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Echocardiographic evaluation of ventricular remodeling in a mouse model of myocardial infarction
Echocardiographic Evaluation of Ventricular Remodeling in a Mouse Model of Myocardial Infarction Shigeto Kanno, MD, PhD, Deborah L. Lerner, MD, Richard B. Schuessler, PhD, Tetsuo Betsuyaku, MD, PhD, Kathryn A. Yamada, PhD, Jeffrey E. Saffitz, MD, PhD, and Attila Kovacs, MD, St. Louis, Missouri
Gene-targeting in mice is a powerful tool to define molecular mechanisms of ischemic heart disease that determine infarct size, postinfarct left ventricular (LV) remodeling, and arrhythmogenesis. Coronary ligation in mice is becoming a widely used model of myocardial infarction (MI), but the pathophysiologic consequences of MI in mice and its relevance to human MI have not been fully elucidated. To characterize structural and functional changes during evolving MI, we analyzed 2-dimensional– based reconstruction of the left ventricle by noninvasive echocardiography obtained 1 day and 1 week after surgical ligation of the left anterior descending coronary artery in mice. Sequential 2dimensional short-axis cineloops of the left ventricle were used to measure LV mass, and LV volumes at end-diastole and end-systole. Echocardiographic infarct size was estimated by measuring the volume of akinetic LV segments. Histologic infarct size was
Despite significant advances in efforts to limit the extent of myocardial infarction (MI) and improve postinfarction remodeling, knowledge of molecular mechanisms in chronic ischemic heart disease remains fragmentary.1 A more detailed understanding of these mechanisms will contribute to development of novel biologically based therapies to improve clinical outcomes in patients with coronary artery disease and MI. Gene targeting and transgenic techniques have From the Departments of Surgery, Medicine, Pediatrics, and Pathology, and The Center for Cardiovascular Research, Washington University School of Medicine. Supported by grant HL-58507 from the National Institutes of Health and grant 12-3040-93200 from the American Heart Association. Reprint requests: Attila Kovacs, MD, Cardiovascular Division, Box 8086, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110 (E-mail: [email protected]). Copyright 2002 by the American Society of Echocardiography. 0894-7317/2002/$35.00 ⫹ 0 27/1/117560 doi:10.1067/mje.2002.117560
measured by planimetry of 9 transverse sections of each heart. There was close correlation between the 2 methods (31% ⴞ 20% of LV mass and 34% ⴞ 17% of LV area, respectively; y ⴝ .83x ⴙ 7.9, r ⴝ 0.96, P < .01). LV volumes at end diastole increased significantly between 1 day and 1 week (51 ⴞ 17 L vs 78 ⴞ 46 L, respectively, P < .05). The relative change in LV volumes at end diastole varied as a function of infarct size (r ⴝ 0.93, P < .01). LV mass and the extent of hypertrophy of noninfarcted segments also varied with infarct size (r ⴝ 0.92, P < .01; r ⴝ 0.90, P < .01, respectively). Thus, echocardiography is an accurate noninvasive tool for determination of infarct size and quantitative characterization of postinfarct remodeling in the mouse model of MI. Alterations in cardiac structure and function after coronary ligation in mice closely resemble pathophysiologic changes in human ischemic heart disease. (J Am Soc Echocardiogr 2002;15:601-9.)
helped define the role of individual gene products in whole organ physiology. Creating human disease models in genetically engineered mice is therefore becoming an increasingly useful strategy to elucidate molecular mechanisms in complex pathophysiologic processes.2 Surgical ligation of the proximal left anterior descending (LAD) coronary artery has been performed in genetically engineered mice to delineate the role of specific molecular pathways in determining infarct size and postinfarct remodeling such as infarct expansion, compensatory hypertrophy, and ischemic cardiomyopathy.3-8 As in any animal model of human disease, it is essential to demonstrate that MI and its sequelae in mice resemble the human condition. Echocardiography has long been invaluable in clinical studies evaluating cardiac structure and function post-MI and in demonstrating clinical benefits of revascularization and pharmacologic therapies. Cardiac ultrasound has been applied increasingly in characterizing structural and functional features of cardiac
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phenotypes and pathophysiologic consequences of surgical procedures in small animal models of human disease.9 Technical advances in imaging systems now provide an opportunity to take full advantage of the spatial and temporal resolution of 2-dimensional (2D) cardiac imaging even in the smallest mammalian hearts.10-13 The purpose of this study was to develop and validate noninvasive, transthoracic echocardiographic methods to measure infarct size and characterize progressive remodeling of LV structure and function in mice with evolving infarcts induced by LAD ligation. We performed serial studies 1 day and 1 week after MI surgery. Consecutive (from base to apex) 2D short-axis images of the left ventricle were used to reconstruct 3-dimensional (3D) LV structure. Detailed histologic evaluation of cardiac structure and infarct size was used to validate the echocardiographic findings. Our results demonstrate that coronary ligation in mice produces acute MI, followed by structural and functional changes that resemble human ischemic heart disease. Two-dimensional echocardiography is highly accurate in the evaluation of infarct size and post-MI remodeling.
MATERIALS AND METHODS MI Surgery Founder mice of uniform genetic background (C57BL/6) were originally purchased from Jackson Laboratories (Bar Harbor, Me) and inbred in a standard barrier facility under veterinary supervision. All protocols were approved by the Animal Studies Committee at Washington University School of Medicine. Animals were anesthetized by intraperitoneal injection of ketamine hydrochloride (87 mg/kg) and xylazine hydrochloride (13 mg/kg). The trachea was exposed through a midline incision and intubated with a 20-gauge intravenous catheter through the oral cavity under visualization. Respiration was controlled by a rodent ventilator at a tidal volume of 0.7 to 1.0 mL and a rate of 130 to 150 strokes per minute. The chest was opened with a left thoracotomy through the 5th intercostal space. A 7-0 prolene suture was tied around the LAD coronary artery immediately distal to the origin of the first diagonal branch. The lungs were re-expanded and the chest was closed. Animals were weaned from the respirator and allowed to recover from anesthesia on a heating mat. Echocardiographic Image Acquisition Transthoracic echocardiography was performed under light anesthesia induced by intraperitoneal injection of 2,2,2-tribromoethanol (Avertin, 2.5% solution, 0.005 mL/g
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body weight; Eastman Chemicals, Eastman Kodak Co, Rochester, NY), which produced a semiconscious state in which the animals breathed spontaneously. Their chests were shaved and the animals were placed on a heating table in a left lateral decubitus position. A commercial echocardiography system (Sequoia, Acuson, Mountain View, Calif) equipped with a 13-MHz linear array ultrasound transducer was used in these experiments. Imaging was performed by direct contact of the transducer with the chest wall (no stand-off was necessary). Care was taken to avoid excessive pressure on the chest. A minimal depth setting of 2 cm and a 1 ⫻ 1 cm zoom area was used to maximize image quality and frame rate (166 frames/s). First, a parasternal long-axis image was acquired with appropriate angulation so that the maximum LV length could be identified. The long-axis view was also used to guide perpendicular angulation of the transducer in acquisition of the short-axis slices. Consecutive 2D short-axis images of the left ventricle were obtained from the parasternal view for several levels of the left ventricle by using the following anatomic landmarks: The most basal images were obtained by first visualizing the base of the aortic root and then apically angulating the transducer until circumferential wall thickening was first visualized; mid myocardial regions were identified by the shift in the location of the papillary muscles from a posterior to a more lateral position within the left ventricle; the most apical images were identified immediately before the level at which no discernable cavity was visualized. Five to 7 short-axis segments were recorded depending on the overall size of the left ventricle or the complexity of the akinetic regions. An effort was made to record with approximately equal spacing between sections. Cineloops of 30 frames covering 1 to 2 heart cycles were stored digitally and analyzed off-line. Echocardiographic Image Analysis Echocardiographic analysis was performed by a single observer blinded to the experimental conditions and to the results of the histologic analysis. Digital images were retrieved and analyzed on the same echocardiography system. On each of the consecutive short-axis images, end-diastolic endocardial and epicardial borders and end-systolic endocardial borders were traced by electronic calipers (Figure 1). This analysis process was facilitated by the image display and analysis capabilities of the echocardiography equipment that included high-speed cineloop display of 3 to 4 end-diastolic or end-systolic frames (improved visual detection of endocardial border) and resizable elliptical calipers that aided the tracing process in areas of echocardiography dropout. To determine infarct size, the full cardiac cycle was displayed in slow motion (⬃15% of native heart rate) and endocardial and epicardial borders of the akinetic segments were traced on each end-diastolic short-axis slices. The distance of each of the short-axis images from the base
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Figure 1 A, Echocardiographic analysis of left ventricular (LV) volumes and infarct size. Sequential short-axis images of left ventricle were acquired from base to apex. B, Endocardial border was traced at end-diastole (Endo-ed) and end-systole (Endo-es) and epicardial border at end-diastole (Epi-ed). Borders of akinetic myocardial segments were also traced. C, Distance between short-axis slices and LV length were measured from long-axis image. D, LV volumes, LV mass, and infarct mass were calculated based on 3D reconstruction of LV geometry by method of disks.
