INTRODUCTION

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THE BARORECEPTOR-HEART RATE REFLEX is of major importance in the short-term regulation of blood pressure (BP). Its gain is decreased in several cardiovascular diseases and particularly in hypertension (8). Although it is known that reversal (7) or prevention (5, 10) of hypertension restores a normal baroreflex function, the precise mechanisms underlying its impairment are not completely understood. Hypertension and associated abnormalities (cardiac hypertrophy, humoral factors) may alter the baroreflex loop at different levels. High BP decreases the gain of the baroreceptor pressure-discharge curve in renal hypertensive rats (19). Humoral factors and especially ANG II can affect baroreflex at the central level (for review, see Ref. 26). Finally, cardiac hypertrophy can modulate the baroreflex gain at the efferent level through a BP-independent mechanism in spontaneously hypertensive rats (SHR) (18). But the scope of these data is limited by methodological considerations. Indeed, the pharmacological method used to assess baroreflex sensitivity in most of these studies may not exactly reflect the physiological baroreflex function because pharmacological intervention 1) usually produces BP variations larger than those occurring spontaneously and 2) can directly alter baroreflex function (13, 25). Several methods have been proposed to study the spontaneous baroreflex sensitivity (SBRS); among them, the most widely used are the sequences method (1) recently adapted in rats (20) and the cross-spectrum analysis (11, 27). We validated in rats the use of another method via a probabilistic approach to determine the SBRS from BP and heart rate (HR) time series without any pharmacological intervention (4, 6).

In Lyon genetically hypertensive rats (LH), previous data using pharmacological methods reported a decreased baroreflex gain (28) compared with their Lyon normotensive (LN) and low-BP (LL) controls, but no data are available concerning SBRS. There is evidence that these animals develop a hypertension that depends on the renin-angiotensin system (RAS) (14). Thus RAS manipulation provides a tool to modify BP levels and cardiac mass and offers the opportunity to gain insight into the mechanisms involved in baroreflex impairment. The present study aimed at determining the SBRS in LH, LN, and LL strains and its variations during angiotensin-converting enzyme (ACE) inhibition and ANG II infusion.

	MATERIALS AND METHODS

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Animals. Ninety-nine three-week-old male rats belonging to the three strains of the Lyon model were housed in controlled conditions (temperature 21 ± 1°C, lighting from 0800 to 2000) and received a standard rat chow (NA A03 UAR, Villemoisson-sur-Orge, France). Some of the rats belonged to a previously published study (14).

BP and HR recordings. Aortic BP was directly measured using a modification of our computerized technique (9). Briefly, two polyethylene catheters were inserted while the rats were under anesthesia (2% halothane in oxygen), one via the left femoral artery into the abdominal aorta and one via the left femoral vein into the inferior vena cava. Catheters were tunneled subcutaneously to the back of the neck and exteriorized. After 24 h of recovery, the arterial catheter was connected to a pressure transducer (Statham P-23 ID, Gould, Cleveland, OH) via a rotating swivel that allows the animals to move freely. Direct BP was digitized online, and beat-to-beat time series were stored by a computer (MVME SYS121, Motorola, Tempe, AZ) before off-line processing was performed using a workstation (Sparc station 1, Sun Microsystems, Mountain View, CA). Continuous BP recordings started 30 min after connection to the pressure transducer.

Cardiac mass measurement. At the end of each cardiovascular study, rats were killed by an overdose of pentobarbital sodium and the left ventricle was dissected out and weighed.

Computation of SBRS. The analysis of the statistical dependence between systolic BP (SBP) and HR was performed as previously reported (6). Briefly, SBP and HR values were taken as probabilistic events, and for each (SBP,HR) couple a coefficient of statistical dependence (Z) was defined as follows

(1)

where P(HR|SBP) represents the estimated conditional probability to observe the HR value if the SBP value is observed and P(HR) is the estimated probability to observe the HR value.

