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J Appl Physiol 97:1746-1754, 2004. doi:10.1152/japplphysiol.00573.2004

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J Appl Physiol 97: 1746 –1754, 2004;
doi:10.1152/japplphysiol.00573.2004.
ANG II in the paraventricular nucleus potentiates the cardiac
sympathetic afferent reflex in rats with heart failure
Guo-Qing Zhu,1 Lie Gao,2 Kuashik P. Patel,2 Irving H. Zucker,2 and Wei Wang1,2
1
Department of Physiology, Nanjing Medical University, Nanjing 210029, Peoples Republic of China; and 2Department
of Cellular and Integrative Physiology, University of Nebraska College of Medicine, Omaha, Nebraska 68198-5850
Submitted 3 June 2004; accepted in final form 24 June 2004
Zhu, Guo-Qing, Lie Gao, Kuashik P. Patel, Irving H. Zucker,
and Wei Wang. ANG II in the paraventricular nucleus potentiates the
cardiac sympathetic afferent reflex in rats with heart failure. J Appl
Physiol 97: 1746 –1754, 2004; doi:10.1152/japplphysiol.00573.
2004.—Chronic heart failure (CHF) is characterized by sympathoex-
citation, and the cardiac sympathetic afferent reflex (CSAR) is a
sympathoexcitatory reflex. Our previous studies have shown that the
CSAR was enhanced in CHF. In addition, central angiotensin II (ANG
II) is an important modulator of this reflex. This study was performed
to determine whether the CSAR evoked by stimulation of cardiac
sympathetic afferent nerves (CSAN) in rats with coronary ligation-
induced CHF is enhanced by ANG II in the paraventricular nucleus
(PVN). Under ␣-chloralose and urethane anesthesia, renal sympa-
thetic nerve activity (RSNA) was recorded. The RSNA responses to
electrical stimulation (5, 10, 20, and 30 Hz) of the CSAN were
evaluated. Bilateral microinjection of the AT1-receptor antagonist
losartan (50 nmol) into the PVN had no significant effects in the sham
group, but it abolished the enhanced RSNA response to stimulation in
the CHF group. Unilateral microinjection of three doses of ANG II
(0.03, 0.3, and 3 nmol) into the PVN resulted in dose-related increases
in the RSNA responses to stimulation. Although ANG II also poten-
tiated the RSNA response to electrical stimulation in sham rats, the
RSNA responses to stimulation after ANG II into the PVN in rats with
CHF were much greater than in sham rats. The effects of ANG II were
prevented by pretreatment with losartan into the PVN in CHF rats.
These results suggest that the central gain of the CSAR is enhanced in
rats with coronary ligation-induced CHF and that ANG II in the PVN
augments the CSAR evoked by CSAN, which is mediated by the
central angiotensin AT1 receptors in rats with CHF.
chronic heart failure; angiotensin II; renal sympathetic nerve activity;
cardiac sympathetic afferent reflex; paraventricular nucleus; angioten-
sin AT1 receptor
IT IS WELL KNOWN THAT THE sympathetic outflow is increased in
human and experimental heart failure, as suggested by an
increase in plasma catecholamine levels and by directly re-
corded muscle sympathetic nerve activity and renal sympa-
thetic nerve activity (11, 25, 30, 40). The chronic sympatho-
excitatory state may contribute to further hemodynamic dete-
rioration, and the degree of sympathoexcitation is prognostic
for survival in the chronic heart failure (CHF) state (5, 7).
However, the origin of sympathoexcitation has still not been
clearly defined. It has been reported that chronic sinoaortic
denervation does not increase mean sympathetic outflow and
blood pressure (10) and that the increase in plasma norepine-
nephrine was not altered in chronically sinoaortic baroreceptor-
Address for reprint requests and other correspondence: W. Wang, Dept.
of Cellular and Integrative Physiology, Univ. of Nebraska College of
Medicine, 985850 Nebraska Medical Center, Omaha, NE 68198-5850
(E-mail: weiwang@unmc.edu).
1746 8750-7587/04 $5.00 Copyright © 2004 the American Physiological Society
denervated dogs with CHF (6, 18). Therefore, a blunted sym-
pathoinhibitory reflex does not completely explain the chronic
elevation in sympathetic outflow in the CHF state. It has been
shown that the cardiac sympathetic afferent reflex (CSAR) is
sympathoexcitatory and contributes to the sympathoexcitation
in dogs with CHF (24, 32). The pathways of this reflex may be
similar to those involved in signaling cardiac pain during acute
ischemia (11, 23). This positive-feedback mechanism may be
deleterious in the CHF state over the long term. Previous
Downloaded from jap.physiology.org on August 12, 2010
studies in our laboratory showed that the discharge of the
cardiac sympathetic afferent nerves was increased in dogs with
CHF (31) and that the CSAR to either electrical stimulation of
cardiac sympathetic afferent nerves or epicardial application of
bradykinin and capsaicin was enhanced in dogs with pacing-
induced heart failure and in rats with coronary artery ligation-
induced CHF (22, 32, 39).
