The analysis of heart rate variability (HRV), blood pressure variability (BPV), and baroreflex sensitivity (BRS) has become a powerful tool for assessing autonomic control. HRV, BPV, and BRS measurements have proved to be independent predictors of sudden cardiac death after acute myocardial infarction, chronic heart failure, or dilated cardiomyopathy.1-4 However, the underlying regulatory mechanisms still are poorly understood.
The renin-angiotensin system (RAS) plays a critical role in cardiac and blood pressure control. Besides angiotensin (Ang) II, other Ang peptides, such as Ang IV [Ang-(3-8)] and Ang-(1-7), also have important biological activities. Ang-(1-7) especially has become an angiotensin of interest because its cardiovascular actions counteract those of Ang II5 and it attenuates the development of heart failure.6 Peripherally, Ang-(1-7) itself produces a potent antidiuretic effect on water-loaded rats7 and potentiates bradykinin.8 Centrally, Ang-(1-7) produces cardiovascular responses when microinjected into the dorsomedial9,10 or ventrolateral medulla,11,12 and intracerebroventricular (ICV) infusion of Ang-(1-7) increases the sensitivity of the baroreceptor reflex (baroreflex bradycardia) in normotensive Wistar rats, in contrast to the decreased sensitivity produced by Ang II or Ang III infusion.13
It is well known that angiotensin converting enzyme (ACE) inhibition decreases the production of Ang II and increases the circulating concentration of Ang-(1-7) in spontaneously hypertensive rats (SHR)14 and humans.15 The Ang-(1-7) buildup after ACE inhibition may result from the accumulation of Ang I or a decrease in its inactivation, or both, because ACE is an important route for the metabolism of Ang-(1-7).16 It was shown that central infusion of the Ang-(1-7) antagonist A779 produces a significant reversal of the improvement of the baroreflex sensitivity induced in SHR by a treatment with ramiprilat.17
Recently we demonstrated that Ang-(1-7) is a functional ligand for the G protein-coupled receptor Mas.18 This receptor is encoded by the Mas protooncogene that was detected first as a result of its tumorigenic activity in in vivo tumor assays.19 High Mas messenger ribonucleic acid (mRNA) expression was shown in testis and found in certain areas of the brain such as the hippocampus, dentate gyrus, piriform cortex, and amygdala.20,21 Low concentrations were observed in the kidney and heart.21,22 It is important to note that Mas-deficient mice are defined by gender-specific alterations of the heart rate and blood pressure variability.23-25
To further elucidate the distinct role of the Ang-(1-7)/Mas axis in blood pressure control of awake animals under physiologic conditions, SHR were chronically treated with the first orally applicable nonpeptide Ang-(1-7) receptor agonist AVE0991.26 Cardiovascular parameters were recorded by radiotelemetry. The HRV and BPV variabilities and spontaneous baroreflex (BR) were analyzed using established methods.27-32
MATERIALS AND METHODS
Ten untreated male SHR (10 months old) and 6 age-matched SHR treated with AVE0991 (20 mg/kg per day; via chow), reaching plasma concentrations of that compound from 0.5 to 0.9 μM, received a telemetry implant (TA11-PA20; DSI, USA) in the aorta and were examined 5 weeks after the start of the treatment. All rats were housed in individual cages in light cycles (8 AM-8 PM) and were allowed access ad libitum to water and a normal diet. The investigation is in agreement with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985).
Analysis of Heart Rate and Blood Pressure Variability
The beat-to-beat values of diastolic and systolic pressure and the beat-to-beat intervals (heart rate) were extracted from the continuous blood pressure signal by pattern-matching cross-correlation analysis. Because arrhythmias and artefacts have a strong influence on the parameters of the BPV and HRV analysis, they were excluded from the time series of variability. This exclusion was made by an adaptive filter algorithm, which takes into account the momentary basic variability.33 The values remaining in the time series are denoted NN-intervals (normal-to-normal intervals). From the resulting 10 minutes of HRV and BPV series, different measures have been calculated with respect to the HRV task force standards.34 From the time domain, the mean deviation, standard deviation, and Shannon entropy of the histogram were determined (Table 1). By means of the Fast Fourier Transform and a Blackman-Harris window, the following frequency domain parameters were calculated from the time series interpolated to 50 ms: the low- and high-frequency power and different ratios (Table 1). Furthermore, several measures of nonlinear dynamics, especially from symbolic dynamics, have been calculated (Table 1).27,28,32,33
Analysis of Spontaneous Baroreceptor Sensitivity
The spontaneous baroreflex can be measured by analyzing the reflecting changes in heart rate on instinctive alterations in blood pressure.35 Here, the dual sequence method (DSM)29,31 was applied for the estimation of the spontaneous BRS. Two kinds of beat-to-beat interval responses were analyzed: bradycardic (blood pressure increase causes heart rate decrease) and tachycardic (blood pressure decrease causes heart rate increase) fluctuations, with the former primarily representing the vagally mediated spontaneous baroreflex. Analysis of the tachycardic fluctuations allows for the investigation of the relation between the vagally (bradycardic) and the sympathetically (tachycardic) mediated fluctuations of the heart rate.
