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Ann Thorac Surg 2002;74:753-760
© 2002 The Society of Thoracic Surgeons
a Harrison Department of Surgical Research, Philadelphia, Pennsylvania, USA
b Departments of Medicine & Pathology, Philadelphia, Pennsylvania, USA
c University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
d Center for Molecular Cardiology, MCP/Hahnemann University School of Medicine, Philadelphia, Pennsylvania, USA
Accepted for publication May 28, 2002.
* Address reprint requests to Dr Gorman, Department of Surgery, 6 Silverstein, Hospital of the University of Pennsylvania, Philadelphia, PA 19104, USA
e-mail: rcgorman{at}uphs.upenn.edu
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Abstract |
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Methods. The distribution of ovine myocardium supplied by the first two diagonal branches of the left homonymous artery were determined in 20 slaughterhouse hearts and eight live sheep using methylene blue and tetrazolium injections, respectively. Seven additional animals had the infarction and underwent serial hemodynamic, microsphere and echocardiographic studies more than 8 weeks and histologic studies at the eighth week. Infarcts represented 24.6% ± 4.7% and 23.9% ± 2.2% of the left ventricular mass in slaughterhouse and live hearts, respectively.
Results. During remodeling, left ventricular end-systolic and end-diastolic volumes increased 115% and 73%, respectively, ejection fraction decreased from 41.2% ± 6.7% to 29.1% ± 5.7%, systolic wall thickening remote from the infarct decreased by 68%, sphericity index increased from 0.465 ± 0.088 to 0.524 ± 0.038, and left ventricular end-diastolic pressure increased from 1.7 ± 1.0 to 8.2 ± 3.5 mm Hg. Serial microsphere measurements documented normal blood flow (1.34 mL/g per minute) to all uninfarcted myocardium and 22% of normal to the infarct. Viable myocardium showed mild interstitial fibrosis.
Conclusions. This ovine model meets all criteria for postinfarction, dilated cardiomyopathy and has the advantages of controlling for variations in coronary arterial anatomy, collateral vascularity, and differences in the numbers, location, and severity of atherosclerotic lesions that confound human studies of the pathogenesis of this disease. This simple model contains only infarcted and fully perfused, hypocontractile myocardium produced by a moderate-sized, regional infarction.
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Introduction |
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The pathogenesis of this process is not well understood, but has been attributed to a change in myocardial material properties due to occlusive or nonocclusive coronary artery disease, myocyte cell loss, acute side-to-side myocyte slippage, chronic myocyte hypertrophy and lengthening, which produce a marked increase in diastolic wall stress [6]. More recent evidence, however, suggests that progressive myocardial cell loss and replacement fibrosis is independent of further ischemia and is secondary to postinfarction neurohormonal responses and regional wall stresses and strains [7].
Animal models of postinfarction congestive heart failure do not faithfully replicate the clinical course of postinfarction, dilated cardiomyopathy. In rats, very large infarctions (50%) are required to produce myocyte hypertrophy and delayed cell loss [5]. Multiple microsphere injections or sequential infarctions can produce cardiac failure in dogs, sheep, and calves [8, 9], but the reproducibility and relevance of these models to the pathogenesis of the human disease is questionable. Other methods of inducing congestive heart failure by rapid pacing, heart block, adriamycin, and pressure overload are not relevant to postinfarction dilated cardiomyopathy.
During a study of the role of papillary muscles in the development of chronic ischemic mitral regurgitation in sheep, Gorman and colleagues [10] observed that ligation of the first two diagonal arteries of the left homonymous (equivalent to the human left anterior descending) artery produced massive ventricular dilatation of the entire ventricle within 8 weeks. This infarct involved only 24% of the left ventricular (LV) mass and did not immediately produce heart failure. The present study confirms and expands these findings and establishes a new, reproducible, ovine model of postinfarction, dilated cardiomyopathy.
