During the last decade, a lively discussion has addressed

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1 Oscillatory Patterns in Sympathetic Neural Discharge and Cardiovascular Variables During Orthostatic Stimulus Raffaello Furlan, MD; Alberto Porta, MS, PhD; Fernando Costa, MD; Jens Tank, MD; Lemont Baker, MS; Richard Schiavi, MS; David Robertson, MD; Alberto Malliani, MD; Rogelio Mosqueda-Garcia, MD, PhD Background We tested the hypothesis that a common oscillatory pattern might characterize the rhythmic discharge of muscle sympathetic nerve activity (MSNA) and the spontaneous variability of heart rate and systolic arterial pressure (SAP) during a physiological increase of sympathetic activity induced by the head-up tilt maneuver. Methods and Results Ten healthy subjects underwent continuous recordings of ECG, intra-arterial pressure, respiratory activity, central venous pressure, and MSNA, both in the recumbent position and during 75 head-up tilt. Venous samplings for catecholamine assessment were obtained at rest and during the fifth minute of tilt. Spectrum and cross-spectrum analyses of R-R interval, SAP, and MSNA variabilities and of respiratory activity provided the low (LF, 0.1 Hz) and high frequency (HF, 0.27 Hz) rhythmic components of each signal and assessed their linear relationships. Compared with the recumbent position, tilt reduced central venous pressure, but blood pressure was unchanged. Heart rate, MSNA, and plasma epinephrine and norepinephrine levels increased, suggesting a marked enhancement of overall sympathetic activity. During tilt, LF MSNA increased compared with the level in the supine position; this mirrored similar changes observed in the LF components of R-R interval and SAP variabilities. The increase of LF MSNA was proportional to the amount of the sympathetic discharge. The coupling between LF components of MSNA and R-R interval and SAP variabilities was enhanced during tilt compared with rest. Conclusions During the sympathetic activation induced by tilt, a similar oscillatory pattern based on an increased LF rhythmicity characterized the spontaneous variability of neural sympathetic discharge, R-R interval, and arterial pressure. (Circulation. 2000;101: ) Key Words: nervous system, autonomic catecholamines spectrum analysis tilt-table test During the last decade, a lively discussion has addressed the physiological interpretation of the rhythmic fluctuations that characterize the variability of cardiovascular signals such as heart period or systolic arterial pressure (SAP). Spectral methodology has been essential in assessing the amplitude and frequency of these oscillations, which in numerous and various experimental approaches, seemed to reflect some patterns of autonomic regulation. 1 One possible view holds that low and high frequency (LF R-R and HF R-R ) spectral components of heart rate variability, despite their mixed origin, 2 when measured in normalized units or as LF/HF ratio (ie, in their reciprocal relationship), furnish quantitative markers of, respectively, cardiac sympathetic and vagal modulation. 2 5 However, when LF R-R is measured in absolute units, it does not furnish an index of sympathetic modulation. 6,7 Instead, the LF component (LF SAP ) of SAP variability is a widely accepted marker of sympathetic vasomotor control. 2,8 Similar rhythmic LF and HF periodicities characterize the discharge activity of medullary and sympathetic preganglionic neurons of anesthetized animals 9,10 and postganglionic sympathetic fibers of humans Recently, direct recordings of muscle sympathetic nerve activity (MSNA) furnished evidence that the LF and HF spectral components of neural discharge variability undergo changes correlated to those undergone by the spectral components of the variability of cardiovascular signals during graded changes in arterial pressure obtained with the infusion of vasoactive drugs. 12 However, it is not clear whether a more physiological stimulus that would induce an increase in sympathetic activity (such as upright tilt) would result in similar oscillatory patterns in sympathetic neural discharge and in the spontaneous rhythmicity of the R-R interval and SAP. Furthermore, no data are available on the modifications of the rhythmic components of MSNA or other indices of sympathetic func- Received June 8, 1999; revision received September 7, 1999; accepted September 23, From Centro Ricerche Cardiovascolari, CNR, Medicina Interna II, Ospedale L. Sacco, and Centro LITA di Vialba, Università degli Studi di Milano, Milan, Italy (R.F., A.P., A.M.); the Division of Clinical Pharmacology, Department of Medicine (F.C., D.R.), and the Department of Biomedical Engeneering (L.B., R.S.), Vanderbilt University, Nashville, Tenn; Clinic Bavaria Kreischa, Kreischa, Germany (J.T.); and the Division of Clinical Pharmacology, Dupont Pharmaceuticals Co, Wilmington, Del (R.M.-G.). Correspondence to Dr Raffaello Furlan, Unità Sincopi e Disturbi della Postura, Medicina Interna II, Ospedale L. Sacco, Università di Milano, Via G.B. Grassi 74, Milano, Italy. raffaellof@fisiopat.sacco.unimi.it 2000 American Heart Association, Inc. Circulation is available at 886