When applying the DSM, the baroreflex sensitivity slopes were determined from 3 consecutive blood pressure and RR-interval values from the original unfiltered data and the mean slope calculated (average slope). Additionally, the total number of baroreflexes (number of bradycardic synchronic slopes) and this number as a percentage of all successive blood pressure changes were calculated (activation).
Group summaries are expressed as median value with interquartile ranges. Data were analyzed using the nonparametric Mann-Whitney U-test. In all tests, the criterion for statistical significance was P < 0.05.
To examine the influence of Ang-(1-7) on the blood pressure control, the analysis was performed in a circadian way considering the sleep/wake phases of the animals. The day period was defined from 8 AM to 8 PM, when rodents are less active. The night period was the remaining time.
Analysis of Blood Pressure Variability
Although the blood pressure during the day did not differ between untreated and AVE0991-treated SHR, the nightly increase in blood pressure as observed in controls and expected in night-active rodents was fully blunted in SHR treated with the Ang-(1-7)-receptor agonist (Table 2). These circadian effects were also reflected by significant alterations of the BPV parameters. During the day, significant BPV parameters allowed for a distinction between the control and AVE0991 groups. Whereas the BPV increased in the untreated rats, as obvious for several linear (sdNN, Shannon) and nonlinear (wpsum02, Wsdvar and polvar6) variability parameters, there was no alteration observed for any BPV parameter in the AVE0991 group. However, the BPV most significantly differed between both groups during the night. The circadian alterations in controls and the missing day-night oscillation in AVE0991-treated animals may also be illustrated graphically, as it was done (eg, for SDNN in Figure 1).
Analysis of Heart Rate Variability
Although the blood pressure did not alter during the day, the AVE group was characterized by an increased HR during the light phase [286.5 bpm (209.4 ms) versus 264.9 bpm (226.5 ms); Table 2]. This alteration was mimicked during the night because of the absence of an increase in blood pressure and less pronounced HR increase [MeanNN: from 226.5 ms at day to 189.6 ms at night (P < 10−12) in the controls; from 209.4 ms at day to 192.2 ms at night (P < 10−3) in the AVE0991 group, see Table 2, Figure 2]. Consequently, the positive chronotropic effect of the Ang-(1-7) receptor agonist can only be detected during the day. Parallel to the BPV, the main and highly significant changes in almost all variability parameters between treated and untreated SHR remained during the night (eg, SdNN, Shannon, Wpsum02, Wsdvar: P < 0.001; LF: P < 0.05; LF/HF, HF/P: P < 0.01). Whereas the controls showed a significantly higher HRV at night (SdNN, Shannon, HF/P: P < 0.05; LF/P: P < 0.01; Wpsum02, Wsdvar: P < 10−5), the day-night analysis in the AVE0991 group did not reveal any significant elevation during the night (all variability parameters not significant) and, consequently, no circadian variability changes (Table 2).
Analysis of Baroreceptor Sensitivity
BRS analysis revealed a qualitatively different pattern of alteration mediated by AVE0991. For both day and night times, strong differences between controls and the compound-treated group were noticed (see Table 2, Figure 3). Whereas the average slope as a standard parameter of BRS did not show any alteration during the day or at night, the number of synchronous bradycardic baroreflex events significantly decreased in the AVE0991-treated group [to 20% during the day (11 versus 52.5) and to 40% during the night (19 versus 46), P < 10−5]. In addition, the baroreflex activation in relation to the slopes in systolic blood pressure (SBP) was reduced by approximately 50% in the AVE0991-treated rats as compared with the control group (activation: 5.66 versus 12.36% at day times; 6.19 versus 11.16% at night). Thus, both parameters indicate a significantly reduced regulatory quantitative baroreflex response of the AVE0991 group.
Short-term blood pressure regulation is accomplished mainly by neural sympathetically and parasympathetically mediated cardiac baroreflexes and peripheral vessel resistance, whereas long-term regulation is achieved by hormonal pathways and by other systems like the renin-angiotensin system.36
To elucidate the relevance of the heptapeptide Ang-(1-7) to the heart rate and blood pressure variabilities and to the baroreflex sensitivity, SHR rats were treated with the recently described nonpeptide Ang-(1-7) receptor agonist AVE0991.26 Its effect in awake SHR after long-term treatment for 24 hours was recorded to visualize the impact of the agonist on the circadian cardiovascular regulation/control.
