INTRODUCTION

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ENZYMATIC DEGRADATION by monoamine oxidase (MAO)-A and -B plays a crucial role in the regulation of tissue levels of serotonin (5-HT), norepinephrine (NE), epinephrine, phenylethylamine, and dopamine. Our laboratory has recently identified (29) a natural mutation of MAO-A occurring in mice with targeted deletion of the MAO-B gene (14). These mice doubly deficient in both MAO isoforms [MAO-A/B knockout (KO)] demonstrate brain levels of 5-HT, NE, dopamine, and phenylethylamine that are increased 8.5-, 2.2-, 1.7-, and 15.7-fold above those noted in adult, wild-type (WT) animals, with levels of the 5-HT metabolite 5-hydroxy-indoleacetic acid essentially undetectable in both the brain and urine.

When these neuroactive compounds are administered acutely, they show cardiac electrophysiological effects, including changes in the dynamics of the sinoatrial node and in the atrioventricular conduction, as well as in the ventricular fibrillation threshold (11, 18, 27, 31, 36). On the other hand, the results of chronic catecholamine elevations observed in patients are more mixed, showing both paroxysmal tachycardia as well as occasional bradyarrhythmias (3, 28, 39). The elevated bioamine levels observed in the MAO-A/B KO mice since early development could certainly alter their cardiovascular autonomic control; however, it is not immediately apparent how the entire system may (or may not) adapt during the animal's lifespan. The purpose of this study was to characterize the cardiac response to this unique amine overload as reflected in heart rate (HR) dynamics.

	METHODS

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Animals

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Surgical Placement of the Telemetry Implants

Recording Protocols

Unperturbed recordings in the home cage. The first group of animals was studied for 36 consecutive hours. A segment of 5 min of continuous ECG was collected each hour.

Novel environment. The same subjects were exposed to a novel environment to investigate possible effects on HR and rhythm. In this paradigm, animals were removed from their home cage and placed into an unfamiliar clear Plexiglas cage (rectangular, 40 × 20 × 16 cm, without bedding, placed under attenuated light). An ECG was recorded during 10 min before, while animals were still in their home cage, as well as during 10 min of residence in the novel environment.

Vagal blockade. Effects of cholinergic blockade were examined in MAO-A/B KO mice and WT mice in their home cages. Twenty minutes of continuous baseline ECG were recorded, after which time the animals were briefly removed and administered 0.9% saline (0.2 ml ip), after which they were returned to their home cages for an additional 60 min of recording. The following day this protocol was repeated, substituting the saline injection for glycopyrrolate (0.1 mg/kg ip), a muscarinic blocker devoid of central effects.

Data Analysis

Temporal statistics. HR variability (HRV) in the time domain was analyzed according to the methods recommended by the task force of HRV in humans (35). For each mouse in its unperturbed home cage environment, mean R-R intervals and standard deviations of the R-R intervals (SD R-R) were calculated over 5 min of recording for MAO-A/B KO mice (n = 8) and WT mice (n = 11). More extended data sampling in four MAO-A/B KO and three WT mice was also undertaken. In these recordings, 5-min windows recorded every hour during 36 consecutive hours were analyzed. The analysis was separated into three night/day/night segments, each of 12-h duration. Mean R-R intervals and SD R-R were then averaged for each mouse across each 12-h period, and group averages (±SE) were calculated across genotypes.

Frequency measures. The spectral domain analysis of the tachogram (35) was done on a frequency range commonly used in rodents (0.03-4 Hz) and was based on ECG recordings of 5-min duration recorded every hour over 50 continuous hours. In brief, the spectral power of the R-R interval was determined from the resampled tachogram using a fast Fourier transform algorithm. To obtain an equidistant time series, the tachogram was resampled by linear interpolation at 40.96 Hz. Spectral power was calculated on 50% overlapped blocks of 4,096 points (i.e., 100 s). Linear trend removal was applied to the time series before calculation of the power to prevent high-frequency (HF) artifacts.

