Am J Physiol Heart Circ Physiol 294: H1581–H1588, 2008.
First published February 1, 2008; doi:10.1152/ajpheart.01000.2007.
Vagal tone dominates autonomic control of mouse heart rate
at thermoneutrality
S. J. Swoap,1 C. Li,1 J. Wess,2 A. D. Parsons,3 T. D. Williams,4 and J. M. Overton3
1
Department of Biology, Williams College, Williamstown, Massachusetts; 2Laboratory of Bioorganic Chemistry, National
Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland; 3Department of Biomedical Sciences, Florida
State University, Tallahassee, Florida; and 4Department of Biology, Emmanuel College, Boston, Massachusetts
Submitted 29 August 2007; accepted in final form 26 January 2008
Swoap SJ, Li C, Wess J, Parsons AD, Williams TD, Overton JM.
Vagal tone dominates autonomic control of mouse heart rate at thermo-
neutrality. Am J Physiol Heart Circ Physiol 294: H1581–H1588, 2008.
First published February 1, 2008; doi:10.1152/ajpheart.01000.2007.—It
is generally accepted that cardiac sympathetic tone dominates the
control of heart rate (HR) in mice. However, we have recently
challenged this notion given that HR in the mouse is responsive to
ambient temperature (Ta) and that the housing Ta is typically 21–
23°C, well below the thermoneutral zone (⬃30°C) of this species. To
specifically test the hypothesis that cardiac sympathetic tone is the
primary mediator of HR control in the mouse, we first examined the
metabolic and cardiovascular responses to rapid changes in Ta to
demonstrate the sensitivity of the mouse cardiovascular system to Ta.
We then determined HR in 1) mice deficient in cardiac sympathetic
tone (“␤-less” mice), 2) mice deficient in cardiac vagal tone [musca-
rinic M2 receptor (M2R⫺/⫺) mice], and 3) littermate controls. At a Ta
of 30°C, the HR of ␤-less mice was identical to that of wild-type mice
(351 ⫾ 11 and 363 ⫾ 10 beats/min, respectively). However, the HR
of M2R⫺/⫺ mice was significantly greater (416 ⫾ 7 beats/min),
demonstrating that vagal tone predominates over HR control at this
Ta. When these mice were calorically restricted to 70% of normal
intake, HR fell equally in wild-type, ␤-less, and M2R⫺/⫺ mice
(⌬HR ⫽ 73 ⫾ 9, 76 ⫾ 3, and 73 ⫾ 7 beats/min, respectively),
suggesting that the fall in intrinsic HR governs bradycardia of calor-
ically restricted mice. Only when the Ta was relatively cool, at 23°C,
did ␤-less mice exhibit a HR (442 ⫾ 14 beats/min) that was different
from that of littermate controls (604 ⫾ 10 beats/min) and M2R⫺/⫺
mice (602 ⫾ 5 beats/min). These experiments conclusively demon-
strate that in the absence of cold stress, regulation of vagal tone and
modulation of intrinsic rate are important determinants of HR control
in the mouse.
telemetry; intrinsic heart rate; cold stress; caloric restriction; sympa-
thetic nervous system
THE FREQUENCY OF CARDIAC CONTRACTION in mammals is
achieved through modulation of heart rate (HR) around its
intrinsic rate (IHR). HR is slowed by parasympathetic nervous
system activity via the muscarinic M2 receptor (M2R) (17) and
elevated by sympathetic nervous system activity via the ␤1-
adrenergic receptor (39). Blockade of these autonomic inputs
indicates that murine IHR is typically about 500 beats/min,
well below the generally reported resting HR of about 600
beats/min [reviewed in Ref. 27, supporting the concept that
cardiac sympathetic tone predominates at rest in the mouse (19,
27)]. However, some recent studies indicate that resting mouse
Address for reprint requests and other correspondence: J. Michael Overton,
Dept. of Biomedical Sciences, 3350-E College of Medicine, Florida State
Univ., Tallahassee, FL 32306-4300 (e-mail: mike.overton@fsu.edu).
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HR is lower than 500 beats/min at rest, calling into question the
sympathovagal balance controlling HR in mice (6, 11, 35).
Our laboratories (48, 54 –56) and others (8, 52) have dem-
onstrated that mouse HR is markedly reduced when experi-
ments are conducted in thermoneutral conditions [ambient
temperature (Ta) ⫽ 30 –32°C]. At this temperature, HR is
generally in the range of 350 – 400 beats/min, in contrast to the
range of 550 – 600 beats/min when studied at the typical Ta
used for in vivo mouse physiology (21–23°C). Our prevailing
hypothesis has been that the tachycardia associated with a cool
Ta reflects the recruitment of sympathetically mediated non-
shivering thermogenesis (13, 23, 33, 51). We have shown that
when female Swiss mice are acclimated to various ambient
temperatures for 48 h, a strong positive and linear relationship
between HR and Ta prevails (48). One purpose of this study
was to test the hypothesis that very rapid changes in Ta would
produce concurrent rapid responses in metabolic rate and HR
in mice. The finding would provide important additional evi-
dence, supporting the need to carefully monitor and control Ta
during the assessment of cardiovascular function in mice.
Caloric restriction is both a strategy voluntarily undertaken
by millions of Americans attempting to lose weight and an
experimental intervention known to extend life span in worms
and rodents (43). Caloric restriction elicits a complex set of
physiological effects consistent with a general homeostatic
survival mechanism in response to reduced energy availability
(9). These responses include reduced sympathetic activity to
most organs including the heart (36, 58), increased cardiac
vagal activity (1, 24), and decreased HR and blood pressure
(15, 57, 59). An additional purpose of this study was to further
analyze the relative contributions of the sympathetic and para-
sympathetic nervous systems to the bradycardia of caloric
restriction.