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Material and methods |
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In vivo studies
In compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1985) 8 Dorsett hybrid sheep between 35 and 45 kg and bred for laboratory use (Animal Biotech Industries, Doylestown, PA) were induced with sodium thiopental (12.5 mg/kg intravenously) and intubated. Anesthesia was maintained with 1% to 2% isoflurane in oxygen. All animals received glycopyrrate (0.4 mg intravenously) 1 hour before incision. All animals underwent left thoracotomy with clean technique and ligation of D1 and D2. Animals received magnesium (1g intravenously) and lidocaine (3 mg/kg intravenously) before infarction and an infusion of lidocaine (2 mg/min) for 60 minutes afterward. The percentage of infarcted myocardium was determined after infusion of triphenyl tetrazolium chloride for 30 minutes as described previously [13].
Assessment of remodeling
Initial instrumentation
Seven additional Dorsett hybrid sheep were intubated and anesthetized as described previously. Surface electrocardiogram and arterial blood pressure were continuously monitored. A sterile left thoracotomy was then performed and the heart exposed. Monofilament nylon snares were loosely passed around the first and second diagonal coronary arteries at their origin from the left anterior descending coronary artery. Coronary snares were briefly tightened. This transient ischemia outlined the sharp border between ischemic and perfused myocardium and identified the location of the future infarct. After marking the infarct borders, the coronary snares were loosened and six myocardial markers (2-mm nonabsorbable polyvinyl chloride beads) were sutured along the mid-ventricular short axis at the left anterior descending coronary artery, at the center of the infarct, at the lateral edges of the infarct, at the posterior descending artery, and at the midpoint between the posterior descending artery and the infarct edge. A left atrial port catheter (Access Technologies, Skokie, IL) was inserted for future injection of fluorescent microspheres and placed in a subcutaneous pocket. Coronary snares were passed through pressure tubing and added to the subcutaneous pocket. The thoracotomy was closed in layers. The left chest was drained with a single chest tube introduced through a separate stab incision. After emergence from anesthesia the chest tube and endotracheal tube were removed. Animals received buphrenorphine (5 mg/kg) just before extubation and flunixin meglumine (1 mg/kg) for postoperative pain relief. Enrofloxacin (10 mg/kg intramuscularly) was administered on postoperative days 1 and 2.
Infarction
Animals were allowed 7 to 10 days to recover. General anesthesia was again induced and the animal was again intubated. Subcutaneous coronary snares were exposed and tightened to produce myocardial infarction. Each animal received magnesium sulfate (1 g intravenously), bretylium (10 mg/kg intravenously), and lidocaine (3 mg/kg intravenous bolus followed by 2 mg/min infusion) before infarction.
Echocardiography
Quantitative two-dimensional subdiaphragmatic echocardiograms were obtained before infarction and at 30 minutes, 2, 5, and 8 weeks after infarction [10]. A sterile midline laparotomy or right or left subcostal incisions were made and subdiaphragmatic two-dimensional echocardiographic images were obtained using a 5-MHz probe (Hewlett Packard 77020A, Palo Alto, CA). Images were recorded on 
-inch videotape at 30 Hz. (Panasonic AG-6300 VHS Recorder, Matsushita Electrical Ind. Co. Ltd, Osaka, Japan). Left ventricular short-axis images at three levels (at the tips of the papillary muscles, at the bases of the papillary muscles, and at the apex) and two orthogonal long-axis views were obtained. Previous reports have validated the reproducibility and effectiveness of this technique for evaluating LV remodeling in sheep [14].
Left ventricular volumes at end-systole and end-diastole were calculated using Simpson’s rule [15]. Ejection fraction was calculated as the ratio of the difference of end-diastolic and end-systolic volumes to the end-diastolic volume. Systolic wall thickening was measured in short-axis views of two regions remote from the infarct, the mid-septum and lateral wall between the papillary muscles, using an offline analysis of echocardiographic images (Tomtec Corp, Chicago, IL). Remote wall thickening is reported as an average of these two values.
Left ventricular myocardial area was calculated in the high papillary muscle short-axis echocardiographic images by subtracting the cavity area from the total epicardial area at end-systole.
Left ventricular sphericity index, as an indicator of LV shape change, was calculated as the ratio of the diastolic short-axis LV internal diameter to the diastolic long-axis LV length (a ratio of unity would represent a sphere) [4]. The circumferential length of the regional wall motion abnormality and total internal ventricular circumference were also measured at each postinfarction time point.
