Vol. 280, Issue 5, H1954-H1962, May 2001
Accuracy of echocardiographic estimates of left ventricular
mass in mice
Keith A.
Collins1,
Claudia E.
Korcarz1,
Sanjeev
G.
Shroff2,
James E.
Bednarz1,
Richard C.
Fentzke1,
Hua
Lin1,
Jeffrey M.
Leiden1, and
Roberto M.
Lang1
1 Noninvasive Cardiac Imaging Laboratory, University of
Chicago, Chicago, Illinois 60637; and 2 Department of
Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania
15261
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ABSTRACT |
Genetically modified mice have created the need for
accurate noninvasive left ventricular mass (LVM) measurements. Recent technical advances provide two-dimensional images adequate for LVM
calculation using the area-length method, which in humans is more
accurate than M-mode methods. We compared the standard M-mode and
area-length methods in mice over a wide range of LV sizes and weights
(62-210 mg). Ninety-one CD-1 mice (38 normal, 44 aortic banded,
and 9 inherited dilated cardiomyopathy) were imaged transthoracically
(15 MHz linear transducer, 120 Hz). Compared with necropsy weights,
area-length measurements showed higher correlation than the M-mode
method (r = 0.92 vs. 0.81), increased accuracy
(bias ± SD: 1.4 ± 27.1% vs. 36.7 ± 51.6%), and
improved reproducibility. There was no significant difference between
end-systolic and end-diastolic estimates. The truncated ellipsoid
estimation produced results similar in accuracy to the area-length
method. Whereas current echocardiographic technology can accurately and reproducibly estimate LVM with the two-dimensional, area-length formula
in a variety of mouse models, additional technological improvements,
rather than refinement of geometric models, will likely improve the
accuracy of this methodology.
disease models; echocardiography; methods
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INTRODUCTION |
MURINE MODELS
have been increasingly used to study a variety of cardiovascular
disorders, such as dilated cardiomyopathy, left ventricular (LV)
hypertrophy, myocardial infarction, and hypertension (3, 4, 10,
20). These transgenic and surgically modified mouse models
require noninvasive imaging methods that would allow accurate
assessment of structural and functional cardiac changes. LV mass (LVM)
is a commonly used and important descriptor of cardiac status. Previous
studies (2, 12, 22) have examined the accuracy of M-mode
and two-dimensional LVM measurement methods in small cohorts of
predominantly normal mice.
Transthoracic two-dimensionally directed, M-mode echocardiography has
yielded estimates of mouse LVM with relatively good correlation
compared with necropsy values (6, 9, 20). However, M-mode
echocardiography is limited in that images are obtained in only one
plane, and thus LVM calculation is subject to greater error than
formulas derived from multiplanar images (6).
Two-dimensional echocardiography has also been limited by
low-frame rate-image acquisition relative to the high heart rate of the
mouse and also by inappropriate frequency transducers for near-field
imaging (12). Recent advances in echocardiographic technology enable better long- and short-axis views as well as more
accurate M-mode measurements (2, 22, 23). The development of high-frame rate imaging and increased probe frequency has
significantly enhanced murine imaging. Newly available probes also have
smaller footprints that are more appropriate to the animal size.
Finally, the creation of high-frequency linear transducers has improved near-field imaging, avoiding the use of acoustic standoffs with their
inherent problems.
In humans, two-dimensional, area-length-based estimates of LVM
have been shown to be more accurate than M-mode-based estimates (1, 17). We hypothesized this to be true in mice as well, particularly when taking advantage of enhanced imaging capabilities. Accordingly, we sought to compare the accuracy of known
echocardiographic formulas for estimating LVM in a large number of mice
relative to previous studies and to determine the sources of error in
these estimates. Specifically, we compared the necropsy LV weights with determined LVM, using M-mode (cubed formula) as well as two-dimensional (area-length and truncated ellipsoid formulas) measurements over a wide
range of LV sizes, cardiac geometries, and weights. Accordingly, our
study was designed to include a chronic model of LV hypertrophy induced
by aortic banding, a transgenic model of dilated cardiomyopathy, and
normal CD-1 mice for control.
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METHODS |
Animal preparation.
