Morning Sympathetic Nerve Activity Is Not Increased in Humans
Implications for Mechanisms Underlying the Circadian Pattern of Cardiac Risk
Background The sympathetic nervous system has been implicated in the circadian pattern of myocardial infarction and sudden death. It has been postulated that sympathetic nerve activity is higher in the morning than at other times of the day and that this increase reflects an endogenous circadian pattern or is triggered by changes in posture and the onset of morning activities.
Methods and Results To test these two concepts, we made microneurographic recordings of muscle sympathetic nerve activity in the morning (6:30 to 8:30 am) and afternoon (2:00 to 4:00 pm) in eight healthy subjects (mean age, 42±4 years), and intraindividual comparisons (paired t tests) were made during (1) supine rest, (2) postural changes simulated by lower body negative pressure (LBNP), and (3) activity produced by sustained handgrip. Plasma cortisol, known to follow a circadian pattern, was measured to assess whether normal circadian patterns were present under experimental conditions. Plasma cortisol exhibited a robust circadian variability (plasma cortisol [mean±SEM], am versus pm: 17±1 versus 9±1 μg/dL, P=.008). In contrast, basal muscle sympathetic nerve activity was not higher in the morning compared with the afternoon (group mean sympathetic nerve activity, am versus pm: 38±6 versus 38±6 bursts per minute, P=NS). Similarly, plasma norepinephrine levels were not higher in the morning compared with the afternoon (plasma norepinephrine, am versus pm: 157±17 versus 173±14 pg/mL, P=NS). During postural stress simulated by LBNP, the magnitude of change in sympathetic nerve activity was not higher in the morning compared with the afternoon (LBNP −20 mm Hg, am versus pm: 103±34% versus 157±31%, P=NS). Finally, the magnitude of change in muscle sympathetic nerve activity during the first minute of handgrip exercise (am versus pm: 11±17% versus 8±11%, P=NS) or the second minute of handgrip exercise (am versus pm: 59±34% versus 60±15%, P=NS) was not higher in the morning compared with the afternoon.
Conclusions These findings challenge the concept that sympathetic nerve activity is higher in the morning either during supine rest or during postural changes and activity. We speculate that if the sympathetic nervous system is involved in the circadian pattern of sudden death, this involvement must reflect exaggerated morning end-organ responsiveness to norepinephrine, not enhanced morning sympathetic outflow.
Several large epidemiological studies have reported a circadian pattern of adverse cardiac events, with a peak incidence of myocardial infarction and sudden cardiac death occurring in the morning (6:00 am to 12:00 noon), after a nadir in these events at night.1 2 3 4 Further characterization of this circadian pattern has identified the hour of awakening, rather than the hour in the day, as being most closely related to the occurrence of adverse cardiac events.5 6 The mechanisms underlying this circadian pattern of adverse cardiac events are not known.
A morning surge in sympathetic nerve activity, either due to an endogenous rhythm or in response to morning activities, has been hypothesized to underlie the circadian pattern in cardiac risk.1 2 3 4 5 6 There is indirect evidence supporting this concept. First, β-adrenergic blockers have been shown in several trials to eliminate the morning peak in sudden death and myocardial infarction, whereas other therapies directed at prevention of these adverse cardiac events do not alter the circadian pattern.7 8 9 Second, plasma norepinephrine, which is at its nadir at night during sleep, increases steeply in the morning in association with awakening and the onset of activities.10 11 It is surprising, however, that plasma norepinephrine levels have not been found to be higher in the morning compared with other times during the day in these studies.10 11 Plasma norepinephrine may be too insensitive an estimate of sympathetic nerve activation in humans, since it represents only a small proportion of norepinephrine released at the sympathetic nerve terminal.12
Microneurography is a highly sensitive and specific method of recording directly muscle sympathetic nerve activity in humans.13 Muscle sympathetic nerve activity correlates closely with cardiac sympathetic activity (as measured by the technique of norepinephrine spillover) during rest and in response to isometric exercise.14 Basal muscle sympathetic nerve activity recorded under reproducible conditions has been shown to vary widely among healthy individuals, yet basal intraindividual muscle sympathetic nerve activity remains stable and reproducible when measured on several occasions separated by months or even years.14 15 16 17
This characteristic stability of basal muscle sympathetic nerve activity in an individual is discrepant with the concept that sympathetic nerve activity is significantly higher in the morning, underlying the increased morning incidence of adverse cardiac events. The purpose of the present study was to determine whether sympathetic nerve activity is higher in the morning compared with other times during the day and whether this increase is present at rest or is related to morning postural changes and the onset of morning activities.
