Effects of ACE Inhibitors on Circulating Versus Cardiac Angiotensin II in Volume OverloadInduced Cardiac Hypertrophy in Rats
Background Cardiac volume overload by an aortocaval shunt increases left ventricular end-diastolic pressure (LVEDP) and plasma and cardiac renin activity and results in LV hypertrophy. To a similar extent, the angiotensin-converting enzyme (ACE) inhibitors enalapril and quinapril prevent the increase in LVEDP. However, only quinapril attenuates the development of LV hypertrophy. We hypothesize that a low affinity of enalapril for cardiac ACE results in continuing generation of cardiac angiotensin II and thus hypertrophic growth of cardiomyocytes.
Methods and Results In the present study, we assessed plasma and cardiac angiotensins I and II 1 and 7 days after aortocaval shunt and the effects of enalapril and quinapril started 3 days before surgery on plasma and cardiac angiotensin I and II at the same time points. Aortocaval shunt increased plasma angiotensin II at 1 day by 180%, but only a small increase (by 40%) persisted at 7 days. Aortocaval shunt increased LV angiotensin II by 100% and 65% at 1 and 7 days, respectively. Both blockers similarly prevented the increase in plasma angiotensin II by aortocaval shunt at both time points. In contrast, only quinapril prevented the rise in LV angiotensin II induced by shunt at 1 and 7 days.
Conclusions Aortocaval shunt increases LVEDP and plasma and cardiac angiotensin II and results in LV hypertrophy. Only prevention of the increase in LVEDP and in plasma and cardiac angiotensin II attenuates the development of LV hypertrophy, consistent with the concept that angiotensin II is involved in the development of cardiac hypertrophy by aortocaval shunt by both hemodynamic and cardiac trophic effects. This study is the first to show that differences in affinity for cardiac ACE may determine the effect of ACE inhibitors on cardiac angiotensin II and therefore cardiac hypertrophy.
Stretch of cardiomyocytes in vitro results in Ang II release and increased protein synthesis.1 2 Ang II appears to mediate this hypertrophic response because the Ang II receptor blocker losartan prevents it.1 Whether in vivo release of angiotensin II by cardiomyocytes in response to stretch also represents the “initiating” mechanism for hypertrophic growth of cardiomyocytes has not yet been evaluated.
In previous studies, we showed that an aortocaval shunt increases LVEDP and plasma and cardiac renin activity and results in LV eccentric hypertrophy and right ventricular hypertrophy.3 The Ang II receptor blocker losartan and the ACE inhibitors enalapril and quinapril attenuate to a similar extent the rise in LVEDP in response to aortocaval shunt and similarly decrease LVPSP.3 4 However, only losartan and quinapril prevent LV hypertrophy and dilation.3 4 The similar effects of quinapril and enalapril on resting LVEDP and LVPSP and a similar blockade of pressor responses to Ang I by the two blockers indicate a similar degree of blockade of plasma or endothelial ACE.4 In these studies, however, changes in plasma Ang II induced by aortocaval shunt and the effects of the two ACE inhibitors on actual plasma Ang II levels were not assessed.
Whereas blockade of circulatory ACE by different ACE inhibitors differs mostly in duration,5 ACE inhibitors show major differences in their affinity for cardiac (and other tissues) ACEs.5 6 In doses equipotent for plasma ACE, enalapril shows minimal (by ≈20%) and short (1 hour) inhibition of cardiac ACE compared with marked (by ≈60%) inhibition lasting 8 hours by quinapril, fosinopril, and zofenopril.5 6 However, whether these differences in affinity for cardiac ACE between ACE inhibitors result in different levels of cardiac Ang II in vivo has not yet been assessed. We hypothesized that a low affinity of enalapril for cardiac ACE compared with quinapril, for example, may result in continuing generation of cardiac Ang II and thus hypertrophic growth of cardiomyocytes.
The objectives of the present study were to assess whether the increases in plasma and cardiac renin activity induced by aortocaval shunt are associated with parallel increases in plasma and cardiac Ang II, to evaluate whether the similar hemodynamic effects of enalapril and quinapril are indeed associated with similar changes in plasma Ang II, and to assess whether the differences in affinity for cardiac ACE between enalapril and quinapril in vitro result in different levels of cardiac Ang II in vivo and therefore may be relevant for the prevention of cardiac hypertrophy.