A total of 91 adult CD-1 mice of both sexes with a mean body weight of
32.5 g (range 16.5-57.4 g) were studied. This included 38 CD-1 normal mice, 44 aortic-banded mice, and 9 transgenic cAMP response
element binding protein (CREB)A133 mice (described below). Image quality was not a criterion for inclusion in this study. Anesthesia was induced by administering isoflurane in a closed chamber
at 5% (Ohmeda Fluotec 3, Matrx Medical; Orchard Park, NY) in 80% room
air-20% O2, followed by 0.5-2.0% isoflurane through a nose cone throughout the experiment. Animals were then secured to a
custom-made water bed in a shallow left lateral decubitus position to
facilitate ultrasound imaging. The bed was connected to a circulating
water bath set at 37°C to prevent hypothermia. Flat pieces of metal
secured to the water bed underneath the paws served as
electrocardiographic electrodes with Redux Crème
(Hewlett-Packard; Andover, MA) to enhance conduction. All procedures
were performed in accordance with the guidelines established by the
American Physiological Society and the Animal Care and Use Committee of the University of Chicago.
Two to three weeks before noninvasive imaging, 49 CD-1 normal mice
underwent aortic banding to induce LV hypertrophy. The mice were
anesthetized, intubated with an 18-gauge angiocatheter, and ventilated
with 1% isoflurane mixed with O2 at 130-150
breaths/min with a 0.8-1.2 ml tidal volume. After a midsternotomy
was performed along the upper third of the sternum, the ascending aorta
was isolated and constricted at midarch with a 7-0 nylon suture to the
size of a 27-gauge needle placed along the vessel. Two weeks after
banding, the aortic pressure gradient was assessed by acquiring Doppler
velocities in the ascending and descending aorta. If the pressure
gradient failed to exceed 15 mmHg, the animal was not included in the
study. Animals that prematurely died before acquiring all images
necessary for comparative analysis were also discarded from the study
for a final count of 44 mice for this subgroup.
The transgenic mice (CREBA133) were generated and
genetically confirmed, as previously described in detail
(3). Briefly, transgenic mice were produced that over
express a dominant-negative form of CREB (CREBA133) under
the transcriptional control of the cardiac-specific 
-myosin heavy
chain promoter. Presence of the CREBA133 transgene was
confirmed by Southern blot analysis of purified tail DNA using the Fast
Track 2.0 kit (Invitrogen; San Diego, CA) in accordance with the
manufacturer's instructions described previously (3).
Although the adult CREBA133 mice were over 16 wk old and
already displayed phenotypic characteristics of congestive heart
failure, such as marked lung congestion and peripheral edema, changes
in cardiac dimensions and function were not necessarily maximal at the
time of imaging.
After imaging was complete, mice were immediately euthanized and their
hearts removed. The left ventricles were carefully isolated by trimming
the atria, the valves, and the right ventricular wall, and then blotted
of excess fluid and weighed (Denver XP-1500 portable balance).
Data acquisition.
Cardiac ultrasound imaging was performed using a high-frequency 15-MHz
linear transducer (Sonos 5500, Agilent; Andover, MA) at a frame rate of
120 frames/s. Parasternal long- and short-axis views were obtained
after adjusting gain settings for optimal epicardial and endocardial
wall visualization. Two-dimensional echocardiographic loops of at least
20 cardiac cycles and M-mode tracings were stored digitally on
magneto-optical disk for offline analysis.
Measurements.
From the short-axis view, epicardial and endocardial LV areas were
measured offline at end systole and end diastole. Images were
considered adequate for measurement when >75% of the epicardial and
endocardial contour could be adequately visualized. In accordance with
the American Society of Echocardiography recommendation, the
short-axis endocardial border was traced on the innermost endocardial
edge, and the epicardial border was traced along the first bright pixel
immediately adjacent to the darker myocardium (Fig.
1). The LV length, defined as the
distance between the apex and the midmitral annulus, was obtained from
parasternal long-axis views in which the mitral annular plane and the
apex were well defined. LV measurements were made from at least three
cardiac cycles at both end systole and end diastole.
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Fig. 1.
Representative echocardiograms and measurements from the left
ventricle of a normal CD-1 mouse. The two-dimensional parasternal
short-axis image (top, left) and long-axis image
(top, right) are shown at end diastole. The
two-dimensional guided M-mode image (bottom) was obtained at
the level of the papillary muscles. T, myocardial wall
thickness; L, left ventricular (LV) length; a,
full major radius; b, minor axis radius; d,
truncated major radius; IVS, interventricular septal thickness; LVID,
LV internal diameter; LVPW, LV posterior wall thickness.
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Two-dimensionally targeted M-mode echocardiographic images were
obtained at the level of the papillary muscles from the parasternal short-axis view and recorded at a speed of 150 cm/s. LV internal diameters and wall thicknesses (leading edge to trailing edge) were
obtained at end systole and end diastole from cross-sectional short-axis views. Heart rate was measured and shortening fraction was
calculated from the M-mode tracing. For both two-dimensional and M-mode
methods, LV end-diastolic measurements were obtained at the peak of the
R wave, whereas end-systolic measurements were obtained at the time of
minimal chamber area.
