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1- and 2-adrenergic-receptor subtypes on heart rate variability
1Department of Pediatrics, Division of Pediatric Cardiology, and the 2Department of Molecular and Cellular Physiology, Stanford University, Stanford, California; and the 3Department of Pediatrics, Veterans General Hospital, Kaohsiung, Taiwan
Submitted 11 January 2005 ; accepted in final form 13 August 2005
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ABSTRACT |
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-Adrenergic receptors (-ARs) play a major role in regulating heart rate (HR) and contractility in the intact cardiovascular system. Three subtypes (1, 2, and 3) are expressed in heart tissue, and the role of each subtype in regulating cardiac function has previously been determined by using both pharmacological and gene-targeting approaches. However, previous studies have only examined the role of -ARs in the macrolevel regulation of HR. We employed three knockout (KO) mouse lines, 1-KO, 2-KO, and 1/2 double KO (DL-KO), to examine the role that -AR subtypes play in HR variability (HRV) and in the sympathetic and parasympathetic inputs into HR control. Fast Fourier transformation (FFT) in frequency domain methods of ECG spectral analysis was used to resolve HRV into high- and low-frequency (HF and LF) powers. Resting HR (in beats/min) was decreased in 1-KO [488 (SD 27)] and DL-KO [495 (SD 12)] mice compared with wild-type [WT; 638 (SD 30)] or 2-KO [656 (SD 51)] (P < 0.0005) mice. Mice lacking 1-ARs (1-KO and DL-KO) had increased HRV (as illustrated by the standard deviation of normal R-R intervals) and increased normalized HF and LF powers compared with mice with intact 1-ARs (WT and 2-KO). These results demonstrate the differential role of -AR subtypes in regulating autonomic signaling.
chronotropy; sympathetic nervous system; parasympathetic nervous system
-ADRENERGIC RECEPTORS (-ARs) are members of the superfamily of seven transmembrane, domain-spanning, G protein-coupled receptors. Three subtypes have been cloned (1, 2, and 3), and all are expressed in heart tissue and highly conserved across species (13). Although the 1-AR and 2-AR are pharmacologically similar and share many signaling properties when expressed in undifferentiated cell lines (12), there is increasing evidence that their signaling properties diverge when expressed in differentiated cells and in vivo (1, 32). The role of -AR subtypes in regulating heart rate (HR) and contractility have previously been studied by using subtype-specific agonists and antagonists, such as metoprolol for the 1-receptor and ICI 118,551 for the 2-receptor, as well as gene-targeted animals. With the use of 1-AR knockout mice (1-KO), Bernstein's laboratory (25) has previously shown that 1-ARs play the dominant role in adrenergic-mediated increases in HR but do not appear to be essential for the maintenance of a normal resting HR. In contrast, 2-ARs appear to play little role in adrenergic-mediated increases in HR in the mouse (7). Recent in vitro data suggest that 2-ARs may couple to the inhibitory G protein Gi, as well as to the stimulatory G protein Gs. In isolated cardiomyocytes, this 2 response appears to be biphasic, first acting through a PKA-independent pathway to increase contraction rate, then through Gi to decrease it (9). Thus 1- and 2-ARs could have potentially opposite effects on cardiovascular autonomic balance and on HR variability (HRV).
Previous studies have been limited to evaluating the role of -ARs in regulating HR at a fairly gross level and have utilized short periods of interrogation, which may not capture more subtle alterations in HR control. With the use of traditional physiological methodologies, it is only possible to gain a small insight into the finer level of control of HRV by measuring gross changes in HR. The use of time-frequency transformations, such as the fast Fourier transformation (FFT), in frequency domain methods of ECG spectral analysis has led to an increased understanding of HRV by resolving its different components into high-frequency (HF) and low-frequency (LF) powers. Because these components are affected by sympathetic and parasympathetic responses differently, FFT analysis allows the quantification of their contributions to overall HRV (HF variability has been thought to be mediated mostly by the parasympathetic system, and LF variability by both sympathetic and parasympathetic systems) (5, 10, 20). Decreased HRV has been shown to be an independent predictor of increased morbidity and mortality in human heart disease, e.g., in patients after myocardial infarction (5, 17, 19, 22).
