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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.jtcvs.2008.08.036 Byline: Jiashing Yu (a), Karen L. Christman (a), Eric Chin (c), Richard E. Sievers (c), Maythem Saeed (b), Randall J. Lee (a)(c) Abbreviations: ECM, extracellular matrix; LV, left ventricular; LVID, left ventricular internal dimension; MI, myocardial infarction; MRI, magnetic resonance imaging; PBS, phosphate-buffered saline; RGD, arginine-glycine-asparagine Abstract: Various approaches to myocardial reconstruction have been developed for the treatment of congestive heart failure resulting from ischemic cardiomyopathy. Author Affiliation: (a) University of California Berkeley and University of California San Francisco Joint Bioengineering Graduate Group, Berkeley/San Francisco, Calif (b) Department of Radiology, University of California, San Francisco, Calif (c) Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, Calif Article History: Received 21 April 2008; Revised 23 July 2008; Accepted 21 August 2008 Article Note: (footnote) Research was partially supported by grants from the Nora Eccles Treadwell Foundation and Symphony Medical, Inc.

Objectives: To determine whether secondhand smoke (SHS) induces pulmonary artery endothelial dysfunction, and whether dietary L-arginine supplementation is preventive. Background: SHS causes coronary and peripheral arterial endothelial dysfunction. Methods: The [...]

Lipid profile changes in heart muscle have been previously linked to cardiac ischemia and myocardial infarction, but the spatial distribution of lipids and metabolites in ischemic heart remains to be fully investigated. We performed desorption electrospray ionization mass spectrometry imaging of hearts from in vivomyocardial infarction mouse models. In these mice, myocardial ischemia was induced by blood supply restriction via a permanent ligation of left anterior descending coronary artery. We showed that applying the machine learning algorithm of gradient boosting tree ensemble to the ambient mass spectrometry imaging data allows us to distinguish segments of infarcted myocardium from normally perfused hearts on a pixel by pixel basis. The machine learning algorithm selected 62 molecular ion peaks important for classification of each 200 μm-diameter pixel of the cardiac tissue map as normally perfused or ischemic. This approach achieved very high average accuracy (97.4%), recall (95.8%), and precision (96.8%) at a spatial resolution of ∼200 μm. In addition, we determined the chemical identity of 27 species, mostly small metabolites and lipids, selected by the algorithm as the most significant for cardiac pathology classification. This molecular signature of myocardial infarction may provide new mechanistic insights into cardiac ischemia, assist with infarct size assessment, and point toward novel therapeutic interventions.

We have developed a robust rat model of myocardial infarction (MI). Here we describe the step-by-step protocol for creating an ischemia-reperfusion rat model of MI. We also describe how to deliver therapeutic injections of mesenchymal stem cells (MSCs) together with fibrin, to show an application of this model. In addition, to confirm the presence of fibrin and cells in the infarct, visualization of MSCs and fibrin by histological techniques are also described. The ischemia-reperfusion MI model can be modified and generalized for use with various injectable polymers, cell types, drugs, DNA and combinations thereof. The model can be created in 7 days or less, depending on the timing of therapeutic intervention.

Current efforts in cardiac tissue engineering center around the use of scaffolds that deliver cells to the epicardial surface. In this study, we examined the effects of fibrin glue as an injectable scaffold and wall support in ischemic myocardium. The left coronary artery of rats was occluded for 17 min, followed by reperfusion. Echocardiography was performed 8 days after infarction. One to 2 days later, either 0.5% bovine serum albumin (BSA) in phosphate-buffered saline, fibrin glue alone, skeletal myoblasts alone, or skeletal myoblasts in fibrin glue were injected into the ischemic left ventricle. Echocardiography was again performed 5 weeks after injection. The animals were then sacrificed and the hearts were fresh frozen and sectioned for histology and immunohistochemistry. Both the fractional shortening (FS) and infarct wall thickness of the BSA group decreased significantly after 5 weeks (p = 0.0005 and 0.02, respectively). In contrast, both measurements for the fibrin glue group, cells group, and cells in fibrin glue group did not change significantly (FS: p = 0.18, 0.89, and 0.19, respectively; wall thickness: p = 0.40, 0.44, 0.43, respectively). Fibrin glue is capable of preserving infarct wall thickness and cardiac function after a myocardial infarction in rats and may be useful as a biomaterial scaffold for myocardial cell transplantation.

