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Am J Physiol Heart Circ Physiol 306: H121H131, 2014.

First published October 25, 2013; doi:10.1152/ajpheart.00534.2013.

Effects of exercise training on SFO-mediated sympathoexcitation during


chronic heart failure
Tamra L. Llewellyn, Neeru M. Sharma, Hong Zheng, and Kaushik P. Patel
Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska
Submitted 16 July 2013; accepted in final form 21 October 2013

Llewellyn TL, Sharma NM, Zheng H, Patel KP. Effects of exercise


training on SFO-mediated sympathoexcitation during chronic heart failure. Am J Physiol Heart Circ Physiol 306: H121H131, 2014. First
published October 25, 2013; doi:10.1152/ajpheart.00534.2013.Exercise training (ExT) has been shown to reduce sympathetic drive during
heart failure (HF). The subfornical organ (SFO) is involved in the neural
control of sympathetic drive. We hypothesized that an activated SFO
contributes to enhanced sympathetic activity in HF. We also postulated
that ExT would reduce the activation of the SFO and its contribution to
the sympathetic drive during HF. Sprague-Dawley rats were subjected to
coronary artery ligation to induce HF. Rats were assigned to ExT for 3 4
wk. Rats with HF had a 2.5-fold increase in FosB-positive cells in the
SFO compared with sham-operated rats, and this was normalized by ExT.
Microinjection of ANG II (100 pmol) into the SFO resulted in a greater
increase in renal sympathetic nerve activity (RSNA), blood pressure, and
heart rate in the HF group than in the sham-operated group. These
responses were normalized after ExT (change in RSNA: 23 3% vs.
8 2%). ExT also abolished the decrease in RSNA in HF rats after the
microinjection of losartan (200 pmol) into the SFO (21 4% vs.
2 3%). Finally, there was elevated mRNA (5-fold) and protein
expression (43%) of ANG II type 1 receptors in the SFO of rats with HF,
which were reversed after ExT. These data suggest that the enhanced
activity of the SFO by elevated tonic ANG II contributes to the enhanced
sympathoexcitation exhibited in HF. The decrease in ANG II type 1
receptor expression in the SFO by ExT may be responsible for reversing
the neuronal activation in the SFO and SFO-mediated sympathoexcitation in rats with HF.
FosB; renal sympathetic nerve activity; angiotensin II; paraventricular
nucleus; subfornical organ

is the leading cause of death in the


United States (28), with 7% of these deaths caused by heart
failure (HF) (23). The American Heart Association reports that
5.3 million adults over the age of 20 yr have HF (23). The
increasing incidence of HF suggests the need for better management as well as understanding the underlying mechanisms
involved. Exercise training (ExT) is an established therapy for
HF that improves the health, quality of life, and clinical
outcome of patients with this disease (3). However, the precise
mechanisms involved in the beneficial effects of ExT remain
unclear.
Enhanced sympathetic nerve activity is a risk factor that
influences the progression of HF and mortality in patients.
Although most therapeutic pharmaceutical strategies target the
peripheral symptoms of the disease, they may not influence the
enhanced sympathetic nerve activity. Indeed, studies (6, 17)
have detailed the role of cardiac and hemodynamic mechanisms involved in the elevated sympathetic drive in HF, but

CARDIOVASCULAR DISEASE

Address for reprint requests and other correspondence: K. P. Patel, Dept. of


Cellular and Integrative Physiology, Univ. of Nebraska Medical Center,
985850 Nebraska Medical Center, Omaha, NE 68198-5850 (e-mail: kpatel
@unmc.edu).
http://www.ajpheart.org

these mechanisms do not fully account for the total sympathoexcitation observed in HF.
In the central nervous system, the paraventricular nucleus
(PVN) of the hypothalamus mediates sympathetic nerve activity and influences the cardiovascular system (1, 18, 19). Our
recent study (15) has shown that the PVN is activated in rats
with HF in conjunction with enhanced glutamatergic tone
within the PVN. Specifically, the neurons of the PVN exhibited
an increased FosB expression, which is a marker of chronic
neuronal activity (30). In addition, renal sympathetic nerve
activity (RSNA) responses were enhanced in rats with HF after
the microinjection of N-methyl-D-aspartate (NMDA) into the
PVN. The activation of the PVN during HF and the exaggerated sympathoexcitatory response to NMDA were both normalized after ExT (15). However, the mechanisms that drive
the enhanced activation of the PVN during HF and mediate the
ExT effect remain to be examined.
Other studies (2, 24, 44) have demonstrated the importance of the subfornical organ (SFO) in sympathetic output
and cardiovascular regulation. The SFO is a highly vascularized circumventricular organ that lacks a functional
blood-brain barrier (37). Therefore, it provides an interface
between peripherally circulating molecules, such as ANG II,
and the brain. Plasma ANG II levels have been shown to
increase in HF and to be restored after ExT (21, 48).
Therefore, the SFO provides a potential link for cross-talk
between peripheral and central cardiovascular regulation. In
fact, our group and others (2, 22, 25, 44) have demonstrated
that the SFO is neuroanatomically connected to the PVN.
Furthermore, electrical stimulation of the SFO produces
action potentials in neurons in the PVN that can be blocked
by AP5 (NMDA receptor blocker), suggesting that the SFO
influences the PVN by a glutamatergic mechanism (24).
Recently, we (22) have shown that ANG II stimulation in
the SFO induces sympathoexcitation via the PVN by a
glutamatergic mechanism. We (15, 48) have also shown that
the PVN is activated in rats with HF in conjunction with
enhanced glutamatergic tone within the PVN.
The mechanisms that drive the enhanced activation of the
PVN during HF and mediate the ExT effect remain to be
examined. We hypothesized that the SFO is activated in HF
and makes critical contributions to the regulation of sympathetic drive in HF. Specifically, we postulated that the
ANG II response and tonic endogenous activation of ANG
II type 1 (AT1) receptors in the SFO are enhanced in HF and
that ExT would reverse these mechanisms. Furthermore, we
predicted that AT1 receptor protein expression would be
enhanced in the SFO during HF and that ExT would restore
its expression leading to the normalization of sympathetic
tone.