LVM was calculated using the following three formulas. The first
formula is the two-dimensional, area-length method: LVM = 1.05 [(5/6) A1(L + T)
(5/6) A2L], where 1.05 is the
specific gravity of muscle, A1 and
A2 are the epicardial and endocardial parasternal short-axis area, respectively, L is the
parasternal long-axis length, and T is the wall thickness
calculated from A1 and A2
(13, 17). The second formula consists of the M-mode (cubic) method: LVM = 1.05 [(IVS + LVID + LVPW)3 (LVID)3], where IVS and LVPW
are the interventricular septal and posterior wall thickness,
respectively, and LVID is the LV internal diameter (1).
The third formula is the truncated ellipsoid method: LVM = 
[(b + T)2 {2/3
(a + T) + d d3/[ 3 (a + T)2]} b2 (2/3
a + d d3/
3a2)], where b is the minor axis
radius of the LV measured at the level of the papillary muscle tip. Its
placement determines the division of the measured LV length
(L) into a full major radius (a) and a
truncated major radius (d). The average T is
calculated from A1 and A2
(18). This analysis was performed in a subgroup of 31 mice, representing the entire spectrum of LVM.
An experienced reader performed all measurements. A subgroup of 30 mice
representing the entire spectrum of LVM was remeasured by the first
reader (C) and by a blinded second reader (J). Intraobserver variability was calculated as (C1 C2) /[(C1 ± C2)/2], where C1 and C2 are the two measurements performed by
the first reader from the same images on different days. Similarly,
interobserver variabilities were calculated as (J1 C1) /[(J1 + C1)/2], where J1 represents the second observer's measurements.
Statistical analysis.
All data are presented as means ± SD. Intergroup
echocardiographic estimates of LVM at end systole and end diastole were
compared by two-way ANOVA. Linear regression analyses were used to
compare LVM estimates with true LV weights determined at necropsy. With the use of the Bland-Altman analysis, the agreement between
echocardiographic LVM and necropsy weight was calculated as the mean
(bias) ± 2 SD (error) of the differences between
echocardiographic and necropsy LVM, and the bias and error for each
method were determined. A P 
0.05 was considered
statistically significant. Sample size computations were also performed
for both M-mode and two-dimensional, area-length methods to determine
the number of animals required to detect significant 15% difference of
the mean LV weight between two or three groups of mice, with a
statistical power of 
0.8 and an of 0.05. The computation assumes
that samples are taken from populations normally distributed with equal
variance and is calculated using the SD of the end-diastolic LVM
measured by either method for the normal and aortic-banded mice groups.
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RESULTS |
Calculation of LVM by the area-length method was possible in 90/91
(99%) mice at end diastole and 89/91 (98%) at end systole. Two-dimensionally targeted M-mode images of the left ventricle were
technically adequate in 83/91 (91%) and 84/91 (92%) mice for
end-diastolic and end-systolic LVM measurements, respectively. The
truncated ellipsoid formula was used in a subgroup of animals (n = 31) due to the difficulties in identifying the
epicardial boundary in the long-axis view.
Measured chamber dimensions as well as heart rates and shortening
fractions for all subgroups are presented in Table
1. Statistically significant increases in
epicardial and endocardial areas, LV length, and posterior wall
thickness were noted at end diastole in aortic-banded mice, compared
with normals. CREBA133 mice had significantly larger cavity
sizes with a tendency toward decreased wall thickness, compared with
normal CD-1 mice. Because of the small sample size of the transgenic
subgroup no significant differences were noted in either heart rate or
shortening fraction.
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Table 1.
Echocardiographic measurements of left ventricular dimensions for
normal CD-1, aortic-banded, and transgenic
CREBA133 mice
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End-diastolic and end-systolic echocardiography-based estimates of LVM
and necropsy weights are presented in Table
2. The LV weights measured at necropsy
for both aortic-banded and CREBA133 mice were significantly
higher than the normal CD-1 mice. The use of the CREBA133
and the aortic-banded mice provided a wide range of LVM (62-210
mg) and end-diastolic chamber dimensions (3.1-5.5 mm). Peripheral
edema characteristic of CREBA133 mice resulted in
significantly higher body weight compared with normal controls
(43.7 ± 5.8 vs. 29.2 ± 8.8 g, P < 0.0001). Thus measurements indexed to body mass are not shown for
comparison between groups.
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Table 2.