Beat-to-beat dynamics. Fast fluctuations of the R-R intervals were examined using Poincaré plots. This plot is a graph in which consecutive R-R interval pairs are plotted with the nth R-R period plotted in the ordinate against the nth+1 R-R period in the abscissa. This graph can be examined, revealing qualitative information about rate-dependent variability. They were quantitatively analyzed as well by calculation of the entropy of the distribution of the R-R interval pairs. This estimates the degree of the two-dimensional spread of the R-R pairs. Thus this measure equals zero for perfect order, for example, in the case of a perfectly steady pacemaker with identical R-R intervals, and is maximum in the extreme of a completely random sequence of intervals. The technique (25) involves computation of the equation

(1)

where H(i, j) is the entropy, log is the logarithm on base 2, and P(i, j) represents the probability of finding an R-R interval of length i followed by an R-R interval of length j. The sum is done over the normalized probabilities in each and all cells in the array. It is necessary to choose a given partition of the R-R intervals and a maximum range for the distribution, for which we used 1 and 256 ms, respectively. These values determined the maximum entropy to be 16 bits.

	RESULTS

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Short-Term HR Dynamics

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Representative time series of electrocardiogram (ECG; middle) and corresponding R-R intervals (bottom) of a monoamine oxidase (MAO)-A/B knockout (KO) mouse. Top: fragment of the ECG record at a faster time base.

Temporal Statistics of Unperturbed Home Cage Recordings

The group SD of the R-R intervals for the same night/day/night segments were as follows: WT (n = 3), 13.8 ± 2.8, 12.7 ± 2.6, and 13.0 ± 1.7 ms; and MAO-A/B KO (n = 4), 17.7 ± 5.5, 18.3 ± 4.9, and 18.7 ± 5.9 ms (mean SD R-R ± SE). There were no significant genotypic differences (P > 0.2). This was confirmed when mean R-R intervals and SD R-R were calculated over 5-min recordings in a larger sample of animals [MAO-A/B KO (n = 8): mean R-R = 97.6 ± 11.4, SD R-R = 5.8 ± 3.0; WT (n = 11): mean R-R = 104.1 ± 12.1, SD R-R = 6.1 ± 2.3; P value for mean R-R = 0.25, P value for SD R-R = 0.84].

Beat-to-Beat Dynamics

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Poincaré plots in which 500 consecutive pairs of R-R intervals are graphed with the nth R-R interval (x-axis) plotted against the nth+1 R-R period (y-axis). Note that increasing heart rate (HR) results in a decrease in variability, seen as a tightening of the scatter along the diagonal line for short R-R intervals and vice versa. Depicted are examples of an animal with a high entropy (A) and a low entropy (B).

Frequency-Domain Analysis of Home Cage Recordings

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Frequency-domain analysis of HR variability (HRV) during the day (7:00-19:00) (A) and night (19:00-7:00) (B). Relative to wild-type (WT) mice (n = 3), the MAO-A/B KO mice (n = 4) exhibited more pronounced fluctuations at all frequencies, but also an enhanced modulation of the 1-4 Hz range. Denoted are the different frequency ranges: high (HF; 1-4 Hz), low (LF; 0.08-1 Hz), and a very-low-frequency range (VLF; 0.03-0.08 Hz). Each spectrum represents the average of 12 segments of 5-min duration each. Note that the axes are logarithmic.

HRV During a Behavioral Stressor

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Genotypic differences in the effect of a novel environment on R-R intervals. Depicted are the mean R-R ± SD (A) and mean locomotor activity ± SD (B) in MAO-A/B KO mice (n = 8) and WT mice (n = 11). The left series of measurements was taken while animals were unperturbed in their home cages, and the right series of measurements were taken immediately after they were placed in a novel environment (a clear Plexiglas cage). Each data point represents the average over 20 s.

Figure 5 demonstrates the dependence of the SD on the averaged R-R intervals, as recorded in mice of both genotypes in their home cages. Data points for the MAO-A/B KO mice obtained for the home cages clustered around a common regression line (slope = 0.48, y-intercept = 36.4, F value of regression = 117.2, P < 0.00001). This behavior persisted despite shortening of the R-R interval when the animals were moved to a novel environment. In contrast, a similar plot from WT mice did not demonstrate a dependence of SD R-R on R-R interval, and data from home cages and the novel environment appear as two distinct clusters with no continuity between them for this genotype. A similar relationship was found between R-R mean and the root mean square of successive differences (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Dependence of the SD of the R-R interval on mean R-R interval during exposure to a novel environment. The same data points as in Fig. 4 are replotted for MAO-A/B KO mice (A) and WT controls (B).

