INTRODUCTION

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IT HAS BEEN PROPOSED that the myocardial renin-angiotensin system (RAS) plays a pivotal and perhaps obligatory role in the initiation and progression of ventricular hypertrophy. Evidence favoring this hypothesis includes the observation that autocrine release of ANG II mediates stretch-induced hypertrophy of fetal cardiac myocytes (33). In both the adult rat heart and cardiac myocytes, stretch activates the early response gene c-fos and antagonists of the ANG II receptor largely attenuate the activation (21, 33). Furthermore, ANG II has been shown to be a potent stimulus of ventricular hypertrophy in vivo, and ANG II-mediated cardiac growth can occur independently of ANG II-mediated increased blood pressure (10, 28, 32, 35, 39). Angiotensin-converting enzyme (ACE) inhibition and ANG II receptor blockade have been shown to have a cardioprotective effect in vivo by preventing or attenuating ventricular hypertrophy (11, 13, 26, 27, 30, 34, 36, 37), even when circulating renin levels are initially depressed (9, 29). Blockade of the RAS also appears to be more effective in reducing human ventricular hypertrophy than other types of antihypertensive therapies (5, 13).

Despite substantial evidence supporting the hypothesis that myocardial RAS plays a pivotal role in both initiation and progression of ventricular hypertrophy, the myocardial RAS may not be the only regulator of ventricular hypertrophy. For instance, it has been pointed out that changes in cardiac preload alone do not necessarily induce cardiac growth signals, and, therefore, extrapolating results from cultured neonatal myocytes may be problematic (21). In one study, growth of contracting adult feline cardiomyocytes was not found to be dependent on activation of ANG II AT₁ receptor (42) and a recent study has shown that knockout mice devoid of AT_1A receptors can still develop left ventricular hypertrophy after aortic banding (17). In addition, not all studies have been able to demonstrate a direct ANG II-induced myocardial growth effect that was independent of changes in cardiac afterload (15). Finally, some studies have found that ACE-inhibition and ANG II receptor antagonists could not prevent or attenuate ventricular hypertrophy (14, 16, 38, 44).

These latter studies do not fully discredit the obligatory involvement of ANG II in regulation of ventricular hypertrophy for three reasons. First, blockade of the RAS was not performed before initiation of ventricular hypertrophy in these studies. Blockade of the myocardial RAS before initiation of ventricular hypertrophy is important to rule out the possibility that the RAS is involved in the initiation rather than the maintenance of ventricular hypertrophy. Second, many previous studies lack measurements of any RAS components in myocardial tissue. Direct measurement of myocardial RAS is especially important in determining the participation of the RAS in the etiology of ventricular hypertrophy, because plasma renin concentrations are often normal or even subnormal in many forms of ventricular hypertrophy and there is speculation that a locally independent RAS exists involving myocardial activation of renin and angiotensinogen gene expression (1, 19, 23-25, 45). Third, there is more than ample evidence to believe that ANG II is an important regulator of ventricular hypertrophy under many conditions.

Therefore, to determine if a local myocardial RAS is always necessary or sufficient to either induce or maintain ventricular hypertrophy in rats, we made measurements of local myocardial renin and angiotensinogen during certain specialized conditions of low plasma renin concentration (DOCA-salt hypertension) and high plasma renin concentration (low-sodium diet) and assessed changes in myocardial mass. In the case of the DOCA-salt hypertension, some rats were pretreated with the ANG II AT₁-receptor antagonist losartan to eliminate the possibility that a local myocardial RAS might be involved in the initiation of ventricular hypertrophy despite falling plasma renin levels.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals and animal protocols. Adult Sprague-Dawley rats were used for these studies. The procedures followed were in accordance with institutional guidelines and the guidelines of the American Association for the Accreditation of Laboratory Animal Care. Separate protocols were used in this study to provide rats with low plasma renin concentrations (with and another without ANG II AT₁-receptor blockade) and rats with relatively high plasma renin concentrations. The following groups were studied: short-term DOCA-treated rats with or without losartan (n = 7, both groups), short-term sham DOCA-treated rats with or without losartan (n = 6, both groups), long-term DOCA-treated rats with (n = 7) or without (n = 8) losartan, long-term sham DOCA-treated rats with (n = 7) or without (n = 9) losartan, short-term low-sodium diet-fed rats and sham-treated controls (n = 5 both groups), and long-term low-sodium diet-fed rats and sham-treated controls (n = 8, both groups).

