Download Data Supplement

Online supplements
Activation of eNOS by a vanadium compound ameliorates pressure overload-induced
cardiac injury in ovariectomized rats.
1Md.
Shenuarin Bhuiyan, 1Norifumi Shioda, 2Masatoshi Shibuya, 2Yoshiharu Iwabuchi
and 1,3Kohji Fukunaga
1
Department of Pharmacology and 2Department of Synthetic Chemistry, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai, Japan and 3Tohoku University 21st
Century COE Program “CRESCENDO,” Sendai, Japan
Corresponding Author:
Dr. Kohji Fukunaga
Department of Pharmacology
Graduate School of Pharmaceutical Sciences, Tohoku University
Aramaki-Aoba, Aoba-ku, Sendai 980-8578, Japan
Tel: 81-22-795-6837
Fax: 81-22-795-6835
E-mail: [email protected]
1
Supplemental materials and data:
Detailed Materials and Methods:
Materials
Reagents and antibodies were obtained from the following sources:
L-[2,3,4-3H]arginine , PerkinElmer Life Sciences, Boston, MA); anti-eNOS antibody (Sigma,
St. Louis, MO); anti-phospho-eNOS antibody (Cell Signaling Technology, Beverly, MA);
anti-phospho-Akt antibody (Ser-437) and total-Akt antibody (Cell Signaling Technology,
Beverly, MA); anti-phospho-Akt antibody (Thr-308) (Upstate Biotechnology, Lake Placid,
NY); anti-phospho-Myosin Light Chain (Ser-19) antibody and total Myosin Light Chain 2
antibody (Cell Signaling Technology, Beverly, MA); anti-Caveolin-3 antibody (BD
Biosciences, San Jose, CA); anti-Heat shock protein-90 antibody (Upstate Biotechnology,
Lake Placid, NY); anti-Calpastatin antibody and anti-dystrophin antibody (Chemicon
International, Inc., Temecula, CA); anti-β tubulin antibody (Sigma, St. Louis, MO);
anti-rabbit antibody (Amersham Biosciences Inc., Piscataway, NJ). Other reagents were of
the highest quality available (Wako Pure Chemicals, Osaka, Japan).
Animals
All procedures for handling animals comply with the Guide for Care and Use of
Laboratory Animals and were approved by the Animal Experimentation Committee of
Tohoku University Graduate School of Pharmaceutical Sciences. Female Wistar rats (6
weeks old) weighing 150-200 gm were obtained from Japan SLC, Inc. (Hamamatsu, Japan).
Rats were housed under climate-controlled conditions with a 12h light/dark cycle and
provided with standard food and water ad libitum. An acclimation period of at least 1 week
was provided before initiating the experimental protocol.
2
Experimental surgical procedure
Female Wistar rats were randomly separated into four treatment groups: 1)
ovariectomy (OVX)(n=8), 2) ovariectomy plus pressure overload plus vehicle treatment
group (OVX-PO-vehicle) (n=8), 3) ovariectomy plus pressure overload plus VO(OPT)
(1.25 mg/kg vanadium) treatment group (V 1.25) (n=8) and 4) ovariectomy plus pressure
overload- plus VO(OPT) (2.5 mg/kg vanadium) treatment group (V 2.5)(n=8). Bilateral
ovariectomy was produced in rats anesthetized with sodium pentobarbital (50 mg/kg, i.p)
(Tokyo Kasei Kogyo, Tokyo, Japan)1,2. A sham operation was performed by exposing the
ovaries without isolation.
Two weeks after the sham operation or ovariectomy, pressure-overload was initiated
in both groups by abdominal aortic banding as we described previously1,3. Briefly, rats were
anesthetized, the abdominal aorta was exposed under sterile conditions through a midline
abdominal incision, and a blunted 25-gauge needle (outside diameter, 0.5 mm) placed
between the right and left renal arteries. A ligature (6-0 silk) was snugly tied around both
the renal artery and the needle. The needle was then removed, leaving the internal diameter
of the aorta approximately equal to that of the needle. Sham-operated animals had an untied
ligature placed in the same location. After surgery, animals were housed under controlled
environmental conditions with food (Purina Formulab Chow 5008) and water ad libitum.
