Echocardiographic assessment of LV hypertrophy and function in aortic-banded mice: necropsy validation

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1 Am J Physiol Heart Circ Physiol 282: H1703 H1708, First published December 13, 2001; /ajpheart Echocardiographic assessment of LV hypertrophy and function in aortic-banded mice: necropsy validation YULIN LIAO, 1 FUMINOBU ISHIKURA, 2 SHINTARO BEPPU, 2 MASANORI ASAKURA, 1 SEIJI TAKASHIMA, 1 HIROSHI ASANUMA, 1 SHOJI SANADA, 1 JIYOON KIM, 1 HISAKAZU OGITA, 1 TSUNEHIKO KUZUYA, 1 KOICHI NODE, 1 MASAFUMI KITAKAZE, 1 AND MASATSUGU HORI 1 1 Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine; and 2 Department of Echocardiography, School of Allied Health Science, Osaka University Faculty of Medicine, Osaka , Japan Received 24 March 2001; accepted in final form 11 December 2001 Liao, Yulin, Fuminobu Ishikura, Shintaro Beppu, Masanori Asakura, Seiji Takashima, Hiroshi Asanuma, Shoji Sanada, Jiyoon Kim, Hisakazu Ogita, Tsunehiko Kuzuya, Koichi Node, Masafumi Kitakaze, and Masatsugu Hori. Echocardiographic assessment of LV hypertrophy and function in aortic-banded mice: necropsy validation. Am J Physiol Heart Circ Physiol 282: H1703 H1708, First published December 13, 2001; /ajpheart We characterized the time course of the left ventricular (LV) geometric and functional changes after aortic banding, validated them by necropsy, and investigated the sensitivity of echocardiographic findings on LV hypertrophy. C57BL/6 mice were subjected to transverse aortic constriction (TAC) or sham operation; echocardiographic assessments were performed before or at 2, 4, 6, and 11 wk after surgery; and some of the mice were euthanized at the corresponding time points. There was a progressive increase in diastolic posterior wall thickness and LV systolic dimension; the percentage of LV fractional shortening (LV%FS) decreased progressively at 4 wk, whereas these parameters remained stable in sham-operated mice. Echo LV mass and LV%FS correlated well with actual whole heart mass and ratio of lung weight to body weight, respectively (r and 0.749, respectively; P ). These results suggest that the development of myocardial hypertrophy and systolic dysfunction is a time-dependent process. Echocardiographic assessment of myocardial hypertrophy and functional changes correlate well with the actual heart mass and lung mass. Echocardiography is sensitive enough to assess myocardial hypertrophy and heart functional changes induced by pressure overload in mice. myocardial; heart failure; left ventricular hypertrophy WITH THE ADVENT OF TRANSGENIC TECHNOLOGY, genetically altered mice with remarkable cardiovascular phenotypes have been commonly used in cardiovascular research, and it is an important approach to integrate a genetically engineered mouse with a pressure overload intervention to study the interplay of genes and pathophysiology of cardiac hypertrophy in vivo. However, the murine model of pressure overload by transverse aortic constriction (TAC) is not widely used because it is difficult to prepare the model in such a small animal; microsurgical techniques are thus required (3, 9, 11). Furthermore, it is critically important to develop approaches for accurate and reproducible measurements of cardiac morphology and function in the intact mice with pressure overload. Transthoracic echocardiography has been reported (5, 8, 14) as a reliable tool for monitoring the changes of cardiac geometry and function in vivo, but serial echocardiographic evaluation of myocardial hypertrophy in mice with TAC has not been extensively performed. Therefore, it is important to confirm the ability of echocardiography to distinguish between differences in severity of cardiac hypertrophy and myocardial function in TAC murine models. In the present study, we hypothesized that echocardiography is able to monitor the changes of left ventricular (LV) hypertrophy and has the sensitivity to distinguish between differences in severity of disease in TAC mice. To test this hypothesis, we established a murine model of TAC and assessed morphometric and functional characteristics with the use of echocardiography. Furthermore, we validated the results of echocardiographic heart mass by necropsy over a time course and checked the correlation between echocardiographic heart function and the lung weight-to-body weight ratio (LW/BW) to confirm the time point of developing heart failure. The sensitivity of echocardiography was determined by monitoring the changes of hypertrophy over time. METHODS TAC model. C57BL/6 male mice (9 10 wk, g) were anesthetized with a mixture of pentobarbital sodium (50 mg/kg ip) and ketamine (25 mg/kg ip). The animal was placed supine and endotracheal intubation was performed rapidly and safely, as described by Brown et al. (1). The cannula was connected to a volume-cycled rodent ventilator with a tidal Address for reprint requests and other correspondence: M. Kitakaze, Dept. of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka , Japan ( kitakaze@zfb.so-net.ne.jp). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /02 $5.00 Copyright 2002 the American Physiological Society H1703

2 H1704 volume of 0.5 ml room air and respiratory rate of 110 breaths/ min. The chest cavity was entered in the second intercostal space at the left upper sternal border through a small incision. With the help of a light source with two flexible fiberoptic arms, the thymus was then gently deflected out of the field of view to expose the aortic arch. After the transverse aorta was isolated between the carotid arteries, it was constricted by a 7-0 silk suture ligature tied firmly against a 27-gauge needle. The latter was promptly removed to yield a constriction of 0.4 mm in diameter. Sham-operated mice underwent a similar surgical procedure without constriction of the aorta. The chest was closed with a 5-0 silk suture, and mice were allowed to recover from anesthesia with light supply to keep the body temperature at 37 C. The surgical preparation was performed without the use of a microscope and was finished in 30 min. All procedures were performed in accordance with the guiding principles of Osaka University School of Medicine with regard to animal care and the Position of the American Heart Association on Research Animal Use. Hemodynamic measurement. Right or left carotid arteries (n 4), respectively, were isolated and cannulated with polyethylene (PE)-10 tubing under a microscope. The catheters were connected to a pressure transducer to measure system blood pressure. These mice were used to confirm the pressure gradient between the carotid arteries after TAC and were not included in experimental groups for echocardiography assessment because the carotid artery was occluded. Noninvasive blood pressure (BP) and heart rate (HR) were measured before and at 4 and 11 wk after surgery by the tail-cuff plethysmography method in unanesthetized mice prewarmed for 10 min at 37 C in a thermostatically controlled heating cabinet (model BP-98A, Softron). Two-dimensional guided M-mode echocardiography. Animals were lightly anesthetized with pentobarbital sodium (30 50 mg/kg ip). Two-dimensional (2-D) guided M-mode echocardiography in the mouse was performed using an echocardiogram (model 5500, Sonos) equipped with a 15- to 6L- MHz transducer (Hewlett-Packard). The heart was imaged in the 2D mode in the parasternal long-axis view with a depth setting of 2 cm. From this view, an M-mode cursor was positioned perpendicular to the interventricular septum and posterior wall of the LV at the level of the papillary muscles. An M-mode image was obtained at a sweep speed of 100 mm/s. Diastolic and systolic LV wall thickness, LV enddiastolic dimensions (LVDD), and LV end-systolic chamber dimensions (LVSD) were measured. All measurements were done from leading edge to leading edge according to the American Society of Echocardiography guidelines (8). The percentage of LV fractional shortening (LV%FS) was calculated as [(LVDD LVSD)/LVDD] 100. LV mass was calculated according to uncorrected cube assumptions with some modifications using the equation LV mass (mg) 1.055[(LVDD PWTD VSTD) 3 (LVDD) 3 ] (2, 10), where is the gravity of myocardium, PWTD is diastolic posterior wall thickness, and VSTD is diastolic ventricular septal thickness. Experimental protocol. Animals were randomly divided into TAC and sham-operation control groups. After the initial assessment using echocardiography, mice were subjected to TAC or sham operation. At 2, 4, 6, and 11 wk after surgery, echocardiography was performed and 4 13 mice from each group were euthanized to obtain body and organ weights. Statistical analysis. Values are expressed as means SE. Two-tailed Student s t-test was used to compare the differences between groups. The least-squares method was used for linear correlation between selected variables. A variable with skewed distribution was transformed with the use of the logarithm