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The Relationship between Autonomic Regulation of Cardiovascular Function and Body Composition

Background We investigated whether the results of autonomic function tests correlate with body composition and shape in healthy young people. Methods We conducted cardiovascular reflex tests (heart rate [HR] and blood pressure [BP] responses to the Valsalva maneuver and HR response to deep breathing) and the tilt table test with 32 subjects (19 males; mean age, 22.1±1.9 years). Participants also completed an anthropometric measurement sequence (weight; height; upper arm, hips, and waist circumference; triceps and subscapular skinfold), bioelectric impedance testing, and hand grip strength measurements. Results Markers of obesity, other anthropometric measures, functional measures, and the basal metabolic rate (BMR) were significantly positively correlated with systolic BP (SBP) and diastolic BP (DBP) in both the supine and tilted positions. There was a positive correlation between the difference in HR (ΔHR) between the tilt and supine body positions and markers of obesity, the functional marker of dominant handgrip strength, and BMR. Participants with a body mass index (BMI) <25 kg/m2 had significantly lower median values of ΔHR, DBP in the tilt-test, SBP at rest, and SBP in the tilt-test than participants who had a BMI ≥25 kg/m2 (10.55 vs. 21.95 bpm, P=0.003; 77.55 vs. 90.05 mmHg, P=0.045; 113.45 vs. 140.55 mmHg, P=0.013; 117.00 vs. 135.25 mmHg, P=0.006, respectively). Body fat percentage was identified as an independent positive predictor (β=0.993; 95% confidence interval [CI], 0.070 to 1.916; P=0.036) and body water percentage was an independent negative predictor of tilted SBP (β=–1.370; 95% CI, –2.634 to 0.106; P=0.035). Conclusion High sympathetic activity, as evaluated by cardiovascular regulation, correlates with a high share of adipose tissue in young healthy persons.


INTRODUCTION
The autonomic nervous system (ANS) regulates many functions, including circulation and metabolism. The ANS exerts direct control of adiposity at the cellular and molecular levels. Both the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS) provide complex homeostatic control to specifically and hypertension, dyslipidemia, and hyperglycemia, which could all appear as a result of prolonged high SNS activity. 3 It has been shown that obesity and central body adiposity correlate with alterations in the ANS, and therefore, people at greater risk for those conditions could be tested to see whether their ANS is distorted. [4][5][6] The ANS and body composition, which provides a specific image of metabolism, are connected and affect the same diseases, such as hypertension, dyslipidemia, hyperglycemia and many other acquired chronic diseases that can be treated only symptomatically. 7 Interventions that affect both metabolism and body composition, such as exercise, act synergistically with pharmacological therapy using beta receptor blockers. 8,9 Research on this topic has included older people 10 and children 11 and focused on conditions such as obesity, 12,13 malnutrition, 14 and pregnancy. 15 In healthy individuals, an increase in the body mass index (BMI) correlates with increased sympathetic activity and lowered parasympathetic activity. 16 The identification of a link between ANS activity and BMI could provide valuable insights into the physiological regulation of body weight (BW) and important clues for the development of effective anti-obesity strategies. 17 Because most previously published studies did not make a thorough nutritional assessment and used only specific tests of the ANS (usually heart rate variability [HRV]), our aim in this study was to determine whether standardized autonomic function tests correlate with body composition and shape, as evaluated with a thorough nutritional assessment, in healthy young people.
The primary objective of this study was to investigate the correlation between the results from ANS tests and body composition and constitution. The secondary objectives were to investigate differences in heart rate (HR), systolic blood pressure (SBP), and diastolic BP (DBP) using a BMI cutoff of 25 kg/m 2 to indicate overweight individuals.

Participants
Thirty-two healthy subjects (19 males and 13 females; mean age, 22.1 ± 1.9 years) took part in this study. The Croatian version of the Composite Autonomic Symptom Score 31 (COMPASS 31) questionnaire was used to evaluate autonomic symptoms. 18 The Godin Leisure-Time Exercise Questionnaire, a self-explanatory, four-item query, was used to evaluate their leisure-time exercise habits. The test group had no illnesses or disabilities, and they were all enlisted as healthy subjects. All participants were acquainted with the study protocol and provided informed consent. This study was approved by the Ethical Committee of University Hospital Center Zagreb (02/21 AG, January 30, 2018).

