MATERIALS AND METHODS
Animals and surgery
All animal procedures were conducted according to the ‘‘Guide for the Care and Use of
Laboratory Animals’’ (NIH Notice Number: NOT-OD-12-020) and were approved by the
Institutional Animal Care and Use Committee at the University of Mississippi Medical Center.
Male and female 3- to 7-month-old C57BL6/J wild type (WT) and MMP-9 null mice were used
in this study. Both WT and MMP-9 null colonies were bred in-house as homozygous colonies
and were maintained in the same room since birth. The MMP-9 null mice were generated in
Zena Werb’s laboratory and backcrossed by Lynn Matrisian’s laboratory and are on the
C57BL/6J background (1,2). Animals were housed at constant temperature (22±2°C) on a 12 h
light/dark cycle, fed standard laboratory mice chow ad libitum, and had free access to tap water.
For the C-1158/59 fragment and MMP-9 time-courses, 5 time points were studied for each
genotype: control (no MI = day 0) and 1, 3, 5, and 7 post-MI. MI was induced by permanent
ligation of the left anterior descending coronary artery, as described previously (3).
For the in vivo treatment studies only WT mice were used. Animals were randomly divided
into 2 groups by an investigator blinded to treatment: MI with vehicle control (saline), and MI
with p1158/59 treatment. At 3 h post-MI, infarction was confirmed by electrocardiography and
echocardiography and osmotic pumps with p1158/59 (Aztec 1007D, 950 μg/day/kg body weight)
or saline were inserted subcutaneously and animals sacrificed 7 days post-MI. A minimal of 6
surviving animals were used per group and time point for all in vivo studies.
1
Echocardiography and necropsy evaluations
Echocardiograms were acquired under spontaneous respiration with 1–2% isoflurane in an
oxygen mix. Images were acquired with the use of a Vevo 2100™ High-Resolution In Vivo
Imaging System (Visual Sonics, Toronto, Canada), at heart rates >400 bpm. Electrocardiogram
and heart rate were monitored throughout the imaging procedure. Echocardiographic
measurements were taken from the two-dimensional parasternal long-axis and short-axis (mmode) recordings of the LV. For each parameter, 3 images from consecutive cardiac cycles were
measured and averaged. The spherical index was calculated as the end-diastolic volume divided
by the volume of a sphere with a diameter equal to the left ventricle (LV) end-diastolic
dimension. One reader analyzed all of the echocardiograms in a blinded manner. Values ±2
standard deviations of mean were considered outliers and all echocardiography parameters
measured post-MI were normalized to LV mass index (4,5).
At necropsy, mice were anesthetized with 1.5-2.5% isoflurane in an oxygen mix. Heparin (4
U heparin/g body weight) was injected intraperitoneally, and after 5 min blood was collected
from the carotid artery. The plasma was isolated by centrifugation and stored with 1x protease
inhibitors (Roche Diagnostics, Indianapolis, IN) at -80°C. After blood collection, the heart was
excised and flushed with cardioplegic solution (6). The LV and right ventricle were separated
and weighed individually. The LV was sectioned into 3 transverse sections and stained with 1%
2, 3, 5-triphenyltetrazolium chloride (Sigma, St Louis, MO) for infarct area determination. The
infarcted LV (LVI) and remote (LVC) regions of the apex and base segments were separated
under a microscope, individually snap frozen, and stored at −80°C for biochemical analysis. The
LV middle-section was fixed in 10% zinc formalin, processed in paraffin, and sectioned at 5 µm
for immunohistochemistry studies.
