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Am J Physiol Heart Circ Physiol 279: H2797-H2806, 2000;
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Vol. 279, Issue 6, H2797-H2806, December 2000

Renin-angiotensin system and sympathetic nervous system in cardiac pressure-overload hypertrophy

Wendell S. Akers1, Andrew Cross2, Robert Speth3, Linda P. Dwoskin2, and Lisa A. Cassis2

Divisions of 1 Pharmacy Practice and Science and 2 Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536; and 3 Department of Veterinary Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington 99163


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiotensin II and norepinephrine (NE) have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. The purpose of this study was to determine the temporal sequence for activation of the renin-angiotensin and sympathetic nervous systems in the rat after 3-60 days of pressure overload induced by aortic constriction. Initially on pressure overload, there was transient activation of the systemic renin-angiotensin system coinciding with the appearance of left ventricular hypertrophy (day 3). At day 10, there was a marked increase in AT1 receptor density in the left ventricle, increased plasma NE concentration, and elevated cardiac epinephrine content. Moreover, the inotropic response to isoproterenol was reduced in the isolated, perfused heart at 10 days of pressure overload. The affinity of the beta 2-adrenergic receptor in the left ventricle was decreased at 60 days. Despite these alterations, there was no decline in resting left ventricular function, beta -adrenergic receptor density, or the relative distribution of beta 1- and beta 2-receptor sites in the left ventricle over 60 days of pressure overload. Thus activation of the renin-angiotensin system is an early response to pressure overload and may contribute to the initial development of cardiac hypertrophy and sympathetic activation in the compensated heart.

beta -adrenergic receptor; norepinephrine; heart failure; left ventricle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC HYPERTROPHY is an adaptive response of the heart associated with several pathological situations, including heart failure, myocardial infarction, and cardiac arrhythmias. The development of left ventricular (LV) hypertrophy enhances contractility and allows for normalization of cardiac wall stress in response to pressure or volume overload (16). The benefits of this adaptive response of the heart may be offset by detrimental effects on both cardiac function and morphology, making cardiac hypertrophy an important cause of increased morbidity and mortality. The mechanisms governing the development of cardiac hypertrophy have been extensively studied; however, they are incompletely understood. A common experimental animal model of cardiac hypertrophy is surgical aortic constriction resulting in sustained pressure overload to the heart (22). Evidence suggests involvement of neurohumoral systems such as the renin-angiotensin system and the sympathetic nervous system in the development of LV hypertrophy from cardiac pressure overload (2, 31, 39).

The sympathetic nervous system has been implicated in the development of cardiac hypertrophy, leading Ostman-Smith (34) to propose that cardiac sympathetic nerves are the final common pathway in the induction of most types of hypertrophy. In the rat aortic constriction model of cardiac pressure overload, LV norepinephrine (NE) content was decreased within 7-14 days (14, 15, 39). Reductions in cardiac NE content were generally associated with elevations in catecholamine turnover in the heart, supporting enhanced cardiac sympathetic neurotransmission (14, 15, 33, 39). Increases in cardiac sympathetic neurotransmission have been suggested to contribute to the development of hypertrophy and alterations in cardiac adrenergic receptor function. However, the majority of studies performed do not support changes in the density and/or affinity of the beta -adrenergic receptor in the heart in response to pressure overload over a 1- to 4-wk period of study (7, 9, 12, 32).

Several lines of evidence suggest that the renin-angiotensin system and ANG II, produced systemically or by an intrinsic cardiac system, are activated and may contribute to cardiac hypertrophy in response to pressure overload. Components of the renin-angiotensin system, including angiotensinogen (11), angiotensin-converting enzyme (ACE) (37), and ANG II (37), are increased in the ventricle in response to pressure overload. However, disparate effects have been reported for the effectiveness of ACE inhibitors or AT1 receptor antagonists in the development of LV hypertrophy after pressure overload. Moreover, abdominal aortic constriction in AT1A receptor knockout mice produced cardiac hypertrophy independent of the AT1 receptor, suggesting that multiple systems are involved in the hypertrophic process, only one of which is the renin-angiotensin system (18, 19).

The intent of this study was to define the temporal sequence for neurohumoral activation in the response to cardiac pressure overload induced by abdominal aortic constriction. Definition of the status of the renin-angiotensin system and the sympathetic nervous system during the development of cardiac hypertrophy was paralleled by chronic measurement of cardiac hypertrophy and function using transthoracic echocardiography.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Male Sprague-Dawley rats weighing 275-325 g (7-9 wk of age; Harlan Sprague Dawley, Indianapolis, IN) were used in all experiments. Rats were housed two per cage with free access to food and water. All studies were approved by the Institutional Animal Care and Use Committee at the University of Kentucky.

