Blockade of the Renin-Angiotensin System in Cardiac Pressure-Overload Hypertrophy in Rats
Abstract Left ventricular hypertrophy in response to pressure overload may be modified by neurohumoral activation. To investigate the contribution of the renin-angiotensin system, we studied rats after banding of the ascending aorta that developed severe left ventricular hypertrophy associated with normal plasma renin but elevated cardiac angiotensin-converting enzyme (ACE) levels. Rats were treated with vehicle, ACE inhibitor (ramipril), angiotensin II type 1 receptor antagonist (losartan), or vasodilator (hydralazine) during weeks 7 through 12 after aortic banding. A significant regression of left ventricular mass index as determined by serial echocardiography was observed in ramipril- and losartan-treated groups during weeks 9 through 12 after banding, whereas hypertrophy further increased in vehicle- and hydralazine-treated groups. Twelve weeks after banding, relative left ventricular weights and myocyte widths were markedly increased in vehicle- and hydralazine-treated groups, whereas ramipril and losartan significantly reduced these parameters. In addition, molecular adaptations in left ventricular hypertrophy, such as upregulation of left ventricular atrial natriuretic peptide and downregulation of sarcoplasmic reticulum Ca2+-ATPase mRNA levels, were blunted by ramipril or losartan treatment. Hypertrophic regression was associated with reduced mortality in rats treated with ramipril (11%) and losartan (13%) versus hydralazine (20%) and vehicle (31%). Thus, the renin-angiotensin system may be involved in the maintenance of chronic left ventricular hypertrophy. Blockade of the system may result in regression of the hypertrophic phenotype and improve survival in rats despite persistent pressure overload.
- hypertrophy, left ventricular
- renin-angiotensin system
- angiotensin-converting enzyme inhibitors
- receptors, angiotensin
Left ventricular hypertrophy (LVH) often develops as an adaptive response that allows normalization of cardiac wall stress in situations of chronic cardiac pressure or volume overload.1 However, the benefits of this physiological adaptation may be offset by detrimental effects on both cardiac function and morphology.2 Maladaptive alterations in chronic pressure-overload LVH may contribute to the substantially elevated cardiovascular risk of patients with this condition.3 Thus, therapeutic efforts, in particular in the treatment of hypertensive heart disease, concentrated on strategies that promoted regression of LVH.4 Interestingly, clinical studies revealed that LVH regression in patients with hypertension is not simply a function of blood pressure control but varies with the antihypertensive agent.5 In particular, medication that lowered neurohormonal activity seemed to be more efficacious, which suggests that mechanisms other than hemodynamic overload contribute to this condition.5 Indeed, experimental studies revealed that chronic angiotensin-converting enzyme (ACE) inhibition may be associated with an amelioration of LVH even when pressure overload persists.6 7
The mechanisms that mediate LVH regression by ACE inhibitors are still unclear. Both the circulating and the local intracardiac renin-angiotensin systems have been proposed to contribute to the development of LVH.8 9 Furthermore, cardiac growth may be modified by genetic variants of the ACE gene.10 Angiotensin II (Ang II), the effector peptide of the system, may induce cardiac protein synthesis and facilitate cardiac growth.11 12 In addition, bradykinin has been suggested to mediate antiproliferative effects and thus may participate in regression of LVH during treatment with ACE inhibitors.13 At present, there remains some controversy as to whether the beneficial effects of ACE inhibitors can be reproduced by Ang II receptor blockade.
The present study was designed to investigate the role of the renin-angiotensin system in chronic pressure-overload hypertrophy. The results suggest that blockade of the system promotes regression of the hypertrophic phenotype on the macroscopic and microscopic as well as the molecular levels. Improvement of LVH was followed by a prolonged survival in rats with aortic stenosis. Finally, ACE inhibition and Ang II type 1 (AT1) antagonism displayed similar effects, which supports a central role of Ang II in the maintenance of LVH in rat hearts with chronic pressure overload.
Male Wistar rats (body weight, 70 to 80 g; aged 3 to 4 weeks) were obtained from Charles River Wiga Inc (Sulzfeld, Germany). Aortic banding was performed by using a silver clip with a 0.6-mm internal diameter as previously described in detail.6 14 15 Rats were maintained on standard rat chow (H1003, Alma KG) and were allowed water ad libitum. All rats were housed individually in a 12-hour dark/light cycle–controlled room. Protocols were in accordance with institutional guidelines.
