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Ann Thorac Surg 2000;69:1351-1357
© 2000 The Society of Thoracic Surgeons
a Division of Cardiovascular and Thoracic Surgery, Duke University Medical Center, Durham, North Carolina, USA
b Division of Cardiology, Duke University Medical Center, Durham, North Carolina, USA
c Department of Radiology, Duke University Medical Center, Durham, North Carolina, USA
d Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA
Address reprint requests to Dr Lowe, Department of Surgery, Duke University Medical Center, Box 3954, Durham, NC 27710
e-mail: lowe0004{at}mc.duke.edu
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Abstract |
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Methods. Miniswine underwent 90% stenosis of the left circumflex coronary artery. Positron emission tomography and dobutamine stress echocardiography were performed 3 and 30 days (n = 6) or 14 days (n = 4) after occlusion to evaluate myocardial blood flow and viability. Triphenyl tetrazolium chloride assessed percent infarction. Electron microscopy was used to identify cellular changes characteristic of hibernating myocardium.
Results. Positron emission tomography (13N-labeled-ammonia) 3 days after occlusion demonstrated a significant reduction in myocardial blood flow in the left circumflex distribution. This reduced flow was accompanied by increased glucose use (18F-fluorodeoxyglucose), which is consistent with hibernating myocardium. Thirty days after occlusion, positron emission tomography demonstrated persistent low flow with increased glucose use in the left circumflex distribution. Dobutamine stress echocardiography 3 days after occlusion demonstrated severe hypocontractility at rest in the left circumflex region. Regional wall motion improved with low-dose dobutamine followed by deterioration at higher doses (biphasic response), findings consistent with hibernating myocardium. The results of dobutamine stress echocardiography were unchanged 30 days after occlusion. Triphenyl tetrazolium chloride staining (n = 6) revealed a mean of 8% ± 2% infarction of the area-at-risk localized to the endocardial surface. Electron microscopy (n = 4) 14 days after occlusion demonstrated loss of contractile elements and large areas of glycogen accumulation within viable cardiomyocytes, also characteristic of hibernating myocardium.
Conclusions. Chronic low-flow myocardial hibernation can be reproduced in an animal model after partial coronary occlusion. This model may prove useful in the study of the mechanisms underlying hibernating myocardium and the use of therapies designed to improve blood flow to the heart.
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Introduction |
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Despite these experimental findings, low-flow myocardial hibernation appears to be a real entity in humans [8–11], and we sought to explore whether this state could be reproduced in an animal model. Prior experimental studies [12] have suggested that substantial collateral recruitment in swine does not occur in the absence of total coronary occlusion. Therefore, we hypothesized that chronic low-flow myocardial hibernation, as assessed using positron emission tomography (PET) and dobutamine stress echocardiography (DES), could be reproduced in a porcine model after partial (90%) left circumflex coronary artery (LCX) occlusion.
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Material and methods |
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Partial coronary occlusion model
All animals underwent induction of anesthesia with ketamine (22 mg/kg intramuscularly), glycopyrrolate (0.003 mg/kg intramuscularly), and diazepam (10 mg intravenously). Orotracheal intubation was performed, and anesthesia maintained with isoflurane (2%) while the animals were mechanically ventilated. Continuous monitoring of the electrocardiogram and pulse oximeter was used throughout the procedure to ensure a stable cardiac rhythm and adequate oxygenation. Cefazolin (1 g intravenously) and bretylium tosylate (5 mg/kg intravenously) were given preoperatively. Initial muscle relaxation was obtained with a once-only injection of pancuronium bromide (2 mg intravenously).
Under sterile conditions, a left anterolateral thoracotomy was performed in the third intercostal space. The pericardium was incised longitudinally, and the left atrial appendage retracted to allow exposure of the LCX. The proximal LCX was dissected free to allow placement of a hydraulic occluder and a 2-mm ultrasonic flow probe (Transonic Systems, Inc, Ithaca, NY) around the vessel. The flow probe was placed distal to the occluder to record downstream flow through the LCX (Fig 1). The pericardium was closed. The occluder and flow probe were then exteriorized through a separate stab incision. A 20F chest tube was placed, and the wound was closed in layers. The chest tube was removed at the conclusion of the procedure. The animals were fitted with specially designed jackets (Lomir Biomedical, Quebec, PQ, Canada) to protect and allow easy access to the externalized hardware.
