The β-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in β1/β2-adrenergic receptor-deficient mice (β1/β2ADR−/−) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; β1/β2ADR−/−, n = 10] by telemetry. Both WT and β1/β2ADR−/− mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 ± 1.81 vs. 92.11 ± 2.62 mmHg) in β1/β2ADR−/− than WT, HR was 16% lower and day-night differences reduced. Both WT and β1/β2ADR−/− mice adapted to changed salt intake without changed MAP. However, the β1/β2ADR−/− mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the β-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice.
- salt intake
- sympathetic nervous system
the remarkable progress in delineating the molecular mechanisms responsible for circadian periodicity in biological functions (as reviewed in Refs. 16 and 27) has not been accompanied by comparable progress in delineating the effector mechanisms that couple oscillations in expression of the primary clock genes with critical cardiovascular functions, such as heart rate and arterial pressure. Although autonomous regulation of clock genes can be demonstrated in peripheral tissues (13), central control mechanisms appear to be critical for the circadian oscillations in arterial pressure, at least in rodents, since the diurnal variation in blood pressure has been reported to be virtually eliminated by lesions of the suprachiasmatic nucleus (14, 21), leading to the inference that the effector mechanisms for circadian arterial pressure regulation are not tissue autonomous but rather require changes in autonomic activity or a circulating humoral factor triggered by central nervous system activity.
A number of pharmacological and ablative strategies have been employed to study the mechanisms for circadian regulation of cardiovascular function (as reviewed in Ref. 14). In the last 5 yr, the availability of genetically modified mouse strains coupled with radiotelemetry has provided a new approach to this problem. No apparent alteration in the circadian rhythms of arterial pressure was detected in homozygous or heterozygous mice with deletions in angiotensin converting enzyme (2), suggesting that cyclic variation in the activity of the renin-angiotensin system is not required for circadian variation in arterial pressure. The vasodilatory effects of endothelial-derived nitric oxide would also appear not to be critical for diurnal blood pressure cycles since the effect of a targeted deletion of the gene for endothelial nitric oxide synthase is amplification, not a reduction, of the dark-light pressure difference (1, 26). Circadian cycles of arterial pressure also persist in mice lacking cycloxygenase 1, although the magnitude of change is blunted modestly (12).
It has been widely assumed that variation in activity of the sympathetic nervous system or circulating catecholamine levels underlie a major portion of circadian cardiovascular variation. The pharmacological evidence for this hypothesis is extensive although not, in fact, definitive (3, 14, 22). The present studies were undertaken to begin an exploration of the role of the adrenergic system in the generation of circadian variations of blood pressure and heart rate. In view of the importance of the β-adrenergic pathway for cardiovascular regulation the present studies investigated circadian rhythmicity in a mouse strain with a targeted deletion in the β1- and β2-adrenergic receptors (β1/β2ADR−/−) (20). A second goal was to determine the impact of loss of this pathway on the response to variation in dietary salt or interruption of the renin-angiotensin system by enalapril. A third goal adopted during the course of the study was to describe and quantify the impact of locomotor activity cycles on blood pressure in mice. The major findings of these studies are that robust circadian rhythms in heart rate and arterial pressure persist in spite of the absence of the β1- and β2-adrenergic signaling pathway, and that spontaneous locomotor activity is strikingly reduced in β1/β2ADR−/− mice.
We used male and female mice deficient in both β1- and β2ADR−/− originally generated by Rohrer et al. (20). Mice were obtained from Jackson Laboratories (Bar Harbor, ME) and interbred to generate subsequent generations. The background of these animals, as described in the original publication, contains contributions from FVB, C57BL/6, and 129SvJ strains. Mixed background mice need to be used with this double-knockout line because it has been observed that β1/β2ADR−/− mice in a pure genetic background show high perinatal mortality (20). To maximize the similarity of background, control animals [indicated throughout this report as wild type (WT)] were generated from the F2 generation of a cross between β1/β2ADR−/− mice and C57BL/6 animals, as recommended by Jackson Laboratories.
Genotyping was done on tail DNA using PCR. All mice were maintained on a 12:12-h light-dark cycle (LD) and were kept on a standard rodent chow or high- or low-salt diets, as described below, and tap water. All diets were provided ad libitum. Animal care and experimentation was reviewed and approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee (protocol #K058-KDB-07) and was carried out in accordance with National Institutes of Health Principles and Guidelines for the Care and Use of Laboratory Animals.
