Background The cardiac renin-angiotensin system (RAS) has been suggested to play an important role in heart failure and cardiac hypertrophy. In the present study, we evaluated the expression of each component of the RAS in hypertrophied heart induced by aortocaval shunt.
Methods and Results The expression levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin II type Ia and Ib receptor (AT1aR and AT1bR) mRNA were determined by the reverse transcription–polymerase chain reaction method owing to the relatively low expression levels of these mRNAs in the ventricle. The expression level of renin or angiotensinogen mRNA in the ventricle was very low, more than 1000-fold lower than that in the kidney or liver, respectively. The expression of ACE mRNA in the ventricle was relatively abundant and was increased in the hypertrophied ventricle in this model, whereas no significant increases in the expression levels of AT1aR and AT1bR mRNA were observed. Administration of lisinopril attenuated the development of left and right ventricular hypertrophy in this model and was accompanied by an attenuation of the upregulation of the ACE, collagen type I-α, and vimentin mRNAs. Because the activity of the circulating RAS in the aortocaval shunt rats was not higher than that in the sham-operated rats, the effects of lisinopril in attenuating the ventricular hypertrophy may be due to inhibition of the increased ACE in the ventricle.
Conclusions The present study supports the importance of ACE expressed in the ventricle in the development of hypertrophy induced by aortocaval shunt.
Many studies have suggested that angiotensins are produced not only in the circulation but also in local tissues, including kidney, adrenal gland, brain, and heart and in the peripheral vasculature.1 2
The involvement of a cardiac RAS in the pathophysiology of cardiac hypertrophy has been based primarily on the results in a cardiac hypertrophy model by pressure overloading. Increased expression of ACE mRNA has been reported in pressure-overload hypertrophy.3 4 The increased expression of ACE has been suggested to increase the generation of angiotensin II in the hypertrophied ventricle because the administration of converting enzyme inhibitor attenuated the development of ventricular hypertrophy.5 6
The involvement of a cardiac RAS in the pathophysiology of cardiac remodeling after myocardial infarction has also been suggested. Increased expressions of ACE mRNA,7 8 AGT mRNA,9 and AT1R8 have been reported in the scar or the intact part of the infarcted heart. The favorable effects of ACE inhibitors in attenuating ventricular dilatation after myocardial infarction10 may reflect the functional importance of these RAS components expressed in the infarcted heart.
In the present study, we evaluated the expression of each component of the RAS in a hypertrophied heart induced by volume overload. We selected this model because the circulating RAS was supposed not to be activated in this model. In this model, it may be possible to dissociate the systemic hemodynamic effects of ACE inhibition on the left ventricle from the effects of blockade of cardiac ACE. Increased expression of ACE mRNA in the ventricles of the heart was found in this volume-overload model, and the favorable effects of an ACE inhibitor on ventricular weight suggest that this increased ACE expression may have some functional significance.
Male Sprague-Dawley rats (Charles River) weighing 250 to 300 g at the time of surgery were kept at a controlled room temperature under a 12-hour light regimen of 6 am to 6 pm and fed regular pelleted rat chow and tap water ad libitum.
In experiments to investigate the effects of drugs on the development of ventricular hypertrophy induced by shunt, hydralazine (10 mg/kg) and lisinopril (10 mg/kg) were dissolved in water and administered orally once a day for 7 days.
AC rats were prepared according to Garcia and Diebold.11 Briefly, the aorta was punctured at the union of a segment two-thirds caudal to the renal artery and one-third cephalic to the aortic bifurcation with an 18-gauge disposable needle. The needle was advanced into the aorta, perforating its adjacent wall and penetrating the vena cava. After the aorta was clamped, the needle was withdrawn, and a drop of cyanoacrylate glue was used to seal the aortic puncture point. The patency of the shunt was verified visually by swelling of the vena cava and mixing of arterial and venous blood. The patency of the fistula shunt was verified the next day with a stethoscope by shunt murmur at the abdomen.