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Three-dimensional frequency histogram (A), Z histogram (B), and contour representation of Z histogram (C) obtained for systolic blood pressure (SBP) and heart rate (HR) couples of values in one Lyon genetically hypertensive (LH) rat. White axes in C define the 2 quarters (baroreflex) containing zones of dependence selected for calculation of spontaneous baroreflex sensitivity (SBRS), and modal class is figured by a black rectangle. Slope of regression line obtained for all these selected values estimates SBRS. bpm, Beats/min.

Effect of a chronic converting enzyme inhibition on SBRS. Starting at 3 wk of age, i.e., immediately after weaning, 11 rats of each strain were treated with the ACE inhibitor perindopril at the dose of 3 mg · kg¹ · 24 h¹ in drinking water (LH-P, LN-P, and LL-P). This treatment was maintained throughout the study (i.e., until 15 wk of age). A similar number of rats remained untreated (LH-C, LN-C, LL-C). At 15 wk of age, aortic BP and HR were measured in freely moving animals during a 1-h period.

Effect of a 4-wk ANG II infusion on the SBRS of perindopril-treated rats. Eleven animals of each strain were treated with perindopril as described in Effect of a chronic converting enzyme inhibition on SBRS. At the age of 12 wk, the rats were anesthetized with halothane (2% in oxygen) and osmotic minipumps (ALZET, 2ML4, ALZA corporation, Palo Alto, CA) were implanted subcutaneously and connected to a polyethylene catheter (PE-60 + PE-10) inserted into the jugular vein. ANG II (Ciba-Geigy, Basel, Switzerland) dissolved in saline was infused via the minipump at a constant rate of 200 ng · kg¹ · min¹ during 4 wk (LH-P-ANG II, LN-P-ANG II, and LL-P-ANG II). During the last week of infusion, aortic BP and HR were directly measured in freely moving animals during 3 h in basal conditions. At the end of this period, an acute intravenous injection of losartan (20 mg/kg) was given and BP was recorded during at least 1 h in stationary conditions (i.e., starting at least 15 min after injection).

Statistical analysis. Values are expressed as means ± SE. Between-strain comparisons within each treatment group used a one-way ANOVA followed by Fisher's test. Between-group comparisons within each strain used an unpaired t-test. Comparisons between losartan and baseline periods used a paired t-test. P < 0.05 was considered statistically significant.

	RESULTS

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Only data from the rats in which the Z analysis allowed determination of the SBRS were included in the results.

As shown in Fig. 2, high BP in LH-C rats was associated with an increased body and left ventricle weight, the latter difference being significant only compared with LL counterparts. Indeed, LN-C rats, which were characterized by a very low body weight, exhibited a higher left ventricle weight when it was corrected for body mass than LL-C despite similar BP levels. The Z analysis allowed reliable determination of the SBRS in 9 LH-C, 10 LN-C, and 7 LL-C rats. Figure 2 indicates that SBRS was lower in LH-C rats compared with LN-C (P < 0.05). The difference between LH-C and LL-C rats did not reach statistical significance (P = 0.06).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   SBP, HR, body weight (BW), index of cardiac mass (LVW/BW), and SBRS obtained in LH, Lyon normotensive (LN), and Lyon low blood pressure (LL) rats in control (C) conditions (LH-C, n = 9; LN-C, n = 10; and LL-C, n = 7), after chronic perindopril (P) treatment (LH-P, n = 11; LN-P, n = 9; and LL-P, n = 9) and after perindopril treatment associated with ANG II infusion (LH-P-ANG II, n = 9; LN-P-ANG II, n = 9; and LL-P-ANG II, n = 9). Values are means ± SE. * P < 0.05 vs. LN rats,  P < 0.05 vs. LL rats. LVW, left ventricular weight.

Perindopril prevented the development of hypertension in LH-P rats. Body weight was decreased in perindopril-treated rats but still differed among hypertensive and normotensive ones. The converting enzyme inhibitor also reversed cardiac hypertrophy, and left ventricle mass became even greater in LN-P than in LH-P rats. Perindopril significantly increased SBRS only in LH-P rats. Thus SBRS no longer differed among the three perindopril-treated strains.