ANS testing
The ANS testing protocol was performed in the Referral Center The test protocol consisted of the following: (1) 10-minute rest on the tilt table (supine phase), (2) three consecutive Valsalva maneuvers with a 3-minute pause after each maneuver, (3) a deep breathing test followed by a 3-minute pause, (4) 10-minute tilt table test, and (5) 5-minute rest period. Throughout the testing, BP and HR were continuously measured beat to beat using a task force monitor (TFM; Medizintechnik AG, Graz, Austria).
For the Valsalva maneuver, the test subject blew into a tube connected to the sphygmomanometer to maintain the pressure on the sphygmomanometer at 40 mmHg for 15 seconds. A little hole was created on the tube to allow the air to leak and prevent the glottis from closing during the test. The test was repeated three times to get a repeatable response. 19 The deep breathing test was performed in the supine position because vagal tone is the highest in that position. That test consists of six respiratory cycles lasting 1 minute.
Each respiratory cycle consists of inhaling deeply and uniformly through nose for 5 seconds and then exhaling uniformly for 5 seconds through mouth. 19 The tilt table test stimulates the ANS by orthostatic provocation.
The test begins with the test subject lying in the supine position for 10 minutes. In controlled conditions the tilt table is inclined to 70° so that the subject's head is up, and that position is maintained for BP and heart frequency were monitored during the test. 19  In this study, we used the Valsalva ratio (VR) and respiratory sinus arrhythmia (RSA) to evaluate impaired cardiovagal function.
VR represents the ratio between the highest heart frequency during phase II and the lowest heart frequency during phase IV of the Valsalva maneuver. RSA is defined as a difference between the end inspiration heart frequency and the end expiration heart frequency.
We calculated RSA for each of the six respiratory cycles of the deep breathing test and used the mean value in our analyses. 20 HRV spectral analyses were performed using Kubios HRV 2.2 software (Department of Applied Physics, University of Eastern Finland, Kuopio, Finland). The autoregressive spectral analysis method was used to analyze the frequency domain parameters.
Data used for the HRV analyses were recorded by TFM with a sampling rate of 1,000 Hz. After the recordings were complete, the data were inspected and edited to correct for any missing data. The quality of the data was ensured by using the medium artefact correction option and smoothness priors-based detrending approach (Lambda = 500). HRV was analyzed in three 5-minute windows from the beat to beat data recorded during the test: the first 5 minutes, the 2nd-7th minute, and the last 5 minutes. During the first 10-minute rest period prior to the tilt-table test an HRV analysis was conducted on the most stable 5-minute interval for each test

Statistical analysis
We used IBM SPSS version 25 (IBM Corp., Armonk, NY, USA) for all statistical analyses. We used the Kolmogorov-Smirnov test to determine whether the data were normally distributed. In the numeric data analysis considering distribution, parametric (t-test for dependent samples) and non-parametric (Mann-Whitney test) statistical methods were used. Correlations between certain variables were examined using Pearson's or Spearman's coefficients.
Univariable and multivariable linear regression analyses were used to determine which variables were statistically significant predictors for the tilted SBP. The level of statistical significance was set at P< 0.05.

Association of autonomic and anthropometric parameters
Separate analyses were performed for the supine phase of ANS testing (Table 1), the tilt phase of ANS testing (Table 2), and differences in HR, SBP, and DBP between the tilt and supine phases of ANS testing overall (Table 3), by BMI (Table 4), and by the VR.
Only statistically significant results are presented.

Markers of obesity correlate positively with supine SBP
Markers of obesity (BMI, BF%, SST, W/H ratio, W/Hip ratio) were significantly and positively correlated with SBP. Other anthropometric measures (BH, BW, WC, HC, MUAC) and nHGS were also positively correlated with SBP. Respondents with higher LBM% and BW% had significantly lower SBP. These numerical results are presented in Table 1. We found no significant correlations for supine DBP.

Markers of obesity correlate positively with tilted SBP and DBP
Similar to the results from the supine phase, markers of obesity These numerical results are presented in Table 2.

Markers of obesity correlate positively with differences in HR and DBP between the tilted and supine phases
There was a positive correlation between difference in ∆HR and markers of obesity, such as BMI ( Supplementary Fig. 1), W/Hip ratio (Fig. 1), and the functional marker dHGS. A significant posi-    These numerical results are presented in Table 3. We found no sig-  (Table 4).  Table 6.

BF% is an independent positive predictor and BW% is an independent negative predictor of tilted SBP
We performed univariable and multivariable regression analyses to determine which anthropometric and body composition measures best predict tilted SBP (Table 5 and Supplementary Table 7).
Because of our small number of participants, we calculated several regression models with a maximum of four variables each. In the first and second models, respectively, BF% and MUAC were positive predictors of tilted SBP independent of age, sex, and BMI ≥ 25 kg/m 2 . In the third model, BW% was a negative predictor of tilted SBP independent of age, sex, and BMI ≥ 25 kg/m 2 .

