Resting heart rate and cardiac autonomic tone during passive head-up tilt: a cross-sectional study in 569 subjects without cardiovascular diseases

Background Resting heart rate (HR) and its variability (HRV) reflect cardiac sympathovagal balance that can stimulated by head-up tilting. HRV is significantly influenced by the level of HR, but how much HRV offers additional information about cardiac autonomic tone than HR alone remains unresolved. We examined the relation of resting HR with short term HRV during passive head-up tilt.Methods Hemodynamics of 569 subjects without medications with direct cardiovascular effects and known cardiovascular diseases were recorded using whole-body impedance cardiography, continuous radial pulse wave analysis and electrocardiography based HRV analysis in supine and upright positions. For statistical analyses the study population was divided into tertiles of resting heart rate according to sexes.Results Higher low frequency to high frequency ratio (LF/HF) of HRV (reflecting sympathovagal balance) was associated with higher HR in supine (p<0.001) and upright positions (p=0.008). The outcome was similar when the HRV analysis was based on HR instead of RR-intervals (p<0.001 supine, p=0.012 upright). The lowest HR tertile presented with higher supine to upright increase in LF/HF than the highest HR tertile (1.1 vs. 0.85, respectively, p=0.037).Conclusion Higher resting HR is related to higher LF/HF in supine and upright positions, reflecting higher cardiac sympathovagal balance. Lower resting HR is associated with lower resting LF/HF, but with a more pronounced increase in both HR and LF/HF during head-up tilt, suggesting greater change cardiac sympathovagal balance in response to upright posture.


Hemodynamic measurements
Trained research nurse carried out hemodynamic measurements in a quiet and temperaturecontrolled laboratory. The study subjects were guided to avoid caffeine products, smoking or heavy meals 4 hours prior to the investigation. Before proper measurements the subjects were resting supine about 10 minutes, during which period a tonometric sensor for pulse wave analysis was placed on left radial pulsation, brachial blood pressure cuff on right upper arm for blood pressure calibration, and electrodes of whole body impedance cardiography were placed on the body surface. The following hemodynamic variables were captured in a beat-to-beat fashion during 5 min periods in supine and passive upright position: HR (1/min), pulse wave velocity (PWV, m/s), systemic vascular resistance index (SVRI, systemic vascular resistance/body surface area, dyn*s/cm 5 /m 2 ), cardiac index (cardiac output/body surface area, l/min/m 2 ) and radial blood pressure (mmHg).
For the whole body impedance cardiography measurement the CircMon R -device (JR Medical Ltd., Tallinn, Estonia) and for pulse wave analysis the SphygmoCor pulse wave monitoring system (SpygmoCor PWMx, Atcor medical, Australia) was used. The measurement protocol is described in detail in our previous publications [22,23] and it has been shown to be repeatable and reproducible [24,25]. In addition, PWV values measured using impedance cardiography show good correlation with the tonometric method (r=0.82, bias 0.02 m/s, 95% CI 0.21 to 0.25) [26], cardiac output values measured using whole body impedance cardiography are congruent with the thermodilution method (bias 0.00 l/min, 95% CI -0.26 to 0.26) and the direct oxygen Fick method (bias -0.32 l/min, 95% CI -0.69 to 0.05) [27], and the correlation between stroke volume recordings using impedance cardiography and 3D-echocardiography has been shown to be good (r=0.781, bias 4.1 ml, 95% CI 2.2 to 10.4) [22].
Heart rate variability analysis Beat-to-beat RR-intervals were captured from electrocardiogram recordings by the impedance cardiography device at 200 Hz sampling rate. Then HRV results were analyzed separately from both RR-intervals and HR by the use of Matlab-software (Natick, Massachusetts, USA). First, normal RRintervals were recognized and an interval was considered ectopic or an artifact if it differed by more than 20 % from the previous ones. The artifact RR-intervals were processed by the cubic spline interpolation method [28].
The following HRV parameters were calculated from the measurements of 5 minutes in the supine and 5 minutes in the upright position: (1) time domain HRV parameters: mean of normal to normal RR (NN)-intervals, SDNN (standard deviation of NN-intervals) and RMSSD (square root of mean squared differences of NN-intervals), and (2) frequency domain parameters by the fast Fourier transformation method [3,29]: power in low frequency range (LF, 0.04-0.15 Hz), power in high frequency range (HF, 0.15-0.40 Hz), LF to HR ratio (LF/HF) and total power.
As described above, the magnitude of HRV depends on the prevailing level of HR. Hence, we calculated the HRV parameters also based on the HR data in addition to the RR-interval data using mean of 60/RR-interval (i.e. HR) instead of pure RR-interval values in the calculations. Furthermore, to weaken the dependence of HRV on HR, the HRV power spectra (from RR-interval data) were divided by average RR-interval (avRRI) to the fourth power and these HRV values, total power, HF power, LF power and LF/HF (TP/avRRI 4 , HF/avRRI 4 , LF/avRRI 4 , LFHF/avRRI 4 , respectively) were included in the analyses [17].

