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(Hypertension. 1997;30:1362-1368.)
© 1997 American Heart Association, Inc.

Articles

Captopril Modifies Gene Expression in Hypertrophied and Failing Hearts of Aged Spontaneously Hypertensive Rats

Wesley W. Brooks; Oscar H. L. Bing; Chester H. Conrad; Lydia O'Neill; Michael T. Crow; Edward G. Lakatta; David E. Dostal; Kenneth M. Baker; ; Marvin O. Boluyt

From the Department of Veterans Affairs Medical Center, Boston, Mass; the Department of Medicine, Boston University School of Medicine, Boston, Mass; the Weis Center for Research, Geisinger Clinic, Danville, Pa; and the Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Md.

	Abstract

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Abstract The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in -myosin heavy chain (-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-₁(III) collagen, and transforming growth factor ß₁ (TGF-ß₁) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normortensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in -MHC, ANF, pro-₁(III) collagen, and TGF-ß₁ gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-ß₁ mRNA by 50% (P<.05) and elevated levels of -MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding -MHC, ANF, TGF-ß₁, pro-₁(III) collagen, and renin-angiotensin system components.

Key Words: angiotensin-converting enzyme inhibition • transforming growth factor-ß₁ • myosin heavy chain • myocardial hypertrophy and failure

	Introduction

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Congestive heart failure is a progressive, debilitating disease with a poor clinical prognosis.¹ The incidence of heart failure increases exponentially with advancing age in humans and is the number one hospital discharge diagnosis among Americans 65 years of age and older.² In the clinical setting, chronic ACEI is the therapy of choice³ and has been shown to benefit patients with heart failure of varyious etiologies.^{4 5 6 7} In animal models of hypertrophy and heart failure, ACEI has been shown to prevent impairment of myocardial function,^{8 9 10 11} but the mechanisms responsible for this effect are not entirely understood.

The SHR has been extensively studied as a model of cardiac hypertrophy that results from genetically induced pressure overload and leads to the development of failure in advanced age.^{12 13 14 15 16} Approximately 50% of male SHR undergo a transition from compensated hypertrophy to failure between 18 and 24 months of age (mean, 21±2 months). During the transition to failure, a significant reduction in -MHC mRNA and a marked increase in procollagen and ANF mRNA levels are observed in ventricles of SHR rats.¹⁷ Potential regulators of these failure-associated changes in gene expression include TGF-ß₁ and Ang II.^{17 18 19} The potential role of Ang II in the fibrosis associated with heart failure is suggested by its marked effects to increase expression of collagen genes in cultured cardiac fibroblasts²⁰ and in the LV of intact rats.¹⁹ Several studies have also indicated a potential role of Ang II in the development of cardiac hypertrophy, since ACEI has been shown to prevent hypertrophy in rats with experimentally or genetically induced pressure overload^{8 21 22 23} and to improve survival in laboratory animals after acute myocardial infarction.²⁴

Historically, the RAS has been viewed as a classic endocrine system consisting of organs that secrete a precursor and various regulatory enzymes into the circulation. The final product, Ang II, is formed when the circulating regulatory enzyme, ACE, cleaves Ang I. Ang II then exerts its influences on distant targets that have the necessary receptors. More recently, it has become apparent that some organs (eg, the heart) synthesize all the necessary components for a local RAS.^{25 26} The development of sensitive techniques to identify the mRNAs for these components has shown that cardiac myocytes possess the necessary components for a localized RAS.^{26 27} At present there is a paucity of information on the possible changes in localized cardiac expression of RAS components in heart failure or in response to ACEI.

The purpose of the present study was twofold. First, we sought to determine whether chronic ACEI would prevent and/or reverse changes in expression of -MHC, ANF, pro-₁(III) collagen, and TGF-ß₁ genes that occur during the transition to failure in the SHR. Second, we investigated the influence of ACEI on serum levels of RAS components as well as cardiac levels of RAS component mRNAs. In separate groups of animals, we initiated chronic ACEI treatment at three different points prior to failure, as well as immediately after signs of failure were evident, to determine whether the time of initiation and length of treatment were important variables in the observed effects on gene expression.

