APPENDIX
Expanded Methods
Experimental model of myocardial infarction (MI).
Before surgical procedures, animals were anesthetized with ketamine (75 mg/kg,
intraperitoneal) and medetomidine (0.5 mg/kg, intraperitoneal). Then, they were
intubated and ventilated with a 16 gauge intravenous catheter and placed in supine
position over a temperature control pad. The animals were monitored by
electrocardiogram (ECG) electrodes connected to the limbs through small needles
inserted subcutaneously. Left-sided thoracotomy was performed by a small incision
between the third and fourth intercostal spaces. The incision was expanded by a blunt
ended retractor in such a manner that the lungs are avoided in the area of retraction. The
pericardial sac surrounding the heart was cut open; the heart was not exteriorized. The
site of ligation of the left anterior descending coronary artery (LAD) was determined 8
mm away from the origin. Using a tapered atraumatic needle, a 6-0 silk ligature was
passed underneath the LAD and tied with three knots. Visible blanching and cyanosis of
the anterior wall of the left ventricle and swelling of the left atrium were indicative of
successful ligation. The procedure was considered successful if the ECG showed STsegment elevation and the anterior wall of the left ventricle became blanched. Ribs and
muscles were closed using 6-0 Vicryl dissolvable sutures leaving a small gap to aspirate
air left in the chest cavity. The air was aspirated by an in-house tube (2 mm diameter), in
a manner that the lungs were not touched. At the time of closure, neomycin powder and
betadine were applied to the muscle and skin stitch sites, respectively. The surgical site
was dressed daily to avoid any infection and to monitor for any dehiscence of the suture
site. The entire procedure was performed within 20 min after induction of anesthesia.
Eight sham operated rats underwent the same procedure without any ligation. After
surgery, animals received four doses of buprenorphine (0.05 mg/Kg, subcutaneous) at
intervals of 8 hours.
Echocardiography.
A transthoracic echocardiographic examination (4-12 MHz phased array sectorial
transducer, HD7 XE Philips) was performed in a blinded fashion at 24 hours after LAD
ligation, in order to confirm the presence of AMI, and at 4 weeks, immediately before
sacrifice. The measurements were obtained according to the recommendations of the
Echocardiography Committee of the Specialty of Cardiology of the American College of
Veterinary Internal Medicine. The arithmetic mean of three measurements was used. LV
volumes and ejection fraction (LVEF) were calculated from the four chambers long axis
view by the Simpson’s method. The wall motion score index (WMSI) was calculated by
using a representation system of 16 segments, obtained from three right parasternal shortaxis views at the level of mitral valve, papillary muscles and apex. Wall motion was
scored as 1=normokinetic, 2=hypokinetic, 3=akinetic, 4=dyskinetic for every segment,
and the WSMI was calculated as the sum of scores divided by the total number of
segments.
RNA extraction and quantitative real-time PCR.
For the isolation of ribonucleic acid (RNA), 20 mg of infarcted or non-infarcted
myocardium was washed with cold phosphate buffered saline and then the
manufacturer´s instructions for the Dynabeads mRNA Direct kit (Amersham Pharmacia
Biotech, Piscataway, NJ, USA) were followed. First–strand complementary
deoxyribonucleic acid was synthesized with the GeneAmp RNA Polymerase Chain
Reaction (RT-PCR) kit (Roche, Branchburg, NJ, USA), as per the manufacturer´s
instructions. Real time RT-PCR was performed with SYBR Premix Ex Taq (Takara
Biotechnology, Dalian, China) following the manufacturer´s instructions in a
LightCycler system (Roche). The genes studied were Gal-3, transforming growth factor
(TGF)-β, Smad-3, IL-33, sST2, collagen type I (Col I), collagen type III (Col III), tissue
inhibitor of metalloproteinase (TIMP)-1, tumor necrosis factor (TNF)-α, IL-6 and
monocyte chemotactic protein (MCP)-1. Glyceraldehyde 3-phosphate dehydrogenase
(GADPH) was used as housekeeping gene. Efficiency for each primer pair was assessed
by using serial dilutions of cDNA. Subsequently, 0.5-3 μl of template cDNA was
amplified by 50 cycles of PCR (10 s at 95 ºC, 10 s at 60 ºC and 15 s at 72 ºC), followed
by a dissociation stage to ensure that only one product was amplified. The primer
sequences used for real time RT-PCR analysis are described in Table S1.