VO(OPT) administration
VO(OPT) was dissolved in 5% glucose. Vehicle (5% glucose) or VO(OPT)
(containing 1.25 and 2.5 mg of vanadium /kg) was administered orally for 14 days (once
daily) in a volume of 0.1 ml/100g of body of rats, started from the 15th days after onset of
aortic banding. During vehicle or VO(OPT) treatment the body weight and food intake of the
3
rats were observed every day.
Haemodynamic measurements
Haemodynamic measurements were done as we described previously 1,4,5,6. Briefly,
rats were anesthetized with sodium pentobarbital (50mg/kg, i.p). The right carotid artery was
cannulated with a polyethylene catheter (PE-50, length 25cm) filled with degassed saline
containing heparin (300 UI/ml). Haemodynamic variables were measured with a pressure
transducer (Nihon Kohden, Tokyo, Japan) connected to a polygraph (Nihon Kohden, Tokyo,
Japan) and recorded using a thermal recorder (Nihon Kohden, Tokyo, Japan). Meticulous
care was taken to ensure that the system remained free of air bubbles during the
experimental period. After arterial blood pressure (AP-601G; Nihon Kohden, Tokyo, Japan)
and heart rate (AT-601G; Nihon Kohden, Tokyo, Japan) measurements were obtained
through the polygraph, the catheter was advanced to the left ventricular cavity. We then
measured LV systolic and end-diastolic pressures (AP-601G; Nihon Kohden, Tokyo, Japan),
the maximal rate of pressure development (+dp/dt), and the rate of relaxation (-dp/dt) of LV
by cardiotachometer (EQ-601G; Nihon Kohden, Tokyo, Japan) using the polygraph.
Western blot analysis
Four weeks after aortic banding, rats were anesthetized, and hearts were excised and
quickly perfused with phosphate-buffered saline to wash out blood from coronary vessels.
Heart tissue was sliced at 2-mm thickness using a slicer (RBS-2; Zivic-Miller Laboratories,
Zelienople, PA). LV tissue samples were then rapidly frozen in liquid nitrogen and stored at
-80°C before use. For assays, each frozen sample was homogenized by methods we
described previously1,7. An equal amount of protein for each sample (25 µg of total protein)
was separated on 7.5-15% SDS-polyacrylamide gels and transferred onto polyvinylidene
4
difluoride membranes (Millipore Corporation, Billerica, MA). After being blocked with 5%
low-fat milk in Tris-buffered saline plus Tween-20, membranes were incubated with specific
primary antibodies overnight at 4°C. This was followed by a donkey anti-rabbit IgG coupled
to horseradish peroxidase, and the blots were developed using the ECL immunoblotting
detection system (Amersham Biosciences) and visualized on X-ray film (Fuji Film).
Autoradiographic films were scanned by densitometry (Lasergraphics, Irvine, CA) and
quantitated using Imagegause V3.41 (Fuji Film). The relative amounts of proteins were
expressed as percent increase over sham.
Isoproterenol administration
Four weeks after aortic banding, a group of rats were treated with chronic
isoproterenol as we described previously1. Vehicle (saline) or DL-isoproterenol (5mg/kg)
(Sigma, St. Louis, MO) in saline was injected intraperitoneally (i.p.) in a volume of 0.1
ml/100 g of body weight once a day for 28 days in the OVX (n=8), OVX-PO-vehicle (n=8),
V 1.25 (n=8) and V 2.5 (n=8) group rats.
Statistical analysis
Values are represented as means ± standard error of the mean (S.E.M.).
Morphometric changes and SDS-PAGE electrophoresis results were evaluated for differences
by one way analysis of variance (ANOVA) combined with Dunnett’s post hoc test. Survival
was analyzed by Kaplan-Meier analysis. A value of P<0.05 was considered statistically
significant.
References:
1.
Bhuiyan MS, Shioda N, Fukunaga K. Ovariectomy augments pressure overload-induced
5
hypertrophy associated with changes in Akt and nitric oxide synthase signaling
pathways in female rats. Am J Physiol Endocrinol Metab. 2007;293:E1606-1614.
2.
Kam KW, Kravtsov GM, Liu J, Wong TM. Increased PKA activity and its influence on
isoprenaline-stimulated L-type Ca2+ channels in the heart from ovariectomized rats. Br
J Pharmacol. 2005;144:972-981.