In addition to alterations in autonomic tone, it is also
possible that Ta could modulate IHR. Several endocrine/para-
crine signals, including thyroid hormones (18), prostanoids
(50), angiotensin (11), and oxyntomodulin (44), influence IHR.
Thus we determined IHR in both thermoneutrality and at
standard laboratory temperatures. To determine the respective
roles of sympathetic and parasympathetic control in murine
HR, we determined HR, HR variability, and IHR for mice
lacking M2 and ␤-adrenergic receptors at both thermoneutral
and standard temperatures. After observing that Ta can influ-
ence IHR, we determined whether the bradycardia of caloric
restriction is also associated with a decrease in IHR. Therefore,
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H1581
 H1582 AUTONOMIC CONTROL OF MOUSE HEART RATE
the primary objective of this work was to gain an enhanced
understanding of the autonomic control of HR in mice. The
results reveal that, in addition to increased sympathetic tone,
reduced cardiac vagal activity and increased IHR contribute to
the increase in mouse HR when studied at Ta below thermo-
neutrality.
MATERIALS AND METHODS
Mice and Surgical Instrumentation
Male C57BL/6J (n ⫽ 7; Charles River) were used for temperature
ramp studies. Male mice lacking all three ␤-adrenergic receptors
(“␤-less” mice), maintained on a C57Bl/6J background (␤-less, n ⫽ 8;
and wild-type controls; n ⫽ 12) were shipped to Florida State
University for physiological assessment (generously provided by Eric
Bachman, Harvard University). Mice lacking M2 receptors main-
tained on a C57Bl/6NTac background (M2R⫺/⫺, n ⫽ 8) and wild-type
controls (n ⫽ 8) were shipped to Williams College for physiological
assessment (provided by J. Wess, National Institute of Diabetes and
Digestive and Kidney Diseases). Mice were studied at 3 to 4 mo of
age. Animal experimentation was approved by local Institutional
Animal Care and Use Committees and was in accord with the
American Physiological Society’s “Guiding Principles in the Care and
Use of Animals.”
Mice were anesthetized (halothane, 1–2% in 95% O2-5% N2
mixture) and instrumented with either an ECG telemetry device
(TA10-F20; Data Sciences, St. Paul, MN) or with a blood pressure
telemetry device, with the catheter placed in the left common carotid
artery (TA11PA-C20; Data Sciences) for cardiovascular assessment
(48, 56). For the implantation into the carotid artery, we used the
procedures described by Butz and Davisson (10). Mice were individ-
ually housed using a 12-h:12-h light-dark cycle and were allowed to
recover from the surgery for a minimum of 10 days. Mice were
provided with ad libitum access to water and standard rodent chow
during all testing with the exception of caloric restriction studies.
Indirect Calorimetry and Activity Monitoring
The apparatus and procedures used for determination of metabolic
rate and locomotor activity have been described previously (57).
Metabolic cages were placed within environmental chambers that
provided computer-controlled Ta and lighting conditions for four
individual cages (37). Individually metabolic cage Ta was monitored
by a thermister mounted on the inside of the polycarbonate cage lid.
Briefly, a computer controls individual cage heaters and the cooling
compressor of the environmental chamber to achieve the desired
individual cage Ta. Oxygen consumption (V̇O2) and carbon dioxide
production (V̇CO2) were measured every 2.5 min by open circuit
respirometry using a modification of the approach described by
Bartholomew et al. (5) to isolate successive samples. The flow rate of
air into the chamber was set at 1 l/min. V̇O2 was calculated every 4
min. Locomotor activity was measured using a custom-designed force
platform as described previously (57). Total activity, measured in
meters, was accumulated in 30-s periods and stored with a 1-cm
resolution.
Telemetry Monitoring
The radiotelemetry signal was continuously detected (1,000-Hz
sample rate) and either analyzed with Data Sciences analysis software,
with HR determined by R-R intervals, or were routed to a pulse
detection unit connected to a computer. Custom-written software
determined arterial blood pressure, and the time between diastolic
nadirs, measured to the nearest millisecond, was used to determine the
interbeat interval and HR. Both methods of HR assessment result in
equivalent measurements of HR.
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To assess cardiovascular variability in the time domain, the stan-
dard deviation of the interbeat interval (SDNN) was calculated from
home cage recordings obtained throughout the light phase of the
circadian cycle. The light phase was selected because it coincides with
a period of generally low locomotor activity and is consistent with the
time period during which analysis of cardiovascular variability is
typically performed in mice (19, 26, 30). Light-phase SDNN was
determined from a minimum of 10 measurements/h calculated over
intervals of 5–30 s each hour. The reported value for total light-cycle
SDNN represents an average of at least 110 determinations per
animal.
IHR Determination
IHR was measured by either intraperitoneal injection of a cocktail
containing metoprolol (10 mg/kg; ␤1-receptor blocker) and atropine
(4 mg/kg; muscarinic receptor blocker), or with the ganglionic
blocker, chlorisondamine (2.5 mg/kg). The IHR was taken as the
minimum HR over the next 30 – 60 min. Both of these methods have
been used to determine IHR, and each method results in an identical
IHR measurement (S. J. Swoap, personal observations).
Protocols
Experiment 1: cardiovascular responses of B6 mice to rapid
changes in Ta. Studies were performed using B6 mice instrumented
with arterial catheters and housed in indirect calorimeters to allow
assessment of mean arterial pressure (MAP), HR, V̇O2, and locomotor
activity. After mice completely recovered from surgery and accli-
mated to initial housing conditions (Ta ⫽ 30°C), a rapid temperature
ramp protocol was initiated without handling or disturbing the mice.
The temperature changes were performed during the middle of the
light phase and were completed in 1.5 to 2 h. The first ramp consisted
of cooling from 30° to 20°C. The Ta was kept at this level for 22 h,
and the ramp was reversed by warming back to 30°C.