was then determined by fitting the slice in the LV cavity along the basal- to apical-axis based on the anterior to posterior dimension of the endocardial tracing. LV length was measured from the parasternal long-axis image at end diastole. LV volumes were calculated with Simpson’s reconstruction at end-diastole and end-systole by adding the intraluminar volumes of each of the short-axis cross-sectional slabs calculated by multiplying each planimetered endocardial area (A) by its height (h), except for the apical region in which the volume was calculated with an ellipsoid-segment formula. The total intraventricular volume was then obtained by summation of volumes of individual sections: V ⫽ A1h1 ⫹ A2h2 ⫹ A3h3 ⫹ A4h4 ⫹ 2 ⫻ A5h5/3 LV mass was calculated at end-diastole by subtracting the volume of LV cavity from total LV volume as measured by epicardial tracing of consecutive short-axis slices and multiplying by the density of myocardium (1.055). Infarct size was calculated from the traced areas of akinetic segments at end-diastole. Each of these areas was multiplied by its appropriate slab height, and corresponding volumes were summated for short-axis sections extending from base to apex of the left ventricle. The total akinetic muscle volume was then multiplied by the density of myocardium to obtain the mass of akinetic myocardium. Infarct size was expressed
as the mass of akinetic myocardial as a proportion of total LV mass. Histologic Analysis of Infarct Size Hearts were excised from anesthetized animals on day 8 after MI surgery (1 day after the final echocardiographic study) and fixed in formalin. Each heart was cut transversely into 3 roughly equal short-axis ventricular slices (apical, middle, and basal portions), embedded in paraffin, and sectioned in its entirety at a thickness of 5 m. Every 20th section was collected on individual glass slides resulting in approximately 5 separate sets of sections per millimeter of tissue thickness and yielding 24 to 30 slides per heart. Three slides containing sections of each of the 3 short-axis ventricular slices (a total of 9 slices from base to apex) were stained with Masson’s trichrome stain for quantitative analysis of infarct size and structure. Quantitative analysis was performed by a single observer blinded to the results of the echocardiographic analysis. Infarct size was measured with a computerized morphometry system and NIH Image 1.49 software. The portions of each of the 9 short-axis ventricular sections occupied by infarct scar and noninfarcted LV free wall and interventricular septum were traced, areas were digitized and summed, and infarct size was calculated as the total area occupied by scar divided by the total ventricular area (infarcted plus noninfarcted areas). The
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was identified histologically on the epicardial surface where the suture had been placed. This localized epicardial injury was apparently related to unsuccessful attempts to ligate the LAD. Although local segmental wall motion abnormalities located at the mid to basal portion of the anterior LV wall were detected in these 3 mice, they were excluded because they did not exhibit MI caused by LAD ligation. Technically adequate echocardiographic images were obtained in all animals. Image quality in mice with MI was reduced compared with echocardiographic images from nonoperated animals of the same strain, likely because of the presence of residual air in the chest cavity and fibrinous exudate 1 day after surgery and development of fibrous adhesions 1 week after surgery. A study was considered technically adequate if at least 5 distinct sequential short-axis slices were obtained, and the endocardial and epicardial borders were sufficiently visualized so that the sizable electronic calipers could be applied to the images. Evaluation of Infarct Size Figure 2 Correlation between echocardiographic and histologic infarct size. A, Representative histologic sections of hearts with small, moderate, and large MI are shown in upper panels. Corresponding 2-dimensional (2D) shortaxis echocardiograms from same animals are displayed below histologic sections. Traced areas represent the akinetic segments of the anterolateral walls. B, Regression plot depicts correlation between relative volume of akinetic myocardium and histologic measurements of infarct size. Internet address for echocardiographic cineloops corresponding to the short-axis images: http://cardiology. wustl.edu/ccrcore.
reliability coefficient for infarct size data both in terms of intraobserver and interobserver variability was 0.98. Statistical Analysis All data are expressed as mean ⫾ SD. Differences between groups were determined with analysis of variance. Specific group differences were determined with paired and unpaired t test when applicable using Bonferroni/Dunn correction for multiple group comparison. Correlation between variables was tested by linear regression analysis. For all testing, P ⬍ .05 was used to determine significance.
RESULTS The overall survival after coronary ligation was 89%. Three mice of 13 survivors were excluded from the final analysis because only minimal myocardial injury
Detection of ischemic myocardial injury by echocardiography is based on virtually instantaneous loss of regional LV wall thickening and endocardial excursion after the onset of ischemia. To validate the accuracy of 2D echocardiography in the noninvasive evaluation of infarct size in mouse hearts, we performed a geometric reconstruction of LV volumes from consecutive 2D short-axis slices of the ventricle and compared the relative size of akinetic segments of the myocardium with the relative infarct size measured histologically in 9 separate transverse LV sections from base to apex. Mean akinetic segment size determined by echocardiography 1 week after MI surgery was 31% ⫾ 20% of LV mass; mean infarct size measured histologically was 34% ⫾ 17% of total LV area. There was no significant difference between these 2 measurements. There was, however, considerable variation in the infarct size among the animals. Figure 2, A, shows representative images of small, moderate, and large size infarcts. Small infarcts were generally located in the mid to apical anterior portion of the left ventricle; moderate infarcts typically involved the mid to apical portions of the anterior and lateral wall and the apex. Large infarcts included most of the anterior and lateral free walls and the entire apex and often extended to the mid and apical portion of the inferior wall. The septum was generally spared, however, even in hearts with the largest infarcts. This variability provided an opportunity to determine the accuracy of our echocardiographic technique in the evaluation of infarct size over a wide
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Figure 3 Structural evidence of postinfarction remodeling in a heart with large myocardial infarct after coronary ligation. A and B, Parasternal long-axis and, C and F, sequential short-axis images of left ventricle (A, C, and D) 1 day and (B, E, and F) 1 week after infarction. Dotted lines represent manual tracings of endocardial and epicardial borders of left ventricle. C and E, Apical short-axis slices demonstrate left ventricular chamber dilation, wall thinning, and infarct expansion. Basal short-axis images show compensatory hypertrophy of noninfarcted segments. Internet address for echocardiographic cineloops: http://cardiology.wustl.edu/ccrcore.