On the day of analysis (1, 7, or 40 days after the operation), rats were anesthetized with pentobarbital, and a PE-50 catheter filled with heparinized saline was inserted into the aorta through the right common carotid artery. Catheters were exteriorized on the necks of the animals. After a 4-hour recovery period, blood pressure was assessed, and blood was collected through the catheter for biochemical analyses.
RNA Isolation and Analysis
RNA was isolated as previously reported.12 The quality of RNA analyzed in the present study was confirmed by ethidium bromide staining and Northern blot analysis of the expression level of GAPDH, as previously reported.12 The rat vimentin and collagen type 1-α cDNA fragments were isolated from the cDNA library of fetal rat ventricle by a differential screening method.13 The expression levels of the renin, AGT, ACE, and AT1aR and AT1bR mRNAs were determined by the RT-PCR method because of relatively low expression levels of these mRNAs in the ventricle of the heart. The expression levels of AT1Rs12 and renin14 mRNAs were determined as previously described.
Rat AGT cDNA15 was synthesized by PCR with the following two primers: 5′-GACCGCGTATACATCCACCCCTTTCATCTC-3′ (nucleotides 73 through 102, exon 2) and 5′-GTCCACCCAGAACTCATGGAGCCCAGTCAG-3′ (nucleotides 882 through 853, exon 3). The synthesized rat AGT cDNA fragment was subcloned into the EcoRV site of pBluescript II KS (+). The resulting plasmid, designated plasmid A, was cut with BamHI, blunt-ended with a Klenow fragment of DNA polymerase, and then cut with Kpn I, which released a Kpn I (in vector)–BamHI (nucleotide 427, blunt-ended) cDNA fragment. This cDNA fragment was subcloned into the Kpn I– and Bal I–cut plasmid A. The resulting plasmid, designated plasmid B, contained a rat AGT cDNA fragment that lacked a region from nucleotide 248 to 425. From this plasmid B, a deletion-mutated cRNA for rat AGT was synthesized with T3 RNA polymerase as previously reported.12 A known amount (5×103 molecules) of the deletion-mutated cRNA for rat AGT was mixed with a sample RNA (1 μg), and this mixture was converted to cDNAs by RT with random primers. The resulting cDNA mixture was amplified PCR with the primers described above. An aliquot of [α32P]dCTP was included in the PCR reaction. Fragments of 810 and 733 bp should be synthesized by PCR from the native rat AGT mRNA and the deletion-mutated cRNA, respectively.
The rat ACE cDNA16 fragment was synthesized by use of the following two primers: 5′-CCTGATCAACCAGGAGTTTGCAGAG-3′ (nucleotides 279 through 303, exon 2) and 5′-GCCAGCCTTCCCAGGCAAACAGCAC-3′ (nucleotides 595 through 571, exon 4). The resulting cDNA fragment was subcloned into the EcoRV site of pBluescript II KS(+). The resulting plasmid was cut with Avr II, blunt-ended with Klenow treatment, and self-ligated. The resulting plasmid contained a rat ACE cDNA fragment that lacked the Avr II site (nucleotide 400). From this plasmid, a mutated cRNA for rat ACE mRNA was synthesized as previously reported.12 A known amount (1.5×106 molecules) of the mutated cRNA for rat ACE was mixed with a sample RNA (1 μg), and this mixture was converted to cDNAs by RT with random primers used as a primer. The resulting cDNA mixture was amplified by PCR with the primers described above. Fragments of 317 and 321 bp should be synthesized by PCR from the native rat ACE mRNA and the mutated cRNA, respectively. An aliquot of [α32P]dCTP was included in the PCR. The PCR products were purified by ethanol precipitation and digested with Avr II restriction enzyme. Digestion of the PCR product from the native ACE mRNA should give 195- and 122-bp fragments.
The PCR amplification profile for AGT and ACE included an initial denaturing step at 94°C for 1 minute and 35 cycles for AGT and 31 cycles for ACE at 94°C for 1 minute, 58°C at 1 minute, and 74°C at 2 minutes. The PCR products were electrophoresed on 5% polyacrylamide gel. The expression levels of the mRNAs were calculated according to the following formula:
where IN and IM represent the intensity of the PCR product from the native mRNA and the mutated cRNA, respectively, and CN and CM represent the content of dCTP in the PCR product from the native mRNA and the mutated cRNA, respectively. The intensity of the PCR products was determined from autoradiography with a densitometer.