Body weight and necropsy- and echocardiography-derived LVM for the
three subgroups and all animals combined
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As seen in Fig. 2A, a good
correlation existed between LVM determined by the area-length method at
end diastole and necropsy LV weight for all mice (y = 0.97x + 3.89; r = 0.91, standard error of estimate (SEE) = 14.1 mg, P < 0.0001). This
correlation was stronger than that of the M-mode calculation of
diastolic LVM and necropsy LV weight (y = 1.15x + 21.63; r = 0.81, SEE = 26.3 mg, P < 0.0001; Fig. 2C). Likewise,
LVM measured at end systole using the area-length method more strongly
correlated with necropsy LV weights than systolic M-mode LVM
(slope = 0.98 vs. 1.15 and r = 0.90 vs. 0.85, respectively; Fig. 2).
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Fig. 2.
Linear regression analyses comparing necropsy LV weights with LV
mass (LVM) calculated by the area-length (AL) method (A and
C) or by the two-dimensionally guided M-mode method (M-mode
LVM, B and D) at end diastole (top) or
end systole (bottom). For the AL and the M-mode methods,
standard error of estimate (SEE) = 14.1 and 26.3 mg, respectively,
at end diastole, and at end systole, SEE = 14.7 and 22.9 mg,
respectively, P < 0.0001. Closed circles, normal CD-1
mice; open circles, transgenic mice; closed triangles, aortic-banded
mice.
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The Bland-Altman analysis was used to compare the echocardiographic
estimates of LV mass with the necropsy LV weights (Fig. 3). The bias (mean difference) between
LVM calculated by the two-dimensional, area-length method in diastole
and necropsy LV weights was significantly smaller than that obtained
using the M-mode method (1.0 vs. 38.4 mg or 1.4% vs. 36.7%,
P < 0.0001; Fig. 3, A and B).
Similarly, the error, defined as 2 SD above and below the mean
difference between the echocardiographic LVM estimate and necropsy LV
weight, was smaller for the two-dimensional, area-length method
compared with the M-mode method (28.0 vs. 53.2 mg or 27.0% vs. 51.6%,
P < 0.0001; Fig. 3, A and B).
Results obtained using data from end systole are also presented in Fig.
3. The biases for both the area-length and M-mode methods were similar
when measured at end diastole or end systole (mean: 1.4% vs. 1.7%,
area length, and 36.7% vs. 23.0%, M mode, respectively). Similarly,
the errors did not differ between end diastole and end systole (27.0%
vs. 28.6%, for two-dimensional, area-length, and 51.6% vs. 42.1%, for M mode, respectively).
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Fig. 3.
Bland-Altman analyses showing the agreement as a percent difference
between necropsy LV weight and LVM calculated by the AL method
(A and C) or by the two-dimensionally guided
M-mode method (B and D) at end diastole
(top) and end systole (bottom). Horizontal
reference lines are zero percent difference (bold), the bias (center
thin line) and 2 SD above and below the bias (outlying thin lines).
Bias ± 2 SD for AL method were 1.4 ± 27.0% vs. 36.7 ± 51.6% for the M-mode method at end diastole and 1.7 ± 28.6%
vs. 23.0 ± 42.1%, respectively, at end systole.
n = 90, 89, 83, and 84 for A, B,
C, and D, respectively. Closed circles, normal
CD-1 mice; open circles, transgenic mice; and closed triangles,
aortic-banded mice.
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In a subgroup of 31 mice, spanning the full range of LVM, a further
estimation of LVM by the truncated ellipsoid method was performed
(Table 3). On average, only the
area-length-derived LV estimates did not significantly differ from LV
necropsy weights; M-mode and truncated ellipsoid methods overestimated
and underestimated LVM, respectively. In linear regression analyses
comparing estimated LVM to necropsy values, the correlation
coefficients of the area-length (r = 0.94) and
truncated ellipsoid methods (r = 0.94) were both greater than the M-mode correlation coefficient (r = 0.88, P < 0.0001). The bias and error values derived
in Bland-Altman analyses for area-length and truncated ellipsoid
methods were also nearly identical, and both were significantly lower
than the corresponding M-mode values (Table 3).
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Table 3.
Comparison of necropsy- and LVM derived by two-dimensional,
area-length, M-mode, and truncated ellipsoid methods
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Sample size computations on the basis of the SDs of the normal CD-1
mice (Table 2) showed that to detect statistically significant differences (power 0.8, of 0.05) of 15% of the mean LV weight between two or three groups of mice, the area-length method would require 20 or 25 mice, respectively, whereas the M-mode method would
require 49 or 60 mice. With the use of the larger SD of the banded mice
(Table 2), sample size computation showed that both methods would
require larger groups (62 or 77 mice for area-length, and 117 or 146 mice for M mode for two or three groups, respectively).