Surgical Induction of Pressure Overload

Rats were randomly assigned to groups [aortic constricted (AC) and sham operated (SO)] and time points (3, 10, 30, or 60 days; n = 8 rats/group at each time point). Rats were anesthetized by ketamine hydrochloride plus acepromazine maleate (90 and 0.02 mg/kg ip, respectively; Fort Dodge Laboratories, Fort Dodge, IA) and prepared for surgery under aseptic conditions. After a midline abdominal laparotomy, pressure overload was induced by suprarenal abdominal aortic constriction using a tantalum Weck hemoclip (Pilling Weck, Research Triangle Park, NC) tightened to the diameter of a 22-gauge needle. Control rats underwent sham surgery consisting of midline laparotomy and isolation of the suprarenal abdominal aorta without constriction. The muscle was sutured, and the skin was closed using surgical wound clips. On the final day of the study, each rat was examined to verify the location of the hemoclip, and both kidneys were weighed to identify the presence of renal atrophy.

Echocardiography

LV function and chamber dimensions were assessed in a subset of rats (n = 5 rats/group at each time point) by transthoracic echocardiography using a diagnostic sonar ultrasound imaging system. Under ether anesthesia, chest wall hair was removed, and rats were held in the left lateral decubitus position. The ultrasound probe was positioned on the chest to obtain a two-dimensional M mode image (short axis) of the LV. The American Society of Echocardiography leading-edge method was used to measure LV anterior (AWT) and posterior (PWT) wall thickness and end-diastolic (EDD) and end-systolic (ESD) diameter. Fractional shortening (FS) was used as an index of contractility and was calculated using the following formula: FS = (EDD - EDS)/EDD.

Hemodynamic Measurements

Mean arterial pressure (MAP), systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were measured for a consecutive 3 min in anesthetized (ketamine-acepromazine, 90 and 0.02 mg/kg ip) rats from each group at each time point. For measurement of these parameters, the right carotid artery was cannulated with a fluid-filled polyethylene (PE-50) catheter connected to a pressure transducer in-line to a Grass polygraph (model 79D; Grass Instrument, Quincy, MA).

Measurement of Contractility in Isolated, Perfused Heart

The isolated, perfused heart was used to examine alterations in the cardiac inotropic responsiveness to isoproterenol. After the administration of ketamine-acepromazine (90 and 0.02 mg/kg ip) and heparin (1,500 IU/kg), the heart was rapidly removed from the thoracic cavity by median sternotomy and immediately placed in ice-cold Krebs-Henseleit buffer (mM: 118 NaCl, 4.8 KCl, 25 NaHCO3, 1.2 MgSO4, 1.2 HK2PO4, 11 glucose, 1.5 CaCl2; 5% CO2-95% O2; O2 tension 600 mmHg; pH 7.4). Hearts were mounted onto an aortic cannula and perfused by retrograde coronary artery perfusion at constant flow with a peristaltic pump. Coronary flow rate was adjusted to achieve a baseline mean coronary perfusion pressure of 65 and 95 mmHg in SO and AC hearts, respectively. These levels of perfusion pressure were chosen based on preliminary data demonstrating an ~30-mmHg increase in the in vivo perfusion pressure in AC (10 days) compared with SO rats. Changes in perfusion pressure were continuously monitored at the level of the aortic root. To determine cardiac function, a latex fluid-filled balloon attached to PE-190 tubing was inserted into the LV. Perfusion pressure and cardiac function were obtained from in-line pressure transducers connected to a Digi-Med blood pressure analyzer and heart performance analyzer, respectively (Micro-Med, Louisville, KY). Hearts were electrically paced at 300 beats/min using two silver Teflon-coated electrodes connected to a Grass model SD9 stimulator (Grass Medical Instruments). LV balloon volume was adjusted to achieve a LV end-diastolic pressure (EDP) of 10 mmHg. After a 20-min stabilization period, baseline measurements of HR, maximal LV pressure (LVP), LVEDP, and positive first derivative of LVP (+dP/dt) were obtained. Subsequently, the contractile response (% change in +dP/dt) to isoproterenol (10 nM) was determined.

