INTRODUCTION

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IN ADDITION TO THE mechanical influences of high cardiac afterload, numerous humoral factors may contribute to the development of the cardiac hypertrophy that accompanies chronic hypertension (33). Recent studies have shown that activation of the renin-angiotensin system (RAS) and formation of angiotensin (ANG) II within the heart may contribute to cardiac hypertrophy and ventricular remodeling (5, 7, 10, 14, 15, 27, 33). Treatment with ANG-converting enzyme (ACE) inhibitors (1, 4, 17, 18, 22) or ANG II receptor blockers (4, 8) is often reported to prevent or reverse cardiac hypertrophy to a greater extent than would result from reduction in arterial pressure alone. Because plasma renin concentrations are often normal or even subnormal in many forms of hypertension or chronic cardiac overload, there is speculation that a locally independent myocardial RAS exists (7, 14, 16, 27) and that local myocardial renin and angiotensinogen gene expression may be activated under certain conditions (1, 2, 15, 16). However, it is not clear whether this local system is powerful enough to have significant anatomic and physiological effects (32).

To gain insight into the possible role of alterations in local myocardial RAS in a model of chronic pressure-overload hypertrophy, we examined the relationship between circulating and myocardial renin and angiotensinogen in rats after subdiaphragmatic aortic constriction. Such a constriction in rats produces an abrupt elevation in cardiac afterload and a rapid cardiac hypertrophic response that are both sustained as long as the constriction is in place (4, 8, 17, 18, 21, 22, 28, 30). Plasma renin activity (PRA) tends to be elevated for at most a few days after the constriction and then returns to control values during the chronic phase of this model (1, 4, 20). However, left ventricular hypertrophy is reported to be attenuated by blockade of the RAS during both the early and the chronic phases of aortic constriction (1, 4, 8, 17, 18). Thus, in the absence of increased systemic RAS activity, local alterations in various components of a myocardial RAS may still be playing important roles in the initiation or maintenance of cardiac hypertrophy.

Therefore, we focused on two specific components of the RAS and tested the hypothesis that myocardial hypertrophy induced by aortic constriction is initiated and/or sustained by local myocardial alterations resulting in increases in myocardial renin or angiotensinogen concentration. Renin and angiotensinogen in the plasma and myocardium of rats subjected to aortic constrictions were measured at 3 or 42 days after surgery, and the ratios of plasma to myocardial levels were determined and compared with those of sham-treated rats. A decrease in this ratio for either substance was taken as support for the above hypothesis. Such a decrease could result either from an increased distribution of these substances within the myocardium, perhaps as a consequence of the myocardial tissue remodeling that accompanies left ventricular hypertrophy (3, 19, 22, 33), or from an increased local production of these substances, perhaps as a consequence of activated myocardial gene expression (1, 15, 16).

In addition, we attempted to determine whether the renin measured in the myocardial tissue was located within the intracellular or extracellular compartment. To this end, renin concentration of isolated cardiac myocyte preparations from hearts of sham and aortic-constricted rats was assessed. An absence of renin in the isolated myocytes was taken as evidence of its being restricted to the extracellular compartment. With the use of the data from the above experiments, an estimation of the ANG I generation rate in the interstitial space was made.

	METHODS

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Adult female Sprague-Dawley rats weighing 175-200 g were used for these studies. Free access to food (Purina Rodent Diet 5001 with sodium content = 0.4%) and water was permitted throughout the experiment.

Aortic constriction procedure. After sedation with xylazine (6 mg/kg ip) and anesthesia with ketamine (30 mg/kg ip), a midline incision was made and a segment of the aorta below the diaphragm and above the renal arteries was exposed. The shaft of a blunted hypodermic needle was laid alongside the vessel, and a ligature of 3-0 silk was passed under the needle and the aorta was tightly tied. (A 21-gauge needle was used for animals that were to survive 3 days and either a 21- or 20-gauge needle was used for those that were to survive for 6-8 wk. The size difference was designed to compensate for the animal's growth in the longer-duration experiments.) The needle was then slipped out of the loop, and the resulting aortic constriction was visually verified. Abdominal muscle layers were individually sutured, and the skin incision was closed with wound clips. Sham treatment consisted of all the above procedures with the exception of placing the ligature.