OBJECTIVES

Proton magnetic resonance (MR) spectroscopy with homonuclear editing was used to assess lactate accumulation in ischemic and anoxic hearts perfused according to the Langendorff method. An increase in the lactate signal was observed after 5 minutes of ischemia and after 10 to 20 minutes of anoxia. This study demonstrates that proton MR spectroscopy can be used to monitor the time course of MR-visible lactate levels in ischemic and anoxic isolated rat hearts.

The present study was designed to evaluate the effects of lovastatin on suppression and regression of atherosclerosis in the lipid-fed rabbit. Fifty-seven New Zealand rabbits in six groups were fed a 0.3 cholesterol diet for 10 weeks. In the progression phase of the study, group CIO served as a control and received 1 ml of DMSO daily by gavage. Two other groups, L10 and H10, received low (L)-dose (10 mg/day) or high (H)-dose (20 mg/ day) lovastatin dissolved in 1 ml of DMSO for 10 weeks. In the regression phase of the study, three groups of rabbits received the high lipid diet for 10 weeks and were then shifted to a normal diet for the second 10 weeks. During the second 10 weeks, the control group C20 received 1 ml of DMSO daily, and groups L20 and H20 received 10 and 20 mg/day of lovastatin by gavage, respectively. In the progression phase of the study, lovastatin significantly attenuated the percent of aortic lesions in groups L10 (8 ± 7) and H10 (9 ± 14) vs. the control group C10 (31 ± 17; p < 0.01). There was a similar reduction in pulmonary lesions in groups L10 (10 ± 6) and H10 (4 ± 5) compared to the control group C10 (30 ± 16; p < 0.01). There was also a reduction in plaque thickness in both the aorta and pulmonary artery, and hence an even greater reduction in estimated plaque volume. In the regression phase of the study, lovastatin also significantly reduced the percent of aortic lesions (groups L20 and H20 vs. C20: 27 ± 18 and 22 ± 7 vs. 40 ± 17; p < 0.05) and pulmonary lesions (21 ± 10 and 17 ± 6 vs. 26 ± 9; p > 0.05 and p < 0.05, respectively); the average maximum plaque thickness of aorta (0.24 and 0.26 mm vs. 0.49 mm; p < 0.01); and the standardized plaque volume per unit area of aorta (4.5 and 3.4 vs. 12.1 mm-; p< 0.05 and p < 0.01, respectively). However, there was no significant difference in the percent of aortic lesions between groups L20 and H20 and group C10 (27 ± 18 and 22 ± 7 vs. 31 ± 17; p > 0.05). In the progression phase of the study, the total serum cholesterol in groups L10 and H10 was lower than that in group C10 (715 ± 465 and 832 ± 461 vs. 1,187 ± 427 mg/dl), suggesting that the beneficial effect seen with lovastatin was due to its cholesterol-lowering effect. In the regression phase of the study, however, there were no differences in cholesterol levels in the control and treated groups. These data demonstrate that lovastatin can effectively suppress aortic and pulmonary atherosclerosis in cholesterol-fed rabbits. In addition to its cholesterol-lowering action, other mechanisms of lovastatin may contribute to this effect.

Previous studies have shown that either fish oil or verapamil can attenuate the development of atherosclerosis in the lipid-fed rabbit. The present study was designed to evaluate the individual and combined effects of these two interventions on regression.