Statistical analyses
IBM SPSS Statistics software (version 25, Armonk, New York, USA) was used for statistical analyses and p-values <0.05 were considered significant. Continuous data was reported as means and 95% CI if normally distributed, and as medians and interquartile range if asymmetrically distributed. For statistical analyses the study subjects were divided into three tertiles according to the mean resting HR, determined from the mean of the last 3 minutes during the 5 minutes of supine measurement.
Because HR and HRV are significantly dependent on sex [4,30], the tertiles were determined separately for men and women but for statistical power men and women were analyzed together.
Differences in study population demographics, hemodynamic measurements and laboratory values between the HR tertile groups were examined using analysis of variances (ANOVA). Tukeys HSD post hoc test was performed for homogeneous and Tamhane's T2 post hoc test for nonhomogeneous variables. Natural logarithms of HRV parameters, C-reactive protein and fasting triglyceride concentrations were used in the statistical analyses to normalize their skewed distributions. The proportions of smoking habits (never, previous or current smoker) were compared using the χ 2 test.
The association of continuously measured HR with HRV during supine and upright positions was investigated using linear regression analysis (with natural logarithms of nonlinear HRV parameters).
The other explanatory factors for linear regression analysis were selected as based on the comparisons between the tertile groups (ANOVA). We previously reported that blood pressure, cardiac index and SVRI are significantly related with the resting level of HR (in both supine and upright positions) [22]. Therefore, mean blood pressure from the hemodynamic variables was selected to the regression analyses rather than cardiac index and SVRI together. Linear regression model for supine and upright HR was calculated using RR-interval -related LF/HF ratio and then the analyses were repeated using LF/HR ratio divided by average RR-interval to the fourth power (to weaken the HR dependence) [17].

Study population
The study subjects were divided into tertiles on the grounds of resting HR according to sexes, thus altogether 187 subjects were allocated to the lowest HR group (HR group 1), 190 subjects to the intermediate HR group (HR group 2) and 192 subjects to the highest HR group (HR group 3). From the traditional cardiovascular risk factors, body mass index and fasting plasma glucose were highest, and HDL cholesterol was lowest in the HR group 3 (p<0.05 for all, Table 1). In addition, fasting plasma triglycerides, C-reactive protein and cystatin C were highest in the HR group 3. The lifestyle habits amount of smoking, weekly alcohol consumption or physical activity did not differ between tertile groups (p>0.05 for all, Table 1). The study population demographics are presented in Table 1 and ranges of HR according to tertile groups in Table 2.

Determinants of blood pressure
Mean resting HR in the whole study population was 64/min (95% CI 63, 64) and there was a statistically significant difference in resting HR between sexes: resting HR among men was 63/min (95% CI 61, 64) and among women 65/min (95% CI 64, 66; p<0.001). Mean HR values according to the HR tertiles are depicted in Table 2 and Figure 1. Both systolic and diastolic radial blood pressures were lower in the HR group 1 when compared with HR group 3 (131/75 vs. 141/83 mmHg, respectively, p<0.001 for systolic and diastolic blood pressure, Table 2). Also cardiac index and PWV were lower in the HR group 1 in comparison with other HR tertiles (p<0.001 for all comparisons), while SVRI was higher in the HR group 1 versus the HR tertiles 2 and 3 (p<0.004, for both comparisons, Table 2).