2 Furlan et al Neural Sympathetic Oscillations During Tilt 887 tion, such as plasma catecholamine level, during orthostatic stress. These aspects are of paramount importance to understand better the possible abnormalities in the central integration of autonomic control of the cardiovascular system that characterize orthostatic intolerance syndromes such as neurally mediated syncope or chronic orthostatic intolerance. 17 In the present experiments, recordings of MSNA were maintained in control supine conditions and during passive head-up tilt in healthy volunteers. It was thus possible to correlate the rhythmic fluctuations occurring in sympathetic outflow with similar oscillations detected from cardiovascular variables during the most natural stimulus leading to sympathetic excitation and vagal withdrawal, that is, the orthostatic position. Methods Ten healthy, normotensive volunteers (4 women and 6 men aged 27 1 years) with no evidence of organic disease (on the basis of the interview and physical examination) were admitted to the study. In every subject, we recorded the ECG with an AC amplifier and direct arterial pressure with an intra-arterial catheter placed into the radial artery of the nondominant arm. The catheter was connected to a pressure transducer positioned at the midchest level to counterbalance hydrostatic modifications during the tilt procedure. Central venous pressure (CVP) was recorded with a miniature (diameter, 3F) catheter tip pressure transducer (Millar) introduced into a vein of the antecubital fossa and advanced near the right atrium. An intravenous line was positioned in the opposite arm for blood sampling. Placement of intra-arterial, CVP, and venous catheters was done under aseptic conditions and local anesthesia. Respiratory activity was evaluated by means of a thoracic bellows positioned at the midchest level and connected to a pressure transducer. MSNA was obtained by the microneurography technique, as reported in detail elsewhere. 18 Briefly, a unipolar tungsten electrode was placed on the right peroneal nerve near the fibular head for multiunit postganglionic sympathetic nerve recording. A reference electrode was inserted subcutaneously, close to the recording needle. The raw neural signal was amplified (1000-fold amplification), fed to a band pass filter (bandwidth between 700 and 2000 Hz), rectified, and integrated (time constant, 0.1 s) by a nerve traffic analyzer system (Bioengineering Dept, University of Iowa). Neural sympathetic activity, ECG, arterial blood pressure, respiratory activity, and CVP signals were recorded on the paper of an optic chart recorder, concomitantly digitized at 300 samples/s by an analogical to digital board (AT-MIO 16E2, National Instrument), and stored on the hard disk of a personal computer. High-performance liquid chromatography with electrochemical detection 19 was used to measure plasma epinephrine and norepinephrine levels on venous blood samples. Protocol After overnight fasting, subjects were placed on an electrically driven tilt table provided with a footrest. They then underwent the instrumentation procedure as described above. Thirty minutes after instrumentation, continuous signal recordings were initiated for baseline resting condition (15 minutes), and a blood sample was withdrawn for plasma catecholamine evaluation. Thereafter, the tilt table angle was advanced at 15 intervals every 3 minutes until the 75 head-up position was reached. That position was maintained for 30 minutes. A second blood sample was obtained after 5 minutes of 75 tilt. The experimental protocol was approved by the Vanderbilt University Institutional Review Board in Human Research, and written informed consent was provided by all subjects. TABLE 1. Hemodynamic and Respiratory Parameters, MSNA Measurements, and Catecholamine Plasma Levels at Rest and During Upright Tilt Rest Tilt HR, bpm * R-R interval, ms * SAP, mm Hg DAP, mm Hg CVP, mm Hg * RESP, cycles/min MSNA, bursts/min * MSNA, bursts/100 beats * NE, pg/ml * E, pg/ml * Data are mean SEM. HR indicates heart rate; DAP, diastolic arterial pressure; RESP, respiratory frequency; NE, norepinephrine; and E, epinephrine. Tilt data refer to the period from the fifth to the tenth minute of passive orthostatism. *P 0.05 vs rest. Data Analysis Microneurography recordings were considered to reflect MSNA in accordance with criteria previously described. 