The interaction between the sympathetic nervous system
and the renin-angiotensin system is well known. It has been
reported that the renin-angiotensin system is activated in
human and experimental CHF (30, 32, 41). Angiotensin II
(ANG II) modulates sympathetic function at several loci,
including the sympathetic ganglia, postganglionic synapses,
and the central nervous system (1). It has been shown that
angiotensin-converting enzyme inhibitors decrease plasma
norepinephrine and improve arterial baroreceptor function
in CHF patients (12, 14). The locus for central ANG II action
is not clear in the CHF state. The hypothalamic paraventricular
nucleus (PVN) is an important integrative site within the brain
in controlling sympathetic outflow and thus cardiovascular
function (2, 9). Activation of neurons in the PVN has been
found to play a major role in the processes leading to sympa-
thetic hyperactivity in rats with coronary ligation-induced CHF
(8, 19, 20, 27, 35–37). It was reported that microinjection of
ANG II into PVN increased blood pressure in normal rats (4)
and that angiotensin-receptor binding in the PVN was in-
creased in rats with chronic high-output heart failure (34).
Recent experiments in our laboratory showed that intracere-
broventricular administration of losartan normalized the en-
hanced CSAR in dogs with pacing-induced CHF (22). How-
ever, it is not known whether ANG II in the PVN is involved
in the enhanced CSAR in CHF. The purpose of the present
study was to determine whether the CSAR is enhanced in the
rats with coronary ligation-induced CHF and whether ANG II
in the PVN is involved in alterations of the central gain of the
CSAR evoked by electrical stimulation of cardiac sympathetic
afferent nerves.
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.
http://www. jap.org
 CARDIAC SYMPATHETIC AFFERENT REFLEX IN RATS
METHODS
Male Sprague-Dawley rats weighing between 350 and 420 g were
used in the experiments. All experiments were approved by the
Institutional Animal Care and Use Committee of the University of
Nebraska and were carried out under the guidelines of the American
Physiological Society and the National Institutes of Health Guide for
the Care and Use of Laboratory Animals.
Model of CHF
CHF was produced by coronary artery ligation as previously
described (26, 35). All rats were anesthetized with pentobarbital
sodium (50 mg/kg ip) and instrumented with sterile techniques. The
trachea was cannulated to facilitate mechanical ventilation. A left
thoracotomy was performed through the fifth intercostal space. After
retraction of the lung, the pericardium was opened to expose the heart.
The left coronary artery was ligated by using 6-0 suture near its
branch point from the aorta, between the pulmonary artery outflow
tract and left atrium. After these maneuvers, the heart was placed in its
original position and the thorax was closed. The air within the thorax
was evacuated, allowing the rats to resume spontaneous respiration
and recover from anesthesia. Analgesics (Nubain-Stadol, 1 ml/kg sc)
were administered after surgery. Mortality was ⬃30%, and death
occurred mainly during the first day after ligation. The rats were caged
in an environment with ambient temperature maintained at 22°C and
humidity at 30 – 40%. Laboratory chow (Purina) and tap water were
available ad libitum. The sham rats were treated the same as the CHF
rats except their coronary arteries were not ligated. The final experi-
ment was carried out 6 – 8 wk after coronary ligation or sham surgery.
Acute Experiments
Each rat was anesthetized with urethane (800 mg/kg ip) and
␣-chloralose (40 mg/kg ip). Supplemental doses of anesthesia were
administered at one-tenth of the initial dose per hour. A midline
incision in the neck was made, and the carotid sinus area was exposed
bilaterally. Each carotid sinus nerve was identified and cut. All other
nerve fibers that were visible in the area of the carotid sinus were also
cut. The carotid bifurcation and the common carotid arteries were
stripped of adventitial tissues from 4 mm below the bifurcation to 4
mm above. The vessels were painted with 10% phenol solution to
destroy any remaining nerve fibers in this area. Each vagus was then
identified in the neck, tied, and sectioned. A carotid artery was
catheterized for measurement of mean artery pressure (MAP) and
heart rate (HR). At the end of each acute experiment, a Millar
transducer-tipped catheter (Millar, Houston, TX) was advanced
through the carotid artery into the left ventricle to determine left
ventricular pressures. The left ventricular end-diastolic pressure and
maximum of the first derivative of left ventricular pressure measure-
ment was determined to provide an index of cardiac contractile
function. The effectiveness of baroreceptor denervation was deter-
mined by recording the change in HR to intravenous injection of
phenylephrine (20 ␮g/kg). This dose evoked an increase in blood
pressure between 25 and 40 mmHg. Baroreceptor denervation was
assumed to be complete if HR did not change more than 5 beats/min
in response to the intervention.