Results are expressed as mean±SD. Statistical analyses were performed with a one- or two-way ANOVA. Linear regression analysis was used to study the relations between variables. Differences were considered statistically significant if P<.05.
Ventricular Hypertrophy by AC
The LV/W and RV/W in the AC rats were significantly higher than those in the sham-operated rats on days 7 and 40 after the operation (Table 1⇓). The plasma renin activity and plasma aldosterone concentration in the AC rats were not significantly different from those in the sham-operated rats (Table 1⇓). The mean blood pressure in the AC rats was significantly lower than that in the sham-operated rats on days 1 and 40. The plasma ANP concentration was significantly higher in the AC rats than in the sham-operated rats from day 1. The plasma ACE activities in the AC rats were not significantly higher than those in the sham-operated rats.
Expression Levels of mRNAs for the RAS Components
The expression levels of the mRNAs for renin, AGT, ACE, and AT1aR and AT1bR were examined in the left and right ventricles of AC and sham-operated rats.
Fig 1⇓ shows typical analyses of the expression levels of renin, AGT, ACE, and AT1R mRNA in the left ventricles of the AC and sham-operated rats. The quality of RNA was confirmed by ethidium bromide staining, in which no degradation of 28s or 18s ribosomal RNA was observed (data not shown).
Table 2⇓ gives the expression levels of the mRNA of the RAS components in the ventricles of the AC and sham-operated rats. No significant differences in the expression levels of renin, AGT, or AT1R mRNA were observed between the AC and sham-operated rats on days 1, 7, and 40. However, the expression levels of ACE mRNA in the left ventricles of AC rats were significantly higher than those in the sham-operated rats from day 7. On day 40, the expression levels of ACE mRNA in the left ventricles of the AC rats were about 3.3 times higher than those in the sham-operated rats. Fig 2⇓ shows the correlations between the LV/W and expression levels of mRNA for renin, AGT, ACE, and AT1R in rats on day 7. A significant correlation was observed only between the expression level of ACE mRNA and LV/W.
The expression levels of mRNAs of the RAS components in the right ventricles were also assessed. Again, only the expression levels of ACE mRNA in the AC rats were significantly higher than those in the sham-operated rats on day 7. The expression levels of AGT mRNA in the right ventricles were significantly higher than those in the left ventricles (P=.025, paired t test).
The expression level of renin mRNA in the ventricle was very low, about 1000-fold lower than that in the kidney (Table 3⇓). The expression level of AGT mRNA in the ventricle was also very low, about 4000-fold lower than that in the liver (Table 3⇓). On the other hand, the expression level of ACE mRNA in the ventricle was relatively high, only about 20- to 40-fold lower than that in the lung (Table 3⇓). The expression level of AT1R (AT1a+AT1b) mRNA in the ventricle was also only about 20-fold lower than that in the liver (Table 3⇓).
The expression levels of renin mRNA in the kidneys of the AC rats were not significantly different from those of the sham-operated rats on day 7 (Table 3⇑).
Effects of an ACE Inhibitor on LVH
The above results indicated that the expression levels of ACE mRNA were increased in AC rats and that circulating RAS activity was not increased. To investigate the possible functional role of this increased ACE expression, the effects of the ACE inhibitor lisinopril on the development of ventricular hypertrophy by AC were examined (Table 4⇓, top). The 7-day administration of lisinopril beginning 1 day after the operation markedly reduced the development of LVH and RVH (P<.05). Another antihypertensive drug, hydralazine, had no attenuating effects on the development of LVH or RVH. No significant differences were observed in the blood pressure levels among the three groups. Lisinopril had no significant effects on ventricular weight in the sham-operated control rats (Table 4⇓, bottom).
The upregulation of the expression of ACE, collagen type 1-α, and vimentin mRNA, which was observed in the AC rats, was significantly attenuated by treatment with an ACE inhibitor (Fig 3⇓ and Table 5⇓). Thus, attenuation of the development of ventricular hypertrophy in AC rats by treatment with lisinopril was accompanied by an attenuation of the upregulation of hypertrophy-associated genes.