DOCA-treated rats and sham DOCA-treated rats without losartan. Rats receiving free access to food (Purina Rodent Diet 5001 with sodium content = 0.4%) and water were sedated with xylazine (6 mg/kg ip) and anesthetized with ketamine (30 mg/kg ip). A unilateral nephrectomy was performed via a left flank incision. A Silastic pellet containing 100 mg DOCA was implanted subcutaneously. Rats were allowed tap water for ~24 h after surgery and then switched to water containing 1% NaCl for the duration of the experiment. Sham treatment in weight-matched animals included all surgical procedures, except excision of the kidney and included implantation of a blank pellet and no addition of NaCl to their drinking water. Animals were killed either 7-8 days after surgery (short-term group) or between 35 and 42 days after surgery (long-term group). Blood pressures (tail cuff method) of restrained conscious rats were assessed weekly or on the day of death.

DOCA-treated rats and sham DOCA-treated rats with losartan. The protocol followed for this model was identical to the protocol described above, with the exception that the AT₁-receptor antagonist losartan was present in the drinking water for the entire length of the protocol starting 2 days before surgery and DOCA pellet implantation. Water was changed at least once every 3 days, and losartan concentrations were adjusted to achieve an intake of ~30 mg · kg¹ · day¹ based on the measured water intakes of animals in each group. Weight-matched DOCA-treated and sham DOCA-treated rats were also given losartan before and after the sham surgery. The effects of these interventions in the sham-treated and DOCA-treated rats were assessed over a short term (8 days) and a long term (35 days). Rats were not weight matched between losartan and no losartan treatments.

The efficacy of the losartan treatment was assessed in a separate group of four rats (2 sham treated, 2 DOCA treated) that had been treated with losartan in their drinking water for 14 days. Arterial pressure was measured in anesthetized rats (pentobarbital sodium 35 mg/kg ip) via a carotid artery cannula. Bolus injections (100 µl) of ANG II in increasing doses from 10 nM to 10 µM were given through the same cannula, and the transient pressor responses were measured and compared with those of rats (n = 5) that had not received losartan.

Low-salt diet-fed rats (no losartan). A moderate sodium-restricted diet was implemented (Harlan Teklad Sodium-Deficient Diet with sodium content ~0.04%) in rats for either a short term (7 days) or a long term (21 days). Weight-matched untreated rats received the regular chow with ~0.4% sodium and served as controls for these groups. Free access to water was provided.

Collection of plasma and cardiac samples for determination of renin and angiotensinogen. All animals were quietly resting and sedated by introduction of carbon dioxide into a closed chamber, followed by rapid decapitation. 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 centrifuged 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 or saline (0 and 10°C) in retrograde fashion at a constant flow rate via the aortic stump for 0.5-2.0 min to remove all residual coronary blood. Thus plasma renin and angiotensinogen did not contaminate myocardial renin and angiotensinogen measurements.

After perfusion, the pericardial tissue, great vessels, and atria were removed, and the hearts were weighed followed by removal of the right ventricular free wall. The left ventricle 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. The other ventricular sections were snap-frozen in liquid nitrogen and stored at 25°C for later analysis.

Ventricular tissue homogenization. Frozen left ventricular samples were homogenized at 0°C with a handheld 7-ml Kontes glass homogenizer in a proteolytic inhibitor buffer (PIB) at a ratio of 1 mg tissue to 5 µl buffer. PIB contained serine-, metallo-, and thiol-protease inhibitors dissolved in a 0.15 M sodium phosphate buffer (pH 7.5) with 1% BSA. The proteolytic inhibitors (followed by their respective final concentrations) were (in mM) 15 EDTA, 2 8-hydroxyquinoline, 10 sodium tetrathionate, 20 benzamidine, 10 N-ethylmaleimide, 3 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), and 10 µM leupeptin and 450 µM aprotinin. Homogenates were split into three aliquots and stored at 25°C. Myocardial homogenates were thawed, centrifuged at 14,000 g for 4 min at 4°C, and the resulting supernatants were assayed for active renin and angiotensinogen.