Duration of the experiments and assessment of hemodynamic load. Animals used for renin and angiotensinogen measurements were killed either 3 or 42 days after aortic constriction or sham surgery. Because reports describing the early changes in plasma renin after aortic constriction indicate a transient change, with peak values occurring within 7 days, and because anesthesia and sham surgery itself will raise plasma renin levels, we chose to wait 3 days to allow the effects of anesthesia and surgery to disappear while still making measurements during the peak transient period (1, 4, 20). At 42 days after the surgery, rats have compensated for the increased cardiac load and the cardiac hypertrophy has stabilized. Only healthy-appearing rats with no signs of congestive failure or any obvious discomfort were used at either time period. Cardiac hypertrophy was determined by assessing the left ventricular weight, the total heart weight, the heart-to-body weight ratio, and in some experiments the cardiac water content. Data from the rats with aortic constrictions were compared with those of sham-treated rats.

Because the methods to measure carotid arterial pressure in anesthetized rats evoke transient elevations in plasma renin, it was not possible to directly assess the hemodynamic load on the heart and the resting plasma renin level in the same group of rats. At 3 days after the aortic constriction, the presence of significant cardiac hypertrophy was taken as evidence of an early increase in cardiac afterload. The sustained long-term presence of increased cardiac load after the aortic constriction was verified in a separate experiment performed on rats with similar aortic constrictions (n = 7) and sham surgeries (n = 12). At 8 wk after surgery, these rats were anesthetized (Inactin, 100 mg/kg ip) and arterial pressure was measured directly by carotid artery cannulation.

Sample collection procedures for the renin and angiotensinogen assessments. On the day of the experiment, heparin (1,000 U/kg ip) was injected 20-30 min before the rats were killed. Quietly resting animals were sedated by introduction of carbon dioxide into the chamber and then rapidly decapitated. Blood draining from the severed neck vessels was collected in 1.5-ml tubes containing 40 µl of 5% EDTA, mixed and stored on ice until centrifugation at 9,000 g for 3 min at 4°C. Separated plasma was snap-frozen in liquid nitrogen and stored at 25°C for later analysis.

Hearts were rapidly removed and perfused with a modified Krebs-Henseleit solution in retrograde fashion at a constant flow rate via the aortic stump for 1.5-2.0 min to remove all residual blood. The perfusate was maintained at ~10°C and contained (in mM) 118 NaCl, 4.7 KCl, 25 NaHCO₃, 3.0 CaCl₂, 1.2 MgSO₄, 0.5 Na-EDTA, and 10 glucose. In addition, the perfusate contained heparin, 100 U/l, and insulin, 10 U/l, and was bubbled with 95% O₂ + 5% CO₂. Flow rates were adjusted to set the perfusion pressure at ~85 mmHg for the sham-treated rats and ~95 mmHg for aortic-constricted rats.

After removal from the perfusion apparatus, the atria were trimmed off and the right ventricular free wall was separated from the left ventricle, which was then cut longitudinally into two sections. All tissues were kept on iced watch glasses while being trimmed. After blotting, right and left ventricular samples were both rapidly weighed and, in some experiments, a portion of the left ventricle was placed in a drying rack for determination of the wet-to-dry weight ratio (cardiac water content). The other ventricular sections were snap-frozen in liquid nitrogen and stored at 25°C for later analysis.

Bilateral nephrectomy. To determine baseline reference values of renin in the myocardium in rats with essentially no renal-derived plasma renin, a separate group (n = 6) of rats was anesthetized with pentobarbital sodium (50 mg/kg ip) and both kidneys were removed. Two days after bilateral nephrectomy (BNX) the animals were killed, and the hearts and plasma were prepared essentially as described above.

Myocyte preparation. Myocytes were prepared according to previously reported methods (31). Briefly, hearts from a separate group of 3-day aortic-constricted (n = 5) or 3-day sham-treated rats (n = 5) were rapidly removed from the decapitated rat and perfused via the aortic stump at 40 mmHg for 5 min without recirculation with a calcium-free physiological salt solution (modified Joklik's solution). This was followed by a 20-min recirculating perfusion with the same solution containing 0.07% collagenase (Worthington, type 2). Hearts were then removed from the perfusion apparatus, minced, and incubated for an additional 10 min in the collagenase solution. The slurry was then strained through cheesecloth, diluted with collagenase-free solution, and allowed to settle for 10 min at room temperature. The supernatant was removed, and the cells were rinsed and repelleted four times in the collagenase-free solution containing 1.0-1.5% bovine serum albumin (radioimmunoassay grade; Sigma). After viability testing during the fourth rinse (93.7 ± 1.1% live cells), the cells were spun down at 1,000 g and the cell pellet was frozen and stored at 20°C for later analysis. Average recovered sample mass was 0.181 ± 0.02 g/heart.