Resting heart rate and its variability analyzed using variance analysis
Median HRV values according to the HR tertile groups are presented in Table 3 Figure 1). Also, SDNN and RMSSD were lower with increasing HR in the tertile groups both supine and upright (p≤0.005 for all group comparisons, Figure 2).
From the frequency domain parameters, total power of variation decreased with increasing HR (p≤0.020, for all group comparisons) and the differences remained similar during posture change from supine to upright (p≤0.034, for all group comparisons, Figure 3A). Both parasympathetic and sympathetic tone related parameters (i.e. power in HF and LF range) were higher among the HR group 1 than in HR groups 2 and 3 (p≤0.004 for all group comparisons, Figure 3B-C). The differences between the HR tertile groups were similar both supine and upright. The LF/HF ratio was lower in the HR group 1 when compared with HR group 3 during supine position (p<0.001 for group comparison, Figure 3D), while the differences between the HR tertiles were not statistically significant during upright position (p=0.116).
The change in LF power from supine to upright position was not significantly different between the groups (p=0.155), but the decrease in HF power was most marked in the HR group 1 and lowest in the HR group 3 (401 ms 2 in group 1, 202 ms 2 in group 2, 117 ms 2 in group 3, p<0.001 for all HR group comparisons). The posture change from supine to upright more increased the LF/HF in the HR group 1 than in the HR group 3 (p=0.037 for the difference). The supine to upright change in total power was not significantly different between the HR groups (p=0.074). The results of the HR tertiles in the analyses of HR based HRV parameters are presented in Supplemental Figures 1-2.
Heart rate variation with the approach to weaken the heart rate dependence HRV parameters analyzed with the approach to weaken the HR dependence (HRV divided by average RR-interval to the fourth power) were all associated with the resting HR tertiles. All of the measured frequency domain HRV parameters (TP/avRRI 4 , HF/avRRI 4 , LF/avRRI 4 , LFHF/avRRI 4 ) were lowest within the HR tertile 1 and highest within the HR tertile 3, both supine and upright (p < 0.001 for all ANOVA tests and post hoc tests, respectively, Figure 4).

Heart rate and heart rate variation in linear regression analyses
The relation of resting and upright HR and HRV was studied in a stepwise linear regression model where the HRV variables (R-R interval related LF/HF and HR relation weakened LFHF/avRRI 4 ) and the statistically significant confounding factors mentioned above were selected as independent variables (i.e. age, PWV, mean blood pressure, sex, BMI, C-reactive protein, triglycerides, HDL cholesterol, fasting plasma glucose and cystatin C). The coefficients of the linear regression analyses are presented in the Supplemental Tables 1-4. Resting HR was positively related with LF/HF (p<0.001 for LF/HF, coefficient Beta for LF/HF 0.299, R 2 =0.237 for the model, Supplemental Table 1). Also upright HR was positively related with LF/HF (p<0.001 for LF/HF, coefficient Beta for LF/HF 0.342, R 2 =0.252 for the model, Supplemental Table 2).
Both supine and upright HR were positively related with the LF/HF ratio also in analyses where the dependence on HR was weakened with the LFHF/avRRI 4 approach (p<0.001, coefficient Beta for LFHF/avRRI 4 0.697, R 2 =0.582 for the model in the supine position, Supplemental Table 3; p<0.001, coefficient Beta for LFHF/avRRI 4 0.690 and R 2 =0.570 for the model in the upright position, Supplemental Table 4).

Discussion
In the present study we showed that lower resting HR predicts greater increase in sympathovagal balance (i.e. LF/HF) during the change from supine to upright posture. In addition, both supine LF and HF power, calculated from RR-intervals, were negatively associated with resting HR level indicating more active autonomic nervous control of HR in subjects with lower resting HR. Even when the dependency of HRV on average HR was weakened by the use of a mathematical method, statistically significant relation with HRV and HR was detected. These findings suggest that lower resting HR is an indicator of more active autonomic nervous system and that HRV is not only a derivative of HR.
On the grounds of the previous studies both HR and HRV are indicators of sympathovagal tone [1,31], and both are also predictors of cardiovascular diseases such as coronary disease [6, 7, 10], heart failure [11, 32-34] and even mortality [7, 12,19,35]. In addition, while the mathematical association of HR with HRV is obvious, their physical relation during stimulation of autonomic nervous tone is not fully understood [13,14,36]. In the present study the relation of HRV with HR was studied in 569 subjects without medications with direct cardiovascular effects or diagnosed cardiovascular diseases during supine and upright position.
There are controversial studies of the relation of HR with HRV [1,13,14,16,18]. The mathematical relation of these two variables is clear, but whether HRV does more represent the level of sympathetic tone than HR alone it is not finally settled. However, our results suggest that low resting HR is associated with decreased LF/HF, e.g. lower sympathetic and higher vagal tonus, independent from the method of calculation. Sacha  High resting HR and decreased HRV are associated with poor cardiovascular health and mortality [5-7, 10-12, 19]. The present study population consisted of rather healthy subjects without cardiovascular diseases and population was divided into tertiles according to the resting HR within each sex. This approach reduced possible differences in the distribution of such cardiovascular risk factors that are significantly influenced by sex. However, subjects with the highest HR also presented with other markers of less favorable prognosis, i.e. decreased HRV (total power) and HDL cholesterol levels, increased triglyceride, fasting plasma glucose, C-reactive protein levels, and higher body mass index (Table 1). In addition, higher resting HR was related with higher blood pressure and pulse wave velocity ( Table 2).