Animals, Treatments, and Surgery
Male Wistar rats weighing 200 to 225 g were obtained from Charles River Breeding Laboratories (Montreal, Quebec, Canada). Rats were housed two per cage, given food and water ad libitum, and kept on a 12-hour light-dark cycle. After an acclimatization period of at least 3 days, they were randomized into six groups: control, control plus enalapril, control plus quinapril, shunt, shunt plus enalapril, and shunt plus quinapril. Treatment with enalapril (250 mg/L in drinking water)3 7 8 and quinapril (200 mg/L in drinking water)9 started 3 days before the shunt or sham surgery and continued for 1 or 7 days. Aortocaval shunt was induced by an 18-gauge needle as described by Garcia and Diebold.10 Control (treated and untreated) rats were subjected to similar surgical procedures with the exception of puncturing the big vessels. On the day of the shunt or sham surgery or 6 days later, under a halothane–nitrous oxide–oxygen anesthesia, a PE-50 catheter (Clay Adams) filled with heparinized saline (100 U/mL) was inserted into the right common carotid artery. Catheters were exteriorized on the neck of the animals. LV and right ventricular weights and plasma and LV Ang I and II were assessed 1 and 7 days after the surgery (Fig 1⇓).
Blood Samples for Assessment of Plasma Ang I and II
On the day of the experiment (ie, 1 and 7 days after the shunt or sham surgery) at approximately 10 am after a 30-minute acclimatization period, 2 mL arterial blood was collected from conscious, unrestrained rats into chilled microcentrifuge tubes containing EDTA-Na2 and 1,10-phenantroline at final concentrations of 5 and 1.25 mmol/L in saline.11 Blood samples were centrifuged at 3000g for 5 minutes, and plasma for assessment of Ang I and II was immediately extracted on a SepPak C 18 cartridge (Millipore).
Cardiac Tissue Sampling for Assessment of Ang I and II
After the collection of blood samples, rats were killed by arterial injection of 1 mol/L KCl. The chest cavity was opened, and the heart was rapidly excised and washed in ice-cold saline. After removal of the atria and great vessels, the ventricles were blotted dry, and the right ventricle was dissected along its septal insertion from the rest of the ventricular mass. The left ventricle was then weighed and placed into boiling 1 mol/L acetic acid.12 13 The whole procedure, from KCl injection until placement of the left ventricle into boiling acetic acid, was practiced extensively and did not last >45 seconds. Tissue samples were boiled for 20 minutes, homogenized with a Polytron homogenizer (Brinkmann Instruments) for 1 minute, and centrifuged for 15 minutes at 7000g.
Plasma and supernatants from the LV homogenates were applied to methanol and water–preconditioned SepPak C 18 cartridges. Cartridges were washed twice with 4 mL of 0.1% trifluoroacetic acid. Angiotensin peptides were eluted by 2 mL of methanol:water:trifluoroacetic acid 80:19.9:0.1. Extracts were evaporated to dryness in a Savant SpeedVac concentrator.
Assessment of Ang I and II
Plasma and LV angiotensins were assessed by RIA after separation on HPLC. For this, plasma and LV samples were dissolved in mobile phase and centrifuged, and supernatant was injected. Angiotensins were separated on a CSC-Spherisorb-ODS2 C18 column, 15×0.46 cm with a 5-μm particle size (CSC), in a gradient system consisting of Waters 510 HPLC pumps controlled by a Waters workstation (Millipore), Rheodyne 7125 injector, and Spectra/Chrom CF-1 fraction collector (Spectrum). The mobile phase was 0.05 mol/L sodium acetate, pH 5.6, and methanol with a linear gradient from 40% to 80% methanol in 24 minutes from injection at a flow rate of 1.4 mL/min. Elution times for Ang II, III, and I were 10.0, 13.0, and 17.5 minutes, respectively. Fractions were collected every 0.5 minutes into polypropylene tubes containing 50 μL of 10% glycerol and 100 μL of 0.05 mol/L Tris buffer, pH 7.4. Subsequently, fractions were dried overnight in a SpeedVac concentrator.14
The tubes were divided into two pools containing the peaks of Ang II and III for Ang II RIA and the peak of Ang I for Ang I RIA (Fig 2⇓).
Both RIAs used the same buffer: 0.05 mol/L Tris, pH 7.4, with 1 mg/mL RIA-grade BSA and 0.02% sodium azide. Ang I and II standards (Sigma Chemical Co) were added to 700 μL or the mobile phase (with 100 μL buffer and 50 μL of 10% glycerol) and dried. For incubation, 200 μL RIA buffer, 100 μL antibody solution in RIA buffer, and 30 μL tracer (125I-Ang I or 125I-Ang II, both from Amersham) in RIA buffer (4000 to 5000 counts per minute) were subsequently added to the dried tube. The antisera (Ang I or II antibody) concentration was adjusted to yield 40% to 45% of specific binding after 24 hours of incubation at 4°C. Ang II antibody had 100% cross-reactivity with Ang II and III and <0.1% cross-reactivity with Ang I. After incubation, bound and free tracers were separated with dextran-coated charcoal (nonspecific binding <1%). The radioactivity of bound tracer was recorded for 4 minutes with a gamma counter (LKB Wallac).