Measurement of Plasma and Tissue Catecholamines

Catecholamines were measured according to previously described methods (27). Briefly, blood (2 ml) was collected on the final day of study from the carotid artery catheter of anesthetized rats into heparin tubes and centrifuged at 1,100 g for 20 min at 4°C. Plasma was stored at -70°C until assay. A sample (50 mg) of LV free wall was frozen in liquid nitrogen. Tissue was homogenized in 0.4 N perchloric acid buffer (1 ml, containing 0.5 mM EDTA and 0.4 mM sodium metabisulfite) on ice for 10 s and centrifuged at 12,365 g for 10 min at 4°C. A fixed amount (524 pg) of dihydroxybenzylamine (DHBA) was added to the supernatant of each sample as an internal standard. An aliquot (1 ml) of plasma was thawed and added to 1 ml of the perchloric acid buffer and internal standard. Catecholamines were extracted from plasma and tissue by the addition of activated alumina (25 mg; Bioanalytical Systems, West Lafayette, IN). The alumina mixture was titrated to pH 8.7 by the addition of 3 M Tris base (pH 10.9) and vortexed for 10 min, followed by centrifugation at 3,091 g for 2 min at 4°C. The supernatant was discarded, and the remaining alumina pellet was washed three times with water. Catecholamines were eluted twice by the addition of 0.15 N of perchloric acid (100 µl). Catecholamine standards [NE (50-200 pg), epinephrine (Epi; 50-200 pg), and DHBA (524 pg)] and samples were quantitated by HPLC with electrochemical detection (Beckman model 116 pump and model 7725 injection valve, Rheodyne, CA; Coulochem model 5100A electrochemical detector and model 5011 analytical cell, ESA, Bedford, MA). Retention times of standards were used to identify NE, Epi, and DHBA, and peak heights were used to quantify amount. The peak height was linear (correlation coefficient > 0.95) to the amount of catecholamine (NE and Epi) up to 200 pg. Extraction recovery for DHBA was >80%, and the sensitivity for catecholamines was 5 pg. All samples were diluted to give peak heights within the range of 50-200 pg and corrected for recovery and dilution.

Measurement of Plasma Angiotensin Peptides

Plasma angiotensin peptide concentration was measured according to a previously described method (6). Blood (5 ml) was collected from the carotid artery catheter of anesthetized rats on the final day of study into tubes containing 125 mM EDTA, 20 mM phenanthroline, 0.2% neomycin, 0.1 mM kallikrein, 2% ethanol, and 2% DMSO (250 µl) to eliminate both the production and breakdown of angiotensin peptides during sample handling. Angiotensin peptides from plasma (2 ml) were extracted using SepPak C-18 column chromatography. Angiotensin peptide concentration in each plasma sample was measured by RIA using a polyclonal ANG II antibody (Dr. A. Freedlender, University of Virginia, Charlottesville, VA) that exhibited minimal cross reactivity to ANG I (2%) and ANG II fragment 5-8 (4%), but 100% cross reactivity to ANG III, ANG II fragment 3-8, and ANG II fragment 4-8. Sensitivity of the RIA was 2 pg/ml.

LV Membrane Preparation

After hemodynamic measurements and blood collection for neurohumoral measurements, hearts were removed and placed in ice-cold Krebs buffer. The LV was dissected free from the atria and right ventricle to obtain absolute cardiac chamber wet weights. LV weight normalized to body weight (LV/BW) was used as an index of cardiac mass for the determination of cardiac hypertrophy. Whole LV, including the interventricular septum, was placed in 30 ml of ice-cold membrane buffer (50 mM NaPO4, 0.25 M sucrose; pH 7.2), homogenized on ice using a Polytron for 20 s, and centrifuged at 1,100 g for 10 min at 4°C. The resulting pellet was discarded, and the supernatant was centrifuged three times at 48,000 g for 10 min at 4°C. The final pellet was resuspended [3 ml of buffer containing 50 mM NaPO4, 0.1 mM EDTA, 28 kallikrein inhibitory units (KIU)/dl aprotinin, and 0.014% bacitracin; pH 7.2], homogenized, and stored at -70°C. Protein concentration was determined by the method described by Bradford (4).

beta -Adrenergic Receptor Binding Assays

Binding assays (saturation isotherm, competition) for beta -adrenergic receptors were performed in membranes prepared from the LV. Saturation isotherms for the beta -adrenergic receptor were performed by adding an increasing concentration (3-400 pM) of (-)-[125I]iodocyanopindolol ([125I]ICYP, nonselective beta -adrenergic receptor antagonist; specific activity 2,200 Ci/mM, Peptide Radioiodination Service Center, Washington State University) to a fixed amount of membrane protein (75 µg) in tubes containing binding assay buffer (50 mM Tris · HCl, 0.1 mM EDTA, 1 mM MgCl2, 28 KIU/dl aprotinin, 0.014% bacitracin, 0.2% bovine serum albumin; pH 7.2). Nonspecific binding was determined at each radioligand concentration by the addition of the nonselective beta -receptor antagonist propranolol (10 µM). Incubation was performed in a total volume of 0.25 ml for 180 min at 25°C and terminated by filtration through Whatman GF/B glass-fiber filters (presoaked in 50 mM Tris · HCl buffer) using a Brandel harvester. Filters were washed three times with ice-cold binding buffer, and the amount of radioactivity retained on the filter was determined in a gamma-counter (model A550, Packard, Downers Grove, IL). Maximal number of binding sites (Bmax) and affinity (Kd) were derived by nonlinear regression analysis using LIGAND software.

Competition studies were performed using the selective beta 1-adrenergic receptor antagonist CGP-20712A. Competition was performed using a fixed concentration of [125I]ICYP (50 pM; 2.5 times Kd) with a range of CGP-20712A concentrations (10-10-10-4 M). The inhibitory constant (Ki) value for CGP-20712A at the beta 1- and beta 2-receptors was calculated according to the equation derived by Cheng and Prusoff (8). One- and two-site models were fit to competition binding data using LIGAND software.