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PET and DSE
Animals underwent PET and dobutamine stress echocardiography DSE to characterize myocardial blood flow, metabolism, and function. This was performed 3 and 30 days after occlusion (postoperative days 7 and 34) (n = 6) or 14 days after occlusion (postoperative day 18) (n = 4). The PET scans were interpreted as showing hibernating myocardium if a flow deficit was noted in the lateral and posteroinferior walls of the left ventricle supplied by the LCX accompanied by normal or increased glucose use in these same regions (both as compared with the nonischemic septum) [13]. Using DSE, viability in the lateral and posteroinferior walls of the left ventricle was defined as an improvement in systolic wall thickening with low-dose dobutamine in myocardial regions with severe hypocontractility at rest [14]. Viable segments were considered ischemic if systolic wall motion deteriorated with stress (biphasic response) [15].
DSE
Dobutamine stress echocardiography was performed in 3-minute stages with incremental doses of dobutamine beginning with 5 µg · kg-1 · min-1 and increasing to 40 µg · kg-1 · min-1. Based on a standard 16-segment model, wall motion was graded as follows: 1 = normal; 2 = hypokinetic (reduced systolic wall thickening); 3 = akinetic (absent systolic wall thickening); and 4 = dyskinetic (outward systolic wall motion). Regional wall motion score index was calculated at rest, low dose, and peak stress. Animals were sedated with ketamine (22 mg/kg intramuscularly) before DSE. The echocardiograms were interpreted by a cardiologist (C.L.D.) with expertise in stress echocardiography, who was blinded to the clinical status of the animals.
PET
After an overnight fast, dynamic PET imaging of the heart was performed at rest for 20 minutes after a 30-second intravenous infusion of 13N-labeled ammonia (15 to 20 mCi). A 40-minute delay followed for decay of 13N activity. Then dynamic imaging of the heart for 60 minutes was done after a 30-second infusion of 18F-fluorodeoxyglucose (10 mCi). All emission images were corrected for photon attenuation using a transmission scan. Time–activity curves of tracer concentration in the left ventricular myocardium were obtained from short-axis views averaged over eight sectors in three short-axis slices (basal, middle, and apical). Previously validated compartmental modeling techniques were applied to the time–activity curves to obtain regional estimates of myocardial blood flow (milliliters per gram per minute) and glucose use (nanomoles per gram per minute) [16, 17]. A "lumped constant" of 1.0 was assumed for the calculation of glucose use from fractional 18F-fluorodeoxyglucose uptake rate [18]. During the PET scans, animals were maintained under general anesthesia and mechanically ventilated as already described. The PET data were interpreted in a blinded manner both qualitatively by a physician (R.E.C.) experienced reading cardiac PET scans, and quantitatively using absolute values for blood flow (milliliters per gram per minute) and glucose use (nanomoles per gram per minute). Absolute values of myocardial blood flow and glucose use were compared within each individual PET study to determine whether viability and ischemia were present in the LCX distribution. For each study, sectors representing the anterior septum (left anterior descending coronary artery distribution) were used as the normal reference segments.
Quantification of myocardial necrosis
After completion of the 30-day studies, animals (n = 6) were sacrificed under general anesthesia as already described, and their hearts were harvested for triphenyl tetrazolium chloride staining to assess extent of infarcted tissue [19]. To determine the anatomic boundaries of the area-at-risk, triphenyl tetrazolium chloride (1%; Sigma Chemical, St. Louis, MO) and Monastral blue (4% solution of stock dye; Sigma Chemical) were infused at 37°C and 120 to 140 mm Hg perfusion pressure into the LCX (after postmortem removal of the occluder) and left main coronary artery using previously described techniques [20]. After fixation in phosphate-buffered 3.7% formalin, the left ventricle was cut into eight transverse slices. These were weighed, and the apical surfaces were photographed. The area-at-risk (stained brick red) and the area of infarction (not stained) were identified, traced from an enlarged projection (magnification x8) of the color slide of each ventricular slice, and quantitated using a digitizing tablet interfaced with a computer using appropriate software (Sigmascan; Jandel Scientific).