The chronic ANG II infusion increased SBP in LH-P-ANG II and LL-P-ANG II rats but not in LN-P-ANG II rats. Body weight did not significantly differ between LH-P-ANG II and LL-P-ANG II but was lower in LN-P-ANG II rats. Cardiac hypertrophy was restored by ANG II infusion in LH-P-ANG II rats compared with both control strains. SBRS was significantly decreased by the ANG II infusion in LH-P-ANG II rats only and became significantly lower than the SBRS of both LL-P-ANG II and LN-P-ANG II. An acute injection of losartan decreased BP in the three strains (119 ± 7, 92 ± 3, and 108 ± 5 mmHg for SBP in LH-P-ANG II, LN-P-ANG II, and LL-P-ANG II, respectively). As shown in Fig. 3, losartan induced a significant increase in SBRS in LH-P-ANG II rats (from 1.1 ± 0.2 to 1.7 ± 0.1 beats · min¹ · mmHg¹) and did not affect SBRS in LL-P-ANG II rats (from 2.8 ± 0.3 to 2.7 ± 0.4 beats · min¹ · mmHg¹). In LN-P-ANG II animals, SBRS could be reliably determined only in five rats, which precluded any valuable statistical comparison.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   SBRS obtained in LH (n = 9) and LL (n = 9) rats treated with perindopril and infused with ANG II before and after acute infusion of losartan. * P < 0.05 vs. preceding period.

	DISCUSSION

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The original findings of the present experiment are that 1) the SBRS is decreased in LH and 2) this impairment is not due to high BP.

The cardiac branch of the arterial baroreflex is a major determinant of the short-term regulation of BP because of its ability to induce bradycardia or tachycardia in response to spontaneous BP variations. It has been reported that this reflex occurs during 25% of the time in conscious cats (1) and 15% of the time in man (24). We developed a method that allows identification among (SBP,HR) couples of those couples related to the baroreflex activity (4, 6), and thus we have been able to estimate its gain. Because no pharmacological intervention is required, this method allows us to describe the spontaneous baroreflex function in physiological conditions, provided that a large number of (SBP,HR) couples is available. SBRS could not be estimated in some rats when the set point of the BP regulation could not be accurately determined. This problem may occur in rats with a high cardiovascular instability, leading to a multimodal (SBP,HR) bivariate distribution. This can be observed after an acute perturbation of the cardiovascular system such as losartan injection, which induces a slow BP decrease. In this case, no clear set point exists and the baroreflex quarters determined with the Z analysis do not contain enough (SBP,HR) couples to significantly fit the regression line. This can be observed regardless of the SBRS level. The percentage of cardiac beats included in the SBRS calculation was ~15% and was highly reduced in case of cardiovascular instability. Therefore, the main limitation of the method is the necessity of the presence of a clear modal class in the (SBP,HR) bivariate distribution, which is usually obtained with 1-h recordings.

Using another nonpharmacological method, the method of sequences, Oosting et al. (21) recently demonstrated that the spontaneous baroreflex function was impaired in SHR compared with their Wistar-Kyoto controls. Our results are consistent with their report because, associated with their hypertension, SBRS was decreased in LH rats compared with both the LN and LL strains. Several mechanisms may account for this impairment: 1) cardiac hypertrophy, which has been shown to decrease the pharmacological baroreflex gain in SHR (10, 18); 2) high BP itself, which is able to decrease baroreflex sensitivity in nitro-L-arginine methyl ester (L-NAME)-induced hypertension (15) even in the absence of significant cardiac hypertrophy; and 3) a direct effect of humoral factors, e.g., ANG II, which can depress baroreflex function by a pressure-independent mechanism (3). The simple comparison of the SBRS in the three strains does not allow us to favor either hypothesis because both BP and cardiac mass were greater in LH-C than in controls even if, in consideration of the latter, the difference did not reach statistical significance compared with LN-C. It was probably because of the low body weight of LN-C animals, which is likely due to a decreased body fat, as suggested by a similar tibia length in LH and LN rats (29). This low body weight led to an overestimation of the relative organ mass.