AT1 Receptor Binding Assay

Saturation isotherms for the AT1 receptor were performed by adding a fixed amount of LV membrane protein (75 µg) to tubes containing the binding assay buffer and an increasing concentration (0.05-5 nM) of 125I-labeled [Sar1Ile8]ANG II (nonselective ANG II receptor antagonist; specific activity 2,176 Ci/mM, Peptide Radioiodination Service Center, Washington State University). The AT2 receptor antagonist PD-123319 (1 µM) was included in the binding buffer to eliminate binding of 125I-labeled [Sar1Ile8]ANG II to the AT2 receptor site. Nonspecific binding was determined at each radioligand concentration by the addition of an excess of unlabeled ANG II (10 µM). Incubation was performed in a total volume of 0.25 ml for 60 min at 26°C and terminated by filtration through Whatman GF/B glass-fiber filters (presoaked in 50 mM sodium phosphate buffer containing 1% polyethylenimine) using a Brandel harvester. Filters were washed three times with ice-cold binding buffer. Bmax and Kd were derived by nonlinear regression analysis using LIGAND software.

Statistical Analysis

Data are presented as means ± SE. For each parameter measured (hemodynamic, LV/BW, plasma NE, plasma ANG II, Bmax, Kd) in the time course study, separate two-way ANOVAs were performed for each parameter with treatment (AC, SO) and time (3, 10, 30, and 60 days) as between-group factors. Serial echocardiographic studies were performed in a subset (n = 5) of AC and SO rats at baseline and 3, 10, 30, and 60 days. A two-way ANOVA (group × time) with time as a repeated measure was performed to determine differences in wall thickness and LV function. The Student-Newman-Keuls test was used for post hoc comparisons of individual parameters across group and time. For data (Epi, NE, Kd, and Bmax) in the 10-day study, a two-tailed t-test was performed to determine differences between groups (AC, SO). P values <0.05 were considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time Course for Alterations in LV Hypertrophy, Cardiac Function, Sympathetic Nervous System, and Renin-Angiotensin System After Pressure Overload

Magnitude of pressure overload. The time course for alterations in blood pressure and heart rate after 3, 10, 30, and 60 days of pressure overload is presented in Table 1. Abdominal aortic constriction resulted in a significant increase in MAP (F1,59 = 135; P < 0.0001), SBP (F1,58 = 111; P < 0.0001), and DBP (F1,58 = 87; P < 0.0001). MAP was increased by ~32 mmHg in AC rats at each time point after pressure overload. HR was not different between AC and SO rats at each time point and within each group across time.

                              
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  		Table 1.   Hemodynamic measurements in AC and SO rats after pressure overload

Body weight and atrial and ventricular weights were analyzed across group and time. There was a significant (F3,60 = 125; P < 0.0001) increase in body weight with time in rats from both groups, with no significant difference between groups (data not shown). LV hypertrophy developed by day 3 and was maintained through 60 days of pressure overload. LV hypertrophy was manifested as an increase in absolute LV weight (F1,60 = 102; P < 0.0001), which remained evident when normalized to body weight (F1,60 = 115; P < 0.0001; Fig. 1A). Left atrial hypertrophy was observed in AC rats from day 10 through day 60 (F1,60 = 41; P < 0.0001). Right ventricle and atrial weights were not different between the two groups at all time points (data not shown). Right and left kidney weights were similar in SO (right 1.29 ± 0.03, left 1.28 ± 0.03 g) and AC (right 1.17 ± 0.03, left 1.31 ± 0.03 g) rats at each time point after pressure overload. Only four rats in the AC group demonstrated right renal atrophy, which occurred in three rats after 60 days of pressure overload.


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  		Fig. 1.   Left ventricular (LV) hypertrophy and fractional shortening after different periods of pressure overload. Time course studies examined the effect of aortic constriction for 3-60 days. A: LV-to-body weight ratio (LV/BW in mg/g). LV wet weight was normalized to body weight in each rat as an index of cardiac hypertrophy (n = 8 rats/group for each time point). Pressure overload resulted in an increase in LV/BW from day 3 to day 60 compared with sham-operated (SO) rats. B: LV fractional shortening. Serial 2-dimensional short-axis images of the LV were assessed by M-mode transthoracic echocardiography in a subgroup of rats (n = 5/group for each time point) at baseline and at 3, 10, 45, and 60 days of pressure overload. Fractional shortening was not altered over 60 days of pressure overload. Data are means ± SE. *P < 0.001, aortic constricted (AC) vs. SO rats at respective time points.

Echocardiographic measurements. EDD and ESD were not different between AC and SO rats at any time point after pressure overload (data not shown). Similarly, LV FS was not different in AC and SO rats at any time point (Fig. 1B). AWT was significantly increased (F1,10 = 16; P < 0.01) in AC compared with SO rats at days 3-60. PWT was not different between AC and SO rats at any time point (data not shown).

