Download Supplementary Material Effect of Ischemia Reperfusion Injury and

SUPPLEMENTARY MATERIAL
EFFECT OF ISCHEMIA REPERFUSION INJURY AND EPOXYEICOSATRIENOIC
ACIDS ON CAVEOLIN EXPRESSION IN MOUSE MYOCARDIUM
Ketul R. Chaudhary1*, Woo Jung Cho2*, Fenghua Yang1, Victor Samokhvalov1,
Haitham E. El-Sikhry1, Edwin E. Daniel3, and John M. Seubert1,3
1Faculty
of Pharmacy and Pharmaceutical Sciences,
2Department
of Cell
Biology, 3Department of Pharmacology, University of Alberta, Edmonton, Alberta.
Corresponding Author:
John M. Seubert, PhD
Faculty of Pharmacy and Pharmaceutical Sciences
University of Alberta,
Edmonton, AB, T6G 2N8
Phone: 780-492-0007
Fax: 780-492-1217
E-mail:[email protected]
SHORT TITLE: Caveolins and EETs in ischemia reperfusion injury.
* Both authors contributed equally to this work.
Supplementary Table 1: Cardiac parameters.
WT
sEH null
WT+11,12-EET
(n=12)
(n=16)
(n=9)
LVDP (cmH2O) (Baseline)
118.4±7.9
119.5±11.3
116.7±7.2
Rate of contraction, dP/dtmax (cmH2O/sec)
(Baseline)
3388±258
3182±517
3375±197
Rate of relaxation, -dP/dtmin (cmH2O/sec)
(Baseline)
-2867±180
-2596±445
-2843±172
HR, perfused (beats/min) (Baseline)
322±16
347±26
354±11
LVDP (cmH2O) (R40)
21.8±2.4
45.7±8.0*
52.8±7.9*
Rate of contraction, dP/dtmax (cmH2O/sec) (R40)
784±87
1271±182
1639±263*
Rate of relaxation, -dP/dtmin (cmH2O/sec) (R40)
-676±86
-1185±204*
-1377±221*
HR, perfused (beats/min) (R40)
303±16
296±17
333±11
Isolated Perfused Heart - Preischemic
Isolated Perfused Heart - Postischemic
Hemodynamic parameters were measured in isolated-perfused hearts. Values represent
mean±SEM, * p<0.05 vs WT. LVDP, left ventricular pressure, HR, heart rate.
SUPPLEMENTARY MATERIALS AND METHODS
Immunohistochemical study
Tissue preparation:
Animals were treated in accordance with the guidelines of Health Science
Laboratory Animal Services, University of Alberta. The investigation conforms
with the Guide for the Care and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85-23, revised 1996). C57BL6
male mice (aged 6-8 weeks) were purchased from Charles River Laboratories
(Pointe Claire, PQ). Under general anesthesia (pentobarbital 100mg/Kg, IP)
hearts were rapidly excised and perfused in Langendorff mode. In the preischemic group, hearts were aerobically perfused for a 20min stabilization period;
while in post-ischemic groups, ischemia-reperfusion was performed for 40 minute
stabilization, 20 minute global no-flow ischemia, followed by 40 minute
reperfusion. Left ventricular myocardium from both the pre-ischemic and postischemic groups was isolated and fixed in 4% paraformaldehyde in 0.1 M sodium
phosphate buffer (PB, pH 7.2–7.4) overnight at 4°C. The fixed hearts were rinsed
and cryoprotected in 30% sucrose in 0.1 M PB overnight at 4°C.
Cryosection:
The cryoprotected hearts were put into disposable embedding molds
(Polysciences, Inc., PA, USA) and filled with Tissue-Tek® optimal cutting
temperature (O.C.T.) compound. Samples were frozen on the pre-cooler of the
cryostat (Leica 1900) for 1 hour. The frozen hearts were cryosectioned at 5μm
thickness. The heart cryosections were attached to glass slides coated with 1.5%
3-aminopropyltriethoxysilane in acetone (Sigma Chemical Co., MO, USA). The
cryosections were dried for 20 minute at room temperature to enhance
attachment.
Immuno-labeling:
The dried cryosections were first rinsed in 0.5% Triton-X 100 in phosphate
buffered saline (pH 7.2–7.4) to remove O.C.T. compound and accelerate
antibody penetration. To reduce artificial staining of non-specific proteins, 10%
normal donkey serum (Calbiochem) was applied before applying primary
antibody. Endogenous mouse IgG in mouse tissue was blocked by mouse IgG
blocking reagent (M.O.M.TM Kit, Vector Laboratories, Inc., CA, USA).
Primary antibodies against Cav-1 and Cav-3 were purchased from BD
Transductions Laboratories (CA, USA) and Abcam Inc. (Cambridge, MA),
respectively. Annexin A4 primary antibody was a gift from Dr. William V. Everson,
University of Kentucky. Cy3-conjugated donkey anti-mouse IgG was from
Jackson ImmunoResearch Labouratories (PA, USA) and Alexa488-conjugated
donkey anti-rabbit IgG was from Invitrogen (OR, USA). During the incubation with
all antibodies, 2% normal donkey serum of total incubation volume was added.
All experimental procedures were performed at room temperature, 22±1°C. To
determine specificity of immunolabelling, primary or secondary antibodies were
omitted.