method. Significance was defined as P RESULTS Mortality. The mortality rate for TAC was 3.7% (7/188) during operation from anesthesia to chest closure, 10.5% (19/181) during the recovery period from anesthesia, and 19.13% (31/162) after recovery from anesthesia to 2 wk. Most of the postoperative deaths can be attributed to acute or subacute heart failure confirmed by significant increase of LW/BW. Ten mice with early deaths were validated by postmortem. The survival days, heart weight-to-bw ratio (HW/BW), and LW/BW were days, mg/g, and mg/g, respectively. The total mortality was 30.32% for TAC and 0% for sham-operated mice. Hemodynamics. The invasive systolic pressure gradient between the right and left carotid artery after TAC was 50.5 mmhg, which was in agreement with previous reports (4, 5). The results of tail-cuff BP and HR, lung weight, and percentage of congestive heart failure before and at 4 and 11 wk after the surgery are shown in Table 1. Congestive heart failure was defined as LW/BW 1.5 times of the mean of LW/BW in sham-operated mice. The lung weight and LW/BW increased markedly in the TAC mice at 4 wk compared with the sham-operated group and also showed a tendency of further increase at 11 wk in the TAC mice, in which no significant difference was found because of the relatively small sample size. Echocardiographic assessment. In vivo 2-D-guided M-mode echocardiograms were obtained in banded mice before and at 2, 4, 6, and 11 wk after TAC (Fig. 1). Table 1. Tail-cuff blood pressure and heart rate and lung weight Baseline (n 15) Sham, 4W (n 10) Sham, 11W (n 9) TAC, 4W (n 13) TAC, 11W (n 5) SBP, mmhg * DBP, mmhg MBP, mmhg HR, beats/min Lung wt, mg LW/BW, mg/g %CHF 0%(0/4) 0%(0/11) 0%(0/9) 46.2%(6/13)* 60.0%(3/5) Values are means SE; n, no. of animals in each group. TAC, transverse aortic constriction; W, week; SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; HR, heart rate; LW/BW, lung weight to body weight ratio; CHF, congestive heart failure. *P 0.05, P 0.01 vs. sham 4W. The number of mice used for lung wt and LW/BW was 4 in baseline and 11 in sham 4W group.

3 H1705 Fig. 1. Representative M-mode echocardiograms of mice before and after 2 11 wk of transverse aortic constriction (TAC), showing a progressive increase in left ventricular (LV) wall thickness and decline in the percentage of LV fractional shortening (LV%FS; systolic dimension increased from 4 wk). Diastolic posterior wall thickness (PWT) is indicated with white lines. As reported in Fig. 2, LV diastolic posterior wall thickness increased progressively from 2 to 11 wk after TAC relative to age-matched sham-operated mice (all P 0.01). Systolic posterior wall thickness increased significantly at 2 wk after TAC and then presented a plateau. LVSD and LVDD slightly but significantly decreased in 2 wk in the TAC mice; LVSD increased noticeably from 4 to 11 wk (P 0.05 or 0.01) whereas LVDD increased significantly only at 11 wk after banding compared with sham-operated mice, which indicates that systolic function was impaired at the early phase. LV%FS was depressed significantly in a time course similar to LVSD. Correlation of echocardiographic parameters and heart mass at necropsy. LV%FS correlated well with LVDD and LVSD (r and 0.918, respectively, both P , n 111), indicating that LV dilation is associated with deterioration of systolic function. In the present study, the depressed ventricular pumping was attributed mainly to the increased LVSD at several time points because the increase in LVSD was greater than that in LVDD. As shown in Fig. 3, the LV mass obtained by echocardiography significantly correlated with the actual heart mass at necropsy, and LV%FS correlated significantly with the LW/BW. Because the variable LW/BW in this study contained outlying values that may influence the value of correlation coefficient, we transformed this variable into a logarithm (LW/BW) and showed a similar result (r 0.750, P ). In addition, the time course of PWTD and HW/BW (Fig. 2), echo LV mass and actual whole heart mass (Fig. 4) were well coincided; Fig. 2. Time course of the changes in LV wall thickness, LV dimensions, FS, and the heart weight-to-body weight ratio (HW/BW). Number of mice analyzed is indicated above or under each data point in A and F; the number of mice in B E is the same as in A. PWTD, LV diastolic PWT; LVSD, LV end-systolic dimension; LVDD, LV end-diastolic dimension. Data are means SE. *P 0.05, **P 0.01 vs. sham-operated mice.

