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Section: Expanded Materials and Methods
Modesti PA et al. Cardiac Growth Factors In Hemodynamic Overload
METHODS ONLINE ONLY
Animals and study design
Thirty-six (n=36) farm pigs of either sex weighing 38±2 kg were used in the study. Animals
were kept and handled in accordance with the recommendations of 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). Infrarenal aortocaval fistula was created in a
first group of animals (n=15) by inserting a Dacron graft (8 mm diameter). Animals in a
second group (n=15) were sham-operated. At baseline and at each experimental time
after surgery (15,30,60,90 days) 3 aortocaval-shunted animals and 3 sham-operated
animals underwent echocardiographic examination and measurements of hemodynamic
parameters. The heart was then removed and two transmural left and right ventricular free
wall specimens, taken midway from base to apex, were immediately placed in liquid
nitrogen for measurement of ET-1, IGF-I, Ang II and hydroxyproline cardiac content and
for RT-PCR studies and in 10% formalin solution for myocyte morphometry.
To investigate the possible role of Ang II in myocardial hypertrophy and collagen
deposition six operated animals were randomized to ACE inhibitor (ramipril 5 mg per day)
or AT1 receptor antagonist (valsartan 80 mg daily orally). Treatment was started 3 days
after surgery and continued for 3 months. The degree of blockade of AT1 receptors and
plasma and endothelial ACE was investigated in instrumented animals as previously
described (1). The dose of valsartan and ramipril were chosen because they significantly
reduced (60% to 40% inhibition at 3 and 24 hours respectively) the increase in mean
arterial pressure following Ang II or Ang I infusion respectively, without causing any
significant effect on resting blood pressure.
Surgical procedures
The animals were premedicated with intramuscular ketamine (10 mg/kg) and diazepam (5
mg/kg). Anesthesia was induced with ketamine (15 mg/kg iv) and the pigs were
subsequently intubated and mechanically ventilated. Anesthesia was maintained with
inhalation of a mixture of 1.5 % isofluorane and oxygen supplemented with 0.1 mg/kg
pancuronium bromide. Peripheral electrocardiography leads were tied. Femoral vessels
were exposed and cannulated for measurement of the hemodynamic parameters and fluid
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Section: Expanded Materials and Methods
Modesti PA et al. Cardiac Growth Factors In Hemodynamic Overload
infusion. Volume overload was induced by creating an infrarenal aortocaval fistula as
previously reported (2). Briefly, the abdomen was opened via a midline incision and the
inferior vena cava and abdominal aorta distal to the renal arteries were cleaned of fat and
adventitia. A side-clamp was used after systemic heparinization (3 mg / kg) and a Dacron
graft (8 mm diameter) was inserted between the aorta and vena cava using a continuous
running suture. The clamps were then released, hemostasis was obtained and the
abdomen was closed. Sham animals underwent a midline incision of the abdomen,
instrumentation and medication in the same way as shunted animals, but they were not
subjected to the application of the Dacron graft.
Ventricular function and hemodynamic measurements
Two 6F pigtail catheters were introduced into the left femoral artery and advanced to
monitor left ventricular pressure. A pulmonary artery catheter was used to measure
capillary wedge pressure (PCWP), pulmonary artery pressure (PAP), right atrial pressure
(RAP), cardiac output (CO), and cardiac index (CI). Echocardiographyc measurements
were
taken
according
to
the
recommendations
of
the
American
Society
of
Echocardiography (3). Left ventricular mass indexed by body weight (LVMI), left ventricular
volumes (v), peak systolic (PSS), end-systolic (ESS) and end-diastolic (EDS) left
ventricular meridional wall stresses, were calculated using standard formulas as previously
reported (1). The contractile state of the left ventricle was evaluated by measuring the
ESS/end-systolic volume index (ESVI) ratio (4). Left ventricular stroke work index was
calculated using the formula:
LVSWI = (mean AoP - PCWP) × SVI
where AoP is aortic pressure and SVI is stroke volume index, which equals CI divided by
heart rate.
Right ventricular stroke work index (RVSWI) was calculated using the formula (5):
RVSWI = (mean PAP - mean RAP) × SVI.
Measurements were analyzed independently by two experienced echocardiographers.
Inter-observer and intra-observer variabilities were 4.1 ± 0.5 % and 2.5 ± 0.3 % for cavity
size and 3.7 ± 0.4 % and 2.1 ± 0.3 % for wall thickness, respectively.