Renin concentration measurement. Somewhat similar renin concentration measurement methodology has been previously described (18, 22). Plasma was diluted 20-fold in PIB to yield renin concentrations similar to myocardial homogenates. Plasma and homogenate samples were, therefore, diluted in the same solvent (PIB) and assayed identically. Duplicate assay tubes containing either 80 µl of a PIB-diluted plasma sample or a sample containing 80 µl of myocardial homogenate in PIB or 80 µl of PIB without a sample (sample blanks) were combined with 150 µl additional PIB and 75 µl of renin substrate prepared from the plasma of rats that had been bilaterally nephrectomized for 48 h. The assay tubes containing 80 µl of PIB without a sample (sample blanks) provide a correction for the small amount of renin contamination present in the renin substrate (angiotensinogen source). However, to remove as much renin as possible from the renin substrate, the 48-h nephrectomized plasma was pretreated with washed pepstatin-A coupled to 4% agrose beads (Sigma) and incubated for 1 h at pH 4.2 in the presence of 500 mM NaCl. Pepstatin beads bound with residual renin were then removed by centrifugation, and the supernatant was titrated to pH 7.5 with 6 M NaOH.

Assay tubes containing samples of plasma, myocardial homogenate, or sample blanks (PIB alone) combined with additional PIB and the pretreated renin substrate, were pipetted into two 100-µl aliquots. One 100-µl aliquot was incubated at 0°C, and the other 100-µl aliquot was incubated at 37°C, both for 18-24 h. Detectable ANG I in the 0°C tube (endogenous immunoreactive ANG I in sample and substrate source) was subtracted from the 37°C tube to calculate a generated ANG I value, although 0°C tube ANG I concentrations were often undetectable. ANG I differences from duplicate assay tubes were averaged. Each assay run contained at least one duplicate pair of sample blanks. The 37°C tube ANG I value minus the 0°C tube ANG I value from the sample blank assay tubes was subtracted from the corresponding ANG I differences of all samples to correct for the small renin contamination of the renin substrate. Sample blank differences ranged from 5 to 33% of the total sample ANG I difference (the 37°C tube ANG I value minus the 0°C tube ANG I value for samples), the higher proportions occurring when very low renin myocardium samples from DOCA-treated animals 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. Renin concentration was expressed as nanograms ANG I per milliliter plasma per hour or nanograms ANG I per gram myocardium per hour.

ANG I was assayed by radioimmunoassay using a modified Du Pont ANG I RIA kit (Wilmington, DE) (18, 22).

Angiotensinogen concentration measurement. Angiotensinogen concentration measurement methodology has been previously documented (18, 22). Briefly, angiotensinogen concentrations from both myocardial homogenate and plasma samples were measured by adding a large excess of exogenous porcine renin to the samples and converting all of the sample angiotensinogen into ANG I. The generation of ANG I after porcine renin addition quickly reaches a plateau after all of the sample angiotensinogen is converted to ANG I (18, 22). (ANG I degradation is prevented by the PIB.) Plateau ANG I concentrations were subsequently determined by RIA for ANG I (18, 22). The ANG I concentration values (µM/l of plasma or µM/kg myocardium) were equal to the corresponding angiotensinogen concentrations, because 1 mol of angiotensinogen is converted to 1 mol of ANG I by renin.

Statistical analysis. Data are reported as means ± SE. Data were evaluated in three separate sets: 1) short-term DOCA-treated rats with or without losartan and their respective sham-treated controls, 2) long-term DOCA-treated rats with or without losartan and their respective sham-treated controls, and 3) short- and long-term low-sodium diet-fed rats and their respective sham-treated controls. Each set of data was initially analyzed by two-way ANOVA. Differences between groups within the set were further assessed by Dunnett's test. (Plasma and myocardial renin values were log transformed before analysis in the DOCA data sets due to the orders of magnitude differences between groups.) All significant differences in heart weight-to-body weight ratios were also significant for corresponding left ventricular weight-to-body weight ratios. In addition, linear regression was used to assess the relationship between plasma renin concentration and myocardial renin concentration and between plasma angiotensinogen concentration and myocardial angiotensinogen concentration. Significant differences were declared at P  0.05.

