Physical exercise is essential for cardiac autonomic regulation in hypertensive patients treated with losartan or enalapril

Background Hypertension treatment with renin-angiotensin system inhibitors (RASi) presents contradictions in relation to the recovery of damage in cardiovascular autonomic control. Conversely, the association of RASi with aerobic physical exercise can be a determining factor for some gains to be achieved. Thus, the objective was to investigate the effects of aerobic physical training on hemodynamic and cardiac autonomic regulation in hypertensive volunteers untreated and treated with RASi. and both in the in the with the and BRS in all groups. However, the association of enalapril with physical training to have prominent with losartan, an AT 1 receptor blocker, and enalapril maleate, an ACEi, inuences cardiovascular autonomic control, focusing on BRS and in the modulation of heart rate (HRV) and blood pressure variability (BPV). We also investigated the aerobic physical training effects compared with pharmacological treatment, as well as the effects of the association of both treatments. in absolute and normalized units compared with their respective groups in the supine position. However, no changes were observed in the losartan group. After aerobic physical training, the control group showed an increase in LF oscillations in normalized units and a reduction in HF oscillations in absolute and normalized units. In turn, the losartan group showed only a decrease in the total variance, while the enalapril group showed a reduction in the HF oscillations in absolute units compared with the results obtained in the supine position. The intergroup comparison of the spectral parameters obtained after orthostatism before aerobic physical training showed that the enalapril group showed a reduction in total variance compared to the control group. It also showed a decrease in HF oscillations in the normalized unit compared to that in the losartan group. In turn, after physical training, we observed that the control group showed an increase in total variance and the losartan group showed an increase in HF oscillations in absolute units, both compared with their respective untrained group, while the enalapril group showed increases in total variance and HF oscillations in absolute and normalized units, in addition to the reduction in LF oscillations in normalized units. We also observed that the trained enalapril group showed an increase in HF oscillations in the normalized unit compared with the trained control group.


Introduction
Until a few decades ago, the treatment of patients with systemic arterial hypertension (SAH) consisted of an approach based mainly on pharmacological therapy. Among the different classes of antihypertensive drugs, type 1 angiotensin II (AT 1 ) receptor blockers and angiotensin-converting enzyme inhibitors (ACEi) have been widely used. These drugs are also associated with other positive cardiovascular effects, such as the in uence on cardiac autonomic regulation, characterized mainly by a reduction in the sympathetic component [1][2][3]. However, these bene ts seem to be associated with short-and mediumterm treatments, since studies that investigated the long-term effects showed contradictory autonomic results, especially in relation to cardiac autonomic modulation and barore ex sensitivity (BRS) [2,[4][5][6]. In this case, it is possible that long-term monotherapeutic treatment with renin-angiotensin system inhibitors results in the impairment of cardiac autonomic regulation, despite the normalisation of the blood pressure (BP) [7,8] More recently, cardiovascular physiotherapy through regular practice of physical exercises, was introduced as an adjunct treatment to pharmacological treatment or as the rst therapeutic choice before drug prescription, especially in patients with mild to moderate hypertension [9,10] This therapeutic approach, based mainly on monitored and supervised aerobic physical training, has many bene ts such as adaptations in cardiac morphology and functionality, increased endothelial vasodilator response and positive effects on cardiovascular autonomic control [11][12][13][14][15][16][17]. Among cardiovascular autonomic effects, there is a reduction in sympathetic autonomic in uence on the heart and vessels and an increase in vagal in uence on the heart [14,15,18]. The cardiovascular autonomic bene ts promoted by aerobic physical training seem to be much more robust and less controversial. Our hypothesis is that aerobic physical training is essential to recovery of cardiovascular autonomic regulation in hypertensive patients, regardless the pharmacological treatment. In addition, it is possible that the combination of both treatments has a more prominent effect on cardiovascular autonomic control. This is important because impairment in cardiovascular autonomic regulation is considered a strong predictor of cardiovascular morbidity/mortality [19][20][21].
Therefore, this study aimed to investigate in volunteers with SAH treated with losartan, an AT 1 receptor blocker, and enalapril maleate, an ACEi, in uences cardiovascular autonomic control, focusing on BRS and in the modulation of heart rate (HRV) and blood pressure variability (BPV). We also investigated the aerobic physical training effects compared with pharmacological treatment, as well as the effects of the association of both treatments.