The telemetry system of Data Sciences International (St. Paul, MN) was used for these experiments. Each transmitter (model TA11PA-C10) was magnetically activated >24 h before implantation. Mice were anesthetized with ketamine and xylazine (90 and 10 mg/kg, respectively), and the left carotid artery was isolated. The tip of the telemeter catheter was inserted into the carotid artery and advanced into the aortic arch with the telemeter body positioned in a subcutaneous pocket on the right flank. One day after surgery each animal was returned to its home cage and provided with ad libitum food and water for the duration of the study. Telemeter calibration was performed following explantation and recorded blood pressure values were corrected for any drift in calibration that may have occurred (25). The telemeter signal was processed using a model RPC-1 receiver, a 20-channel data exchange matrix, APR-1 ambient pressure monitor, and a Data Quest ART Silver 2.3 acquisition system (Data Sciences International). The implanted telemeter was activated on the morning of the 7th–10th day, with recording periods of a minimum of 4 days for each animal in each condition. The sampling intervals used for most of the studies presented here were as follows: at 2-min intervals, the system was set to sample systolic, diastolic, and mean arterial blood pressure, pulse pressure, heart rate, and activity over a 10-s interval and to record their average values. In most studies, the recording room was maintained at 21–22°C with an LD cycle. In 4 WT and 4 β1/β2ADR−/− mice, LD conditions were followed by exposure to 24-h darkness for 7 days (DD). Measurements were made on days 5–7 of DD.
At the time of telemetry implantation, WT mice averaged 28.6 ± 2.3 g and were 17.4 + 1.9 wk old, while β1/β2ADR−/− mice averaged 29.3 + 1.2 g and were 16.7 + 0.2 wk of age. Telemetry studies were undertaken in seven control animals (5 female, 2 male) and 10 β1/β2ADR−/− mice (7 female, 3 male).
Salt diets and enalapril administration.
After baseline studies were obtained, five mice of each genotype were placed randomly on either a high- (8.0%) or low (0.04%)-NaCl diet in a random order. All diets were provided for a 1-wk period prior to telemetry study. In three WT and four β1/β2ADR−/− mice, an additional telemetric study was performed in mice maintained on a low-salt diet for 1 wk with enalapril added to the drinking water (150 mg/l). This regimen provides an average of ∼10 mg/kg/day of the drug.
Initial telemetry data analyses were performed using the analysis program of Dataquest A.R.T. 2.3 (Data Sciences International). Data are expressed as means ± SE. Statistical comparisons were done by paired and unpaired Student's t-test for comparisons of mean arterial pressure (MAP), systolic arterial pressure, diastolic arterial pressure, heart rate, pulse pressure, and mean activity between WT and β1/β2ADR−/− mice. P values <0.05 were considered to indicate a significant difference.
Circadian rhythm of arterial pressure and heart rate.
Both WT and β1/β2ADR−/− mice demonstrated robust circadian variations of arterial pressure and heart rate. A representative recording from each strain is shown in supplemental Fig. S1, and average hourly values are plotted in Fig. 1. While circadian periodicity was maintained, MAP and heart rate were significantly reduced in the β1/β2ADR−/− mice, as summarized in Table 1 and shown in Fig. 2 for 24-h mean values of individual animals. Average 24-h values for MAP were 10% lower, and values for heart rate were 16% lower in β1/β2ADR−/− mice than WT mice. Of note, while circadian rhythms were present, the reduction in the absolute magnitude of the dark-light differences in mean, systolic, and diastolic arterial pressure did achieve significance (see Table 1). As shown in Fig. 3, differences in MAP, heart rate, and activity between objective night time (6 PM–6 AM) and objective day time (6 AM–6 PM) were not different under LD and DD conditions.
A significant reduction in heart rate has been documented previously in the β1/β2ADR−/− mice by telemetry (6). A small reduction in arterial pressure was also observed in the initial phenotypic description of the β1/β2ADR−/− mice (20), but it did not achieve significance, probably due to methodological differences; those measurements were made without the lengthy undisturbed observation possible with telemetry.