Experiment 2: influence of Ta on resting HR and IHR in control,
␤-less, and M2R mice. All mice were housed at 23°C for 4 days.
Resting HR was derived from a 10-h average of light-cycle data
obtained from the final day of housing at 23°C. IHR was then
measured, using either double autonomic blockade or ganglionic
blockade, 4 h before the onset of the dark cycle of the fifth day. Mice
were then housed at thermoneutrality (Ta ⫽ 30°C) for 4 days.
Light-cycle HR was recorded, and IHR was again measured.
Experiment 3: influence of caloric restriction on resting HR
and IHR. To determine the relative contribution of decreased sympa-
thetic and increased vagal tone to caloric restriction-induced brady-
cardia in mice, we measured the HR, IHR, and SDNN in the same
groups of mice before and after caloric restriction (consuming 70% of
ad libitum food intake) at 30°C. Baseline caloric intake was measured
during the 4 days at Ta ⫽ 30°C. Mice were then calorically restricted
to 70% of ad libitum food intake for 10 days. A final measurement of
light-cycle HR and IHR was obtained 4 h before the onset of the dark
cycle.
Data Analysis and Statistics
The physiological parameters measured of the littermate controls
for the ␤-less mice were not significantly different from those mea-
sured from the littermate controls of the M2R⫺/⫺ mice (both control
groups were B6 mice). Hence, both sets of control mice were pooled
into a single control group. All results are reported as means ⫾ SE.
Differences in baseline variables between knockout mice and wild-
type controls were assessed by Student’s t-tests. The effects of
increased Ta and subsequent caloric restriction were statistically
assessed by repeated-measures ANOVA. Tukey post hoc tests were
used to determine significant differences between means. Significance
levels of P ⬍ 0.05 were accepted.
294 • APRIL 2008 • www.ajpheart.org
 AUTONOMIC CONTROL OF MOUSE HEART RATE
RESULTS
Experiment 1: Mouse HR and MAP Exhibit Rapid Response
to Changing Ta
In the first phase of this experiment, the Ta within the
metabolic cage was cooled from 30° to 20°C in 1 h. Figure 1
shows that HR (Fig. 1A), V̇O2 (Fig. 1C), and MAP (Fig. 1E)
responded rapidly to decreasing Ta. Within 90 min, HR in-
creased from 300 to 600 beats/min, V̇O2 increased from 0.75 to
1.5 ml/min, and MAP increased from 75 to 105 mmHg. These
responses were generally independent of locomotor activity
(Fig. 1G). The elevated levels of HR, V̇O2, and MAP achieved
promptly after cooling were sustained for the subsequent 20 h
as evident in Figs. 1, B, D, and F. In the second phase of this
experiment, Ta within the metabolic cage was warmed from
20° to 30°C in 2 h. HR, V̇O2, and MAP rapidly decreased in
these mice as the Ta was warmed from 20° to 30°C, with those
changes being independent of locomotor activity (Fig. 1H).
Experiment 2: Vagal Tone Suppresses HR Below Intrinsic
Levels at Thermoneutrality
Figure 2A depicts HR over dark and light cycles at a Ta of
23°C in control B6 mice, mice lacking sympathetic influence to
the heart (␤-less), and in mice lacking parasympathetic influ-
ence to the heart (M2R⫺/⫺). At this cool Ta, control mice
exhibited the expected high light-cycle HR of 604 ⫾ 10
beats/min. M2R⫺/⫺ mice also displayed a high light-cycle HR
at this same Ta (602 ⫾ 5 beats/min). However, ␤-less mice had
significantly lower light-cycle HR (442 ⫾ 14 beats/min),
consistent with other reports of lower resting HR in mice
lacking ␤-receptors (14). Dark-cycle and 24-h HR averages
were also significantly lower in ␤-less mice at 23°C (data not
shown). When Ta was raised to 30°C, the light-cycle HR fell
significantly in all groups (control mice, 351 ⫾ 11; ␤-less,
363 ⫾ 10; and M2R⫺/⫺, 416 ⫾ 7 beats/min). The light-cycle
HR of ␤-less mice was identical to that of control mice (P ⬎
0.05). However, the light-cycle HR in M2R⫺/⫺ mice was
significantly elevated (Fig. 2B). These data suggest that ␤-ad-
renergic receptor signaling is required at cool housing temper-
atures (e.g., 23°C) to achieve a normal light-cycle HR, whereas
muscarinic receptor signaling is required at 30°C to achieve a
normal light-cycle HR.
We also examined IHR after housing mice for 4 days at
either 23° or 30°C to determine whether there was a funda-
mental shift in autonomic balance of HR control at thermo-
neutrality. A typical HR tracing for a B6 control mouse with
IHR measurement is shown in Fig. 3A. When Ta was raised
from 23° to 30°C, the IHR of control mice fell significantly
from 486 ⫾ 14 to 403 ⫾ 7 beats/min (Fig. 3B). As Fig. 3B
shows, the IHR of M2R⫺/⫺ mice was significantly lower than
that of control mice at 23°C (436 ⫾ 2 beats/min). Similar to
that in control mice, IHR in the M2R⫺/⫺ mice fell significantly
with exposure to thermoneutrality (377 ⫾ 5 beats/min). The
percent drop in IHR with the increase in Ta was not signifi-
cantly different between control and M2R⫺/⫺ mice (17 ⫾ 3%
vs. 14 ⫾ 2%, respectively). The IHR of ␤-less mice at 30°C
was 418 ⫾ 9 beats/min, which was not significantly different
from that in control mice (Fig. 3B). However, when we
attempted to measure IHR in ␤-less mice at 23°C in a pilot
study, we were unable to assess an IHR at this cool temperature
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H1583
because the HR of these mice did not stabilize after ganglionic
blockade. These mice experienced continuous and progressive
bradycardia to well below 300 beats/min. In fact, these mice
are cold sensitive (28), and we presume the mice became
hypothermic due to a disruption of sympathetic activity to
brown adipose tissue.