range. The correlation between the relative volume of akinetic myocardium and histologic measurements of infarct size is shown in Figure 2, B. A close correlation was observed between the 2 techniques with no significant overestimation or underestimation of infarct size by echocardiography (y ⫽ 0.83x ⫹ 7.9, r ⫽ 0.96, SEE ⫽ 2.77, P ⬍ .01). Echocardiographic Evaluation of Post-MI Remodeling Postinfarct remodeling leading to infarct expansion, alterations in LV chamber dimensions and hypertrophy of noninfarcted regions occurs in human MI. To characterize the progression of remodeling in the mouse model of MI, we performed serial echocardiographic studies 1 day and 1 week after coronary ligation (Figure 3). The size of the akinetic region and the extent to which global parameters of LV structure and function changed between 1 day and 1 week varied considerably. Therefore, to determine the effect of infarct size on the extent of postinfarct remodeling, we divided the animals into groups with small, moderate, and large infarcts involving less than 25%, 25% to 45%, and greater than 45% of LV mass, respectively, as determined by echocardiography. Changes in the structural parameters of postinfarct
remodeling are summarized in Table 1. No significant changes in end-diastolic or end-systolic LV volumes, LV mass, or wall thickness of remote segments occurred between 1 day and 1 week post-MI in the group with small infarcts. In contrast, a small but significant decrease in relative infarct size and absolute infarct mass occurred in this group during the same period. Given the close correlation between echocardiographic and histologic measurements of infarct size, these data indicate that the mass of muscle exhibiting segmental akinesis seen 1 day after coronary ligation overestimates the eventual infarct size. This finding is consistent with the well-recognized difference between area-at-risk and infarct size. In the group with moderate infarcts, LV chamber size and infarct mass were significantly larger at both time points compared with the group with small infarcts and there was a trend toward an increase in LV volumes and LV mass within the same animals from 1 day to 1 week. Postinfarct remodeling was most marked in the group with large infarcts (Figure 3). Both end-diastolic and end-systolic LV volumes and infarct mass were increased at 1 day compared with small infarct group, and more importantly, there was a marked increase in these parameters from 1 day to 1 week after coronary ligation. The marked increase in
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Table 1 Echocardiographic characterization of left ventricular structure and function 1 day and 1 week after left anterior descending ligation in subgroups of mice with small, moderate, and large MI Small
LVVed (L) LVVes (L) LVM (mg) MI mass (mg) IS (%) WTh base (mm) RWT base EF (%) SV (L) CO (mL/min)
Moderate
Large
1 d post MI
1 wk post MI
1 d post MI
1 wk post MI
1 d post MI
1 wk post MI
39 ⫾ 11 18 ⫾ 5 59 ⫾ 10 12 ⫾ 5 20 ⫾ 5 0.80 ⫾ 0.10 0.6 ⫾ 0.1 54 ⫾ 3 21 ⫾ 6 13 ⫾ 4
40 ⫾ 10 17 ⫾ 6 62 ⫾ 12 9 ⫾ 5* 14 ⫾ 6* 0.78 ⫾ 0.12 0.6 ⫾ 0.1 59 ⫾ 6 23 ⫾ 5 14 ⫾ 3
61 ⫾ 6† 32 ⫾ 1† 78 ⫾ 20 31 ⫾ 2† 41 ⫾ 7† 0.73 ⫾ 0.08 0.6 ⫾ 0.1 47 ⫾ 4 29 ⫾ 5 18 ⫾ 2
91 ⫾ 15† 58 ⫾ 13† 97 ⫾ 13† 36 ⫾ 3† 37 ⫾ 2† 0.84 ⫾ 0.06 0.5 ⫾ 0.1 36 ⫾ 4*† 33 ⫾ 2 21 ⫾ 2†
65 ⫾ 14‡ 47 ⫾ 7‡ 76 ⫾ 20 40 ⫾ 5‡ 55 ⫾ 10‡ 0.82 ⫾ 0.04 0.7 ⫾ 0.1 26 ⫾ 5‡§ 17 ⫾ 6 10 ⫾ 4
133 ⫾ 30*‡ 102 ⫾ 21*‡ 131 ⫾ 34*‡ 71 ⫾ 11*‡§ 55 ⫾ 8‡§ 1.07 ⫾ 0.08*‡ 0.5 ⫾ 0.1 23 ⫾ 3‡§ 31 ⫾ 10* 20 ⫾ 6*
Values are mean ⫾ SD. MI, Myocardial infarctions; LVVed, left ventricular enddiastolic volume; LVVes, left ventricular endsystolic volume; LVM, left ventricular mass; IS, infarct size as a percent of LVM; WTh, wall thickness at the base of the left ventricular; RWT, relative wall thickness at the base of the left ventricular; EF, ejection fraction; SV stroke volume; CO cardiac output. *P ⬍ .05 1 d vs 1 wk after MI; †P ⬍ .05 moderate vs small infarcts; ‡P ⬍ .05 large vs small; §P ⬍ .05 large vs moderate infarcts.
Figure 4 Relative changes in (A) end-diastolic left ventricular volume (%⌬LVVed) and (B) LV mass (%⌬LVMass) from 1 day to 1 week as a function of infarct size and correlation between ejection fraction and (C) infarct size at 1 week.
LV mass between 1 day and 1 week was because of both an increase in infarct mass (infarct expansion) and compensatory hypertrophy of noninfarcted regions as evidenced by a significant increase in the mass of noninfarcted LV segments and an increase in inferobasal wall thickness. Ejection fraction, stroke volume, and cardiac output were all in the normal range 1 day post-MI in the group with small infarcts and remained unchanged over time. The moderate infarct group showed a mild decrease in ejection fraction at 1 week but maintained normal stroke volume and cardiac output throughout the study interval. The most significant changes were seen in the large infarct group that exhibited severely depressed ejection fraction at both time points. Stroke volume and cardiac output were also depressed 1 day post-MI but normalized after 1 week. These changes are consistent with the compensatory effects of the Frank-Starling mechanism and development of compensatory hyper-
trophy of noninfarcted segments, which maintained and restored cardiac function in the presence of marked wall motion abnormalities. Regression analysis was performed to determine the relationship between structural and functional parameters of postinfarct remodeling and infarct size. As shown in Figure 4, there was a significant correlation between the relative increases in LV volumes and mass and echocardiographically measured infarct size, and there was a strong inverse correlation between ejection fraction and infarct size at 1 week.
DISCUSSION Postinfarct remodeling is a progressive process involving LV chamber dilatation, infarct wall thinning, and compensatory thickening of noninfarcted regions.14 During the early phase of MI, infarct expan-
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sion and regional dilatation contribute to ventricular chamber enlargement. In the current study, infarct expansion defined by significant increase in infarct mass, was seen only in the animals with large infarcts (⬎45% of LV mass). This group also showed the greatest degree of compensatory hypertrophy as evidenced by increases in LV mass and wall thickness in segments remote from the infarct (base of the heart) between 1 day and 1 week after MI surgery. Compensatory hypertrophy was observed only in animals that developed marked deterioration in LV ejection fraction. The combined effects of a large infarct and markedly depressed systolic function initiates a vicious circle in which progressive ventricular dilatation and insufficient compensatory hypertrophy create marked heterogeneity of segmental wall stress (increased chamber sphericity and radius) and wall motion.15 This results in thinning of infarcted segments, the most extreme form of which is development of ventricular aneurysm. Despite variability in infarct size within groups, studies of animals with small, moderate, and large infarcts facilitated analysis of the relationship between the amount of myocardium initially damaged and the resultant remodeling and compensatory processes. In fact, it is widely recognized that parameters of ventricular remodeling are a function both of the extent of myocardial damage and the time after infarction.14 Simultaneous noninvasive measurement of infarct mass, LV volumes, and myocardial mass of noninfarcted regions, as described in the current study, allows accurate evaluation of the relationship among these parameters at a given time point in the remodeling process. Previous studies using the same mouse model have focused on the time course of structural and functional consequences of MI and showed marked changes during the first 1 to 2 weeks post-MI, followed by more modest deterioration during the ensuing weeks.3-8 These studies have used a variety of invasive and noninvasive in vivo and in vitro techniques and showed a significant, albeit weak correlation between infarct size and some parameters of remodeling (LV volume,4 septal mass,5 fractional shortening6) but no correlation with others (LV mass,4,6 end-diastolic LV dimension).6 The lack of correlation with some parameters of remodeling is likely a result of methodologic limitations of obtaining anatomic and physiologic data with different techniques at different time points. Our data clearly demonstrate that a complex interplay between structural and functional aspects of the remodeling process occurs during the first week after coronary ligation, the major determinant of which is the extent of the initial myocardial injury. Given the close correlation observed between infarct