Left ventricular tissue homogenization. Frozen left ventricular samples or myocytes were homogenized at 0°C with a hand-held 7-ml Kontes glass homogenizer in a proteolytic inhibitor buffer (PIB) at a ratio of 1 mg tissue to 5 µl buffer. Myocytes were also sonicated for 5 s. PIB contained serine-, metallo-, and thiol-protease inhibitors in a 0.15 M sodium phosphate buffer with EDTA (15 mM), 8-hydroxyquinoline (2 mM), sodium tetrathionate (10 mM), benzamidine (20 mM), N-ethylmaleimide (10 mM), 4-(2-aminoethyl)-benzenesolfonylfluoride hydrochloride (PEFABLOC) (1 mM), leupeptin (10 µM), aprotinin (450 µM), and bovine serum albumin (1.0%). Homogenates were split into three aliquots and stored at 25°C. Myocardial homogenates were thawed and centrifuged at 14,000 g for 4 min at 4°C, and the resulting supernatants were assayed for active renin, angiotensinogen, and, in some cases, for active renin glycoforms.

Renin measurement. Plasma was diluted 20-fold in PIB to yield renin concentrations similar to myocardial homogenates (13). Plasma samples and homogenates were therefore diluted in the same solvent (PIB) and assayed identically as follows. Duplicate assay tubes containing 80 µl of PIB-diluted plasma, myocardial or myocyte homogenate in PIB, or PIB (sample blanks) were combined with 150 µl additional PIB and 75 µl of unfractionated plasma from rats that had been bilaterally nephrectomized 48 h before (providing an angiotensinogen source). PIB therefore accounted for ~70-75% of the volume of each assay tube. For each duplicate, one 100-µl aliquot was incubated at 0°C and another 10-µl aliquot was incubated at 37°C, both for 18-24 h. Detectable average ANG I in the 0°C tubes (endogenous immunoreactive ANG I in sample and substrate source) was subtracted from the 37°C tube average to calculate generated ANG I, although 0°C tube ANG I concentrations were often undetectable. Results from duplicates were averaged. The corresponding differences in the sample blank tubes were also subtracted from the generated ANG I value to correct for endogenous renin in the substrate source. Sample blanks ranged from 4 to 40% of the generated ANG I, the higher proportions occurring when 48-h BNX myocardium or myocytes were assayed. During the 37°C incubation, ANG I was generated in direct proportion to the sample renin concentration. Only 4% or less of the total available angiotensinogen was converted to ANG I, and linear generation of ANG I over time was verified throughout the study (11, 23). Thus the enzymatic activity measured from the average of duplicate 37°C tubes, after 0°C tube and sample blank corrections, was directly proportional to renin concentration. Renin concentration was expressed as nanogram ANG I per milliliter plasma per hour, nanogram ANG I per gram myocardium per hour, or nanogram ANG I per gram myocytes per hour incubation.

All remaining myocyte supernatants not previously assayed for myocyte renin concentration were used to measure myocyte membrane renin concentration. Supernatants were further centrifuged at 100,000 g at 4°C, and the pellet (membrane fraction) was resuspended in 1 ml PIB, sonicated with three 5-s cycles, recentrifuged at 100,000 g at 4°C, and the washed pellet (membrane fraction) was resuspended in 230 µl of PIB and 75 µl of unfractionated BNX plasma (angiotensinogen source) both with and without 1% Triton X-100 to determine ANG I generation from myocyte membranes.

ANG I was assayed by radioimmunoassay using a modified DuPont ANG I radioimmunoassay kit (Wilmington, DE). Major modifications included preparation of ANG I standards with the same solute contents as samples. Radioimmunoassay data were linearized with a log-logit transformation, resulting in a correlation coefficient of 0.99 or better. The average labeled ANG I bound 55-60% to the primary antibody without competition. The average percent of error for predicting the standard concentration from the transformed curve was <6%.

