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Am J Physiol Heart Circ Physiol
279: H2456–H2463, 2000.
Preserved ventricular contractility in infarcted mouse
heart overexpressing ␤2-adrenergic receptors
XIAO-JUN DU, XIAO-MING GAO, GARRY L. JENNINGS,
ANTHONY M. DART, AND ELIZABETH A. WOODCOCK
Baker Medical Research Institute and Alfred Heart Centre,
Alfred Hospital, Melbourne 8008, Victoria, Australia
Received 8 March 2000; accepted in final form 2 June 2000
echocardiography; heart failure; hemodynamics; transgenic
mice
rimental in the development of HF remains controversial.
Clinical studies provide evidence that excessive
␤-adrenergic activity is detrimental in the heart with
compromised structure and function (5). Long-term
␤-blockade treatment significantly improves cardiac
function and prognosis in HF patients (5, 11, 18), and
hence a downregulated ␤-adrenergic system could be
interpreted as adaptive and self-protective. However,
treatment with ␤-blockers simultaneously improves
both contractile function and ␤-adrenergic signaling (4,
11–13, 16), suggesting the possibility that restoration
of the latter, with or without upregulation of ␤1-AR,
might contribute to the improved function. The controversy extends to the experimental situation. Transgenic strains that overexpress ␤1-AR and Gs␣ have a
cardiac phenotype of hypertrophy and dysfunction (3,
8, 14, 23). In contrast, transgenic (TG) mice that overexpress ␤2-AR, ␤ARK inhibitor, or AC have markedly
enhanced cardiac contractility without significant cardiac pathology at least up to 8 mo of age (9, 15, 19, 25,
29). While overexpression of ␤2-AR at a high level
accelerates the development of HF induced genetically
or by pressure overload (6, 7, 24), expressing ␤2-AR at
a low level (⬃30-fold) or expressing a ␤ARK inhibitor
prevented cardiac hypertrophy and dysfunction in two
genetic HF models (6, 24).
TG mice (TG4) with cardiac-specific overexprssion of
␤2-AR have constitutively activated ␤-adrenergic signaling due to an ⬃200-fold increase in ␤2-AR concentration (19). In this study, we sought to determine
whether overexpression of ␤2-AR was beneficial or detrimental following experimentally induced myocardial
infarction (MI).
␤-ADRENERGIC SYSTEM plays a key role in heart failure (HF) (5). In response to chronically elevated cardiac sympathetic drive, ␤-adrenergic signaling is suppressed with 30–50% loss of ␤1-adrenergic receptors
(␤1-AR), reduced adenylyl cyclase (AC) activity, and a
lower ratio of Gs/Gi proteins (5). Meanwhile, the expression and the activity of ␤-AR kinase 1 (␤ARK1),
which is involved in ␤-AR downregulation and desensitization, are elevated (5). However, whether these
changes in ␤-adrenergic activity are beneficial or det-
Mice, genotyping, and ␤2-AR binding. Parent TG mice
(TG4) that overexpress ␤2-AR by ⬃200-fold were generated
at the Howard Hughes Medical Institute, Duke University
Medical Center (Durham, NC) (19). TG mice were crossed
with F1 mice from C57BL ⫻ SJL strains. The genomic DNA
was extracted from tail biopsy, and expression of the trans-
Address for reprint requests and other correspondence: X.-J. Du,
Baker Medical Research Institute, St Kilda Road Central, PO Box
6492, Melbourne 8008, Victoria, Australia (E-mail: xiaojun.du
@baker.edu.au).
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.