20 CVP values are referenced to atmospheric pressure; no atrial transmural pressure values are provided because intrathoracic esophageal pressure was not evaluated concomitantly. Figure 1. Spontaneous fluctuations involving postganglionic neural sympathetic activity (MSNA) and hemodynamic parameters in a healthy human at rest and during 75 head-up tilt. Note during tilt that neural discharge activity is grouped after a LF pattern with a period of 10 s, which is coupled with analogous rhythmic fluctuations in blood pressure (BP; ie, Mayer waves) and heart rate (HR; measured in beats/min). A different oscillation at HF (period of 3 s), synchronous with modifications of respiratory activity (RESP), characterized CVP signal major fluctuation. EKG indicates electrocardiogram.

3 888 Circulation February 29, 2000 Figure 2. Examples of spectrum and crossspectrum analyses of sympathetic nerve discharge rhythmicity and respiratory activity. Note 2 oscillatory components in spectrum of MSNA variability. A coherence (K 2 ) value 0.5 is obtained in HF region of cross-spectrum, suggesting breathing periodicity plays an important role in modulating sympathetic neural traffic. PSD indicates power spectrum density; RESP, respiratory activity; and a.u., arbitrary units. Analog data were analyzed offline after analog-to-digital conversion at 300 samples per second per channel. The principles of the software for data acquisition and spectrum and cross-spectrum analysis of R-R interval and SAP variability and respiratory activity have been described in detail elsewhere. 3,21 The methodology we used for signal processing and autoregressive and bivariate spectrum analysis of MSNA variability were previously used to assess the rhythmic content of thoracic efferent preganglionic sympathetic activity in anesthetized cats. 9 Briefly, the integrated sympathetic nerve signal was low-pass filtered by a 2400 coefficients finite impulse response filter with a cut frequency at 0.5 Hz. Thereafter, the neural signal was sampled once per cardiac cycle synchronously with the R-wave peak of the ECG from which the neurogram was obtained. This neurogram, in turn, underwent autoregressive spectrum and cross-spectrum analyses. The power of the LF and HF oscillatory components of R-R interval variability is provided in both absolute (ms 2 ) and normalized units, in accordance with the suggestions of the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. 1 A similar approach was used to express results of MSNA variability spectrum analysis. Absolute values of each component were computed as the integral of the oscillatory components LF (mean, Hz; frequency bands were between 0.04 and 0.12 Hz) and HF (mean, Hz; frequency bands were between 0.16 and 0.38 Hz). Normalization was obtained by dividing the absolute power of each component by total variance (minus the power of the very-low-frequency component) and then multiplying by The use of normalized units for the LF and HF components of R-R interval and MSNA variability was necessary because of the marked changes in these variances when comparing different subjects and experimental conditions. The LF R-R /HF R-R ratio, which is nondimensional, may furnish a frame of reference for studies using different methodologies, and it represents a further global index to explore the sympathovagal modulation of sinoatrial node spontaneous activity. 3 The squared coherence function (K 2 ) was computed as the square of the cross-spectrum normalized by the product of the spectra of the 2 signals. 