Six weeks after surgery, rats with aortic stenosis were randomized to receive treatment with ramipril (10 mg/kg per day, n=35), hydralazine (20 mg/kg per day, n=35), losartan (40 mg/kg per day, n=16), or vehicle (n=36). The smaller number in the losartan-treated group was because of the limited availability of this drug. Sham-operated control rats (n=36) received no medication. Pilot studies in rats with aortic stenosis revealed that doses used in this study were equipotent with regard to the magnitude of blood pressure reduction. Drugs were added to the drinking water every second day. Concentrations were individually adjusted to drinking behavior to ensure adequate administration of each agent.16 During the 6-week treatment period, rats were monitored daily for determination of survival rate. Subsequently, 10 additional rats with aortic stenosis that received no long-term medication were included in this study for in vivo measurement of transstenotic pressure gradients.
In Vivo Hemodynamic Monitoring
The time course of development and regression of LVH was studied in vivo by transthoracic echocardiographic examinations 4 weeks after surgery (before drug treatment), as well as 9 and 12 weeks after surgery, ie, after 3 and 6 weeks of drug treatment, respectively. Rats were anesthetized with methohexital sodium (30 mg/kg IP). Echocardiography was performed using a Hewlett-Packard Sonos 1500 system with a 5-MHz electronic probe. Left longitudinal imaging was performed at an angle of approximately 45° through the left parasternal rib space with a maximal imaging depth of 40 mm. After obtaining a good-quality two-dimensional image, a two-dimensionally guided motion mode (M-mode) image of the left ventricle was recorded on VHS videotape and on paper at 100 mm/s; special care was taken to optimize endocardial borders. Images of reasonable quality from the apical site could not be obtained in most rats. Furthermore, echocardiography did not allow high-quality imaging earlier than 4 weeks after banding, ie, before the age of 7 weeks. From the recordings, left ventricular (LV) end-diastolic diameter (IDd) and end-diastolic LV posterior wall thickness (PWd) were analyzed. Left ventricular mass index (LVMI) was calculated according to a modified formula of Devereux and Reicheck17 as
There was a good correlation between calculated LVMI and postmortem relative LV weight (r=.70; standard error of the estimate [SEE]=0.272; P<.0001).
Indirect Systolic Blood Pressure
Indirect systolic blood pressure was determined by the tail-cuff method16 by using an automated cuff inflator-pulse detection system (BP recorder No. 8005, W+W Electronic AG). Unanesthetized rats were placed in a restraining holder from which the tail protruded. Vasodilation was achieved by local warming of the tail with an infrared bulb. Cuff and transducer were placed around the tail, and the cuff was inflated until the pulse disappeared. When the cuff was deflated, the point of reappearance of the pulse indicated the value of systolic blood pressure. The reported values are the mean of four to six recordings performed at the same time of day by the same investigator on 3 consecutive days in weeks 8 and 12 after surgery, ie, in weeks 2 and 6 of drug treatment.
Transstenotic Pressure Gradients
Transstenotic pressure gradients were determined in untreated anesthetized (50 mg/kg IP thiopental sodium) and mechanically ventilated rats with aortic stenosis 12 weeks after surgery. Arterial pressure was measured directly after preparation of the right carotid artery and placement of PE-50 tubing in the ascending aorta distal to the clip. LV pressure was recorded after thoracotomy and intraventricular placement of a 20-gauge tube through the apex of the left ventricle. Both tubes were connected to pressure transducers (P23XL, Statham Instruments), and pressures were recorded simultaneously on a multichannel recorder (Recomed, PPG Hellige GmbH). Transstenotic gradients were calculated by subtraction of aortic systolic pressure from peak LV pressure. To study the effect of blood pressure reduction on LV load, pressure tracings were recorded before and 10 minutes after intravenous (left jugular vein) injection of hydralazine (1 mg/kg, n=5) or losartan (2 mg/kg, n=5).