Sampling
Fifty-four men, aged between 40 and 60 years, who did not practice regular physical exercises, were studied. These volunteers were divided into three groups ( Figure 1); untreated hypertensive (control group; N=16), hypertensive treated with losartan (losartan group; N= 21), and hypertensive treated with enalapril maleate (enalapril group; N= 17). All volunteers were screened at the Laboratory of Physiology and Cardiovascular Physiotherapy (Laphy-Carphy) of the Ribeirão Preto Medical School (FMRP-USP). The volunteers were diagnosed with SAH for at least 2 years, classi ed into stages I and II, with low to moderate cardiovascular risk [9]. The treatment was monotherapy with antihypertensive drugs with AT 1 receptor blocker (losartan, 25-50 mg/day) or ACEi (enalapril maleate, 10-20 mg/day) for at least six months to two years before being included in the study.
The choice to include only men was based on previous studies, which showed that men and women have differences in cardiac autonomic modulation balance. In this case, the men had a greater predominance of the sympathetic autonomic component [22][23][24].
None of the volunteers performed regular practice of physical exercise, none presented cognitive disorders, musculoskeletal disorders, endocrine, metabolic and cardiovascular diseases (except SAH), or had any other disease or physical limitation that compromises the performance in the tests. We also did not include smokers or volunteers with SAH who were using combined pharmacological therapy or who used any other medication that interfered with cardiac function and cardiovascular autonomic control.

Ethical aspects
All volunteers were interviewed, in addition to checking their respective medical records that contain all the data related to identi cation and disease treatment.

Experimental protocols
Data were collected during three visits between 7-10 am, with an interval of 48h between visits. During the rst visit, anthropometric measurements and blood samples were taken to analyse the metabolic pro les at the Laboratory of the Clinical Research Support Centre of the Ribeirão Preto Medical School's Hospital (HCFMRP/USP). During the second visit, the cardiorespiratory functional test was performed, while on the third and last visits, electrocardiographic and pulsatile BP records were performed for the analysis of the cardiovascular autonomic modulation, both at the Laboratory of Physiology and Cardiovascular Physiotherapy (Laphy-Carphy) of the Ribeirão Preto Medical School. Each visit lasted ~2h, and all volunteers were instructed not to drink alcohol and caffeine, not perform strenuous physical exercises, and to maintain their usual diet for 48h before the evaluations. The volunteers in the drug groups were instructed not to interrupt the pharmacological treatment on their own and were advised to sleep at least 7 or 8h on the night before the visits.
The protocols were repeated after 16 weeks of supervised aerobic physical training on a motorised treadmill. In addition, for the nal evaluation, an interval of 48h was established between the last session of physical training and the rst day of revaluation.

Anthropometric parameters
Body weight and height were obtained using an analogue scale with an altimeter (Welmy), while the body mass index (BMI) values were obtained using the formula W/H 2 , where W is the weight in kilogrammes and H is the height of the subject in metres. Body composition was evaluated using the bioelectrical impedance method (Quantum BIA 101; Q-RJL Systems, Clinton Township, Michigan, USA).

Laboratory exams
Blood samples (3.5 mL, BD Vaccutainer® EDTA -Becton, Dickin, and Company, Franklin Lakes, NJ, USA) were used to analyse the fasting glycaemia (hexoquinase-UV), triglycerides (desidrogenase), and total cholesterol and fractions (esterase-oxidase). All volunteers were asked to fast for 12 hours prior to the assessments.

Cardiorespiratory Function Test
An incremental treadmill exercise test was performed along with a submaximal test, established with the heart rate (HR) corresponding to the sum of the baseline HR and 85% of the reserve HR (maximum HR -basal HR), following the previously described Balke protocol [25]. Electrical activity was monitored by an electrocardiogram (ECG) with one lead (CM5). Oxygen and dioxide carbon uptake (VO 2 and VCO 2 ) were obtained using a metabolic analyser (Ultima™