0363-6135/14 Copyright 2014 the American Physiological Society

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MATERIALS AND METHODS

Induction of HF. Male Sprague-Dawley rats weighing 180 200 g


(Sasco Breeding Laboratories, Omaha, NE) were fed and housed
according to institutional guidelines. The Institutional Animal Care and
Use Committee of the University of Nebraska Medical Center
approved all protocols. Rats were randomly assigned to sham-operated (sham) and HF groups. Left coronary artery ligation surgery was
performed to induce HF. Each rat was anesthetized with pentobarbital
sodium (50 mg/kg ip), and the trachea was intubated to assist ventilation. A left thoracotomy was performed, and the left coronary artery
was ligated between the pulmonary artery outflow tract and the left
atrium. Sham rats received only the thoracotomy and heart manipulation. The thorax was closed, the tracheal tube was removed, and the
rat was allowed to recover from anesthesia.
Six weeks after surgery, echocardiography parameters were measured under light isoflurane anesthesia in a subset of rats. The left
ventricular (LV) end-systolic dimension (LVESD) and LV end-diastolic dimension (LVEDD) were visualized, measured, and used to
calculate fractional shortening and ejection fraction. Before euthanization, LV end-diastolic pressure (LVEDP) was measured (PowerLab, AD Instruments, Colorado Springs, CO) using a conductance
catheter (Millar) inserted into the LV via the right carotid artery. To
anatomically assess the extent of HF, infarct size of the LV was
calculated. The LV was isolated from the heart and longitudinally cut
and laid flat. Infarct size (in %) was measured by dividing the size of
the infarcted area by the total size of the LV using ImageJ software
[National Institutes of Health (NIH)]. Rats with LVEDP 15 mmHg
and infarct size 30% were considered to be in HF.
Urinary norepinephrine excretion measurements. Urinary norepinephrine excretion was measured as an index of overall sympathetic
activation. Six to eight weeks after surgery, rats from all groups were
placed in metabolic cages, and urine was collected for 24 h. The 50-ml
collecting tubes contained mineral oil to prevent evaporation losses.
After collection, urine was centrifuged, transferred to Eppendorf tubes
containing 1 N HCl to prevent the autooxidation of catecholamines,
and then stored at 80C. The urinary norepinephrine concentration
of thawed samples was measured using a commercially available
ELISA kit (Labor Diagnostika Nord, Nordhorn, Germany) following
the manufacturers instructions. The limit of detection of the assay
was 1.5 ng/ml norepinephrine in urine. The urinary excretion of
norepinephrine was calculated using the urine flow rate.
ExT protocol. Three weeks after surgery, sham and HF rats were
randomly assigned to complete a moderate ExT regimen or to remain
sedentary (Sed). ExT groups ran on a motorized treadmill (Columbus
Instruments, Columbus, OH) 5 days/wk for 3 4 wk. The ExT protocol
began with a familiarization period and progressed in duration and
intensity to 20 25 m/min, for 60 min/day, at a 510% grade according
to a modified protocol by Musch and Terrell (27). Five rats that did
not acclimate to running on the treadmill were excluded from the
study. The aerobic training effect was verified in all groups of rats by
measuring citrate synthase activity in the soleus muscle after euthanization according to the Srere method (38). At this time point (6 wk
after surgery), echocardiography and experimentation were carried
out.
Hemodynamic and RSNA measurements. The preparation for measuring hemodynamic and RSNA parameters was performed as previously described (22). Briefly, HF and sham rats were anesthetized
with urethane (0.75 g/kg ip) and -chloralose (70 mg/kg ip). The left
femoral vein and artery were cannulated, and the arterial line was
connected to a pressure transducer (Gould P23 1D) for data recording
and analysis (PowerLab, AD Instruments) of mean arterial pressure
(MAP) and heart rate (HR). Next, the left kidney was exposed by a
retroperitoneal flank incision, and a branch of the renal nerves was
isolated, placed on thin bipolar platinum electrodes, and secured with
a Wacker Silgel mixture (604 and 601). The electrical signal was
amplified with high- and low-frequency cutoffs of 1,000 and 100 Hz,

