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CARDIOVASCULAR DISEASE
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.
H121
H122
H123
Reference Sequence
Forward Primer
Reverse Primer
NM_030985
BC_085760
NM_031103
5=-TATCACAGTGTGCGCGTTTCA-3=
5=-CAGCTTCATCATCCAGTTCC-3=
5=-CCCCAATGAAACCAACGAAA-3=
5=-TGGTAAGGCCCAGCCCTAT-3=
5=-CTAGGAAGAGCAGCACCCAC-3=
5=-ATGGACAGTCACAGGCTTC-3=
HF Sed 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.
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00534.2013 www.ajpheart.org
H124
H125
120
SHAM HF
EXT EXT
FosB
40 kDa
GAPDH
37 kDa
0.8
100
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
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
-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
1s
HF EXT
HR
(BPM)
BP
(mmHg)
-0.3
-0.3
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.
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00534.2013 www.ajpheart.org
H126
25
Sham Sed
20
20
10
HR (beats/min)
15
MAP (mmHg)
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.
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
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
1s
HF EXT
600
HR
(BPM)
RSNA 0.6
(Microvolts)
-0.6
-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).
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00534.2013 www.ajpheart.org
H127
-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)
*
-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.
H128
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.
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
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
H130
H131
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