A left flank incision was made, and a retroperitoneal dissection was
used to expose the renal artery and nerves. The renal sympathetic
nerves were identified and dissected free of the surrounding connec-
tive tissue. The nerve was immersed in a warm mineral oil bath and
was placed on a pair of platinum-iridium recording electrodes. The
signal was amplified with a Grass direct current preamplifier (model
P18D, Astro-Med, West Warwick, RI) with low-frequency cutoff set
at 30 or 100 Hz and high-frequency cutoff at 1 or 3 kHz. The
amplified discharge was monitored on a storage oscilloscope (model
121 N, Tektronix, Beaverton, OR) and then imported to a computer
J Appl Physiol • VOL 97 • NOVEMBER 2004 •
1747
system with other parameters. A voltage integrator (model 1801,
Buxco Electronics, Sharon, CT) was used for quantifying the raw
renal sympathetic nerve activity (RSNA). Background noise was
determined when nerve activity was completely inhibited by increas-
ing arterial pressure (phenylephrine 20 ␮g/kg iv) before sinoarotic
denervation or after section of the central end of the renal nerve at the
end of the experiment. This value was subtracted from all the
integrated values of RSNA. The raw nerve activity, integrated nerve
activity, arterial pressure, and HR were recorded on a PowerLab data
acquisition system (model 16S, ADInstruments, Mountain View, CA)
and stored on disk until analyzed.
The chest was opened through the left second intercostal space. The
left ventral ansa, which contains cardiac sympathetic afferent nerves
was identified, tied, and ligated. A pair of stainless steel stimulating
electrodes was placed on the central end of this nerve. The stimulus
was delivered with a stimulator (model S88, Grass, West Warwick,
RI) and a stimulus isolation unit. The frequencies of stimulation
varied at 5, 10, 20, and 30 Hz at a constant voltage of 10 V. The pulse
width was kept at 1 ms, and each stimulus train lasted 30 s. Stimuli
were delivered in random sequences in each experimental protocol.
The time period between each stimulus was 1–2 min.
Downloaded from jap.physiology.org on August 12, 2010
The rats were placed in a stereotaxic instrument (Stoelting, Chicago,
IL), and the skull was exposed through an incision on the midline of the
scalp. After the bregma was identified, cannulas were positioned in the
PVN. The coordinates for the PVN were determined from the Paxinos
and Watson rat atlas (9), which are 1.8 mm posterior to, 0.4 mm lateral
to the bregma, and 7.9 mm ventral to the zero level. A cannula (outer
diameter 0.5 mm and inner diameter 0.1 mm) connected to a microsy-
ringe (0.5 ␮l; model 7000.5, Hamilton, Reno, NV) was advanced into the
PVN with a manipulator (model 310, Stoelting). The volume of micro-
injection was 100 nl (100 nl in 1 min), and the controls for each group
were injected with isotonic saline (100 nl).
At the end of the experiment, the rat was euthanized with an
overdose of anesthetic (pentobarbital sodium 100 mg/kg iv). The brain
was removed from the skull and placed in 10% formalin. The brains
were sectioned, and the microinjection site was verified (Fig. 1). Only
the data of rats whose microinjection sites were within the boundaries
of the PVN were used for analysis.
Experimental Protocols
RSNA response to stimulation in sham and CHF rats. The RSNA
response to electrical stimulation of the central end of the cardiac
sympathetic afferent nerves were determined and compared in sham
rats (n ⫽ 12) and CHF rats (n ⫽ 12).
Microinjection of losartan into the PVN in sham and CHF rats.
Bilateral microinjections of losartan (50 nmol for each) or saline into
the PVN were carried out in sham rats (n ⫽ 8) and CHF rats (n ⫽ 8).
One minute later, the RSNA responses to electrical stimulation were
determined and compared.
Dose-response relationship of ANG II in CHF rats. Three doses of
ANG II (0.03, 0.3, and 3 nmol) and saline were unilaterally microin-
jected into the PVN at random in CHF rats (n ⫽ 6). The time period
between each injection was at least 10 min after complete recovery.
One minute after the injection, the RSNA responses to electrical
stimulation were determined.