respectively (Grass amplifier). The rectified output from the amplifier


(RC filtered, time constant: 0.5 s) was displayed using the PowerLab
system (8si, AD Instruments) to record and integrate the raw renal
nerve discharge. Background noise was determined at the end of the
experiment after the administration of hexamethonium (30 mg/kg iv).
Overall integrated renal nerve activity was calculated by subtracting
the background noise from the recorded value.
ANG II and losartan microinjection into the SFO. Anesthetized rats
were placed in a stereotaxic apparatus (David Kopf Instruments,
Tujanga, CA) for microinjections into the SFO. A longitudinal incision was made on the head to expose the bregma, and a small burr
hole was made in the skull to access the SFO. The coordinates of the
SFO were 0.9 mm posterior to the bregma, 5.6 mm ventral to the dura,
and on the midline (33). A thin needle (outer diameter: 0.2 mm)
connected to a 0.5-l microsyringe (Hamilton, Reno, NV) was lowered into the SFO. RSNA, MAP, and HR were recorded before and
after the microinjection of 100 200 pmol (50 100 nl) of exogenous
ANG II (Sigma-Aldrich, St. Louis, MO) or losartan (Merck) dissolved
in artificial cerebrospinal fluid into the SFO. Injections were randomized and given at 30 45-min intervals. Vehicle injections of artificial
cerebrospinal fluid were given to control for volume responses. Three
to four microinjections were made into the SFO in each experiment.
Microinjection sites were confirmed at the end of each experiment by
histology. Sympathoexcitation after drug injection was calculated as
the percent change in RSNA from baseline and as absolute changes in
MAP and HR from baseline. Baseline and peak responses were
averaged over a 30-s time interval.
Brain histology and immunohistochemistry for FosB and the AT1
receptor. A separate cohort of the four groups of rats was anesthetized
with pentobarbital (65 mg/kg ip) and perfused transcardially with 150
ml of heparinized saline followed by 300 ml of freshly prepared 4%
paraformaldehyde in 0.1 M of sodium phosphate buffer. The brain
was removed from each rat, postfixed at 4C for 4 h in 4% paraformaldehyde, and then placed in 30% sucrose for 72 h. The brain was
sectioned (30 m) in the coronal plane with a cryostat. After being
washed with PBS, sections were blocked with 10% goat serum and
then incubated with goat anti-FosB primary antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) for 2 days at 4C. After being
washed with PBS, sections were incubated with biotinylated anti-goat
secondary antibody for 2 h, incubated with avidin-biotin complex
(1:200, Vectastain Elite ABC kit, Vector Laboratories, Burlingame,
CA) for 1 h and then rinsed in PBS for 30 min. Sections were stained
with diaminobenzidine solution (Vector Laboratories) for 10 min,
washed with PBS, and mounted for visualization with light microscopy (100, Leica, Buffalo Grove, IL). ImageJ software (NIH) was
used to quantify FosB-positive cells in the SFO. The number of nuclei
stained with FosB within a 200-m radius from the center of the SFO
was counted, blindly, using the Find Maxima function in ImageJ,
which was validated by manual counting of the nuclei. Four to five
sections of the SFO were averaged for each rat.
Immunofluorescence was used to assess the localization of AT1
receptor expression in the SFO. SFO sections were obtained as
described above and incubated with 10% goat serum and 0.02%
Triton X-100 in PBS for 1 h at room temperature. Tissues were then
incubated with anti-rabbit AT1 receptor primary antibodies (1:500,
Santa Cruz Biotechnology) overnight at 4C. After being washed with
PBS, sections were incubated with Cy3-conjugated goat anti-rabbit
antibody (1:500, Jackson ImmunoResearch) for 3 h at room temperature. After being washed with PBS, sections were mounted on slides,
and coverslips were placed with fluoromounting-G (SouthernBiotech). The distribution of immunofluorescence within the SFO was
viewed using an Olympus fluorescence microscope equipped with a
digital camera (Qimaging). Quantification of the intensity of the
fluorescence was done using ImageJ software (NIH).
Plasma levels of ANG II. At the time of death, whole blood was
collected via the left renal vein from rats from all four groups. The
needle was coated in EDTA (80 mg/ml), and the collection vial

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Table 1. Primer sequences for real-time PCR


Gene

Reference Sequence

Forward Primer

Reverse Primer

ANG II type 1 receptor


Angiotensin-coverting enzyme
Ribosomal protein L19

NM_030985
BC_085760
NM_031103

5=-TATCACAGTGTGCGCGTTTCA-3=
5=-CAGCTTCATCATCCAGTTCC-3=
5=-CCCCAATGAAACCAACGAAA-3=

5=-TGGTAAGGCCCAGCCCTAT-3=
5=-CTAGGAAGAGCAGCACCCAC-3=
5=-ATGGACAGTCACAGGCTTC-3=

contained 10 mg/ml EDTA. The angiotensinase inhibitor bestatin was


added to blood samples immediately after collection. Blood samples
were centrifuged at 4,000 rpm at 4C for 5 min. Blood plasma was
carefully transferred into Eppendorf tubes and stored at 80C. The
concentration of plasma ANG II was then measured using a commercially available ELISA kit (Cayman Chemical, Ann Arbor, MI)
following the manufacturers instructions.
Micropunch of the SFO. Animals were euthanized with pentobarbital (150 mg/kg ip), and the brain was removed and immediately
frozen on dry ice. Five serial coronal sections (100 m) were cut
using a cryostat, and, following the Palkovits technique (31), the SFO
was punched using a diethylpyrocarbonate-treated blunt 18-gauge
needle attached to a syringe.
Real-time PCR to determine mRNA expression of AT1 receptors in
the SFO. RNA for real-time PCR was extracted from SFO punches
according to the TriReagent method following the manufacturers
instructions (MRC, Cincinnati, OH). In brief, the tissue homogenate
was separated into organic and aqueous phases by the addition of
bromochloropropane and centrifugation. The RNA, contained in the
aqueous phase, was precipitated with isopropanol, washed with ethanol, dried, and solubilized in 10 l of nuclease-free water. After the
extraction of mRNA, samples underwent reverse transcription for 40
min at 37C in the presence of 1.5 M random hexamers and 100 units
of Moloney murine leukemia virus reverse transcriptase. Real-time
RT-PCR measurements were made using the iCycler iQ Multicolor
Real-Time Detection System with output to a computer-based acquisition system (Bio-Rad). The protocol consisted of denaturation (95C
for 3 min), amplification and quantification repeated 50 times (95C
for 10 s, 55C for 45 s), denaturation at 95C for 1 min, reannealing
at 55C for 1 min, and a melt curve (5595C with a heating rate of
0.5C/10 s). The reaction mixture consisted of SYBR Green Supermix
(Bio-Rad), 10 nM of forward primers, 10 nM of reverse primers,
nuclease-free water, and the cDNA template of interest. Primer
sequences for the AT1 receptor, angiotensin-converting enzyme
(ACE), and ribosomal protein L19 are shown in Table 1. The relative
expression of AT1 receptors and ACE were calculated according to
the 2CT method, where CT is threshold cycle.
Western blot analysis to measure AT1 receptor protein expression.
SFO tissue punches were added to 80 l of lysis buffer (pH 7.5 at
4C) and homogenized by sonication for 15 s. After the protein
concentration was quantified with a BCA protein assay kit (Pierce
Chemical, Rockford, IL), 20 g of protein mixed with SDS-PAGE
buffer were loaded and fractionated on a 7.5% polyacrylamide gel.