21 K 2 quantifies the amount of linear link between oscillations with the same frequency contained in 2 different signals. Values of coherence higher than 0.5 were considered significant. Cross-spectrum analyses between respiratory activity and R-R interval, SAP, and MSNA variabilities were also used to identify the respiratory-linked oscillatory component of those signals, thus excluding a possible breathing entrainment of the LF oscillations. Data are expressed as mean SEM. Student s t test for paired observations was used to evaluate the changes produced by the tilt maneuver. Differences were considered significant at P Results Table 1 shows the effects produced by the tilt maneuver on hemodynamics and on the biochemical and neurophysiological estimates of sympathetic activity. During tilt, no major changes could be observed in systemic blood pressure and respiratory rate, although R-R interval and CVP values were reduced. MSNA and plasma norepinephrine and epinephrine levels increased, as expected. Figure 1 illustrates the spontaneous rhythmic oscillations of sympathetic neural discharge and the hemodynamic signals recorded in a subject at rest and during upright tilt. The

4 Furlan et al Neural Sympathetic Oscillations During Tilt 889 TABLE 2. Spectral Measurements of MSNA, R-R Interval, and SAP Variability at Rest and During Upright Tilt Rest Tilt MSNA LF, nu * HF, nu * LF/HF * R-R interval Variance, ms * LF, ms LF, nu * HF, ms * HF, nu * LF/HF * SAP Variance, mm Hg * LF, mm Hg * HF, mm Hg * Data are mean SEM. nu indicates normalized units. *P 0.05 vs rest. neural activity was clustered in series of bursts that tended to occur with a periodicity of 10 s, which mirrored spontaneous LF fluctuations of blood pressure (Mayer waves). A second spontaneous oscillation at the respiratory frequency characterizes CVP variability. The crucial role played by respiratory activity in modulating sympathetic neural traffic is depicted in Figure 2. A large oscillatory component at the same frequency of breathing is evident in the spectrum of MSNA while the subject is recumbent. The linear relationship between the spontaneous periodicity of respiration and MSNA is presented in the coherence diagram (bottom) showing a K 2 value that is 0.5 only in the HF respiratory band. Results of the frequency domain analysis of spontaneous R-R interval, SAP, and MSNA variability are summarized in Table 2. During tilt, the normalized power of the LF oscillatory component of MSNA variability (LF MSNA ) increased, whereas HF MSNA decreased, thus paralleling similar changes in the R-R interval and systolic blood pressure spectral profiles (Figure 3). In addition, the amount of increase in sympathetic neural discharge in response to tilt was correlated to the increase of the normalized power of LF MSNA (Figure 4). The linear relationships between R-R interval, SAP, and MSNA spontaneous variability, as assessed in the LF and HF bands by the peak coherence values, are summarized in Figure 5. At rest in the LF region, 7 of 10 subjects showed K 2 values 0.5 between MSNA and R-R interval variability (mean, ), whereas the K 2 between MSNA and SAP variability was 0.5 in 9 of the 10 subjects (mean, ). During the tilt maneuver, K 2 between MSNA and R-R interval and SAP variability increased (P 0.05) in the LF band in all subjects ( and , respectively), suggesting enhanced coupling between the LF spontaneous fluctuations of each pair of signals. Conversely, during tilt, K 2 decreased in the HF band compared with values in the recumbent position. Finally, peak coherence values in the HF band between respiratory activity and MSNA were unaffected by orthostatic stimulus. Discussion In the present study, we addressed the hypothesis that a common oscillatory pattern might characterize the variability of neural sympathetic discharge, heart rate, and blood pressure in healthy humans during the sympathetic activation that attends orthostatic stress. Our findings suggest that an increase in the LF component of all these variability signals is the hallmark characterizing the enhanced sympathetic modulation that occurs during upright tilt. They also indicate that the physiological increase of sympathetic activity produced by tilt enhanced the coupling between the LF component of sympathetic discharge and the LF components of R-R interval and SAP variability, leaving unchanged the relationship between respiratory activity and MSNA in the HF component. Effects of Tilt on LF Component of MSNA Variability The results of the present study indicate that, at rest, the pattern of sympathetic neural discharge was characterized by 2 major oscillatory components at 0.1 and 0.27 Hz, which were synchronous with analogous fluctuations in R-R interval and SAP spontaneous variability. This observation agrees with previous findings obtained from both animal and clinical studies suggesting that the sympathetic preganglionic 9 and postganglionic discharge activity also display LF and HF oscillatory patterns. In our group of healthy subjects, upright tilt