Although our study population was composed of a wide range of LV sizes,
shapes, and weights, a single geometric construct was used to obtain
two-dimensional, area-length-based LVM estimates (half ellipsoid and
half cylinder) (13, 17). With the use of multiple linear
regression analysis, we assessed whether differences in end-diastolic
size [parasternal short-axis chamber diameter, i.e., LV internal
diameter (LVID); parasternal long-axis length (L)], shape
(LVID/L), and relative hypertrophy (parasternal
short-axis thickness-to-radius ratio, 2T/LVID) could
explain the error values (two-dimensional, area-length estimate of
LVM necropsy LV weight). This analysis was performed for each
group of animals separately and for all animals combined. As seen in
the APPENDIX, there was no difference in the error pattern
among the three groups (normal, banded, and transgenic). Two covariates
(2T/LVID and L) were identified as being
significant, with the following regression equation: %Error = 5.7 + 6.5 (2T/LVID) + 4.1 (L);
r = 0.46; P < 0.0001. Because both
slope coefficients are positive, two-dimensional, area-length method
overestimates LVM at high values of 2T/LVID (relative
hypertrophy) and/or L (length) (APPENDIX). This
regression relationship, although significant, could explain only 21%
of the total variation in error values.
Intraobserver variability for both the area-length method and the M
mode-based calculations was small (9.1 ± 8.7% vs.
11.9 ± 13.4%, respectively, end diastole, Fig.
4). No differences in intraobserver
variabilities were noted when measuring at end systole versus end
diastole. Interobserver variability was significantly larger with
M-mode-based measurements than those obtained in area-length-based calculations at end diastole (20.9 ± 15.9% vs. 6.1 ± 6.3%, P < 0.0001) and at end systole (17.4 ± 15.4% vs. 8.7 ± 6.5%, P < 0.05).
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Fig. 4.
Percent intraobserver (A) and interobserver
variabilities (B) for LVM calculated using the AL or the
M-mode method. Variability, defined as percent error between the
measured LVM and necropsy weight, is shown for LVM calculated at end
diastole (solid bars) and at end systole (open bars). Error bars denote
means ± SD. *P < 0.05; and **P < 0.0001, compared with the AL method, at end systole and end
diastole, respectively.
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DISCUSSION |
Genetic modifications are frequently used to produce mice models
to investigate the molecular basis of cardiac growth and development
(3, 4, 7). These technologies have led to a proliferation
of transgenic and knockout mice models displaying a variety of
cardiovascular phenotypes. To noninvasively study these phenotypes in a
serial manner, it is necessary to develop accurate and reproducible
measurements of cardiac morphology and function. This study evaluated
the accuracy and reproducibility of echocardiographic M-mode and
two-dimensional, area-length estimates of LVM in mice over a wide range
of chamber sizes and wall thicknesses. Our main findings were the
following: 1) the M-mode method systematically overestimated
LVM; 2) mass estimates by the two-dimensional, area-length method were better than those obtained by the M-mode method
(significantly reduced bias and error and higher correlation); and
3) although the two-dimensional, area-length method yielded
mass estimates that correlated highly with necropsy weights
(r = 0.90) and had insignificant bias (<2%), the
error of individual estimates was ~25% (from Bland-Altman analysis).
Attempts to reduce this inaccuracy by using a more realistic geometric
model (truncated ellipsoid) or by covariance analysis of individual
errors were unsuccessful.
M-mode estimates of LVM.
Although transthoracic M-mode echocardiography has been widely used for
the assessment of LVM in a variety of animal models (8, 11,
19), it has important limitations that originate from the
unidimensional nature of this technique. In our study, M-mode LVM
measurements significantly overestimated necropsy weights in most cases
(Table 2). Early studies using M-mode echocardiography in mice
estimated LVM using the parasternal long-axis view (7, 12), resulting in frequently overestimated measurements, most likely due to the ultrasound beam not being perpendicular to the parasternal long-axis plane. Even with the use of two-dimensional guidance from the parasternal short-axis view at the level of the
papillary muscles (2), investigators have reported either overestimation (9, 20) or underestimation of LVM
(6), compared with necropsy values.
Overestimation of M-mode mass measurements might also be due to the
severe geometric constraints imposed by the cubed formula: both
endocardial and epicardial surfaces are full ellipsoids, each having a
long-to-short axis ratio of 2:1. In our study, the long-to-short axis
ratio was <2.0 (1.75 ± 0.17, end diastole), consistent with a
systematic overestimation of LVM. Similarly, Youn et al.
(23) recently found a long-to-short axis ratio <2:1 in 10 normal mice. The assumption of equal long-to-short axis ratio for
endocardial and epicardial surfaces results in increased wall thickness
at the apex, inconsistent with the mouse LV morphology. This erroneous
assumption also contributes to the overestimation of LVM.