THE
H2456
METHODS
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society
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Du, Xiao-Jun, Xiao-Ming Gao, Garry L. Jennings,
Anthony M. Dart, and Elizabeth A. Woodcock. Preserved ventricular contractility in infarcted mouse heart
overexpressing ␤2-adrenergic receptors. Am J Physiol Heart
Circ Physiol 279: H2456–H2463, 2000.—Effects of cardiac
specific overexpression of ␤2-adrenergic receptors (␤2-AR) on
the development of heart failure (HF) were studied in wildtype (WT) and transgenic (TG) mice following myocardial
infarction (MI) by coronary artery occlusion. Animals were
studied by echocardiography at weeks 7 to 8 and by catheterization at week 9 after surgery. Post-infarct mortality, due to
HF or cardiac rupture, was not different among WT mice,
and there was no difference in infarct size (IS). Compared
with the sham-operated group (all P ⬍ 0.01), WT mice with
moderate (⬍36%) and large (⬎36%) IS developed lung congestion, cardiac hypertrophy, left ventricular (LV) dilatation,
elevated LV end-diastolic pressure (LVEDP), and suppressed
maximal rate of increase of LV pressure (LV dP/dtmax) and
fractional shortening (FS). Whereas changes in organ
weights and echo parameters were similar to those in infarcted WT groups, TG mice had significantly higher levels of
LV contractility in both moderate (dP/dtmax 4,862 ⫾ 133 vs.
3,694 ⫾ 191 mmHg/s) and large IS groups (dP/dtmax 4,556 ⫾
252 vs. 3,145 ⫾ 312 mmHg/s, both P ⬍ 0.01). Incidence of
pleural effusion (36% vs. 85%, P ⬍ 0.05) and LVEDP levels
(6 ⫾ 0.3 vs. 9 ⫾ 0.8 mmHg, P ⬍ 0.05) were also lower in TG
than in WT mice with large IS. Thus ␤2-AR overexpression
preserved LV contractility following MI without adverse consequence.
␤2-AR OVEREXPRESSION AND MYOCARDIAL INFARCTION
occlusion of the aorta. This procedure was repeated three
times, and the averages of the peak levels of dP/dtmax and
LVSP were used as indexes for the maximal contractile
reserve.
Morphometry and organ weights. Animals were killed by
pentobarbitone overdose. The chest was opened to determine
whether pleural effusion was present before isolation of the
heart. The heart was immersed in saline on ice. The LV, right
ventricle (RV), and atria were separated and weighed. When
organic thrombus was present in the left atrium, the weight
of thrombus was subtracted. The lungs and liver were
weighed, and the tibial length was measured. LVs were fixed
in 10% formalin in PBS for sectioning.
Infarct size determination. The LV was embedded in paraffin and serially cut from the apex to the base. A 5-␮m
transverse section was collected every 0.8 mm, and 5–7
sections were obtained from each LV. Sections were stained
with hematoxylin and eosin, and images were digitized. The
lengths of the entire endo- and epicardial circumferences and
portions of infarcted segments from both sides were measured using the Optimas 6.5 program. Percentages of infarcted LV of the endo- and epicardial circumferences were
calculated, and the averages were used (10, 21).
Statistics. Results are expressed as means ⫾ SE or as
percentages. For parametric data, between-group comparison was made by analysis of variance followed by unpaired
Student’s t-test. The chi-square test or Fisher’s exact test was
used to compare percentages between groups. The leastsquare method was used for linear correlation and regression.
RESULTS
Outcome of surgery. Of 125 operated mice, 20 (11 WT
and 9 TG) died within 24 h due to surgical reasons. All
sham-operated mice survived until the time of functional study. Of 83 mice with coronary artery occlusion
that survived longer than 24 h, 35 (21 WT and 14 TG)
Fig. 1. Transverse sections of the left ventricle (LV) from mice
without (A) and with (B and C) myocardial infarction (MI) for 9 wk.
D: left atrium with chronic thrombi from a mouse with large infarct.
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gene was detected by Southern blot hybridization with the
use of a 32P-labeled Hinc II fragment of the transgene construct (19). Both male and female animals, ages 12–18 wk,
were used.