Quantification of growth factor mRNA and peptide levels in the myocardium
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Section: Expanded Materials and Methods
Modesti PA et al. Cardiac Growth Factors In Hemodynamic Overload
Myocardial levels of ppET-1, angiotensinogen, and IGF-I transcripts were quantified using
RT-PCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standard,
as previously reported (6). Total mRNA was isolated from homogenized frozen samples
using TRIzol reagent (BRL-Life Technologies), as indicated by the manufacturer, and
reverse-transcribed using oligo dT(20). PCR primers for angiotensinogen, ppET-1 and
IGF-I were purchased from Pharmacia. To ensure that different amounts of PCRs on
myocardial biopsies were not due to markedly different mRNA starting concentrations,
PCR analysis was performed for the internal control mRNA (GAPDH) on serial twofold
dilutions of cDNA for each sample. The last dilution giving a positive reaction for GAPDH
was used to equalize the amount of cDNA used in each PCR. PCR reactions were
performed in a DNA Thermal Cycler (Perkin Elmer Cetus), band densities were analyzed
using a computer image densitometer (Qwin, Leica) and the densitometric growth
factor/GAPDH ratio was expressed as a percentage of the values obtained in sham
animals.
Measurements of Ang II in cardiac homogenated tissue was performed by RIA assay
according to van Kats et al. (7). Briefly, after preliminary acid-alchool exctraction, samples
were concentrated on Sep-Pak cartridges for HPLC separation as previously described
(1). Cross reactivities for Ang II antiserum (Bachem) were 67% with Ang III, 70% with
Ang-(3-8) exapeptide, 91% with Ang-(4-8) pentapeptide, 0.1% with Ang I, and 0.2% with
Ang-(2-10) nonapeptide. Intra- and inter-assay variation coefficients were 7.7% and
13.6%.
Cardiac ET-1 was determined according to Wei et al. (8). Briefly the specimens were
pulverized, boiled for 5 minutes in 10 vol of 1 mol/L acetic acid/20 Mmol/L hydrochloric
acid solution to abolish intrinsic proteolytic activity and then homogenized. The
homogenate was centrifuged for 30 min at 15000 rpm at 4 °C. The supernatant was then
lyophilized, reconstituted with RIA buffer and analyzed by specific RIA assay kit (Bachem).
Cross reactivities for ET-1 antiserum were 7% with endothelin-2, 7% with endothelin-3,
17% with big ET. The coefficients of intra- and inter-assay variations were 4% and 10%.
IGF-I was assayed with radioimmunoassay in homogenated tissue according to Jalil et al.
(90). IGF-I antiserum (Bachem) did not cross-react with IGF-II (0.02%), epidermal growth
factor (0%), growth hormone (0%), insulin (0%), or somatostatin (0%). Intra- and interassay variabilities were 3.5% and 10.3%.
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Section: Expanded Materials and Methods
Modesti PA et al. Cardiac Growth Factors In Hemodynamic Overload
Myocardial collagen content
Myocardial collagen concentrations were measured by the hydroxyproline assay according
to Woessner (10). Briefly, transmural specimens (1 g) were homogenized and aliquotes
(100 mg) hydrolyzed in 6N HCl at 110°C for 24 hours. The pH of hydrolyzed material was
neutralized by NaOH 2.5 N and reconstituted in 2 mL H2O. Hydrolysate was mixed with
chloramine T buffer and oxidized for 20 minutes at room temperature. Chloramine T was
destroyed by adding perchloric acid 3.15 mol/L solution and the oxidized product was
reacted with p-dimethylaminobenzaldehyde 20% solution at 60°C for 20 min. The resulting
chromophore was quantified spectrophotometrically at 577 µm against a hydroxyproline
standard curve. Because hydroxyproline is incorporated only into collagen and assuming
that collagen contains 14% hydroxyproline, the total collagen content per wet weight can
be calculated.
Myocyte morphometry
Myocyte and sarcomere lengths were measured on myocytes isolated from formalin-fixed
tissue by the potassium hydroxide digestion method according to Tamura et al. (11). In
brief, tissue was rinsed in PBS, cut into small pieces and put into 12.5 M KOH solution for
24 hours. Subsequently, the pieces were transferred to PBS, vortexed for 10 min and
poured through nylon mesh (250 µm). Rod cells with apparently normal sarcomere
structures and no visible membrane damage were judged to be undamaged myocytes.
Cell and sarcomere lengths were measured in the first 100 undamaged single myocytes
encountered in each preparation (400X magnification).
Statistical analysis
Data are expressed as mean ± SD. Comparisons were performed using one-way ANOVA
and Student’s t test, followed by the Tukey multiple-range comparison test, as appropriate.
Univariate linear relations were analyzed with the Pearson correlation. The significance
level was set at 0.05. All calculations were performed using BMDP statistical software
(BMDP Statistical Software Inc.).
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Section: Expanded Materials and Methods
Modesti PA et al. Cardiac Growth Factors In Hemodynamic Overload
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Section: Expanded Materials and Methods
Modesti PA et al. Cardiac Growth Factors In Hemodynamic Overload
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