	RESULTS

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Losartan efficacy. Chronic (14 day) losartan treatment (30 mg · kg¹ · day¹ via the drinking water) shifted the injected ANG II-blood pressure response curve ~100-fold to the right compared with that of control animals (Fig. 1). Thus the dosing conditions used in this experiment effectively antagonized the AT₁-receptor-mediated pressor responses to ANG II.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chronic (14 day) losartan treatment (30 mg · kg1 · day1 via drinking water) shifted injected ANG II-blood pressure response curve ~100-fold to right (dotted line, n = 4) compared with that of animals not receiving losartan (solid line, n = 5). Mean change in blood pressure is plotted for each of 4 separate doses of ANG II via bolus injection.

Short-term DOCA treatment without and with losartan. Short-term (7 days after surgery) DOCA treatment had no significant effect on blood pressure of the rats, as shown in Table 1. However, short-term treatment of sham-treated rats (animals not receiving combined DOCA, uninephrectomy, and 1% NaCl) with losartan induced a significant decrease in blood pressure compared with sham-treated rats without losartan. Short-term DOCA-treated rats given losartan did not experience a decrease in blood pressure (Table 1).

Plasma and myocardial renin concentrations (PRC and MRC, respectively) of the short-term-treated rats are also shown in Table 1. DOCA treatment significantly decreased PRC and MRC in both the absence and presence of losartan. Losartan alone resulted in dramatic increases in both PRC and MRC levels.

The plasma and myocardial angiotensinogen concentrations (PAC and MAC, respectively) were not influenced by the short-term DOCA treatment, as shown in Table 1, but they were decreased by the losartan exposure. This reduction was counteracted by the DOCA treatment of the rats receiving losartan.

The effect of short-term DOCA treatment in the absence and presence of losartan on the various weight determinations is also shown in Table 1. Over the 7 days, DOCA-treated rats either lost weight or grew more slowly than their respective sham-treated controls. Treatment with losartan prevented this loss in body weight and promoted weight gain in the sham-treated group. Water content of the hearts from short-term DOCA-treated rats (78 ± 0.6%) was not significantly different from the short-term sham-treated rats (77.5 ± 0.3%). Heart weights of the various groups over the short term did not vary significantly, and the observed increase in heart-to-body weight ratio of the short-term DOCA-treated rats reflects the decrease in body weight.

Thus, over short-term reduction in plasma and myocardial renin concentrations in the DOCA-treated groups, there was no significant decrease in absolute heart weights. Even though blood pressure did not rise over short-term DOCA treatment, some signal was present that maintained heart weight at a time when body weight decreased. The presence of a similar pattern in the losartan-treated group also indicates that the RAS is not involved in the maintenance of heart weight in these groups.

Long-term DOCA treatment without and with losartan. Long-term (35-42 days after surgery) DOCA treatment was associated with a significant increase in arterial pressure, which was partially attenuated by the presence of losartan, as is shown in Table 2. Blood pressures of the long-term sham-treated rats with losartan were not significantly different from those of sham-treated rats not exposed to losartan. This finding differs from that observed in the short-term situation.

The plasma and myocardial renin concentrations of the long-term-treated rats are also shown in Table 2. As with the short-term experiments, DOCA treatment significantly decreased both the plasma and myocardial renin concentrations in the absence of losartan. DOCA treatment also inhibited the increase in both plasma and myocardial renin concentration caused by losartan. The plasma angiotensinogen concentration was significantly reduced by the long-term DOCA treatment and by the long-term losartan exposure, as is shown in Table 2, but DOCA treatment had no further effect on plasma or myocardial angiotensinogen concentrations in the rats exposed to losartan.