Plasma NE and ANG II concentration. Statistical analysis of plasma NE concentration after pressure overload revealed a significant effect of group (F1,36 = 7; P < 0.05) and time (F3,36 = 6; P < 0.05). Plasma NE concentration was not different in AC and SO rats at day 3; however, plasma NE concentration was increased in AC rats by day 10 and remained elevated through day 60 (Fig. 2A). Statistical analysis of plasma ANG II concentration revealed a significant effect of time (F3,38 = 22; P < 0.0001), no between-group effect, and a significant interaction between time and group (F3,38 = 4; P < 0.05). Plasma ANG II concentration was increased by 63% in AC versus SO rats at day 3. Moreover, plasma ANG II concentration at day 3 in both groups was significantly increased compared with days 10-60 (Fig. 2B).


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  		Fig. 2.   Plasma norepinephrine (NE) and ANG II concentration after different periods of pressure overload. Time course studies examined the effect of aortic constriction for 3-60 days. A: plasma NE concentration. NE was extracted from plasma by the addition of activated alumina followed by resolution and quantification by HPLC with electrochemical detection. Plasma NE increased in AC rats at 10 days of pressure overload and remained elevated through 60 days. B: plasma ANG II concentration. Plasma was partially purified using SepPak C-18 column chromatography, and ANG II immunoreactivity was measured by RIA using a polyclonal ANG II antibody. Plasma ANG II was increased in both groups at day 3 compared with days 10-60. In AC rats at day 3, plasma ANG II was increased compared with control, followed by a return to control levels through day 60. Data are means ± SE (n = 6/group for each time point). *P < 0.05, AC vs. SO rats at respective time points.

LV beta -adrenergic receptors. Initial binding experiments using [125I]ICYP were performed in control rats to determine optimal membrane protein and time to equilibrium (data not shown). A representative saturation binding isotherm with corresponding Scatchard plot for specific [125I]ICYP binding in rat LV membranes prepared from AC and SO rats after 60 days of pressure overload is illustrated in Fig. 3A. Specific binding of [125I]ICYP was saturable and best described by a one-site model. The affinity and density for [125I]ICYP binding were not influenced by time in either group (Table 2). Moreover, there was no effect of pressure overload on the affinity or density for [125I]ICYP binding in LV membranes over the time course examined.


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  		Fig. 3.   Saturation isotherm and CGP-20712A competition of (-)-[125I]iodocyanopindolol ([125I]ICYP) binding in LV membranes from 60-day AC and SO rats. A: saturation isotherms were performed by adding a fixed amount of membrane protein to increasing concentrations of [125I]ICYP. Inset, Scatchard transformation of saturation isotherm data. [125I]ICYP bound to a single class of sites with high affinity in membranes from AC and SO rats, with no between-group differences. B, bound; B/F, bound/free. B: competition of [125I]ICYP binding by increasing concentrations of the selective beta 1-adrenergic receptor antagonist CGP-20712A. CGP-20712A displaced [125I]ICYP in a concentration-dependent manner; competition data fit a 2-site model, with a high- and a low-affinity site for CGP-20712A displacement. Data are means ± SE (n = 4 rats/group).


                              
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  		Table 2.   Left ventricular beta -adrenergic receptor binding of [125I]ICYP in AC and SO rats after pressure overload

Competition of [125I]ICYP binding with the beta 1-receptor antagonist CGP-20712A in LV membranes from AC and SO rats was concentration dependent and best described by a two-site model (Fig. 3B). A high-affinity (Ki1, beta 1) and a low-affinity (Ki2, beta 2) site were defined by CGP-20712A competition (28). Statistical analysis revealed a significant effect of time on the Ki1 (F2,12 = 7; P < 0.01) and Ki2 (F2,12 = 35; P < 0.0001) for CGP-20712A in AC and SO rats (Table 2). Derived Ki constants for the high- and the low-affinity site were significantly increased (P < 0.05) in AC and SO rats at day 60 of pressure overload compared with days 3 and 10. Moreover, the Ki2 value for CGP-20712A was significantly increased (P < 0.001) in AC compared with SO rats after 60 days of pressure overload. In LV membranes from SO rats, the relative proportion (% beta 1 subtype) of beta 1- and beta 2-receptors averaged 54% (Table 2). The proportion of beta 1- and beta 2-receptors in LV was not significantly influenced by pressure overload at any time point.