	Methods

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Animals
Male retired breeder SHR and WKY rats (6 to 9 months old) were purchased from Taconic Inc and boarded in the animal facility at the Boston Department of Veterans Affairs Medical Center until the time of study (18 to 24 months of age). All animals received proper care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). One hundred and eight male SHR and 12 WKY rats were initially included in the study. All groups of rats were housed two per container, fed standard rat chow, and allowed free access to water. Groups of animals were treated by adding 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.⁸ Groups of animals had treatment initiated at 12, 18, and 21 months of age. An additional group of SHR was administered captopril when evidence of impaired cardiac function was detected clinically and by echocardiography.⁹ Control groups included untreated SHR and age-matched WKY and treated WKY rats. Animals were monitored with measurements of BW and blood pressure. Animals were also observed several times a week for evidence of tachypnea and labored respiration; when these findings were clearly in evidence, animals were either killed and studied or treated with captopril. Animals not demonstrating respiratory difficulties were studied at 24 months of age. At autopsy, animals were examined for pleural or pericardial effusions, atrial thrombi, and right ventricular hypertrophy. Individual chambers were quickly dissected, and chamber weights were recorded. Cardiac tissue samples were rapidly frozen and stored in liquid nitrogen for later mRNA analysis (performed at the Laboratory of Cardiovascular Science, Gerontology Research Center, National Institutes of Health, Baltimore, Md).

Cardiac mRNA Analysis
Frozen samples of left and right ventricular tissue were processed and cardiac RNA extracted for Northern blot analysis, using methods previously published.¹⁷ Briefly, RNA was isolated from cardiac tissue by the method of Chomczynski and Sacchi.²⁸ Ten micrograms of total RNA were size-fractionated by electrophoresis through 1% agarose gels, transferred electrophoretically at 5 V/cm to a nylon (Duralon) membrane, and hybridized with ³²P-radiolabeled probes overnight at 63.5°C for cDNA probes and 60°C for oligonucleotide probes.²⁹ Hybridization intensity was quantified in disintegration per minute directly from blots with a Betascope 603 (Betagen Corp). This apparatus contains an electronic sensor that responds to ß radiation from all areas of the blot directly with an efficiency of at least 15%. Signals visualized on a computer screen were identified by position relative to 18S and 28S rRNA migration and quantified. Each blot was subsequently stripped and reprobed. The signal from each sample was normalized to the signal obtained with a probe specific for the 18S ribosomal RNA.

Probes
Complementary DNA probes were synthesized from a template by the random-prime method.³⁰ The template for the ANF probe was a 700-bp cDNA sequence, obtained by PCR with primers complementary to the published sequence for rat mRNA.³¹ The probe for pro-₁(III) collagen was cDNA kindly provided by M.-L. Chu.³² The probe for TGF-ß₁ was generously provided by M. Sporn.³³ Probes for -MHC and 18 S ribosomal RNA were end-labeled synthetic oligonucleotides.³⁴