Angiotensinogen measurement. Angiotensinogen concentrations from left ventricular myocardial homogenate supernatants and plasma were measured by adding a large excess of exogenous porcine renin, thereby converting all the angiotensinogen present to ANG I (verified by ANG I generation plateau). ANG I concentrations were subsequently determined by radioimmunoassay and converted to angiotensinogen concentrations based on a 1:1 molar relationship between ANG I generation and angiotensinogen depletion. Plasma was diluted 70-fold in PIB to yield comparable angiotensinogen concentrations to myocardial homogenates. Plasma samples and homogenates were therefore diluted in the same solvent (PIB) and assayed identically as follows. Seven milliunits of Porcine renin (Sigma, St. Louis, MO) in 325 µl PIB was added to 25 µl diluted plasma or myocardial homogenate at 0°C and then incubated in duplicate at 37°C for 20 min; another set of duplicates was incubated for 40 min. Care was taken to ensure that the samples were not warmed prior to the incubation step. Appropriate combinations of porcine renin and PIB, plasma and PIB, homogenates and PIB, or PIB alone yielded no detectable ANG I. In previous work (13) the average percent difference between the ANG I concentration determined from the 20- and 40-min time points was 0.36 and 4.04% for plasma and myocardial homogenates, respectively, indicating that an ANG I generation plateau had been reached by 20 min and that ANG I is relatively stable in the assay.

Myocardial versus plasma ANG I generation rate. An approximation of the myocardial interstitial space ANG I generation rate allows an estimation of the myocardial ANG I generation rate that can then be compared with the plasma ANG I generation rate. Approximate interstitial myocardial renin and angiotensinogen concentrations were derived from the average myocardial renin and angiotensinogen values measured from 3-day sham and aortic-constricted rats and on the assumption that all measured myocardial renin and angiotensinogen was confined to an interstitial space of 25 ml/100 g with a density of ~1 g/ml (25, 26, 29). Therefore, measured control myocardial renin and angiotensinogen average values (originally expressed per g of whole heart tissue) were multiplied by four to estimate their respective concentrations per milliliter of interstitial volume. These approximate interstitial concentrations were then combined in vitro, and the resultant ANG I generation rate was measured. Measured plasma renin and angiotensinogen concentrations were also combined in vitro, and the corresponding plasma ANG I generation rate was measured. Porcine renin was used as the renin source, and 48 BNX plasma served as the angiotensinogen source in these experiments. In vitro generation rates were measured over 10 min at 37°C in the presence of 50-85% PIB at pH 7.4.

Shallow gradient isoelectric focusing of renin glycoforms. In one rat from each group, active renin was resolved into five major glycoforms (I-V) using shallow gradient isoelectric focusing (SIEF). The methods were similar to those previously used (12, 13, 24). Six hundred microliters of plasma was incubated with 15 mg SiO₂ (to remove lipid and fibrin) at 20°C for 30 min followed by centrifugation at 100,000 g at 4°C for 30 min. Approximately 20 µl of the plasma supernatants diluted in 180 µl SIEF upper-reservoir anolyte (0.05 histidine) were applied to the focusing gels. Twelve hundred microliters of ventricular homogenate was incubated with 30 mg SiO₂ at 20°C for 30 min followed by centrifugation at 100,000 g at 4°C for 30 min. The homogenate supernatant was then dialyzed [molecular weight cutoff (MWCO) 25,000] against the SIEF upper-reservoir anolyte (0.05 histidine) at 4°C for 18 h 30 min, transferred to a second dialysis container (MWCO = 6,000-8,000) and dialyzed against solid polyethylene glycol to concentrate the sample approximately fourfold. The concentrated sample was again centrifuged at 100,000 g at 4°C for 30 min, and 200 µl of the homogenate sample was applied to the focusing gels.

Statistical analysis. Data are reported as means ± SE. Comparisons between data obtained from sham and aortic-constricted animals within and between groups were performed using Student's t-tests, linear regression, and analysis of variance. Statistical significance was assumed at P < 0.05 (2 tailed).