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size and the extent of remodeling post-MI, we would propose the use of this integrated noninvasive approach that allows simultaneous acquisition of structural and functional data as a function of infarct size and time. This approach provides more sensitive quantitative evaluation of the remodeling process than other previously reported methods. Numerous studies have demonstrated that because of the segmental nature and variable location of infarcts, M-mode, or even 2D imaging from a single plane (2D long-axis) is less accurate for measuring LV volumes than imaging multiple 2D planes.16 Previous studies in mice using M-mode analysis showed weak or absent correlation between infarct size measured anatomically and M-mode based measurements of LV volumes and infarct size.4,6 With M-mode echocardiography, the well-recognized inaccuracy of the cube formula for volume measurements is compounded by the fact that the standard placement of the M-mode line in the parasternal view does not include the typical infarct area in both basal-to-apical and septal-to-lateral directions. In our experience, infarcts of various sizes created in mice by LAD occlusion are typically located in the mid to apical anterolateral segments with sparing of mid to basal inferior and virtually all septal segments. Given the usual orientation of the heart in the mouse chest, M-mode echocardiograms traverse the left ventricle through the anteroseptal and inferoposterior segments. In fact, we often observed that small anterolateral infarcts were not visualized on the parasternal long-axis views. Two-dimensional echocardiography has been used extensively for quantitative assessment of infarct size.17-19 There is general agreement that quantitative analysis of regional wall motion from 2D echocardiograms consistently overestimates the extent of infarction evident on pathologic examination.19 Commonly cited explanations for this discrepancy include tethering of nonischemic myocardium adjacent to infarcted myocardium, delayed mechanical recovery of function in viable regions surrounding the infarct zone, and methodologic limitations in the quantification of regional dyssynergy. In contrast, our results indicate a lack of overestimation of infarct size when measurements are based on more accurate reconstruction of LV geometry. This finding is in agreement with the study by Yao et al20 who demonstrated in a dog model of acute MI that infarct mass measurement by volume-rendered 3D echocardiography correlated closely with actual infarct mass without significant overestimation or underestimation of infarct size. We concur with Yao et al on the likely explanation for the improved accuracy of these tech-
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niques. First, we used strict criteria for the quantification of regional dysfunction (only akinetic or dyskinetic segments were included). Second, the number of short-axis slices (5 to 7 per heart) of the mouse left ventricle with an average length of 6 mm allows a more detailed analysis of LV structure and infarct size than could be accomplished with 4 to 5 slices in larger hearts with an average length 20 times that of the mouse heart. Third, most of the infarcts in our study were transmural and associated with marked thinning of the infarcted segments and clear demarcation of the scar from the adjacent noninfarcted segments. A similar echocardiographic method for the evaluation of myocardial injury was recently reported by Scherrer-Crosbie et al13 who described acute changes in LV volumes and systolic function associated with ligation of the LAD in an open-chest mouse preparation. They observed an increase in end-diastolic and end-systolic LV volumes and a decrease in LV ejection fraction after coronary ligation and found a significant correlation between echocardiographyderived and Doppler flow probe-based measurements of cardiac output. They described the extent of segmental wall motion abnormalities but there was no histologic validation of the method. In a separate publication, Scherrer-Crosbie et al21 performed myocardial perfusion studies with the use of intravenously injected echocardiographic contrast agent in the same acute LAD occlusion preparation. They found a close correlation between the size of perfusion defect detected on the contrast images and the area-at-risk measured by postmortem dye perfusion technique. Dyssynergy of segmental wall motion overestimated the area-at-risk. Although we did not attempt to characterize the relationship between infarct size and area-at-risk in the current study, we have previously observed that myocardial contrast echocardiography is a highly effective technique for the noninvasive evaluation of myocardial perfusion under various pathologic conditions (infarct size and area-at-risk; coronary occlusion and reperfusion; coronary stenosis and myocardial ischemia; nonischemic wall motion abnormalities) (unpublished data). Taken together, these studies provide strong evidence for the usefulness of echocardiography in the comprehensive noninvasive evaluation of structural and functional consequences of MI. Limitations The major limitation of the study is that the image acquisition method is technically demanding and the analysis is labor intensive. This is partly because of the less than optimal visualization of the endocardial
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borders and partly because of the large number of manual tracings required for the analyses. Contrast echocardiography, automatic border detection, or 3D image acquisition may greatly improve this technique in the future. We thank Karen Green for excellent technical assistance. REFERENCES 1. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000;101:2981-8. 2. Lin MC, Rockman HA, Chien KR. Heart and lung disease in engineered mice. Nat Med 1995;1:749-51. 3. Li B, Li Q, Wang X, Jana KP, Redaelli G, Kajstura J, et al. Coronary constriction impairs cardiac function and induces myocardial damage and ventricular remodeling in mice. Am J Physiol 1997;273:H2508-19. 4. Patten RD, Aronovitz MJ, Deras-Mejia L, Pandian NG, Hanak GG, Smith JJ, et al. Ventricular remodeling in a mouse model of myocardial infarction. Am J Physiol 1998;274: H1812-20. 5. Michael LH, Ballantyne CM, Zachariah JP, Gould KE, Pocius JS, Taffet GE, et al. Myocardial infarction and remodeling in mice: effect of reperfusion. Am J Physiol 1999;277:H660-8. 6. Gao XM, Dart AM, Dewar E, Jennings G, Du XJ. Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice. Cardiovasc Res 2000;45:330-8. 7. Sam F, Sawyer DB, Chang DL, Eberli FR, Ngoy S, Jain M, et al. Progressive left ventricular remodeling and apoptosis late after myocardial infarction in mouse heart. Am J Physiol Heart Circ Physiol 2000;279:H422-8. 8. Lutgens E, Daemen MJ, de Muinck ED, Debets J, Leenders P, Smits JF. Chronic myocardial infarction in the mouse: cardiac structural and functional changes. Cardiovasc Res 1999;41:586-93. 9. Christensen G, Wang Y, Chien KR. Physiological assessment of complex cardiac phenotypes in genetically engineered mice. Am J Physiol 1997;272:H2513-24. 10. Kovacs A, Yee R, Da´ vila-Roma´ n VG, Barzilai B. Comparison of M-mode and 2D based measurements of LV geometry in mice [abstract]. J Am Soc Echocardiogr 1998;11:538. 11. Mor-Avi V, Korcarz C, Fentzke RC, Lin H, Leiden JM, Lang RM. Quantitative evaluation of left ventricular function in a transgenic mouse model of dilated cardiomyopathy with 2D contrast echocardiography. J Am Soc Echocardiogr 1999;12: 209-14. 12. Youn HJ, Rokosh G, Lester SJ, Simpson P, Schiller NB, Foster E. Two-dimensional echocardiography with a 15-MHz transducer is a promising alternative for in vivo measurement of left ventricular mass in mice. J Am Soc Echocardiogr 1999;12:70-5. 13. Scherrer-Crosbie M, Steudel W, Hunziker PR, Liel-Cohen N, Ullrich R, Zapol WM, et al. Three-dimensional echocardiographic assessment of left ventricular wall motion abnormalities in mouse myocardial infarction. J Am Soc Echocardiogr 1999;12:834-40. 14. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction: experimental observations and clinical implications. Circulation 1990;81:1161-72. 15. Pfeffer MA, Pfeffer JM. Ventricular enlargement and reduced
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survival after myocardial infarction. Circulation 1987; 75(suppl 4):IV93-7. 16. Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, Feigenbaum H, et al. Recommendations for quantitation of the left ventricle by 2D echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of 2D Echocardiograms. J Am Soc Echocardiogr 1989;2:358-67. 17. Weiss JL, Bulkley BH, Hutchins GM, Mason SJ. Two-dimensional echocardiographic recognition of myocardial injury in man: comparison with postmortem studies. Circulation 1981; 63:401-8. 18. Wyatt HL, Meerbaum S, Heng MK, Rit J, Gueret P, Corday E. Experimental evaluation of the extent of myocardial dys-
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synergy and infarct size by 2D echocardiography. Circulation 1981;63:607-14. 19. Force T, Kemper A, Perkins L, Gilfoil M, Cohen C, Parisi AF. Overestimation of infarct size by quantitative 2D echocardiography: the role of tethering and of analytic procedures. Circulation 1986;73:1360-8. 20. Yao J, Cao QL, Masani N, Delabays A, Magni G, Acar P, et al. Three-dimensional echocardiographic estimation of infarct mass based on quantification of dysfunctional left ventricular mass. Circulation 1997;96:1660-6. 21. Scherrer-Crosbie M, Steudel W, Ullrich R, Hunziker PR, Liel-Cohen N, Newell J, et al. Echocardiographic determination of risk area size in a murine model of myocardial ischemia. Am J Physiol 1999;277:H986-92.