The difference between resting light-cycle HR and IHR
reflects the autonomic control of HR and, therefore, the balance
between sympathetic and parasympathetic control of the heart.
As Fig. 3C shows, light-cycle HR at 30°C in control and ␤-less
mice was well below IHR (⬃40 and 70 beats/min, respec-
tively). In contrast to the relationship between HR and IHR in
control mice, the light-cycle HR of M2R⫺/⫺ mice was well
above IHR (⬃40 beats/min; Fig. 3C). In fact, we never observed
a HR at any time in these knockout mice that was below IHR.
Since assessment of HR variability in the time domain may
provide insight into cardiac autonomic activity, we calculated
SDNN in response to increasing Ta in all mice. As reported
previously (48, 54), increasing Ta toward thermoneutrality
increased SDNN in control mice (Fig. 4). SDNN in ␤-less mice
was not significantly different from SDNN in control mice at
either 23° or 30°C (Fig. 4). In contrast, the SDNN of M2R⫺/⫺
mice was significantly lower than control mice at 23°C and did
not rise in response to elevated Ta.
Experiment 3: IHR is Decreased by Caloric Restriction
Caloric restriction resulted in an ⬃10% decrease in body
weight and similar magnitude of light-cycle bradycardia (⬃75
beats/min) in all groups (Fig. 5A). Decreased caloric intake in
control mice increased SDNN in control mice (Fig. 5B), as
seen previously (54). This increase in HR variability was
significantly blunted in ␤-less mice and completely absent in
M2R⫺/⫺ mice (Fig. 5B). Furthermore, we observed that caloric
restriction-reduced IHR by ⬃30 – 40 beats/min in all groups of
mice (Fig. 5A).
DISCUSSION
The results support new and important concepts in our
understanding of HR control in mice. We have previously
demonstrated that Ta is an important determinant of murine HR
and blood pressure (48, 56). In this study, we report for the first
time concurrent measures of metabolism, HR, and blood pres-
sure during rapid shifts in Ta. The findings provide further
strong support for the concept that housing conditions of mice
are an important determinant of cardiac sympathetic and para-
sympathetic outflow, which in turn has a major impact on HR
and HR variability. In addition, novel findings from mutant
mice lacking M2R and ␤-receptors convincingly demonstrate
that cardiac vagal tone dominates autonomic control of resting
HR in mice studied at thermoneutrality. Finally, we provide
new data demonstrating that both Ta and caloric balance
modulate IHR in mice. The finding adds to a growing list of
factors that can modulate IHR.
Ta and Cardiovascular Physiology in Mice
It is generally accepted that normal mice have a resting HR
of about 550- 600 beats/min, exhibit minimal vagal tone, and
have an IHR that is below resting HR (19, 27). Our new
findings support previously published work and convincingly
demonstrate that these descriptors of the mouse physiological
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 H1584 AUTONOMIC CONTROL OF MOUSE HEART RATE

Fig. 1. Heart rate [HR in beats/min (bpm); A and B], oxygen consumption (V̇O2; C and D), mean arterial pressure (MAP; E and F), and locomotor activity (G
and H) responses to decreasing ambient temperature (Ta) cooling from 30° to 20°C (A, C, E, and G) and increasing Ta from 20° to 30°C. (B, D, F, and H). Data
were obtained from B6 mice (n ⫽ 7) and are plotted as means ⫾ SE (solid line). For each graph, Ta is also plotted (dashed line).

state are strongly influenced by Ta. The physiological demands ations (21), we and others have clearly documented the meta-
of thermoregulation are substantial in mice studied at a Ta bolic and cardiovascular consequences of the mild cold stress
comfortable for humans. While housing factors such as type of that exist under standard laboratory conditions (8, 48, 51, 52,
bedding and number of mice per cage are important consider- 56). Nonshivering thermogenesis is activated at temperatures
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 AUTONOMIC CONTROL OF MOUSE HEART RATE H1585
tibility to diet-induced obesity (2), indicating impaired sympa-
thetically mediated facultative thermogenesis. At standard
housing temperatures, ␤-less mice exhibit a 10 –15% reduction
in metabolic rate and reduced body temperature but have
normal thyroid hormone levels and responsiveness to hyper-
thyroidism (2, 3, 28). Although these mice tolerate standard
laboratory conditions well, the possibility of unrecognized
compensatory adaptations that contribute to the phenotype in
unconditional knockout mice must be acknowledged (53).
Mice lacking the ␤1-receptor have markedly impaired exer-
cise- and isoproterenol-induced elevation in HR (38, 39) but
were reported to have normal resting HR (Ta was not reported
in these studies). More recent findings (14) are consistent with
our observations (Fig. 2) of significantly lower resting HR in
␤-less mice studied at Ta ⫽ 23°C. Furthermore, brief, nonin-
vasive ECG recordings indicated that that ␤-less mice have a
HR of ⬃150 beats/min lower than wild-type mice (3), although
HR levels obtained with the noninvasive ECG method (550
beats/min) were about 100 beats/min greater than we observed
using telemetry (450 beats/min, Fig. 2). The magnitude of

Fig. 2. HR in control mice, in mice deficient in cardiac sympathetic tone

(␤-less), and in mice deficient in cardiac vagal tone [muscarinic M2 receptor
(M2R⫺/⫺)]. A: HR for mice of each genotype averaged over 30-min time
intervals at an Ta of 23°C. Note the substantially lower HR at all time points
in the ␤-less mouse. B: average light-cycle HR at Ta values of 23° and 30°C.
All genotypes exhibit greatly reduced HR at thermoneutrality (Ta ⫽ 30°C).
However, the HR of M2R⫺/⫺ mice at 30°C is significantly higher than that of
controls or ␤-less mice. *P ⬍ 0.05 vs. control mice.

