Important differences in cardiovascular autonomic control of hypertensive middle-aged women and men – a critical appraisal


 BackgroundNormotensive women before menopause showed a vagal predominance of cardiac autonomic modulation, while age-matched men showed a sympathetic modulation predominance. However, some women develop systemic arterial hypertension (SAH) even with preserved ovarian function. Our hypothesis is that these women may have cardiovascular autonomic parameters like those of hypertensive men, even when subjected to pharmacological treatment. We aim to investigate the cardiovascular autonomic modulation balance and baroreflex sensitivity (BRS) in hypertensive women with preserved ovarian function and age-matched men.MethodsOne hundred volunteers between 18 and 45 years of age, were assigned to two groups of fifty: Hypertensive group, with a SAH history for at least 6 months (25 men and 25 women) treated with monotherapy (losartan, 25-50mg/kg) and normotensive group (25 men and 25 women). Anthropometric, hemodynamic, metabolic, and autonomic cardiovascular assessments were performed focusing: BRS, autonomic modulation of heart rate variability (HRV) and blood pressure variability (BPV).ResultsThe results showed that HRV analysis, women showed higher values ​​of HF oscillations in absolute and normalized units, and lower values ​​of LF in normalized units and LF/HF ratio compared to men. When normotensive and hypertensive groups were compared, hypertensive groups showed lower values ​​of total variance, LF and HF bands in absolute units. There were similar in BPV. However, hypertensive groups showed lower BRS values when compared to normotensive group.ConclusionThe results indicate that hypertensive groups even with blood pressure controlled through pharmacological treatment, continued to have reduced HRV than normotensive, and hypertensive women have minor cardiovascular autonomic impairment than men.


Introduction
Systemic arterial hypertension (SAH) is a multifactorial disease with high prevalence in the adult population worldwide [1][2][3]. It is accompanied by impairments in cardiovascular homeostasis resulting from several factors, mainly endothelial dysfunction and changes in cardiac autonomic balance [4][5][6]. Autonomic changes are determined by an increased predominance of sympathetic autonomic component and/or a decrease in parasympathetic (vagal) component and, if not controlled and/or reversed, could predispose the individual to a higher occurrence of acute myocardial infarction, vascular accident brain disease and heart failure [1][2][3].
Considering the different age groups affected by SAH, women up to until climacteric period have a lower hypertension prevalence, when compared to agematched men. However, this condition is not observed after menopause [1][2][3]. The prevalence differences between the sexes have been attributed, in part, to the ovarian hormones action [7,8], mainly estrogens. Estrogens play a key role in the dilating factors release, derived from the vascular endothelium, as well as the cardiac autonomic control, by promoting a greater vagal autonomic participation and/or reduction of the sympathetic in uence in cardiac autonomic regulation [6,7,9,10].
Despite the ovarian hormones' cardiovascular protection, some women precociously develop cardiovascular diseases, especially SAH. The causes for the SAH diagnosis, before physiological ovarian failure, are uncertain, however, it is possible that important changes in cardiovascular autonomic control might play a role. It is well known that cardiac autonomic balance regulation is different between the sexes, while women's is characterized by a vagal autonomic component predominance, there is a sympathetic predominance in men, especially when the heart rate variability (HRV) modulation is considered [10][11][12]. In addition, it is possible that SAH, and its complications, may cancel the cardiovascular protective effect from ovarian hormones, since there are no sex differences in pathophysiological cardiovascular autonomic modulation, such as the ones reported for acute myocardial infarction and heart failure [13][14][15], contrary to what is observed in normotensive individuals [10,11]. However, most studies have been carried out on women after menopause, a period known to be characterized by substantial reduction in HRV and barore ex sensitivity (BRS) [7]. Thus, we investigated the cardiovascular autonomic control in hypertensive men and women with preserved ovarian function, focusing on BRS and the balance of cardiovascular autonomic modulation.