We measured blood flow within each of eight segments of the left ventricular myocardium in dogs by an Ultrafast CT scanner. The results were compared with flow determined by radiolabeled microspheres. Computed tomography (CT)flow was measured by an intravenous injection of nonionic contrast agent done simultaneously with the left atrial injection of microspheres. We calculated flow from the CT data by obtaining CT number versus time curves for regions of interest in the myocardium and by using a formula that related flow to both the time and value of the peak enhancement. Measurements were obtained in five dogs at rest and during hyperpeffusion induced by chromonar. Based on 169 regional measurements, the Ultrafast CT and microsphere-determined flows correlated moderately (r=0.68) over a range of 0.4 to 8 mL/min/g. However, when the data were divided into resting and hyperperfusion (ie, 20 to 30 minutes after the injection of the chromonar) states, a significant (P< 0.01) increase in regional flow was determined from the CT measurements. The conclusion was that Ultrafast CT can distinguish between low and high myocardial flow states in dogs and has considerable potential for evaluating coronary flow reserve.

Lazaroid U-74500A, a 21-aminosteroid that inhibits iron-dependent lipid peroxidation, was previously shown to have protective effects on neuronal viability in animal models of ischemic neurologic injury. To test the hypothesis that lazaroid enhances myocardial salvage after ischemia/reperfusion, lazaroid-pretreated rats (2 mg/ kg intravenously) (n = 26) and control rats pretreated with vehicle (n = 22) were subjected to 45-min left coronary (LCA) occlusion followed by 120 min of reflow. Lazaroid-pretreated rats and controls had similar ischemic risk areas (AR), as determined by phthalocyanine blue dye staining (47.2 ± 2.3 vs. 48.2 ± 3.0, p = NS, mean ± SEM). Infarct size, measured by triphenyltetra-zolium staining, was markedly reduced in lazaroid-treated rats as compared with controls (6.0 ± 1.5 vs. 22.2 ± 3.9, p = 0.001, infarct size/ischemic RA × 100). There were no differences in systolic blood pressure (SBP), heart rate (HR), rate-pressure product (RPP), or maximum rate of left ventricular systolic pressure development (LVdP/dt) between groups during ischemia or reperfusion. Lazaroid pretreatment enhances myocardial salvage after 45-min LCA occlusion and 2-h reflow in this animal model. This beneficial effect is independent of the hemodynamic determinants of myocardial oxygen demand.

Ischemic left ventricular failure was produced in eight acutely instrumented, anesthetized dogs to study the contribution of changing myocardial compliance and pericardial pressure to shifts in right and left ventricular diastolic pressure-volume relations. Right and left ventricular and pericardial volumes were measured by ungated computed tomography. Cardiac volumes were manipulated by infusion of saline solution, hemorrhage, phenylephrine infusion and, during failure only, nitroglycerin administration. During both control and failure periods, these interventions shifted the left and right ventricular pressure-volume relations by changing pericardial pressure only; that is, these interventions caused no change in the ventricular transmural pressure-volume relation. The induction of failure as such increased pericardial pressure only minimally and did not change the left ventricular or right ventricular transmural pressurevolume relations significantly. Volume loading during the control period caused an apparent pericardial creep which attenuated the pericardial effect on ventricular pressure-volume relations. During failure, volume loading caused an increase of right ventricular volume, but tended to decrease left ventricular volume; this was associated with a leftward displacement of the interventricular septum.

Atrioventricular (AV) block is a useful substrate for the study of cardiac physiology. The objective of this investigation was to develop a straightforward and reproducible model of permanent AV block in rats. Working through a sternotomy, we used an epicardial fat pad between the aortic root and the right atrial wall of the rat as a landmark for the site for injection of 70% ethanol (5–10 μl) into the myocardium 3 mm below the epicardial surface. Stable, complete heart block was produced in 23 of 28 rats (82%) with a success rate of 100% in the last 16 rats of the series. Saline injection produced no heart block in 15 rats. A separate group of 14 animals was allowed to recover. Chronic heart block was achieved in all ethanol-injected animals for up to 7 days before death. The survival rate in the recovered rats was 90% in the ethanol-injected group and 100% in the saline-injected control group. Acute hemodynamic changes following the production of heart block consisted of an increase in central venous pressure, a decrease in systolic blood pressure, a decrease in left ventricular pressure, and a decrease in change in pressure over time. Chronic hemodynamic changes demonstrated a return to baseline of the central venous pressure, a persistent decrease in systolic blood pressure, and a decrease in left ventricular pressure. After the rats were killed and the hearts were dissected, discrete areas of myocardial damage were identified histologically in the atrial septum near the AV conduction axis tissue in the ethanol-injected hearts. Complete heart block was associated only with lesions extending into the specialized muscle of the AV node or His bundle. Focal mild hemorrhage, inflammation, and damaged myocardial fibers were observed in the acute stage, whereas healing lesions were characterized by granulation tissue and fibrosis replacing conduction tissue. The simple technique described provides a reproducible model for permanent, complete heart block and the study of cardiac function.