Assessment of myocardial perfusion
Serial microsphere injections were performed at baseline and 2, 5, and 8 weeks after infarction to determine regional myocardial perfusion at each time point. Fifteen million, color-coded, 15.5-µm diameter NuFlow Fluorescent microspheres (IMT Laboratories, Irvine, CA) were injected at each time interval through the left atrial port. A reference blood sample was withdrawn using a constant withdrawal syringe (Harvard Apparatus Co, Cambridge, MA) from the right femoral artery beginning 5 seconds before microsphere injection and continuing for 80 seconds to allow measurements of regional perfusion.
After the end of the 8-week study animals were euthanized and hearts explanted. Myocardial markers placed at the time of initial instrumentation were used to define the infarct borders. Samples were obtained from myocardium between adjacent myocardial markers yielding samples from within the infarct, the myocardium within 1 cm of the infarct (border zone myocardium), and remote LV myocardium immediately adjacent to the posterior descending artery. Myocardial samples and reference blood samples were analyzed using flow cytometry for microsphere content by IMT Laboratories. Regional perfusion was calculated using the following formula: 
, where Qm= myocardial blood flow per gram (mL/min per gram) of sample, Cm = microsphere count per gram of tissue in sample, Qr = withdrawal rate of the reference blood sample (mL/min), and Cr = microsphere count in the reference blood sample.
Hemodynamic measurements
The surface electrocardiogram and arterial blood pressure were continuously monitored (Sonometrics Inc, London, Ontario, Canada) and recorded during all data collection procedures. A high-fidelity pressure transducer (Spc-350, Millar Instruments Inc, Houston, TX) was inserted from the femoral artery into the left ventricle for continuous LV pressure monitoring (Hewlett-Packard 78534c monitor). A pulmonary artery catheter was also placed for each data collection procedure. Thermodilution cardiac output was measured in triplicate at each serial time point for each animal.
Histology
Sections of the myocardium from the infarct, border zone, and remote myocardium were fixed in 5% buffered formalin, paraffin embedded, cut at 3 to 4 µm, and stained with either hematoxylin and eosin or Masson’s trichrome. Light microscopic analysis of these specimens permitted qualitative assessment of fibrosis, myocyte hypertrophy, and myocyte degeneration in the infarct, border zone, and remote myocardium.
Statistics
For all measurements at the same time points, values are reported as means and standard deviations. Differences between baseline measurements and measurements at subsequent times were compared using analysis of variance with repeated measures.
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Results |
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Hemodynamics
Hemodynamic data are summarized in Table 1.
Left ventricular end-diastolic pressure increased from 1.7 ± 1.0 mm Hg at baseline to 8.2 ± 3.5 mm Hg 8 weeks after infarction (p 
0.05). Maximum LV dp/dt decreased from 1,445 ± 255 mm Hg/s at baseline to 1,141 ± 357 mm Hg/s 8 weeks after infarction (p 0.05).
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0.005) or 73.2% at 8 weeks after infarction. Similar increases were seen in LV end-systolic volume, which increased from 34.5 ± 6.6 mL at baseline to 67 ± 4.6 mL (115.3%) at 8 weeks (p 0.005) (Fig 2).
The increase in LV volumes involved the entire ventricle and no regional or asymmetrical areas of dilatation were observed.
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0.05) (Fig 3).
A decrease in LV systolic muscle-to-cavity area ratio was also observed; the ratio decreased from 1.74 ± 0.34 before infarction to 1.22 ± 0.16 at 8 weeks. The circumferential length of the wall motion abnormality increased from 3.38 ± 0.78 cm immediately after infarction to 5.62 ± 1.19 cm at 8 weeks (p 0.05). The ratio of the wall motion abnormality-to-ventricular circumference also increased significantly by 8 weeks (Table 2).
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0.05). Remote systolic wall thickening decreased progressively from 2.50 ± 0.5 to 0.80 ± 0.8 mm (p 0.05) (Fig 4).
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0.005 infarct versus uninfarcted myocardium at each time point). Figure 5
summarizes the regional blood flow of the myocardial samples.