Electron microscopy
After completion of the 14-day studies, animals (n = 4) were sacrificed under general anesthesia as previously described. Immediately before sacrifice, four transmural biopsies of the beating heart using a Tru-cut needle were taken at random from the ischemic LCX distribution and nonischemic anterior septum for electron microscopic analysis. Sections were immediately divided into epicardial and endocardial halves, immersed in 4% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer at pH 7.4, minced, and stored for 24 hours in glutaraldehyde for fixation. Tissue was processed and stained for electron microscopy as described previously [21]. Sections were studied for the presence or absence of myocardial necrosis and electron microscopic findings considered characteristic of ischemic, viable myocardium such as sarcomere loss, increased numbers of small mitochondria, and glycogen accumulation [22]. Electron microscopic sections were examined by a cardiac pathologist (C.S.), blinded to the location from which the tissue samples were derived.
Statistical analysis
All data are presented as the mean ± the standard error. Myocardial blood flow and glucose use by PET, and wall motion scores by DSE were compared using a paired Student t test. A p value of less than 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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PET
The PET perfusion images in all animals at all time points (3, 14, and 30 days after occlusion) showed decreased 13N-labeled ammonia accumulation in the lateral and posteroinferior wall myocardium and normal to increased F18-fluorodeoxyglucose accumulation in the area of perfusion abnormality, findings consistent with hibernating myocardium (Fig 2) [13]. Quantitative measurement of myocardial blood flow by PET both 3 and 30 days after occlusion (n = 6) confirmed a highly significant decrease in myocardial perfusion to the lateral and posteroinferior walls of the left ventricle supplied by the partially occluded LCX coronary artery compared with the corresponding nonischemic septal regions (Table 1). Myocardial viability was confirmed by the finding of increased glucose use in the regions of decreased blood flow [13] compared with the corresponding control septal regions (Table 2). Similar results were seen in the group of animals (n = 4) undergoing PET 14 days after occlusion (data not shown).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Unlike prior short-term studies, however, this study demonstrates sustained low-flow perfusion-contraction matching, as shown by PET and DSE, in an animal model. Reduced myocardial blood flow, assessed using 13N-labeled ammonia, and increased glucose use, measured with 18F-fluorodeoxyglucose, were present in the myocardial regions supplied by the partially (90%) occluded LCX up to 30 days after occlusion. This perfusion-metabolism mismatch is characteristic of hibernating myocardium [13]. Dobutamine stress echocardiography both 3 and 30 days after occlusion demonstrated severe hypocontractility at rest in the myocardial regions supplied by the subtotally occluded LCX. The function in these regions improved with low-dose dobutamine and subsequently deteriorated with higher doses (biphasic response), again findings consistent with ischemic, viable myocardium [14, 15]. Electron microscopy of sections from the hibernating regions revealed loss of contractile material in otherwise viable cardiomyocytes. The areas previously occupied by the myofilaments were filled with glycogen and numerous small mitochondria. These structural changes, along with the increase in myocardial glucose use seen with PET, have been described as hallmarks of hibernating myocardium [7].
Although this study demonstrates resting left ventricular dysfunction and reduced transmural myocardial blood flow in the LCX distribution in association with preserved glucose uptake and inotropic reserve consistent with hibernating myocardium, these findings do not rule out the possibility of repetitive stunning occurring concomitantly with low-flow hibernation. Shen and Vatner [5] demonstrated reversible left ventricular systolic dysfunction after periods of excitement associated with increased heart rate and maximum rate of rise of left ventricular pressure in miniswine with ameroid constrictors about the LCX. This transient dysfunction occurred despite normal myocardial blood flow at rest in the ameroid distribution. They concluded that the reversible periods of left ventricular dysfunction resulted from myocardial stunning after episodes of increased oxygen demand.