Microinjection of ANG II into the PVN in sham and CHF rats.
Unilateral microinjections of ANG II (3 nmol) or saline into the PVN
were carried out in sham rats (n ⫽ 6) and CHF rats (n ⫽ 6). One
minute later, the RSNA responses to electrical stimulation were
determined and compared.
Pretreatment with losartan into the PVN in CHF rats. This series
of experiments were carried out in 10 CHF rats and included three
interventions: 1) unilateral microinjection of saline (100 nl) into the
PVN as control, 2) unilateral microinjection of losartan (50 nmol)
followed by ANG II (3 nmol) into the PVN, and 3) unilateral
microinjection of ANG II (3 nmol) into the PVN. One minute later
www.jap.org
1748 CARDIAC SYMPATHETIC AFFERENT REFLEX IN RATS
after each microinjection, the RSNA responses to electrical stimula-
tion were determined and compared. The time period between inter-
ventions 1 and 2 was at least 15 min, and the period between
intervention 2 and 3 was at least 120 min, well after the acute effects
of losartan returned to normal.
Fig. 1. Schematic representations of serial sections from the rostral (⫺1.4) to
the caudal (⫺2.12) extent of the region of the paraventricular nucleus. A: ⫺1.4.
B: ⫺1.8. C: ⫺2.12. F, Sites of termination of the microinjections in both sham
and heart failure rats that are considered to be within the paraventricular
nucleus; 䊐 are the sites of termination of the microinjections, which are
considered to be outside of the paraventricular nucleus. 3V, third ventricle.
J Appl Physiol • VOL 97 • NOVEMBER 2004 •
Drugs
ANG II was obtained from Sigma Chemical. Losartan was a gift
from Merck. All drugs were made fresh on the day of the experiment.
Infarct Size Determination
At the conclusion of the acute experiment, the heart was dissected
free of adjacent tissues and lungs. The ventricles were separated from
the atria, and the right ventricular free wall was dissected from the
septum. The atria and both ventricles were rinsed, blotted, and
weighed. The left ventricle was opened with an incision along the
septum from base to apex. Incisions were made in the left ventricle so
that the tissue could be pressed flat. The circumferences of the left
ventricle and the region of infracted tissue were outlined on a clear
photograph taken by a digital camera. Infarct size was calculated and
expressed as a percentage of left ventricular surface area on the basis
of the surface areas measured by the SigmaScan program (SPSS
Science, Chicago, IL).
Data and Statistical Analysis
The RSNA was expressed as the percent change from control
Downloaded from jap.physiology.org on August 12, 2010
(before stimulation). The percent changes in the RSNA induced by
cardiac sympathetic afferent nerve stimulation were plotted in each
group and were used as an index of the central sensitivity of the
CSAR. The slope of the linear relationship between the RSNA
response and frequency of stimulation was also calculated by linear
regression. Baseline parameters were determined by averaging 10 s of
the integrated RSNA, MAP, and HR immediately before cardiac
sympathetic afferent stimulation. The last 10 s of the stimulus re-
sponse were compared with the baseline. A two-way repeated-mea-
sures ANOVA followed by the Newman-Keuls test for post hoc
analysis was used when multiple comparisons were made. All statis-
tical analyses were done using computer software (SigmaStat, SPSS).
All data are expressed as means ⫾ SE. P ⬍ 0.05 was considered
statistically significant.
RESULTS
Baseline Hemodynamics After Coronary Artery Ligation
The baseline hemodynamics, heart weight, and infarction
size were measured at 6 – 8 wk after coronary ligation or sham
surgery (Table 1). Coronary-ligated rats showed an average
infarct size of 33.7 ⫾ 1.9%. The heart weight and the ratio of
heart weight to body weight were significantly increased in
CHF rats, suggesting compensatory hypertrophy of the nonin-
farcted region of the myocardium. The baseline systolic arterial
pressure, pulse pressure, left ventricle peak systolic pressure,
and maximum of the first derivative of left ventricular pressure
were deceased, and the left ventricular end-diastolic pressure
was increased significantly in CHF rats. There were no statis-
tical differences in baseline MAP, diastolic arterial pressure,
and HR between the sham and CHF rats. These histological
and functional data show the presence of myocardial damage
and suggest a decreased contractile function in CHF rats.