The expression level of ACE mRNA in the ventricle increased with the introduction of AC, although the activity of the circulating RAS was not increased. Administration of the ACE inhibitor lisinopril attenuated the development of LVH induced by AC. These results may be strong evidence of the importance of increased ACE expressed in the hypertrophied ventricle. However, we should evaluate our current results more closely to assess the possible role of a cardiac RAS.
Characteristics of AC Rats
The characteristics of AC rats may depend on the size of the AC.17 Notable features of the AC rats in the present study were that RVH and LVH were evident on day 7, plasma ANP levels were markedly increased from day 1, and neither plasma renin activity nor plasma aldosterone concentration was increased, even on day 40. These characteristics of the AC rats in the present study indicate that our model reflected stable compensated hypertrophy and not decompensated heart failure.
Expression Levels of mRNAs of RAS Components
Although the local generation of angiotensins was demonstrated previously,1 it remains unclear whether angiotensins are produced by locally synthesized components or plasma-borne components of the RAS. It is also unclear which components of the RAS are rate-limiting in the generation of angiotensins in peripheral tissues. The expressions of each component of the RAS (renin, AGT, ACE, AT1aR, and AT1bR) in the ventricle were confirmed by others3 9 12 18 and the present study. However, the expression level of renin or AGT mRNA in the ventricle was very low, <1000-fold lower than that in the kidney or liver. Therefore, it is unlikely that locally synthesized renin or AGT influences the local concentration of these substances. In fact, we recently demonstrated that plasma-derived, but not locally synthesized, renin contributes to the generation of angiotensins in the peripheral vasculature.19 Moreover, Jan Danser et al20 recently reported that angiotensin production in the ventricle depends on plasma-derived renin and plasma-derived AGT stored in the interstitial fluid. The lack of any significant modulation of these genes in hypertrophied ventricle by AC suggests that these components synthesized in the ventricles may not play a primary role in the initiation and maintenance of ventricular hypertrophy in this model.
On the other hand, the expression level of ACE mRNA in the ventricles was relatively high. In addition, the expression level of ACE mRNA was increased in hypertrophied ventricle by AC. However, this does not necessarily mean that the modulation of ACE expression affects the generation of angiotensins in the ventricle. It has been claimed that the quantity of ACE is vast and is not likely to be rate-limiting. Indeed, plasma angiotensin I is readily converted to angiotensin II in a single pass through the pulmonary circulation. In local tissues, however, the expression level of ACE is not necessarily excessive, and modulation of the expression of ACE may affect the generation of angiotensin II locally. Indeed, Schunkert et al3 reported that an increased cardiac ACE expression caused by pressure overloading of the ventricle by aortic stenosis resulted in an increased intramyocardial conversion of angiotensin I to angiotensin II.
Measuring the angiotensin II concentration in the hypertrophied ventricle might provide a definitive answer as to whether increased ACE in the ventricles of AC rats affects the local generation of angiotensin II. However, this might prove technically difficult to accomplish.21 Therefore, as an alternative, we investigated the effects of lisinopril on the development of LVH induced by AC. Administration of lisinopril attenuated the development of LVH and RVH and was accompanied by the attenuation of upregulation of hypertrophy-associated genes, such as collagen type I-α and vimentin. No significant reduction in blood pressure was observed with the administration of lisinopril. The plasma renin activity and plasma aldosterone concentration in AC rats were not higher than those in sham-operated rats. No significant effects of lisinopril on heart weight were observed in sham-operated rats. Therefore, it is tempting to speculate that the attenuating effect of lisinopril on ventricular hypertrophy occurs through the inhibition of the increased ACE in the ventricles of AC rats. The increased expression of ACE in the hypertrophied ventricle by AC may lead to increased angiotensin II generation or bradykinin degradation and may contribute to the maintenance of hypertrophy by AC.