The β1/β2ADR−/− mice also demonstrated a striking reduction in locomotor activity that has not previously been reported. A histogram depicting the distribution of activity counts is provided in Fig. 4 (individual activity profiles for five WT and five β1/β2ADR−/− mice are given in supplemental Fig. S2). It can be seen that the most striking difference is the proportion of high activity intervals; the β1/β2ADR−/− mice show no observation intervals with activity counts exceeding 40 (see Fig. 4, inset).
Activity was reduced in both night and day phases of the circadian cycle (see Fig. 2 and Table 1). While some dark-light difference in activity persisted, the magnitude of the change was markedly reduced (Table 1).
Effect of locomotor activity on arterial pressure.
Since β1/β2ADR−/− mice showed markedly reduced locomotor activity, the impact of activity on blood pressure and blood pressure variability was assessed. We calculated the linear regression relationship between activity and MAP, using log(activity + 0.1) as the independent variable. The log transformation reduces the skewing of the activity distribution and addition of 0.1 allows log transformation of values with zero activity (26). Results were as follows: WT, MAP = 8.9 log(activity + 0.1) + 92.3 (r2 = 0.387) and β1/β2ADR−/−, MAP = 6.3 log(activity+0.1) + 99.5 (r2 = 0.096).
The WT slope (8.9 mmHg/log activity) had a confidence interval 8.7 to 9.1, and differed significantly at the P < 0.01 level from the shallower slope (6.4 mmHg per log activity, confidence interval 6.0 to 6.7) observed in the β1/β2ADR−/− mouse, and the contribution of activity to variability was reduced in knockout animals.
We also compared the distribution of MAP measurements in all observation intervals and in intervals in which no locomotor activity was observed. This analysis was possible in five animals of each genotype in which recordings were made with an identical sampling protocol. When all observations were included, most WT animals showed a clear bimodal pattern, as has been observed previously (26), with the higher MAP mode predominant in the dark or active phase and the lower MAP mode dominant in the light phase (Fig. S2, left ). A tendency to bimodality was also present in several of the β1/β2ADR−/− mice but was less prominent (Fig. S.3, right). The bimodal pattern was substantially less prominent when only intervals without activity were considered (Fig. S.4) consistent with the earlier suggestion (25, 26) that the high-MAP mode reflects the impact of periods of high activity. Median values from these analyses are provided in Table 2.
Of significance for the present studies, when only inactive intervals were considered, the magnitude of the circadian shift in MAP was substantially reduced, suggesting that activity is a significant determinant of circadian blood pressure differences. Table 2 summarizes median values. In the WT animals, the circadian shift in median MAP was reduced from a dark-light difference of 18.9 ± 1.5 (all observations) to 7.6 ± 2 mmHg (inactive intervals only). The change in β1/β2ADR−/− mice was more modest, reflecting lower activity levels from 11.6 ± 2.1 to 9.1 ± 2.1 mmHg. During the inactive intervals there was no significant difference between the two genotypes (see Table 2). These data suggest no direct effector role for the β-adrenergic system in circadian regulation of vascular variables, aside from for the indirect consequences of altered locomotor activity.
Impact of low- and high-salt diet and low-salt diet plus enalapril.
Fig. 5 depicts the circadian pattern for MAP, heart rate, and activity measured in both genotypes after animals were maintained on a low- or high-salt diet for 1 wk. Table 3 summarizes mean 24-h values. Neither the WT nor the β1/β2ADR−/− mice demonstrated salt sensitivity of arterial pressure with a high-salt diet, and both strains compensated for a low-salt dietary intake without a fall in MAP. Of note, the low-salt diet provoked a significant increase in nocturnal activity in the WT and a high-salt diet significantly reduced heart rate, but neither change occurred in the β1/β2ADR−/− mice, suggesting that both responses reflect altered β-adrenergic activation. Other variables did not change significantly.
We also assessed the ability of animals of both genotypes to compensate for the additional stimulus of renin-angiotensin system blockade with enalapril in addition to a low-salt diet (Fig. 6). MAP fell to a similar level in the two strains, and circadian variation was maintained.