Results are expressed as mean±SEM. Differences between groups at a given treatment period were evaluated by ANOVA and Duncan’s multiple-range test. Differences were considered significant at P<.05.
Assessment of Ang I and II
The sensitivity of detection of the method used to assess Ang I and II was 0.5 and 0.2 pg per tube, respectively. Recovery of angiotensins from plasma and tissue was determined by spiking of plasma or LV samples with 0 to 100 pg synthetic Ang I and II. Spiked samples were processed the same way as regular samples, including boiling, homogenization, SepPak extraction, HPLC, and RIA. Overall recoveries of Ang I and II were 60% to 80% from cardiac tissue and 80% to 100% from plasma. There was no significant difference in recovery between Ang I and II.
Plasma Ang I and II
Aortocaval shunt increased plasma Ang I by ≈150% (P<.05) 1 day after surgery compared with control rats (Table 1⇓). By 7 days after the shunt, plasma Ang I in rats with aortocaval shunt had returned to close to the values of control rats (Table 1⇓). The time course of changes in plasma Ang II in response to aortocaval shunt showed the same trend as described for Ang I: plasma Ang II increased by ≈180% at day 1 after the shunt surgery, but only a small increase (≈40%, P<.05) persisted at 7 days after surgery compared with control rats (Table 1⇓).
Enalapril and quinapril increased plasma Ang I in control rats compared with untreated control rats at both time points of follow-up (Table 1⇑). This increase in plasma Ang I by enalapril and quinapril was more pronounced (P<.05) in rats with aortocaval shunt at day 1 but was no longer pronounced at day 7 after the shunt surgery compared with their respective treated control rats (Table 1⇑).
Enalapril and quinapril caused only small (P=NS) changes in plasma Ang II at days 1 and 7 in control rats (Table 1⇑). In contrast, both blockers completely prevented the increase in plasma Ang II by aortocaval shunt at days 1 and 7 after the shunt surgery (Table 1⇑).
LV Ang I and II
Aortocaval shunt caused only small (P=NS) increases in LV Ang I at days 1 and 7 after surgery (Table 1⇑). In control rats, enalapril and quinapril similarly increased LV Ang I at day 1 (Table 1⇑). This increase persisted in quinapril-treated but not enalapril-treated control rats at day 7 (Table 1⇑). In rats with aortocaval shunt, enalapril increased LV Ang I at both time points. Quinapril caused no significant changes in LV Ang I at days 1 and 7 after the shunt surgery (Table 1⇑).
Aortocaval shunt resulted in an increase in LV Ang II of about 100% (P<.05) and 65% (P<.05) at days 1 and 7 after shunt surgery, respectively (Fig 3⇓). Enalapril and quinapril did not cause significant changes in LV Ang II in control rats at either time point of follow-up (Fig 3⇓). Quinapril completely prevented the increase in LV Ang II at days 1 and 7 after the shunt surgery. In contrast, enalapril failed to prevent the increase in LV Ang II at day 1 after surgery (Fig 3⇓). At 7 days after the shunt, enalapril resulted in a minor (P=NS) decrease in LV Ang II compared with untreated shunt rats (Fig 3⇓).
Because changes in central and peripheral hemodynamics are the primary determiners of the hypertrophic response of the heart, we show previously published results on hemodynamics. Aortocaval shunt decreased LVPSP at days 1 and 7 after surgery (Table 2⇓). Enalapril and quinapril decreased LVPSP in control rats by ≈10 mm Hg but had no effect on LVPSP in rats with aortocaval shunt (Table 2⇓).
Aortocaval shunt increased LVEDP at both days 1 and 7 after surgery compared with control rats (Table 2⇑). Enalapril and quinapril attenuated to a similar extent the increase in LVEDP in response to aortocaval shunt (Table 2⇑).
Body Weight and LV Weight
There were no differences in body weight between the groups at 1 and 7 days after surgery (Table 2⇑). Aortocaval shunt increased LV weight compared with control rats by ≈20%. Enalapril and quinapril caused only small (P=NS) decreases in LV weight in control rats (Table 2⇑). Rats with aortocaval shunt receiving enalapril developed LV hypertrophy similar to that in untreated rats. In contrast, quinapril significantly attenuated the development of LV hypertrophy compared with untreated rats with aortocaval shunt (Table 2⇑).