2
Identification of the novel collagen/MMP-9 cleavage site
A human recombinant collagen Iα1 (60220CL, R&D) that contains the collagen Iα1 Nterminal, 60 G-X-Y repeats without the common MMP-9 cleavage site, and the C-terminal
(overall residues Gln23-Lys277 + Gly1094-Leu1464) was incubated with MMP-9 catalytic
domain (PF040, Calbiochem) in a 1:5 enzyme/substrate ratio. The reaction was performed at
37°C overnight with 1x zymogram buffer (BioRad). Cleavage was inactivated by adding
ethylenediaminetetraacetic acid (EDTA, 25 mM final concentration) for 10 min at room
temperature. Cleavage products were separated by 1-D SDS-PAGE electrophoresis in a 4-12%
Bis-Tris gel and stained with EZBlue™ gel staining reagent (Sigma Aldrich), which is a
Coomassie brilliant blue-based dye compatible with MS analysis. New cleavage products were
excised from the gel, de-stained and dehydrated, and the proteins digested in situ with trypsin
(Promega). The digests were analyzed by capillary HPLC-electrospray ionization tandem mass
spectrometry (HPLC-ESI-MS/MS) on a Thermo Fisher LTQ Orbitrap Velos mass spectrometer
fitted with a New Objective Digital PicoView 550 NanoESI source. On-line HPLC separation of
the digests was accomplished with an Eksigent/AB Sciex NanoLC-Ultra 2-D HPLC system:
column, PicoFrit™ (New Objective; 75 μm i.d.) packed to 15 cm with C18 adsorbent (Vydac;
218MS 5 μm, 300 Å). Precursor ions were acquired in the Orbitrap in profile mode at 60,000
resolution (m/z 400); data-dependent collision-induced dissociation (CID) spectra of the 6 most
intense ions in the precursor scan above a set threshold were acquired sequentially in the linear
trap at the same time as the precursor ion scan.
Mascot (version 2.3.02; Matrix Science) was used to search the uninterpreted CID spectra
against a combination of the mouse subset of the NCBInr database [Mus. (145,083 sequences)]
and a database of common contaminants (179 sequences). Methionine oxidation was considered
3
as a variable modification; semitrypsin was specified as the proteolytic enzyme, with 1 missed
cleavage allowed. The Mascot data files were combined in Scaffold (Proteome Software; version
3) for a subset search of the CID spectra using X! Tandem, cross correlation of the X! Tandem,
Mascot results, determination of protein, and peptide identity probabilities. The thresholds for
acceptance of peptide and protein assignments in Scaffold were 95% and 99%, respectively, and
minimum of 2 unique peptides.
Peptides and antibody design and synthesis
We designed a “spanning peptide” (peptide sequence Ac-CGPPGPRGRTGDSGP-NH2) that
contains the identified MMP-9/collagen cleavage site (site 1158/59) in the middle of the
sequence. This peptide was used to check for MMP cleavage, identify the cleavage site, and for
generation of a cleavage-site specific antibody (spanning peptide and antibody synthesized by
New England Peptides Inc., Gardner, MA). The antibody was designed against the amino acids
downstream of the 1158/59 site and binds to the unique sequence RTGDSPAGC that does not
repeat throughout the remaining molecule. An “activity peptide” was designed and synthesized
containing the 15 amino acids most downstream of the cleavage site (p1158/59, sequence AcRTGDSGPAGPPGPPG-NH2, CPC Scientific Inc.). Peptide p1158/59 was used for all the in
vitro and in vivo activity studies.
Tissue, protein, and peptide cleavage assays
Protein extracts (5 µg) from the LVI or remote LVC regions from MMP-9 null mice at day 7
post-MI were incubated with MMP-9 as described above. Cleavage assays were previously
optimized by testing different enzyme concentrations and several incubation times
(enzyme:protein ratios tested 1:2, 1:5, 1:10, and 1:20, from 3 h up to 72 h). Recombinant
4
collagen Iα1 (R&D, 1 µg) was incubated with MMP-2, -8, -11 (all R&D) or MMP-9
(Calbiochem) and cleavage assays were performed for 24 and 48 h.
The spanning peptide was dissolved with distilled water to a stock solution concentration of 1
mg/mL. Pro-form MMP-2 and -8 were activated with 1 mM p-aminophenylmercuric acetate
(APMA). One microgram of the stock solution peptide was mixed with 1 µL of 10X zymogen
buffer and 0.5 µL of active MMP-2, -8, -9, or -11 recombinant protein (100 ng/µL) and made up
to 10 µL with distilled water in order to obtain a final 1:20 enzyme:peptide ratio. Negative
control had all but no enzyme and blank had all but no protein. Reactions were mixed and
incubated at 37°C for 24 h.