Quantitation of Renin and Ao mRNA by Reverse Transcriptase-PCR
Total RNA was extracted from cardiac tissue using the method of Chomczynski and Sacchi.²⁸ DNA was removed from RNA samples by incubating 40 pg of total RNA with 1 U of RQ1 DNase (Promega) in 50 µL of 1x transcription buffer for 15 minutes at 37°C, after which RNA were phenol-chloroform-ethanol–extracted. Multiplex quantitative reverse transcriptase-PCR was performed by adding known amounts of deletion-mutant competitor RNA to RNA samples. Eight aliquots of DNA-free total RNA (250 ng/aliquot), each containing one of seven serial dilutions of competitor RNA for renin, Ao, or EF-1, was reverse-transcribed with 10 U/pL Maloney murine leukemic virus (Gibco-BRL) in the presence of 2.5 µmol/L random hexamers (40 minutes, 40°C). A duplicate aliquot having maximum concentrations of competitor RNA was incubated in the absence of reverse transcriptase and used as a control to detect for possible DNA contamination. After reverse transcription, levels of renin and Ao mRNA were quantified by performing multiplex PCR as described previously.²⁷ First-strand cDNA (50 µL per tube) was amplified in the presence of Ampli-Taq® DNA polymerase, 200 pmol/L primers, and 5 µCi of [-³²P]dCTP. The sense primer, 5'-CCT-CGC-CTC-TCT-GGA-CTT-ATC-3' located in exon 2 (bases 737 to 756), and the antisense primer, 5'-CAG-ACA-CTG-AGG-TGC-TGT-TG-3' located in exon 3 (bases 962 to 941) were used to generate a 226-bp product from rat Ao cDNA.³⁵ The sense primer for the detection of rat renin cDNA spanned oligonucleotide bases 1033 to 1052 (5'-CTG-CCA-CCT-TGT-TGT-GTG-AG-3') on exon 6, and the antisense primer spanned bases 1296 to 1277 (5'-CCA-GTA-TGCACA-GGT-CAT-CG-3') on exon 9 to produce a 264-base product.³⁶ The sense primer, 5'-GGA-ATG-GTG-ACA-ACA-TGC-TG-3' (bp 635 to 654), and antisense primer, 5'-CGT-TGA-AGC-CTA-CAT-TGT-3'(bp 982 to 963) generated a 347-bp product from EF-1 cDNA.³⁷ To prevent depletion of substrate by amplification of housekeeping cDNAs (EF-1), PCR primers for EF-1 were added at 12 cycles after Ao and renin primers were added to the reaction mix. Samples were "hot-started" by incubating at 96°C for 8 minutes and cooled to 63°C, after which 10 µL of PCR reaction mix (2 mmol/L MgCl₂, 50 mmol/L KCI, and 2.5 U of Ampli-Taq® DNA polymerase [Perkin-Elmer Corp]) were added. The reaction profile included 35 cycles (renin, Ao) or 23 cycles (EF-1) of denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, and primer extension at 72°C for 90 seconds.

After multiplex titration assay, 15-µL aliquots of PCR product were separated using 6% polyacrylamide gel electrophoresis. The gel was dried, and the quantity of radioactivity in bands of interest was determined by using a PhosphorImager (Molecular Dynamics). Data analysis was performed by plotting the log ratios of target and internal standard PCR product against the log of the input competitor RNA. The concentration of competitor RNA, which corresponds to zero on the ordinate, represents an amount of target mRNA equal to exogenously added competitor RNA. This calculated value was corrected to account for differences in the number of [³²P]dCTPs incorporated in the target versus deletion-mutant cDNAs.²⁷ The final value was normalized to a standard amount of product amplified from the housekeeping mRNA to account for differences in RNA loading.

Measurement of Renin and Angiotensin in Plasma
On the day of study, blood was collected by decapitation and placed in chilled tubes containing ammonium EDTA and o-phenanthroline which were added to prevent degradation of angiotensin peptides in plasma. The plasma was separated by centrifugation at 3000 rpm for 15 minutes at 4°C and stored at -70°C until use. Plasma samples were applied to a Sep-Pak plus C18 cartridge (Waters). The Sep-Pak C18 cartridge was pretreated consecutively with 10 mL of methanol, 5 mL of tetrahydrofuran, 5 mL of hexane, 10 mL of methanol, and 10 mL of 0.1% trifluoroacetic acid. Samples of plasma (approximately 1. 5 mL) were then applied to the cartridge and washed with 10 mL of 0.1% trifluoroacetic acid and then 10 mL of a mixture of methanol/water/trifluoroacetic acid (10/89.9/0.1% by volume). Ang I and Ang II were measured in fractionated samples by immunoreactive radioimmunoassay (ALPCO). The plasma renin concentration was measured as the rate of generation of Ang I at 37°C by radioimmunoassay (DuPont).

Statistical Analysis
Two-way ANOVA was used to evaluate differences among groups (ie, strain and treatment effects), and post hoc comparisons between groups were performed by Tukey's procedure or Newman-Keuls multiple comparison test. Differences were considered significant at P<.05.