Methods
One hundred volunteers between 18 and 45 years of age, were assigned to two groups of fty: Hypertensive, with a history of SAH for at least 6 months (25 men and 25 women with preserved ovarian function) and normotensive (25 men and 25 women). All were screened at the Laboratory of Exercise Physiology and Cardiovascular Physiotherapy (LAFFIC), of the Ribeirão Preto Medical School (FMRP-USP). The hypertensive volunteers had a history of essential arterial hypertension stages I and II, with low to moderate cardiovascular risk [2]. All hypertensive volunteers have been treated only with a monotherapy using AT 1 receptor blocker (Losartan, 25-50 mg/day) for at least 6 months. None of the volunteers smoked, neither presented the following: cognitive disturbances, pregnancy, musculoskeletal disorders, metabolic and cardiovascular diseases (except for SAH) or any other disease that compromise the assessments. Finally, this study was approved by the Ethics Committee of the Ribeirão Preto Medical School's Hospital (HCFMRP/USP) under the protocol number Protocols Data was collected in the morning during two laboratory visits, between 07:00 and 10:00 am, with a 48-hour interval between the visits. All women had a regular menstrual cycle and all the collected data were in follicular phase of their cycle. The rst assessment included anthropometric measurements and blood collection, at the Laboratory of the Clinical Research Support Centre of the Ribeirão Preto Medical School's Hospital (HCFMRP/USP).
The second visit took place at the Laboratory of Exercise Physiology and Cardiovascular Physiotherapy of the Ribeirão Preto Medical School. During the second visit, the following protocols were completed: cardiovascular autonomic analysis and cardiorespiratory function test. Each visit lasted approximately 2 hours.
All volunteers were asked to avoid drinking alcoholic beverages, as well as exercising, and maintain their usual diet for 48 hours prior to the assessments. They were also instructed to maintain their regular medication intake (Losartan, 25-50 mg) and advised to sleep at least 7 or 8 hours, the night before the visits.

Anthropometry and body composition
Evaluations followed the International Society for the Advancement of Kinanthropometry recommendations. The weight and height values were obtained using the Welmy analog scale with altimeter (Welmy, Santa Bárbara d'Oeste, São Paulo, Brazil), the volunteers body mass index (BMI) was calculated using the formula weight / height², with weight in kilogram and height in meters.
Body fat percentage was obtained through skin folding, performed by an experienced evaluator. The adipometer used was the Sanny®, with a 0.1 mm scale, mandibular pressure of 9.8 g/mm 2 and contact area (surface) of 97mm 2 , according to the manufacturer's speci cations. The skin folds were obtained at the following points: triceps, subscapular, axilla (midaxillary), supra iliac, abdomen, thigh, and chest. From the data obtained, the following parameters were calculated: sum of the seven DC (ΣDC) (mm) and the body fat percentage (% BF), estimated by age group. [16] Cardiorespiratory Function Test An incremental treadmill exercise test (Super ATL Millenium®, Inbramed/Inbrasport, Porto Alegre, RS, Brazil) was performed following the Balke protocol, previously described. [17] Electrical activity was monitored by electrocardiogram (ECG) with nine leads (CM5, DI, DII, V1-V6). Oxygen and dioxide carbon uptake (VO 2 and VCO 2 , respectively) were performed using the metabolic analyser (Ultima ™ CardiO2, Medical Graphics Corp., St. Paul, Minneapolis, USA).

Heart Rate Variability and Blood Pressure Variability Analysis
Heart rate variability (HRV) data was obtained using the RR intervals (iRR) from the electrocardiographic record (ECG), through the modi ed CM5 shunt, at a sampling frequency of 2000 Hz. The blood pressure variability (BPV) values were obtained from the SBP data recorded beat-to-beat, using the digital plethysmography recording equipment (Finometer Pro, Finapres Medical System, Amsterdam, Netherland), with a cuff positioned on the middle nger of the right upper limb. The data interface to the microcomputer was performed using the PowerLab4 / 35 device (ADInstruments, Australia). The data was recorded and stored (Software LabChart 8.0, ADInstruments, Australia) for further analysis. Volunteers were instructed to remain in supine position for approximately 10 minutes to stabilize the cardiovascular parameters. After this period, the ECG and arterial pulse pressure were recorded, simultaneously, for another 10 minutes. The temperature (22 °C) and ambient lighting were controlled, and the sessions were performed in a noise-free environment.
For the standardization of the biological signals acquisition and recording, the BP plethysmography recording equipment was calibrated before each test, using physiological calibration and return-to-ow (RTF), in addition to the photoplethysmography height sensor. This procedure allowed the adjustment of the peripheral pressure values (cuff on the middle nger), when compared to the brachial artery pressure values (cuff positioned on the upper region of the ipsilateral arm). Biological signals uninterrupted recording started after this calibration phase. BPV and HRV analyses were performed using custom computer software (CardioSeries v2.4, http://sites.google.com/site/cardioseries) developed by Dias, DPM of the University of São Paulo, Brazil [18]. The values of the R-R (RRi) and SBP intervals were redesigned in 3 Hz cubic spline interpolation, to normalize the time interval between the beats. The series of interpolated RRi and SBP were divided into half-overlapping sets of 256 data points, overlapping 50% (Welch Protocol). Stationary segment was visually inspected and those with artifacts or transients were excluded. Each RRi and SBP stationary segment was submitted to spectral analysis by Fast Fourier Transform (FFT), after the HRV normalized values were obtained by calculating the percentage of LF and HF power, related to the total spectrum power, minus the very low-frequency band (VLF; < 0.04 Hz). In addition, the normalization procedure was performed to minimize total power variations in the absolute values of LF and HF [19]. To assess the sympathovagal balance, LF/HF ratio of RRi variability was also calculated [20].
Spontaneous Barore ex Sensitivity Analysis BRS was assessed in time-domain, using the sequence technique [21]. The computer software CardioSeries v2.4 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 found. The number of barore ex sequences found (per 1,000 beats) and the mean individual slope of signi cant SAP/PI relationship, obtained by averaging all slopes computed within the test period, were calculated and used as a measure of spontaneous BRS.

