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ONLINE METHODS
Evaluation of Cardiac Function
Cardiac function was evaluated by echocardiography under light sodium pentobarbital anesthesia
with spontaneous respiration. [1-3] An echocardiography system (SSD5000; Aloka, Tokyo,
Japan) with a dynamically focused 5-MHz convex transducer was used, and M-mode tracings
from the short-axis view at the level of the papillary muscle were recorded. Left ventricle (LV)
end-diastolic diameter (LVDd), LV end-systolic diameter (LVDs), and LV wall thickness
(LVWT), calculated as the sum of the thickness of the interventricular septum (IVS) and the
posterior LV wall (PWT), were measured, and percent fractional shortening (%FS) was
calculated as follows: %FS = (LVDd-LVDs)/LVDd x 100. Echocardiography was performed at 0,
5, 10, and 15 weeks after banding.
Hemodynamic Measurement by Millar Catheter
The rats were anesthetized with sodium pentobarbital (50 mg/kg; intraperitoneal injection) and
artificially ventilated. Left ventricular pressures were measured using a Millar Micro Tip
Catheter Transducer (model SPR-320, 2F, Millar Instruments, Houston, TX). The catheter was
inserted into the right carotid artery and advanced to the left ventricle. Arterial pressures were
measured in the carotid artery. LV end-diastolic pressure (LVEDP), maximum rate of pressure
increase (dP/dtmax), maximum rate of pressure reduction (negative dP/dtmax), aortic pressure
(AoP) and heart rate (HR) were measured.
Evaluation of Sympathetic Activity
Sympathetic activity was evaluated by measuring 24-h urinary norepinephrine (U-NE) excretion
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using high-performance liquid chromatography, as previously described. [4, 5]
NRG-1β treatment protocol
The mini-osmotic pump, filled with vehicle or NRG-1β, was implanted subcutaneously in the
back and connected to a polyethylene tube (PE10). A small hole was then made in the
atlanto-occipital membrane that covers the dorsal surface of the medulla, and the tip of the tube
was placed intracisternally and fixed in place with tissue adhesive. After full recovery from
anesthesia, the rats were free to move about in their cages. Chronic intracisternal or
intraperitoneal infusion of NGR-1β (Ray Biotech, Norcross, GA) was performed at 5 weeks after
banding for 2 weeks using a subcutaneously or intraperitoneally implanted mini-osmotic pump
(Alzet model 1002; Durect Corporation, Cupertino, CA). The surgical procedures were
performed as described previously. [6] Infusion was stopped 14 days after initiation. Rats were
divided into six groups as follows: (1) sham-operated rats (sham); (2) nontreated banding rats
(AB); (3) low-dose (0.05 μg/kg/day) NRG-1β intracisternal infusion (IC low); (4) low-dose (0.05
μg/kg/day) NRG-1β intraperitoneal infusion (IP low); (5) high-dose (0.5μg/kg/day) NRG-1β
intracisternal infusion (IC high); (6) high-dose (0.5μg/kg/day) NRG-1β intraperitoneal infusion
(IP high). The doses were determined based on previous studies. [7, 8]
Western Blot Analysis
Rabbit immunoglobulin G (IgG) monoclonal antibodies against NRG-1 (1:1000), ErbB2,
(1:1000), and ErbB4 (1:1000) were used as the primary antibodies. All antibodies were
purchased from Santa Cruz Biochemical (Santa Cruz, CA). The animals were anesthetized using
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sodium pentobarbital (100 mg/kg; intraperitoneal injection) and perfused transcardially with
phosphate-buffered saline (PBS). The brains and whole hearts were removed quickly. The tissues
were homogenized and then sonicated in lysis buffer containing 40 mmol/L HEPES, 1% Triton
X-100, 10% glycerol, 1 mmol/L phenylmethanesulfonyl fluoride, and 1 protease inhibitor
cocktail tablet (Roche Diagnostics, Indianapolis, IN). The tissue lysate was centrifuged in a
microcentrifuge at 6000 rpm for 5 min at 4°C. The lysate was collected, and the protein
concentration determined using a bicinchoninic acid protein assay kit (Pierce Chemical,
Rockford, IL). Aliquots of protein (10 μg) from each sample were separated on a 7.5% sodium
dodecyl sulfate-polyacrylamide gel. Subsequently, separated proteins were transferred onto
polyvinylidene difluoride membranes (Immobilon-P membrane; Millipore, Billerica, MA). The
membranes were incubated with rabbit IgG monoclonal antibody for 24 to 48 h. The membranes
were then washed and incubated with horseradish peroxidase-conjugated horse anti-rabbit IgG
antibody (1:10,000) for 40 min. Immunoreactivity was detected using autoradiography with
enhanced chemiluminescence and a Western blotting detection kit (GE Healthcare, Tokyo,
Japan).