Cardiac Renin-Angiotensin System and Sympathetic Nervous System After 10 Days of Pressure Overload

In a separate study, cardiac catecholamine content, AT1 receptor density, and cardiac function were examined at 10 days of pressure overload. This time point was chosen to determine whether increases in plasma ANG II concentration (day 3) subsequently influenced the cardiac AT1 receptor and to determine whether elevations in plasma NE concentration (day 10) were associated with alterations in cardiac catecholamine content and cardiac dysfunction. In agreement with results from the time course study, there was a significant increase in LV/BW at 10 days of pressure overload (SO 2.1 ± 0.1, AC 3.3 ± 0.1; P < 0.05). LV NE content was not significantly altered after 10 days of pressure overload (SO 635 ± 60, AC 521 ± 86 ng/g tissue). In contrast, LV Epi content was significantly increased in AC rats (SO 487 ± 96, AC 860 ± 110 ng/g tissue; P < 0.05).

Radioligand binding assays for the AT1 receptor were performed in LV membranes prepared from AC and SO rats after 10 days of pressure overload. Saturation isotherms demonstrated that specific binding of 125I-labeled [Sar1Ile8]ANG II in LV membranes was saturable and best described by a one-site model, with no differences in binding affinity between AC (2.2 ± 0.7 nmol/l) and SO rats (1.3 ± 0.1 nmol/l). However, the density of AT1 receptor sites in LV was increased fivefold in AC rats after 10 days of pressure overload (SO 8.7 ± 0.8, AC 42.2 ± 9 fmol/mg protein; P < 0.05).

In a separate group of rats subjected to 10 days of pressure overload, the contractile (+dP/dt) response to a single EC50 (10 nM) of isoproterenol was determined in the isolated, perfused heart (Fig. 4). There was a significant increase in LV/BW in AC compared with SO rats (SO 2.9 ± 0.1, AC 4.2 ± 0.2; P < 0.05). There was no difference between SO and AC rats in baseline +dP/dt (SO 2,772 ± 259, AC 2,729 ± 618 mmHg/s; P > 0.05). Isoproterenol increased contractility (% increase in +dP/dt) in hearts from both SO and AC rats; however, the contractile response to isoproterenol was significantly reduced (by 35%) in AC compared with SO rats (Fig. 4).


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  		Fig. 4.   The effect of isoproterenol on cardiac contractility in isolated, perfused hearts from 10-day AC and SO rats. The isolated, perfused heart from 10-day AC and SO rats was prepared as described in MATERIALS AND METHODS. A single concentration (10 nM) of isoproterenol was added to the perfusate, and the positive 1st derivative of LV pressure (+dP/dt) was determined. Isoproterenol increased contractility in hearts from both SO and AC rats; however, the % increase in contractility was significantly decreased (by 35%) in AC compared with SO rats. Data are means ± SE (n = 3 rats/group).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrate that neurohumoral activation of the renin-angiotensin system and the sympathetic nervous system are elicited in a temporally defined sequence in response to pressure overload (Fig. 5). Initially on pressure overload, there is transient activation of the systemic renin-angiotensin system coinciding with the appearance of LV hypertrophy, followed by a marked increase in AT1 receptor density in the LV, generalized increases in sympathetic nervous system activity, and a decline in the response to inotropic challenge. At 60 days of pressure overload, alterations in the affinity of the beta 2-adrenergic receptor site for CGP-20712A were demonstrated in the LV. Despite these initial alterations, there was no decline in resting LV function, as defined by echocardiography, the density of beta -adrenergic receptor sites, or the relative distribution of beta 1- and beta 2-receptor sites in the LV over 60 days of pressure overload. Thus activation of the systemic renin-angiotensin system is an early response to aortic constriction and may initiate development of cardiac hypertrophy and sympathetic activation. In contrast, activation of the sympathetic nervous system appears to underlie the progression and maintenance of cardiac hypertrophy in response to pressure overload. Rapid activation of these neurohumoral systems and the development of cardiac hypertrophy result in compensated heart function at rest but rapid declines in the responsiveness to inotropic challenge.


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  		Fig. 5.   The temporal sequence for activation of the renin-angiotensin system and the sympathetic nervous system in cardiac hypertrophy from pressure overload. Increases in plasma ANG II occur early after suprarenal aortic constriction and are not maintained. LV hypertrophy (LVH) is evident within 3 days of pressure overload, coinciding with transient elevations in plasma ANG II. By day 10, plasma NE concentration is elevated, AT1 receptor density and epinephrine (Epi) content are increased in the ventricle, and LVH is maintained. Through day 60, LVH and elevations in plasma NE are maintained, but fractional shortening and ventricular beta -receptor density are normal. On day 60, the affinity of the beta 2-receptor in the ventricle is decreased. RBF, renal blood flow.