With the use of three gene KO mouse lines, 1-KO, 2-KO, and 1/2 double KO (DL-KO), we have examined the roles of -AR subtypes in HRV and in regulating cardiovascular autonomic balance. Based on our previous data on macroregulation of HR, we hypothesized that the 1-AR subtype would be the dominant -AR subtype in regulating HRV. The use of targeted gene disruption for these studies is an important adjunct to standard pharmacological methods, for even with highly selective -AR antagonists it is extremely difficult to block one receptor subtype for prolonged periods in vivo. Chronic administration of -antagonists may result in -AR upregulation, confounding our ability to determine a specific role for -AR signaling. Additionally, the dose of a subtype selective antagonist (e.g., metoprolol) required to completely block its high-affinity receptor (e.g., 1) from the relatively high concentration of norepinephrine released from a sympathetic varicosity (
1 mM) will likely cause some inhibition of the low-affinity receptor (e.g., 2), as well. Finally, -AR antagonists and their metabolites may have significant affinities for nonadrenergic G protein-coupled receptors as well as nonreceptor sites of action. Thus these studies should nicely complement our understanding of HRV obtained by classical pharmacological methods.
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METHODS |
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1-KO, 2-KO, and 1/2-DL-KO mice (7, 23, 24) were constructed by gene-targeting techniques as previously described and compared with wild-type controls (WT). 1-KO and 2-KO mice were bred on a congenic FVB background; DL-KO mice were on a mixed FVB/C57/129 background. In preliminary studies, we did not find significant differences in HRV indexes between these two WT strains. To eliminate the potential effect of gender, we studied only female mice of each genotype. The mean age at the time of study was 16 (SD 4.0) wk, and the mean weight was 27 (SD 3) g before telemetry unit implantation. Mice were housed in cages at 24°C with normal light-dark cycles in full compliance with the animal welfare policy of the Public Health Service and the American Association for the Accreditation of Laboratory Animal Care. The animal research protocol was approved by the Administrative Panel for Laboratory Animal Care at Stanford University. Animal preparation and surgery. ECG recordings were obtained with an implantable telemetric unit (PhysioTel, Data Sciences International, St. Paul, MN). Mice were anesthetized with 2% inhaled isoflurane, and an abdominal midline incision was made on the ventral surface. Two other smaller incisions, one on each side, were made in the pectoral region for suturing of the leads. Subcutaneously, these leads were directed cranially from the abdominal incision with a trochar sleeve and sutured into the pectoral muscles after the sleeve was removed. The 3.5-g wireless radiofrequency transmitter was then inserted into the abdominal cavity. To anchor the implant in place, it was sutured to the abdominal muscles. Skin incisions were sutured, and a warming lamp maintained body temperature for recovery.
Study protocol. Animals were allowed to recover for 2 wk after surgery. ECG recordings (24 h) were obtained in an unrestrained, temperature-controlled environment with the mice housed in separate, isolated cages far away from the stimulation of other animals and with normal light-dark cycles. Mice were free to eat and drink during the recording.
Data acquisition and analysis. ECG signals were recorded from the telemetric unit with the use of an under-cage receiver (Data Sciences International), digitized at a sampling rate of 1 kHz, and fed into a microcomputer-based data acquisition system (MacLab System, AD Instruments, Milford, MA). ECG signal processing was performed with the software program Chart v4.0 and HRV analysis with the HRV plug-in for Chart v4.0 (AD Instruments). Events in each 120-s segment of the recording were detected by using a threshold algorithm. Only stable sinus rhythm was included in the subsequent analysis, and ectopic beats were excluded. We based our algorithm for ectopic beat exclusion on a previous report (29). All epochs were visually analyzed for the integrity of the tracing. Ectopic beats were defined as those that are above or below extreme thresholds (2–3 times above or below the average R-R interval). The R-R values that were in these extremes are not included in the analysis. No averaged or interpolated artificial beats replaced them. HRV measurement and analysis were conducted following published guidelines (29), and standard time- and frequency-domain indexes were derived (Table 1).
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Frequency-domain measures. In the frequency domain, the power spectral density was calculated by applying the FFT to overlapping segments of the resampled data and by averaging the spectral results (29). The FFT was calculated by using 512 points and half overlap with a Hann window. Cutoff frequencies divided the power spectrum into two main parts, LF (0.4–1.5 Hz) and HF (1.5–4.0 Hz) powers (Fig. 1) and were determined by multiplying the standard frequencies used in human studies by 10 to account for HR differences between mice and humans, as recommended by Gehrmann et al. (10). LF and HF powers were normalized (nLF and nHF) to account for differences in total power (TP) between animals by multiplying the power region of interest by 100 and dividing by the difference between TP and very LF (VLF) power (0.0–0.4 Hz) (29).