No significant increases in the expression levels of the AT1aR and AT1bR mRNAs were observed in the hypertrophied ventricle in the present model. This finding may be in contrast to those observed in a hypertrophied ventricle caused by pressure overloading, in which the increased expression of AT1aR has been reported.22 The expression level of AT1aR mRNA was reportedly higher in cardiac fibroblast than in cardiomyocyte.23 The AC in the present study may not have been sufficient to cause enough fibrosis in the hypertrophied ventricles to increase AT1R mRNA levels.
ACE in Cardiac Tissue and Hypertrophy
Cells responsible for ACE expression in the cardiac tissue of infarcted ventricles have been identified as predominantly fibroblastlike cells that can produce type I collagen.24 Many studies confirmed that ACE binding or activity is anatomically coincident with connective tissue formation.8 24 25 Thus, marked upregulation of ACE mRNA in the AC rats on day 40 may reflect a progression of fibrosis in the ventricle.
Treatment with lisinopril markedly attenuated the development of ventricular hypertrophy in the AC rats and was accompanied by attenuation of the upregulation of ACE, collagen type I-α, and vimentin mRNA. This indicates that lisinopril may prevent the proliferation of the fibroblastlike cells that are responsible for the increased ACE expression. Angiotensin II is not only a vasoactive peptide but also a growth-promoting factor and has been reported to promote fibroblast proliferation.26 27 Bradykinin, on the other hand, has been reported to reduce collagen synthesis and augment collagenase activity in adult cardiac fibroblast.28 Because angiotensin II antagonist has been reported to be effective in attenuating hypertrophy by shunt,17 the mechanism by which lisinopril attenuates hypertrophy by shunt may involve primarily its effects in blocking angiotensin II generation rather than in potentiating bradykinin. In the tissue RAS or the tissue angiotensin II–generating system, it is not yet clear what the rate-limiting component is for angiotensin II generation. The fact that lisinopril attenuated hypertrophy in the present AC model in which only the expression levels of ACE mRNA were increased strongly suggests that increased ACE in the hypertrophied ventricle may be rate-limiting or at least may have some effect on the generation of angiotensin II in situ.
Comparison With Other Studies on AC
Recently, Ruzicka et al17 showed that the angiotensin II receptor antagonist losartan but not the ACE inhibitor enalapril attenuated the development of ventricular hypertrophy by AC and suggested the presence of an alternative pathway for the generation of angiotensin II. However, subsequent studies29 by the same authors showed that the efficacy of ACE inhibitors in attenuating ventricular hypertrophy by AC seems to depend on their efficacy in cardiac ACE inhibition. Because enalapril shows only minimal and short-lived inhibition of cardiac ACE, it failed to attenuate the development of ventricular hypertrophy by shunt. On the other hand, quinapril, which markedly inhibits cardiac ACE, could attenuate the development of hypertrophy by shunt, even though it has hemodynamic effects similar to those of enalapril. Because lisinopril has effects on the inhibition of cardiac ACE that are almost identical to those of quinapril,30 our finding that lisinopril was effective in attenuating hypertrophy by shunt is consistent with those of Ruzicka and Leenen.29
As noted earlier regarding the characteristics of the AC rats in the present study, the model in the present study reflects stable compensated hypertrophy, not decompensated heart failure. In this AC fistula model of stable compensated hypertrophy, only the expression of ACE mRNA was increased, and no significant modulation of the expression of other RAS components was observed. However, this may not apply to decompensated heart failure or other types of ventricular hypertrophy. In fact, the expression of renin mRNA was temporarily markedly increased in the ventricles of isoproterenol-induced myocardial necrosis (our unpublished observation).
Selected Abbreviations and Acronyms
|AT1R||=||angiotensin II type I receptor|
|LV/W||=||left ventricular weight per body weight|
|LVH||=||left ventricular hypertrophy|
|PCR||=||polymerase chain reaction|
|RV/W||=||right ventricular weight per body weight|
|RVH||=||right ventricular hypertrophy|
This study was supported by a grant-in-aid from the Japanese Ministry of Education, Science, and Culture.
- Received November 30, 1994.
- Revision received March 13, 1995.
- Accepted May 25, 1995.
- Copyright © 1995 by American Heart Association
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