Statistical analysis
Electronic spreadsheets were prepared, and the information was analyzed using the electronic program Sigma-Stat ®, version 11. Variables were analyzed using parametric and non-parametric tests, when required. The effect of sex and hypertension were analyzed by Two-Way ANOVA and, when appropriate, a post-hoc comparison was performed using the Student-Newman-Keuls method. Student's t-test was used for comparisons between groups. P value was set at ≤ 0.05. Table 1 shows the characteristics and hemodynamic parameters of the studied groups. Women, in both groups, had lower values of body weight and height, when compared to men. However, BMI and body fat percentage were similar. As for hemodynamic and cardiorespiratory tness parameters, men showed higher values of VO 2peak , systolic, diastolic, and mean blood pressure, when compared to women. However, when hypertensive and normotensive groups were compared, the hypertensive groups showed higher values of systolic and mean arterial pressure. Women had lower values of fasting blood glucose, triglycerides, LDL, total cholesterol, and higher HDL values, when compared to men, as shown in Table 2. On the other hand, when hypertensive and normotensive groups were compared, fasting blood glucose values were higher in hypertensive volunteers.   Table 3 and Fig. 1, comparing women to men, the HRV analysis, obtained in the supine position, women showed higher values of HF oscillations in absolute and normalized units, as well as lower values of LF in normalized units and LF/HF ratio. When normotensive and hypertensive groups were compared, hypertensive women and men showed lower values of total variance and LF and HF bands in absolute units. There were no sex or hypertension differences for BPV, however, hypertensive men and women showed lower BRS values (barore ex gain, ms/mmHg), when compared to normotensive (Table 3). Additionally, we also observed that hypertensive men had lower BRS values, when compared to hypertensive women. Data presented as mean ± SEM. RRi -intervals R-R do ECG, ms -milliseconds, LF -low frequency band, HF -high frequency band, nu -normalized units, mmHg mercury millimeters, BEI -barore ex effectiveness index, F -factor, d.f. -degrees of freedom.