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-AR KO mice to the WT and other KO mice was performed by ANOVA with Scheffé's post hoc testing. In addition, to study the interaction between -AR subtypes, a multiple ANOVA (MANOVA) was performed by assigning two of the following four variables to each mouse, based on the presence or absence of each receptor subtype: 1+ (including 2-KO and WT genotypes), 2+ (including 1-KO and WT), 1– (including 1-KO and DL-KO), and 2– (including 2-KO and DL-KO). Thus, for example, a 2-KO mouse was assigned the variables 1+ and 2– for this analysis. Data for both ANOVA and MANOVA are means (SD). Differences were found significant at P < 0.05. |
RESULTS |
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1-AR (both 1-KO and DL-KO) had an overall lower HR compared with mice with an intact 1-AR (WT or 2-KO), which was statistically significant by ANOVA (Fig. 2). In contrast, the absence of the 2-AR alone (2-KO) had no significant effect on HR. There was also no evidence (by MANOVA) of a receptor interaction between 1- and 2-ARs in determining this phenotype; HR was increased in mice with an intact 1-AR independent of the presence or absence of the 2-AR.
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1-ARs (Fig. 3). 2-KO mice had similar or only slightly greater standard deviations compared with WT mice, whereas 1-KO and DL-KO mice had the greatest standard deviations.
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1-AR (both 1-KO and DL-KO) increased SDNN compared with WT or 2-KO mice (P < 0.05 by ANOVA), and this was independent of the presence or absence of the 2-AR (Fig. 4A). In contrast, the 2-AR was important in the regulation of SDANN (Fig. 4B). Absence of the 2-AR (2-KO and DL-KO) increased SDANN compared with WT and 1-KO mice (P < 0.05 by ANOVA), and this increase was independent of the presence or absence of the 1-AR, suggesting the importance of the 2-AR in LF variations in HR.
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2-AR (2-KO and DL-KO) resulted in a significant increase in TP, which was independent of the presence or absence of the 1-AR (Fig. 5A). In the frequency domain, we can also gain a better understanding of how the parasympathetic and sympathetic nervous systems differentially contribute to HRV, as appreciated in the LF and HF ranges. Absence of the 2-AR appeared to confer significant increases in LF and HF power, independent of the presence or absence of the 1-AR (Table 2). However, because these increases in LF and HF powers may have been the result of the increase in TP, it was necessary to correct for this difference in TP by normalizing the LF and HF powers. The results for nLF and nHF illustrate the importance of this correction. In contrast to the uncorrected data, for nLF and nHF, it is the 1-AR that is responsible for the differences seen in these normalized power ranges. In the absence of the 1-AR (1-KO and DL-KO), nLF power was decreased and nHF power increased (Fig. 5, B and C).
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1-KO and DL-KO that did not reach statistical significance (P = 0.07). By MANOVA, however, the LF-to-HF ratio was significantly decreased in mice lacking the 1-AR (Fig. 5D). |
DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-AR subtypes in regulating HRV with the use of a gene-targeted approach. Studies with combinations of subtype-specific agonists and antagonists are another means of addressing this question but are potentially limited by the variable degree of subtype selectivity of these pharmacological agents. Furthermore, there is the difficulty of evaluating the effects of chronic receptor blockade given the receptor desensitization that would occur during chronic administration. Previous investigators have determined the feasibility and value of HRV measurements in mice, both in the anesthetized (3) and conscious states (11, 21, 30), as well as under physiological sympathetic, parasympathetic, and complete autonomic blockade (10, 11), but little was known about the individual effects of each -AR subtype on these variables.
The first significant finding of the current study is that the absence of the 1-AR results in a decrease in average HR, when measurement of HR is performed over a full 24-h period. This finding contrasts with our previous data, wherein short (1 min) measurement intervals were used, which suggested that the resting HR of 1-KO mice was unchanged vs. WT mice (24). Thus, over the longer time of interrogation of the present study, a significant difference in HR related to the presence or absence of the 1-AR was revealed. This demonstrates the value of longer acquisition times in revealing subtle phenotypic differences in genetically altered mice.
In the time domain, absence of the 1-AR also increased SDNN, independent of the presence or absence of the 2-AR. This is consistent with a previous study in the mouse wherein nonspecific -antagonists were used (10). In general, a lower HR, as seen in mice lacking the 1-AR, results in increased total autonomic variability, as measured by the SDNN.