Protein was transferred to a polyvinylidene difluoride membrane


(Millipore, Billerica, MA), blocked with 5% dry milk in Tris-buffered
saline and Tween 20 (TBST), and incubated with rabbit anti-AT1
receptor (Santa Cruz Biotechnology), rabbit anti-FosB (Cell Signaling Technology, Danvers, MA), or rabbit anti-GAPDH (Santa Cruz
Biotechnology) primary antibodies overnight at 4C. After three
washes with TBST, the membrane was incubated with an anti-rabbit
peroxidase-conjugated secondary antibody (Thermo Scientific, Rockford, IL) for 1 h. Signals were visualized using an enhanced chemiluminescence substrate (Thermo Scientific) and detected by a
Worklab digital imaging system. Protein band signals were quantified
by ImageJ software (NIH) and normalized to the intensity of the
GAPDH signal. Previously, we have confirmed the absence of crossnonspecific binding of AT1 receptor antibody in two ways (36, 47) to
address concerns regarding the specificity of commercially available
antibodies (4). First, we used two different antibodies from two
different companies, with the two different antibodies targeting different ends of the AT1 receptor protein, showing that they resulted in
similar bands (47). Second, we tested the human embryonic kidney
cell line lacking the AT1 receptor as a negative control and the same
cell line transfected with human AGTR1 cloned in a pcDNA 3.1
expression vector (UMR cDNA Resource Center, University of Missouri, Rolla, MO) as a positive control. Using immunoblot detection,
we were able to detect the AT1 receptor signal in human embryonic
kidney-293 cells transfected with AGTR1 expression plasmid but not
in empty cells (36).
Statistical analysis. Data were subjected to two-way ANOVA to
compare differences between HF and sham rats subjected to ExT or
Sed protocols. Tukeys honestly significant difference test was used in
post hoc analysis to make comparisons between groups. Statistical
significance was set at P 0.05.
RESULTS

Characterization of HF and ExT. Rats that underwent


coronary artery ligation were excluded if they did not meet
the criteria for HF as defined in MATERIALS AND METHODS.
Compiled data for infarct size, LVEDP, LVEDD, LVESD,
ejection fraction, fractional shortening, urinary norepinephrine, and citrate synthase activity from the four groups of
rats are shown in Table 2. Briefly, infarct size was 30%
in both HF groups, whereas there was no infarct in either

Table 2. Effects of ExT on characteristics of HF


Characteristic

Sham Sed Group

HF Sed Group

Sham ExT Group

HF ExT Group

Infarct size, % of LV
LV end-diastolic pressure, mmHg
Mean arterial pressure, mmHg
Ejection fraction, %
Fractional shortening, %
LV end-diastolic dimension, mm
LV end-systolic dimension, mm
Urinary norepinephrine, g/day
Citrate synthase activity, molg1min1

0
4.1 0.5
122.6 6.7
69.2 0.9
40.2 0.7
7.4 0.4
4.4 0.4
3.6 0.3
11.5 1.5

34.1 2*
24 3.9*
109.6 6.1
40.9 3*
21.4 1.9*
10.3 0.7*
8.3 0.8*
8.1 0.3*
12.8 0.7

0
3.6 0.4
104.3 9.4
75.15 1.6
45.39 1.3
7.2 0.8
4.0 0.7
3.5 0.2
18.2 2.9*

32.6 2*
9.6 1.5*
85.1 5
42.25 3.6*
22.48 2.4*
10.1 1.2*
7.9 1.5*
3.8 0.1
16.3 1.8*

Values are means SE; n 8 rats/group. ExT, exercise training; HF, heart failure; sham, sham operation; Sed, sedentary; LV, left ventricle. *P 0.05 vs.
the sham Sed group; P 0.05 vs. the HF Sed group.
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EXERCISE IMPROVES SFO-MEDIATED SYMPATHO-EXCITATION IN HF

sham group. LVEDP was also elevated in the HF Sed group


compared with the sham Sed group, suggesting impaired LV
function. ExT improved LVEDP during HF but did not
normalize it to sham Sed levels. Further characterization by
echocardiography revealed that ejection fraction was reduced in the HF Sed group compared with the sham Sed
group, and ExT did not improve this marker for HF. A
similar pattern was seen for fractional shortening of the LV.