After incubation, C18 ziptip was used for peptide desalting and 12 ng of the cleaned peptide
was analyzed by LC-MS. MS/MS spectra were searched with SEQUEST using Proteome
Discoverer (version 1.3; Thermo Fisher) against the mouse RefSeq database (Date 11/03/2013)
containing 27186 sequences. For this database search, the precursor mass tolerance and fragment
mass tolerance were set at 10 ppm and 0.02 Da, respectively. No protease was specified. A decoy
version of the RefSeq mouse database was used to estimate peptide and protein false discovery
rate. Analyses were performed on Q Exactive (Thermo Fisher, Waltham, MA) coupled with a 15
cm x 75 µm C18 column (5 µm particles with 100 angstrom pore size). A nano UPLC at 300
nL/min with a 20 min linear acetonitrile gradient (from 5-45% B over 20 min; A = 2%
acetonitrile and 0.2% formic acid in water, B = 0.2% formic acid in 90% acetonitrile) was used.
A top 2 data dependent MS/MS with exclusion for 25 sec was set. The samples were run with
higher-energy C-trap dissociation (HCD) fragmentation at normalized collision energy of 30 and
an isolation width of 2 m/z. A lock mass of the polysiloxane peak at 371.1012 was used to
5
correct the mass in MS and MS/MS. Target values in MS were 1e6 ions at a resolution setting of
70,000 and in MS2 1e5 ions at a resolution setting of 17,500.
Cardiac fibroblast isolation
We used C57BL/6 WT mice 3-6 months old, both male and female (n=10/sex). The hearts
were washed in phosphate saline buffer (PBS) and the LV separated from the right ventricle. The
LV was minced and digested in a collagenase solution (collagenase type 2 600U/mL and DNase
I 60U/mL in HBSS) for 45 min at 37ºC. Cell aggregates were mechanically dissociated by
pipetting in between incubation. Cell lysate was centrifuged at 250 xg for 5 min and cells
resuspended in fibroblast medium (DMEM/F12 supplemented with 10% fetal bovine serum
(FBS) and 1% antibiotic-antimycotic solution). Cardiac fibroblasts were cultured at 37ºC with
5% CO2 and used up to passage 3.
Cytotoxicity, proliferation, and migration assays
Cellular cytotoxicity was assessed at passage 2 using the Quanti-Chrom™ LDH cytotoxicity
assay kit (Bioassay Systems). In summary, cells were plated in a 24-well plate at a concentration
of 1x105 cells/mL until semi-confluence (80% confluence), cells were then starved for 16 h, and
stimulated in serum free + p1158/59 (1 nM, 10 nM, 100 nM, and 1 µM) or spanning peptide for
24 h. Cytotoxicity levels were measured according to the manufacturers’ instructions.
Cell proliferation was assessed in a similar fashion by use of a colorimetric immunoassay,
based on the measurement of bromodeoxyuridine (BrdU) incorporation during DNA synthesis
(Roche Ltd.), according to the manufacturer’s instructions. Cells grown in 10% FBS medium
were used as a positive control.
6
Wound healing was analyzed using Electric Cell-substrate Impedance Sensing (ECIS,
Applied Biophysics). Cells at passage 3 were plated in an ECIS-wound 96-well plate (4.0x104
cells/mL, quadruplicates/condition). Cells were allowed to proliferate until stable impedance
values were observed (~48 h). The fibroblast media was removed and replaced with the
following conditions: 1) negative control, serum free media; 2) positive control, fibroblast
medium; 3) serum free media + 100 nM p1158/59; 4) serum free media + 1 µM p1158/59; and 5)
serum free media + 1 µM spanning peptide. Cells were wounded for 10 sec at 1200 uA, 40,000
Htz. After wounding, impedance values were recorded for 48 h and normalized to respective
value pre-wound to account for cell number differences.
Fibroblast stimulation assays
Cardiac fibroblasts were stimulated using the conditions described above for the wound
healing assay. Cells were plated in 6-well plates (passage 2, 40,000 cells/mL,
duplicates/condition) and allowed to reach semi-confluence. Cells were starved in serum free
media for 16 h, after which media was replaced by appropriate stimulus condition for 24 h.
Conditioned media and cells were collected separately and frozen at -80°C.
HUVEC tube formation assay
Human umbilical vein endothelial cells (HUVEC) were purchased from American Type
Culture Collection (ATCC, PCS-100-010™) and cultured until passage 3. The angiogenic
properties of p1158/59 were tested using the Cultrex In Vitro Angiogenesis Assay kit Tube
Formation (R&D). Cells (2 x 104 cells/ well) were incubated with 1 μM p1158/59, spanning
peptid, or saline in vascular basal media (ATCC, PCS-100-030™) for 6 h, per manufacturers’
instructions. Tube formation was visualized using an inverted light microscope and
7
representative images were acquired at 10X. All images were identically processed using the
photocopy tool.