was characterized by the expected reduction of CVP and a marked increase in heart rate, MSNA, and plasma epinephrine and norepinephrine levels, which is suggestive of a remarkable enhancement in overall sympathetic activity. However, we did not assess the contribution of norepinephrine spillover and clearance during tilt 22 in sustaining such high plasma levels of norepinephrine. Similar to what was observed during hypoglycemia, 23 our findings indicate that a generalized increase in neural sympathetic traffic develops as a consequence of a gravitational stimulus. In addition, they also highlight the flexibility of the sympathetic response, which in other peculiar conditions, such as during mental stress 24 and hyperinsulinemia 25 in humans or hemorrhage 26 in animals, may selectively activate only some compartments. In the present study, the tilt maneuver was attended by a clear modification in the pattern of sympathetic neural discharge; this consisted of an increased number of bursts clustered with a periodicity of 10 s, which were detected by power spectral analysis as a marked increase of LF MSNA. Therefore, during tilt, LF MSNA became predominant in the autospectrum, mimicking analogous changes in R-R interval and SAP variability. Importantly, the increase in the sympathetic neural discharge induced by tilt was accompanied by a proportional enhancement of the relative power of LF MSNA,

5 890 Circulation February 29, 2000 Figure 3. Effects produced by tilt on power spectra of MSNA, R-R interval (RR), SAP variability, and respiratory activity (RESP). At rest, 2 oscillatory components characterize spectra of MSNA, R-R interval, and SAP variability. During sympathetic activation induced by tilt, LF component of MSNA increases, thus resembling changes obtained in same oscillatory component of R-R interval and SAP variability. Although HF MSNA amplitude is unaffected by passive orthostatism, its relative power is reduced during tilt. Differences in y-axis scales between rest and tilt in power spectrum density (PSD) of R-R interval variability reflect marked reduction of total R-R interval variance induced by orthostatic stress. a.u. indicates arbitrary units. indicating a link between this oscillatory component and the amount of sympathetic neural firing. These observations agree with the results of previous studies in humans in which baroreflex unloading induced by nitroprusside administration was associated with an increase in MSNA and in the LF component of its variability. 11,12 A 0.1 Hz discharge rhythmicity linked with LF spontaneous oscillations of R-R interval 9,10 and blood pressure has been recorded in anesthetized animals from sympathetic neurons belonging to different areas of the nervous system involved in cardiovascular regulation. Results compatible with ours were obtained in several studies in which stimuli eliciting an enhancement of sympathetic efferent activity, such as inferior cava vein, 9 common carotid bilateral occlusions, 29 or an increase of cerebrospinal fluid pressure, 28 were characterized by the onset of a typical LF oscillatory pattern in sympathetic neural discharge, coupled with pronounced LF fluctuations of systemic arterial pressure. Interestingly, those oscillations persisted after baroreflex afferent inflow was abolished (by a stabilizer device connected to the arterial system of the animal 29 or in dogs with spinal section after bilateral vagal denervation 28 ). These observations suggest that baroreflex activity is not necessary to the genesis of LF fluctuations of both preganglionic sympathetic efferent discharge and blood pressure and that these fluctuations may be the result of the activity of a central oscillator. In keeping with this, the results of a recent study by Cooley et al 30 challenged the necessary role of arterial baroreceptors in generating LF oscillations of R-R interval variability. Indeed, in 2 patients with intractable heart failure, the abolition of spontaneous fluctuations of systemic blood pressure by a left ventricular assist device was characterized by persistent LF components in the power spectrum of R-R interval variability. 30 However, in humans, the importance of baroreflex inhibitory activity on tonic MSNA is well established. In addition, theoretical 31 and experimental 32,33 models have hypothesized a role of baroreceptor mechanisms in the genesis of the LF oscillatory components of both R-R interval and SAP variability. 33 Thus, a limitation of the present study is that we could not differentiate the relative contribution exerted by an intrinsic LF rhythmicity inherent in any sympathetic excita-