Discussion
Cardiovascular autonomic control has been widely studied in different pathophysiological conditions, and these studies have guided diagnoses, treatments and used as predictors of cardiovascular morbidity and mortality [19,22]. For SAH, an increase in sympathetic participation and/or reduction of vagal participation in the cardiac autonomic balance is frequently observed in several parameters evaluated [4,5]. In most cases, these changes result in a reduction in BRS [4,23]. In turn, when indices that quantify cardiac autonomic modulation are used, it is common to observe a decrease in HRV, and together with a reduction in BRS, they indicate an important loss in autonomic mechanisms of cardiovascular protection.
On the other hand, normotensive adult men and women, although sharing the same autonomic mechanisms of cardiovascular autonomic regulation, they show a signi cant difference in cardiac autonomic modulation regulation. In this case, women have a vagal predominance of cardiac autonomic modulation, while men have a predominance of sympathetic modulation [10,11]. However, little is known about these differences in pathophysiological conditions, such as cardiovascular diseases. Thus, it is common for studies to include men and women, without considering these particularities of cardiac autonomic control due to sex. In this case, our results showed that women and men with SAH have differences in cardiac autonomic regulation, speci cally, a predominance of vagal autonomic modulation was seen for women and sympathetic autonomic modulation for men. However, the absolute HRV values found in the present study are much lower when compared to the normotensive groups, mainly for the HF oscillations in the men group. In this case, the results corroborate the statement that SAH leads to the HRV reduction, an important autonomic change. Interestingly, the reduction HRV did not affect the balance of autonomic modulation, therefore, the predominance was similar to that observed in normotensive women and men. The reason for maintaining the vagal component predominance in the cardiac autonomic balance in hypertensive women is uncertain, however, the sexual characteristics, mainly the sexual female hormones, might be playing a role in this. Estrogens might be the main hormones at play, since experimental and clinical studies have shown that they play an important role in autonomic regulation, increasing vagal autonomic modulation and reducing cardiac sympathetic autonomic modulation [7]. Furthermore, other studies indicate that estrogens might in uence the vascular tone regulation and, consequently, arterial blood pressure by different mechanisms such as the release of vasodilating factors from the endothelium, antioxidant and anti-in ammatory factors [6,24,25], down regulation of angiotensin-converting enzyme [26], reduced activity of angiotensin II (AT1) receptors [27], which together would cause a positive in uence on cardiac contractility [9]. Corroborating this information, our results showed that women had lower BP values, even if they underwent identical pharmacological treatment (losartan).
Studies in the literature have shown that male hormones also have in uence on the cardiovascular autonomic control [28]. In men, the excess of androgens seems to cause vagal cardiac dysfunction, in addition to disturbances in ventricular regulation [29]. Another important observation of the differences in cardiac autonomic modulation between the sexes can be exempli ed by women with Polycystic Ovary Syndrome. In this group of women, there is a signi cant increase in the main male hormone, testosterone, positively correlated with the increased sympathetic autonomic component in uence [30]. Thus, testosterone levels in men, and estrogens in women, are variables that could explain the sex differences found in the autonomic modulation of HRV, including in patients with SAH.
Other aspects that may in uence the sympathetic autonomic modulation predominance in men involve body composition and the investigated blood parameters. Regarding body composition, we know that men have a higher volume of skeletal muscle mass. This greater muscle volume may result in a greater participation of sympathetic autonomic component in cardiovascular regulation [31]. The literature also shows that men would have a greater number of sympathetic ganglion neurons, when compared to women [32]. In our study, despite the almost similar anthropometric results, men tended to have higher BMI values and lower body fat percentage values, suggesting that this group had a higher percentage of lean mass. On the other hand, blood parameters were quite different. Women had higher values of HDL, and lower values of triglycerides and fasting glucose. In fact, these differences could justify the reduction of autonomic modulation in hypertensive men, but not in hypertensive women.  [10]. Everything indicates that SAH appears to induce greater damage in men, evidenced by a signi cant reduction in vagal participation in cardiovascular autonomic regulation. The second nding is that for the treatment with losartan, despite the blood pressure (BP) normalization, the cardiac autonomic modulation parameters were far below the desirable level, as noted above. This is worrisome, since HRV indexes are often used as morbidity and mortality predictors. [19] However, as our ndings demonstrate, men are less protected, mainly because they exhibit an excessive drop in HF oscillations that correspond to vagal autonomic modulation. Finally, the signi cant reduction in the HF component of HRV may also be due, at least in part, to the metabolic mis t found in hypertensive men.

Perspective And Signi cance
Hypertensive women and men, even when treated with losartan, show reductions in HRV, when compared to normotensive volunteers. Despite systemic arterial hypertension, women and men have a different predominance of autonomic components over cardiac modulatory balance. Women are more dependent on vagal modulation, while men have a greater prevalence of the sympathetic autonomic component. Therefore, according to these ndings, it is possible to suggest that men have higher cardiovascular risks, and that the possible causes of the differences found are the characteristics of sex hormones. However, further studies are needed to identify the precise mechanisms responsible for these ndings.

Declarations
Authors' contribution SVP and HCDS participated in the study design and conceptualization, data collection, data analysis and manuscript preparation for the original draft and review and editing. TPF participated in the data collection and project administration, data analysis and manuscript preparation for the original draft. ACG participated in the study design and conceptualization and manuscript preparation for review and editing. THF and KPR participated in the study design and conceptualization, data collection, data analysis and manuscript preparation for the original draft.
Ethical approval and consent to participate Included in the "Methods" section. Availability of data and materials The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interest
The authors declare that they have no con ict of interest.

Consent for publication
Not applicable.