Heart Rate Variability and Blood Pressure Variability Analysis
The spectral analysis of HRV was recorded between 8 am and 10 am according to the following protocol: after remaining in a supine rest position on an orthostatic bed for 20 min, the volunteers were head up tilted passively (75° angle) for an additional 10 min. HRV for the supine and head up tilted positions (i.e., tilt test) was recorded using an electrocardiogram (ADInstruments, Bella Vista, Australia), and a time series of RR interval (RRi) was determined. The HRV was obtained using the RRi from the electrocardiographic record (ECG) through the modi ed CM5 shunt at a sampling frequency of 1000Hz. The BPV data values were obtained from the systolic BP (SBP) recorded beat-to-beat using the digital plethysmography recording equipment (Finometer Pro, Finapress Medical System, Amsterdam, Netherlands) using a cuff positioned on the middle nger of the right upper arm. The BPV and HRV analyses were performed as previously described using custom computer software (CardioSeries v2.7, http://sites.google.com/site/cardioseries) [22,23,26,27].
2.8. Spontaneous barore ex sensitivity BRS was assessed in the time domain using the sequence technique. The computer software CardioSeries v2.7 scanned the beat-to-beat time series of pulse interval (PI) and SBP values, searching for sequences of at least 3 consecutive beats, in which progressive increases in SBP were followed by progressive increases in PI (up sequence) and progressive decreases in SBP were followed by progressive decreases in PI (down sequence), with a correlation coe cient (r) between PI and SBP (values higher than 0.8). Spontaneous BRS was determined by the mean slope of the linear regression line between the SBP and PI values of each sequence. The number of barore ex sequences found (per 1000 beats) and the mean individual slope of the signi cant SBP/PI relationship, obtained by averaging all slopes computed within the test period, were calculated, and used as a measure of spontaneous BRS [23,26,28,29].
The sequence method also presents the barore ex effectiveness index (BEI). It is the ratio of the number of sequences and the total number of SBP ramps.
The BEI shows how many SBP changes are effectively translated into a change in PI, independent of its magnitude.

Aerobic physical training
The sessions were supervised, monitored, and performed three times per week for 16 weeks. The training intensity was calculated as the sum of HR at rest and 70-80% of reserve HR, obtained by means of the following equation: HR recorded at the peak of the cardiopulmonary testing -HR at rest. The training sessions lasted for 1 h, divided into three phases as follows: 5 min of warm-up using an intensity lower than the target HR training range (50-65% of reserve HR), 50 min of training using the training HR (70-80% of reserve HR) and 5 min of cool-down using an intensity lower than the training HR (40-50% of reserve HR). There was an adaptation period during the rst two weeks of the study, during which participants went through 20-30-min sessions for familiarization and adaptation to the training protocol. The intensity used was equivalent to the sum of HR at rest and 50% -60% of the reserve HR, followed by increases in the intensity and duration in the subsequent weeks, until the volunteers reached the training HR as described. HR was monitored throughout the sessions using a pulse frequency meter (Polar RS810).

Statistical analysis
The results are presented as mean ± standard deviation (SD). Variables were analysed using parametric and nonparametric tests, when required. Age and height were assessed using one-way analysis of variance (ANOVA). When appropriate, post hoc comparisons were performed using Tukey's test. The effects of laboratory examinations, hemodynamic parameters, cardiopulmonary functional tests, HRV, BPV, and spontaneous BRS were assessed by two-way ANOVA. When appropriate, post hoc comparisons were performed using the Student-Newman-Keuls method. Student's t test was used for comparisons between groups, and paired Student's t test for intragroup comparison (before and after training). Differences were considered signi cant at P<0.05. All statistical tests were performed using SigmaPlot 11.0 software (Systat Software Inc., San Jose, CA, USA). The SigmaPlot 11.0 software was used for sample size calculation, con dence level was set at 95%, and power at 80%, with the LF and HF variables in normalized units. The sample size was set to 16 participants per group.