cooler than the lower critical limit of the thermoneutral zone,

which is about 30°C for mice (22). Sympathetic control of
brown adipose tissue metabolic activity is an important mech-
anism of heat generation in small rodents (see Ref. 42 for
review). Indeed, V̇O2 rapidly and promptly changes inversely
with Ta between 20 –30°C in a range from 0.75–1.5 ml/min.
We also report for the first time the rapid responsiveness of
mouse blood pressure and HR to changes in Ta. In C57 mice,
we find that MAP decreases from about 100 to about 80 mmHg
when Ta is warmed from 20 –30°C. The finding reinforces the
need to monitor and control for Ta during blood pressure
measurements in rodents (32). Not surprisingly, HR is directly
related to metabolic rate during changes in Ta. However, the
sensitivity of mouse HR to small changes in Ta is striking.
The magnitude of change in HR is 250 –300 beats/min between
the Ta of 20 –30°C. The HR at thermoneutrality (⬃350 beats/
min) is similar to values reported previously (56). These HR Fig. 3. Intrinsic HR (IHR) measurement. A: typical HR tracings are shown for
responses are clearly not due to alterations in locomotor activ- the same control mouse at Ta values of 23° and 30°C. IHR was measured after
ity (Fig. 1, G and H) and thus are likely regulated by the administration of metoprolol (a ␤1-receptor blocker) and atropine (a musca-
autonomic nervous system. rinic receptor blocker) to block autonomic input to the heart. The IHR of this
mouse at 23°C was 517 and at 30°C was 403 beats/min. Note that at 23°C,
Cardiac Autonomic Activity in Mice resting HR is higher than IHR, whereas at 30°C, resting HR is lower than IHR.
B: IHR is lower in M2R⫺/⫺ mice than in B6 mice irrespective of Ta (P ⬍ 0.05).
To further analyze the autonomic mechanisms regulating IHR falls with elevated Ta in control and M2R⫺/⫺ mice. IHR was not
HR, we measured HR, IHR, and HR variability in the time determined (ND) in ␤-less mice at 23°C (see RESULTS for explanation). *P ⬍
0.05 vs. same strain at 23°C. C: the resting HR of control and ␤-less mice is
domain at multiple Ta values and during caloric restriction in significantly lower than IHR at an Ta of 30°C. M2R⫺/⫺ mice, which lack
␤-less mice and in mice lacking the M2R. ␤-less mice are endogenous vagal influence to the heart, exhibit resting HRs significantly
known to exhibit cold intolerance (28) and increased suscep- above IHR. NS, not significant.

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 H1586 AUTONOMIC CONTROL OF MOUSE HEART RATE

caloric restriction: 1) a depressed IHR and 2) an elevation of

vagal activity at the heart. The potential mechanisms of the
decrease in IHR with caloric restriction are discussed below.
IHR is Influenced by Many Factors
An additional major concept we provide here is that IHR is
heavily influenced by Ta and caloric availability. IHR is com-
monly determined by pharmacological blockade of sympa-
thetic and parasympathetic nerve activity and thus presumably
represents the spontaneous depolarization rate of sinoatrial
myocytes. Regulation of the hyperpolarization-activated cyclic
nucleotide-gated channel (HCN), which is responsible for the
diastolic, depolarizing “funny” current, is clearly one mecha-
Fig. 4. HR variability, as measured by standard deviation of the interbeat nism by which IHR could be modulated. Interestingly, the
interval (SDNN), increased in control and ␤-less mice when Ta was elevated HCN channel gene expression is known to be depressed in
from a mild cold stress (23°C) to thermoneutrality (30°C). This increase in mice with depressed thyroid signaling and bradycardia (20).
variability was not observed in M2R⫺/⫺ mice, demonstrating that it is M2 Clearly, thyroid hormone activity is regulated by nutritional
receptor signaling, and hence vagal tone, that is responsible for the elevated
SDNN with thermoneutrality. *P ⬍ 0.05 vs. control.
status (42), thus this is one mechanism by which nutritional
status could regulate IHR. However, it must be acknowledged
that the ultimate pacemaker activity is determined by multiple
tachycardia associated with decreasing Ta below thermo- ionic currents (41) and that much remains to be learned
neutrality is markedly diminished in ␤-less (Fig. 2), suggesting concerning the mechanisms by which IHR is determined (12).
that increased sympathetic activity produces the elevated HR Previously, only a few interventions have been shown to
observed in at Ta ⫽ 23°C. Nonetheless, ␤-less mice still exhibit alter IHR, including nucleus ambiguus lesions or renal hyper-
significant bradycardia upon exposure to thermoneutrality. The tension (34) that may be mediated by the actions of angiotensin
finding suggests that increased vagal tone or decreased IHR are II on IHR (7, 11). We show here that IHR fell by 20% with a
also involved in the thermoneutrality-induced slowing of HR. simple elevation of Ta by 7°C. This fall in IHR accounts for a
The bradycardia induced by vagal efferent stimulation or by substantial portion of the drop in resting HR associated with
methacholine administration is mediated by the M2 receptor housing at thermoneutrality. In addition, we provide evidence
(17). We found that M2R knockout mice have a normal resting that reductions in IHR may be responsible for about 50% of the
HR at Ta ⫽ 23°C (Fig. 2) yet exhibit minimal HR variability bradycardia associated with caloric restriction. Several hor-
(Fig. 4). This is consistent with the prevailing view that the mones known to be altered by energy balance, including
major determinant of beat-to-beat variability in HR is vagal thyroid hormone (18, 29, 40), insulin (25), and glucagon-like
activity (16, 19). At Ta ⫽ 30°C, we observed that in contrast to
control and ␤-less mice, the IHR of M2R mice was always less
than resting HR (Fig. 3). This is clear evidence that control of
HR in mice when housed near thermoneutrality requires the M2
receptor, and thus, HR at thermoneutrality is mediated predom-
inantly by vagal tone. In control and ␤-less mice, we observed
an increase in HR variability with exposure to thermoneutrality
(Fig. 4). In contrast, M2R⫺/⫺ mice failed to increase HR
variability with either elevated Ta (Fig. 4) or with caloric
restriction (Fig. 5). The interpretation of these findings is
somewhat limited by a lack of adequate sampling to perform
variability analysis in the frequency domain. However, even
with this information, it is recognized that caution is required
when extrapolating from measures of HR variability to activity
of the autonomic nervous system (45, 46).