The effect of long-term DOCA treatment in the absence and presence of losartan on the various weight determinations are also shown in Table 2. DOCA-treated rats were significantly smaller than the respective sham-treated rats. Because the initial weight of the losartan group of rats was smaller than that of the group not treated with losartan (data not shown), they were also somewhat smaller at the end of the experiment. However, the growth rates (as indicated by the changes in body weight in Table 2) of the losartan groups were somewhat greater than those of the rats without losartan, consistent with the short-term experiments. Water content of the hearts from the DOCA group (78.0 ± 0.1%) was again not significantly different from that of the sham-treated group (77.8 ± 0.1%). Because the absolute heart weights of the DOCA-treated rats in each group are significantly greater than those of their respective sham-treated controls despite a loss of body weight and no change in cardiac water content, the significant increase in heart-to-body weight ratios of the DOCA-treated rats reflects true cardiac hypertrophy.

Thus long-term reduction in plasma and myocardial renin concentrations did not decrease myocardial mass in the presence of DOCA-salt-induced hypertension. In fact, the presence of significant cardiac hypertrophy despite the very low PRC and MRC and the presence of losartan indicates that the RAS is not involved in the initiation or maintenance of cardiac hypertrophy in this model.

Short- and long-term sodium-restricted diet. As shown in Table 3, both short- and long-term dietary sodium restriction produced significant increases in PRC and MRC but did not influence angiotensinogen concentrations. Whereas sodium restriction resulted in a significant slowing of growth of the animals, it also resulted in a decrease in heart weight that was significant in the animals with long-term sodium restriction. No difference was seen in the cardiac water content between the long-term sodium-restricted (75.0 ± 0.4%) and the control (77.1 ± 0.6%) animals. There were no significant increases in the heart-to-body weight ratio, and the small increases in the low-sodium diet-fed groups reflect the decrease in body weight and not cardiac hypertrophy. Sodium-restricted animals were somewhat hyperexcitable, such that reliable measurements of blood pressure could not be obtained.

Thus short- and long-term elevation in plasma and myocardial renin concentrations induced by dietary sodium restriction did not induce an increase in cardiac mass. In fact, in this situation of long-term sodium restriction, cardiac mass actually decreased.

Relationships between myocardial and plasma values of renin and angiotensinogen. Figure 2A shows the relationship between PRC and the corresponding MRC for all rats used in this study. Linear regression analysis showed the overall relationship to be highly significant with r² = 0.95 over three to four orders of magnitude (note log scale). Figure 2B shows the relationship between PAC and the corresponding MAC for all rats. Overall regression analysis indicated a high degree of significance with r² = 0.66. Seventy-five percent of the separate animal groups were significant (P  0.05) for the plasma renin vs. myocardial renin regression, 50% of the separate groups were significant (P  0.05) for the plasma angiotensinogen vs. myocardial angiotensinogen regression.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Myocardial renin vs. plasma renin for all animals in study (P < <0.01, r2 = 0.95; A, note log scale). Regression analysis for each group (P value; r2): long-term-treated animals: without losartan, sham (0.002; 0.78); without losartan, DOCA (0.16; 0.3); with losartan, sham (0.05; 0.57); with losartan, DOCA (0.002; 0.87); low salt, sham (0.05; 0.43); low salt (0.14; 0.32). Short-term-treated animals: without losartan, sham (0.02; 0.77); without losartan, DOCA (0.03; 0.63); with losartan, sham (0.001; 0.95); with losartan, DOCA (0.05; 0.62); low salt, sham (0.1; 0.6); low salt (0.7; 0.06). Also shown are myocardial angiotensinogen vs. plasma angiotensinogen values for all animals in study (P < <0.01, r2 = 0.66; B). Regression analysis for each group (P value; r2): long-term-treated animals: without losartan, sham (0.009; 0.65); without losartan, DOCA (0.004; 0.8); with losartan, sham (0.09; 0.46); with losartan, DOCA (0.02; 0.67); low salt, sham (0.0002; 0.91); low salt (0.02; 0.43). Short-term-treated animals: without losartan, sham (0.55; 0.09); without losartan, DOCA (0.2; 0.3); with losartan, sham (0.04; 0.69); with losartan, DOCA (0.5; 0.12); low salt, sham (0.11; 0.62); low salt (0.35; 0.29).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