DISCUSSION
The results of this study show statistically significant correlations between autonomic cardiovascular regulation, as measured by changes in SBP, DBP, and HR during the tilt-table test, HR response to the Valsalva maneuver, and HRV, and anthropometric and body composition measures. During the first 10 minutes in the supine position during the tilt-test, SBP correlated significantly and positively with markers of obesity (BMI, BF%, SST, W/H ratio, W/Hip ratio). Contrarily, we found a statistically significant negative correlation between SBP and LBM% and BW%, which are indicators of a high percentage of muscle mass, as well as between DBP and BW%. Therefore, we speculate that in the supine rest position, fat tissue affects SBP. There are several possible explanations for our observed association between BP and body fat composition.
One is the direct influence of fat tissue endocrine regulation on the SNS or blood vessels. 2 On the other hand, higher sympathetic ac- tivity could cause an increase in SBP and fat accumulation. 23 Our observational study cannot determine the causality of the association. Moreover, exogenous factors such as low levels of physical activity also affect SBP and fat accumulation and could explain this observation. 24 We observed several correlations between the tilt-test parameters and stimulation of the heart muscle. 26 Higher ∆DBP than ∆SBP thus confirms the described theory. Because ∆DBP and ∆HR during tilting were significantly and positively correlated with several markers of obesity, we found no significant correlation between obesity markers and ∆SBP.
Another measure frequently used in evaluations of the ANS is the Valsalva maneuver, which is divided into phases that consider the predominance of the SNS and PNS. The VR, indicating cardiovagal activation, is the ratio between the highest pulse during phase II and the lowest pulse during phase IV. 27 The VR correlated significantly and positively with height and dHGS. That could be an indicator of larger chest volume and muscle mass. Mechanical pressure stimulation of skeletal muscles can induce reflex changes in HR and BP, although the neural mechanisms underlying this effect are unclear. 28 A recently published study showed that cardiac sympathetic nerve activity regulates HR responses to muscle mechanical pressure stimulation and that the direction of HR responses depends on the tonic level of the nerve activity, i.e., bradycardia occurs when the tonic activity is high, and tachycardia occurs when the activity is low. 28 On the other hand, we found no correlation between the VR and anthropometric measurements, which are more specific markers of obesity.
The third part of this study evaluated HRV, which is considered to be a positive predictor in the prevention of cardiovascular diseases. It is highest in children and adults with good cardiorespiratory fitness status. 29 We found a correlation between HRV and anthropometric measurements during both the rest and tilt phases of the tilt-test. From the HRV, we calculated SDNN and r-MSSD as markers of HRV in long cycles, pNN50 and HF as markers of the PNS, and the LF/HF which is partly a marker of the PNS but dominantly a marker of the SNS. 21 The negative correlation between ABSI and the HRV markers SDNN and LF during rest indicates that PNS activity might be connected with a lower percentage of adipose tissue. ABSI is affected by BMI and height, but it mostly reflects WC, so it is better marker of obesity than BMI. The importance of these correlations is emphasized by a recent study showing that twelve weeks of aerobic exercise in women with obesity was associated with a significant improvement in HRV parameters, indicating an improvement in ANS function. 30 However, connecting the decrease in PNS activation with obesity must be done with care because other measurements of cardiovagal tonus (e.g., r-MSSD, HF, VR, RSA) were not statistically significantly correlated with markers of obesity in our study.
Finally, we wanted to see whether HR, SBP, and DBP differ depending on BMI. 31 In our study, the tilt-test induced almost double the increase in HR (∆HR) in the group of subjects whose BMI was ≥ 25 kg/m 2 than it did in the group whose BMI was < 25 kg/m 2 .
The group with a BMI ≥ 25 kg/m 2 also had significantly higher SBP during rest and higher SBP and DBP during the tilt-up phase.
That is evidence of a relationship between being overweight and having a higher sympathetic/parasympathetic activity ratio, which has been already described in the literature. 3 Although BMI is simple to calculate, and therefore often used, it does not provide infor- growth hormone secretion peaks compared with people with a similar BMI but lower W/Hip ratio. A higher W/Hip ratio is also connected with elevated pulse pressure and HR. 32 We showed here that ∆HR during tilting also correlated positively with the W/Hip ratio, which again indicates that focusing on changes in HR and DBP during tilting is important. Several previously published studies observed similar connections between obesity markers and ANS parameters. Hillebrand et al. 16 observed that body fat, especially abdominal body fat, is associated with mean HR, corrected QT time, and the ventricular gradient magnitude. Furthermore, in the Olivetti Heart Study performed with 1,079 men, the multiple regression analysis showed that WC remained the strongest independent predictor of BP after adjustment for confounders. 33 In results similar to ours, a study performed with medical students from different populations found that the WC and W/Hip ratio correlated with BP values. 34,35 Taken together, these results indicate that abdominal obesity correlates with higher sympathetic activity and hormonal and metabolic dysregulation through the hypothalamicpituitary-adrenal axis.
This study has several limitations. It is a descriptive, cross-sectional study with a relatively small number of participants. Because of the low number of subjects, it was difficult to address the issue of plausible confounders. Nevertheless, we used a complete, standardized battery of ANS tests and thereby expanded what is known about the ways in which body composition influences ANS.
In conclusion, we found a relationship between higher sympathetic activity, as evaluated by monitoring cardiovascular regulation, and a higher amount of adipose tissue in young healthy persons. The most prominent correlation is a positive one between SBP and waist and HC. In addition, SNS activation, as mediated by the baroreflex during the tilt-up phase of the tilt-test, is also positively correlated with markers of obesity. Therefore, we suggest that in addition to measuring HR and SBP during the tilt-up stress test, clinicians and researchers should also focus on changes in DBP, which could provide additional indications of increased SNS activity in otherwise healthy persons. Remarkably, all the changes in autonomic cardiovascular regulation reported here were already found in young and healthy people, so those parameters could be used as the earliest appearing predictive markers for cardiovascular disease.