RSNA Responses to Electrical Stimulation in Sham
and CHF Rats
The RSNA responses to varying frequencies of stimulation
of the cardiac sympathetic afferent nerves were used to eval-
uate the central gain of the CSAR in 12 sham rats and 12 CHF
rats. A significant increase was found at 10, 20, and 30 Hz of
stimulation in CHF rats. In most rats, RSNA increased imme-
diately after stimulation were delivered and reached its maxi-
www.jap.org
 CARDIAC SYMPATHETIC AFFERENT REFLEX IN RATS 1749
Table 1. Heart weight, infarct size, and baseline The slope of the RSNA responses to varying frequency of
hemodynamics after 6 – 8 wk of coronary ligation or sham stimulation was also increased significantly after unilateral
surgery in rats microinjection of high and middle doses of ANG II into the
PVN.
Sham CHF

n 20 30
Response to ANG II in the PVN After Pretreatment
BW, g 412.4⫾5.2 407.6⫾4.3 With Losartan
HW, g 1.32⫾0.03 1.65⫾0.03*
HW/BW, g/kg 3.21⫾0.06 4.06⫾0.08* To determine the contribution of AT1 receptors to the
IS, %LV area 0 33.7⫾1.9* enhanced response to stimulation in CHF rats, losartan was
SAP, mmHg 115.2⫾4.0 95.9⫾2.4* unilaterally microinjected into the PVN before measurment of
DAP, mmHg 66.9⫾3.5 70.5⫾2.7 the effect of administration of ANG II into the PVN. As shown
PP, mmHg 48.3⫾3.2 25.4⫾1.9*
MAP, mmHg 85.0⫾3.4 82.0⫾2.5
in Fig. 5, losartan abolished the effect of ANG II. There were
HR, beats/min 334.6⫾18.2 388.9⫾9.4* no significant differences between the saline group and losartan
LVSP, mmHg 126.2⫾5.3 99.5⫾4.1* plus ANG II group.
LVEDP, mmHg 1.6⫾0.7 15.5⫾1.1*
dP/dtmax, mmHg/s 3,497⫾94 1,894⫾97* Unilateral Microinjection of ANG II into the PVN in Sham
Values are as means ⫾ SE; n, no. of animals. BW, body weight; HW, heart
and CHF Rats
weight; IS, Infarct size; LV, left ventricle; SAP, systolic arterial pressure;
DAP, diastolic arterial pressure; PP, pulse pressure; MAP, mean arterial
Three doses of ANG II (0.03, 0.3, and 3 nmol) or saline were
pressure; LVSP, LV peak systolic pressure; LVEDP, LV end-diastolic pres- microinjected into the PVN in rats with CHF. Figure 6 shows

Downloaded from jap.physiology.org on August 12, 2010

sure; dP/dtmax, maximum of the first differentiation of LV pressure. *P ⬍ 0.05 dose-related responses. The RSNA responses were signifi-
compared with sham rats. cantly enhanced after microinjection of the 0.3- and 3-nmol
doses of ANG II in rats with CHF. To compare with sham rats,
the effects of unilateral microinjection of ANG II (3 nmol) into
mal level within 15 s. However, the RSNA response to stim- the PVN were determined in six sham rats and six CHF rats
ulation did not increase significantly in sham rats. Figure 2
shows the difference of the CSAR between sham and CHF rats.
RSNA responses to stimulation were enhanced in CHF rats. A
significant difference of the RSNA responses between the two
groups appeared at 20 and 30 Hz. The linear slope of the
RSNA responses to varying frequencies of stimulation were
also increased in CHF rats (Fig. 2B).
Bilateral Microinjection of Losartan Into the PVN in Sham
and CHF Rats
The effects of bilateral microinjection of losartan (50 nmol
for each) into the PVN were determined in eight sham rats and
eight CHF rats (Fig. 3). Losartan normalized the enhanced
RSNA responses to varying frequency of stimulation in CHF
rats. The significant inhibition appeared at 20 and 30 Hz. The
slope of the RSNA responses to varying frequency of stimu-
lation was also decreased significantly in this group. However,
this inhibition was not seen in sham rats. Bilateral microinjec-
tion of ANG II in the PVN did produce significant effects on
baseline RSNA and MAP in both sham and CHF rats (Table 2).
Dose-Response Relationship of ANG II in CHF Rats
Unilateral microinjection of three doses of ANG II (0.03,
0.3, and 3 nmol) and saline into the PVN were carried out at
random in six CHF rats. The RSNA responses to electrical
stimulation of the central end of the left cardiac sympathetic
nerve and the central gain of the CSAR were measured after
microinjection of saline or ANG II. A representative recording
is shown in Fig. 4. As can be seen, the RSNA response to the
30 Hz stimulation was enhanced after microinjection of ANG
II (3 nmol). Figure 5 shows average RSNA responses to
electrical stimulation of the left cardiac sympathetic nerve in
control (saline) and after 3 nmol ANG II. Figure 6 shows the Fig. 2. Renal sympathetic nerve activity (RSNA) response to varying frequen-
cies of electrical stimulation of cardiac sympathetic afferent nerves in sham
responses for each dose of ANG II. Both high and middle and chronic heart failure (CHF) rats. A: percent change. B: slope. Values are
doses of ANG II augmented the RSNA responses to stimula- means ⫾ SE. RSNA response to stimulation was significantly enhanced in
tion. The significant enhancement appeared at 20 and 30 Hz. CHF rats. *P ⬍ 0.05 compared with sham rats; †P ⬍ 0.05 compared with control.