An index of overall sympathetic activation, measured by


urinary norepinephrine excretion, was increased in the HF
Sed group compared with the sham Sed group and was
normalized by ExT. Finally, to characterize the training
effect from exercise, citrate synthase activity in the soleus
muscle was measured. Both sham ExT and HF ExT groups
exhibited a similar increase in citrate synthase activity
compared with their respective Sed control groups.

Fig. 1. Effects of exercise training (ExT) on


FosB immunohistochemistry in the subfornical organ (SFO) during heart failure (HF).
A and B: representative images of FosB
immunostaining from sham-operated (sham)
sedentary (Sed) and HF Sed rats (A) and
sham ExT and HF ExT rats (B). Arrows
indicate examples of FosB-positive staining.
Bars 100 m.

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EXERCISE IMPROVES SFO-MEDIATED SYMPATHO-EXCITATION IN HF

120

SHAM HF
EXT EXT

FosB

40 kDa

GAPDH

37 kDa

0.8

100

Fig. 2. Effects of ExT on activation of the SFO


during HF. A: summary data for FosB-positive
cells in the SFO (as shown in Fig. 1). Data are
presented as means SE; n 8 rats/group.
B: representative Western blot of FosB (top) and
summary data (bottom). n 4 rats/group. *P
0.05, sham Sed group vs. HF Sed group; P
0.05, HF Sed group vs. HF ExT group.

0.7
80

FosB : GAPDH

FosB-Positive Cells

SHAM HF
SED
SED

60

40

20

0.6
0.5
0.4
0.3
0.2
0.1
0

SHAM
SED

HF
SED

SHAM
EXT

HF
EXT

SHAM HF SHAM HF
SED SED EXT EXT

FosB immunohistochemistry and protein expression in the


SFO. The number of cells in the SFO that stained positive for
the neuronal activation marker FosB was increased in the HF
Sed group (101 9 cells) compared with the sham Sed group
(29 2 cells; Figs. 1 and 2A). This enhanced activation was
attenuated in HF ExT rats (36 7 cells). Western blot data,
using the more specific FosB antibody, showed a similar

pattern for activation of the SFO; FosB expression was


increased 33% in the HF Sed group (Fig. 2B). Interestingly,
both sham and HF groups demonstrated little FosB expression in the SFO after ExT. Altogether, these data show that the
SFO is in an activated state during HF, perhaps contributing to
the enhanced sympathetic activity. Interestingly, after ExT, the
activation in the SFO is attenuated, paralleling the decrease in

SHAM SED
HR
(BPM)
MAP
(mmHg)
BP
(mmHg)

600

600

600

600

100

100

100

100

160

160

160

160

40

40

40

40

140

140

140

140

20

20

20

20

0.06

0.06

0.06

0.06

0.01

0.01

0.01

0.01

0.3

0.3

0.3

0.3

IRSNA
(Microvolts/s)
RSNA
(Microvolts)

HF SED

-0.3

-0.3

Baseline

-0.3

ANG II (100 pmol)

-0.3

Baseline

SHAM EXT
600

600

600

600

100

100

100

100

MAP
(mmHg)

160

160

160

160

40

40

40

40

140

140

140

140

20

20

20

20

IRSNA 0.06
(Microvolts/s)

0.06

0.06

0.06

0.01

0.01

0.01

0.01

0.3

0.3

0.3

0.3

RSNA
(Microvolts)

-0.3

-0.3

Baseline

1s

ANG II (100 pmol)

1s

HF EXT

HR
(BPM)

BP
(mmHg)

ANG II (100 pmol)

-0.3

-0.3

ANG II (100 pmol)

Baseline

Fig. 3. Representative tracings of renal sympathetic nerve activity (RSNA) and hemodynamic parameters before (baseline) and after microinjection of ANG II
(100 pmol) into the SFO in four groups of rats (sham Sed, HF Sed, sham ExT, and HF ExT). HR, heart rate [in beats/min (BPM)]; MAP, mean arterial pressure;
BP, blood pressure; iRSNA, integrated RSNA.
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EXERCISE IMPROVES SFO-MEDIATED SYMPATHO-EXCITATION IN HF


20

25

Sham Sed

20
20

10

HR (beats/min)

15

MAP (mmHg)

RSNA (% of basal value)

30

10

HF Sed
Sham Ext
HF Ext

15

10

5
5

Fig. 4. Summary data for the effect of ExT on RSNA and hemodynamic responses to ANG II microinjection into the SFO during HF. Data are presented as
means SE; n 5 rats/group. *P 0.05, sham Sed vs. HF Sed group; P 0.05, HF Sed group vs. HF ExT group.

overall sympathetic activation as measured by urinary norepinephrine excretion.


ANG II and losartan microinjection into the SFO. Stimulation of the SFO by microinjection of ANG II increased RSNA
in both sham Sed and HF Sed groups. The percent change in
RSNA from basal values was enhanced in the HF Sed group
(22.7 3.1%) compared with the Sham Sed group (9.0
1.3%; Figs. 3 and 4). Similarly, MAP and HR responses to
ANG II were elevated in the HF Sed condition (13 4 vs.
3 1 mmHg and 17 3 vs. 5 1 beats/min, respectively).

