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ONLINE RESOURCE

Attenuation of Increased Secretory Leucocyte Protease Inhibitor, Matricellular Proteins and Angiotensin II and Left Ventricular Remodeling by Candesartan and Omapatrilat During Healing After Reperfused Myocardial Infarction.

Ariv Palaniyappan, PhD,† Richard R.E. Uwiera, DVM, PhD,‡ Halliday Idikio, MD,§ Vijay Menon, MSc,*Catherine Jugdutt, MCSP,* and Bodh I. Jugdutt, MBChB, MSc, DM.*†

*Division of Cardiology and Department of Medicine, †Cardiovascular Research Group, ‡Health Sciences Laboratory Animal Services, and §Department of Pathology and Laboratory Medicine University of Alberta, Edmonton, Canada.


Online Methods

Animal preparation. All experiments were approved by our institutional animal welfare committee and conformed to the Guide for the Care and Use of Animals (NIH Publication No.85-23, revised 1996). Healthy male Sprague-Dawley rats were premedicated, ventilated, and underwent mid left anterior descending coronary (LAD) occlusion followed by reperfusion for chronic RMI under general anesthesia and a limited left lateral thoracotomy [1,2]. Unlike the Selye technique [3], we directly visualized left coronary artery anatomy [4]. All animals were monitored during and after recovery for 3 weeks.

Echocardiograms and hemodynamics. As described previously [1,2,5], two-dimensional echocardiograms (2D-Echo) and transmitral and tissue Doppler, electrocardiograms (ECGs) and blood pressures (tail cuff) were recorded at repeated intervals over 3 weeks (baseline before treatments on day-2; after treatments on day-23, as well as day-7, day-14 and day-25 or 2 days after cessation of therapy) in RMI and sham rats. The 2D-Echo images were recorded using a standard protocol (long-axis, 3 short-axis and 2 apical planes), with transmitral and tissue Doppler in the apical 4-chamber plane. Myocardial injury was confirmed clinically on day-1, asynergy on 2D-Echo on day-2, and typical changes on ECGs. Coded 2D-Echos were analyzed off-line, in blinded fashion and digitized using customized software for LV remodeling and systolic function (6), including the extent of total LV asynergy as percent surface area of the endocardial area, end-systolic and end-diastolic volumes by modified Simpson’s method, biplane ejection fraction, infarct expansion index (ratio of infarct to non-infarct segment lengths) and thinning (ratio of infarct to non-infarct wall thicknesses) on the mid-papillary end-diastolic short-axis 2D-Echo section. Segment lengths were measured between midpoints of papillary muscle landmarks. For diastolic function, transmitral Doppler and Tissue Doppler Imaging (TDI) recordings were analyzed for the peak early (E) to late (A) filling velocity ratio (E/A), the E-wave deceleration time (DT) as described before [1,2,7], and the mitral annulus (septal) tissue Doppler for e¢ velocity and the E/e¢ ratio, as described elsewhere [5,6]. Differences in measurements between observers (B.I.J., A.J., V.M) were resolved by consensus. Interobserver and intraobserver errors in measurements were small (<1% in marking asynergy, segment length and wall thickness; <5% in areas of outlines) [1,2,6].

Tissue sampling, infarct-scar size and apoptosis. After final recordings and thoracotomy under anesthesia (day-25), the LAD was reoccluded and monastral blue dye (1 ml/kg) was injected into the left atrium to define the risk area. Diastolic arrest was then produced (1M potassium chloride) and hearts were quickly removed, rinsed in saline, weighed and cut into five transverse sections. Biopsy samples were rapidly taken from the reperfused ischemic zone (free wall, excluding visible scar) and the non-ischemic zone (septum) from the middle section, flash-frozen and stored in liquid N2. The other transverse sections were incubated in TTC to better delineate infarction. Outlines of LV rings, risk and infarct areas were made on plastic overlays, and infarct size was measured by computerized planimetry [1,2,6,7], and cardiomyocyte and interstitial cell apoptosis (8) by the TUNEL assay combined with a-sarcomeric actin staining.

Zymographic MMP-9 and MMP-2 activity. As described previously [1,2,6,7], zymographic degradative activities of these gelatinases were measured. Briefly, LV samples were mixed with sodium dodecyl sulfate buffer without reducing agent and applied to 7% polyacrylamide gels copolymerized with 2 mg/ml of gelatin (Sigma, St. Louis, MO). After electrophoresis, the gels were washed free of the sulfate buffer, rinsed with incubation buffer and incubated overnight at 37°C. The gels were then stained (2% Coomassie Brilliant blue G-250, Sigma) and destained. Gelatinolytic activities were detected and quantified using a GS-800 calibrated densitometer (BioRad). Band intensities were analyzed and expressed in arbitrary units using Quantity-one software (Bio-Rad). Conditioned medium from untreated HT1080 cells was used as MMP-2 and MMP-9 reference standards.