␤2-AR density in the left ventricle (LV) was measured in a
separate group of TG and WT mice (n ⫽ 6 each). Saturation
curves were generated by incubating myocardial membrane
proteins with [125I]-(l)-iodocyanopindalol (125I-CYP; 2,200 Ci/
mmol; NEN) at 12–400 pmol/l for 1 h to determine the
affinity of receptors. Nonspecific binding was determined by
the presence of l-isoproterenol at 20 ␮mol/l. The binding
assay was performed by incubating membrane proteins with
100 pmol/l 125I-CYP for 1 h in the presence of ICI-118551
(Sigma) at 10⫺11 –10⫺5 mol/l. ␤2-AR density was 300-fold
higher in TG than in WT hearts (1,506 ⫾ 274 vs. 5 ⫾ 2
fmol/mg protein).
Microsurgery. Experimental procedures were approved by
the local animal experimentation ethics committee. Mice
were anesthetized (mixture of 8 mg/100 g ketamine, 2 mg/
100 g xylazine, 0.06 mg/100 g atropine, and 0.1 mg/100 g
temgesic as a pain reliever), intubated, and ventilated. Under a surgical microscope, a left thoracotomy was performed
to expose the heart. The location of the left coronary artery
was identified and then occluded with a 7-0 silk suture, as
described previously in detail (10). Sham-operated mice underwent similar surgery without occlusion of the coronary
artery.
Echocardiography. Transthoracic echocardiography was
performed with the use of a Hewlett-Packard Sonos 5500
ultrasound machine with a 12-MHz phased-array transducer
(0.5- to 0.7-cm standoff added), as described previously (10).
Mice were anesthetized with the anesthetic mixture as used
for surgery and placed on a heating pad. A standard lead II
electrocardiogram was recorded for heart rate (HR) measurement. After a short-axis two-dimensional (2D) image of the
LV was obtained at a level close to the papillary muscles, a
2D guided M-mode image crossing the anterior and posterior
walls was recorded (sweep speed 100 mm/s). Parameters
measured digitally on the M-mode trace were LV inner dimensions of diastole and systole (LVIDd, LVIDs), LV external
dimension of diastole (LVEDd), and fractional shortening
[FS ⫽ (LVIDd ⫺ LVIDs)/LVIDd] (10).
Cardiac catheterization. Cardiac function was assessed by
cardiac catheterization. Mice were anesthetized (pentobarbitone 8 mg/100 g and atropine 0.06 mg/100g ip) and placed
supine on a heating pad. A 1.4-F Millar microtipped transducer catheter was inserted into the LV via the right carotid
artery. The aortic blood pressure, LV pressures, and the
maximal rate of increase or decay of LV pressure (dP/dtmax or
dP/dtmin, respectively) were recorded. HR was derived from
pulse signals.
To further characterize the murine MI model of HF, in
sham-operated and infarcted WT mice, a ␤1-agonist, dobutamine, was infused intravenously at increasing doses of 7.5–
480 ng/min per mouse for 1–3 min (0.028–1.8 ␮g/kg), and the
functional response was monitored.
To assess the cardiac functional reserve, in some WT and
TG mice with MI, the peak levels of LV systolic pressure
(LVSP) and dP/dtmax were measured during a brief occlusion
of the aorta by following the method previously applied to the
rat (21). While the Millar catheter remained in the LV,
mechanical ventilation was commenced, and the chest was
opened by a midline incision via the sternum. The ascending
aorta was dissected, and a fine suture enclosed the aorta.