	Results

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Effect of Captopril on LV Weight and Plasma Renin and Angiotensin Concentrations
The LV/BW ratio was markedly increased in both failing SHR (SHR-F) and nonfailing SHR (SHR-NF) in comparison to normotensive WKY rats (Fig 1 and Table 1). Overall, captopril treatment of SHR groups decreased LV/BW by 35% relative to SHR-F, but LV/BW remained elevated relative to WKY rats. LV/BW of SHR-NF in which treatment was initiated at 21, 18, and 12 months of age was decreased 25%, 37%, and 32%, respectively (all values P<.01 versus SHR_NoRx). In the SHR-F, in which captopril therapy was initiated at the time of failure and studied 2 to 4 months after captopril treatment, LV/BW was 2.9±0.2 or 21% less (P<.01 versus SHR-F). In the WKY, captopril treatment for 12 months had no significant effect on LV/BW (1.86±0.17 versus 1.74±0.22 for WKY and WKY_Rx, respectively). Table 1 also shows data for the plasma RAS components for untreated WKY, SHR-F, and captopril-treated SHR (SHR_Rx). Plasma renin concentration in untreated SHR-F appeared slightly higher than untreated WKY, but this was not statistically significant. On average, 6 to 12 months of captopril treatment of SHRs increased the plasma renin concentration by 16-fold (P<.05) and fourfold (NS) relative to WKY and SHR-F groups, respectively. Plasma Ang I levels of SHR-F were not different from those levels observed in the WKY rats. In contrast, plasma Ang I levels were markedly elevated during chronic ACEI therapy. In captopril-treated SHR, Ang I levels were significantly higher than untreated WKY and SHR-F (P<.05) groups. Surprisingly, circulating levels of Ang II were not elevated in SHR-F relative to WKY rats and were not reduced by ACEI therapy, suggesting that long-term treatment with captopril may have resulted in a rebound in Ang II to pretherapy levels, perhaps mediated via non-ACEI pathways.

View larger version (40K): [in this window] [in a new window]   Figure 1. LV/BW ratio from WKY, SHR-NF, and SHR-F without and following captopril treatment. Treatment was initiated in SHR at 12, 18, and 21 months of age (SHR-NFRx 12, 18, or 21, respectively), and animals were studied at 24 months. In the treated SHR-F group (SHR-FRx), treatment for 2 to 4 months was begun at the time of failure. Captopril treatment significantly reduced hypertrophy as indicated by the LV/BW ratio in SHR-NF and SHR-F compared with the untreated counterparts (P<.05; 2-way ANOVA). However, there was no significant change in the LV/BW ratio with captopril in normotensive WKY. Values are mean±SE. *P<.05 vs WKY; §P<.05 vs SHR-NF. P<.05 versus SHR-F (Newman-Keuls multiple comparison test).

View this table: [in this window] [in a new window]   Table 1. Effect of Captopril Treatment on LV/BW Ratio and Plasma Renin, Ang I, and Ang II Levels

Effect of Captopril on Gene Expression in LV Tissue
The relative amount of Ao mRNA in the LV determined by quantitative multiplex PCR analysis was not significantly different in the SHR-NF in comparison to untreated WKY rats (Fig 2, Table 2). However, in the SHR-F the level of Ao mRNA in the LV was decreased by 75% relative to WKY and SHR-NF groups. Renin mRNA levels were not significantly different in the LV of SHR-NF or SHR-F in comparison to WKY rats. Although there was no significant difference in Ao mRNA in SHR-NF with captopril treatment of any duration, renin mRNA levels increased more than twofold in the LV of captopril-treated rats relative to untreated control. Captopril treatment initiated at the time of failure (SHR-F_Rx) and continued for 2 to 4 months did not significantly increase Ao mRNA or renin mRNA levels in the LV relative to untreated SHR-F.

View larger version (79K): [in this window] [in a new window]   Figure 2. A representative multiplex PCR titration assay showing EF-1, renin, and Ao products. Serial dilutions of competitor RNA for renin (Renin-C, 8.15 to 0.13 fg), Ao (Ao-C, 78 to 1.22 fg), and EF-1 (EF-1-C, 8.15 to 0.13 pg) were reverse transcribed (+RT) with 250-ng aliquots of total RNA of cardiac tissue from an 18-month normotensive WKY. One aliquot of RNA was used as a negative control (-RT) in which reverse transcriptase (RT) was omitted from the reaction. Sense and antisense primers were added for renin and Ao, and cDNAs were coamplified for 12 cycles after which primers for EF-1 were added. The PCR was stopped at 35 cycles, and amplification products were separated on a 6% polyacrylamide gel. Amounts of competitor RNA added to each reaction are indicated in the table below each lane of the autoradiograph.