3.
Jouannot P, Hatt P. Rat myocardial mechanics during pressure-induced hypertrophy
development and reversal. Am J Physiol Cell Physiol. 1977;232:C355-C364.
4.
Bhuiyan MS, Shibuya M, Shioda N, Moriguchi S, Kasahara J, Iwabuchi Y, Fukunaga K
Cytoprotective effect of bis(1-oxy-2-pyridinethiolato)oxovanadiun(IV) on myocardial
ischemia/reperfusion injury elicits inhibition of Fas ligand and Bim expression and
elevation of FLIP expression. Eur J Pharmacol. 2007;571:180-188.
5.
Bhuiyan MS, Takada Y, Shioda N, Moriguchi S, Kasahara J, Fukunaga K.
Cardioprotective effect of vanadyl sulfate on ischemia/reperfusion-induced injury in rat
heart in vivo is mediated by activation of protein kinase B and induction of
FLICE-inhibitory protein. Cardiovasc Ther. 2008;26:1-14.
6.
Bhuiyan MS, Fukunaga K. Inhibition of HtrA2/Omi ameliorates heart dysfunction
following ischemia/reperfusion injury in rat heart in vivo. Eur J Pharmacol.
2007;557:168-177.
7.
Takada Y, Hashimoto M, Kasahara J, Aihara K, Fukunaga K. Cytoprotective effect of
sodium orthovanadate on ischemia/reperfusion-induced injury in the rat heart involves
Akt activation and inhibition of fodrin breakdown and apoptosis. J Pharmacol Exp Ther.
2004;311:1249-1255.
6
Table S1: Effect of VO(OPT) on morphometric parameters
Parameters
No of animals (n)
OVX
OVX-PO
V 1.25
V 2.5
8
7
8
8
Body weight (gm)
258.0±3.5
245.7±5.1
219.5±5.9‡||
212.0±5.8‡||
Heart weight (gm)
0.95 ±0.03
1.15±0.03‡
0.95±0.05||
0.84±0.04¶
0.76±0.02
0.92±0.03†
0.78±0.05§
0.7±0.03||
0.19±0.02
0.23±0.02
0.16±0.01§
0.15±0.01||
1.6±0.03
1.84±0.02‡
1.59±0.06¶
1.46±0.03*¶
Left ventricle
weight (gm)
Right ventricle
weight (gm)
Lung weight (gm)
Data are expressed as means ± S.E.M. *, P<0.05, †, P<0.01 & ‡, P<0.001 versus the OVX
group; §, P<0.05, ||, P<0.01 & ¶, P<0.001 versus the OVX-PO-vehicle treated group.
OVX, ovariectomy;
OVX-PO, ovariectomy plus pressure overload plus vehicle treatment group;
V 1.25, ovariectomy plus pressure overload plus VO(OPT) (1.25 mg/kg vanadium) treatment
group;
V 2.5, ovariectomy plus pressure overload plus VO(OPT) (2.5 mg/kg vanadium) treatment
group;
7
Table S2: Effect of VO(OPT) treatment on sham and OVX animals
Parameters
No of animals (n)
sham
sham-V 2.5
OVX
OVX-V 2.5
8
5
8
5
Body weight (gm)
231.4±2.3
212.7±7.8
258.3±3.5 †
251.2±7.9
Heart weight (gm)
0.9±0.02
0.8±0.04
0.95±0.03 *
0.82±0.02
Left ventricle weight (gm)
0.71±0.01
0.61±0.03
0.76±0.02
0.67±0.02
Right ventricle weight (gm)
0.2±0.01
0.16±0.01
0.19±0.02
0.16±0.01
Lung weight (gm)
1.48±0.04
1.5±0.03
1.6±0.03
1.54±0.03
Data are expressed as means ± S.E.M. *, P<0.05, and †, P<0.01 versus the sham group;
sham-V 2.5, sham plus VO(OPT) containing 2.5 mg/kg vanadium treatment group;
OVX, ovariectomy;
OVX-V 2.5, ovariectomy plus VO(OPT) containing 2.5 mg/kg vanadium treatment group;
8
5
HW/BW (mg/gm)
A
4
3
2
1
B
LW/BW (mg/gm)
0
8
7
6
5
4
3
2
1
0
sham
sham-V2.5
OVX
OVX-V2.5
sham
sham-V2.5
OVX
OVX-V2.5
Figure S1: Effect of VO(OPT) (2.5mg/kg vanadium) on HW/BW(A) and LW/BW(B)
ratio in the sham and OVX rats. Each bar represents the mean ± S.E.M.