Heart rate variability measurements based on RR-interval
Within the present study population resting HR was inversely related with both sympathetic During posture change from supine to upright vagal tone (HF power) decreased significantly independent from the resting HR level. This finding is consistent with two previous studies where HRV was recorded from 22 and 12 healthy subjects, respectively, during head-up tilt [2,39]. Moreover, SDNN and total power, which represent of total autonomic tone, were negatively related with resting HR during supine and upright recordings, the findings of which are congruent with previous studies [13]. In our study there was no statistically significant change in sympathetic tone (LF power) during posture change from supine to upright in any of the HR groups. Hence, the change in the autonomic tone in response to head-up tilt seems to be more mediated by an alteration in vagal than the sympathetic tone regardless of the resting HR level. Not only during resting position but also during head-up tilt, subjects with lower HR were characterized by more reactive autonomic nervous tone.
Previously Tsuji et al. studied the relation of HRV with cardiovascular events in 2501 patients with a mean age 53 years. They found that reduced LF, HF and total power were significantly related with increased risk for cardiovascular events when the other risk factor were taken into consideration (p<0.05) [35]. In the present study, lower supine LF/HF ratio and the greater change in this variable in response to upright posture may hence reflect better cardiovascular health in the subjects with lower HR.

Information provided by heart rate variability measurements based on heart rate
In addition to common RR-interval based HRV determinations, we also calculated the HRV parameters from the prevailing HR instead of RR-interval values to strengthen the knowledge of the relation between HR and HRV. In the HR based HRV analysis, resting HR was related with lower LF power (sympathetic tone) but HF power (vagal tone) did not differ between the resting HR tertiles during supine position (Supplemental Figure 2). During the head-up tilt a similar decrease in HF power (vagal tone) was detected in all HR groups, but LF power increased more in the lowest HR group when compared with the higher HR groups. Thus, during the upright position the HRV differences almost disappeared within the HR tertiles. The HR based HRV evaluation may more represent cardiac sympathetic influences than the RR-interval based approach. As far as we know, there are no previously published results of the HR based HRV measurements.
The different HRV measurement methods generated opposite results about the association of resting HR with HRV. This can be explained by the negative mathematical relation between HR and RRinterval. In the RR-interval based HRV analyses, the evaluated vagal tone significantly differed between the HR groups ( Figure 1C), but in the HR based HRV analyses there were no differences in vagal tone between the HR groups.
Importantly, regardless of the HRV measurement method (RR-interval based or HR based), lower resting HR was related to a lower supine LF/HF ratio, and a more pronounced change in this variable during head-up tilt, indicating a greater change in the sympathovagal balance in response to upright posture.

Study limitations
It is known that age has effect on autonomic tone In the present study, the mean age was same within the resting HR tertiles (Table 1) and only 22 subjects (8%) were over 60 years of age (range 18-72). All of the study subjects were included in the analyses, and the possibility remains that the effect of age on HRV was not totally eliminated even though age was included as one of the confounding factors in the regression analyses. Also sex has marked effect on autonomous tone and reduced HRV has been observed male subjects [4,20,30]. The sex-specific HR tertiles were constructed in order to avoid powerful confounding of sex on the results.
Many medications, especially beta blockers, have direct effect on HR and other hemodynamic variables and also on HRV [42,43]. In the present study subjects with cardiovascular medications were all excluded. However, not only cardiovascular medications but also for example antidepressants have effects on HRV [44] and also mental illnesses have been associated with reduced HRV [8,9]. The present study included subjects who were medicated with antidepressants but all were in the stable phase of the disease. In addition to posture, breathing frequency is associated with HRV via autonomous activity [4]. In the present study the respiratory frequency heart rate variability in evaluating cardiovascular regulation. A critical appraisal.      Heart rate variability calculated from RR-intervals: natural logarithm (Ln) of supine and upright total power (A), low frequency power (B), high frequency power (C), and low to high frequency power ratio (D) according to the resting heart rate (HR) tertiles; n=569, †p<0.05, *p<0.001, analysis of variance with Tukey's HSD post hoc test.

Figure 4
Heart rate dependence on heart rate variability weakened by dividing heart rate parameters by average RR-intervals to the fourth power [17]: natural logarithm (Ln) of supine and upright total power (A), low frequency power (B), high frequency power (C), and low to high frequency power ratio (D) according to the resting heart rate (HR) tertiles; n=569, *p<0.001, analysis of variance with Tukey's HSD post hoc test.

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