There are three main conclusions from this study. The first conclusion is that the myocardial RAS is not always necessary to initiate or maintain cardiac hypertrophy. The second conclusion is that the myocardial RAS is not always sufficient to induce cardiac hypertrophy. The third conclusion is that renin and angiotensinogen found in the myocardium directly correspond to their respective plasma values. This may be the result of both renin and angiotensinogen being in equilibrium between the myocardium and plasma. The present study focuses on special situations involving the RAS and the development of ventricular hypertrophy and is not indicative of all situations resulting in ventricular hypertrophy. The main purpose of the present study is to show that in certain situations, the RAS is neither necessary nor sufficient to initiate or maintain cardiac hypertrophy, despite the fact that in many other instances, the RAS is a critical cardiac growth factor (5, 9-11, 13, 21, 26-30, 32-37, 39).

Data supporting the first conclusion that the myocardial RAS is not always necessary to induce or maintain cardiac hypertrophy include the observations that DOCA treatment resulted in initiation and progression of cardiac hypertrophy despite lower than normal MRC and that the AT₁-receptor antagonist losartan did not interfere with either the initiation or the maintenance of DOCA-induced cardiac hypertrophy. These results are in general agreement with other studies showing a failure of AT₁-receptor blockade or ACE inhibition to attenuate mineralocorticoid salt-induced cardiac hypertrophy (16, 44). However, other studies indicate that RAS blockade can attenuate cardiac hypertrophy in low plasma renin states (9, 29), consistent with the possibility of local myocardial RAS mediation of cardiac hypertrophy. In the present study, measurement of myocardial renin and angiotensinogen (after removal of coronary blood), showed significantly reduced renin and normal or reduced angiotensinogen in the heart during initiation and maintenance of mineralocorticoid salt-induced cardiac hypertrophy.

DOCA treatment both without and with concurrent losartan exposure reduced the rats' gain in body weight. Such inhibition of overall growth can confuse the interpretation of a change in the ratio of heart to body weight. However, in this study, cardiac mass was consistently greater in the DOCA-treated rats than in their sham-treated controls. Thus the hypertrophy was present both in absolute terms as well as in relative terms of increased heart-to-body weight ratio. Furthermore, because the water content of the hearts of DOCA-treated rats was not different from that of the sham-treated rats, the cardiac weight gain was not due to cardiac edema secondary to DOCA treatment, although both DOCA- and sham-treated rats may have experienced some increase in cardiac weight gain due to the lack of oncotic pressure in the retrograde saline perfusion. The mechanism(s) responsible for cardiac hypertrophy in the DOCA model is not well understood. The increased mechanical load resulting from the increased blood pressure may itself evoke hypertrophic responses independent of any humoral or local RAS factors. It is also possible that DOCA may directly cause cardiac hypertrophy, independent of the RAS. For instance, aldosterone causes increased myocardial fibrosis (43) possibly mediated through increased intracellular calcium in cardiac myofibroblasts (31).

Even though losartan had no effect on DOCA-induced cardiac hypertrophy, it did appear to partially lower blood pressure in the long-term DOCA-treated rats (from 176 ± 7 to 128 ± 9 mmHg). A possible explanation for the antihypertensive effect of losartan in low renin DOCA-treated rats is that the area postrema has been implicated in the mediation of some of the blood pressure actions of ANG II. Fink et al. (12) showed that ablation of the area postrema did not allow development of DOCA-induced hypertension and, more recently, Collister and Osborn (4) demonstrated that area postrema-lesioned rats exhibited an attenuated response to the hypotensive effects of losartan. Therefore losartan-mediated lowered blood pressure in DOCA-treated rats may have been due to losartan effects on the area postrema. Interestingly, acute treatment with losartan was reported to have no effect on blood pressure in DOCA-treated rats, whereas acute treatment with the ACE inhibitor captopril did lower blood pressure via kininase II inhibition and subsequent increased kinin levels (3).