However, after ExT, these parameters were all normalized to


those observed in sham Sed rats. These data show that in HF,
there is an exaggerated response to ANG II stimulation in the
SFO, suggesting a hyperactive state in this region. Further,
ExT restored the response to ANG II stimulation, suggesting
that the ExT mechanism reestablishes the ANG II response in
the SFO, similar to the sham Sed group.
To elucidate the endogenous tonic contribution of ANG II,
the AT1 receptor blocker losartan was microinjected into the
SFO in all four groups of rats. After losartan microinjection,

SHAM SED

HF SED

600

600

600

600

100

100

100

100

MAP 120
(mmHg)

120

120

120

HR
(BPM)

60

60

60

60

BP
160
(mmHg)

160

160

160

40

40

40

40

IRSNA 0.15
(Microvolts/s)

0.15

0.15

0.15

0.02

0.02
0.6

0.02
0.6

0.02
0.6

RSNA 0.6
(Microvolts)

Baseline

-0.6

-0.6

-0.6

-0.6

Los (200 pmol)

Baseline

SHAM EXT
600

600

600

100

100

100

100

MAP 120
(mmHg)

120

120

120

60

60

60

60

BP
160
(mmHg)

160

160

160

40

40

40

40

IRSNA 0.15
(Microvolts/s)

0.15

0.15

0.15

0.02
0.6

0.02
0.6

0.02
0.6

0.02

-0.6

-0.6

Baseline

1s

Los (200 pmol)

1s

HF EXT

600
HR
(BPM)

RSNA 0.6
(Microvolts)

Los (200 pmol)

-0.6

Los (200 pmol)

-0.6

Baseline

Fig. 5. Representative tracings of RSNA and hemodynamic parameters before (baseline) and after microinjection of losartan (Los; 200 pmol) into the SFO in
four groups of rats (sham Sed, HF Sed, sham ExT, and HF ExT).
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EXERCISE IMPROVES SFO-MEDIATED SYMPATHO-EXCITATION IN HF

-10

-15

-20

10

-5

-10

-15

-10
Sham Sed

Sham Ext

*
-25

HF Sed

-20

HF Ext

-30

-20

-25

-30

HR (beats/min)

-5

MAP (mmHg)

RSNA (% of basal value)

*
-40

Fig. 6. Summary data for the effect of ExT on RSNA and hemodynamic responses to losartan microinjection into the SFO during HF. Data are presented as
means SE; n 5 rats/group. *P 0.05, sham Sed group vs. HF Sed group; P 0.05, HF Sed group vs. HF ExT group.

there was a significantly greater decrease in RSNA (21.6


4.9% of the basal value), MAP (17 4 mmHg), and HR
(28 6 beats/min) from baseline in the HF Sed group,
whereas there was little change from baseline in the sham Sed
group (Figs. 5 and 6). After ExT, responses to losartan in the
HF group were similar to sham groups. These data suggest that
there is enhanced ANG II-mediated endogenous tone in the HF
Sed state in the SFO. Furthermore, ExT restored the sympathetic and hemodynamic responses to losartan and ANG II,
indicating that ExT improves sympathoexcitation via the SFO
during HF by decreasing the endogenous ANG II-mediated
tone or activity in the SFO.
Expression of the ANG II system during HF. Circulating
levels of ANG II were increased in the HF Sed group nearly
twofold (53.2 8.3 vs. 29.5 1.9 pg/ml; Fig. 7A). ANG II
levels were restored to sham levels after ExT in HF rats
(29.3 1.8 pg/ml), which has been previously reported (21,
48). Interestingly, ACE mRNA expression in the SFO was
unchanged during HF and after ExT (sham Sed group: 1, HF
Sed group: 1.03 0.18, sham ExT group; 0.71 0.23, and HF
ExT group: 0.67 0.22, n 4 rats/group), suggesting that
circulating ANG II, rather than centrally produced ANG II, is
responsible for ANG II-mediated activation of the SFO during
HF. In the SFO of HF Sed rats, AT1 receptor mRNA expression was increased fivefold (Fig. 7B) and protein expression
was increased 43% compared with sham Sed control rats (Fig.
7C). Additionally, immunofluorescence corroborated the enhanced AT1 receptor expression in the SFO during HF and its
reduced expression after ExT (Fig. 8). These results validate
the functional data and suggest that the SFO is overactive
during HF due to the increased AT1 receptor expression. After
ExT, mRNA and protein expression of the AT1 receptor in the
SFO were restored to sham Sed levels.
DISCUSSION

Our study demonstrates that neuronal activity in the SFO is


elevated during HF with a concomitant increase in systemic
sympathetic output, as indicated by the elevated excretion of
norepinephrine in the urine. Additionally, sympathoexcitatory
and hemodynamic responses to both endogenous and exogenous ANG II in the SFO were exaggerated in HF, suggesting
that the SFO contributes to the increased sympathoexcitation