At the end of treatment, rats were killed by decapitation. Trunk blood was collected for determination of plasma renin activity (PRA), aldosterone, and atrial natriuretic peptide (ANP) concentrations as well as serum ACE activity. The hearts were excised, rinsed with saline, and blotted dry. The right ventricle was isolated by dissection along its septal insertion before left and right ventricle were balanced. Specimens for determination of cardiac ACE activity (interventricular septum) and cardiac ACE density by autoradiography (cross section) as well as mRNA measurements (free wall) were snap-frozen in liquid nitrogen within 3 minutes and stored at −80°C until analysis. For morphometric analysis, isolated hearts were perfusion fixed with Karnofsky’s solution in a Langendorff apparatus at a coronary perfusion pressure of 80 mm Hg in sham-operated and 100 mm Hg in LVH hearts.14 15
Morphometry of LV myocytes was performed by an investigator (D.G.) who was blinded for modality of treatment by using a modification of the methods of Anversa et al,18 as previously described.14 Three 1-mm-thick tissue blocks from the lateral mid–free wall of the left ventricle were postfixed in osmium tetroxide, dehydrated in graded ethanol, and embedded in a low-viscosity epoxy resin (ERL 4206). Semithin sections (1.0 μm) with transversely cut myofibers were stained with methylene blue–azur II. Myocytes were selected for determination of diameters if a visible nucleus was present and the cellular membrane was unbroken. Myocyte width was measured with a personal computer–assisted digital analysis device (Olympus; magnification ×160). Approximately 50 cells per heart were counted.
PRA, aldosterone, and ANP concentrations in plasma were determined by radioimmunoassay using commercially available kits according to the manufacturers’ instructions (PRA: RENK, No. 2510; plasma aldosterone concentration: ALDOCTK-2, No. 2714; Sorin Biomedica AG; plasma ANP concentration: RIK, No. 9103, Peninsula Laboratories GmbH).
ACE Activity and ACE Density
Serum and cardiac ACE activities were measured by a modified fluorometric method as previously described.15 Because of possible dissociation of ACE inhibitors from cardiac ACE in the fluorometric assay,19 cardiac ACE density in sham-operated, vehicle-treated, and ACE-inhibited rats was also determined by in vitro autoradiography to assess the local inhibition of the cardiac ACE by ramipril, as previously described.14
Total RNA from left ventricles was isolated according to the method of Chirgwin et al.20 For Northern blot analysis, equal aliquots of total RNA (20 μg) were denatured and size-fractionated by electrophoresis on 1% agarose gels under denaturing conditions. RNA was transblotted to nylon membranes (GeneScreen Plus, NEN) and immobilized by ultraviolet irradiation. Blots were prehybridized and hybridized using standard protocols.15 16 21 DNA probes used in this study were (1) ANP, a synthetic 84-nucleotide–long oligonucleotide complementary to the coding region of the rat ANP22 ; (2) sarcoplasmic reticulum (SR) Ca2+-ATPase, a 2.1-kb EcoRI fragment generated from cDNA clone RHCa 39 containing a coding region and the 3′ untranslated region specific for rat slow–cardiac SR Ca2+-ATPases23 ; and (3) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a 1.3-kb Pst I fragment from cDNA clone pUC-GAPDH13 containing the entire coding region and a part of the 3′ untranslated region of the rat GAPDH.24 At the end of hybridization, filters were washed and exposed at −80°C to x-ray films (XAR-5, Eastman Kodak) by using intensifying screens. Different exposures of all autoradiograms were obtained to ensure that laser scanning (Molecular Dynamics Personal Densitometer No. 50301) was performed within the linear range of densitometry. The densitometric scores of ANP and SR Ca2+-ATPase mRNAs were normalized by that of GAPDH mRNA, which encodes a constitutively expressed glycolytic enzyme as an internal control.
All results are expressed as mean±SEM. Multiple comparisons between three or more groups were carried out by two-way ANOVA and Fisher’s exact test for post hoc analyses. Survival was analyzed by the standard Kaplan-Meier analysis using likelihood ratio and χ2 tests. Statistical significance was accepted at a value of P<.05.