View this table: [in this window] [in a new window]   Table 2. Effects of ACEI on Steady State Levels of mRNA in LV of WKY and SHR

Heart failure was associated with a 4.5-fold decrease in -MHC mRNA compared with the WKY group (Table 3). Although LV -MHC mRNA levels in SHR-NF were more than threefold higher in comparison to SHR-F (similar to our previously published findings),¹⁷ this difference was not statistically significant in the present study. Captopril treatment resulted in a significant upregulation -MHC mRNA levels in WKY, SHR, and SHR-F in comparison to untreated SHR-F values (P<.05). In normotensive WKY rats, captopril treatment resulted in a twofold increase in -MHC mRNA levels relative to untreated WKY rats. In SHR, captopril treatment increased -MHC mRNA levels two- to threefold in comparison to untreated SHR-NF. Among captopril-treated SHR-NF groups, there was no significant difference in -MHC mRNA levels regardless of the time captopril treatment was initiated.

View this table: [in this window] [in a new window]   Table 3. Effects of ACEI on Steady State Levels of mRNA in LV of WKY and SHR

ANF mRNA levels were more than fivefold increased in the LV of SHR-F hearts in comparison to WKY hearts and more than 60% greater than SHR-NF hearts (P<.05; Table 3). In contrast, captopril significantly reduced ANF mRNA levels in WKY and SHR treated groups in comparison to untreated SHR-F (P<.05). Captopril treatment reduced ANF mRNA levels in SHR-F by 31% (corresponding to the 21% decrease in LV/BW), although this difference in ANF mRNA was not significant. When treatment was initiated at 12, 18, and 21 months of age, ANF mRNA levels in SHR were significantly reduced relative to SHR-NF levels. Although the level of ANF mRNA in captopril-treated WKY rats was reduced by approximately 28% of untreated WKY rat levels, this difference was not statistically significant. The well-documented relationship between the level of ANF mRNA and LV hypertrophy is also strong in the present study (r=.86, P=.013).

The level of pro-l(III) collagen mRNA was increased nearly fourfold in the LV of failing hearts in comparison to WKY hearts and elevated approximately 2.5-fold in comparison to SHR-NF (P<.05; Table 3). Captopril treatment of WKY rats had no significant influence on the level of pro-l(III) collagen mRNA in comparison to untreated WKY rats. In SHR treated with captopril beginning at 12 months of age, there was a >60% decrease in the level of pro-l(III) collagen mRNA relative to untreated SHR-F. Levels of pro-l(III) collagen mRNA were not determined in SHR-NF treated at 18 and 21 months of age. Captopril treatment of SHR with evidence of heart failure did not significantly reduce the elevated level of pro-l(III) collagen mRNA in the LV.

Levels of TGF-ß₁ mRNA did not differ in the LV between SHR-NF and WKY groups. However, there was a 42% increase in TGF-ß₁ mRNA levels in the LV of SHR-F compared with WKY rats (Table 3). The difference between SHR-NF and SHR-F groups, although similar in magnitude to our previous finding,¹⁷ was not significant in the present study. Captopril treatment significantly reduced the TGF-ß₁ mRNA levels in WKY, SHR-NF, and SHR-F groups in comparison to their untreated counterparts (P<.05).

	Discussion

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Our previous work has shown that the transition from stable hypertrophy to heart failure in the SHR is characterized by functional impairment, reduced myofibrillar mass, and increased fibrosis of LV myocardium^{13 14 38 39} and that these functional and structural changes are accompanied by marked alterations in the expression of genes encoding pro-l(III) collagen, -MHC, ANF, and TGF-ß₁.¹⁷ In the present study, we report that each of the aforementioned changes in gene expression is prevented when chronic ACEI therapy is initiated prior to the onset of heart failure. Furthermore, the present data show that when ACEI therapy is initiated after signs of failure become evident, the failure-associated reduction in the levels of -MHC mRNA and the failure-associated increase in TGF-ß₁ mRNA are completely reversed. The failure-associated increases in levels of pro-l(III) collagen and ANF mRNA are also attenuated (not statistically significant) when ACEI therapy is begun in SHR with heart failure. These findings demonstrate that ACEI therapy can strongly and selectively influence expression of genes that are important to the function and structure of the heart. Circulating levels of Ang II were not lowered by chronic ACEI, suggesting that the beneficial effects of captopril may be attributed instead to either changes in local tissue production of Ang II or to non-ACEI effects of captopril.