The second main conclusion of this study that the myocardial RAS is not sufficient to induce cardiac hypertrophy is based on the observation that, although sodium restriction increased myocardial renin concentration, it had no effect on cardiac mass and did not induce cardiac hypertrophy. Buttrick et al. (2) also demonstrated that a very low salt intake resulted in high plasma renin activity without cardiac hypertrophy, although myocardial renin was not measured. Furthermore, in that study, the animals on the low-sodium diet experienced a weight loss that might have influenced the development of cardiac hypertrophy. In the present study, the moderate sodium restriction did allow weight gain in the rats, although less than that observed in rats fed the control diet. Swoap et al. (40) showed that 50% calorie restriction significantly blunted left ventricular hypertrophy in DOCA-treated rats, but the calorie restriction was apparently severe enough to also prevent weight gain.

The third major finding in this study is that myocardial renin and myocardial angiotensinogen are significantly and highly correlated with corresponding plasma values. It has been previously reported that after bilateral nephrectomy, plasma and cardiac renin levels fall (6, 22) and plasma and cardiac angiotensinogen values increase (6, 22). These previous studies indicate that most myocardial renin and angiotensinogen may be derived from the plasma compartment. In this report, this same general relationship between plasma and myocardial renin and angiotensinogen was also found to hold over very wide ranges of renin and angiotensinogen in situations other than bilateral nephrectomy. The linear relationship between plasma and myocardial renin was quite striking (r² = 0.95 for all groups combined), and, when considered with both the previously mentioned bilateral nephrectomy data and a report showing a strong correlation between plasma and myocardial renin (7) in failing human hearts, indicates that plasma renin of renal origin distributes within the myocardium in proportion to plasma renin concentration. Given the very high correlation between plasma and myocardial angiotensinogen (r² = 0.66 for all groups combined), plasma angiotensinogen also distributes within the myocardium in proportion to its plasma concentration; somewhat similar findings were previously reported (22, 24). These observations (Fig. 2) do not necessarily preclude the possibility of local activation of either myocardial renin or angiotensinogen gene expression (1, 23, 24) or of cardiac tissue binding of renin (7, 8).

Details of the plasma and myocardial renin and angiotensinogen assays employed in this report have been recently published (18, 22). We were very careful to remove all blood from the hearts before myocardial renin and angiotensinogen measurements were made, thus eliminating plasma renin and angiotensinogen contamination of the myocardial measurements. We were also very careful to ensure that the renin activity measurements were not partly due to other similar proteases, such as cathepsin D. For instance, the ANG I generation step was performed at pH 7.5, with >1% BSA in the ANG I generation tubes. These conditions inhibit cathepsin D activity (41). In our assay, porcine cathepsin D activity is inhibited over 97% when assayed at pH 7.5 vs. 4.5. In a previous study using similar assay methodology, the isoelectric focusing profile of rat myocardial renin matched the focusing profile of rat plasma renin and did not match the focusing profile of rat cathepsin D (22). A combination of serine-, metallo-, and thiol-protease inhibitors was employed during ANG I generation, preventing interference by many other nonspecific proteases. Additionally, myocardial renin activity was tightly correlated with plasma renin, an unlikely result for a nonrenin myocardial enzyme.

A possible deficiency in this study is that myocardial ANG II and AT₁ receptors were not measured. Although it seems unlikely that myocardial ANG II levels were elevated when myocardial renin and angiotensinogen levels were reduced, it is possible that AT₁ receptors were upregulated and helped mediate DOCA-induced cardiac hypertrophy. However, the use of losartan to block myocardial ANG II makes it especially unlikely that ANG II was responsible for ventricular hypertrophy in the DOCA-treated rats. The dose of losartan used (30 mg · kg¹ · day¹) should have been more than sufficient to block cardiac hypertrophy if DOCA-induced hypertrophy were mediated by the cardiac or systemic RAS. For instance, at doses between 3 and 30 mg · kg¹ · day¹, losartan significantly blocked or reduced cardiac hypertrophy in other rat models of cardiac hypertrophy, including aortic coarctation (11), the Dahl salt-sensitive rat (9), the transgenic rat, TGR(mRen2)27 (37), after myocardial infarction (34), and in the spontaneous hypertensive rat (36).

In summary, this study demonstrates that the myocardial RAS is not always necessary nor sufficient to initiate or maintain cardiac hypertrophy and that under the various experimental conditions of the present report, both myocardial renin and angiotensinogen concentrations are in an apparent equilibrium with corresponding plasma concentrations.

Perspectives