The data obtained show a lower blood pressure during the night in AVE0991-treated rats. However, this results from the lack of circadian rhythm and, thus, from the missing blood pressure increase during the night, as it was expected for night-active rodents (not a decreasing but a missing increase in blood pressure during the night). Loss of regulation of BPV and HRV parameters indicates a loss of circadian rhythm, which leads to highly significant differences of linear and nonlinear parameters during the night. Because the heart rate in the AVE0991 group is the same during the night but higher during the day, the compound acts as a tachycardiac substance. This effect is missing during the night as a result of the lack of up-regulating BP, HR, and variability parameters. Notably, one variability parameter, “polvar6,” which proved to be very powerful in previous studies,32,37 suggested changes in HRV during daytime (increase in polvar6 means decrease in the short-term HRV). Thus, the AVE0991 subjects are characterized by more intermittently decreased phases of HRV indicating a higher risk of cardiovascular diseases.32,37
Because increased BPV is a general phenomenon in hypertension and SHR are also characterized by an increased BPV,38,39 AVE0991 does not have any restoring capacity, which is illustrated best by the unaltered BPV parameters during the day.
Taking all blood pressure and heart rate parameters into account, it has to be concluded that AVE0991 completely suppresses the circadian rhythm. Although it has not yet been described for Ang-(1-7), the impact of the renin-angiotensin system on the circadian cardiovascular regulation is well documented by a variety of data showing that Ang II application can suppress the circadian rhythm. Baltatu and co-workers demonstrated that Ang II infusion in Sprague-Dawley rats inverted the 24 hour rhythm of blood pressure, whereas the 24 hour heart rate rhythm remained unaltered.40 This regulatory pattern of exogenous Ang II was also observed in aged Wistar rats.41 In the first study using telemetry to evaluate the effects of Ang-(1-7) infusion (subcutaneously for 7 days) on cardiovascular parameters, Ang-(1-7) did not produce significant changes in the circadian rhythm.42
However, it has to be noted that AVE0991 is not the first compound blunting the circadian rhythm in SHR rats. Oosting et al43 also reported the blunting capacity of prazosin and hexamethonium. Because these compounds cannot be related easily to the Ang-(1-7) signalling pathways and, thus, may not act via the same mechanism, upcoming studies will have to investigate the cardiovascular day-night regulation either in response to Ang-(1-7) or in Mas-deficient animals lacking heptapeptide actions. Only then can the observed AVE0991 results be related to endogenous Ang-(1-7) actions.
Apart from the highly significant impact of AVE0991 on the circadian rhythm of cardiovascular regulation, the effect of the Ang-(1-7) agonist on baroreceptor activation was most impressive. SHR animals are characterized by an impaired spontaneous44 and induced BRS.38,45 AVE0991 was not able to improve it, although the blood pressure is lower in AVE991-treated animals during the night and it has been shown that reduced blood pressure restores impaired BRS in hypertensive subjects.45
Although the baroreceptor sensitivity measured as the average slope did not alter under treatment, both the number of bradycardic synchronous slopes and the percentage of baroreceptor events in response to a blood pressure increase were significantly decreased under AVE0991. Because the activation is halved, but the number of bradycardic synchronic slopes is reduced by more than 75% in AVE-treated animals (during the day), more than an increase in baroreflex threshold has to be responsible for the alterations. The second parameter determining the number of events is the amount of stimuli (sequences of 3 consecutively increased BP values). The quantitative analysis of these sequences during the day revealed a more than halved number of these triplicates in AVE-treated rats as compared with their untreated controls (AVE: 194.3 versus control: 424.7). However, it has to be noted that all BRS differences detected showed circadian independency and, thus, the effect of AVE0991 on baroreflex activation seems to be independent of its modulation of circadian rhythm.
The AVE0991 effects seem to contradict previous findings, according to which Ang-(1-7) improves BRS, whereas A779 impairs it in SHR.46 However, these effects acted on the induced baroreceptor reflex and after peptide injection into the nucleus tractus solitarii. Therefore, a direct comparison should be avoided. Nevertheless, the general impact of Ang metabolites on the BRS has been described widely.
Although the beneficial cardiovascular effects of Ang-(1-7) have been demonstrated in a variety of experiments,6,47 our data obtained with respect to the Ang-(1-7) agonist AVE0991 also suggest nonbeneficial effects of this heptapeptide on the circadian rhythm and spontaneous baroreflex activation. Further studies comparing AVE0991 and Ang-(1-7) application in a head-to-head setting will have to find out whether AVE0991 may mediate Ang-(1-7)-independent effects.
We thank the Humboldt Foundation for providing a fellowship to S Heringer-Walther and Aventis AG (Frankfurt, Germany) for providing the AVE0991 compound.
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