In rats with aortic stenosis, echocardiographically determined LVMI was significantly elevated 4 weeks after banding of the ascending aorta, which demonstrated a rapid onset of LV hypertrophy in this model (Fig 1A⇓). Further progression of LVH was noted in vehicle-treated rats during the entire time course up to 12 weeks after banding. The effects of medication on LVMI are shown in Fig 1B⇓. Three weeks of treatment with the ACE inhibitor ramipril, the AT1 antagonist losartan, or the vasodilator hydralazine failed to regress LVH. During weeks 9 through 12 after banding (weeks 3 through 6 of treatment), however, a reduction of the LVMI was observed in rats with a blocked renin-angiotensin system (P<.05, ramipril and losartan at 6 weeks versus 3 weeks). In contrast, LVMI increased further in rats receiving hydralazine (Fig 1B⇓). Neither treatment affected LV end-diastolic inner diameter (data not shown).
To study whether drug-related regression of LVH may be explained by long-term blood pressure reduction, systolic tail-cuff blood pressures were measured at weeks 7 and 11 after aortic banding (weeks 1 and 5 of treatment, n=8 to 10 per group). Reduction of systolic tail-cuff blood pressure was similar in all drug treated groups (Table 1⇓). In particular, no significant differences were noted among ramipril-, losartan-, and hydralazine-treated groups. Twelve weeks after aortic banding, heart rate in the vehicle-treated group was increased by 8% compared with sham-operated control rats. Although ramipril and losartan tended to lower heart rates slightly, the differences between treatment groups were not significant.
To study the effect of a moderate blood pressure reduction on ventricular load in rats with aortic stenosis, LV pressures and transstenotic pressure gradients were measured directly in additional vehicle-treated rats. Short-term intravenous administration of hydralazine (1 mg/kg) or losartan (2 mg/kg) resulted in arterial blood pressure reductions similar to those in rats with aortic stenosis that received long-term treatment (Tables 1⇑ and 2⇓). A systolic blood pressure reduction of 24 mm Hg was accompanied by a 6% fall in peak LV systolic pressure, whereas transstenotic pressure gradients increased slightly (Table 2⇓).
Regression of compensatory hypertrophy was related to improved survival in experimental rats with aortic stenosis (Fig 2⇓). After randomization, 11 of 36 rats died in the vehicle-treated group (31%) compared with 4 of 35 rats in the ramipril-treated group (11%), resulting in significantly reduced mortality in the ramipril-treated group (P<.05 versus vehicle-treated group), as calculated by Kaplan-Meier analysis. In contrast, survival was not improved in hydralazine-treated rats (7 of 35, 20%; P=NS versus vehicle-treated group). The mortality rate in losartan-treated rats (2 of 16, 13%) was similar to that in ACE-inhibited rats, but the number of rats was too small to show statistical significance (P=.16 versus vehicle-treated rats). Of the 24 rats that died during follow-up, 21 had pleural effusions (n=19), ascites (n=3), or pulmonary (n=2) or hepatic (n=4) congestion. Three rats had no macroscopic evidence of heart failure. None of the rats in the sham-operated group died.
Extent of Hypertrophy
In surviving rats, long-term aortic banding resulted in significantly increased LV weight–to–body weight (LV/BW) ratios in vehicle-treated (1.8-fold, P<.001) and hydralazine-treated (1.9-fold, P<.001) rats compared with sham-operated control rats (Fig 3⇓). The LV/BW ratio increase was blunted in the ramipril-treated rats (1.5-fold, P<.001 versus sham-operated rats) and losartan-treated rats (1.4-fold, P<.001 versus sham-operated rats). Thus, despite identical clip size, LVH was significantly reduced by ACE inhibition (P<.001 versus vehicle-treated rats) and by AT1 blockade (P<.001 versus vehicle-treated rats). In addition, LV/BW ratios were significantly lower in the ramipril-treated (20%, P<.001 versus hydralazine-treated rats) and losartan-treated (27%, P<.001 versus hydralazine) groups compared with hydralazine-treated rats that were characterized by similar systolic blood pressures. The difference in cardiac weights between study groups could not be explained by differences in body weights. Hydralazine-treated rats (386±16 g) were slightly smaller than vehicle-treated (431±19 g), ramipril-treated (435±18 g) or losartan-treated (410±26 g) rats, which may result in even later onset of pressure overload and a less severe gradient across the 0.6-mm aortic clip.