After stabilization, the aortic blood flow was stopped by
tightening the suture for 3 s, and LV pressure and dP/dt were
continuously recorded immediately before and during the
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␤2-AR OVEREXPRESSION AND MYOCARDIAL INFARCTION
H2458
Table 1. Body weight, infarct size, and organ weights in mice with sham-operation and with MI for 9 wk
Group (n, M/F)
Wild type
Sham (12, 4/8)
m-IS (11, 4/7)
l-IS (13, 6/7)
Transgenic
Sham (10, 4/6)
m-IS (12, 5/7)
l-IS (11, 4/7)
IS, %LV
BW1,
g
BW2,
g
Tibia
Length,
mm
28.3 ⫾ 2.2
46.4 ⫾ 1.3‡
24 ⫾ 2
25 ⫾ 1
28 ⫾ 1
26 ⫾ 2
27 ⫾ 1
28 ⫾ 1
17.9 ⫾ 0.2
17.8 ⫾ 0.1
17.6 ⫾ 0.2
25.0 ⫾ 2.5
44.3 ⫾ 1.8‡
24 ⫾ 1
27 ⫾ 1
25 ⫾ 2
27 ⫾ 1
29 ⫾ 1
27 ⫾ 1
17.7 ⫾ 0.2
18.1 ⫾ 0.1
17.8 ⫾ 0.2
LV
Weight,
mg
RV
Weight,
mg
Atria
Weight,
mg
Lung
Weight, mg
Pleural
Effusion
122 ⫾ 8
164 ⫾ 7*
195 ⫾ 7*‡
89 ⫾ 6
119 ⫾ 4*
136 ⫾ 5*‡
22 ⫾ 2
28 ⫾ 2*
37 ⫾ 3*‡
12 ⫾ 1
17 ⫾ 2*
22 ⫾ 2*‡
133 ⫾ 4
178 ⫾ 14*
203 ⫾ 15*
0/12
2/11
11/13*
127 ⫾ 8
158 ⫾ 9*
184 ⫾ 12*‡
93 ⫾ 6
116 ⫾ 7*
124 ⫾ 8*
22 ⫾ 1
25 ⫾ 1
36 ⫾ 4*‡
12 ⫾ 1
17 ⫾ 1*
25 ⫾ 3*‡
142 ⫾ 4
154 ⫾ 5
209 ⫾ 21*‡
Heart
Weight, mg
0/10
2/12
4/11*†
Values are means ⫾ SE; n ⫽ no. of animals. M/F, male/female; IS, infarct size; BW1 and BW2, body weight at the time of surgery and 9
wk afterward, respectively; LV and RV, left and right ventricle; m-IS and l-IS, moderate (⬍36%) and large (⬎36%) IS; MI, myocardial
infarction. * P ⬍ 0.05 vs. respective sham group; † P ⬍ 0.05 vs. respective wild-type group; ‡ P ⬍ 0.05 vs. respective m-IS group.
Cardiac function in WT mice. Echocardiography was
performed at 7–8 wk, and LV catheterization was done
at 9 wk after surgery. In WT and TG mice, HR levels
recorded at echo test were higher than those recorded
during catheterization (Table 2), most likely because of
the different anesthetic regimes used (see METHODS).
LV dimensions of systole and diastole were significantly increased, and FS was reduced in infarcted mice
compared with control levels (Table 2 and Fig. 2). At 9
wk after surgery, mean arterial pressure (MAP) and
LV end-diastolic pressure (LVEDP) were significantly
elevated (Table 2) and LVSP, dP/dtmax, and dP/dtmin
were reduced in MI groups versus controls (Fig. 3A).
These changes were more pronounced in animals with
large IS (Table 2).
We tested the LV functional response to dobutamine
given intravenously. In sham-operated animals, dobutamine induced a dose-dependent increase in dP/dtmax,
LVSP, and HR. The inotropic responses were significantly depressed in mice with moderate and large IS
(Fig. 4), whereas HR responses were attenuated only in
the large IS group. These results indicate downregulation of ␤-adrenergic signaling in the infarcted mouse
heart.