9
400
HR (beats/min)
A
300
200
100
0
sham
sham-V2.5
OVX
OVX-V2.5
160
MABP (mmHg)
B
††
120
80
40
0
sham
sham-V2.5
OVX
OVX-V2.5
Figure S2: Effect of VO(OPT) (2.5mg/kg vanadium) on heart rate and mean arterial
blood pressure in the sham and OVX rats. Both sham and OVX rats were orally treated
with VO(OPT) (containing 2.5 mg/kg vanadium) once day for 14 days and heart rate (HR
(A) and mean arterial blood pressure (MABP)(B) were measured after the treatment period.
Each bar represents the mean ± S.E.M. *, P<0.05, versus the sham group; †, P<0.01 versus
the OVX group.
10
sham
OVX
V 1.25
Body weights (gm)
A
sham-V2.5
OVX-V2.5
V 2.5
300
200
100
0
0
B
1
2
3
Sham
OVX
V 1.25
30
Food intake (gm)
5
Days
8
11
14
sham-V2.5
OVX-V2.5
V 2.5
20
10
0
0
3
6
9
12
15
Days
Figure S3: Effect of VO(OPT) on body weight (A) and food intake (B). Rats were treated
with VO(OPT) (containing 2.5mg/kg vanadiaum) for 14 days and body weight and food
intake were observed each day. Values expressed as mean ± S.E.M. for body weights and
mean food intake per rats.
11
4
HW/BW (mg/gm)
A
3
2
OVX
1W
2W
3W
4W
7
LW/BW (mg/gm)
B
**
**
6
5
4
OVX
1W
2W
3W
4W
Figure S4. Time course of cardiac hypertrophy. Heart-to-body weight ratio (HW/BW) (A)
and lung-to-body weight ratio (LW/BW) (B) increased time-dependently. Number of rats in
OVX, TAC 1 week (1W), TAC 2 week (2W), TAC 3 week (3W) and TAC 4 week (4W) are
4,4,4,5and 5 respectively. **, P<0.01 versus the OVX group.
12
A
pAkt (Ser 473)
Total Akt
OVX
pAkt (% of sham)
250
1W
2W
3W
4W
*
200
150
100
50
**
0
OVX
1W
2W
3W
4W
B
peNOS (Ser 1179)
eNOS
OVX
1W
2W
peNOS
120
NOS (% of sham)
3W
4W
eNOS
100
*
80
*
60
40
20
0
OVX
1W
2W
3W
4W
Figure S5. Time course of LV Akt and eNOS activity. A, left ventricular Akt
phosphorylation (Ser 473) increased 1 week after pressure overload and there after decreased
time-dependently with no change in total Akt level. B, left ventricular eNOS and Akt
mediated eNOS phosphorylation (Ser 1179) decreased time-dependently. Number of rats in
OVX, TAC 1 week (1W), TAC 2 week (2W), TAC 3 week (3W) and TAC 4 week (4W) are
4,4,4,5and 5 respectively. *, P<0.05 and **, P<0.01 versus the OVX group.
13
Figure S6: Putative mechanism of VO(OPT) mediated cardioprotection. In normal
female heart, eNOS is localized to the caveolae through its interaction with caveolin 3 and
compartmentalized with L-type Ca2+ channel and β-aderenergic receptor. Activation of Akt
signaling ultimately leads to eNOS phosphorylation, eNOS activation and thereby activates
14
nitric oxide signaling pathways. The resulting combination of these effects subsequently
confers ventricular dilation and cardioprotection. Pressure overload-induced hypertrophy
severely impairs eNOS and Akt signaling pathways, thereby imbalances the NO mediated
cardioprotective action. Treatment with VO(OPT) activate the Akt activity and enhances Akt
mediated eNOS activity and subsequently confers cardioprotection against myocardial
hypertrophy.
15