After written informed consent was obtained, eight healthy subjects (six men and two women; mean age, 42±4 years) were enrolled in the study. The study protocols were approved by the UCLA Human Subject Protection Committee. Subjects were screened with a medical history and physical examination; all were healthy nonsmokers who did not take medications. Subjects abstained from caffeine ingestion for 24 hours before the experimental studies, but their diet was otherwise uncontrolled.
Measurements and Procedures
Sympathetic nerve activity was recorded directly from the peroneal nerve using microneurography.13 Multiunit postganglionic muscle sympathetic nerve recordings were made using a tungsten microelectrode with a tip diameter of 5 to 15 μm. The signals were amplified by a factor of 50 000 to 100 000 and band-pass filtered (700 to 2000 Hz). For recording and analysis, nerve activity was rectified and integrated (time constant, 0.1 second) to obtain a mean voltage display of sympathetic nerve activity that was recorded on paper. A recording of muscle sympathetic activity was acceptable when (1) weak electrical stimulation through the electrode produced muscle twitch without paresthesias and (2) the mean voltage neurogram revealed narrow-based, pulse-synchronous bursts (signal-to-noise ratio of >2:1) that did not increase during arousal stimuli. Only nerve recordings with similar intraindividual signal-to-noise ratios in the morning and afternoon studies were accepted for analysis.
To detect potential differences in intravascular volume that may underlie differences in sympathetic activation, central venous pressure was recorded directly from a polyethylene catheter inserted into an antecubital vein, with the tip advanced to a vein in the thorax. Position was confirmed by the waveform of the recorded signal and changes in this waveform in response to deep inspiration. The catheter remained in place until the morning and afternoon experimental sessions were completed.
Forearm blood flow was measured using venous occlusion plethysmography. The arm was elevated above the heart level to ensure adequate venous drainage. A mercury-filled Silastic tube attached to a low-pressure transducer was placed around the forearm and connected to a plethysmograph (Hokanson).
Sphygmomanometer cuffs were placed around the wrist and upper arm. The wrist cuff was inflated to suprasystolic levels for 1 minute before flow measurement. At 15-second intervals, the upper arm cuff was inflated above venous pressure for 7 to 8 seconds. The rate of increase in strain reflects the rate of increase in forearm volume and arterial blood flow. Forearm blood flow (in mL · min−1 · 100 mL tissue−1) was determined based on a minimum of six separate readings. Forearm vascular resistance (in U) was calculated by dividing mean arterial pressure (one third of pulse pressure plus diastolic pressure) by forearm blood flow.
Blood pressure was monitored noninvasively with an automatic blood pressure cuff. Heart rate was monitored continuously through lead II of the ECG.
Venous blood samples for norepinephrine and cortisol level measurements were obtained from an indwelling antecubital line. Norepinephrine samples were collected in iced tubes, centrifuged at −5°C, and frozen at −80°C. Norepinephrine concentrations were measured by liquid chromatography with electrochemical detection.18 Plasma cortisol was measured by competitive protein binding.19
Graded lower body negative pressure was used to simulate postural changes. The legs and lower abdomen of the subject were sealed in the lower body negative pressure chamber (University of Iowa, Bioengineering). Negative pressure was sequentially supplied at −5, −10, −15, and −20 mm Hg for 3 minutes at each level.
Sustained handgrip was used to produce exercise. Before the study, maximal voluntary contraction (in kg) was measured in the nondominant arm of each subject. During the study, the subject performed static exercise at 30% of maximal voluntary contraction for 2 minutes.
Each experimental protocol consisted of two sessions performed within 24 hours: (1) morning, between 6:30 and 8:30 am, and (2) afternoon, between 2:00 and 4:00 pm. Subjects were admitted to the UCLA Clinical Research Center the night before the morning study. Before the morning session of each of the three protocols, the subject was not permitted to sit or stand and remained fasting until the session was completed. Before the onset of the afternoon session, the subject lay supine for 30 minutes and was fasting for at least 2 hours. Between the sessions, the subjects were encouraged to engage in usual daily activities, including walking, reading, watching television, and visiting. All experimental protocols were conducted in the same quiet, semidark, thermoneutral (22°C) room.