Protein extraction
For the in vitro assays, the conditioned media was concentrated for 4 h in a speed vacuum
(Savant SPD121P), and all samples were adjusted to a final volume of 200 µL using serum free
fibroblast media. For the in vivo studies, the tissue wet weight was recorded and samples
homogenized in Protein Extraction Reagent Type 4 (Sigma) and 1x proteinase inhibitors cocktail
(PI, Roche) as described previously (7). Protein quantification was performed using a Coomassie
Brilliant Blue G-250-based assay (Quick Start™ Bradford Protein Assay, Bio-Rad). All samples
were stored at -80°C until use.
Immunoblots
An aliquot of each sample (10 µg protein) was loaded onto a 4-12% Bis-Tris gel and
separated by 1-D SDS-PAGE electrophoresis. Proteins were transferred to a nitrocellulose
membrane, which was treated with the MemCode™ Reversible Protein Stain Kit (Pierce,
Thermo Scientific), to check for efficiency of protein transfer and for use as a loading control.
De-stained membranes were blocked for 1 h at room temperature with 5% non-fat milk (BioRad) and hybridized overnight at 4°C with primary antibodies against the following: collagen I
primary antibody (Cedarlane, #CL50141AP-1, 1:2000), MMP-9 primary antibody (Chemicon,
#AB804, 1:1000 ), C-1158/59 primary antibody (New England Peptides, 1:500), and biotinylated
Griffonia simplicifolia lectin 1 (Vector Laboratories, #B-1105, 1:50). After 1 h incubation with a
secondary antibody, positive signaling was detected by chemiluminescent using an ECL
substrate (GE Healthcare). Immunoblots were densitometrically analyzed using GE Image Quant
8
LAS4000 luminescent image analyzer (GE Healthcare). The signal intensity of each sample was
normalized to the total protein in its respective lane.
Gene expression assays
RNA extraction from cells and tissue was performed using TRIzol® Reagent and Purelink®
RNA (Invitrogen) according to the manufacturer’s instructions. RNA levels were quantified
using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Reverse transcription of
equal RNA content (500 ng) was performed using the RT² First Strand Kit (Qiagen). Real-Time
RT2-PCR gene array for extracellular matrix and adhesion molecules (Qiagen PAMM-013A)
was performed to quantify mRNA levels. Forkhead box O3 (Foxo3) and tissue inhibitor of
matrix metalloproteinase-4 (Timp4) gene expression were quantified using Taqman primers (Life
Technologies, Mm01185722_m1 and Mm01184417_m1). Gene assays were performed and
analyzed according to the Minimum Information for Publication of Quantitative Real-Time PCR
Experiments (MIQE) guidelines. While we measured 5 housekeeping genes (glucuronidase beta,
Gusb; hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1); heat shock protein 90 alpha
(cytosolic), class B member 1, Hsp90ab1; glyceraldehyde-3-phosphate dehydrogenase, Gapdh;
and beta actin, Actb) all data was normalized only to the 1 reference gene Hprt1 since the
expression of the other 4 genes changed in the post-MI LV (Table S1). Genes that showed no
statistical difference between groups are displayed in Table S2.
9
Table S1. Hprt1 was the only housekeeping gene not altered post-MI. Ct values for all 5
housekeeping genes analyzed. At the top are the Day 0 no MI controls (normal heart, no injury)
and at the bottom are infarcted hearts at Day 7 post-MI [n=6 mice, mean and standard deviation
(SD)].
Day 0
#10806 #10807 #10809 #10810 #10811 #10812 Mean
SD
25.3
24.6
25.1
25.1
24.9
25.2
Gusb
25.0
0.26
22.8
22.5
22.9
22.6
22.8
22.8
Hprt1
22.7
0.18
19.5
18.9
18.9
18.9
18.8
19.0
Hsp90ab1
19.0
0.26
18.7
17.9
18.3
18.1
17.9
18.3
Gapdh
18.2
0.30
20.3
19.5
20.2
19.2
19.9
19.9
Actb
19.8
0.42
Day 7
#10911 #10888 #10735 #10889 #11025 #11165 Mean
SD
23.3
22.6
22.3
23.9
23.6
22.2
Gusb
23.0
0.70
22.7
22.4
21.6
23.8
23.3
22.7
Hprt1
22.8
0.74
20.3
19.6
19.1
20.4
19.7
19.6
Hsp90ab1
19.8
0.47
20.9
20.4
18.4
22.1
20.9
21.0
Gapdh
20.6
1.21
18.1
17.2
16.8
19.9
19.8
18.3
Actb
18.3
1.29
T.TEST
0.0001
0.9750
0.0046
0.0009
0.0226
Histology and immunohistochemistry
Collagen deposition was evaluated by picrosirius red staining as described previously (8).