Gene-targeting in mice is a powerful tool to define molecular mechanisms of ischemic heart disease that determine infarct size, postinfarct left ventricular (LV) remodeling, and arrhythmogenesis. Coronary ligation in mice is becoming a widely used model of myocardial infarction (MI), but the pathophysiologic consequences of MI in mice and its relevance to human MI have not been fully elucidated. To characterize structural and functional changes during evolving MI, we analyzed 2-dimensional– based reconstruction of the left ventricle by noninvasive echocardiography obtained 1 day and 1 week after surgical ligation of the left anterior descending coronary artery in mice. Sequential 2dimensional short-axis cineloops of the left ventricle were used to measure LV mass, and LV volumes at end-diastole and end-systole. Echocardiographic infarct size was estimated by measuring the volume of akinetic LV segments. Histologic infarct size was
Despite significant advances in efforts to limit the extent of myocardial infarction (MI) and improve postinfarction remodeling, knowledge of molecular mechanisms in chronic ischemic heart disease remains fragmentary.1 A more detailed understanding of these mechanisms will contribute to development of novel biologically based therapies to improve clinical outcomes in patients with coronary artery disease and MI. Gene targeting and transgenic techniques have From the Departments of Surgery, Medicine, Pediatrics, and Pathology, and The Center for Cardiovascular Research, Washington University School of Medicine. Supported by grant HL-58507 from the National Institutes of Health and grant 12-3040-93200 from the American Heart Association. Reprint requests: Attila Kovacs, MD, Cardiovascular Division, Box 8086, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110 (E-mail: [email protected]). Copyright 2002 by the American Society of Echocardiography. 0894-7317/2002/$35.00 ⫹ 0 27/1/117560 doi:10.1067/mje.2002.117560
measured by planimetry of 9 transverse sections of each heart. There was close correlation between the 2 methods (31% ⴞ 20% of LV mass and 34% ⴞ 17% of LV area, respectively; y ⴝ .83x ⴙ 7.9, r ⴝ 0.96, P < .01). LV volumes at end diastole increased significantly between 1 day and 1 week (51 ⴞ 17 L vs 78 ⴞ 46 L, respectively, P < .05). The relative change in LV volumes at end diastole varied as a function of infarct size (r ⴝ 0.93, P < .01). LV mass and the extent of hypertrophy of noninfarcted segments also varied with infarct size (r ⴝ 0.92, P < .01; r ⴝ 0.90, P < .01, respectively). Thus, echocardiography is an accurate noninvasive tool for determination of infarct size and quantitative characterization of postinfarct remodeling in the mouse model of MI. Alterations in cardiac structure and function after coronary ligation in mice closely resemble pathophysiologic changes in human ischemic heart disease. (J Am Soc Echocardiogr 2002;15:601-9.)
helped define the role of individual gene products in whole organ physiology. Creating human disease models in genetically engineered mice is therefore becoming an increasingly useful strategy to elucidate molecular mechanisms in complex pathophysiologic processes.2 Surgical ligation of the proximal left anterior descending (LAD) coronary artery has been performed in genetically engineered mice to delineate the role of specific molecular pathways in determining infarct size and postinfarct remodeling such as infarct expansion, compensatory hypertrophy, and ischemic cardiomyopathy.3-8 As in any animal model of human disease, it is essential to demonstrate that MI and its sequelae in mice resemble the human condition. Echocardiography has long been invaluable in clinical studies evaluating cardiac structure and function post-MI and in demonstrating clinical benefits of revascularization and pharmacologic therapies. Cardiac ultrasound has been applied increasingly in characterizing structural and functional features of cardiac
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phenotypes and pathophysiologic consequences of surgical procedures in small animal models of human disease.9 Technical advances in imaging systems now provide an opportunity to take full advantage of the spatial and temporal resolution of 2-dimensional (2D) cardiac imaging even in the smallest mammalian hearts.10-13 The purpose of this study was to develop and validate noninvasive, transthoracic echocardiographic methods to measure infarct size and characterize progressive remodeling of LV structure and function in mice with evolving infarcts induced by LAD ligation. We performed serial studies 1 day and 1 week after MI surgery. Consecutive (from base to apex) 2D short-axis images of the left ventricle were used to reconstruct 3-dimensional (3D) LV structure. Detailed histologic evaluation of cardiac structure and infarct size was used to validate the echocardiographic findings. Our results demonstrate that coronary ligation in mice produces acute MI, followed by structural and functional changes that resemble human ischemic heart disease. Two-dimensional echocardiography is highly accurate in the evaluation of infarct size and post-MI remodeling.
MATERIALS AND METHODS MI Surgery Founder mice of uniform genetic background (C57BL/6) were originally purchased from Jackson Laboratories (Bar Harbor, Me) and inbred in a standard barrier facility under veterinary supervision. All protocols were approved by the Animal Studies Committee at Washington University School of Medicine. Animals were anesthetized by intraperitoneal injection of ketamine hydrochloride (87 mg/kg) and xylazine hydrochloride (13 mg/kg). The trachea was exposed through a midline incision and intubated with a 20-gauge intravenous catheter through the oral cavity under visualization. Respiration was controlled by a rodent ventilator at a tidal volume of 0.7 to 1.0 mL and a rate of 130 to 150 strokes per minute. The chest was opened with a left thoracotomy through the 5th intercostal space. A 7-0 prolene suture was tied around the LAD coronary artery immediately distal to the origin of the first diagonal branch. The lungs were re-expanded and the chest was closed. Animals were weaned from the respirator and allowed to recover from anesthesia on a heating mat. Echocardiographic Image Acquisition Transthoracic echocardiography was performed under light anesthesia induced by intraperitoneal injection of 2,2,2-tribromoethanol (Avertin, 2.5% solution, 0.005 mL/g
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body weight; Eastman Chemicals, Eastman Kodak Co, Rochester, NY), which produced a semiconscious state in which the animals breathed spontaneously. Their chests were shaved and the animals were placed on a heating table in a left lateral decubitus position. A commercial echocardiography system (Sequoia, Acuson, Mountain View, Calif) equipped with a 13-MHz linear array ultrasound transducer was used in these experiments. Imaging was performed by direct contact of the transducer with the chest wall (no stand-off was necessary). Care was taken to avoid excessive pressure on the chest. A minimal depth setting of 2 cm and a 1 ⫻ 1 cm zoom area was used to maximize image quality and frame rate (166 frames/s). First, a parasternal long-axis image was acquired with appropriate angulation so that the maximum LV length could be identified. The long-axis view was also used to guide perpendicular angulation of the transducer in acquisition of the short-axis slices. Consecutive 2D short-axis images of the left ventricle were obtained from the parasternal view for several levels of the left ventricle by using the following anatomic landmarks: The most basal images were obtained by first visualizing the base of the aortic root and then apically angulating the transducer until circumferential wall thickening was first visualized; mid myocardial regions were identified by the shift in the location of the papillary muscles from a posterior to a more lateral position within the left ventricle; the most apical images were identified immediately before the level at which no discernable cavity was visualized. Five to 7 short-axis segments were recorded depending on the overall size of the left ventricle or the complexity of the akinetic regions. An effort was made to record with approximately equal spacing between sections. Cineloops of 30 frames covering 1 to 2 heart cycles were stored digitally and analyzed off-line. Echocardiographic Image Analysis Echocardiographic analysis was performed by a single observer blinded to the experimental conditions and to the results of the histologic analysis. Digital images were retrieved and analyzed on the same echocardiography system. On each of the consecutive short-axis images, end-diastolic endocardial and epicardial borders and end-systolic endocardial borders were traced by electronic calipers (Figure 1). This analysis process was facilitated by the image display and analysis capabilities of the echocardiography equipment that included high-speed cineloop display of 3 to 4 end-diastolic or end-systolic frames (improved visual detection of endocardial border) and resizable elliptical calipers that aided the tracing process in areas of echocardiography dropout. To determine infarct size, the full cardiac cycle was displayed in slow motion (⬃15% of native heart rate) and endocardial and epicardial borders of the akinetic segments were traced on each end-diastolic short-axis slices. The distance of each of the short-axis images from the base
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Figure 1 A, Echocardiographic analysis of left ventricular (LV) volumes and infarct size. Sequential short-axis images of left ventricle were acquired from base to apex. B, Endocardial border was traced at end-diastole (Endo-ed) and end-systole (Endo-es) and epicardial border at end-diastole (Epi-ed). Borders of akinetic myocardial segments were also traced. C, Distance between short-axis slices and LV length were measured from long-axis image. D, LV volumes, LV mass, and infarct mass were calculated based on 3D reconstruction of LV geometry by method of disks.