In concurrence with studies in humans and dogs (6, 18, 28), our data lend evidence to the hypothesis that the LF component of the HRV power spectrum in mice is at least partially modulated by the sympathetic nervous system, specifically by 1-ARs. In all of our KO models, the LF and HF components are both affected. The fact that the HF component, widely accepted as a marker for parasympathetic modulation, is also altered in our KO mice is most likely attributable to unopposed parasympathetic effects but could reflect the choice of 1.5 Hz as the LF-to-HF boundary (10). Perhaps the best evidence for sympathetic modulation of LF power is in the absence of the 1-AR, where the nLF power was decreased and the nHF power counteractively increased. Previous studies in mice have also delineated the general contribution of sympathetic and parasympathetic input into HRV, in both time and frequency domains, with the use of acute sympathetic blockade. Gehrmann et al. (11) showed that the nonspecific -blocker propranolol increased TP and both LF and HF components. Whereas nLF increased, nHF decreased and the LF-to-HF ratio increased. In a separate study, Gehrmann et al. (11) showed that the administration of propranolol in combination with the muscarinic agonist carbachol increased all time- and frequency-dependent parameters. These responses were blunted in transgenic mice with a reduction in membrane-bound G
protein, suggesting that G plays a role in parasympathetic-mediated HRV. Wickman et al. (31) examined the effect on HRV of deletion of Gi protein receptor kinase-4, the gene encoding the atrial ACh-activated potassium channel (IKACh), which has been implicated in vagal control of HR. In the KO animals, power was reduced in both LF and HF domains.
In studies of enhanced -adrenergic signaling, mice overexpressing Gs
manifested decreases in time-domain and both components of frequency-domain (LF and HF) responses (30). Similarly, mice overexpressing 1-ARs in the atrium exhibit a decrease in variability in both LF and HF components (21).
However, no prior studies in mice have examined the specific roles of 1 versus 2-AR subtypes in HR variability. A human study (2) with a mix of both specific and nonspecific -blockers in patients with heart failure showed increases in both time- and frequency-domain variables with -blockade, but specific receptor effects were not analyzed. Another study (26) in humans evaluated the 1-specific antagonist metoprolol versus the nonselective -blocker carvedilol in a small number of heart failure patients and found that although both -blockers slowed HR, neither affected any of the other time- or frequency-domain variables.
Although our previous data suggest that the 1-AR exclusively mediates autonomic control of HR on a macroscopic level, both 1- and 2-ARs appear to play a role in sympathetic regulation of HRV. Absence of 1- and/or 2-ARs increases total autonomic variability, the 1-AR through both time- and frequency-domain indexes and the 2-AR through the frequency-domain indexes, TP, and ultra-LF power. However, normalization of the data is important for interpreting these results. Although it would appear that the 2-AR influences both HF and LF powers, normalizing to account for differences in TP across KO strains reveals that it is actually the 1-AR, as opposed to the 2-AR, that significantly alters the balance between the two major power spectrum components and, therefore, the parasympathetic and sympathetic relationship.
Some previous studies have questioned the validity of the LF-to-HF ratio in the mouse. Just et al. (16) found that parasympathetic blockade resulted in a dramatic reduction in pulse interval (PI) across all frequencies, although this was most profound in the LF range. Increasing parasympathetic tone similarly increased PI variability in all ranges. Inhibition of sympathetic tone (expected to be similar to the loss of the 1-receptor) resulted in an increase in the PI spectrum in the range above 1.5 Hz, which is similar to our current findings. Increases in sympathetic tone resulted in a decrease in PI variability that the authors state was "at almost all frequencies," although examination of their data shows that this effect was predominantly in the VLF and LF ranges, the only points where it reached statistical significance. Janssen et al. (14) found that atropine resulted in a decrease in mostly LF variability, whereas metoprolol resulted in an increase in variability in the upper end of the LF and the lower end of the HF range up to 3 to 4 Hz. Taken together, these studies indicate that the removal of -stimulation (now known to be specifically 1-AR stimulation) appears to increase the HF range, which agrees with our results.