J Appl Physiol • VOL 97 • NOVEMBER 2004 • www.jap.org

1750 CARDIAC SYMPATHETIC AFFERENT REFLEX IN RATS

Fig. 3. Effects of bilateral microinjection of

losartan into the paraventricular nucleus on
the RSNA response to varying frequencies of
electrical stimulation of cardiac sympathetic
afferent nerves in sham rats and CHF rats. A
and B: percent change. C and D: slope. Values
are means ⫾ SE. Losartan had no significant
effects in sham rats, but it normalized the
enhanced cardiac sympathetic afferent reflex
in CHF rats. *P ⬍ 0.05 compared with sham
rats.

Downloaded from jap.physiology.org on August 12, 2010

(Fig. 7). ANG II not only augmented the RSNA responses to sham and CHF rats; however, the baseline MAP change was
stimulation in sham rats but also enhanced the RSNA re- not significantly different between the two groups.
sponses to stimulation in CHF rats compared with control
(saline), respectively. The significant enhancement appeared at DISCUSSION
20 and 30 Hz in both sham and CHF rats. Although the RSNA
responses were enhanced in CHF rats, much greater responses The primary findings in this study were that 1) the CSAR
to stimulation after microinjection of ANG II into the PVN in evoked by stimulating cardiac sympathetic afferent nerves was
CHF rats were observed compared with sham rats after micro- enhanced in rats with CHF, 2) bilateral microinjection of the
injection of ANG II. Similarly, the slope of the RSNA re- angiotensin AT1-receptor antagonist losartan into the PVN
sponses to varying frequency of stimulation was increased normalized the enhanced CSAR in CHF rats, 3) unilateral
significantly after microinjection of ANG II into the PVN in microinjection of ANG II into the PVN augmented the en-
both sham and CHF rats. The slope of the RSNA responses to hanced CSAR in sham and CHF rats, and 4) pretreatment with
stimulation was significantly greater in CHF rats than in sham losartan abolished the effects of ANG II.
rats either before or after administration of ANG II. In these studies, we used the coronary ligation technique to
As shown in Table 2, microinjection of ANG II into the produce CHF. This model has been extensively used to inves-
PVN significantly increased the baseline RSNA in sham and tigate CHF in rats (15, 28, 35). Coronary-ligated rats showed
CHF rats. However, the baseline RSNA change was greater in an average infarct size of 33.7% of the left ventricle. The heart
CHF rats than in sham rats (22.0 ⫾ 3.4 vs. 10.7 ⫾ 2.1%; P ⬍ weight and heart weight-to-body weight ratio were signifi-
0.05). ANG II also significantly increased baseline MAP in cantly greater in CHF rats than in the sham rats, suggesting
compensatory hypertrophy of noninfarcted regions of the myo-
cardium. In rats with coronary ligation, systolic arterial pres-
Table 2. Baseline change after microinjection of ANG II and sure, pulse pressure, left ventricular peak systolic pressure, and
losartan into PVN maximum of the first derivative of left ventricular pressure
Sham CHF
were decreased, and left ventricular end-diastolic pressure was
increased. These changes indicated that the rats with coronary
n ⌬RSNA, % ⌬MAP, mmHg ⌬RSNA, % ⌬MAP, mmHg
ligation had decreased myocardial contractile function and CHF.