Two-dimensional, area-length estimates of LVM.
On the basis of previous comparative human and animal studies (7,
9, 17, 23), we anticipated that the two-dimensional, area-length
method, in which the LVM is assumed to be bullet-shaped (half ellipsoid
with a cylinder on the top), might be more accurate for determining LVM
in murine hearts of various sizes and shapes. In this study, the LVM
estimations by the two-dimensional, area-length method correlated more
closely to necropsy weights than the M-mode calculations. In addition,
compared with M-mode estimates, linear regression analysis of
area-length LVM produced slopes much closer to one and intercepts
closer to zero. Although the transgenic animal group was too small to
make definitive conclusions specifically for this subgroup, our study
provides data on a large number of animals over a wide range of LVM
sizes, and the results are consistent with recent findings obtained in
a small number of normal (23) and hypertrophied mice
(2). Importantly, the bias for the two-dimensional, area-length method was significantly smaller than that of the M-mode measurements.
We hypothesized that because the endocardium of the interventricular
septum and posterior walls is usually best visualized at end systole,
wall thickness measurements performed at this time in the cardiac cycle
could possibly result in improved accuracy. Moreover, any error in
measuring the larger end-systolic thicknesses would be a smaller
proportion of that measurement. However, irrespective of the method,
LVM estimates were similar at end systole and end diastole.
In the present study, two-dimensional, area-length-based mass estimates
were highly reproducible, as reflected by intraobserver variabilities
around 10% and even lower interobserver variabilities, which were also
less than one-half those of M-mode-based values. The M-mode variability
for LVM estimation between readers in our study was similar to that
found by Hoit et al. (7) and Tanaka et al.
(20) who found an attributable error of 25-30%,
predominantly as a result of wall thickness measurement error.
We suspected that recent technological advances contributed to the
superior performance of the two-dimensional, area-length method. To
obtain high-quality echocardiographic images in mice, it is necessary
to use high-frequency ultrasound transducers that have higher spatial
resolution than predecessor probes and are expected to result in
reduced errors (17). The linear probe used in this study
has the additional advantage of not requiring a standoff, which by
itself may become a source of error (4). Finally, the
higher frame rate obtained with this linear transducer (~120 Hz)
allowed increased sampling throughout the cardiac cycle, which is
critical in small animals with high heart rates. As our results show,
these technological improvements provided an image quality suitable for
LVM measurements in nearly all mice.
Attempts to improve the two-dimensional area-length estimates of
LVM.
Despite the fact that the two-dimensional, area-length method was more
accurate than the M-mode method in a variety of mouse models,
significant individual errors were still noted between necropsy weights
and estimated LVM. We examined whether individual errors could be
improved using two approaches. First, to allow more geometric
flexibility, the truncated ellipsoid formula, which places the minor
axis more basally, was applied. We found this method underestimated LVM
and did not improve error values significantly (Table 3) thus providing
no obvious advantage over the two-dimensional, area-length method and
having the disadvantage of more complex data acquisition. Second,
multiple linear regression was performed to identify covariates that
could account for observed errors. Although we identified two
significant covariates, they accounted for only 21% of the total
variance in the error. In brief, both approaches to improve the
performance of the two-dimensional, area-length method did not yield
positive results, suggesting that the errors originate from basic
measurement inaccuracies. Indeed, the theoretical analysis, described
in detail in the APPENDIX, predicted an error of 20% due
to measurement inaccuracy, in agreement with the errors we obtained.
Implications for future studies.
On the basis of the above analysis of sources of error, the major
implication of our results is that further improvement in accuracy
would come from improvements in measurement instrumentation. Furthermore, our sample size calculations indicate that the lower levels of variability of the area-length method provide an additional advantage over the M-mode method, such that future studies would require smaller animal groups to detect significant differences. However, the specific group sizes should be interpreted cautiously and
adjusted accordingly when the effects of surgical or genetic treatment
on LVM may not be uniform in all animals, resulting in
larger-than-natural variance.
Alternative methods.
Noninvasive assessment of LVM, as well as cardiac function, can
certainly be accomplished in mice by other means. Magnetic resonance
imaging and ultrafast computed tomography scanning offer accurate
estimation of LVM (5, 14, 21), but their use may be
limited by expense and availability. Three-dimensional
echocardiography, a fervent goal of cardiac imaging, offers a
theoretical potential for assessing hearts of widely varying sizes and
pathologies (16). Transesophageal imaging of the murine
heart with an intracardiac probe has been used but is limited by
low-frame rates (15).