Hypertension and related cardiovascular damages depend on the RAS in LH rats (14). Indeed, after a chronic treatment with perindopril, BP was no longer different among the three strains. Left ventricle mass was similar in LH-P and LL-P rats but significantly lower than in LN-P rats, confirming that the correction for body weight overestimated cardiac mass in the LN strain. The spontaneous baroreflex function was improved in LH rats, and, as a consequence, SBRS did not differ among the three strains. This result agrees with pharmacological estimations of the cardiac baroreflex sensitivity that also indicate a normalization of cardiac baroreflex function after treatment with ACE inhibitors (5). However, it is the first time, to our knowledge, that this effect can be confirmed without any pharmacological intervention in genetically hypertensive rats.

The use of an ANG II infusion in perindopril-treated animals provided three different situations of potential interest to determine the involvement of high BP and cardiac hypertrophy in baroreflex impairment apart from high ANG II levels: 1) high BP with cardiac hypertrophy in LH rats, 2) moderately high BP without cardiac hypertrophy in LL rats, and 3) low BP in LN rats. Because baroreflex function was impaired only in the LH strain, it can be concluded that SBRS does not depend directly on the BP level but rather on either cardiac hypertrophy or on the direct effect of ANG II on baroreflex components. If ANG II was the sole factor involved, then the SBRS should have been reduced as well in LN and LL rats, but this was not the case. One of the reasons could be a greater central effect of ANG II in hypertensive rats than in LN rats. There is evidence that the central modulation by ANG II is due to an action at the area postrema, a circumventricular organ located in the medulla oblongata accessible to circulating ANG II (23); this effect is mediated by the angiotensin AT₁ receptor subtype (30). A difference in the central response to ANG II between strains is plausible because it has also been disclosed at the vascular and kidney levels in the Lyon model (14, 16). To further address this question, an additional AT₁ blockade was performed in acute conditions. This treatment led to an improvement of SBRS in LH-P-ANG II rats but not to a restoration to the level of ACE-inhibited LH. This may have two explanations: 1) ANG II does not completely account for the decreased SBRS in LH-P-ANG II animals or 2) a longer duration of AT₁ blockade might have been required. Indeed, Brooks (3) has shown that resetting of the cardiac baroreflex after the withdrawal of a chronic ANG II infusion was rapid (within 30 min) whereas the gain remained decreased at that time. Malpas et al. (17) showed that 48 h are required to observe an improvement of the gain after the interruption of an ANG II infusion.

The alternative hypothesis implicates a predominant role of cardiac hypertrophy. Accordingly, the SBRS level fits in with the variations of cardiac mass after ACE inhibition and ANG II infusion in LH rats. Head and Minami (10, 18) have extensively studied this relation in SHR. They found that cardiac hypertrophy strongly decreased HR range and, consequently, baroreflex gain by modifying the curvature of the BP-HR sigmoidal relationship, a possible mechanism being a modification of cardiac receptor activity. There is evidence that cardiac receptors can modulate baroreflex function (22). Their activity could be altered in pathological situations characterized by changes in left ventricular end-diastolic pressure like congestive heart failure (2). It is conceivable that cardiac hypertrophy may also lead to a modulation of this ventricular mechanoreflex and, consequently, to baroreflex impairment. However, the hypothesis of a link between cardiac mass and baroreflex gain is not confirmed by experiments in pharmacologically induced hypertension, e.g., in ANG II-infused rabbits (17) or in L-NAME-induced hypertension in rats (15). This remark emphasizes the fact that, together with the different models of hypertension, there are probably different mechanisms involved in baroreflex impairment.

In conclusion, we showed, using a determination of SBRS, that 1) SBRS is altered in LH rats and 2) this alteration depends on the RAS because it can be prevented by ACE inhibition and reproduced by ANG II infusion. This probably occurs not because of high BP levels but more likely because of either cardiac hypertrophy or a direct effect of ANG II on the baroreflex loop.

Perspectives