Saline 8 0.8⫾1.8 1.7⫾0.6 ⫺1.1⫾1.5 ⫺0.7⫾0.5 The mechanism by which sympathetic function is enhanced
Losartan in the CHF state has been a topic of intense investigation for
(50 nmol) 8 ⫺1.2⫾1.6 ⫺1.0⫾0.8 ⫺5.9⫾2.1 ⫺4.7⫾1.6 many years. The precise cause is still not completely under-
Saline 6 ⫺1.2⫾2.8 ⫺0.7⫾0.4 ⫺2.7⫾4.0 ⫺0.9⫾0.5
ANG II stood because of its multifactorial nature. The chronic eleva-
(3 nmol) 6 10.7⫾2.1* 8.5⫾2.2* 22.0⫾3.4*† 11.6⫾2.4* tion in sympathetic outflow in this disease cannot be com-
pletely explained by blunted sympathoinhibitory reflexes, be-
Values are means ⫾ SE; n, no. of animals. ANG II, angiotensin II; PVN,
paraventricular nucleus; ⌬RSNA, change in renal sympathetic nerve activity;
cause chronic sinoaortic denervation does not increase
⌬MAP, change in mean arterial pressure. *P ⬍ 0.05 compared with pread- sympathetic outflow or arterial pressure (6, 10, 18). A previous
ministration. †P ⬍ 0.05 compared with sham. study from our laboratory indicated that the CSAR was en-
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 CARDIAC SYMPATHETIC AFFERENT REFLEX IN RATS
Fig. 5. Effects of microinjection of saline, ANG II (3 nmol), and losartan (50
nmol) ⫹ ANG II (3 nmol) into the paraventricular nucleus on the RSNA
responses to varying frequencies of stimulation of cardiac sympathetic afferent
nerves in CHF rats. A: percent change. B: slope. Values are means ⫾ SE.
RSNA response was significantly enhanced after microinjection of ANG II
into the paraventricular nucleus. Pretreatment with losartan completely abol-
ished the effects of ANG II. *P ⬍ 0.05 compared with saline. †P ⬍ 0.05
compared with losartan ⫹ ANG II.
J Appl Physiol • VOL 97 • NOVEMBER 2004 •
1751
Fig. 4. Tracings showing the effect of uni-
lateral microinjection of angiotensin II
(ANG II; 3 nmol in 100 nl) on the RSNA
response to cardiac sympathetic afferent
stimulation (10 V, 0.5 ms, and 30 Hz) in a
CHF rat. ABP, arterial blood pressure; MAP,
mean arterial pressure. Microinjection of
ANG II into the paraventricular nucleus en-
hanced the RSNA response to stimulation.
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Fig. 6. Effects of microinjection of 3 doses of ANG II (0.03, 0.3, and 3 nmol)
into the paraventricular nucleus on the RSNA responses to varying frequencies
of stimulation of cardiac sympathetic afferent nerves in CHF rats. A: percent
change. B: slope. Values are means ⫾ SE. RSNA responses were significantly
enhanced after microinjection of the 0.3- and 3-nmol doses of ANG II.
*Significant difference compared with saline, P ⬍ 0.05; †P ⬍ 0.05 compared
with control.
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1752 CARDIAC SYMPATHETIC AFFERENT REFLEX IN RATS
Fig. 7. Comparison of the effects of microinjection of ANG II (3 nmol into
paraventricular nucleus) on the RSNA responses to varying frequencies of
stimulation of cardiac sympathetic afferent nerves in CHF rats with sham rats.
A and B: percent change. Values are means ⫾ SE. HF, heart failure (CHF).
Losartan abolished the enhanced RSNA responses evoked by ANG II. *Sig-
nificant difference compared with sham, P ⬍ 0.05. †P ⬍ 0.05 compared with
control.
hanced in dogs with pacing-induced CHF (31, 32). It is known
that the CSAR is a sympathoexcitatory reflex. Stimulation of
cardiac sympathetic afferents results in an increase in blood
pressure, HR, and sympathetic outflow. This excitatory CSAR
is activated by an increase in cardiac pressure and dimension
and by various substances that may be augmented in the
myocardium during ischemia or CHF (17, 24). In the present
study, the RSNA responses to electrical stimulation of cardiac
sympathetic afferent nerves were determined in CHF and sham
rats. Because the stimulus was delivered to the afferent limb
(bypassing the cardiac receptors) and the responses were re-
corded in the efferent limb of the CSAR arc, the ratio of
changes in RSNA to different frequencies of stimulation rep-
resents the central gain of this reflex. Because these experi-
ments were carried out in sinoaortic-denervated and vagoto-
mized rats, the possibility of contribution from arterial and
cardiopulmonary baroreflexes secondary to changes in arterial
and cardiac pressures were eliminated. The present study
showed the RSNA responses to electrical stimulation of car-
diac sympathetic afferent nerves were enhanced in rats with
coronary ligation induced CHF, which is consistent with our
laboratory’s previous findings in dogs with pacing-induced
CHF (22). This particular finding suggested that central mech-
anisms are involved in the augmented CSAR in the CHF state.