In general, our results are in agreement with findings from previous studies examining the time course for cardiac hypertrophy and function in response to pressure overload. In this study, pressure overload induced by suprarenal abdominal aortic constriction increased MAP proximal to the site of aortic constriction. The magnitude of the pressure increase was sufficient to produce sustained LV and left atrial hypertrophy within a 3-day time frame. In agreement with these findings, an increase in LV/BW was demonstrated within 3 days of aortic constriction (39). In other studies, an increase in ventricular RNA content was detected after 3 days of aortic constriction (14); however, LV-to-BW ratios were not significantly increased until day 14, suggesting that increases in ventricular protein synthesis precede the development of cardiac hypertrophy. Echocardiographic analysis was consistent with postmortem measurements in this study demonstrating an increase in AWT with normal chamber dimensions in AC rats after 3-60 days of pressure overload. Previous investigators demonstrated that pressure overload is initially characterized by the development of concentric LV hypertrophy with compensated LV contractile performance (29, 31). Our results demonstrate that hypertrophy of the LV within 3 days of aortic constriction compensates to maintain baseline cardiac function over an 8-wk time frame of pressure overload.

Previous studies demonstrated that subdiaphragmatic aortic constriction resulted in a rapid elevation in plasma renin activity, which returned to control values during the chronic phase of pressure overload (2). We measured plasma ANG II concentration after pressure overload to determine the status of the systemic renin-angiotensin system. In both groups of rats, plasma ANG II concentration was elevated at day 3 compared with levels at days 10-60, suggesting that the stress of surgery initially activated the renin-angiotensin system. However, at 3 days of pressure overload, plasma ANG II concentration was elevated in AC compared with SO rats. Our results extend previous findings by demonstrating an increase in plasma ANG II at 3 days after suprarenal abdominal aortic constriction. Plasma ANG II concentrations in AC and SO rats beyond day 3 were not different and were in agreement with previously published values in the rat (38). Acute increases in plasma ANG II after suprarenal aortic constriction in the present study are consistent with increases in the secretion of renin from the juxtaglomerular cells of the kidney in response to the initial reduction in renal perfusion pressure (17). Brilla et al. (5) reported a normal plasma ANG II concentration in rats subjected to infrarenal aortic constriction from 1 to 8 wk. In contrast, plasma ANG II concentration increased within 1 wk and remained elevated for 8 wk after suprarenal aortic banding with constriction of the right renal artery and subsequent atrophy of the right kidney. Thus our results of a transient increase in plasma ANG II concentration after pressure overload induced by suprarenal aortic constriction without coexisting renal atrophy demonstrate that differences in plasma ANG II concentration between various studies using the pressure-overload model of aortic constriction most likely result from variability in the placement of the vascular constriction in relation to the renal arteries.

In agreement with previous results (35), a modest density of AT1 receptor sites was observed in the LV of SO control rats. After 10 days of pressure overload, a marked increase (5-fold) in AT1 receptor density was observed in the LV. In an aortocaval shunt model of volume overload with associated cardiac hypertrophy, an increase in AT1 receptor density was previously demonstrated (23). Together, these results demonstrate an increase in cardiac AT1 receptor density in cardiac hypertrophy resulting from volume or pressure overload. Thus alterations in neurohumoral mediators including systemic and/or cardiac ANG II may contribute to hypertrophy independent of the hemodynamic stress associated with elevated blood pressure. In support of this, Heller et al. (20) demonstrated a positive correlation between plasma or LV renin concentration and the degree of cardiac hypertrophy at 3 days of pressure overload but not at 42 days of pressure overload, despite sustained elevations in systolic pressure. In contrast to results from our study, Lopez et al. (30) reported a reduction in cardiac AT1 receptor density after 8 wk of pressure overload. Moreover, in AT1A receptor knockout mice subjected to abdominal aortic constriction for 2 (19) or 3 (18) wk, LV hypertrophy was unabated, demonstrating that hypertrophy can develop independent of AT1 receptor stimulation. We suggest that elimination of one of these neurohumoral systems, such as in the AT1A receptor knockout, is compensated by other neurohumoral mediators capable of initiating LV hypertrophy and the maintenance of cardiac function. In this study, pressure overload resulted in temporally defined activation of the renin-angiotensin system and the sympathetic nervous system, despite consistently elevated systolic pressure. Moreover, our results of a marked increase in cardiac AT1 receptor density at 10 days of pressure overload are consistent with a direct growth-promoting effect of ANG II contributing to the early increase in cardiac mass.

In addition to the neurohumoral influences of ANG II on cardiac mass, previous investigators demonstrated significant coronary vascular and myocardial lesions as early as 1 wk after pressure overload induced by aortic constriction (36). Previous studies suggested that coronary vascular and myocardial lesions may be related to neurohumoral factors induced by aortic constriction. For example, increases in plasma ANG II concentration were suggested to contribute to cardiomyocyte necrosis and coronary vascular damage (41). In the animal model of chronic ANG II infusion, widespread multifocal areas of myocyte necrosis were observed within 2 days and were accompanied by significant cellular infiltration (24). These effects of ANG II on myocardial necrosis were prevented by the administration of the AT1 receptor antagonist losartan. The potential role of ANG II-mediated myocardial necrosis is consistent with findings from this study demonstrating an increase in cardiac AT1 receptor density early after pressure overload. Moreover, consistent with results from this study, ANG II-mediated myocyte necrosis has been specifically linked to interactions with cardiac sympathetic neurons (21).