HRV During Vagal Blockade

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Prolonged tachycardia and increased HRV after cholinergic blockade in MAO-A/B KO mice. Depicted are R-R intervals before and after either intraperitoneal glycopyrrolate (A) or intraperitoneal saline administration (C). The raw data of all R-R intervals of MAO-A/B KO mice (n = 4) and WT mice (n = 8) were acquired over a 20-min baseline, followed by injection (time = 0) and subsequent data acquisition over 60 min. The R-R interval frequency histograms for each postglycopyrrolate (B) or postsaline period (D) are also shown.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Mean R-R intervals and SD before and after administration of either glycopyrrolate (A) or saline (B) (same data as in Fig. 6, A and C). Averages were computed over the entire 20-min baseline or over the entire 60 min after drug administration. Compared with baseline conditions, glycopyrrolate produced a significant (*P = 0.019) reduction in R-R intervals. All other differences were nonsignificant.

	DISCUSSION

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Several relevant differences in HR dynamics were identified in this study between WT and MAO-A/B KO mice. First, in the unperturbed environment of their home cages, spectral analysis of the R-R intervals revealed more pronounced fluctuations at all frequencies in the mutant mice compared with controls, with a particular enhancement of modulation in the range of 1-4 Hz. Temporal statistics obtained in the home cage of the animals showed no significant genotypic difference in HRV or entropies calculated from the respective Poincaré plots. Second, during exposure to the stress of a novel environment, HR increased and HRV decreased in both genotypes. However, a different time course of HR changes was seen in WT mice (sustained tachycardia throughout the observation period) than in MAO-A/B KO mice (initial tachycardia with subsequent return to baseline). That such modulation may be the result of an enhanced vagal tone was suggested by observations that MAO-A/B KO mice responded to cholinergic blockade with a decrease in HRV, as well as a prolonged tachycardia, greater than that observed in WT mice.

The autonomic effects of chronic elevations of the catecholamines or indoleamines remain controversial. Because the genetic mutation in the MAO-A/B KO mice exists from the earliest stages of embryogenesis throughout development, phenotypes examined in adulthood will likely include adaptive changes that occur in response to the absence of the gene (16). The presence of physiological adaptation is increasingly being described in a number of genetic mutant mice, including reports of compensatory changes in the localization, density (30), and sensitivity (7) of postsynaptic receptors, and changes in cell metabolism (38). Reports have, however, usually focused on isomodal changes, that is, changes that occur within the same neurochemical system as that of the genomic ablation. For example, deletion of the MAO-A gene in MAO-A-deficient mice results in a significant isomodal increase in its preferred substrate 5-HT (6, 19) as well as a downregulation of postsynaptic 5-HT_1A, 5-HT_2A, and 5-HT_2C receptors (33). Compensatory changes, however, may also occur in separate neurochemical systems (cross-modal). Sympathetic and parasympathetic regulation of the cardiac rhythm are tightly coupled. The results of the current study suggest that MAO-A/B KO mice compensate their high adrenergic state by an enhanced vagal tone. During behavioral activation elicited by the animal's exploration of a novel environment, both MAO-A/B KO and WT mice demonstrated an initial increase in HR to near maximal levels, with a concomitant decrease in HRV. Over the course of the 10-min observation period, mutant mice displayed a more rapid return to baseline HR along with a marked increase in HRV. We hypothesized that such changes resulted from an exaggerated vagal recruitment whose effect was to decelerate cardiac rhythms in face of the elevated adrenergic tone. In a separate paradigm in which behavioral activation was elicited by the stress of administration of an intraperitoneal injection, suppression of vagal activity with glycopyrrolate completely eliminated this compensatory effect in the mutant mice. In fact, HR remained high (short R-R intervals) and HRV remained low for the duration of 1 h after drug injection. In contrast, WT mice slowed their HR (increased R-R interval), probably by a decrease in adrenergic tone, a mechanism most likely compromised in MAO-A/B KO mice due to their inability to rapidly metabolize excessive tissue levels of catecholamines and indoleamines. Thus the sudden decelerations of HR observed in MAO-A/B KO mice (Fig. 1) were most likely due to enhanced vagal tone rather than to reduced adrenergic tone. This may be relevant to understanding the tachy- as well as bradyarrhythmias reported in patients with pheochromocytoma, in whom chronic elevations of catecholamines are associated with compensatory elevations in vagal tone (10, 24).

HR is well known to be dependent on locomotor activity. In our study, significant differences in the HR response were observed during exposure to the stress of a novel environment despite the absence of a significant genotypic difference in locomotor activity. Although locomotor activity tended to be lower in the mutant animals, differences in the R-R interval persisted even at times toward the end of the exposure, when activity counts were similar. Preliminary results from our laboratory suggest that differences in the cardiac response of MAO A/B KO mice seen in the current study are similar to that obtained when animals are immobilized in a Plexiglas tube maintained on a heating pad set at 36.5°C (personal communication). Such observations suggest that the animal's HR responses to stress are independent of differences in locomotor activity.