J Appl Physiol • VOL
It has been shown that ANG II in the central nervous system
affects sympathetic outflow and cardiovascular function (16,
29). Previous studies in our laboratory indicated that intrave-
nous and intracerebroventricular administration of losartan
significantly attenuated the augmented CSAR in dogs with
CHF (22) and that chronic intracerebroventricular infusion of
ANG II enhanced the central sensitivity of the CSAR signifi-
cantly in normal dogs. The latter response was abolished by
losartan (21). These results suggest that elevation of central
ANG II can sensitize the CSAR via central AT1 receptors and
that central ANG II plays an important role in the enhanced
responses in dogs with heart failure. However, the specific sites
where ANG II acts in the central integration of this reflex are
still not known.
The PVN is an important integrative site within the brain to
control cardiovascular function (2, 9). It is known that the PVN
contains neurons that project to the intermediolateral cell
column of the thoracolumbar spinal cord and the rostral ven-
trolateral medulla, areas involved in controlling sympathetic
Downloaded from jap.physiology.org on August 12, 2010
nerve activity and blood pressure (3, 9). Microinjection of
ANG II into the PVN resulted in an increase in RSNA, MAP,
and HR in rats, and the response was significantly attenuated
after systemic administration of losartan (4). ANG II receptors
are densely distributed in the PVN (33). Furthermore, ANG
II-mediated excitatory projections to the RVLM has been
reported recently (13). In view of our studies, we suggest that
ANG II may also be a mediator of the enhanced CSAR in this
hypothalamic nucleus. Therefore, we tested this hypothesis by
determining the effects of microinjection of ANG II and
losartan into the PVN on the CSAR in CHF and sham rats.
Cardiac sympathetic afferent reflex responses were signifi-
cantly augmented by exogenous ANG II injected unilaterally
into the PVN. To block tonic ANG II in the PVN, bilateral
microinjections of the AT1-receptor antagonist losartan was
carried out. In the present experiments, bilateral microinjection
of losartan into the PVN had no effect in sham rats, but it
normalized the enhanced RSNA response to electrical stimu-
lation in rats with CHF. Although the RSNA response to
sympathetic afferent stimulation was enhanced in CHF rats,
unilateral microinjection of ANG II into the PVN further
potentiated this response. The effects of ANG II were abol-
ished by pretreatment with losartan. These results suggest that
the enhanced central gain of the CSAR in the CHF state is
related to the elevated sensitivity or increased number of AT1
receptors in the PVN. Recently, Yoshimura et al. (34) reported
that ANG II receptors in the PVN was increased in rats with
chronic high-output heart failure. Similar results were found in
dogs with pacing-induced CHF in our laboratory (unpublished
data). Taken together, these data suggest that an increased
number of AT1 receptors in the PVN contributes to the en-
hanced central gain of the CSAR. In addition, our laboratory’s
previous study showed that the cerebrospinal fluid concentra-
tion of ANG II was significantly increased in dogs with CHF
(30). In the present study, unilateral microinjection of ANG II
into the PVN potentiated the CSAR in both sham and CHF
rats. Furthermore, the RSNA responses were larger in CHF rats
than in sham rats. This suggests that the elevated level of ANG
II in the PVN may also be involved in the sympathoexcitation
of CHF. In the present study, baseline RSNA and MAP were
not significantly decreased after administration of losartan into
the PVN (Table 2). One would expect that if ANG II or AT1
97 • NOVEMBER 2004 • www.jap.org
 CARDIAC SYMPATHETIC AFFERENT REFLEX IN RATS
receptors were involved in setting the tonic level of sympa-
thetic outflow in this CHF model, we would observe a signif-
icant decrease in RSNA after losartan. Indeed, we demon-
strated this using intracerebroventricular losartan in dogs with
CHF (38). The explanation for this finding compared with our
previous study may be related to the fact that we injected the
losartan unilaterally into only one nucleus that sends projec-
tions to sympathetic premotoneurons compared with the drug
reaching a variety of pertinent sites after intracerebroventricu-
lar injection (38, 39). Be that as it may, it is not clear why
baseline RSNA and MAP did not decrease significantly after
PVN losartan.
In summary, the CSAR induced by electrical stimulation of
the cardiac sympathetic afferent nerves was enhanced in the
rats with coronary ligation-induced CHF. Microinjection of
ANG II into the PVN potentiated the enhanced CSAR, and
losartan normalized the enhanced CSAR in rats with CHF.
These data strongly suggest that ANG II and AT1 receptors in
the PVN play an important role in the enhanced central gain of
the CSAR in rats with CHF.
ACKNOWLEDGMENTS
Losartan was kindly provided by the DuPont-Merck Co.
GRANTS
This research was supported by Grant-in-Aid from the American Heart
Association and National Heart, Lung, and Blood Institute Grants RO1
HL-077691 and PO-1 HL-62222.
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