Myocyte cross-sectional diameters were determined in three to four rats from each group. Fig 4⇓, top, shows photomicrographs from representative LV myocardium. In rats with aortic stenosis receiving vehicle or hydralazine, the increase in relative LV weight was reflected in 2.5-fold higher myocyte widths (P<.001 versus sham-operated control rats, Fig 4⇓, bottom). Compared with vehicle- and hydralazine-treated rats, long-term ACE inhibition and AT1 blockade resulted in smaller myocyte width (P<.001 versus vehicle-treated rats, Fig 4⇓, bottom). In particular, rats in the ramipril- and losartan-treated groups displayed cell widths that were 39% to 49% smaller than those in vehicle- or hydralazine-treated rats (P<.001). Cell widths in rats from the losartan-treated group were not statistically different from those in sham-operated control rats.
Measurement of Cardiac ANP and SR Ca2+-ATPase mRNA
To study molecular markers of cardiac hypertrophy, LV ANP and SR Ca2+-ATPase mRNAs were quantified by Northern blot analyses and normalized to GAPDH mRNA (n=12 to 14 per group). A representative RNA blot is shown in Fig 5A⇓. Aortic banding resulted in 15- and 14-fold increases in ANP mRNA levels in vehicle- and hydralazine-treated rats, respectively (P<.001 versus sham-operated control rats, Fig 5B⇓). Compared with vehicle- and hydralazine-treated rats, ANP mRNA levels were significantly lower in ramipril- and losartan-treated rats.
Long-term aortic banding resulted in a 44% reduction of SR Ca2+-ATPase mRNA in rats in the vehicle-treated group compared with sham-operated control rats (P<.01, Fig 5C⇑). There was a trend toward higher SR Ca2+-ATPase signals in the ACE-inhibited group; however, the differences were not significant. AT1 blockade by losartan significantly elevated SR Ca2+-ATPase signals compared with rats in the vehicle-treated group (P<.05). The difference between rats in the losartan- and ramipril-treated groups, on the other hand, was not significant. Overall, there was an inverse correlation between the levels of ANP and SR Ca2+-ATPase mRNAs (r=−.51, P<.0001).
ANP plasma levels were significantly elevated in the vehicle-treated (P<.001 versus sham-operated rats) and hydralazine-treated (P<.005 versus sham-operated control rats) rats with long-term aortic banding. Treatment with ramipril or losartan significantly attenuated the increase in ANP plasma levels (P<.01 versus vehicle-treated rats, Table 3⇓). A significant correlation between LV ANP mRNA levels and plasma ANP concentrations was observed in rats with long-term LV pressure overload (r=.54, P<.005).
Table 3⇑ displays plasma hormones 12 weeks after surgery (after 6 weeks of treatment). PRA was similar in the sham-operated and vehicle-treated rat groups. In contrast, PRA was significantly elevated in ramipril- and losartan-treated rats, which indicated negative feedback regulation by effective blockade of the renin-angiotensin system.21 Interestingly, hydralazine-treated rats displayed an activation of PRA as well. The increase in PRA in the hydralazine-treated rat group was accompanied by elevated plasma aldosterone levels that were significantly higher in hydralazine-treated compared with sham-operated, ramipril-treated and losartan-treated rats. With the exception of ACE inhibited rats, serum ACE activities were unaffected by aortic stenosis or drug treatment (Table 3⇑).
In contrast, cardiac ACE activities were markedly increased (Table 3⇑), which resulted in a close correlation between cardiac ACE and LV/BW ratio (r=.67, P=.0001) as measured by fluorometric assay. Induction of cardiac ACE in rats with aortic stenosis was also evident by in vitro autoradiography (Fig 6⇓). Ramipril treatment, on the other hand, resulted in approximately 70% inhibition of cardiac ACE activity (Fig 6⇓ bottom). Thus, cardiac ACE density in ramipril-treated rats with aortic stenosis was similar to that seen in untreated sham-operated control rats (Fig 6⇓ bottom).