	RESULTS

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Characteristics of rats with short- and long-term aortic constriction. After 3 days of elevated afterload, left ventricular weight and total heart weight as well as left ventricular-to-body weight and heart-to-body weight ratios of the rats with aortic constrictions (n = 9) were significantly elevated above those of the sham-treated rats (n = 7) (Table 1). However, right ventricular weights and right ventricular-to-body weight ratios of the 3-day aortic-constricted rats were not significantly different from those of the sham-treated rats. Left ventricular water content, as assessed by the wet-to-dry wt ratio of the 3-day aortic-constricted rats (79.7 ± 0.04%) was not different from that of the sham-treated rats (79.5 ± 0.04%). The same pattern of hypertrophic growth was still evident 42 days after the aortic constriction (n = 7) compared with the sham-treated rats (n = 6).

The carotid arterial pressure of a separate group of rats with aortic constrictions of 8-wk duration (193 ± 9 mmHg, n = 7) was significantly (P < 0.05) higher than that of rats receiving sham-operations 8 wk before (139 ± 6 mmHg, n = 12). Although the body weight of the aortic-constricted rats (259 ± 9 g) was not different from that of the sham-operated rats (260 ± 5 g), the total heart weight and the heart-to-body weight ratio of these aortic-constricted rats (1,389 ± 92 mg and 5.40 ± 0.41 mg/g, respectively) were also significantly greater than those of the sham-operated rats (800 ± 21 mg and 3.07 ± 0.05 mg/g, respectively). These data verify the sustained presence of elevated hemodynamic load on the heart imposed by long-term aortic constriction and the continued presence of cardiac hypertrophy.

Renin and angiotensinogen levels in rats with short- and long-term aortic constriction. Three days after aortic constriction, neither plasma nor myocardial renin concentration were significantly different from sham controls (P = 0.2 and 0.1, respectively), and the ratio of plasma to myocardial renin was nearly identical to the sham controls (Table 2). There were significant correlations between plasma renin and heart-to-body weight ratios (r = 0.49, P = 0.05) and between left ventricular renin and heart-to-body weight ratios (r = 0.65, P = 0.006) for all rats at 3 days, and between left ventricular renin and heart-to-body weight ratios for 3-day aortic-constricted rats (r = 0.8, P = 0.005). Plasma and myocardial angiotensinogen of the aortic-constricted rats were also not significantly different from sham controls (P = 0.3 for both), and again the ratio of plasma to myocardial angiotensinogen was nearly identical (Table 2).

Forty-two days after aortic constriction, plasma and myocardial renin concentrations were not significantly different from the corresponding sham controls, and the ratio of plasma to myocardial renin was similar in both groups and not statistically different from either 3-day group. Plasma and myocardial angiotensinogen concentrations as well as the ratio of plasma to myocardial angiotensinogen of the 42-day aortic-constricted group were not different from the 42-day sham controls (Table 2). There were no significant correlations between plasma or left ventricular renin concentrations and heart-to-body weight ratios days after the surgery.

Figure 1 shows an example of myocardial and plasma active renin glycoform profiles for a 42-day sham and aortic-constricted animal used to help confirm that the reninlike enzymatic activity measured in the myocardium was indeed renin. Five major rat renin glycoforms (I-V) were clearly evident in both plasma and myocardial samples. The glycoforms focused at previously published isoelectric points [pH(I)] of 5.7, 5.4, 5.2, 5.0, and 4.8. Yields were ~100%.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Isoelectric focusing of active renin glycoform profiles of plasma (A and B) and left ventricle (C and D) homogenates in a sham (A and C) and aortic-constricted (AC; B and D) animal. , Representative pH gradient for focusing gels (A). Renin concentration units are ng ANG I · ml elution buffer for each gel slice1 · 24 h incubation1.