This study has the following primary findings. Aortocaval shunt increases plasma and LV Ang II. Enalapril and quinapril prevent to a similar degree the increase in plasma Ang II. In contrast, only quinapril prevents the increase in cardiac Ang II induced by aortocaval shunt.
Assessment of the Method Used to Evaluate Ang I and II
Separation of angiotensins on HPLC and assessment of angiotensins by RIA represent standard biochemical techniques.12 13 14 15 16 The sampling part of the method, however, is critical to preserve angiotensins at the level that most likely reflects the (patho)physiological processes. We eliminated the effect of anesthesia, decreased the total time of cardiac ischemia to about 45 seconds, and stopped ischemia-activated proteases linked to Ang I and II generation or degradation by immediately placing tissue samples into boiling acetic acid and collecting blood directly into chilled tubes containing inhibitors. This approach resulted in recovery of >80% plasma Ang I and II as assessed from spiking of plasma samples with Ang I and II. These data are consistent with others reporting similar recoveries for plasma Ang I and II.17 18 Recoveries for LV Ang I and II were about 60% to 80%. These recoveries are lower compared with plasma, possibly reflecting losses related to degradation of Ang I and II by tissue proteases during boiling in acetic acid and to (technical) losses from separation of supernatant and centrifugate after the homogenization of the tissue. Campbell et al16 showed recoveries for Ang I and II from cardiac and other tissues of ≈40% to 60%. They did not boil the tissue samples or use any other preventive measures from collection to homogenization to prevent generation or degradation of angiotensins. Our baseline levels for Ang I and II in control rats showed only small variability (see the SEM) and high reproducibility. Others reported similar data on plasma and cardiac Ang I and II in rats under physiological circumstances.15 19
Effects of Aortocaval Shunt on Plasma and LV Ang I and II
Aortocaval shunt increased both Ang I and II at day 1 after surgery. Both Ang I and II had decreased to close to control values at 7 days after the shunt. This time course of changes reflects the changes in plasma renin activity that we previously reported: peak values 1 day after surgery and then a gradual decrease.3 These increases in plasma Ang II appear to cause arteriolar vasoconstriction and thus maintain blood pressure within the normal range during the early stages of an aortocaval shunt.20 21 Although the total peripheral resistance is reduced in shunt animals,3 22 23 resistance is increased in certain vascular beds—at least in part—as a result of high levels of Ang II.21 22 23 In addition, an increased plasma Ang II could contribute through its renal effects (eg, increases in renal sodium and fluid reabsorption) to volume expansion and cardiac output.23 We are not aware of any study on changes in plasma Ang I and II in response to aortocaval shunt in rats.
In the doses used, enalapril and quinapril had similar effects on LVEDP and LVPSP4 and similarly shifted the pressure–Ang I dose-response curve to the right.4 Consistent with these findings, enalapril and quinapril prevented to a similar extent the increase in plasma Ang II by aortocaval shunt. As a result of the blockade of Ang I to II conversion, plasma renin activity3 and consequently Ang I increased in enalapril- and quinapril-treated animals.
The previously reported increases in LV renin activity 1 and 7 days after the shunt3 also suggested increases in LV angiotensins. Indeed, LV Ang I and II increased by ≈50% and 100%, respectively, at day 1 after the shunt. Although LV Ang I returned to the level seen in control rats, LV Ang II remained significantly increased compared with control rats. As for plasma Ang I and II, the changes in LV angiotensins in response to aortocaval shunt have not yet been assessed. However, similar increases in LV Ang II after 7 days of isoproterenol infusion (4.2 mg·kg−1·d−1), 16.2±2.0 pg/g tissue in isoproterenol-treated rats compared with 7.3±0.8 pg/g tissue in control rats, were associated with LV hypertrophy by 16%.15
In contrast to complete prevention of the increase in plasma Ang II 1 and 7 days after aortocaval shunt by both blockers, only quinapril also completely prevented the increase in LV Ang II at days 1 and 7 after aortocaval shunt. Because quinapril prevented the increase in both plasma and cardiac Ang II at both time points of follow-up, it appears that Ang II generation is driven by ACE rather than by nonspecific Ang II–forming enzyme(s). The nearly complete blockade of the increase in plasma Ang II by enalapril and lack of blockade of the increase in cardiac Ang II indicate a low affinity of enalapril for cardiac ACE. These results also suggest that regulation of Ang II generation in cardiac tissue can be independent of circulatory Ang II.