Growing evidence suggests that hemodynamic and neurohormonal factors closely interact in the regulation of myocardial growth and hypertrophy.11 25 In the present study, we investigated the contribution of the renin-angiotensin system to the maintenance of LVH. The model of long-term banding of the ascending aorta in the rat was selected to study whether LVH can be modulated despite persisting pressure overload of the heart. The study extends previous investigations in this model,6 7 14 15 26 which showed that long-term blockade of the renin-angiotensin system may improve survival and ameliorate LVH on the macroscopic, microscopic, and molecular levels. In contrast, vasodilation with hydralazine or doxazosin27 did not affect the progression of the disease and resulted in severe LVH that was not different from that in vehicle controls.
Serial echocardiography in individual rats was performed to study the time course of treatment effects. The data suggest that long-term blockade of the renin-angiotensin system did not simply arrest cardiac growth but rather resulted in regression of established LVH. Interestingly, serial echocardiography revealed that more than 3 weeks of medication was required to detect treatment effects in rats with a banded ascending aorta, a finding that may help to explain negative results in trials of shorter duration.28
The in vivo echocardiographic data were corroborated by measurements that demonstrated significantly smaller relative LV weights and myocyte widths, as well as an amelioration of molecular cardiac alterations in rats with aortic stenosis treated with drugs that blocked the renin-angiotensin system. Thus, the data suggest that blockade of the renin-angiotensin system interferes with central hypertrophic responses activated by cardiac pressure overload. The mechanisms responsible for these drug-related effects on LVH regression are incompletely understood. In particular, there is some controversy as to whether this response is related (1) to blockade of Ang II or augmentation of bradykinin effects, (2) to systemic or local inhibition of the renin-angiotensin system, or (3) to improved hemodynamics or direct effects on myocyte growth. Several observations presented here may add new information to this ongoing debate.
Ang II Blockade Versus Bradykinin Augmentation
Ang II and bradykinin have been implicated in the modulation of cardiac growth.11 12 29 Thus, ACE inhibitor–related LVH regression may be mediated by interaction with either of these neurohormones. A recent study by Linz and Schölkens30 suggests that ACE inhibitor–related reduction of LVH in rats with abdominal aortic banding may be antagonized by bradykinin receptor blockers. Furthermore, Garr and coworkers31 provide data that demonstrate better efficacy of ACE inhibitors than losartan with regard to postinfarction remodeling. In contrast, in the present study as well as others,32 33 AT1 antagonism and ACE inhibition displayed similar efficacy in regression of LVH. Thus, the present data support the hypothesis that growth-related effects of ACE inhibitors are at least partially mediated by Ang II acting on the AT1 receptor.
Systemic Versus Local Inhibition of the Renin-Angiotensin System
The present data as well as previous reports34 35 did not reveal a stimulation of the endocrine or circulating renin-angiotensin system in rats with long-term cardiac pressure overload. Both PRA and serum ACE activity were not elevated in aortic banded rats. Thus, it remains unclear whether Ang II formation by the circulating renin-angiotensin system is involved in the pathogenesis of pressure-overload LVH. The intracardiac renin-angiotensin system may be yet another target for ramipril or losartan. In this regard, it may be of interest that augmented expression of cardiac ACE, angiotensinogen, and Ang II receptor subtypes has been demonstrated in experimental as well as clinical LVH.14 15 34 36 37 Elevated cardiac ACE expression and enzyme activity in pressure-overload LVH were related to accelerated intracardiac conversion of Ang I to Ang II.14 On the other hand, inhibition of the cardiac ACE decreased local Ang II generation and improved cardiac function of hypertrophied hearts.14 38 39 In addition to these studies, the present data demonstrate a close positive correlation of cardiac ACE activity and severity of LVH, which increases the evidence that suggests an induction of the intracardiac renin-angiotensin system in long-term pressure overload of the heart.