Protocol 1: Resting Sympathetic Nerve Activity
The goal of this protocol was to compare basal, resting sympathetic nerve activity in the morning versus that in the afternoon. Eight subjects participated in this study, with the morning session performed first. To control for the possibility that the order of the sessions would influence the results, four subjects repeated the study on a different day in the reverse order, ie, afternoon session first. To ensure that subjects were acclimated to the new surroundings, six subjects repeated the study after spending two consecutive nights in the UCLA Clinical Research Center. In this subset, plasma cortisol, which is well established to follow a circadian pattern,20 was measured to ensure that the circadian variability was preserved under experimental conditions. In addition, plasma norepinephrine levels were obtained as an additional measure of sympathetic nerve activity.
The central venous catheter was inserted into an antecubital vein. The blood pressure cuff and ECG electrodes were positioned. The arm was positioned for venous plethysmography, and the leg was positioned for microneurography. After an adequate nerve recording site was obtained, the subject rested quietly for 10 minutes. Then, sympathetic nerve activity, blood pressure, forearm blood flow, and heart rate were recorded for 10 minutes. Baseline muscle sympathetic nerve activity was calculated as the mean during the last 5 minutes.
Protocol 2: Postural Changes Simulated by Lower Body Negative Pressure
The goal of this protocol was to compare sympathetic responses triggered by postural changes in the morning versus the afternoon. Six subjects participated in this protocol.
The subject was positioned in the lower body negative pressure chamber. A central venous catheter was inserted into an antecubital vein. The blood pressure cuff and ECG electrodes were positioned. The arm was positioned for venous plethysmography, and the leg was positioned for microneurography. After an adequate nerve recording site was obtained, the subject rested quietly for 10 minutes. Baseline sympathetic nerve activity, forearm blood flow, blood pressure, and heart rate were recorded for 5 minutes. Graded negative pressure was applied for 3 minutes at each level (−5, −10, −15, and −20 mm Hg). Sympathetic nerve activity, forearm blood flow, blood pressure, and heart rate were recorded during the last 2 minutes at each level for analysis.
Protocol 3: Exercise Simulated by Sustained Handgrip
The goal of the present study was to compare sympathetic responses triggered by exercise in the morning versus the afternoon. Five subjects participated in this protocol.
The blood pressure cuff and ECG electrodes were positioned. The leg was positioned for microneurography. After an adequate nerve recording site was obtained, the subject rested quietly for 10 minutes. Baseline sympathetic nerve activity, blood pressure, and heart rate were recorded for 5 minutes. Sustained handgrip (30% maximum voluntary capacity) was performed for 2 minutes; sympathetic nerve activity, blood pressure, and heart rate were recorded continuously.
Muscle sympathetic neurograms were analyzed independently by both investigators, who were blinded to the subject’s identity and time of the study session. Sympathetic bursts were identified by visual inspection. Interobserver variability was low (correlation coefficient, .9). Muscle sympathetic nerve activity was expressed as burst frequency (bursts per minute) and total activity (U per minute). Statistical analysis was performed using paired Student’s t tests. Absolute changes and percent changes were analyzed, and the results were not different. Values are reported as mean±SEM. Values of P<.05 were considered statistically significant.
Protocol 1: Resting Basal Sympathetic Nerve Activity and Hemodynamic Measurements
In one subject, an adequate sympathetic nerve recording site was not obtainable in the afternoon session; in another subject, morning and afternoon nerve recordings had markedly different signal-to-noise ratios. These subjects successfully completed the protocol on a repeat occasion, in which acceptable sympathetic nerve recordings were obtained during morning and afternoon sessions.
Mean arterial blood pressure, central venous pressure, and forearm vascular resistance were not different in the morning compared with the afternoon, but heart rate was significantly lower in the morning (Table⇓).
Fig 1⇓ shows original records of morning and afternoon muscle sympathetic nerve activities recorded in three supine, resting subjects. Individual data from all patients are shown in Fig 2⇓. Basal sympathetic nerve activity was not higher when measured in the morning compared with the afternoon (group mean sympathetic nerve activity, am versus pm: 38±6 versus 38±6 bursts per minute, P=NS).
We considered the possibility that the order of the sessions would influence the results. Four subjects repeated the study in the reverse order. Basal sympathetic nerve activity was also not higher in the morning when the sessions were performed in the reverse order (am versus pm: 36±3 versus 34±3 bursts per minute, P=NS).