6 Furlan et al Neural Sympathetic Oscillations During Tilt 891 Figure 4. Relationship between modifications in MSNA produced by tilt maneuver and concomitant increase in power of LF oscillations of sympathetic discharge activity. Note that increase in sympathetic neural discharge is associated with concomitant increase of power of its LF rhythmicity. n.u. indicates normalized units. tion 2 from the likely concomitant reflex mechanisms occurring during baroreflex unloading. Effects of Tilt on HF Component of MSNA Variability The results of the present study also indicated that the normalized power of HF MSNA decreased during tilt. This pattern suggests that recordings of sympathetic activity might also offer a window to the likely link between HF MSNA Figure 5. Relationship between R-R interval (RR), SAP, and MSNA rhythmic fluctuations, as assessed by coherence function (K 2 ) in LF and HF regions, both at rest (white bars) and during tilt (dashed bars). Tilt increases coupling between signals in LF band, suggesting a preferential pattern of spontaneous fluctuations of signals during sympathetic activation on basis of frequency at 0.1 Hz. HF oscillatory component shows opposite pattern. Tilt does not affect relationship between respiration and MSNA variability. *P 0.05 vs tilt. rhythmicity and parasympathetic central regulation. In keeping with this hypothesis is the previous finding of an increase in HF MSNA during the baroreflex-mediated enhancement of parasympathetic activity obtained by increasing systemic blood pressure with phenylephrine administration. 12 In addition, a reduction of neural sympathetic discharge and a concomitant enhancement of the normalized power of HF MSNA were evident after low-dose atropine infusion; this directly increases central parasympathetic activity by activating central muscarinic receptors. 13 However, the finding in the present study of a high peak coherence between MSNA and the respiratory signal, which was unaffected by modifications in the autonomic profile induced by tilt, suggests a strong link between respiration and sympathetic efferent discharge, which might be mediated by vagal afferents. 34 Regarding this aspect, our results agree with previous observations emphasizing the role of respiratory activity in generating rhythmic oscillations in heart rate and MSNA. 35 Clinical Implications The definition of a pattern of MSNA variability during the gravitational stimulus in healthy subjects may have important clinical implications by furnishing a frame of reference in those conditions characterized by orthostatic intolerance. In fact, manipulation of sympathetic activity by centrally acting drugs such as clonidine and yohimbine worsened or, conversely, ameliorated orthostatic tolerance in patients with neurally mediated syncope. 16 In a group of subjects affected by chronic orthostatic intolerance syndrome, the exaggerated tachycardia in the absence of changes in mean blood pressure during standing have been attributed to a central abnormality in the distribution of sympathetic activity to the vessels and to the heart. 17 It is, therefore, conceivable that in these and related diseases, 36 abnormalities in the sympathetic drive to different cardiovascular target organs may coexist with concomitant impairments in sympathetic discharge rhythmicity. Conclusions The results of the present study indicate that the overall sympathetic excitation induced by upright tilt is characterized by an increase of the discharge activity of the sympathetic peroneal fibers and by a proportional modification of their LF rhythmic pattern of firing. These changes were associated with concomitant relative reductions of HF MSNA and were mirrored by analogous modifications in R-R interval and SAP variability. Thus, a common oscillatory pattern can be detected from the variability of the sympathetic outflow and the cardiovascular target functions. These findings are consistent with the hypothesis of a central-pattern push-pull organization, according to which sympathetic excitation and vagal withdrawal are linked to an enhancement of LF oscillation and, conversely, vagal excitation and sympathetic inhibition are linked to an increase in the HF component. 2 References 1. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability: standards of measurements, physiological interpretation, and clinical use. Circulation. 1996;93:

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