Results
The BP and HR values are presented in Table 1. The control group had lower HR values and higher BP values than the losartan and enalapril groups. After aerobic physical training for 16 weeks, all groups showed similar values, characterized by a reduction in baseline HR and BP. Anthropometric characteristics, cardiorespiratory tness, and blood parameters evaluated are also shown in Table 1. The results showed that there were no differences in anthropometric characteristics before and after aerobic physical training between the studied groups. The VO 2peak showed similarities between the groups, even after the increase resulting from aerobic physical training. Regarding laboratory tests, the groups showed similar values for almost all parameters evaluated before and after aerobic physical training. In this case, only total cholesterol levels were signi cantly lower after aerobic physical training. Figure 2 and Table 2 show the parameters of HRV obtained in the supine position, before and after aerobic physical training. The results show that the losartan group had lower values of HF oscillation in absolute units compared to the control group. After 16 weeks of aerobic physical training, the control and enalapril groups showed increases in total variance and in LF and HF oscillations in absolute units, while the losartan group only showed increases in total variance and HF oscillations in absolute units. Values expressed as means ± SD. HR, heart rate; LF, low frequency; HF, high frequency, ms, milliseconds; nu, normalized units; SBP, systolic blood pressure; mmHg, millimetres of mercury; BRS, barore ex sensitivity; BEI, barore ex effectiveness index. a P < 0.05 vs. Control before training; b Losartan before training; c Enalapril before training. The intergroup comparison of the spectral parameters obtained after orthostatism before aerobic physical training showed that the enalapril group showed a reduction in total variance compared to the control group. It also showed a decrease in HF oscillations in the normalized unit compared to that in the losartan group. In turn, after physical training, we observed that the control group showed an increase in total variance and the losartan group showed an increase in HF oscillations in absolute units, both compared with their respective untrained group, while the enalapril group showed increases in total variance and HF oscillations in absolute and normalized units, in addition to the reduction in LF oscillations in normalized units. We also observed that the trained enalapril group showed an increase in HF oscillations in the normalized unit compared with the trained control group. Values expressed as means ± SD. HR, heart rate; LF, low frequency; HF, high frequency; ms, milliseconds, nu, normalized units; SBP, systolic blood pressure; m millimetres of mercury; BRS, barore ex sensitivity; BEI, barore ex effectiveness index. a P < 0.05 vs. Control before training; b Losartan before training; c Enala before training. Table 2 shows the autonomic parameters of BPV and spontaneous BRS in all groups, obtained in the supine position, before and after aerobic physical training. BPV results showed intergroup differences after aerobic physical training, and the enalapril group showed an increase in variance compared to the losartan group. In turn, the BRS results showed that all groups had similar responses before aerobic physical training. After 16 weeks of aerobic physical training, all groups showed an increase in BRS, characterized by an increase in the total gain for bradycardic (Up) and tachycardic (Down) responses in relation to their respective values obtained before aerobic physical training. In addition, the losartan group showed a reduction in the BEI, and number of ramps compared to the control group, while the enalapril group showed a reduction only in the BEI compared to the control group. Table 3 also shows the BPV and BRS parameters in all groups, but obtained during the tilt test, and before and after aerobic physical training. BPV results showed intergroup differences before aerobic physical training, in which the enalapril group showed a decrease in variance compared to the control group. In turn, the BRS results showed that the enalapril group had a reduction in BEI and ramp numbers in relation to the control group, while the losartan group showed a reduction only in ramp numbers when compared with the control group. After aerobic physical training, the number of ramps remained reduced in the losartan and enalapril groups compared with the control group. In turn, during the tilt test, no differences were observed before and after aerobic physical training in any of the BRS parameters.