We had previously shown that the switch from -MHC (V₁ isoform) to ß-MHC protein (V₃ isoform) during the transition to failure is complete³⁹ and that the disappearance of -MHC protein is due primarily to a reduction in the level of -MHC mRNA, with little change in the level of ß-MHC mRNA.¹⁷ The present results show that ACEI therapy not only prevents these changes when initiated prior to failure but reverses them when administered to SHR after the onset of failure. Since myosin isozyme composition has been shown to influence shortening velocity, we examined the relationship between relative changes in LV -MHC mRNA levels and shortening velocity (at 0.5-g load) of LV papillary muscles obtained from the same hearts of untreated and captopril treated animals.⁹ When the mean for each groups is plotted, shortening velocity is positively correlated (r=.85, P<.05) with the level of -MHC expression (Fig 3). This relationship between relative proportions of - and ß-MHC, first demonstrated in cardiac muscle by Schwartz and coworkers,⁴⁰ appears to be evident at the mRNA level as well. Although the difference between SHR-NF and SHR-F in terms of the relative proportion of -MHC protein is small (16% versus 0%; V₁+1/2 V₂, calculated from Reference 39³⁹ , Table 3), the biological effect may be important, considering that 6 of 12 captopril-treated SHR survived through 24 months of age when captopril therapy was initiated after the onset of failure (19 to 22 months of age).⁹

View larger version (26K): [in this window] [in a new window]   Figure 3. Levels of -MHC mRNA plotted against LV papillary muscle shortening velocity in untreated (open symbols) and captopril-treated groups (filled symbols). Shortening velocity and isometric force data have recently been reported9 and are used here to examine the relationship between -MHC mRNA gene expression and myocardial function. There is a significant positive correlation between contractile shortening and -MHC mRNA expression (r=.85, P<.05), suggesting that shortening velocity can be directly regulated by LV -MHC gene expression in hypertrophied and failing SHR as well as in normotensive WKY rats. Moreover, this relationship can be modulated by ACEI therapy.

The known influence of TGF-ß₁ on MHC,⁴¹ coupled with the strong correlation observed between the levels of TGF-ß₁ and -MHC mRNA (r=-.85, P<.05) in the present study, suggests that the changes in myocardial expression of -MHC during the transition to heart failure and reversal of those changes by ACEI may be mediated by changes in TGF-ß₁ expression. On the other hand, a dissociation of the expression of TGF-ß₁ and -MHC is observed in response to 1, 3, or 7 days of Ang II infusion,¹⁹ suggesting that the influence of TGF-ß₁ on -MHC expression may be more complicated in the SHR. Future studies will need to measure active and latent forms of TGF-ß₁ to determine whether TGF-ß₁ influences expression of -MHC in vivo. Detailed analyses such as those performed by Schneider and coworkers⁴² on the skeletal -actin gene will be required to determine how TGF-ß₁ exerts its influence on the -MHC gene.