Objectives. We evaluated the influence of passive smoking on experimental atherosclerosis in cholesterol-fed rabbits.

To evaluate the effects of dietary fish oil on cholesterol-induced atherosclerosis, 36 New Zealand rabbits in four groups were fed a 0.3% cholesterol diet for 10 weeks. One group served as control, whereas groups I, II and III received 1, 2 and 3 ml/day, respectively, of fish oil (Protochol, eicosapentaenoic acid, 180 mg, and docosahexaenoic acid [DHA], 120 mg/ml). The percent of aortic and pulmonary atherosclerosis was measured by planimetry of sudanophilic lesions. The percent of aortic lesions in the control group was 59 ± 22%. The two higher dose fish oil groups showed a significant reduction in aortic lesions: group I (40 ± 26%, p = NS), group II (18 ± 11%, p < 0.01) and group III (36 ± 22%, p < 0.05). Area of pulmonary artery lesions was significantly higher in the control group (48 ± 22%) as compared with group I (15 ± 13%, p < 0.01), group II (4 ± 3%, p < 0.01) and group III (8 ± 9%, p < 0.01).

Objectives. We sought to assess the effects of second-hand smoke (SHS) and gender on infarct size in young rats exposed in utero or in the neonatal to adolescent period, or both.

Objectives. To assess the effects of interaction of sex hormones, hypercholesterolemia (HC) and environmental tobacco smoke (ETS) exposure on endothelium-dependent relaxation, we examined vascular reactivity in vitro in an animal model of atherogenesis.

Infarct size reduction after ischemic preconditioning has been reported as an all-or-nothing response, with a threshold of 2 to 5min of preconditioning ischemia time. Furthermore, infarct size reduction is associated with glycogen depletion before prolonged ischemia. We sought to determine whether increasing preconditioning ischemia time results in progressive myocardial glycogen depletion and infarct size reduction and if so, whether infarct size reduction correlates with glycogen depletion before prolonged ischemia. Therefore, ventilated rats underwent a single episode of preconditioning ischemia lasting 1.25 (n=18), 2.5 (n=20), 5 (n=9), or 10 (n=16) min, or multiple episodes cumulating in 10 (2×5min, n=17) or 20 (4×5min, n=13 or 2×10min, n=14) min of preconditioning ischemia time, all followed by 5min of reperfusion. Control rats (n=22) were not preconditioned. In separate groups, glycogen was determined before whereas infarct size was determined after prolonged ischemia (45min) and reperfusion 120min). Myocardial glycogen levels and infarct size are shown as a function of preconditioning ischemia time.

