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Online Data Supplement

Eicosapentaenoic acid suppresses adverse effects of C-reactive protein overexpression on pressure overload-induced cardiac remodeling

Toshiyuki Nagai, MD, PhD1,2, Toshihisa Anzai, MD, PhD1, 2, Yoshinori Mano, MD1,

Hidehiro Kaneko, MD1, Atsushi Anzai, MD1, Yasuo Sugano, MD, PhD3,

Yuichiro Maekawa, MD, PhD1, Toshiyuki Takahashi, MD, PhD1,

Tsutomu Yoshikawa, MD, PhD1, Keiichi Fukuda, MD, PhD1

1 Division of Cardiology, Department of Medicine, Keio University School of Medicine,

Tokyo, Japan

2 Department of Cardiovascular Medicine, National Cardiovascular Center, Osaka, Japan

3 Cardiovascular Center, International University of Health and Welfare Mita Hospital,

Tokyo, Japan

Correspondence: Toshihisa Anzai, MD, PhD

Department of Cardiovascular Medicine, National Cerebral and Cardiovascular Center

5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan

Phone: +81-6-6833-5012 (ext. 2321), Fax: +81-6-6833-9865, E-mail:


Methods

Mouse model of transverse aortic constriction (TAC) and experimental study protocol

Fourteen TAC/CRPtg and 14 TAC/CON were administered the standard food. Twelve TAC/CRPtg were administered the eicosapentaenoic acid (EPA) (7% of the total energy) food diet (TAC/CRPtg/EPA) for two weeks before TAC and then up to four weeks after TAC. Mice aged 9-12 week old were anesthetized with xylazine (5 mg/kg, SC) and ketamine (100 mg/kg, IM). The chest was opened and the transverse aorta of CRPtg (TAC/CRPtg; n = 14, TAC/CRPtg/EPA; n = 12) and non-transgenic littermates (TAC/CON; n=14) was ligated between the truncus brachiocephalicus and the left common carotid artery by tying a 6-0 silk suture against a 25-gauge needle. Sham-operated CRPtg (Sham/CRPtg; n = 14) and non-transgenic littermates (Sham/CON; n = 14) underwent the same procedure without ligation of the aorta. All procedures were performed in accordance with the Keio University animal care guidelines, which conform to 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).

Diet

All food was custom-manufactured (Sankyo Laboratory Service Corporation, Inc., Tokyo, Japan). The standard food was similar to typical commercial rodent food, with 65% of the total energy from carbohydrate (75% from cornstarch, 15% maltodextrin, and 10% sucrose by energy), 15% of energy from fat (78% from cocoa butter, 22% soybean oil), and 20% from protein (casein supplemented with L-cysteine). The EPA diets had identical protein and carbohydrate composition to the standard food, and had 15% of total energy from fat. The EPA diets derived 2.3% of total energy from soybean oil and 7% of total energy as EPA from fish oil (Oriental Yeast Co., Ltd., Tokyo, Japan).

Echocardiography and hemodynamic measurements

Echocardiographic and hemodynamic studies were performed one week and four weeks after the operation. For echocardiography, mice were anesthetized with isoflurane. M-mode tracings derived from the short axis of the left ventricular (LV) were recorded, using an echocardiographic machine (8.5-MHz linear transducer; EnVisor C, Philips Medical Systems, Andover, MA, USA), to measure anterior wall thickness, posterior wall thickness, LV end-diastolic diameter (LVEDD), and LV end-systolic diameter (LVESD). Aortic ejection time (ET) was obtained from Doppler flow measurements of LV outflow tract velocity. LV systolic function was assessed by fractional shortening (FS = LVEDD-LVESD/LVEDD) and the velocity of circumferential fiber shortening (circ/sec = FS/ET).

Cardiac catheterization studies were performed using a 1.4 French micromanometer-tipped catheter (SPR-671, Miller Instruments, Houston, TX, USA) under sedation with ketamine (50 mg/kg, IP) and 2% xylazine (6 mg/kg, IP) with spontaneous respiration as previously described.[1] LV systolic pressure and LV end-diastolic pressure were measured using analysis software (PowerLab, AD Instruments, Bella Vista, NSW, Australia). In addition, this catheter was inserted into the femoral artery to determine the pressure gradient across the aortic constriction.

Histological Analyses

The ventricular tissue was fixed in formalin and embedded in paraffin using standard histological procedures . The tissue was cut to yield 5-µm thick cross sections. The sections were subsequently stained with hematoxylin-eosin (HE) to determine cardiomyocyte short axis cross-sectional area (CSA) in the LV, or Masson’s trichrome stain to determine the degree of fibrosis. CSA and percent area fraction (%AF) of fibrosis were measured using ImageJ software (version 1.38x, National Institutes of Health, Bethesda, MD, USA).

Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from heart tissue by a modification of the acid guanidinium thiocyanate and phenol/chloroform extraction method as previously described [1]. Quantitative real-time RT-PCR of each sample was carried out with TaqMan Gene Expression Assays kits and an ABI Prism™ 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA), based on methods described previously [1]. Gene expression of glutathione peroxidase-3 (Mm00492427_m1), 47-kDa a-subunit of nicotinamide adenine dinucleotide phosphate oxidase (Mm00447921_m1), interleukin-6 (Mm00446190_m1), transforming growth factor (TGF)-β1 (Mm00441724_m1) was determined. The CT value for each sample was divided by that of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to generate the standardized CT value. Primer pairs and the probe for GAPDH were: forward primer: AACTCCCTCAAGATTGTCAGCAA; reverse primer: GTGGTCATGAGCCCTTCCA; TaqMan probe: CTGCACCACCAACTGCTTAGCCCC.

Immunohistochemical analysis

Sections to be stained for TGF-β1 were incubated for 30 minutes at room temperature in 1% hydrogen peroxide in methanol to quench endogenous peroxidase activity. The sections were incubated with the primary antibody (sc-146, Santa Cruz Biotech., Santa Cruz, CA, USA) for 1 hour. The sections were then incubated in a mixture containing 25 µL normal mouse serum, 5 µL biotinylated anti-rat IgG (BA-9400, Vector Laboratories, Burlingame, CA, USA) and 500 µL phosphate buffered saline (PBS) for 30 minutes.

Measurement of serum C-reactive protein (CRP) levels

A blood sample was taken from the right ventricle at the time of sacrifice. Serum CRP was measured by latex photometric immunoassay (Nonopia® CRP, Sekisui Medical Co., Tokyo, Japan).

Metabolic and biochemical variables

Plasma was analyzed for free fatty acids and triglycerides using enzymatic spectrophotometric methods. Plasma adiponectin and leptin were measured by enzyme-linked immunosorbent assay (ELISA). LV tissue was analyzed for phospholipid fatty acid composition by gas chromatography.

Reference

1. Sugano Y, Anzai T, Yoshikawa T, Maekawa Y, Kohno T, Mahara K, Naito K, Ogawa S (2005) Granulocyte colony-stimulating factor attenuates early ventricular expansion after experimental myocardial infarction. Cardiovasc Res 65:446-456