exhibited in HF via an ANG II mechanism. The AT1 receptor,


which is well known to excite neurons upon ANG II binding
(39), is increased in terms of mRNA and protein expression in
the SFO during HF. We propose that the elevated AT1 receptor
expression contributes to the neuronal activation of the SFO
during HF. These results demonstrate the critical role for the
SFO in relaying humoral signals such as ANG II, which are
elevated in HF, to downstream cardiovascular centers, such as
the PVN, during HF. Our study also revealed the therapeutic
effects of ExT during HF and provide insights into how the
SFO contributes to improving the elevated sympathetic tone
during HF, likely through an ANG II/AT1 receptor-dependent
mechanism.
The present study found that the SFO is activated in HF. In
HF Sed rats, there were threefold more FosB-positive cells in
the SFO compared with sham Sed rats. In addition, HF Sed rats
exhibited greater protein expression of FosB, which is a
stable splice variant of FosB that is specifically upregulated
during chronic neuronal activation (29, 30). These findings
support the hypothesis that the increased activation of the
neurons in the SFO during HF functionally alters sympathetic
drive. Another study (40) has reported SFO activation using
Fra-like immunofluorescence at only 2 wk but not after 4 wk
after the induction of HF. Furthermore, ANG II infusion has
also been shown to activate the SFO (5, 11). These studies are
consistent with our findings because circulating ANG II levels
are elevated in HF (32, 48). In addition, a time course study (5)
examining inducible transcription factors in the SFO found that
expression of the neuronal activity marker cFOS was rapidly
increased after an intracerebroventricular injection of ANG II.
Therefore, as the SFO can sense circulating peptides, it could
be inferred that the enhanced FosB expression is related to the
increased levels of plasma ANG II during HF.
Additionally, HF Sed rats exhibited an increased sympathetic and hemodynamic response to acute activation of the
SFO by ANG II microinjection. In light of the FosB results,
which suggest that the SFO is activated during HF, may be due
to the exaggerated sympathoexcitatory response to ANG II.
Consistent with these findings, there was a sharp decrease in
sympathetic activity in response to losartan in the SFO in HF
Sed rats, whereas this response was minimal or absent in the
sham Sed group. These results imply that there is a tonic high

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EXERCISE IMPROVES SFO-MEDIATED SYMPATHO-EXCITATION IN HF

Plasma Angiotensin II
(pg/ml)

70

60
50
40
30
20
10
0

SHAM
SED

HF
SED

SHAM
EXT

HF
EXT

B
AT1R mRNA Expression
(Normalized to RPL 19)

6
5
4
3
2
1
0

SHAM
SED

SHAM SED

HF
SED

HF SED

SHAM
EXT

SHAM EXT

HF
EXT

HF EXT

AT1R

43 kDa

GAPDH

37 kDa

1.2

AT1R : GAPDH

1
0.8
0.6
0.4
0.2
0

SHAM
SED

HF
SED

SHAM
EXT

HF
EXT

Fig. 7. A and B: effects of ExT on plasma levels of ANG II in four groups (A;
n 5 rats/group) and mRNA expression of the ANG II type 1 (AT1) receptor
(AT1R) in the SFO in four groups (B; n 4 rats/group). C: representative
Western blots (top) and summary data (bottom) of AT1R protein expression in
the SFO in four groups (sham Sed, HF Sed, sham ExT, and HF ExT). n 6
rats/group. GAPDH was used as a loading control. *P 0.05, sham Sed group
vs. HF Sed group; P 0.05, HF Sed group vs. HF ExT group.

level of ANG II present at the level of the SFO during HF,


which may be contributing to the state of activation of the SFO.
Previous studies (2, 24, 44) have demonstrated the importance
of the SFO in cardiovascular regulation. However, most studies
have examined the pressor response to ANG II injection in the
SFO (16), and little work has been done regarding its contribution to sympathetic regulation specifically. Previously, we
(22) have shown that ANG II stimulation in the SFO induces

sympathoexcitation via the PVN by a glutamatergic mechanism. Therefore, the ANG II-induced activation of the SFO
influences the neuronal activity in the PVN and thereby regulates sympathetic activity. Others have elucidated some of the
cellular mechanisms within neurons that contribute to ANG
II-mediated effects in HF and other cardiovascular diseases,
such as hypertension. First, it has been demonstrated that
superoxide is involved in increasing neuronal activity via ANG
II inhibition of neuronal K channels (45). Additionally, ANG
II-mediated hypertension can be abolished by scavenging intracellular superoxide in the SFO (35, 49). The enhanced
inflammatory milieu during HF and high-ANG II states may
also act upon the SFO and contribute to its neuronal activation
(8, 12, 43). The superoxide and inflammatory mechanisms
likely contribute to the neuronal activation in the SFO and the
downstream sympathetic activation observed during highANG II and HF conditions.
Consistent with our findings of an overactive sympathoexcitatory state in the SFO during HF, the AT1 receptor was
upregulated during HF in the SFO. Previous work has also
shown that AT1 receptor mRNA and protein (42, 46), and AT1
receptor binding densities (46) are elevated in the SFO during
HF. The AT1 receptor has also been found to be elevated in
other important cardiovascular regulatory regions during HF,
such as the PVN and rostral ventrolateral medulla (20, 47). Due
to its permeable blood-brain barrier, elevated ANG II in
circulating blood during HF could increase sympathetic activation via its AT1 receptor in the SFO. Interestingly, ANG II
exhibits positive feedback on its own receptor, upregulating its
expression during HF. It has been shown that ANG II upregulates AT1 receptors in cultured neurons via an NF-B mechanism (26) and in the SFO and PVN through an ANG IIdependent MAPK pathway (41, 42). Here, we also found that
ACE mRNA expression was unchanged in the SFO during HF,
suggesting that local ANG II production in the SFO may not be
influencing SFO activation to the same extent as circulating
ANG II. Ultimately, the elevated AT1 receptor expression in
the SFO is improved by ExT, and further research is warranted
to determine the precise source of the ANG II.
Although ExT did not improve cardiac function significantly, overall sympathetic drive was attenuated, as indicated
by a decrease in excretion of urinary norepinephrine. Reducing
norepinephrine is very important, as HF patients with lower
levels of plasma norepinephrine are given a better prognosis
(7). These results suggest that ExT may alter the activity of
neurons in central cardiovascular regulatory sites, such as the
SFO, thereby contributing to the improvement in sympathetic
tone. Indeed, our results show that ANG II-mediated sympathetic and hemodynamic responses were improved as well as
AT1 receptor expression in the SFO. We and others have
previously shown that ExT improves ANG II signaling in
downstream regions of the brain, such as the PVN (10, 13, 48),
and rostral ventrolateral medulla (13, 50). Our present evidence
adds that the SFO also displays significant ANG II signaling
dependency during HF and in mediating the effects of ExT.
However, it was not explicitly established here whether the
downstream centers are dependent on changes in the SFO in
HF. Additionally, components of the nonclassical axis of the
ANG system were not investigated here but have been linked
to the central effects of ExT and losartan in the treatment of HF
(13, 50). Future studies should investigate the influence of