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Comment |
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TopFootnotesAbstractIntroductionMaterial and methodsResultsCommentReferences |
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In sheep similar-sized infarcts in other locations remodel to produce chronic ischemic mitral regurgitation and anteroapical LV aneurysm [13, 17]. These models and the present model are unique in that the only intervention is a moderate-sized infarction of 21% to 24% of the LV mass [13, 17]. The only difference between models is the location of the infarction. Together these three ovine models reproduce the three major clinical causes of postinfarction congestive heart failure. The controlled variables of all three models are the lack of collateral blood flow to the infarcted area, maintenance of normal blood flow to uninfarcted areas, and the moderate size of the infarcted myocardium. These variables are uncontrolled and confounding variables in patients and in other animal models.
Coronary artery disease is now the most common cause of heart failure. Cardiomyopathy due to coronary arterial disease may develop from multiple infarctions of varying size in different locations throughout the myocardium [3, 18] but often results after a single moderate-sized infarct that is initially well tolerated hemodynamically [4]. The heterogeneity of human coronary anatomy, variable collateral vascularity, and differences in the location, number, and degree of atherosclerotic obstructions produce the chronic ischemic syndromes of hibernating and stunned myocardium that contribute to global myocardial dysfunction. Stunned myocardium is defined as perfused, hypocontractile myocardium produced by a prior episode of ischemia and is reversible [19]. Hibernating myocardium is defined as chronically poorly perfused, hypocontractile myocardium, which is also reversible [20]. Techniques have been developed that effectively identify and treat chronically ischemic myocardium, but these strategies have not produced consistent results in reversing or arresting the progressive heart failure associated with postinfarction cardiomyopathy [21].
Recently, clinical and experimental evidence has defined a third type of viable but impaired myocardium, termed remodeled myocardium. Remodeled myocardium, previously termed border zone myocardium, is defined as fully perfused, hypocontractile myocardium adjacent to an infarct [22]. Narula and colleagues [23], using a novel nuclear imaging protocol, have documented that approximately 30% of hypocontractile segments in ischemic cardiomyopathic patients could be classified as remodeled myocardium. Jackson and colleagues (personal communication) have demonstrated stretched, hypocontractile fully perfused myocardium, "remodeled myocardium," in an experimental model of anteroapical LV aneurysm. This new sheep model of ischemic cardiomyopathy further advances the concept of remodeled myocardium and suggests that a regional infarct can initiate a myopathic process that can spread beyond the border zone to involve the entire ventricle. Whether the myopathic mechanism responsible for remodeled myocardium is reversible or not is unknown. The inconsistent results of salvage surgical procedures designed to restore normal ventricular geometry and reduce ventricular wall stress suggest that the myopathic process may not be reversible [24–27].
In the absence of measurable ischemia outside the infarct region, this sheep model of postinfarction, dilated cardiomyopathy does not include either stunned or hibernating myocardium and, therefore, is a pure model of remodeled myocardium. The simplicity of the model—myocardium is either infarcted or normally perfused—and the absence of confounding variables provide an opportunity to elucidate the mechanism of the progressive myopathic process whereby fully perfused, normally contractile myocardium becomes hypocontractile after regional transmural infarction. The presence of vacuolized myocytes in normally perfused myocardium at the infarct border suggests a mechanism for the contractile dysfunction that develops in this model. Narula and colleagues (personal communication) have shown that the majority of vacuolized myocytes, also termed myofibrillarlytic cells, are apoptotic and are associated with an upregulation cytoplasmic caspase-3. Both myofibrillarlytic [28] and apoptotic processes [29] in myocytes have been proposed to be reversible phenomena. The association between myocyte apoptosis and remodeled myocardium is unique and provides a new therapeutic target for the treatment or prevention of heart failure secondary to postinfarction LV remodeling.[30]
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Footnotes |
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The editor thanks Dr Bartley P. Griffith for managing the blinded peer review.
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= baseline; 
= perfusion at 2 weeks; 
= perfusion at 5 weeks; 
= perfusion at 8 weeks. Error bars represent standard error. p 