4 H1706 Fig. 3. Correlation between actual whole heart mass and echo-calculated LV mass (A), LV%FS and lung weight (LW)/BW ratio (B). meanwhile, no differences were found in BW between the TAC mice and sham-operated controls (Fig. 4A). Actual heart mass correlated significantly with the PWTD (r 0.717, P 0.01, n 77). The appearance of hypertrophied heart, congestive lung, and histological findings of myocardial hypertrophy are shown in Fig. 5. Sensitivity of echocardiography to monitor the changes of LV hypertrophy. To determine the ability of echocardiography to discriminate between differences in severity of cardiac hypertrophy, we investigated whether echocardiography could differentiate between mice with a 30% change in myocardial hypertrophy. As presented in Fig. 4, actual heart mass increased 30% between every two adjacent time points (Fig. 4B); the corresponding echo LV mass also significantly increased (Fig. 4C). In addition, we divided the 77 mice into 4 groups according to their actual heart mass and controlled the increase value not 30% between every two groups; the corresponding echo LV mass also showed a similar increase tendency (Fig. 6). In addition, we also examined the sensitivity of echocardiography to reflect the changes on cardiac function. Forty-one mice with postmortem data of LW and echocardiographic data of LV%FS were divided into three groups according to their LW/BW ratio; when logarithm of LW/BW increased from (n 31) to (n 4) (a 34% increase), the corresponding LV%FS significantly decreased from to %, a 26% decrease reached (P 0.01), whereas an increase of 15.1% of logarithm of LW/BW from to (n 6) did not cause a significant decrease in corresponding LV%FS (from to %, a 10.8% decline achieved, P 0.05). Therefore, we implied that LV%FS could detect a 30% change in logarithm of LW/BW. DISCUSSION Fig. 4. Graphs displaying a similar time course of BW between aorticbanded mice and sham-operated mice (A, the number of mice in each time point is the same as in C), and a similar changing tendency of echo-calculated LV mass and actual whole heart mass in both banded and sham-operated mice (B and C). Number of mice analyzed is indicated above the data points in B and C. Data are means SE. One of the main findings of this study is that the degree of myocardial hypertrophy assessed with echocardiography increased progressively along with the time of banding and was validated by necropsy at different time points. The tendencies of wall thickness and echo LV mass increasing are in accordance with that of the actual whole heart mass. Furthermore, we have checked the sensitivity of echocardiography to discriminate between severity of cardiac hypertrophy and myocardial function in this model and showed that echocardiography could differentiate between mice

5 H1707 Fig. 5. Representative photographs showing hypertrophied heart (right) and age-matched sham-operated mouse heart (left, top), histological findings of myocardium stained with hematoxylin and eosin (middle), and the congestive lung and normal lung in a banded mouse with chronic heart failure and a sham-operated mice (bottom), 11 wk after surgery. with a 30% change in LV hypertrophy and logarithm of (LW/BW). The ability to distinguish between differences in severity of cardiac hypertrophy and heart function will be important as attempts are made to manipulate dysfunction in these murine models. There are reports that showed that in ascending aortic constriction (3) and TAC (13) mice actual heart mass increased over time, but they did not analyze the relation between echocardiographic measurements and actual heart mass. A recent study (4) of nine ascending aortic-banded mice reported that echo LV mass peaked at 3 wk and presented a plateau thereafter until 8 wk but had no validation by postmortem examination at different time points. Hill et al. (7) validated echocardiographic calculated LV mass by necropsy in six mice of different ages (not aortic-banded mice), but they did not check the correlation between the findings by echocardiography and the postmortem results in TAC model at different time points. Therefore, our study maybethefirst to validate the echocardiographic measurements by necropsy on different time points with relatively long time course in TAC murine model. In the present study, significant depressed systolic function developed as early as 4 wk after the banding and gradually deteriorated over time, which was validated by pulmonary congestion. Gardin et al. (6) also reported that LV%FS inversely correlated with the ratio of lung mass to body mass in 15 mice (r 0.79), which is similar to our results. We also showed that heart function declined markedly from 4 wk, which was validated by pulmonary congestion. Meanwhile, LV%FS also decreased significantly from the 4 wk after TAC, and a similar tendency presented in LVSD, indicating that the impaired systolic function should be attributed largely to the increase in systolic dimension because the diastolic dimension did not increase at