was then determined by fitting the slice in the LV cavity along the basal- to apical-axis based on the anterior to posterior dimension of the endocardial tracing. LV length was measured from the parasternal long-axis image at end diastole. LV volumes were calculated with Simpson’s reconstruction at end-diastole and end-systole by adding the intraluminar volumes of each of the short-axis cross-sectional slabs calculated by multiplying each planimetered endocardial area (A) by its height (h), except for the apical region in which the volume was calculated with an ellipsoid-segment formula. The total intraventricular volume was then obtained by summation of volumes of individual sections: V ⫽ A1h1 ⫹ A2h2 ⫹ A3h3 ⫹ A4h4 ⫹ 2 ⫻ A5h5/3 LV mass was calculated at end-diastole by subtracting the volume of LV cavity from total LV volume as measured by epicardial tracing of consecutive short-axis slices and multiplying by the density of myocardium (1.055). Infarct size was calculated from the traced areas of akinetic segments at end-diastole. Each of these areas was multiplied by its appropriate slab height, and corresponding volumes were summated for short-axis sections extending from base to apex of the left ventricle. The total akinetic muscle volume was then multiplied by the density of myocardium to obtain the mass of akinetic myocardium. Infarct size was expressed
as the mass of akinetic myocardial as a proportion of total LV mass. Histologic Analysis of Infarct Size Hearts were excised from anesthetized animals on day 8 after MI surgery (1 day after the final echocardiographic study) and fixed in formalin. Each heart was cut transversely into 3 roughly equal short-axis ventricular slices (apical, middle, and basal portions), embedded in paraffin, and sectioned in its entirety at a thickness of 5 m. Every 20th section was collected on individual glass slides resulting in approximately 5 separate sets of sections per millimeter of tissue thickness and yielding 24 to 30 slides per heart. Three slides containing sections of each of the 3 short-axis ventricular slices (a total of 9 slices from base to apex) were stained with Masson’s trichrome stain for quantitative analysis of infarct size and structure. Quantitative analysis was performed by a single observer blinded to the results of the echocardiographic analysis. Infarct size was measured with a computerized morphometry system and NIH Image 1.49 software. The portions of each of the 9 short-axis ventricular sections occupied by infarct scar and noninfarcted LV free wall and interventricular septum were traced, areas were digitized and summed, and infarct size was calculated as the total area occupied by scar divided by the total ventricular area (infarcted plus noninfarcted areas). The
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was identified histologically on the epicardial surface where the suture had been placed. This localized epicardial injury was apparently related to unsuccessful attempts to ligate the LAD. Although local segmental wall motion abnormalities located at the mid to basal portion of the anterior LV wall were detected in these 3 mice, they were excluded because they did not exhibit MI caused by LAD ligation. Technically adequate echocardiographic images were obtained in all animals. Image quality in mice with MI was reduced compared with echocardiographic images from nonoperated animals of the same strain, likely because of the presence of residual air in the chest cavity and fibrinous exudate 1 day after surgery and development of fibrous adhesions 1 week after surgery. A study was considered technically adequate if at least 5 distinct sequential short-axis slices were obtained, and the endocardial and epicardial borders were sufficiently visualized so that the sizable electronic calipers could be applied to the images. Evaluation of Infarct Size Figure 2 Correlation between echocardiographic and histologic infarct size. A, Representative histologic sections of hearts with small, moderate, and large MI are shown in upper panels. Corresponding 2-dimensional (2D) shortaxis echocardiograms from same animals are displayed below histologic sections. Traced areas represent the akinetic segments of the anterolateral walls. B, Regression plot depicts correlation between relative volume of akinetic myocardium and histologic measurements of infarct size. Internet address for echocardiographic cineloops corresponding to the short-axis images: http://cardiology. wustl.edu/ccrcore.
reliability coefficient for infarct size data both in terms of intraobserver and interobserver variability was 0.98. Statistical Analysis All data are expressed as mean ⫾ SD. Differences between groups were determined with analysis of variance. Specific group differences were determined with paired and unpaired t test when applicable using Bonferroni/Dunn correction for multiple group comparison. Correlation between variables was tested by linear regression analysis. For all testing, P ⬍ .05 was used to determine significance.
RESULTS The overall survival after coronary ligation was 89%. Three mice of 13 survivors were excluded from the final analysis because only minimal myocardial injury
Detection of ischemic myocardial injury by echocardiography is based on virtually instantaneous loss of regional LV wall thickening and endocardial excursion after the onset of ischemia. To validate the accuracy of 2D echocardiography in the noninvasive evaluation of infarct size in mouse hearts, we performed a geometric reconstruction of LV volumes from consecutive 2D short-axis slices of the ventricle and compared the relative size of akinetic segments of the myocardium with the relative infarct size measured histologically in 9 separate transverse LV sections from base to apex. Mean akinetic segment size determined by echocardiography 1 week after MI surgery was 31% ⫾ 20% of LV mass; mean infarct size measured histologically was 34% ⫾ 17% of total LV area. There was no significant difference between these 2 measurements. There was, however, considerable variation in the infarct size among the animals. Figure 2, A, shows representative images of small, moderate, and large size infarcts. Small infarcts were generally located in the mid to apical anterior portion of the left ventricle; moderate infarcts typically involved the mid to apical portions of the anterior and lateral wall and the apex. Large infarcts included most of the anterior and lateral free walls and the entire apex and often extended to the mid and apical portion of the inferior wall. The septum was generally spared, however, even in hearts with the largest infarcts. This variability provided an opportunity to determine the accuracy of our echocardiographic technique in the evaluation of infarct size over a wide
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Figure 3 Structural evidence of postinfarction remodeling in a heart with large myocardial infarct after coronary ligation. A and B, Parasternal long-axis and, C and F, sequential short-axis images of left ventricle (A, C, and D) 1 day and (B, E, and F) 1 week after infarction. Dotted lines represent manual tracings of endocardial and epicardial borders of left ventricle. C and E, Apical short-axis slices demonstrate left ventricular chamber dilation, wall thinning, and infarct expansion. Basal short-axis images show compensatory hypertrophy of noninfarcted segments. Internet address for echocardiographic cineloops: http://cardiology.wustl.edu/ccrcore.