Direct injection of marrow cells into injured myocardium improves cardiac function, however, repair may be limited by the number of stem cells that home and persist in the injured myocardium. Using nude rats that received a 17 min transient ligation of their left anterior descending artery, we tested whether anti-CD3 activated human T cells (ATC), G-CSF primed peripheral blood mononuclear cells (G-PBMC), or G-CSF primed purified CD34+ cells would target MI-specific injury antigens ICAM-1 or rat myosin light chain (MLC) when injected intra-jugularly 48 hrs after myocardial injury. ATC, G-PBMC, or CD34+ cells were armed (50 ng/106 cells) with anti-CD3 x anti-rat ICAM-1 or anti-CD45 x anti-MLC. Nude rats were euthanized at 24 hours or 5 weeks after treatment and hearts were frozen, sectioned, fixed, and stained accordingly. In a pilot experiment, ATC armed with OKT3 x anti-ICAM were detected at the MI sites by Cy3 staining but not in the adjacent normal myocardium 24 hrs after infusion; rats given unarmed ATC or ATC armed with anti-CD3 x anti-CD20 (irrelevant BiAb) did not stain. High numbers of cells in the MIs in rats given anti-human CD45 x anti-rat MLC-armed G-PBMC stained positive by tyramide-amplified IHC for the mouse IgG or human CD45 cells. Human cells stained for HLA-DR (>5000 cells/injured HPF) in MIs of rats treated with BiAb-armed cells compared to unarmed-treated rats (<500/injured HPF) by 24 hrs post-infusion. By 5 weeks, the persistent human marker was HLA-Class I in the MIs whereas only a few HLA-Class II cells remained; the number of Class I cells in the MIs of rats treated with BiAb-armed cells (45.3±4.9 Class I+ cells/HPF) was significantly greater compared to MIs of rats treated with unarmed cells (25.9±1.7 Class I+ cells/HPF). In order to determine whether Isolex column-purified CD34+ cells (99% CD34, 70% CD33, and negative for CD-4,-7,-10,-16,-19,-20, and -23) obtained from G-PBMC would lead to cells expressing Class I and troponin, actin, or VE-cadherin, the hearts from rats that received CD34+ cells alone (3 rats) or CD34+ cells armed with anti-CD45 x anti-MLC (6 rats) were harvested 5 weeks after the infusion, fresh frozen, and double stained for each antigen-pair. These double stains as well as confocal images on the same slides show evidence for myocytes staining for human Class I and the respective cardiac antigens. After 5 weeks, ECHOS showed that the fractional shortening of the armed group was significantly improved compared to the unarmed group (0.33 ± 0.06 vs 0.19 ± 0.03; P = 0.02). Additionally, average LV size of the armed group was significantly less dilated compared to the unarmed group (0.47 ± 0.12 vs 0.67 ± 0.04; p = 0.04). Our studies show that arming CD34+ cells with BiAbs can target SC to MIs. The targeted SC express surface markers for human Class I and the muscle proteins troponin, actin, and cadherin; ECHO data suggest improved cardiac function. Studies are underway to maximize BiAb-armed CD34+ cell delivery to provide optimal recovery of cardiac function and to further characterize the cells that develop from the CD34+ cells.

We previously showed that environmental tobacco smoke (ETS) increased myocardial infarct size in a rat model of ischemia and reperfusion. If reduced reperfusion was caused by endothelial cell damage and increased vascular tone, we postulated that L-arginine (ARG) would increase nitric oxide and better protect the heart. 60 rats were randomly divided into 4 groups: ETS or Control (C) with and without ARG (2.25% ARG in drinking water). The ETS groups were exposed (4 Marlboro cigarettes per 15 minutes. 6 hours a day) for 6 weeks. During ETS-exposure, average air nicotine, carbon monoxide and total particulate concentrations were 1304 μg/m3, 78 ppm and 31 mg/m3, respectively. After 6 weeks, all rats were subjected to 35 min LAD occlusion (0) and 120 min reperfusion, with hemodynamic monitoring via the carotid artery. Aortic rings were harvested to evaluate vascular reactivity. Infarct size (infarct mass/risk area x 100%) decreased significantly in the ETS with ARG group compared to the ETS without ARG group. There were no significant differences among groups in heart rate (HR), systolic pressure (SP), and rate pressure product. Tlere were positive correlations between infarct size and heart rates from baseline to reperfusion 120 min (r = 0.4-0.6. p = 0.01-0.001). There was no relationship between vascular reactivity and infarct size.GroupNo. of RatsInf/LV (%)Inf/RA (%)0-35’HR (beats/m)0-35'SP (mmHg)Max Relax (%)C1125±351±6408±11120±784±11C+ ARG1025±252±3415±10103±11112±15ETS1034±464±6427±16108±8128±16ETS + ARG1122±3*42±6*410±17106±10127±18Values are Means±SEM*p&lt;0.05, p values from two-way ANOVA