Renin determination after 48 h BNX. Left ventricles from 48 h BNX rats (n = 6) were assayed for renin concentration to determine the contribution of renal sources to myocardial renin and to establish a baseline reference for cardiac-derived renin. Plasma renin concentration in the 48-h anephric rats fell ~98% to 0.07 ± 0.003 ng ANG I · ml¹ · h¹ compared with ~4 ng ANG I · ml¹ · h¹ for both sham groups obtained from the experiments reported in Table 2. Myocardial renin concentration after nephrectomy fell ~90% to 0.11 ± 0.03 ng ANG I · g myocardium¹ · h¹, compared with ~1 ng ANG I · g myocardium¹ · h¹ for these same sham groups (Table 2 and Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Renin concentration measured from left ventricular homogenates (ng ANG I · g myocardial wall1 · h1) from 3- and 42-day post sham (open bars) or AC (hatched bars) animals and from bilateral nephrectomy (BNX) (filled bar) animals. Cardiac myocyte renin concentration (ng ANG I · g myocytes1 · h1) prepared from 3-day post sham (open bars) or aortic constriction (hatched bars) animals are shown on far right. Both BNX and cardiac myocyte renin concentration were significantly less (* P < 0.0001) than the myocardial renin concentration (ng ANG I · g myocardial wall1 · h1) measured from left ventricular homogenates.

Renin determination in isolated cardiac myocytes. Cardiac myocytes were prepared from rats 3 days after aortic constriction or sham surgery (n = 5/group) to determine whether significant intracellular renin concentration could be detected. The myocytes were washed four times, and no renin was detectable in the final wash. Although there was detectable reninlike enzymatic activity in both sham and 3-day aortic-constricted myocytes (0.22 ± 0.05 and 0.19 ± 0.07 ng ANG I · g myocytes¹ · h¹, respectively), the levels were far lower than concentrations obtained in myocardial tissue of either sham or aortic-constricted rats used for experiments (Fig. 2). No renin was detected in the washed myocyte membrane fraction with or without Triton X-100.

Myocyte reninlike concentration was approximately equal to the 48-h BNX myocardium sample, especially after accounting for the fact that myocyte renin concentration is expressed per gram of myocytes and not per gram of myocardium, the latter of which includes extracellular space. The reninlike concentrations measured in the left ventricle tissue 48 h after nephrectomy and in the isolated cardiac myocytes from the sham and 3-day aortic-constricted rats were too low to apply to gels to determine whether the enzymatic activity was due to renin or to reninlike enzymatic activity such as cathepsin D.

In vitro incubation of renin and angiotensinogen at the calculated interstitial concentrations (see METHODS) as well as at the measured plasma concentrations was evaluated in three separate assays, each performed in duplicate. The estimated myocardial interstitial fluid ANG I generation in 3-day sham animals was 3.1 ± 0.2 ng ANG I · ml¹ · h¹ and was approximately 24% of the corresponding plasma ANG I generation of 12.91 ± 0.5 ng ANG I · ml¹ · h¹. The estimated myocardial interstitial fluid ANG I generation in 3-day aortic-constricted animals was 5.3 ± 0.07 ng ANG I · ml¹ · h¹ and was ~27% of the corresponding plasma ANG I generation of 19.4 ± 0.8 ng ANG I · ml¹ · h¹.

	DISCUSSION

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The data presented here do not support the hypothesis that myocardial hypertrophy induced by aortic constriction is either initiated or sustained by local myocardial alterations resulting in increases in renin or angiotensinogen within the myocardium. Instead, it appears more likely that cardiac renin and angiotensinogen may participate in a diffusional steady state between the myocardial interstitium and plasma during both the acute (3 day) and chronic (42 day) phases of cardiac overload. This conclusion is based primarily upon five observations. 1) The constancy of the plasma-to-left ventricle concentration ratio of both renin and angiotensinogen during the sham and aortic-constricted treatments (Table 2) suggests that a diffusional balance exists for these molecules between the plasma and the myocardium. 2) Myocyte renin concentration was not elevated in the aortic-constricted group, indicating that there was not an increased local production of renin within the myocyte during the initiation of left ventricle hypertrophy. 3) Two-day BNX of normal animals reduced left ventricular renin concentrations by ~90% (Fig. 2). This is consistent with a renal origin of most myocardial renin as opposed to local production. 4) Because myocardial wall renin measurements were performed after removal of all coronary vessel blood and myocyte renin-like enzymatic activity levels were quite low compared with myocardial wall renin, most myocardial renin probably resides within the interstitium. 5) Left ventricular renin concentrations were always ~25% of the plasma levels. This is consistent with renin distribution in an interstitial volume of ~25 ml/100 g wet wt, which agrees with previously determined myocardial interstitial values of 18-25 ml/100 g wet wt (25, 26, 29). Left ventricular angiotensinogen concentrations were ~4% of plasma, consistent with both a smaller volume of myocardial distribution compared with renin due to the larger size of angiotensinogen and the very probable destruction of myocardial angiotensinogen by myocardial renin (13).