range. The correlation between the relative volume of akinetic myocardium and histologic measurements of infarct size is shown in Figure 2, B. A close correlation was observed between the 2 techniques with no significant overestimation or underestimation of infarct size by echocardiography (y ⫽ 0.83x ⫹ 7.9, r ⫽ 0.96, SEE ⫽ 2.77, P ⬍ .01). Echocardiographic Evaluation of Post-MI Remodeling Postinfarct remodeling leading to infarct expansion, alterations in LV chamber dimensions and hypertrophy of noninfarcted regions occurs in human MI. To characterize the progression of remodeling in the mouse model of MI, we performed serial echocardiographic studies 1 day and 1 week after coronary ligation (Figure 3). The size of the akinetic region and the extent to which global parameters of LV structure and function changed between 1 day and 1 week varied considerably. Therefore, to determine the effect of infarct size on the extent of postinfarct remodeling, we divided the animals into groups with small, moderate, and large infarcts involving less than 25%, 25% to 45%, and greater than 45% of LV mass, respectively, as determined by echocardiography. Changes in the structural parameters of postinfarct
remodeling are summarized in Table 1. No significant changes in end-diastolic or end-systolic LV volumes, LV mass, or wall thickness of remote segments occurred between 1 day and 1 week post-MI in the group with small infarcts. In contrast, a small but significant decrease in relative infarct size and absolute infarct mass occurred in this group during the same period. Given the close correlation between echocardiographic and histologic measurements of infarct size, these data indicate that the mass of muscle exhibiting segmental akinesis seen 1 day after coronary ligation overestimates the eventual infarct size. This finding is consistent with the well-recognized difference between area-at-risk and infarct size. In the group with moderate infarcts, LV chamber size and infarct mass were significantly larger at both time points compared with the group with small infarcts and there was a trend toward an increase in LV volumes and LV mass within the same animals from 1 day to 1 week. Postinfarct remodeling was most marked in the group with large infarcts (Figure 3). Both end-diastolic and end-systolic LV volumes and infarct mass were increased at 1 day compared with small infarct group, and more importantly, there was a marked increase in these parameters from 1 day to 1 week after coronary ligation. The marked increase in
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Table 1 Echocardiographic characterization of left ventricular structure and function 1 day and 1 week after left anterior descending ligation in subgroups of mice with small, moderate, and large MI Small
LVVed (L) LVVes (L) LVM (mg) MI mass (mg) IS (%) WTh base (mm) RWT base EF (%) SV (L) CO (mL/min)
Moderate
Large
1 d post MI
1 wk post MI
1 d post MI
1 wk post MI
1 d post MI
1 wk post MI
39 ⫾ 11 18 ⫾ 5 59 ⫾ 10 12 ⫾ 5 20 ⫾ 5 0.80 ⫾ 0.10 0.6 ⫾ 0.1 54 ⫾ 3 21 ⫾ 6 13 ⫾ 4
40 ⫾ 10 17 ⫾ 6 62 ⫾ 12 9 ⫾ 5* 14 ⫾ 6* 0.78 ⫾ 0.12 0.6 ⫾ 0.1 59 ⫾ 6 23 ⫾ 5 14 ⫾ 3
61 ⫾ 6† 32 ⫾ 1† 78 ⫾ 20 31 ⫾ 2† 41 ⫾ 7† 0.73 ⫾ 0.08 0.6 ⫾ 0.1 47 ⫾ 4 29 ⫾ 5 18 ⫾ 2
91 ⫾ 15† 58 ⫾ 13† 97 ⫾ 13† 36 ⫾ 3† 37 ⫾ 2† 0.84 ⫾ 0.06 0.5 ⫾ 0.1 36 ⫾ 4*† 33 ⫾ 2 21 ⫾ 2†
65 ⫾ 14‡ 47 ⫾ 7‡ 76 ⫾ 20 40 ⫾ 5‡ 55 ⫾ 10‡ 0.82 ⫾ 0.04 0.7 ⫾ 0.1 26 ⫾ 5‡§ 17 ⫾ 6 10 ⫾ 4
133 ⫾ 30*‡ 102 ⫾ 21*‡ 131 ⫾ 34*‡ 71 ⫾ 11*‡§ 55 ⫾ 8‡§ 1.07 ⫾ 0.08*‡ 0.5 ⫾ 0.1 23 ⫾ 3‡§ 31 ⫾ 10* 20 ⫾ 6*
Values are mean ⫾ SD. MI, Myocardial infarctions; LVVed, left ventricular enddiastolic volume; LVVes, left ventricular endsystolic volume; LVM, left ventricular mass; IS, infarct size as a percent of LVM; WTh, wall thickness at the base of the left ventricular; RWT, relative wall thickness at the base of the left ventricular; EF, ejection fraction; SV stroke volume; CO cardiac output. *P ⬍ .05 1 d vs 1 wk after MI; †P ⬍ .05 moderate vs small infarcts; ‡P ⬍ .05 large vs small; §P ⬍ .05 large vs moderate infarcts.
Figure 4 Relative changes in (A) end-diastolic left ventricular volume (%⌬LVVed) and (B) LV mass (%⌬LVMass) from 1 day to 1 week as a function of infarct size and correlation between ejection fraction and (C) infarct size at 1 week.
LV mass between 1 day and 1 week was because of both an increase in infarct mass (infarct expansion) and compensatory hypertrophy of noninfarcted regions as evidenced by a significant increase in the mass of noninfarcted LV segments and an increase in inferobasal wall thickness. Ejection fraction, stroke volume, and cardiac output were all in the normal range 1 day post-MI in the group with small infarcts and remained unchanged over time. The moderate infarct group showed a mild decrease in ejection fraction at 1 week but maintained normal stroke volume and cardiac output throughout the study interval. The most significant changes were seen in the large infarct group that exhibited severely depressed ejection fraction at both time points. Stroke volume and cardiac output were also depressed 1 day post-MI but normalized after 1 week. These changes are consistent with the compensatory effects of the Frank-Starling mechanism and development of compensatory hyper-
trophy of noninfarcted segments, which maintained and restored cardiac function in the presence of marked wall motion abnormalities. Regression analysis was performed to determine the relationship between structural and functional parameters of postinfarct remodeling and infarct size. As shown in Figure 4, there was a significant correlation between the relative increases in LV volumes and mass and echocardiographically measured infarct size, and there was a strong inverse correlation between ejection fraction and infarct size at 1 week.