Immunoblot Analysis. as described previously [1,2,6,7,9], frozen LV samples were processed as follows. Equal amounts of protein from the extracts were applied to 12% acrylamide gels and electrophoresed. The samples were then electroblotted onto polyvinylidene difluoride membranes (BioRad, Hercules, CA) and probed with antibody (at specified dilutions) against: SLPI (V-17; SC-10538; 1:1000), SPARC (C-15; SC-13324; 1:500), OPN (K-20; SC-10591; 1:250), TGF-β1 (1:200), Smad-2/P-Smad-2, and IL-10 (1:200) from Santa Cruz Biotechnology (CA); MMP-2 (1:1000), MMP-9 (1:2000), TIMP-1 (1:1000), TIMP-3 (1:1000), IL-6 (1:200), ADAM-10 (AB19026; 1:1000 ) and ADAM-17 (AB19027; 1:1000) from Chemicon (Temecula, CA); eNOS (1:2500), iNOS (1:5000) and nNOS (1:2500) from BD Biosciences (ON, Canada); TNF-a (1:100) from R&D Systems (MN); and actin (ab-3280; 1/1000) from Abcam (Cambridge, MA). Following incubation with primary antibody, the membranes were washed and a secondary peroxidase-conjugated antibody (species-dependent on the primary antibody) was applied (1:5000) and incubated for 1 h at room temperature. After washing, immunoreactive signals were detected using enhanced chemiluminescence (Amersham, NJ) and a calibrated densitometer, quantified using Quantity-One software (BioRad) and expressed in arbitrary units. Appropriate loading procedures, confirmation of gel transfer efficiency and retention, molecular weight markers and commercially available positive controls were used.

RNA isolation and RT-PCR analysis. As described previously [1,6], RNA isolation and quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for SLPI, SPARC, OPN were done on the LV samples. RNA was isolated by RNeasy Midi Kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer’s protocol. Total RNA was quantified and mRNA samples were reverse transcribed into cDNA by using the SuperScript first-strand synthesis system (Invitrogen, Burlington, ON, Canada). mRNA levels of SLPI, SPARC, OPN were measured via real-time PCR using the iCycler system (Bio-Rad). Primer sequences designed for rat, to span at least one exon-exon junction of the target of mRNA to prevent the amplification of any contaminating genomic DNA, were used (See online Table 1). The mRNA levels detected by SYBR Green (Qiagen) analysis were normalized to GAPDH mRNA levels. PCR efficiency was examined by serially diluting the template cDNA, and the melting curve data were collected to assess PCR specificity. Each cDNA sample was run in triplicate, and a corresponding sample that had not been subjected to reverse transcription was included as a negative control in each run. Relative mRNA levels were calculated according to the critical threshold cycle comparative method.

Myeloperoxidase (MPO) activity. As described previously [1,6,10], frozen LV samples were homogenized in a solution containing 20 mM potassium phosphate to 1:10 (w:v), centrifuged for 30 min (20,000g at 4°C), and the pellets frozen for 12 h. After thawing, each pellet was added to buffer (0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer, pH 6) containing 30 U/ml aprotinin. Each sample was then sonicated (1 min at 4°C), centrifuged for 30 min (at 40,000g, 4°C), and an aliquot of the supernatant reacted with o-dianisidine dihydrochloride (0.167 mg/ml) and 0.0005% hydrogen peroxide solution. The rate of change in absorbance was measured spectrophotometrically at 405 nm. MPO activity, a quantitative index of neutrophil infiltration [10], was defined as the quantity of enzyme degrading 1 μM of peroxide/min at 37°C.

Morphometric and biochemical analysis of fibrosis, inflammation and collagens.

As described previously [1,6,11-13], 4 mm sections of formalin-fixed samples were stained with hematoxylin-eosin and Masson’s trichrome for infarct scar, and picrosirius red for collagen volume fraction. Immunostaining (H.I.) was done on paraffin-embedded tissues, using i) a-smooth muscle antibody (Dako, CA) for myofibroblasts, and ii) granulocyte MPO (1/200) and macrophage MAC 387 (1/100, marker of reactive infiltrating macrophages/monocytes) and CD68 (1/400, universal marker of macrophages/monocytes) antibodies (Thermo-Scientific, CA) for monitoring resolution of post-RMI inflammation (Nikon Coolscope digital microscope, Tokyo, Japan). Staining density was quantitatively assessed by ImagePro software. Biochemical assays (A.P.) were done on frozen samples for total soluble collagen, insoluble cross-linked collagen, hydroxyproline in hydrolyzed soluble and insoluble collagen, insoluble/soluble collagen ratio (an indirect index of cross-linking), and direct cross-linking as pyridinoline [1,14].

Regional myocardial blood flow and no-reflow.

Regional myocardial blood flow was measured by injecting colored microspheres (15-m; Triton, San Diego, USA) into the left atrium [6,15,16] at 90-min post LAD occlusion and at 10-min and 10-h post reperfusion. After removing the hearts, transmural samples from the center of the ischemic zone (TTC-negative) and center of the non-ischemic zone (TTC-positive) in a transverse section at the low-papillary level were divided into inner and outer halves, weighed, digested in 4 mol/L KOH, and eluted microspheres analyzed on a spectrophotometer. No-reflow zones by absence of thioflavin-S (TFL) on analysis of inner and outer ischemic zones (from a duplicate low-papillary transverse section) under violet light (6,13,17). Ischemic flows were corrected to allow for ~25% increase in wet weight due to tissue edema associated with reperfusion post-infarction [18] and underestimation from microsphere loss [19,20]. TFL-negative and TTC-negative areas in photographed transverse sections were planimetered [6].

Plasma atrial natriuretic peptide (ANP). Levels of ANP were measured as described elsewhere [21].

Online Results

Online Table 1. RT-PCR primer sequences of matrix proteins.

Online Table 2. Group characteristics.

Online Table 3. Hemodynamic measurements at day-2, day-23 and day-25.

Online Figures and Figure Legends

Online Figure 1: Angiotensin pressor responses and ANP levels. A, angiotensin II. B, angiotensin I. C, plasma ANP levels.

Online Figure 2: Changes in hemodynamics in sham, placebo-control, candesartan (CN) and omapatrilat (OMA) groups. A, changes in heart rate. B, changes in mean arterial pressure (MAP). n = 15/group. D2 = day-2; D23 = day-23; D25 = day-25.

Online References

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