There are several reasons why our data on frequency-domain responses may differ from these previous studies. First, we normalized our frequency-domain results for changes in TP. Initially, when not correcting for changes in TP, it appeared that the absence of the 2-receptor resulted in increases in LF and HF responses. However, when normalized for the significant changes in TP, the absence of the 1-receptor was responsible for decreases in nLF and increases in nHF responses. Second, Just et al. studied their animals only 48 h after instrumentation. Bernstein's laboratory (8) and others (14, 15) have shown persistent effects of surgery and anesthesia on sympathetic drive and HRV measurements persisting for a minimum of 4 days after surgical procedures. This may have altered some of the HRV responses in the study by Just et al., in particular (as noted by the authors) "the balance between sympathetic and parasympathetic tone." Third, in the study of Just et al., HRV was calculated from the arterial blood pressure tracing, which was obtained with a fluid-filled catheter rather than from a pressure transducer-tipped catheter. In our study, HRV analysis was obtained directly from the ECG. It is possible that the loss of signal from a very small lumen, fluid-filled catheter was enough to affect HRV data, particularly in the HF realm. Finally, the use of a tethered system may have either inhibited spontaneous activity or increased stress similar to the effects of restraint. This is critically important and may explain some of the discrepancy in the literature over the true balance of resting sympathetic and parasympathetic tone in the mouse. Data from Bernstein's laboratory (8) have consistently found true resting HRs in the mouse that have been lower than many that have been reported, as a result of the exceptional care to avoid stressing the animals and to record resting values only when the animal is truly at rest (while not grooming, eating, or engaged in other activity). This is why for the current study we utilized chronically implanted, telemetry-derived ECG tracings that provide the highest possible frequency response and the lowest level of behavioral perturbation. Thus our results suggest that the LF-to-HF ratio may be useful in the mouse, provided that chronic instrumentation with appropriate frequency response is utilized and that results are normalized for changes in TP.
There are several potential limitations of our study. We did not measure blood pressure concomitantly with HRV. However, we have previously performed extensive studies of blood pressure in 1, 2, and 1/2-KO mice and have not found any differences during the conditions experienced during the present study. Thus it is unlikely that blood pressure differences could have affected our HRV analysis. The additional instrumentation required to accurately measure blood pressure during the time epochs studied (24-h recordings) would have likely altered sympathetic tone and confounded our results. Previous studies (27) have suggested that there are strain differences in HRV indexes that could have affected our results, although only for the DL-KO mice. We performed preliminary studies to evaluate strain differences between the two WT strains used in our study and found that the strain-based differences were minor compared with the marked differences between both WT and DL-KO strains. We must also caution the reader to keep in mind potential species differences in -AR regulation of cardiac function. For example, the 2-receptor does not appear to be coupled to adenylyl cyclase in mice, whereas pharmacological studies suggest that it is coupled to adenylyl cyclase in humans. However, given the proliferation of murine models of heart failure in which one of the primary derangements is often -receptor downregulation, it is equally important for us to know the role of these receptors in murine models, even if we exercise caution about their extrapolation to humans.
HRV has been shown to be a strong independent predictor of postmyocardial infarction morbidity and mortality (4, 17, 19), and similar results have been shown for other heart diseases (22), but little is known about the molecular mechanisms of this phenomenon. -AR-blocking drugs have become standard therapy in patients postinfarction and in those with chronic heart failure. By demonstrating that the chronic absence of specific -AR subtypes increases HRV, this study provides a mechanism potentially linking the two clinical observations. Our results, demonstrating differential roles of 1 and 2-ARs in modulating HRV, may be useful in the study of murine models of human cardiovascular disease and in designing future pharmacological therapies, although these results must be placed in the context of species differences in receptor biology.
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FOOTNOTES |
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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.
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REFERENCES |
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 Z. Zuberi, L. Birnbaumer, and A. Tinker The role of inhibitory heterotrimeric G proteins in the control of in vivo heart rate dynamics Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2008; 295(6): R1822 - R1830. [Abstract] [Full Text] [PDF]
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S. M. Kim, Y. Huang, Y. Qin, D. Mizel, J. Schnermann, and J. P. Briggs Persistence of circadian variation in arterial blood pressure in {beta}1/{beta}2-adrenergic receptor-deficient mice Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1427 - R1434. [Abstract] [Full Text] [PDF]
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S. J. Swoap, C. Li, J. Wess, A. D. Parsons, T. D. Williams, and J. M. Overton Vagal tone dominates autonomic control of mouse heart rate at thermoneutrality Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1581 - H1588. [Abstract] [Full Text] [PDF]
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N. Spielmann, A. S. Leon, D. C. Rao, T. Rice, J. S. Skinner, T. Rankinen, and C. Bouchard Genome-wide linkage scan for submaximal exercise heart rate in the HERITAGE family study Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3366 - H3371. [Abstract] [Full Text] [PDF]
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P < 0.05,