The bradycardia of negative energy balance is observed in
both humans (1, 9, 31) and mice (47, 49, 56) undergoing
weight loss. Studies using pharmacological blockade and anal-
ysis of HR variability suggest that the primary mechanism for
caloric restriction-induced bradycardia is increased vagal tone
(1). In this study, caloric restriction reduced HR in all mice but
did so using different mechanisms (Fig. 5). In control and
␤-less mice, increased vagal tone (inferred from the increase in
SDNN) and reduced IHR were engaged (Fig. 5). Caloric
restriction-induced bradycardia in M2R⫺/⫺ mice was not ac- Fig. 5. A: 70% caloric restriction causes bradycardia in the light cycle of all
genotypes tested. All genotypes had an equivalent drop in both light-cycle HR
companied by an increase in SDNN but was accompanied by and IHR. B: the elevated SDNN with caloric restriction seen in control mice
a decrease in IHR. Collectively, these data suggest that there was blunted in ␤-less mice and not observed in M2R⫺/⫺ mice. *P ⬍ 0.05 vs.
are two major components to bradycardia associated with control.

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 AUTONOMIC CONTROL OF MOUSE HEART RATE
peptide 1 (4), influence IHR. In addition, Sowden et al. (44)
recently demonstrated that an acute administration of the an-
orexigenic hormone oxyntomodulin can dramatically increase
IHR. Hence, it is likely that the circulating status of these
hormones, as well as other hormones that reflect energy status
(e.g., leptin or ghrelin), plays a major role in the reduction of
IHR during a bout of caloric restriction. The relative contribu-
tion of each of these hormones remains to be determined.
Taken together, our findings indicate that, in addition to vagal
and sympathetic tone, IHR is an important component of the
adaptive response to altered Ta and reduced energy availability
and should be considered as an important regulatory arm of HR
control.
Conclusion
These experiments demonstrate that mice exhibit sensitive
metabolic and cardiovascular responses to variations in Ta
below the thermoneutral zone. Furthermore, our findings indi-
cate that when studied at thermoneutrality, cardiac autonomic
function of mice more closely resembles that of humans in that
both species exhibit dominant vagal tone and subsequent rest-
ing HR levels below IHR. In contrast, in the mild cold stress of
standard laboratory conditions, mice exhibit tachycardia due to
a withdrawal of cardiac vagal tone, increased sympathetic tone,
low HR variability, and elevated IHR. The findings further
emphasize the need for careful attention to controlling Ta
during the assessment of cardiovascular physiology in mice.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of Charles Badland,
Molly Gutilla, and Ashley Christman.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants R15-HL-081101-01 (to S. J. Swoap) and R01-HL-56732 (to J. M.
Overton).
REFERENCES
1. Aronne LJ, Mackintosh R, Rosenbaum M, Leibel RL, Hirsch J.
Autonomic nervous system activity in weight gain and weight loss. Am J
Physiol Regul Integr Comp Physiol 269: R222–R225, 1995.
2. Bachman ES, Dhillon H, Zhang CY, Cinti S, Bianco AC, Kobilka BK,
Lowell BB. Beta AR signaling required for diet-induced thermogenesis
and obesity resistance. Science 297: 843– 845, 2002.
3. Bachman ES, Hampton TG, Dhillon H, Amende I, Wang J, Morgan
JP, Hollenberg AN. The metabolic and cardiovascular effects of hyper-
thyroidism are largely independent of ␤-adrenergic stimulation. Endocri-
nology 145: 2767–2774, 2004.
4. Barragan JM, Rodriguez RE, Blazquez E. Changes in arterial blood
pressure and heart rate induced by glucagon-like peptide-1-(7-36) amide in
rats. Am J Physiol Endocrinol Metab 266: E459 –E466, 1994.
5. Bartholomew GA, Vleck D, Vleck CM. Instantaneous measurements of
oxygen consumption during preflight warm up and postflight cooling in
sphinged and saturniid moths. J Exp Biol 90: 17–32, 1981.
6. Baudrie V, Laude D, Elghozi JL. Optimal frequency ranges for extract-
ing information on cardiovascular autonomic control from the blood
pressure and pulse interval spectrograms in mice. Am J Physiol Regul
Integr Comp Physiol 292: R904 –R912, 2007.
7. Bealer SL. Systemic angiotensin II alters intrinsic heart rate through
central mechanisms. Brain Res Bull 58: 61– 65, 2002.
8. Bissonnette JM, Knopp SJ, Maylie J, Thong T. Autonomic cardiovas-
cular control in methyl-CpG-binding protein 2 (Mecp2) deficient mice.
Auton Neurosci 136: 82– 89, 2007.
9. Brozek J, Chapman CB, Keys A. Drastic food restriction: effect on
cardiovascular dynamics in normotensive and hypertensive conditions.
JAMA 137: 1569 –1574, 1948.
AJP-Heart Circ Physiol • VOL
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (180.252.107.236) on December 22, 2017.
Copyright © 2008 American Physiological Society. All rights reserved.
H1587
10. Butz GM, Davisson RL. Long-term telemetric measurement of cardio-
vascular parameters in awake mice: a physiological genomics tool. Physiol
Genomics 5: 89 –97, 2001.