6 H1708 Fig. 6. Sensitivity of echocardiography to assess cardiac hypertrophy. Four groups were divided according to heart mass, which increased 27 30% between group B and A, C and B, and D and C; the corresponding LV mass calculated from echocardiography also showed significant increases. The number of mice from group A to D is 25, 19, 21, and 12, respectively. Data are means SE. least before 7 wk after TAC. The systolic wall thickness did not increase progressively after 2 wk, also suggesting impaired systolic function. These findings imply that the impairment of systolic heart function can be developed before the occurrence of eccentric hypertrophy. The normal values of LV dimensions and wall thickness in mice have been reported differently in small sampling size (5, 8, 13 16). We obtained the normal values using a relatively large sample size (n 92), which is consistent with other studies (5, 14, 15). In conclusion, the present study provides strong evidence that the development of myocardial hypertrophy and systolic dysfunction in the murine model of TAC is a time-dependent process. Echocardiography is a reliable and sensitive means to study the process of LV remolding and functional changes induced by pressure overload. REFERENCES 1. Brown R, Walters DM, Greenberg RS, and Mitzner W. A method of endotracheal incubation and pulmonary functional assessment for repeated studied in mice. J Appl Physiol 87: , De Simone G, Wallerson DC, Volpe M, and Devereux RB. Echocardiographic measurement of left ventricular mass and volume in normotensive and hypertensive rats: necropsy validation. Am J Hypertens 3: , Ding B, Price RL, Goldsmith EC, Borg TK, Yan X, Douglas PS, Weinberg EO, Bartunek J, Thielen T, Didenko VV, and Lorell BH. Left ventricular hypertrophy in ascending aortic stenosis mice. Circulation 101: , Fard A, Wang CY, Takuma S, Skopicki HA, Pinsky DJ, Di Tullio MR, and Homma S. Noninvasive assessment and necropsy validation of changes in left ventricular mass in ascending aortic banded mice. J Am Soc Echocardiogr 13: , Gao XM, Dart AM, Dewar E, Jennings G, and Du XJ. Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice. Cardiovasc Res 45: , Gardin JM, Siri FM, Kitsis RN, Edwards JG, and Leinwand LA. Echocardiographic assessment of left ventricular mass and systolic function in mice. Circ Res 76: , Hill JA, Karimi M, Kutschke W, Davission RL, Zimmerman K, Wang Z, Kerber RE, and Weiss RM. Cardiac hypertrophy is not a required compensatory response to short-term pressure overload. Circulation 101: , Manning WJ, Wei JY, Katz SE, Litwin SE, and Douglas PS. In vivo assessment of LV mass in mice using high-frequency cardiac ultrasound: necropsy validation. Am J Physiol Heart Circ Physiol 266: H1672 H1675, Meguro T, Hong C, Asai K, Takagi G, McKinsey TA, Olson EN, and Vatner SF. Cyclosporine attenuates pressure-overload hypertrophy in mice while enhancing susceptibility to decompensation and heart failure. Circ Res 84: , Pollick C, Hale SL, and Kloner RA. Echocardiographic and cardiac Doppler assessment of mice. J Am Soc Echocardiogr 8: , Rockman HA, Ross RS, Harris AN, Knowlton KU, Steinhelper ME, Field LJ, Ross JRJ, and Chien KR. Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy. Proc Natl Acad Sci USA 88: , Sahn DJ, DeMaria A, Kisslo J, and Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements. Circulation 58: , Sakata Y, Hoit BD, Liggett SB, Walsh RA, and Dorn GW. Decompensation of pressure-overload hypertrophy in G q-overexpressing mice. Circulation 97: , Tanaka N, Dalton N, Mao L, Rockman HA, Peterson KL, Gottshall KR, Hunter JJ, Chien KR, and Ross J. Transthoracic echocardiography in models of cardiac disease in the mouse. Circulation 94: , Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE, and Carretero OA. Echocardiographic assessment of cardiac function in conscious and anesthetized mice. Am J Physiol Heart Circ Physiol 277: H1967 H1974, Youn HJ, Rokosh G, Lester SJ, Simpson P, Schiller NB, and 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 12: 70 75, 1999.