We considered the possibility that difficulty sleeping in new surroundings would alter normal circadian rhythms. Six subjects repeated the protocol after sleeping for two consecutive nights in the UCLA Clinical Research Center. Morning and afternoon plasma cortisol and norepinephrine levels were obtained in this subset of patients on the day of the experimental protocol. In one subject, an adequate nerve recording site was not obtainable; only hemodynamic and humoral data are presented for this subject. Plasma cortisol was higher in the morning compared with the afternoon, indicative of preservation of the circadian pattern under experimental conditions (Fig 3⇓). Again, mean arterial blood pressure (am versus pm: 83±2 versus 85±4 mm Hg, P=NS) and forearm vascular resistance (am versus pm: 24±3 versus 24±3 U, P=NS) were not different in the morning compared with the afternoon, but heart rate was significantly lower in the morning (am versus pm: 60±3 versus 65±3 beats per minute, P=.05). Basal sympathetic nerve activity was not higher when measured in the morning compared with the afternoon (group mean sympathetic nerve activity, am versus pm: 30±9 versus 29±8 bursts per minute, P=NS). Similarly, plasma norepinephrine levels were not higher in the morning compared with the afternoon (Fig 4⇓).
Protocol 2: Postural Changes Simulated by Graded Lower Body Negative Pressure
The magnitude of change in heart rate, mean arterial blood pressure, central venous pressure, and forearm vascular resistance triggered by lower body negative pressure was not higher in the morning compared with the afternoon (Fig 5⇓). The magnitude of the increase in sympathetic nerve activity triggered by lower body negative pressure was not higher in the morning compared with the afternoon (Fig 6⇓).
Protocol 3: Exercise Produced by Sustained Handgrip
In response to sustained handgrip, the magnitudes of change in heart rate and mean arterial pressure measured at peak handgrip exercise were not higher in the morning compared with the afternoon (Fig 7⇓). Similarly, the magnitude of the increase in sympathetic nerve activity during sustained handgrip was not higher in the morning compared with the afternoon (Fig 8⇓).
The major new findings of the present study are that in healthy humans, (1) basal muscle sympathetic nerve activity is not higher in the morning compared with the afternoon, (2) the sympathetic nerve activation triggered by postural changes is not greater in the morning compared with the afternoon, and (3) sympathetic nerve activation during exercise is not greater in the morning compared with the afternoon. These findings that sympathetic nerve activity is not increased in the morning compared with the afternoon are present despite a simultaneous marked increase in morning compared with afternoon plasma cortisol levels, consistent with intact normal circadian patterns under study conditions.20
Previous investigators have examined the circadian pattern of sympathetic nerve activity by studying target organs, such as coronary and peripheral blood vessels, at different times during the day.21 22 23 Panza and colleagues21 measured forearm blood flow in the morning, before postural changes or activity, compared with forearm blood flow in the afternoon or evening in healthy humans. Basal resting forearm vasoconstriction was highest in the morning in 10 of the 12 healthy subjects studied. This effect was presumably mediated by increased morning sympathetic α-vasoconstrictor activity, since infusion of the α-receptor antagonist phentolamine resulted in greater vasodilatation in the morning compared with afternoon or evening. In a follow-up study in patients with coronary artery disease, these investigators found that the ischemic threshold, which was strongly linked to forearm vascular resistance, was lowest in the morning compared with the afternoon and evening.22 These investigators concluded that basal resting sympathetic nerve activity is increased in the morning.21 22
In contrast, Parker and colleagues23 investigated the relationship of morning coronary ischemic events to the time of onset of morning activities. Ischemic events were diagnosed by ambulatory ECG. The number of ischemic events in the morning was not increased during supine rest but rose sharply with the onset of morning activities and the accompanying increase in heart rate. In contrast to Panza and colleagues,21 these investigators concluded that the increased incidence of morning ischemia was directly related to activity and not attributable to a basal increase in morning coronary tone.23
In the present study, we found that basal, resting sympathetic nerve activity was not increased in the morning compared with the afternoon. Similarly, plasma norepinephrine levels were not increased in the morning compared with the afternoon. These findings are consistent with prior microneurography studies that demonstrate the reproducibility over time of muscle sympathetic nerve activity recorded in resting, supine humans14 15 16 17 and with the previously reported studies of plasma norepinephrine, in which resting plasma norepinephrine was not significantly higher in the morning compared with the afternoon.10 11 In the present study, basal resting heart rate was significantly lower in the morning compared with the afternoon, but blood pressure was not different. These results are similar to those of other investigators,23 who have found that the heart rate increases in the morning only after the onset of activities. In many studies, blood pressure has been found to be lowest at night during sleep but not different in the morning compared with the afternoon.24 25 In contrast to the findings of Panza and colleagues,21 we did not find an increase in basal forearm vascular resistance in the morning.