11. Chen Y, Joaquim LF, Farah VM, Wichi RB, Fazan R Jr, Salgado HC,
Morris M. Cardiovascular autonomic control in mice lacking angiotensin
AT1a receptors. Am J Physiol Regul Integr Comp Physiol 288: R1071–
R1077, 2005.
12. Dobrzynski H, Boyett MR, Anderson RH. New insights into pacemaker
activity: promoting understanding of sick sinus syndrome. Circulation
115: 1921–1932, 2007.
13. Dulloo AG. A sympathetic defense against obesity. Science 297: 780 –
781, 2002.
14. Ecker PM, Lin CC, Powers J, Kobilka BK, Dubin AM, Bernstein D.
Effect of targeted deletions of ␤1- and ␤2-adrenergic-receptor subtypes on
heart rate variability. Am J Physiol Heart Circ Physiol 290: H192–H199,
2006.
15. Ernsberger P, Nelson DO. Effects of fasting and refeeding on blood
pressure are determined by nutritional state, not by body weight change.
Am J Hypertens 1: 153S–157S, 1988.
16. Fazan R Jr, de Oliveira M, da Silva VJ, Joaquim LF, Montano N,
Porta A, Chapleau MW, Salgado HC. Frequency-dependent baroreflex
modulation of blood pressure and heart rate variability in conscious mice.
Am J Physiol Heart Circ Physiol 289: H1968 –H1975, 2005.
17. Fisher JT, Vincent SG, Gomeza J, Yamada M, Wess J. Loss of vagally
mediated bradycardia and bronchoconstriction in mice lacking M2 or M3
muscarinic acetylcholine receptors. FASEB J 18: 711–713, 2004.
18. Foley CM, McAllister RM, Hasser EM. Thyroid status influences
baroreflex function and autonomic contributions to arterial pressure and
heart rate. Am J Physiol Heart Circ Physiol 280: H2061–H2068, 2001.
19. Gehrmann J, Hammer PE, Maguire CT, Wakimoto H, Triedman JK,
Berul CI. Phenotypic screening for heart rate variability in the mouse.
Am J Physiol Heart Circ Physiol 279: H733–H740, 2000.
20. Gloss B, Trost S, Bluhm W, Swanson E, Clark R, Winkfein R, Janzen
K, Giles W, Chassande O, Samarut J, Dillmann W. Cardiac ion channel
expression and contractile function in mice with deletion of thyroid
hormone receptor alpha or beta. Endocrinology 142: 544 –550, 2001.
21. Gordon CJ. Effect of cage bedding on temperature regulation and
metabolism of group-housed female mice. Comp Med 54: 63– 68, 2004.
22. Gordon CJ. Relationship between autonomic and behavioral thermoreg-
ulation in the mouse. Physiol Behav 34: 687– 690, 1985.
23. Gordon CJ. Temperature Regulation in Laboratory Rodents. New York:
Cambridge University Press, 1993.
24. Hall JE, Brands MW, Dixon WN, Smith MJ Jr. Obesity-induced
hypertension. Renal function and systemic hemodynamics. Hypertension
22: 292–299, 1993.
25. Hicks KK, Seifen E, Stimers JR, Kennedy RH. Effects of streptozoto-
cin-induced diabetes on heart rate, blood pressure and cardiac autonomic
nervous control. J Auton Nerv Syst 69: 21–30, 1998.
26. Janssen BJ, Leenders PJ, Smits JF. Short-term and long-term blood
pressure and heart rate variability in the mouse. Am J Physiol Regul Integr
Comp Physiol 278: R215–R225, 2000.
27. Janssen BJ, Smits JF. Autonomic control of blood pressure in mice: basic
physiology and effects of genetic modification. Am J Physiol Regul Integr
Comp Physiol 282: R1545–R1564, 2002.
28. Jimenez M, Leger B, Canola K, Lehr L, Arboit P, Seydoux J, Russell
AP, Giacobino JP, Muzzin P, Preitner F. ␤1/␤2/␤3-Adrenoceptor knock-
out mice are obese and cold-sensitive but have normal lipolytic responses
to fasting. FEBS Lett 530: 37– 40, 2002.
29. Johansson C, Vennstrom B, Thoren P. Evidence that decreased heart
rate in thyroid hormone receptor-␣1-deficient mice is an intrinsic defect.
Am J Physiol Regul Integr Comp Physiol 275: R640 –R646, 1998.
30. Just A, Faulhaber J, Ehmke H. Autonomic cardiovascular control in
conscious mice. Am J Physiol Regul Integr Comp Physiol 279: R2214 –
R2221, 2000.
31. Kollai M, Bonyhay I, Jokkel G, Szonyi L. Cardiac vagal hyperactivity in
adolescent anorexia nervosa. Eur Heart J 15: 1113–1118, 1994.
32. Kurtz TW, Griffin KA, Bidani AK, Davisson RL, Hall JE. Recom-
mendations for blood pressure measurement in humans and experimental
animals. Part 2: blood pressure measurement in experimental animals: a
statement for professionals from the subcommittee of professional and
public education of the American Heart Association Council on High
Blood Pressure Research. Hypertension 45: 299 –310, 2005.
33. Lowell BB, Spiegelman BM. Towards a molecular understanding of
adaptive thermogenesis. Nature 404: 652– 660, 2000.
294 • APRIL 2008 • www.ajpheart.org
 H1588 AUTONOMIC CONTROL OF MOUSE HEART RATE
34. Machado BH, Brody MJ. Contribution of neurogenic mechanisms to
control of intrinsic heart rate. Am J Physiol Regul Integr Comp Physiol
256: R231–R235, 1989.
35. Pelat M, Dessy C, Massion P, Desager JP, Feron O, Balligand JL.
Rosuvastatin decreases caveolin-1 and improves nitric oxide-dependent
heart rate and blood pressure variability in apolipoprotein E⫺/⫺ mice
in vivo. Circulation 107: 2480 –2486, 2003.