Discussion
Page 8/12 The HRV indices and BRS gain re ect the cardiovascular system's ability to reorganize itself due to variations in the demands of the internal environment.
These autonomic parameters are recognized as important predictors of morbidity and mortality [19][20][21]. In this study, the results indicated that aerobic physical training is a therapeutic tool that plays a fundamental role to positively regulates the cardiovascular autonomic control in hypertensive patients regardless of pharmacological treatment.
Long-term treatment with losartan and enalapril was not accompanied by improved cardiovascular autonomic control, although BP was maintained at values considered normal. In fact, when compared with untreated hypertensive volunteers, the results suggest that pharmacological treatments with reninangiotensin system (RAS) inhibitors in the long-term seem to promote a reduction in the autonomic modulation of HRV, attenuating the variation intensity of each autonomic component, including during the tilt test. Some parameters, such as the HF oscillations of HRV, in the losartan group had lower values than the untreated hypertensive group [7,8]. Moreover, when this group was subjected to a tilt test, the autonomic parameters evaluated did not differ from those in supine position in absolute units, which is not common. This suggests that in addition to the absence of positive cardiovascular autonomic effects, treatment with losartan appears to decrease the autonomic regulatory e cacy. Likewise, treatment with enalapril alone did not result in major autonomic bene ts, but it appears that enalapril has some advantages over losartan. The reasons for these observations are uncertain. However, it is important to highlight the mechanisms of action of these two antihypertensive drugs. While enalapril reduces the formation of angiotensin II by inhibiting the angiotensin-converting enzyme (ACE), it also contributes to the reduction of bradykinin degradation. The greater bioavailability of bradykinin promotes greater formation of nitric oxide (NO) and vasodilating prostaglandins, further reducing the BP, and promoting its effects on the vascular beds, heart, and kidneys [30][31][32]. However, this increase in bradykinin concentration also seems to be responsible for a greater activation of the cardiac sympathetic afferent re ex, resulting in the attenuation of cardiovascular autonomic modulation [33]. In turn, losartan has been pointed out in the literature as being related to an important factor that interferes with the trophic effect of angiotensin II on sympathetic autonomic modulation [34][35][36][37].
Thus, blocking AT 1 receptors appears to result in cardiac autonomic effects that are slightly different from that of enalapril. An experimental study with spontaneously hypertensive rats (SHRs) treated with losartan or enalapril maleate showed that both had reduced BP but did not promote any change in cardiac autonomic modulation. Regarding BRS, only enalapril group showed an increase in gain. When these animals were trained, a better cardiac autonomic response was observed, and the association of aerobic physical training with enalapril resulted in greater gains. The authors pointed out that these differences would involve two aspects: rst is related to a greater decrease in angiotensin II levels, and second to the improvement of endothelial function. In the rst case, the aerobic training effects was to interfere with the pathways of Elastase-2 and chymase, which are alternative forms of angiotensin II production that do not depend on ACE [26]. This action would further decrease the concentration of angiotensin II, including the central nervous system, potentiating the positive effects of enalapril. Other studies have shown an increase in Elastase-2 in the arteries, lungs, and heart of SHRs [38,39], as well as a higher concentration of mRNA levels for chymase in these animals compared to normotensive rats [40]. In addition, in vascular and cardiac pathways, elastase-2 and chymase become potentially activated in SHRs treated with enalapril maleate due to ACE inhibition [39,41]. However, new approaches are needed to elucidate the interaction between aerobic physical training effects and these mechanisms. The second aspect is related to a relationship with endothelial function recovery, which responds with greater production of NO and other dilating factors. This can be caused by the reduction of angiotensin II, as well as by the increase in bradykinin concentration, both of which results from aerobic physical training, through greater vascular shear stress [42][43][44]. In this condition, NO seems to have an effective action in reducing sympathetic participation and increasing BRS [45,46].
In fact, our study reinforces the importance of aerobic physical training as a fundamental therapy for the treatment of hypertensive patients. We know that its therapeutic effects are broad and systemic [12,13,47]. More speci cally, on the cardiovascular system, the bene ts related to cardiac morphological and functional adaptations are relevant, making the heart more e cient and less dependent on sympathetic autonomic in uence, especially during rest and activities of daily living [11,16,17]. Gains on BRS are also observed, which is the result of adaptations in the autonomic re ex arc involving even the central neural nuclei of cardiovascular control, resulting in a more e cient cardiac regulation, characterized by a more balanced autonomic rearrangement. This autonomic rearrangement can be seen through increases in HRV and BRS, resulting in a greater adaptive capacity during physiological demands [48][49][50][51]. This new autonomic status induced by physical training can contribute to a more favourable positive outcome, not only in hypertensive patients, but also in patients with different conditions, mainly cardiovascular, metabolic, and immunological.
For all these bene ts, the association of aerobic physical training with RAS inhibitors, mainly with enalapril, proved to be more advantageous for the control of hypertension and cardiac autonomic regulation. Regarding the differences found between enalapril and losartan before and after aerobic physical training, it is important to point out that this is a relevant nding and needs to be investigated, especially the observations obtained during the tilt test. The 'freezing' in HRV values of the spectral parameters observed in losartan group was surprising and needs to be explained, as it represents the main difference between the treatment with losartan and enalapril.
Thus, our ndings con rm the aerobic physical training as an important tool that must be used in the hypertensive patients before and during pharmacological treatment. In this case, the association with pharmacological treatment is fundamental when aiming beyond BP reduction and gains in cardiovascular autonomic regulation. Furthermore, the cause of the differences found in cardiovascular autonomic regulation between losartan and enalapril are uncertain and need to be investigated.