Relevance of LV Ang II for the Development of Cardiac Hypertrophy
As results from this study show, aortocaval shunt increases LVEDP and plasma and cardiac Ang II and causes LV hypertrophy.3 4 23 Attenuation of the increase in LVEDP and plasma and cardiac Ang II by quinapril or losartan (blocker of Ang II receptors) attenuates the development of LV hypertrophy.3 4 In contrast, this study also shows that enalapril prevents an increase in LVEDP and plasma Ang II but does not prevent an increase in cardiac Ang II or the development of LV hypertrophy.3 4 These data are consistent with the concept that Ang II is involved in the development of cardiac hypertrophy in response to cardiac volume overload by aortocaval shunt in two ways. First, plasma Ang II is a determiner of cardiac preload (by venoconstriction and sodium and water retention), cardiac afterload (by arterial vasoconstriction), and LV function (ventricular relaxation).3 23 24 25 Second, an increase in cardiac Ang II mediates the hypertrophic response of cardiomyocytes to cardiac volume overload because LV hypertrophy develops when an increase in cardiac Ang II is not prevented (eg, by enalapril).
This concept of the role of cardiac Ang II as a trophic factor in the development of cardiac hypertrophy in vivo is consistent with previously reported data by Sadoshima et al1 2 showing that Ang II released in vitro by cardiomyocytes in response to stretch mediates their hypertrophic response.
In the model of cardiac volume overload induced by an aortocaval shunt, enalapril and quinapril prevent an increase in plasma Ang II, but only quinapril prevents an increase in cardiac Ang II. These data reflect the effects of the two blockers at their peak (about 4 to 6 hours after the drinking period). The biological half-life of enalapril is substantially shorter (14 to 18 hours) and dissociates faster from the ACE active side compared with quinapril (30 to 36 hours).5 6 26 Because rats drink only 12 hours per day, quinapril probably provides adequate blockade of plasma and LV ACE at the end of the dosing interval, whereas enalapril provides neither of these. This may result in higher LVEDP and plasma and cardiac Ang II at the end of the dosing interval, changes that probably would contribute to the development or persistence of cardiac hypertrophy.
Possible Clinical Implications
This study is the first to show that differences in affinity for cardiac ACE between ACE inhibitors in vitro5 6 result in different levels of cardiac Ang II in vivo. These differences in affinity for cardiac ACE between ACE inhibitors appear to determine their effect on cardiac hypertrophy when cardiac Ang II mediates or potentiates the growth of cardiomyocytes. In patients with end-stage renal disease, obese hypertensive patients, and patients who have had myocardial infarction, cardiac volume overload clearly contributes to the development of cardiac hypertrophy.27 28 If cardiac Ang II also mediates or potentiates the development or maintenance of cardiac hypertrophy in these patients, one may hypothesize that these patients would benefit more from treatment with ACE inhibitors with high affinity for cardiac ACE than from treatment with those showing low affinity for cardiac ACE. This, however, remains to be assessed.
In conclusion, because enalapril fails to prevent an increase in cardiac Ang II and the development of cardiac hypertrophy in this model of volume overload, it appears that (1) besides the affinity for plasma ACE, affinity for cardiac ACE determines the effect of ACE inhibitors on cardiac hypertrophy when Ang II mediates or potentiates the growth of cardiomyocytes and (2) increases in both plasma and cardiac Ang II potentiate the hypertrophic response of the heart to cardiac volume overload by aortocaval shunt by both hemodynamic and direct trophic effects.
Selected Abbreviations and Acronyms
|HPLC||=||high-performance liquid chromatography|
|PSP||=||peak systolic pressure|
This study was supported by an operating grant from the Heart and Stroke Foundation of Ontario and by Parke-Davis, Division of Warner-Lambert Co, Canada. Enalapril maleate was a generous gift from Merck, Sharp & Dohme, Kirkland, Quebec, Canada. Quinapril hydrochloride was a generous gift from Parke-Davis, Division of Warner-Lambert Co, Ann Arbor, Mich. Antibody against Ang I was provided by Dr D. Osmond, Toronto, Ontario, Canada. Antibody against Ang II was a generous gift from Dr P. Admiraal, University Hospital Dijkzigt, Rotterdam, the Netherlands.
- Received May 10, 1995.
- Revision received June 19, 1995.
- Accepted August 8, 1995.
- Copyright © 1995 by American Heart Association
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