There has been some debate as to whether long-term administration of ACE inhibitors may effectively inhibit the local cardiac renin-angiotensin system. For example, Hirsch et al40 failed to detect cardiac ACE inhibition in rats with myocardial infarction treated with either captopril or enalapril by using the fluorometric assay. Negative-feedback regulation as well as hypertrophy-mediated induction of ACE expression may account for this finding.15 21 41 In addition, technical aspects in measurements of tissue ACE activity need to be considered.19 In the present study, in vitro autoradiography was used to assess tissue ACE density in ACE inhibitor–treated rats.41 By using this technique, we found that ramipril treatment resulted in substantial inhibition of cardiac ACE, which suggests that local Ang II formation may be reduced by this intervention.14
Reduction of Hemodynamic Load Versus Local Growth Inhibition
Blockade of the renin-angiotensin system may result in hemodynamic improvements, such as a fall in peripheral and pulmonary resistance. Since mechanical load is a central mechanism in the regulation of cardiac growth, similar effects might explain regression of LVH in rats with aortic stenosis. Short-term administration of hydralazine or losartan in rats with aortic stenosis only minimally affected ventricular systolic pressures. Unfortunately, long-term measurements of LV load are not feasible. Repeated measurement of tail-cuff blood pressures revealed a similar decrease in ramipril-, losartan-, and hydralazine-treated rats with aortic stenosis. Because of the aortic ligature, however, arterial blood pressure reduction as measured by tail cuff may have only a small effect on LV afterload. Thus, given the present hemodynamic data, it seems unlikely that reduction of afterload is the only factor responsible for the regression of LVH observed in only losartan- and ramipril-treated rats. On the other hand, we cannot exclude the possibility that slight hemodynamic improvements may contribute to this drug-related effect.
Direct growth-promoting effects of Ang II may also contribute to maintenance of pressure-overload LVH.25 The importance of locally synthesized Ang II was emphasized by the studies of Sadoshima et al.42 These investigators provided strong evidence that Ang II is stored in cardiac myocytes and released upon short-term imposition of cellular stretch. Indeed, these data on neonatal rat myocytes suggest that Ang II is part of the signaling pathway activated by short-term mechanical growth stimulation and initiation of LVH. The importance of Ang II in the maintenance of pressure-overload LVH is emphasized by the present in vivo data. Treatment with either an ACE inhibitor or AT1 antagonist resulted in regression of LVH in rats with long-term aortic stenosis. Decreased heart weights could not be attributed to cell loss or changes in cellular composition of the myocardium. Rather, losartan and ramipril blunted the hypertrophic phenotype of cardiac myocytes.
Long-term ACE inhibition resulted in a significant survival benefit in rats with fixed aortic stenosis despite regression of adaptive hypertrophy. The mechanisms that account for this finding cannot be derived from our data. Studies in patients with chronic heart failure suggest that the beneficial effects of ACE inhibition on survival may be attributed to either improvement of peripheral hemodynamics or reduction of sudden cardiac death.43 44 In the present experimental model, ACE inhibition was unlikely to improve survival by peripheral vasodilatation since fixed ascending aortic stenosis prevented significant drug-related unloading of the heart. Postmortem analyses of rats that died prematurely revealed pulmonary and hepatic congestion, pleural effusion, and consecutive right ventricular hypertrophy, which suggest that death was related to advanced cardiac failure rather than to arrhythmias. Given the fact that peripheral hemodynamic and “antiarrhythmic” effects are unlikely to explain the improved outcome in rats treated with an ACE inhibitor, it may be hypothesized that ACE inhibition may delay the transition of hypertrophy to cardiac failure on the myocardial level.
Several changes in cardiac gene expression, such as induction of ventricular atrial natriuretic factor or downregulation of SR Ca2+-ATPase, have been associated with this critical transition of LVH to heart failure.26 ACE inhibition and AT1 antagonism blunted these molecular adaptations in rats with fixed aortic stenosis. Furthermore, ANP levels were markedly lowered by ramipril and losartan. Taken together, the data provide indirect evidence that blockade of the renin-angiotensin system may prolong survival and ameliorate heart failure by affecting myocardial structure or function in response to long-term pressure overload.
The authors would like to thank Drs A. Kurtz and K.-H. Wrobel for support of these studies and Dr H.-W. Hense for calculation of the Kaplan-Meier analysis. The SR Ca2+-ATPase cDNA probe was genereously provided by Dr K.R. Boheler. The authors also thank T. Cornelius for critical discussion, as well as S. Kürzinger, I. Kirst, and Dr B. Ackermann for excellent technical assistance.
Reprint requests to Dr Schunkert, Klinik und Poliklinik für Innere Medizin II, Universität Regensburg, Franz-Josef-Strauß-Allee 11, D-93053 Regensburg, FRG.
- Received July 26, 1994.
- Revision received August 31, 1994.
- Accepted October 3, 1994.
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