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EXERCISE IMPROVES SFO-MEDIATED SYMPATHO-EXCITATION IN HF

H129

A
AT1R

SHAM
SED

HF
SED

SHAM
EXT

HF
EXT
Fig. 8. Effects of ExT on immunofluorescence
staining of the AT1R in the SFO during HF.
A: representative images from each of the four
groups (sham Sed, HF Sed, sham ExT, and
HF ExT). B: quantification of AT1R immunofluorescence intensity. n 4 rats/group. Bars
100 m. *P 0.05, sham Sed group vs. HF
Sed group; P 0.05, HF Sed group vs. HF
ExT group.

B
Fluorecence Intensity (a.u.)

2000

*
1500

1000

500

SHAM
SED

HF
SED

SHAM
EXT

HF
EXT

ACE2 and the Mas receptor, as they may be involved in


mediating the ExT-induced changes in the SFO during HF.
The present study offers insights into the mechanism of the
positive effects of ExT during HF. To date, the mechanisms for
the normalization of sympathetic outflow by ExT during HF
have not been fully elucidated. We (15, 48) have previously
shown that glutamatergic and angiotensinergic activation in the
PVN are restored after ExT during HF; however, it was not
clear what upstream mechanisms may be mediating the improvement after ExT. Here, we found that ExT normalizes the
activation of the SFO during HF, likely through an ANG
II-dependent mechanism. ExT reversed FosB activation of the
SFO during HF as well as ANG II-mediated sympathoexcitation. Additionally, there was little response to losartan micro-

injection after ExT in HF rats, suggesting the restoration of


ANG II tonicity in the SFO by ExT.
The ANG system was also normalized to sham Sed levels
after ExT. Specifically, plasma ANG II and AT1 receptor
mRNA and protein expression were reduced in the HF ExT
group. The mechanism responsible for decreasing ANG II after
ExT during HF has still not been elucidated. While there are
local ANG systems present in the brain, our finding that there
was no change in ACE expression in the SFO during HF or
after ExT suggests that local ANG II production in the SFO
may not contribute to SFO activation but, rather, circulating
ANG II. Indeed, current evidence indicates that plasma renin
levels are reduced after ExT in normal subjects (9), cardiac
ANG II levels and cardiac ACE activity are decreased after

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EXERCISE IMPROVES SFO-MEDIATED SYMPATHO-EXCITATION IN HF

ExT in rats with HF (34), and ACE expression is decreased,


whereas ACE2 expression is increased in the rostral ventrolateral medulla of rabbits with HF after ExT (13). It is also
possible that other neurohumoral factors are altered with ExT
and influence the SFO, such as cytokines and the endothelin
system, which have also been shown to be involved in HF (14,
43). Additionally, further investigation is needed to determine
if neuronal mechanisms in the SFO are improved, such as
oxidative and inflammatory stress, which is present during
high-ANG II and HF states.
Our study describes the role of the SFO as a sensory organ
involved in sympathetic drive during HF. Because the SFO
lacks a functional blood-brain barrier, it has a close interaction
with the circulation and is influenced by peripheral factors such
as circulating ANG II. Here, we found that the SFO is activated
in HF, and we propose that the high levels of peripheral ANG
II contribute to the chronic activation of the SFO. It is known
that activation of the SFO by ANG II leads to glutamatergic
release at downstream centers like the PVN (22), and, because
the SFO positively influences the activity of the PVN, it likely
contributes to the elevated sympathetic drive during HF.
Therefore, the SFO participates in setting the elevated sympathetic activity during HF or hypertension through sensing
neurohumoral factors such as ANG II, cytokines, endothelin-1,
and many others (37).
In conclusion, the increased activation of the SFO during HF
contributes to the enhanced sympathetic drive exhibited in HF,
and the SFO mediates the ExT effect of restoring sympathetic
activity during HF. Additionally, we conclude that the restoration of plasma ANG II levels during ExT reverses SFO
activation as well as systemic sympathetic activity and SFOmediated sympathetic drive observed in the HF condition.
ACKNOWLEDGMENTS
The surgical assistance of Dr. Kurtis Cornish and the technical assistance of
Johnnie Hackley are greatly appreciated.
GRANTS
This work was supported by University of Nebraska Medical Center Skala
and Patterson Fellowships (to T. L. Llewellyn) and National Heart, Lung, and
Blood Institute Grant HL-62222.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.L.L., H.Z., and K.P.P. conception and design of
research; T.L.L. and N.M.S. performed experiments; T.L.L. and N.M.S.
analyzed data; T.L.L., N.M.S., H.Z., and K.P.P. interpreted results of experiments; T.L.L., N.M.S., and K.P.P. prepared figures; T.L.L. and K.P.P. drafted
manuscript; T.L.L., H.Z., and K.P.P. edited and revised manuscript; T.L.L.,
N.M.S., H.Z., and K.P.P. approved final version of manuscript.
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