In so far as HR and body temperature are closely linked, an important aspect is the thermal response of the animals. A limitation of our current study is that we did not specifically measure temperature regulation or cardiac responses to such factors as cold-induced stress. We believe that possible differences in temperature regulation are not primary in determining the HR alterations observed in the current study. This is supported by the fact that genotypic differences obtained in the spectral analysis were seen both at night as well as during the day, suggesting that such differences are observable independent of the animal's circadian temperature regulation. Furthermore, recent telemetry recordings from our laboratory show that genotypic differences in HR are observed at times during the light phase, when MAO-A/B KO and WT mice show no difference in core temperature or locomotor activity (personal communication).

Changes in sinoatrial rhythm and vagal tone in the MAO-A/B KO mice might occur via several possible mechanisms. Such changes may reflect functional and/or structural abnormalities at the level of the central nervous system, the peripheral nervous system, the vasculature, or sinoatrial node. Increases in parasympathetic tone may be due directly to increased recruitment of existent cholinergic fibers or indirectly through activation of noncholinergic fibers that are pre- and postsynaptic to vagal terminals. It has been suggested, for instance, that activation of presynaptic 5-HT receptors may change neurotransmitter release from vagal afferent neurons and thus may modulate the cardiac reflex responses through mechanisms linked to alterations in vagal tone (17, 22, 32). Furthermore, it is well known that vagal slowing of the HR increases during noradrenergic stimulation. Thus, during infusion of NE characterized by accentuated sympathovagal antagonism, HR becomes remarkably unstable, an observation attributed primarily to sudden vagal bursts or withdrawals (37). ACh and NE at the level of the sinoatrial node show different temporal influences, and it has been suggested that the faster cholinergic response may relate to the more direct G protein-mediated channel pathway of vagal effects as opposed to the slower response of the noradrenergic effects acting through a slower cAMP-protein kinase pathway (4).

Spectral analysis of HRV has been used in the past as a qualitative measure to assess sympathetic/parasympathetic balance. Whereas HF power is widely accepted as a marker of cardiac parasympathetic control, modulated by breathing, low-frequency (LF) power has contributions from both vagal and sympathetic inputs (1, 26, 34). Increased sympathetic activity is characterized by a shift in favor of the LF component, whereas during increases in vagal activity the HF component predominates (12, 20). These HF and LF components present in R-R intervals are highly coherent with muscle sympathetic nerve activity as well as systolic arterial pressure variabilities (12, 23). Alterations in the LF-to-HF ratio with pharmacological blockade suggests that the control of HRV is similar in mice and humans, with the mouse LF component (0.4-1.5 Hz) regulated by both sympathetic and vagal inputs and the mouse HF component (1.5-4 Hz) predominantly parasympathetically mediated (13). MAO-A/B KO mice compared with WT mice showed increased power at all spectral frequencies, with a particular enhancement of modulation in the range of 1-4 Hz, consistent with an overall increase in parasympathetic tone. The increases in the 1- to 4-Hz band are in the range of frequencies of murine respiration (9) and likely reflect an altered respiratory modulation of HRV. Diaphragmatic dysfunction may impair the breathing pattern and, hence, artificially alter respiration as a basic constituent of neurocardiovascular control (13). Recent research has shown that MAO-A-deficient mice (MAO-A KO) demonstrate abnormalities in morphology and activity of the phrenic motoneurons (2, 5). These abnormalities likely originate from an excess of 5-HT, because prenatal treatments inhibiting 5-HT synthesis or treatment with a 5-HT_2A receptor antagonist reinstitute normal phrenic motoneuron morphology and activity in MAO-A KO mice. Whether similar neuronal abnormalities can be observed in MAO-A/B-deficient mice remains to be clarified. Furthermore, spectral differences between MAO-AB KO and WT mice will require more extensive study to delineate, given the small number of animals used in our study.

In conclusion, this study shows that mice deficient in MAO-A and -B demonstrate altered HR dynamics during a behavioral challenge, whereas less pronounced genotypic differences were exhibited in the home cage environment. Pharmacological infusion studies suggest that this differential response might be related to a compensatory enhanced cholinergic tone. The uncovered differences between mutant mice and their WT counterparts may further our understanding of the several pathophysiological consequences of states with high sympathetic/parasympathetic tone.