In the present study we utilized radiotelemetry to explore the role of the β-adrenergic pathway as a potential effector mechanism in circadian rythmicity of cardiovascular function. The most important finding is the persistence of strong diurnal cyclical variations in arterial pressure and heart rate in both male and female β1/β2ADR−/− animals, the β-ADR classes most critical for cardiovascular regulation. The initial hypothesis of these studies, that the sympathetic nervous system, through mediation of β-ADRs, is a critical direct effector of the circadian variation of arterial pressure, either through direct effects on vascular tone or by alteration in heart rate and cardiac output, was supported by substantial indirect evidence. A number of experimental approaches have established that sympathetic activity is greater in the active phase of the circadian cycle, including direct measurement of stellate ganglion activity (11) and spectral analysis of heart rate variability in humans and rodents (7, 28). Circulating catecholamines are higher in the active phase, during the day in humans and during the night in rodents (4, 8). Many pharmacological studies that have compared the effect of administration of β-blockers on arterial pressure in different phases of the circadian cycle have demonstrated that the agents produce larger effects in the active phase (9, 14). Nevertheless, while we also demonstrated a somewhat greater dark-light difference in arterial pressure between the WT and the genetically “β -blocked” animals, the persistence of robust rhythmic variations in pressure and heart rate indicates that there must be other major effector pathways. Our data indicate that locomotor activity is an important determinant of the circadian variation in arterial pressure in animals of both genotypes, and that the modest reduction in the amplitude of circadian arterial pressure cycles in the β1/β2ADR−/− mice is largely a consequence of reduced locomotor activity.
While diurnal cycles in arterial pressure and heart rate were only modestly affected by genetic interruption of the β-adrenergic pathway, locomotor activity was strikingly reduced in the β1/β2ADR−/− mice, particularly in the active phase. The mechanisms responsible for the relative inactivity are unclear. Specifically, our studies do not permit inferences about whether the reduced locomotor activity in the β1/β2ADR−/− mice is a central or peripheral effect. Nonetheless, it is of note that a previous study, which carefully assessed exercise in the β1/β2ADR−/− mouse found no defect in maximal exercise capacity (20). Review of the neurophysiological literature finds substantial support for central effects of the β-adrenergic system on the regulation of wakefulness and activity. β-receptor blocking agents have been shown to depress motor activity in rats and primates (15, 24). Several laboratory groups have examined the effect of disruption of the dopamine β-hydroxylase gene on locomotor activity, sleep phases, and the transitions between wakefulness and sleep (10, 19, 23). This genetic manipulation appears to produce increased non-REM sleep (19) but inconsistent effects on locomotor activity with a telemetric study demonstrating decreased activity (23), while videometric monitoring did not detect differences in activity (19). We did not undertake to assess sleep phases, but would infer, based on the absence of bouts of relatively high locomotor activity in the β1/β2ADR−/− mice, that the differences in activity are not solely accounted for by differences in the portion of time spent asleep.
In the present studies, we observed that changes in dietary salt intake were surprisingly well compensated in the face of genetic interruption of β-adrenergic signaling, with little resultant effects on MAP, heart rate, or circadian variation after 1 wk of dietary manipulation. The mixed background animals used as control for this strain also showed no evidence of salt-induced elevations of blood pressure, suggesting that the absence of salt sensitivity in the knockout mice is not due to absence of β-ADR signaling. In view of the higher C57BL/6 component of WT compared with knockout mice this conclusion is somewhat tentative, since the salt effects on blood pressure in mice are likely to be influenced by genetic background. When we superimposed interruption of the renin-angiotensin system by administration of enalapril, MAP fell to comparable levels in the two genotypes, with preserved circadian rhythms, again indicating the robustness and redundancy of the regulatory pathways controlling arterial pressure.
Perspectives and Significance
Our primary goal in the present studies was to determine the role of the β-adrenergic pathway in circadian variations of arterial pressure. Circadian cycles in arterial pressure are of substantial clinical interest; observational studies in patients indicate that loss of the normal fall in arterial pressure with sleep (nondipping status) is associated with poorer health outcomes, for example, with increased stroke and cardiovascular mortality (5, 18) and increased progression of diabetic renal disease (17). It is therefore important to understand the effector mechanisms for circadian blood pressure cycles, since preservation of normal daily rhythms may potentially offer health benefits. The present studies provide evidence that even complete absence of β-ADRs does not have significant impact on the circadian rhythm of arterial pressure. Whether α-ADRs may play a role in diurnal blood pressure variations needs to be explored in future studies.
This work was supported by the Intramural Research Program of National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). S. M. Kim and Y. Qin are recipients of a Visiting Fellowship from the NIDDK.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 the American Physiological Society