The present study, performed on a large number of mice with a wide
range of chamber sizes, demonstrates that transthoracic echocardiographic imaging of the murine heart, using improved transducer technology, can be used to accurately and reproducibly estimate LVM with the two-dimensional, area-length formula. This methodology will allow serial assessment of cardiac phenotypic changes
in a variety of mouse models. Our results suggest that even in animals
with uniform ventricular geometry, the use of the two-dimensional,
area-length technique should be positively considered in view of its
increased accuracy, markedly decreased intra- and interobserver
variabilities as well as potentially smaller sample sizes necessary to
demonstrate differences between groups. Further improvements in
accuracy are likely to come from modification in instrumentation rather
than from refinement of geometric models.
| |
APPENDIX |
Predicted Uncertainty in LV Mass Estimates Due to Measurement
Inaccuracies: A Theoretical Analysis
The two-dimensional, area-length formula (see text) uses three
measured variables to calculate the LV mass (LVM) estimate: chamber
length (L), epicardial short-axis area
(A1), and endocardial short-axis area
(A2). Thus the measurement inaccuracy in each of
these measurements will contribute to the uncertainty in LVM estimate.
As a first approximation, assume that the short-axis areas
(A1 and A2) are related
to linear dimensions (D1 and
D2) via a circular geometry (i.e.,
A1 = D
/4 and A2 = D
/4). With this assumption, the computed wall thickness (T), is equal to
(D1 D2)/2. The
two-dimensional, area-length formula can now be written as follows
|
(A1)
|
Algebraic simplification of Eq. A1 yields
|
(A2)
|
LVM is a function of three variables (D1,
D2, and L), and therefore the
predicted uncertainty in LVM (
LVM) can be expressed in terms of the
measurement inaccuracies of D1,
D2, and L
(D1, D2, and
L, respectively) as follows
|
(A3)
|
where | | denotes absolute value and
LVM/D1,
LVM/D2, and LVM/L are
partial derivatives given by (from Eq. A2):
|
(A4)
|
|
(A5)
|
|
(A6)
|
With the use of Eqs. A4-A5, one can calculate
the partial derivatives at nominal values of L,
A1 (equivalently, D1),
and A2 (equivalently, D2)
(Table 1). These derivative values can then be substituted in Eq. A3 to obtain the uncertainty in LVM. Typically, the lateral resolution in a two-dimensional image is much lower than the axial resolution, resulting in larger uncertainties in
D1 and D2 (which are
calculated from A1 and
A2) than that in L. However, as a
first approximation, we assume that uncertainties in all three
variables are the same and equal to the axial resolution corresponding
to the imaging frequency (D1 = D2 = L = axial
resolution). This will yield an optimistic estimate of LVM uncertainty;
the real value will be greater.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: R. M. Lang and S. G. Shroff, Univ. of Chicago Medical Center, M.C. 5084, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail:
rlang{at}medicine.bsd.uchicago.edu and sshroff{at}pitt.edu).
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.
Received 25 August 2000; accepted in final form 27 November 2000.
| |
REFERENCES |
1.
Devereux, RB,
Alonso DR,
Lutas EM,
Gottlieb GJ,
Campo E,
Sachs I,
and
Reichek N.
Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings.
Am J Cardiol
57:
450-458,
1986[ISI][Medline].
2.
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:
582-587,
2000[ISI][Medline].
3.
Fentzke, RC,
Korcarz CE,
Lang RM,
Lin H,
and
Leiden JM.
Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart.
J Clin Invest
101:
2415-2426,
1998[ISI][Medline].
4.
Fentzke, RC,
Korcarz CE,
Shroff SG,
Lin H,
Sandelski J,
Leiden JM,
and
Lang RM.
Evaluation of ventricular and arterial hemodynamics in anesthetized closed-chest mice.
J Am Soc Echocardiogr
10:
915-925,
1997[ISI][Medline].
5.
Franco, F,
Dubois SK,
Peshock RM,
and
Shohet RV.
Magnetic resonance imaging accurately estimates LVM in a transgenic mouse model of cardiac hypertrophy.
Am J Physiol Heart Circ Physiol
274:
H679-H683,
1998[Abstract/Free Full Text].
6.
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:
907-914,
1995[Abstract/Free Full Text].
7.
Hoit, BD,
Khoury SF,
Kranias EG,
Ball N,
and
Walsh RA.
In vivo echocardiographic detection of enhanced left ventricular function in gene-targeted mice with phospholamban deficiency.
Circ Res
77:
632-637,
1995[Abstract/Free Full Text].
8.
Magid, NM,
Young MS,
Wallerson DC,
Goldweit RS,
Carter JN,
Devereux RB,
and
Borer JS.