ELISA
A mini-osmotic pump, filled with vehicle or NRG-1β, was implanted intraperitoneally. Rats were
divided into three groups (n=3 for each), including control (vehicle), IP-low (NRG-1β: 0.05
μg/kg/day), and IP-high (NRG-1β: 0.5 μg/kg/day). Serum samples were collected by drawing
blood from the carotid artery 2 weeks after the initiation of treatment. We used an enzyme-linked
immunosorbent assay (ELISA) kit (TSZ Scientific LLC, Framingham, MA) specific for feline
NRG1 to quantify NRG1 protein according to the manufacturer's protocol. Briefly, protein
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samples from rat serum were diluted and added to a 96-well plate precoated with the primary
antibody to NRG1; the plate was then covered tightly and incubated for 30 min at room
temperature. After washing, horseradish peroxidase conjugate was added to each well in
duplicate. The plate was incubated for 30 min, washed, and chromogen substrates A and B were
added to each well. After 30 min incubation, the reaction was stopped with the stop solution and
the plate was analyzed in an Emax microplate reader (Molecular Devices) at 450 and 570 nm
within 15 min. The NRG-1β concentration was expressed in picograms of NRG-1β per milliliter
of total serum.
Statistical Analysis
All values are expressed as the mean ± SEM. Changes in the %FS, LVDd, LVDs, and LVWT
values by echocardiography were compared using a two-way analysis of variance (ANOVA).
Expression levels of NRG-1, ErbB2, and ErbB4 in the heart were compared using a paired t-test.
The other values were compared using a one-way ANOVA. In the ANOVA, comparisons
between any two mean values were performed using Bonferroni’s correction for multiple
comparisons. Differences were considered significant when the P value was less than 0.05.
Study limitations
This study has some technical limitations. First, rhNRG-1β was administered by intracisternal
infusion. We previously reported that rhNRG-1β signaling is important in the RVLM. We cannot,
however, exclude possible effects of rhNRG-1β at other brain sites. It is technically difficult to
chronically administer drugs into the RVLM. In this regard, further studies are necessary to
determine the role of NRG-1/ErbB signaling in other nuclei on HF. Second, we demonstrated
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reduction in urinary norepinephrine excretion as sympathetic nerve activity. This method,
however, cannot distinguish between cardiac sympathetic nerve activity and renal sympathetic
nerve activity. For this purpose, it would be preferable to measure cardiac and renal
norepinephrine spillover. We believe that NRG-1 affects systemic sympathetic nerve activity due
to its central effect. At this time, however, we cannot determine whether mainly cardiac or renal
sympathetic nerve activity is affected.
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REFERECES
1. Ito K, Hirooka Y, Sunagawa K. Acquisition of brain Na sensitivity contributes to salt-induced
sympathoexcitation and cardiac dysfunction in mice with pressure overload. Circ Res.
2009;104:1004-1011.
2. Ito K, Hirooka Y, Sunagawa K. Blockade of mineralocorticoid receptors improves salt-induced
left-ventricular systolic dysfunction through attenuation of enhanced sympathetic drive in
mice with pressure overload. J Hypertens. 2010;28:1449-1458.
3. Ito K, Hirooka Y, Matsukawa R, Nakano M, Sunagawa K. Decreased brain sigma-1 receptor
contributes to the relationship between heart failure and depression. Cardiovasc Res.
2012;93:33-40.
4. Kishi T, Hirooka Y, Sakai K, Shigematsu H, Shimokawa H, Takeshita A. Overexpression of
eNOS in the RVLM causes hypotension and bradycardia via GABA release. Hypertension.
2001;38:896-901.
5. Sakai K, Hirooka Y, Matsuo I, Eshima K, Shigematsu H, Shimokawa H, Takeshita A.
Overexpression of eNOS in NTS causes hypotension and bradycardia in vivo. Hypertension.
2000;36:1023-1028.
6. Kimura Y, Hirooka Y, Sagara Y, Ito K, Kishi T, Shimokawa H, Takeshita A, Sunagawa K.
Overexpression of inducible nitric oxide synthase in rostral ventrolateral medulla causes
hypertension and sympathoexcitation via an increase in oxidative stress. Circ Res.
2005;96:252-260.
7. Liu X, Gu X, Li Z, Li X, Li H, Chang J, Chen P, Jin J, Xi B, Chen D, Lai D, Graham RM,
Zhou M. Neuregulin-1/erbB-activation improves cardiac function and survival in models of
ischemic, dilated, and viral cardiomyopathy. J Am Coll Cardiol. 2006;48:1438-1447.
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8. Matsukawa R, Hirooka Y, Nishihara M, Ito K, Sunagawa K. Neuregulin-1/ErbB signaling in
rostral ventrolateral medulla is involved in blood pressure regulation as an antihypertensive
system. J Hypertens. 2011;29:1735-1742.
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ONLINE FIGURE LEGENDS
ONLINE FIGUARE 1 Western blot of (A) NRG-1, (B) ErbB2, and (C) ErbB4 in the
brainstem of sham rats. Western blot was performed at 0 (before sham operation), 5, 10,
and 15 weeks (W) after sham operation. The densitometric average was normalized to
the values obtained from the analysis of β-tubulin as an internal control. Expression is
shown relative to that at 0 W, which was assigned a value of 1. Values are expressed as
mean ± SEM (n=5-6 for each).
ONLINE FIGURE 2 Effects of aortic banding and low dose of rhNRG-1βon the heart. A,
Example of the heart at 15 weeks after banding. B, Representative M-mode
echocardiography at 15 weeks after banding.
ONLINE FIGURE 3 Expression of NRG-1 (A), ErbB2 (B), and ErbB4 (C) in the heart at
15 weeks (W) after banding. The densitometric average was normalized to the values
obtained from the analysis of β-tubulin as an internal control. Expression is shown
relative to that in sham, which was assigned a value of 1. Values are expressed as mean
± SEM. *, P < 0.05 (vs. sham; n=6 for each)