DISCUSSION Postinfarct remodeling is a progressive process involving LV chamber dilatation, infarct wall thinning, and compensatory thickening of noninfarcted regions.14 During the early phase of MI, infarct expan-
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sion and regional dilatation contribute to ventricular chamber enlargement. In the current study, infarct expansion defined by significant increase in infarct mass, was seen only in the animals with large infarcts (⬎45% of LV mass). This group also showed the greatest degree of compensatory hypertrophy as evidenced by increases in LV mass and wall thickness in segments remote from the infarct (base of the heart) between 1 day and 1 week after MI surgery. Compensatory hypertrophy was observed only in animals that developed marked deterioration in LV ejection fraction. The combined effects of a large infarct and markedly depressed systolic function initiates a vicious circle in which progressive ventricular dilatation and insufficient compensatory hypertrophy create marked heterogeneity of segmental wall stress (increased chamber sphericity and radius) and wall motion.15 This results in thinning of infarcted segments, the most extreme form of which is development of ventricular aneurysm. Despite variability in infarct size within groups, studies of animals with small, moderate, and large infarcts facilitated analysis of the relationship between the amount of myocardium initially damaged and the resultant remodeling and compensatory processes. In fact, it is widely recognized that parameters of ventricular remodeling are a function both of the extent of myocardial damage and the time after infarction.14 Simultaneous noninvasive measurement of infarct mass, LV volumes, and myocardial mass of noninfarcted regions, as described in the current study, allows accurate evaluation of the relationship among these parameters at a given time point in the remodeling process. Previous studies using the same mouse model have focused on the time course of structural and functional consequences of MI and showed marked changes during the first 1 to 2 weeks post-MI, followed by more modest deterioration during the ensuing weeks.3-8 These studies have used a variety of invasive and noninvasive in vivo and in vitro techniques and showed a significant, albeit weak correlation between infarct size and some parameters of remodeling (LV volume,4 septal mass,5 fractional shortening6) but no correlation with others (LV mass,4,6 end-diastolic LV dimension).6 The lack of correlation with some parameters of remodeling is likely a result of methodologic limitations of obtaining anatomic and physiologic data with different techniques at different time points. Our data clearly demonstrate that a complex interplay between structural and functional aspects of the remodeling process occurs during the first week after coronary ligation, the major determinant of which is the extent of the initial myocardial injury. Given the close correlation observed between infarct
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size and the extent of remodeling post-MI, we would propose the use of this integrated noninvasive approach that allows simultaneous acquisition of structural and functional data as a function of infarct size and time. This approach provides more sensitive quantitative evaluation of the remodeling process than other previously reported methods. Numerous studies have demonstrated that because of the segmental nature and variable location of infarcts, M-mode, or even 2D imaging from a single plane (2D long-axis) is less accurate for measuring LV volumes than imaging multiple 2D planes.16 Previous studies in mice using M-mode analysis showed weak or absent correlation between infarct size measured anatomically and M-mode based measurements of LV volumes and infarct size.4,6 With M-mode echocardiography, the well-recognized inaccuracy of the cube formula for volume measurements is compounded by the fact that the standard placement of the M-mode line in the parasternal view does not include the typical infarct area in both basal-to-apical and septal-to-lateral directions. In our experience, infarcts of various sizes created in mice by LAD occlusion are typically located in the mid to apical anterolateral segments with sparing of mid to basal inferior and virtually all septal segments. Given the usual orientation of the heart in the mouse chest, M-mode echocardiograms traverse the left ventricle through the anteroseptal and inferoposterior segments. In fact, we often observed that small anterolateral infarcts were not visualized on the parasternal long-axis views. Two-dimensional echocardiography has been used extensively for quantitative assessment of infarct size.17-19 There is general agreement that quantitative analysis of regional wall motion from 2D echocardiograms consistently overestimates the extent of infarction evident on pathologic examination.19 Commonly cited explanations for this discrepancy include tethering of nonischemic myocardium adjacent to infarcted myocardium, delayed mechanical recovery of function in viable regions surrounding the infarct zone, and methodologic limitations in the quantification of regional dyssynergy. In contrast, our results indicate a lack of overestimation of infarct size when measurements are based on more accurate reconstruction of LV geometry. This finding is in agreement with the study by Yao et al20 who demonstrated in a dog model of acute MI that infarct mass measurement by volume-rendered 3D echocardiography correlated closely with actual infarct mass without significant overestimation or underestimation of infarct size. We concur with Yao et al on the likely explanation for the improved accuracy of these tech-
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niques. First, we used strict criteria for the quantification of regional dysfunction (only akinetic or dyskinetic segments were included). Second, the number of short-axis slices (5 to 7 per heart) of the mouse left ventricle with an average length of 6 mm allows a more detailed analysis of LV structure and infarct size than could be accomplished with 4 to 5 slices in larger hearts with an average length 20 times that of the mouse heart. Third, most of the infarcts in our study were transmural and associated with marked thinning of the infarcted segments and clear demarcation of the scar from the adjacent noninfarcted segments. A similar echocardiographic method for the evaluation of myocardial injury was recently reported by Scherrer-Crosbie et al13 who described acute changes in LV volumes and systolic function associated with ligation of the LAD in an open-chest mouse preparation. They observed an increase in end-diastolic and end-systolic LV volumes and a decrease in LV ejection fraction after coronary ligation and found a significant correlation between echocardiographyderived and Doppler flow probe-based measurements of cardiac output. They described the extent of segmental wall motion abnormalities but there was no histologic validation of the method. In a separate publication, Scherrer-Crosbie et al21 performed myocardial perfusion studies with the use of intravenously injected echocardiographic contrast agent in the same acute LAD occlusion preparation. They found a close correlation between the size of perfusion defect detected on the contrast images and the area-at-risk measured by postmortem dye perfusion technique. Dyssynergy of segmental wall motion overestimated the area-at-risk. Although we did not attempt to characterize the relationship between infarct size and area-at-risk in the current study, we have previously observed that myocardial contrast echocardiography is a highly effective technique for the noninvasive evaluation of myocardial perfusion under various pathologic conditions (infarct size and area-at-risk; coronary occlusion and reperfusion; coronary stenosis and myocardial ischemia; nonischemic wall motion abnormalities) (unpublished data). Taken together, these studies provide strong evidence for the usefulness of echocardiography in the comprehensive noninvasive evaluation of structural and functional consequences of MI. Limitations The major limitation of the study is that the image acquisition method is technically demanding and the analysis is labor intensive. This is partly because of the less than optimal visualization of the endocardial
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borders and partly because of the large number of manual tracings required for the analyses. Contrast echocardiography, automatic border detection, or 3D image acquisition may greatly improve this technique in the future. We thank Karen Green for excellent technical assistance. REFERENCES 1. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000;101:2981-8. 2. Lin MC, Rockman HA, Chien KR. Heart and lung disease in engineered mice. Nat Med 1995;1:749-51. 3. Li B, Li Q, Wang X, Jana KP, Redaelli G, Kajstura J, et al. Coronary constriction impairs cardiac function and induces myocardial damage and ventricular remodeling in mice. Am J Physiol 1997;273:H2508-19. 4. Patten RD, Aronovitz MJ, Deras-Mejia L, Pandian NG, Hanak GG, Smith JJ, et al. Ventricular remodeling in a mouse model of myocardial infarction. Am J Physiol 1998;274: H1812-20. 5. Michael LH, Ballantyne CM, Zachariah JP, Gould KE, Pocius JS, Taffet GE, et al. Myocardial infarction and remodeling in mice: effect of reperfusion. Am J Physiol 1999;277:H660-8. 6. Gao XM, Dart AM, Dewar E, Jennings G, Du XJ. Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice. Cardiovasc Res 2000;45:330-8. 7. Sam F, Sawyer DB, Chang DL, Eberli FR, Ngoy S, Jain M, et al. Progressive left ventricular remodeling and apoptosis late after myocardial infarction in mouse heart. Am J Physiol Heart Circ Physiol 2000;279:H422-8. 8. Lutgens E, Daemen MJ, de Muinck ED, Debets J, Leenders P, Smits JF. Chronic myocardial infarction in the mouse: cardiac structural and functional changes. Cardiovasc Res 1999;41:586-93. 9. Christensen G, Wang Y, Chien KR. Physiological assessment of complex cardiac phenotypes in genetically engineered mice. Am J Physiol 1997;272:H2513-24. 10. Kovacs A, Yee R, Da´ vila-Roma´ n VG, Barzilai B. Comparison of M-mode and 2D based measurements of LV geometry in mice [abstract]. J Am Soc Echocardiogr 1998;11:538. 11. Mor-Avi V, Korcarz C, Fentzke RC, Lin H, Leiden JM, Lang RM. Quantitative evaluation of left ventricular function in a transgenic mouse model of dilated cardiomyopathy with 2D contrast echocardiography. J Am Soc Echocardiogr 1999;12: 209-14. 12. Youn HJ, Rokosh G, Lester SJ, Simpson P, Schiller NB, Foster E. Two-dimensional echocardiography with a 15-MHz transducer is a promising alternative for in vivo measurement of left ventricular mass in mice. J Am Soc Echocardiogr 1999;12:70-5. 13. Scherrer-Crosbie M, Steudel W, Hunziker PR, Liel-Cohen N, Ullrich R, Zapol WM, et al. Three-dimensional echocardiographic assessment of left ventricular wall motion abnormalities in mouse myocardial infarction. J Am Soc Echocardiogr 1999;12:834-40. 14. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction: experimental observations and clinical implications. Circulation 1990;81:1161-72. 15. Pfeffer MA, Pfeffer JM. Ventricular enlargement and reduced
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survival after myocardial infarction. Circulation 1987; 75(suppl 4):IV93-7. 16. Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, Feigenbaum H, et al. Recommendations for quantitation of the left ventricle by 2D echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of 2D Echocardiograms. J Am Soc Echocardiogr 1989;2:358-67. 17. Weiss JL, Bulkley BH, Hutchins GM, Mason SJ. Two-dimensional echocardiographic recognition of myocardial injury in man: comparison with postmortem studies. Circulation 1981; 63:401-8. 18. Wyatt HL, Meerbaum S, Heng MK, Rit J, Gueret P, Corday E. Experimental evaluation of the extent of myocardial dys-
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