We measured plasma NE concentration to determine the status of the sympathetic nervous system after pressure overload. Plasma NE concentration of SO rats was similar to reported values in anesthetized rats (3, 27). Siri (39) reported a progressive increase in plasma NE concentration in AC rats that reached statistical significance within 7 days. Our results extend these findings by demonstrating increased plasma NE concentration after 10 days of pressure overload that remained elevated over 60 days. Interestingly, acute elevations in circulating ANG II were demonstrated to cause increases in plasma NE concentration (10, 25, 40). In the present study, increases in plasma ANG II preceded elevated plasma NE concentration. Given the interrelationships between these two systems, these results are consistent with observations suggesting that initial increases in plasma ANG II stimulate the noradrenergic nerve terminals of the sympathetic nervous system and raise plasma NE concentration. We suggest that the time course for increases in plasma ANG II and NE support the activation of the systemic renin-angiotensin system as the initial mechanism for elevations in plasma NE, whereas increased activity in tissue renin-angiotensin systems or other neurohumoral mediators may contribute to sustained elevations in plasma NE during prolonged periods of pressure overload (11, 37).

Previous investigators demonstrated that aortic constriction increased (1-7 days) plasma Epi concentration (39). This is the first study to demonstrate elevations in cardiac Epi content after pressure overload. Cardiac Epi content is derived from adrenal-released Epi taken up from the plasma (26). Future studies must be undertaken to determine whether aortic constriction results in stimulation of the release of Epi from the adrenal medulla or, alternatively, enhanced cardiac uptake of Epi. Interestingly, evidence demonstrates that Epi can facilitate NE release from sympathetic nerve terminals through actions at presynaptic beta 2-adrenergic receptors (1). Thus increases in cardiac Epi content after pressure overload may contribute to elevations in cardiac sympathetic neurotransmission. A limitation of the present study is the possible influence of anesthetic on plasma NE and Epi concentrations, which may differ between hypertensive and normotensive rats.

Despite sustained increases in circulating NE concentration from 10 to 60 days of pressure overload, the density of beta -adrenergic receptor sites in the LV was not altered over 60 days of pressure overload. These results are in agreement with previous studies demonstrating that cardiac beta -adrenergic receptor density was not altered at 3 (14) and 4 (7) wk of pressure overload. In addition, our results extend these findings by demonstrating that the relative beta 1- to beta 2-receptor subtype distribution was not altered after pressure overload. Despite normal beta -adrenergic receptor density and subtype distribution in the LV, the response to inotropic challenge with isoproterenol was reduced in the isolated, perfused heart from rats subjected to 10 days of pressure overload. These results are in agreement with previous studies demonstrating that despite normal baseline cardiac function, inotropic responsiveness is depressed in the aortic constriction model of pressure overload (13). Thus, rather than receptor downregulation, desensitization of the cardiac beta -adrenergic receptor may be an early response to elevations in systemic and cardiac sympathetic nerve activity.

An unexpected finding in this study was that the affinity of CGP-20712A for the beta 1- and beta 2-receptor sites in LV was shifted to a lower affinity in both groups of rats at 60 days. Moreover, the Ki2 for the beta 2-site was significantly increased (lower affinity) in ventricle membranes from 60-day AC rats compared with controls. This may be related to the increase in cardiac Epi, which is capable of acting on the beta 2-adrenergic receptor.

In conclusion, results from this study demonstrate that transient increases in plasma ANG II concentration precede elevations in the systemic concentration of NE after cardiac pressure overload. LV hypertrophy was evident within 3 days of pressure overload, coincident with increases in plasma ANG II and preceding sympathetic activation. At 10 days of pressure overload, marked increases in cardiac AT1 receptor density, elevated cardiac Epi content, and impaired responsiveness to inotropic challenge were evident. All of these changes occurred in the absence of detectable alterations in cardiac beta -adrenergic receptor density or declines in resting cardiac function. These results demonstrate that activation of the systemic (plasma ANG II) and cardiac (AT1 receptor density) renin-angiotensin system occur early in the development of LV hypertrophy. In contrast, the time course for sympathetic stimulation suggests that initial increases in ANG II may contribute to subsequent sympathetic stimulation and the continued maintenance and progression of LV hypertrophy.


    ACKNOWLEDGEMENTS

The technical expertise of Victoria King and Michael Fettinger contributed to the completion of these studies.


    FOOTNOTES

This research was supported by National Institutes of Health Grant 52934 and by an internal pilot research grant on the biology of aging (Dr. P. Wise).

Address for reprint requests and other correspondence: L. A. Cassis, Div. of Pharmaceutical Science, Coll. of Pharmacy, Univ. of Kentucky, Lexington, KY 40536-0082 (E-mail: lcassis{at}pop.uky.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 February 2000; accepted in final form 14 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(6):H2797-H2806
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