not bradykinin plays a role in reduction of collagen is not known. However, a similar increase in bradykinin has been observed with lisinopril treatment. Therefore, bradykinin alone does not appear to play a key role in modulation of myocardial collagen.

Previous studies have shown that the Type I to Type III collagen ratio decreases in hypertrophied myocardium [31]. These studies have shown that there exists a specific temporal pattern of expression of the interstitial collagens. The functional significance of changes in the relative proportions of Type I and Type III collagens in the interstitium has not been absolutely determined [31].

A number of studies have shown the effect of various chemical factors and growth factors on collagen gene expression, including the transforming growth factor β family [32–34]. Cell density [35] and extracellular matrix [36] for extracellular environment have also been demonstrated to influence collagen synthesis in vitro. The present study demonstrates unique new information that the effect of captopril on collagen biosynthesis appears not to be exclusively due to AII formation. However, if it is not AII formation, then the factors that are responsible for modulating collagen biosynthesis, both at the transcription and translation levels, have yet to be determined.

Time for primary review 19 days.

	Acknowledgements

 
This study was supported in part by National Institutes of Health grant HL 27838 and a grant from Pfizer, Inc. We are also grateful to Dr. Preenie Senanayake for determination of angiotensin II levels in plasma and tissues in her core facility at the Cleveland Clinic Foundation.

We are also grateful to Dr. Crombrugghe for providing us the collagen probes. We are grateful to JoAnne Holl for typing the manuscript and to Christine Kassuba for editorial assistance.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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