Hypertrophic and functional response to experimental chronic aortic regurgitation.
J Mol Cell Cardiol
20:
239-246,
1988[ISI][Medline].
9.
Manning, WJ,
Wei JY,
Katz SE,
Litwin SE,
and
Douglas PS.
In vivo assessment of LVM in mice using high-frequency cardiac ultrasound: necropsy validation.
Am J Physiol Heart Circ Physiol
266:
H1672-H1675,
1994[Abstract/Free Full Text].
10.
Patten, RD,
Aronovitz MJ,
Deras-Mejia L,
Pandian NG,
Hanak GG,
Smith JJ,
Mendelsohn ME,
and
Konstam MA.
Ventricular remodeling in a mouse model of myocardial infarction.
Am J Physiol Heart Circ Physiol
274:
H1812-H1820,
1998[Abstract/Free Full Text].
11.
Pollack, PS,
Bailey BA,
Budjak R,
Fernandez E,
and
Houser SR.
Progressive feline pressure-overload: noninvasive assessment correlates with abnormalities in single cells.
Am J Physiol Heart Circ Physiol
264:
H1307-H1314,
1993[Abstract/Free Full Text].
12.
Pollick, C,
Hale SL,
and
Kloner RA.
Echocardiographic and cardiac Doppler assessment of mice.
J Am Soc Echocardiogr
8:
602-610,
1995[Medline].
13.
Reichek, N,
Helak J,
Plappert T,
Sutton MS,
and
Weber KT.
Anatomic validation of left ventricular mass estimates from clinical two-dimensional echocardiography: initial results.
Circulation
67:
348-352,
1983[Abstract/Free Full Text].
14.
Ruff, J,
Wiesmann F,
Hiller KH,
Voll S,
von Kienlin M,
Bauer WR,
Rommel E,
Neubauer S,
and
Haase A.
Magnetic resonance microimaging for noninvasive quantification of myocardial function and mass in the mouse.
Magn Reson Med
40:
43-48,
1998[ISI][Medline].
15.
Scherrer-Crosbie, M,
Steudel W,
Hunziker PR,
Foster GP,
Garrido L,
Liel-Cohen N,
Zapol WM,
and
Picard MH.
Determination of right ventricular structure and function in normoxic and hypoxic mice: a transesophageal echocardiographic study.
Circulation
98:
1015-1021,
1998[Abstract/Free Full Text].
16.
Scherrer-Crosbie, M,
Steudel W,
Hunziker PR,
Liel-Cohen N,
Ullrich R,
Zapol WM,
and
Picard MH.
Three-dimensional echocardiographic assessment of left ventricular wall motion abnormalities in mouse myocardial infarction.
J Am Soc Echocardiogr
12:
834-840,
1999[ISI][Medline].
17.
Schiller, NB,
Shah PM,
Crawford M,
DeMaria A,
Devereux R,
Feigenbaum H,
Gutgesell H,
Reichek N,
Sahn D,
and
Schnittger I.
Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms.
J Am Soc Echocardiogr
2:
358-367,
1989[Medline].
18.
Schiller, NB,
Skioldebrand CG,
Schiller EJ,
Mavroudis CC,
Silverman NH,
Rahimtoola SH,
and
Lipton MJ.
Canine left ventricular mass estimation by two-dimensional echocardiography.
Circulation
68:
210-216,
1983[Abstract/Free Full Text].
19.
Schwarz, ER,
Pollick C,
Dow J,
Patterson M,
Birnbaum Y,
and
Kloner RA.
A small animal model of nonischemic cardiomyopathy and its evaluation by transthoracic echocardiography.
Cardiovasc Res
39:
216-223,
1998[Abstract/Free Full Text].
20.
Tanaka, N,
Dalton N,
Mao L,
Rockman HA,
Peterson KL,
Gottshall KR,
Hunter JJ,
Chien KR,
and
Ross J, Jr.
Transthoracic echocardiography in models of cardiac disease in the mouse.
Circulation
94:
1109-1117,
1996[Abstract/Free Full Text].
21.
Wiesmann, F,
Ruff J,
Hiller KH,
Rommel E,
Haase A,
and
Neubauer S.
Developmental changes of cardiac function and mass assessed with MRI in neonatal, juvenile, and adult mice.
Am J Physiol Heart Circ Physiol
278:
H652-H657,
2000[Abstract/Free Full Text].
22.
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,
1999[Abstract/Free Full Text].
23.
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[ISI][Medline].
Am J Physiol Heart Circ Physiol 280(5):H1954-H1962
0363-6135/01 $5.00
Copyright © 2001 the American Physiological Society
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ABSTRACT
INTRODUCTION