Because these same periods of excitement, such as at the time of feeding, likely occurred in our animals as well, repetitive stunning may indeed coexist with chronic hibernation in our animal model. However, the 35% reduction in transmural myocardial blood flow demonstrated by PET 30 days after occlusion in the present study is equivalent to a 55% to 60% reduction in subendocardial blood flow (at this time, PET cannot determine subendocardial flow because it lacks adequate spatial resolution), a degree of reduction that consistently results in regional left ventricular dysfunction in animal studies [11].
The most widely used animal model of chronic ischemia has been the ameroid constrictor [5, 6, 12, 25–27]. These constrictors have been used most often in swine because their coronary anatomy, with minimal preexisting coronary collateral vessels, is similar to that of humans [28]. Ameroid constrictors are constructed of casein, a material that absorbs water and produces total coronary occlusion over a period of 14 to 30 or more days [25, 26]. Inherent limitations to the use of these occluders include an inability to control the rate or degree (sometimes incomplete) of coronary occlusion [5, 12, 26]. In addition, there is a large variation in the reported percent infarction of the area-at-risk (5% to 100%) [6]. As a model of human coronary artery disease, the ameroid model is limited as well. Myocardial blood flow and function at rest after ameroid-induced coronary occlusion in swine are typically normal because of extensive coronary collateral development [6, 27]. However, coronary flow reserve is impaired, and the model is essentially one of stress-induced ischemic dysfunction.
We think our model overcomes many of these limitations: both the rate and degree of coronary occlusion are controlled, there is resting myocardial ischemia with viability (hibernation) in the distribution of the coronary stenosis, and only a small degree of subendocardial infarction. All of these features make the model potentially appealing for studies involving experimental coronary stenosis or interventions to improve blood flow or function in the chronically ischemic heart.
Limitations of the present study are several. First, as already noted, there was an approximately 35% reduction in transmural flow on PET 30 days after inflation of the occluder at a time when the flow probe immediately downstream from the occluder was measuring a 90% reduction in antegrade LCX flow. This discrepancy likely results, at least in part, from the effects of spillover from adjacent normal segments on PET [29]. A second possibility is that there may be some degree of collateral recruitment to the ischemic area that is able to maintain myocardial viability, although not to a degree necessary to preserve either rest or stress function. We have performed coronary angiography (see Fig 1) on a large number of animals at intervals out to 6 months after production of LCX stenosis as described in this study with little evidence for epicardial collateralization seen (unpublished data). However, swine, like humans, often form small endomural and subendocardial collateral vessels that may not be visible angiographically [28]. These findings differ from the ameroid constrictor model of LCX occlusion where large epicardial collaterals originating from the left anterior descending coronary artery are seen [26].
Another limitation of the study is the lack of demonstration of reversibility in the observed left ventricular dysfunction. Vanoverschelde and associates [7] noted that mechanical recovery of left ventricular dysfunction after revascularization, ie, reversible dysfunction, is an integral part of the definition of hibernating myocardium. We were unable to demonstrate this in the present study because of the need to sacrifice the animals in the hibernating state for both triphenyl tetrazolium chloride staining and microscopic analysis. However, we [30] have recently shown improved wall motion 6 months after transmyocardial laser revascularization using this model. Consequently, the model appears to meet all of the criteria used to describe hibernating myocardium: chronic left ventricular dysfunction at rest, response of the dysfunctional myocardium to inotropic stimulation, and reversibility on revascularization [7].
In summary, this study demonstrates that low-flow myocardial hibernation is a real entity that can be reproduced in miniswine after experimental high-grade LCX stenosis. We hope that the development of this model will prove useful in the study of the mechanisms underlying hibernating myocardium and of the use of therapies designed to improve blood flow to the chronically ischemic heart.
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Acknowledgments |
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References |
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