A large and growing literature documents the importance of both peripheral and local RAS components in myocardial physiology and pathology.^{23 25 43} Weber and coworkers^{44 45} have hypothesized that both the circulating and local RAS play an important role in the increased synthesis of extracellular matrix components in cardiac hypertrophy and heart failure. The present study demonstrates that ACEI regresses cardiac hypertrophy in the SHR and prevents heart failure that normally occurs in the last quartile of the SHR lifespan. Most notably, it does so without lowering the circulating levels of Ang II, although the accumulation of renin and Ang I in ACEI-treated rats indicates that the converting enzyme is indeed inhibited. While our current data do not provide any direct evidence for either circulating or local RAS involvement in fibrosis, some findings are suggestive in this regard. For example, the level of Ao mRNA was negatively correlated with that of pro-1-collagen (r=-.92, P=.03) and with connective tissue area (r=-.77, P=.04). Although speculative, low levels of Ao mRNA may reflect negative feedback from elevated tissue levels of Ang I. Given the complexity being uncovered in local RAS function, there are many other ways in which both circulating and local RAS components might have promoted the fibrosis observed in the failing hearts. First, there may have been changes in circulating Ang II that were not detected by our one-time measurement. Second, we did not measure levels of circulating aldosterone, which may be more important in stimulating fibrosis than circulating levels of Ang II.^{44 45} Third, levels of circulating Ang I were elevated in ACEI-treated rats. Since carboxypeptidase A in the interstitial fluid may convert Ang I to Ang 1–9, which in turn inhibits local ACE activity,⁴⁶ the increase in circulating levels of Ang I may be a significant mechanism by which ACEI therapy indirectly inhibits local ACE activity. Although ACEI did not regress fibrosis when administered beginning at 21 month of age or after the onset of failure (see Reference 9⁹ , Table 5), this may simply reflect the extremely slow turnover rates of some extracellular matrix components. Further study, in which multiple components of both tissue and plasma RAS are quantitated before and after failure such as the study by Kohara et al,⁴⁷ is warranted to more directly address this issue. Since angiotensin AT₁ receptors have been implicated in fibrosis⁴⁸ and bradykinin B₂ receptors have been implicated in the effects of ACEI, the role of kinins and angiotensin receptor subtypes in the various effects of ACEI must be considered in future studies.^{43 49} Finally, the recent finding that ACEI decreases the incidence of apoptosis in the SHR⁵⁰ offers another plausible mechanism for the beneficial effects of ACEI in this model.

Fibroblasts are the primary source of collagen types I and III in the heart. Studies of fibroblasts in culture may therefore offer insights into regulation extracellular matrix accumulation, although caution must be exercised in extrapolating from the culture to the in vivo setting. It has been shown, for example, that Ang II and norepinephrine induce proliferation of fibroblasts and increase collagen synthesis in cultured cardiac fibroblasts.^{19 51} Elevated expression of TGF-ß₁, mRNA has been reported to precede increases in collagen mRNA in the in vivo heart.¹⁸ Ang II and norepinephrine each augment, up to threefold, expression and secretion of latent TGF-ß₁ and TGF-ß₂ in neonatal cardiac fibroblasts,^{52 53} Thus, Ang II-induced activation of TGF-ß₁ gene expression may be a primary mechanism by which collagen genes are stimulated in fibroblasts in an autocrine manner resulting in increased extracellular matrix production, while myocyte growth and gene expression are influenced by paracrine actions. In keeping with these results from cultured fibroblasts, ACE activity has been observed to be focused in areas rich in fibroblasts in the heart.⁴⁵ The present study provides no further evidence for this relationship between Ang II and TGF-ß₁, since levels of TGF-ß₁ were not correlated with either fibrosis or Ao mRNA. Levels of renin mRNA were negatively correlated with those of TGF-ß₁ mRNA (r=-.88, P=.008), but given the complexity of local RAS regulation, it is difficult to interpret the meaning of this without data on tissue levels of other RAS components. Future studies to localize the RAS components in the failing hearts of SHR would seem to be warranted.

	Selected Abbreviations and Acronyms

 

ACEI = angiotensin-converting enzyme inhibition ANF = atrial natriuretic factor Ang = angiotensin Ao = angiotensinogen BW = body weight EF-1 = elongation factor 1 LV = left ventricle, left ventricular MHC = myosin heavy chain PCR = polymerase chain reaction RAS = renin-angiotensin system SHR = spontaneously hypertensive rat(s) TGF = transforming growth factor WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This study was supported by Medical Research Funds from the Department of Veterans Affairs (W.W.B., O.H.L.B, C.H.C), the National Research Council (M.O.B.), and NIH grant HL-44883 (K.M.B., D.E.D.). K.M. Baker is an Established Investigator of the American Heart Association. The authors thank Mon-Li Chu and Michael Sporn for generously providing collagen and TGF-ß₁ probes, respectively, and Anna Kempinski for technical assistance associated with quantitative polymerase chain reaction experiments. The authors are grateful for the assistance of Leslie Heckendorf in the preparation of the manuscript.

	Footnotes

 
Reprint requests to Wesley W. Brooks, DSc, Research Service (151), Boston VA Medical Center, 150 South Huntington Ave, Boston, MA 02130.

Received February 26, 1997; first decision March 21, 1997; accepted July 16, 1997.

	References

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