Twenty-seven intact male Yorkshire swine (Parsons Research, Amherst, MA) were fed a high-cholesterol diet (500g once daily, Sinclair Research, Columbia, MO) starting at four weeks of age and continuing for the duration of the experiment. At 8 weeks age, swine underwent left circumflex (LCx) ameroid placement (Research Instruments SW, Escondido, CA) to induce chronic ischemia, and were then divided into three groups. One group (HCC, n=9) continued on the high-cholesterol diet alone, the second (HCW, n=9) received high-cholesterol diet supplemented with 375 mL of red wine daily (Black Mountain pinot noir, 12.5% alcohol v/v, 0.3–0.5 µg/mL resveratrol, Haro Hills, CA), and the third group (HCV, n=9) received high-cholesterol diet supplemented with 112 mL of vodka daily (Rubinoff vodka, 40% alcohol v/v, Somerville, MA). Resveratrol content in this particular variety of pinot noir was quantified using liquid chromatography-mass spectroscopy. The doses of beverage were selected to provide equal amounts of alcohol to both treated groups, and the beverages were consumed mixed with chow. All three groups were provided with water ad libitum. One animal from the HCW group died prior to the end of the experiment, presumably from cardiac arrhythmia, resulting in a final n=8 for the HCW group. Animals were assigned a unique ID number at the start of the experiment, and all subsequent analyses were carried out using only these identifying numbers to eliminate observational bias.
Anesthesia was induced with intramuscular telazol (4.4 mg/kg) and maintained with 3.0% isoflurane. After intubation, titanium ameroid constrictors (1.75–2.25 mm internal diameter, sized to LCx diameter) were placed around the proximal LCx via left thoracotomy. Aspirin (325 mg/day) was administered one day prior to the procedure and continued for 5 days afterwards to prevent peri-procedural thrombosis. Alcohol supplementation was begun on the first postoperative day.
Two weeks prior to myocardial harvest, six animals from each group were briefly anesthetized with intramuscular telazol (2.2 mg/kg) one hour postprandially, and whole blood was drawn from the external jugular vein for serum alcohol quantification and platelet aggregation studies.
Seven weeks after ameroid placement, all swine were once again anesthetized and intubated. An arterial sheath was placed into the right femoral artery via cutdown, and blood samples were drawn and analyzed for total and HDL cholesterol (Beckman DXC 800 chemistry analyzer, Brea, CA). Coronary angiography was performed. After midline sternotomy, hemodynamic and functional measurements were performed, followed by cardiac harvest. 1-cm thick transverse slices were taken through the LV, and the resulting rings were divided into 8 sections each. Myocardial samples were rapidly frozen in liquid nitrogen (molecular studies), placed in 4 °C Krebs solution (microvessel studies) or dried at 60 degrees (microsphere analysis).
All experiments were approved by the Rhode Island Hospital Institutional Animal Care and Use Committee. Animals were cared for in accordance with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ (NIH publication no. 5377-3 1996).
X-ray coronary angiography with iohexol (GE Healthcare, Princeton, NJ) was carried out via femoral artery approach to verify LCx occlusion at the terminal surgery. A 5-French Amplatz R1 catheter (Cordis Corporation, Bridgewater, NJ) was advanced into the right and left coronary artery ostia and 4 mL of contrast injected per side to visualize coronary vessels. The resulting angiograms were read by a blinded cardiologist. Angiographic collateral formation was assessed according to the Rentrop grading system of 0 to 3, depending on the presence and extension of the collateral filling of coronary epicardial vessels. Myocardial perfusion was scored using the blush scoring system (also 0 to 3)13
Measurement of Global and Regional Myocardial Function
Heart rate (HR), mean arterial pressure (MAP), developed left ventricular pressure (DLVP), first derivative of LV pressure (+dP/dt), and regional myocardial contractility in the ischemic area at risk (AAR) were recorded prior to cardiac harvest using intraventricular and intra-aortic single-sensor pressure catheters (Millar Instruments, Houston, TX) and the Sonometrics system (Sonometrics Corp. London, ON, Canada) as previously described14
Myocardial Perfusion Analysis
Myocardial perfusion was measured via isotope-labeled microspheres (BioPAL, Worcester, MA). 1.5×107
gold-labeled microspheres were injected during temporary LCx occlusion at to identify the AAR. Lutetium (resting heart rate) and Europium (pacing to 160 beats/minute) labeled microspheres were injected at the final procedure while simultaneously withdrawing arterial blood from the femoral artery catheter. Harvested LV samples were completely dried in a 60° C oven, then exposed to neutron beams and microsphere densities measured (BioPAL). Myocardial blood flow in the AAR was determined using the following equation:
- Blood flow = (withdrawal rate/tissue weight) × (tissue microsphere count/blood microsphere count)
Coronary arterioles (80–180µm diameter) from the AAR were isolated and placed in a microvessel chamber. Vessels were maximally preconstricted with thromboxane-A2 analog U46619 (0.1–1.0 µM), then treated with endothelium-dependent vasodilator adenosine-5’-diphosphate (ADP, 10−9 to 10−4 mol/L) and endothelium-independent vasodilator sodium nitroprusside (SNP, 10−9 to 10−4 mol/L). Responses were defined as percent relaxation of the preconstricted diameter. All reagents were obtained from Sigma-Aldrich (St Louis, MO).