Staining for Griffonia simplicifolia lectin 1 (GSL-1, Vector Laboratories, #B-1105, 1:50) were
performed in serial sections of zinc-formalin fixed LV. Tissue sections were deparaffinized,
dehydrated, and heat-incubated with antigen retrieval solution (Dako) for approximately 15 min.
Tissue was blocked with horse serum (Vector) and incubated with primary antibody in blocking
serum at room temperature for 1 h. The avidin-biotinylated enzyme complex was added,
followed by DAB chromogen.
Representative images for all staining methods were acquired using an Olympus BX43
microscope. A minimum of 5 random fields at 40x magnification from each section were
scanned and quantified. Positive staining was quantified by using Image-Pro software (Media
Cybernetics) to calculate the percentage of positive stained area (red staining for collagen and
brown DAB staining for GSL-1) per total section area. The data are presented as the area of
10
positive staining per total area. All histology mages were displayed using the same saturation,
tone, and contrast intensities. Representative images for collagen deposition were acquired under
polarized light.
Quantification of C-1158/59 in human plasma
Written informed consent was collected from all patients under protocols approved by the
ethics committee of the University of Mississippi Medical Center (IRB protocol #2013-0164).
Plasma was obtained by centrifugation of blood samples collected 24-48 h post-MI diagnostic
(n= 8 control patients, no-MI, median age 56 years old; n=20 MI patients, median age 59 years
old). Patient characteristics are listed on supplemental Table S3. Equal volume loading was used
to separate proteins by SDS-PAGE electrophoresis. Immunoblot was performed as described
above using the C-1158/59 primary antibody (New England Peptides, 1:100 dilution).
Statistical analysis
Data are reported as mean±SEM. Multiple group comparisons were analyzed by one-way
ANOVA, followed by the Student Newman-Keuls when the Bartlett’s variation test passed, or
by the Kruskall-Wallis non-parametric test when the Bartlett’s variation test did not pass, with
the Dunn’s multiple comparison post-test used when differences were observed. Comparisons
between the saline and p1158/59 groups were performed by unpaired Student t-test. A p<0.05
was considered significant.
Literature Cited in Supplemental Methods
1.
2.
Lindsey ML, Escobar GP, Dobrucki LW et al. Matrix metalloproteinase-9 gene deletion
facilitates angiogenesis after myocardial infarction. Am J Physiol Heart Circ Physiol
2006;290:H232-9.
Vu TH, Shipley JM, Bergers G et al. MMP-9/gelatinase B is a key regulator of growth
plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998;93:411-22.
11
3.
4.
5.
6.
7.
8.
Ma Y, Halade GV, Zhang J et al. Matrix metalloproteinase-28 deletion exacerbates
cardiac dysfunction and rupture after myocardial infarction in mice by inhibiting M2
macrophage activation. Circ Res 2013;112:675-88.
Armstrong AC, Gidding S, Gjesdal O, Wu C, Bluemke DA, Lima JA. LV mass assessed
by echocardiography and CMR, cardiovascular outcomes, and medical practice. JACC
Cardiovascular imaging 2012;5:837-48.
Reffelmann T, Kloner RA. Transthoracic echocardiography in rats. Evalution of
commonly used indices of left ventricular dimensions, contractile performance, and
hypertrophy in a genetic model of hypertrophic heart failure (SHHF-Mcc-facp-Rats) in
comparison with Wistar rats during aging. Basic Res Cardiol 2003;98:275-84.
Michael LH, Ballantyne CM, Zachariah JP et al. Myocardial infarction and remodeling in
mice: effect of reperfusion. Am J Physiol 1999;277:H660-8.
de Castro Brás LE, Ramirez TA, DeLeon-Pennell KY et al. Texas 3-step
decellularization protocol: looking at the cardiac extracellular matrix. J Proteomics
2013;86:43-52.