Ultrastructural study
Two of each pre-IR and post-IR hearts were examined for the
ultrastructural study. The hearts were sagittally opened and judged as to whether
the clog remained in the tissue or not. If the clog was present the tissue was not
used in the ultrastructural study. The left ventricle was selected and pre-fixed into
a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M sodium
cacodylate buffer, pH 7.2 - 7.4, for 2 hrs at 4°C. The pre-fixed left ventricle was
cut to 1 mm3 without physical damage using two razor-blades and shortly rinsed
in the cacodylate buffer. Post-fixation was performed with 1% OsO4 (Electron
Microscopic Sciences, PA, USA) in 0.05 M cacodylate buffer for 2 hours at 4°C.
The pre- and post-fixed heart samples were dehydrated by ethyl-alcohol series,
substituted by propylene oxide, and infiltrated by a mixture of Araldite512Embed812. The samples were heat polymerized with accelerator 1.5% DMP-30
[2,4,6-tri-(dimethylaminomethyl) phenol] (Electron Microscopic Sciences, PA,
USA) for 48 hours at 60°C. Semi-thin sections of 1 μm were cut, stained with 1%
toluidine blue and evaluated for ultra-thin sections. Ultra-thin sections of 60-70nm
were cut and loaded using perfect loop (Electron Microscopic Sciences, PA,
USA) on 300-mesh copper grid. Sections were stained with 4% uranyl acetate
(Canemco & Marivac Inc., QC, Canada) in 50% ethanol and Reynold's lead
citrate. The grids were examined using a Philips 410 transmission electron
microscope equipped with a charge-coupled device camera (MegView III) at 80
kV.
Isolated Heart Perfusions
Hearts were perfused in the Langendorff mode in retrograde fashion at
constant pressure (100cmH2O) [1]. Continuously aerated (95%O2/5%CO2)
Krebs-Henseleit buffer was used to perfuse the isolated hearts. A balloon-tipped
catheter was inserted into the left ventricle through the left atrium. Pressure was
recorded using pressure transducer connected to the catheter. A PowerLab
system (AD Instruments) was used to process data. Hearts were perfused with
buffer for 40min of stabilization period and then subjected to 30min global no-flow
ischemia, followed by 40min reperfusion. For 11,12-EET treated WT hearts,
hearts were perfused with 11,12-EETs (1 μM) throughout the reperfusion period.
Percent of left ventricular developed pressure at 40min of reperfusion, as
compared to base line, was taken as a marker for functional recovery. Stock
solution for 11,12-EET was prepared in 100% ethanol. Appropriate vehicle
controls were performed for each group.
Tissue Homogenization and Subcellular Fractionation
For preparation of subcellular fractions, tissues were minced and
homogenized in homogenization buffer containing (sucrose 250 mM, TrisHCL
10 mM, EDTA 1 mM, sodium orthovanadate 1 mM, sodium flouride 1 mM,
aproptinin 10 μg/L, leupeptin 2 μg/L, pepstatin 100 μg/L) [2, 3]. The homogenate
was centrifuged at 700 × g for 10 min to remove the debris and the supernatant
was again centrifuged to 10,000 × g for 20 min at 4 °C. The resulting supernatant
and pellet were separated and the pellet was used as the mitochondrial fraction.
The supernatant obtained from further centrifugation of the S-9 fraction at
100,000 × g for 1 h at 4 °C was used as the cytosolic fraction. The pellete
obtained after 700 × g for 10 min centrifugation was re-suspended in
homogenization buffer and centrifuged at 250 × g for 10 min. This step was
repeated two more times and all three supernatant were combined. Combined
supernatant was centrifuged at 10,000 × g for 30 min. Pellete was discarded and
supernatant was further centriguged at 100,000 × g for 1 hr. Supernatant was
discarded and resulting pellete was used as the plasma-membrane fraction after
suspending in homogenization buffer. The protein concentration of these
fractions was determined colorimetrically using a Bio-Rad BCA protein assay kit
using bovine serum albumin as a standard.
Immunoblot Analysis
SDS-polyacrylamide gel electrophoresis of cytosolic or mitochondrial
proteins (100 µg and 25 µg, respectively) were performed with 4% stacking and
12% separating gels [3]. Following transfer of the separated proteins to
nitrocellulose membranes (Boi-Rad), blots were blocked with 5% skimmed milk
powder dissolved in TBS (0.1 M Tris-Cl, 0.9% NaCl, pH 7.4). After blocking, blots
were washed three times for 10 min with TBS-T (TBS containing 0.1% Tween
20) and were probed with Cav-1 and Cav-3 to analyze protein expression
changes in subcellular fractions. Prohibitin, GAPDH and Kir6.2 were used as
loading controls for mitochondrial, cytosol and plasma-membrane fraction,
respectively. All primary antibodies were prepared in TBS. The blots were
washed for three times for 15 min with TBS-T. Following this, appropriate
horseradish
peroxidase-conjugated
anti-rabbit,
anti-mouse
or
anti-goat
secondary antibody in 5% skimmed milk/TBS-T was incubated with the blot.
Washing was performed as for the primary antibody. Chemiluminescent
detection was carried out using ECL reagent (GE health care, UK) and exposure
to x-ray film [1]. The exact same blot was used to assess protein of interest and
loading controls. Stripping was performed using Stripping buffer (Thermo
Scientifics, IL) as per manufactures protocol. The band images were acquired
using the HP scanjet 7400c and relative band intensities were assessed by
densitometry using Image J (NIH, USA). All blots analyzing protein expression
and loading control were scanned at the exact same time. Protein expression in
vehicle treated controls were taken as 100% and compared with treated group.
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Sambrook J FE, Maniatis T. Molecular Cloning: A Laboratory Manual.
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