Collagen volume quantification
Tissue samples obtained from the infarcted and non-infarcted LV were fixed in 10%
buffered formalin, embedded in paraffin, sectioned, and mounted on glass slides.
Samples were sectioned at a thickness of 5 μm and stained with Masson’s trichrome.
Collagen area fraction was assessed in images acquired at ×40 total magnification by
blinded digital color separation and thresholding to calculate the ratio of green (collagen)
to nonwhite (total tissue) pixels in 5 fields per animal.
Supplemental Table 1. Primers used for quantitative RT-PCR analysis
Forward primer (5´→3´)
Reverse primer (5´→3´)
CTGCACCACCAACTGCTTA
CAGAGGTGCCATCCAGAGTT
Gal-3 signaling related genes
Gal-3
TGF-β
Smad-3
CCCGACTGGACCACTGACA
CACCCGCGTGCTAATGGT
CACCAGTGCTACCTCCAGTGT
CAGCATGCGAGGCATGACT
GGCACTGCTTCCCGAATG
TAGTGTTCTCGGGGATGGAA
IL-33/ST2 signaling related genes
IL-33
sST2
GTGCAGGAAAGGAAGACTCG
GGTGTGACCGACAAGGACT
TGGCCTCACCATAAGAAAGG
TTGTGAGAGACACTCCTTAC
Fibrosis-related genes
Collagen I
Collagen III
TIMP-1
CATGTTCAGCTTTGTGGACCT
TCCCCTGGAATCTGTGAATC
CAGCAAAAGGCCTTCGTAAA
GCAGCTGACTTCAGGGATGT
TGAGTCGAATTGGGGAGAAT
TGGCTGAACAGGGAAACACT
Inflammation-related genes
TNF-α
IL-6
MCP-1
ATACACTGGCCCGAGGCAAC
CCACATCTCGGATCATGCTTTC
CCACTTCACAAGTCGGAGGCTTA GTGCATCATCGCTGTTCATACAATC
TTCCTTATTGGGGTCAGCAC
CAGTTAATGCCCCACTCACC
Rat mRNA
Housekeeping gene
GAPDH
Supplemental Table 2. Body weight and renal parameters
BW (gr)
ΔBW (g)
Urea (mg/dl)
Creatinine
(mg/dl)
Sham
230±25
80±16
MI
236±14
87±5
Eple
237±7
88±4
Spiro
242±12
85±6
49 ± 4.6
44 ± 3.8
41 ± 3.0
0.5 ± 0.02
0.4 ± 0.03
0.5 ± 0.02
42 ±2 .2vs
p=0.023
sham
0.4 ±0 .01
Sodium (mEq/L)
153 ± 1.2
158 ± 3.9
159 ± 2.9
p<0.000vsM
150 I± 2.7
Potassium
(mEq/L)
3.7 ± 0.3
3.3 ± 0.2
4.4 ± 0.5
4.8 ± 1.1
Data showed as mean ± SEM. Plasma levels were measured 4 weeks
post-MI. BW indicates body weight the day of the surgery; ΔBW,
increase of body weight after 1 month; Eple; eplerenone. Spiro;
spironolactone.
Supplemental Figure 1. Fibrosis determination in the non-infarcted myocardium.
Representative photomicrographs (40x) illustrating the Masson’s trichrome staining in
absence of MI (Sham) or in the non-infarcted myocardium from MI rats. Bar graph
shows quantitative analysis of interstitial fibrosis expressed as collagen volume fraction
respect to control. Eple; eplerenone. Spiro; spironolactone.