Quantification of Fibrosis
12-µm thick sections from the AAR were fixed and trichrome stained. Digital images of the stained slices were captured using Aperio slide scanning software (Aperio Technologies, Vista, CA). The amount of blue-stained collagen was quantified in a blinded fashion for three randomly selected 10X fields per section using Image J software (NIH, Bethesda, MD) and expressed as a percentage of the total section area. Measurements from the three fields were averaged to obtain representative % fibrosis for each section.
Sixty micrograms of total protein from AAR homogenates were fractionated by SDS-PAGE (Invitrogen, San Diego, CA) and transferred to PVDF membranes (Millipore, Bedford, MA). Membranes were incubated with antibodies against endothelial nitric oxide synthase (eNOS), phospho-eNOS, mammalian target of rapamycin (mTOR), phospho-mTOR, FOXO1, phospho-FOXO1 (Cell Signaling Technology, Danvers, MA), Sirt-1 (Santa Cruz Biotechnology, Santa Cruz CA), and vascular endothelial growth factor (VEGF) (Calbiochem, San Diego, CA) at dilutions recommended by the manufacturer, followed by the appropriate HRP-linked secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Immune complexes were detected with chemiluminescence (Amersham, Piscataway, NJ) and photographed using GeneSnap software (Syngene, Cambridge, England). Densitometry was performed using Image J software. α-tubulin (Cell Signaling Technology) was used as a loading control. Expression of phosphorylated proteins was expressed as a ratio of phosphorylated:total protein.
Protein Oxidative Stress
Dinitrophenylhydrazine-derivatized tissue homogenates containing 30 µg of total protein from the AAR were separated as above. Membranes were incubated with primary antibody to dinitrophenylhydrazine, followed by HRP-linked secondary antibody per manufacterer’s recommendations (Millipore, Billerica, MA). Immune complexes were visualized with chemiluminescence. Densitometric analysis of entire lanes was performed using Image J software.
Immunostaining for Capillary and Arteriolar Density
12 µm-thick frozen sections of myocardium from the AAR were formalin fixed, then incubated with antibodies against porcine endothelial marker CD-31 (R&D Systems, Minneapolis, MN) and smooth muscle actin (SMA, Sigma Aldrich), followed by the appropriate alexa-fluor conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Photomicrographs of three random 20X fields per section were taken with a Zeiss Axiolab microscope (Carl Zeiss Inc, Thornwood, NY). Capillaries, defined as CD-31 positive structures between 5–25 µm2 in cross-sectional area, and arterioles, defined as structures co-staining for both SMA and CD-31, were counted using Image J software. Results were averaged for three slices per section and are presented as vessels/mm2.
Whole blood samples drawn 1 hour postprandially were collected directly into tubes containing 3.2% trisodium citrate. Platelet function studies were performed using a Platelet Lumi-Aggregometer (Chronolog Corporation, Havertown, PA). Aggregation response to the agonists ADP (10 µM) and arachidonic acid (0.5 mM) was measured by impedance aggregation and ATP secretion using the firefly luciferin-luciferase system as previously described15
All results are presented as mean ± SEM. Microvessel responses were analyzed using two-way, repeated-measures ANOVA using the Bonferroni method to compare all pairwise contrasts between treatment-group means (K=3). Student’s t test was used to compared blood alcohol content between the HCW and HCV groups. All other comparisons were carried out using one-way ANOVA with a Neuman-Keuls post-hoc test to compare between groups using GraphPad Prism 5.0 Software (GraphPad Software Inc., San Diego, CA). Differences with a p value < 0.05 were considered statistically significant.