In one study, 12% of myocardial renin was found to be in the membrane fraction of cardiac homogenates (6). However, in the present study, we were unable to detect renin in washed membranes prepared from myocytes taken from either 3-day sham or aortic-constricted hearts. If renin was present bound to membranes of myocytes or nonmyocyte cells, 12% of additional myocardial renin would not cause the renin plasma-to-left ventricular homogenate ratio to be significantly altered from the results of this study.

In the present study, we compared the generation rate of myocardial interstitial ANG I to the plasma generation rate. Despite the relatively small amount of myocardial angiotensinogen found in this study, the estimated myocardial interstitial fluid ANG I generation was between 24 and 27% of the corresponding plasma ANG I generation. Many assumptions must be made to actually compare the two ANG I generation rates. Given that ACE may be present in the myocardial interstitium (6) and that generation of ANG II in the immediate vicinity of its receptor is potentially more likely to result in receptor occupancy than plasma-generated ANG I or ANG II, both sites of ANG I generation are probably important.

We attempted to rule out the possibility that a portion of the measured myocardial renin concentration was not due to renin and instead may have been due to reninlike enzymatic activity, such as cathepsin D activity. This possibility appears unlikely for four reasons. 1) Unfractionated BNX plasma was used as an angiotensinogen source at pH 7.5 during ANG I generation, conditions that inhibit cathepsin D activity (32). In our assay conditions cathepsin D activity is over 97% inhibited when assayed at pH 7.5 versus pH 4.5. 2) The isoelectric focusing profile of myocardial renin concentration matched the focusing profile of plasma renin and did not match the focusing profile of rat cathepsin D [rat cathepsin D isoelectric points range from 6.0 to 6.2 in a focusing system that focuses rat renin between 5.35 and 5.65 (9)]. 3) A combination of serine-, metallo-, and thiol-protease inhibitors was employed during ANG I generation, preventing interference by many other nonspecific proteases. 4) Myocardial renin concentration fell after BNX, an unlikely result for a nonrenin myocardial enzyme. However, despite these precautions, it is possible that some or all of the reninlike enzymatic activity measured in either 2-day BNX myocardium or in cardiac myocytes was not due to renin. This is because reninlike activity from these samples was too low to be confirmed as renin by isoelectric profiling, and its presence after 2 days BNX is consistent with cathepsin D activity. A more detailed analysis of myocardial renin after BNX was recently published (13).

Focus and limitations of the study. The presence and role of a locally derived myocardial RAS is somewhat controversial (32). Myocardial gene expression of all constituents of the RAS has been demonstrated (15) and myocardial renin gene expression has been reported to increase secondary to volume overload (cardiac stretch) (2). Furthermore, changes in ACE or in ANG II receptors during cardiac hypertrophy may contribute to an increase in concentration of ANG II or in its efficacy. In the present study we focused on measurement of only two constituents of the RAS (renin and angiotensinogen) and the conclusion that locally initiated myocardial alterations in their levels do not occur does not preclude the possibility that ANG II receptors or local generation of ANG II may have been regulated in a cardiac tissue-specific manner. The primary reason we chose to measure renin and angiotensinogen rather than ANG I or ANG II is that, during sample collection, it is less likely that these larger molecules will diffuse out of the myocardium or be destroyed or produced to the same extent as ANG I or ANG II. Furthermore, demonstration of the presence of mRNA for renin or angiotensinogen does not allow quantization of the actual amounts of these proteins with the myocardium. It is clear, however, that all components involved in each step of the cascade need to be examined to fully assess the participation of a myocardial RAS during left ventricular hypertrophy.

We conclude from this study that, in aortic-constricted rats, 1) myocardial and plasma renin appear to be in a diffusional steady state during both acute and chronic phases of this aortic-constricted chronic pressure-overload model, 2) there is no evidence in this model of either increased local distribution (relative to plasma) or local production of renin or angiotensinogen within the hypertrophied myocardium or its myocytes and, 3) myocardial renin may be confined to the interstitium, and interstitial generation of ANG I within the myocardium may significantly contribute to total myocardial ANG I.

Perspectives