In the present study, postural changes and exercise increased sympathetic nerve activity, but the magnitude of this increase was not greater in the morning compared with the afternoon. Similarly, the magnitude of the increase in heart rate and blood pressure during postural changes and exercise was not greater in the morning compared with the afternoon, consistent with the findings of other investigators.26 In summary, the finding that sympathetic nerve activity recorded at rest and in response to postural changes and exercise is the same in the morning and afternoon challenges the concept that higher morning sympathetic nerve activity underlies the circadian pattern of cardiac risk.
Although in previous studies plasma norepinephrine levels have not been found to be higher in the morning compared with other times of the day, these studies have demonstrated a significant nadir in norepinephrine occurring at night during sleep.10 11 With the use of microneurography, sympathetic nerve activity directly recorded in humans during stage IV non–rapid-eye-movement sleep was less than half the level recorded during wakefulness, thus confirming this early observation based on humoral data that sympathetic activity reaches its nadir at night during sleep.27 28 29 These observations coupled with our findings that sympathetic nerve activity is not higher in the morning compared with the afternoon suggest a different hypothesis to explain the circadian pattern of cardiac risk. We speculate that after the hours of relative sympathetic withdrawal that occur during sleep, the morning restoration of basal sympathetic nerve activity in susceptible individuals leads to a magnified effect compared with the same level of sympathetic nerve activity at other times during the day. Investigations of human vascular and platelet responsiveness to catecholamines support this hypothesis. Hogikyan and Supiano30 reported up-regulation of α-adrenergic responses after short-term pharmacological sympathetic suppression in healthy humans. Tofler and colleagues31 32 measured platelet sensitivity to catecholamines in healthy subjects and found that catecholamine-induced platelet aggregability was increased in the morning after the assumption of upright posture compared with other times of the day.
We studied healthy subjects without known cardiac disease. It is possible that patients with cardiac disease have higher levels of sympathetic nerve activity in the morning compared with other times during the day. This seems unlikely, however, since the prior work that demonstrated heightened platelet aggregability and vascular constriction in the morning was conducted in healthy humans without coronary artery disease.21 31
We studied sympathetic activity in only one vascular bed—skeletal muscle. It is possible that other organs exhibit circadian patterns in sympathetic nerve activity not present in skeletal muscle. The most relevant organ to consider is, of course, the heart. In the present study, however, heart rate was significantly lower in the morning compared with the afternoon, not higher as one would predict if cardiac sympathetic nerve activity were increased. Furthermore, muscle sympathetic nerve activity has been found to correlate highly with cardiac sympathetic nerve activity measured in norepinephrine spillover studies when compared at rest and during handgrip exercise.14 Plasma norepinephrine, which reflects systemic sympathetic activity, was not increased in the morning in the present study. Nevertheless, sympathetic nerve activity is known to exhibit regional differentiation, and it is possible that vascular beds other than the skeletal muscle of the lower extremity do exhibit a circadian pattern of sympathetic nerve activation.
In summary, our findings that sympathetic nerve activity is not increased in the morning compared with the afternoon, either at rest or in response to postural changes or exercise, challenge the concept that higher morning sympathetic nerve activity underlies the circadian pattern of cardiac risk. These findings suggest that if sympathetic nerve activity is involved in the circadian pattern of cardiac risk, this involvement reflects increased morning sensitivity to catecholamines. Possible mechanisms include a potentiation of norepinephrine effects through (1) an up-regulation of adrenergic receptors or postreceptor signal transduction systems30 ; (2) synergy with cortisol, which is known to follow a circadian pattern peaking in the morning and which increases the effects of norepinephrine10 33 ; or, conversely, (3) a morning decrease in counterregulatory systems that normally oppose norepinephrine effects. Further investigations focusing on the cellular mechanisms of circadian patterns in end-organ sensitivity to catecholamines are warranted.
This study was supported in part by US Public Health Service grants 5 MO1-RR-00865-20 and 3 M01-RR-00865-20S1A1; grant-in-aid from the American Heart Association, Greater Los Angeles Affiliate; and the Committee on Research of the Academic Senate of the Los Angeles Division of the University of California. The authors are grateful to the nurses and staff of the UCLA Clinical Research Center for their assistance in these studies.
- Received November 7, 1994.
- Accepted December 12, 1994.
- Copyright © 1995 by American Heart Association
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