Chiao YA, Ramirez TA, Zamilpa R et al. Matrix metalloproteinase-9 deletion attenuates
myocardial fibrosis and diastolic dysfunction in ageing mice. Cardiovasc Res
2012;96:444-55.
12
Table S2. List of extracellular matrix and associated adhesion molecules that did not show gene expression
differences between p1158/59 treated mice and the saline group, at day 7 post-MI (n=6/group).
Gene name
Protein encoded
p value
Adamts1
A disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 1
0.441
Adamts5
Adamts type 1 motif, 5 (aggrecanase-2)
0.096
Adamts8
Adamts type 1 motif, 8
0.204
Cdh1
Cadherin 1
0.467
Cdh2
Cadherin 2
0.399
Cdh3
Cadherin 3
0.057
Cdh4
Cadherin 4
0.979
SCntn1
Contactin 1
0.146
Col1a1
Collagen type I, alpha 1
0.112
Col2a1
Collagen type II, alpha 1
0.097
Col4a3
Collagen type IV, alpha 3
0.566
Col6a1
Collagen type VI, alpha 1
0.135
Ctnna1
Catenin (cadherin associated protein), alpha 1
0.065
Ctnna2
Catenin (cadherin associated protein), alpha 2
0.091
Fbln1
Fibulin 1
0.590
Foxo3
Forkhead box O3
0.168
Hapln1
Hyaluronan and proteoglycan link protein 1
0.819
Hc
Hemolytic complement
0.555
Itga2
Integrin, alpha 2
0.121
Itgae
Integrin, alpha e
0.562
Itgal
Integrin, alpha l
0.142
Itgav
Integrin, alpha v
0.085
Itgax
Integrin, alpha x
0.439
Itgb4
Integrin, beta 4
0.126
Lama2
Laminin, alpha 2
0.120
Lama3
Laminin, alpha 3
0.432
Lamb2
Laminin, beta 2
0.265
Lamb3
Laminin, beta 3
0.251
Mmp1a
Matrix metallopeptidase 1
0.181
Mmp2
Matrix metallopeptidase 2
0.109
Mmp3
Matrix metallopeptidase 3
0.189
Mmp7
Matrix metallopeptidase 7
0.224
Mmp9
Matrix metallopeptidase 9
0.121
Mmp10
Matrix metallopeptidase 10
0.361
Mmp12
Matrix metallopeptidase 12
0.099
Mmp13
Matrix metallopeptidase 13
0.522
Mmp15
Matrix metallopeptidase 15
0.610
Ncam1
Neural cell adhesion molecule 1
0.054
Ncam2
Neural cell adhesion molecule 2
0.259
Pecam1
Platelet/endothelial cell adhesion molecule 1
0.088
Postn
Periostin
0.085
Sell
Selectin, lymphocyte
0.254
Spock1
Sparc/osteonectin, cwcv and kazal-like domains proteoglycan 1
0.166
Syt1
Synaptotagmin I
0.118
Thbs1
Thrombospondin 1
0.307
Thbs2
Thrombospondin 2
0.050
Timp1
Tissue inhibitor of metalloproteinase 1
0.068
Timp2
Tissue inhibitor of metalloproteinase 2
0.992
Timp3
Tissue inhibitor of metalloproteinase 3
0.134
Timp4
Tissue inhibitor of metalloproteinase 4
0.186
Vtn
Vitronectin
0.258
Table S3. Patient characteristics. Values are mean±standard
deviation.
Controls
n=8
MI patients
n=20
Age, years
55±14
61±12
Sex, female/male
5/3
6/14
Race, African American/Caucasian
3/5
8/12
Previous myocardial infarction, MI
NA
4/20
Hypertension
NA
13/20
Diabetes mellitus
NA
8/20
Body mass index
NA
30±9
Systolic blood pressure
NA
125±27
Diastolic blood pressure
NA
75±16
Glucose
NA
158±110
Hemoglobin
NA
34±90
White blood cells count
NA
11±4
Red cell distribution width
NA
14±1
Peak troponin
NA
3±5
Beta blocker
NA
6/20
ACEI or ARB
NA
6/20
Aspirin
NA
6/20
Statin
NA
5/20
ACEI= angiotensin-converting enzyme inhibitor, ARB= angiotensin
II receptor antagonist, NA- not applicable or available.