36. Rappaport EB, Young JB, Landsberg L. Initiation, duration and dissi-
pation of diet-induced changes in sympathetic nervous system activity in
the rat. Metabolism 31: 143–146, 1982.
37. Rashotte ME, Basco PS, Henderson RP. Daily cycles in body temper-
ature, metabolic rate, and substrate utilization in pigeons: influence of
amount and timing of food consumption. Physiol Behav 57: 731–746,
1995.
38. Rohrer DK, Desai KH, Jasper JR, Stevens ME, Regula DP Jr, Barsh
GS, Bernstein D, Kobilka BK. Targeted disruption of the mouse beta1-
adrenergic receptor gene: developmental and cardiovascular effects. Proc
Natl Acad Sci USA 93: 7375–7380, 1996.
39. Rohrer DK, Schauble EH, Desai KH, Kobilka BK, Bernstein D.
Alterations in dynamic heart rate control in the ␤1-adrenergic receptor
knockout mouse. Am J Physiol Heart Circ Physiol 274: H1184 –H1193,
1998.
40. Safa-Tisseront V, Ponchon P, Laude D, Elghozi JL. Autonomic con-
tribution to the blood pressure and heart rate variability changes in early
experimental hyperthyroidism. J Hypertens 16: 1989 –1992, 1998.
41. Schram G, Pourrier M, Melnyk P, Nattel S. Differential distribution of
cardiac ion channel expression as a basis for regional specialization in
electrical function. Circ Res 90: 939 –950, 2002.
42. Silva JE. Thermogenic mechanisms and their hormonal regulation.
Physiol Rev 86: 435– 464, 2006.
43. Sinclair DA. Toward a unified theory of caloric restriction and longevity
regulation. Mech Ageing Dev 126: 987–1002, 2005.
44. Sowden GL, Drucker DJ, Weinshenker D, Swoap SJ. Oxyntomodulin
increases intrinsic heart rate in mice independent of the glucagon-like
peptide-1 receptor. Am J Physiol Regul Integr Comp Physiol 292: R962–
R970, 2007.
45. Stauss HM. Power spectral analysis in mice: what are the appropriate
frequency bands? Am J Physiol Regul Integr Comp Physiol 292: R902–
R903, 2007.
46. Stauss HM, Persson PB. Cardiovascular variability and the autonomic
nervous system. J Hypertens 24: 1902–1905, 2006.
AJP-Heart Circ Physiol • VOL
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (180.252.107.236) on December 22, 2017.
Copyright © 2008 American Physiological Society. All rights reserved.
47. Swoap SJ. Altered leptin signaling is sufficient, but not required, for
hypotension associated with caloric restriction. Am J Physiol Heart Circ
Physiol 281: H2473–H2479, 2001.
48. Swoap SJ, Overton JM, Garber G. Effect of ambient temperature on
cardiovascular parameters in rats and mice: a comparative approach. Am J
Physiol Regul Integr Comp Physiol 287: R391–R396, 2004.
49. Swoap SJ, Weinshenker D, Palmiter RD, Garber G. Dbh⫺/⫺ mice are
hypotensive, have altered circadian rhythms, and have abnormal responses
to dieting and stress. Am J Physiol Regul Integr Comp Physiol 286:
R108 –R113, 2004.
50. Takayama K, Yuhki K, Ono K, Fujino T, Hara A, Yamada T,
Kuriyama S, Karibe H, Okada Y, Takahata O, Taniguchi T, Iijima T,
Iwasaki H, Narumiya S, Ushikubi F. Thromboxane A2 and prostaglan-
din F2␣ mediate inflammatory tachycardia. Nat Med 11: 562–566, 2005.
51. Talan MI, Kirov SA, Kosheleva NA. Nonshivering thermogenesis in
adult and aged C57BL/6J mice housed at 22 degrees C and at 29 degrees
C. Exp Gerontol 31: 687– 698, 1996.
52. Wernstedt I, Edgley A, Berndtsson A, Faldt J, Bergstrom G, Walle-
nius V, Jansson JO. Reduced stress- and cold-induced increase in energy
expenditure in interleukin-6-deficient mice. Am J Physiol Regul Integr
Comp Physiol 291: R551–R557, 2006.
53. Williams RS, Wagner PD. Transgenic animals in integrative biology:
approaches and interpretations of outcome. J Appl Physiol 88: 1119 –1126,
2000.
54. Williams T, Chambers J, Roberts L, Henderson R, Overton J. Diet-
induced obesity and cardiovascular regulation in C57BL/6J mice. Clin Exp
Pharmacol Physiol 30: 769 –778, 2003.
55. Williams TD, Chambers JB, Gagnon SP, Roberts LM, Henderson RP,
Overton JM. Cardiovascular and metabolic responses to fasting and
thermoneutrality in A(y) mice. Physiol Behav 78: 615– 623, 2003.
56. Williams TD, Chambers JB, Henderson RP, Rashotte ME, Overton
JM. Cardiovascular responses to caloric restriction and thermoneutrality
in C57BL/6J mice. Am J Physiol Regul Integr Comp Physiol 282:
R1459 –R1467, 2002.
57. Williams TD, Chambers JB, May OL, Henderson RP, Rashotte ME,
Overton JM. Concurrent reductions in blood pressure and metabolic rate
during fasting in the unrestrained SHR. Am J Physiol Regul Integr Comp
Physiol 278: R255–R262, 2000.
58. Young JB, Landsberg L. Suppression of sympathetic nervous system
during fasting. Science 196: 1473–1475, 1977.
59. Young JB, Mullin D, Landsberg L. Caloric restriction lowers blood
pressure in the spontaneously hypertensive rat. Metabolism 27: 1711–
1714, 1978.
294 • APRIL 2008 • www.ajpheart.org