Differences between TG and WT mice. Pleural effusion, a sign of left HF (7), was less frequent in TG than
in WT mice with large IS (36% vs. 85%, P ⬍ 0.05, Table
1). Increases in weights of heart or LV, RV, and atria
were similar between WT and TG mice with MI. In
Table 2. Functional measures by echocardiography and by catheterization in mice with sham-operation or MI
Group
Wild type
Sham
m-IS
l-IS
Transgenic
Sham
m-IS
l-IS
HR1,
beats/min
LVIDd, mm
LVIDs, mm
FS, %
LVEDd,
mm
HR2,
beats/min
MAP,
mmHg
LVSP,
mmHg
310 ⫾ 16
384 ⫾ 21*
380 ⫾ 21*
3.3 ⫾ 0.2
4.6 ⫾ 0.2*
5.8 ⫾ 0.3*‡
1.8 ⫾ 0.2
3.6 ⫾ 0.3*
4.8 ⫾ 0.4*‡
46 ⫾ 2
23 ⫾ 4*
17 ⫾ 3*‡
5.2 ⫾ 0.2
6.2 ⫾ 0.2
7.2 ⫾ 0.3*‡
346 ⫾ 20
332 ⫾ 15
327 ⫾ 13
72 ⫾ 3
58 ⫾ 5*
61 ⫾ 4*
96 ⫾ 3
86 ⫾ 2*
80 ⫾ 4*
3 ⫾ 0.3
6 ⫾ 0.8*
9 ⫾ 0.8*‡
499 ⫾ 29†
528 ⫾ 26†
528 ⫾ 26†
3.6 ⫾ 0.2
4.6 ⫾ 0.3*
5.2 ⫾ 0.3*
2.0 ⫾ 0.2
3.3 ⫾ 0.4*
4.2 ⫾ 0.3*
47 ⫾ 3
31 ⫾ 5*
21 ⫾ 3*‡
5.2 ⫾ 0.2
6.1 ⫾ 0.2*
6.8 ⫾ 0.3*‡
458 ⫾ 15†
461 ⫾ 9†
431 ⫾ 23†
84 ⫾ 5
67 ⫾ 2*
66 ⫾ 3*
112 ⫾ 5†
91 ⫾ 2*
87 ⫾ 4*
3 ⫾ 0.4
5 ⫾ 0.6*
6 ⫾ 0.3*†‡
LVEDP,
mmHg
Values are means ⫾ SE. HR1 and HR2, heart rates measured at the time of echocardiography (7–8 wk) and cardiac catheterization (9 wk),
respectively; LVIDd and LVIDs, LV internal dimension at diastole or systole, respectively; FS, fractional shortening; MAP, mean arterial
pressure; LVSP, LV systolic pressure; LVEDP, LV end-diastolic pressure. * P ⬍ 0.05 vs. respective sham group; † P ⬍ 0.05 vs. respective
wild-type group; ‡ P ⬍ 0.05 vs. respective m-IS group.
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died of cardiac rupture or acute and chronic HF with a
mortality of 46% for WT and 38% for TG groups (P ⫽
not significant). Mice that died of HF had a threefold
increase in lung weight versus sham-operated mice
(405 ⫾ 15 vs. 137 ⫾ 3 mg, P ⬍ 0.001), and the incidences of atrial thrombus and pleural effusion were
87% and 100%, respectively. Infarct size (IS) was measured in the 27 mice that died and was 52.0 ⫾ 2.5%
(n ⫽ 17) in the mice that died of HF and 41.4 ⫾ 2.4%
(n ⫽ 10) in the mice that died of rupture.
Infarct size and grouping of surviving mice. All mice
that survived coronary artery occlusion had a transmural infarct localized to the LV free wall and apex.
The RV and septum were not involved (Fig. 1). IS
ranged from 8% to 54% of LV and was not significantly
different between WT and TG groups (36 ⫾ 2% vs. 33 ⫾
3%). To compare cardiac function of WT and TG mice
with different IS, we divided animals into subgroups
with moderate and large IS by the median IS of 36%
(Table 1).
Organ weights in WT mice. Since body weights and
tibial length were similar in all groups of WT and TG
mice, the organ weights are presented as absolute
values (Table 1). At 9 wk, weights of whole heart or LV,
RV, and atria were significantly increased in WT mice,
with MI indicating the development of hypertrophy in
noninfarcted myocardium. Lung weights increased significantly, implying chronic pulmonary congestion. The
degree of these changes was dependent on IS (Table 1).
␤2-AR OVEREXPRESSION AND MYOCARDIAL INFARCTION
H2459
Fig. 2. Two-dimensional echocardiographic images (A
and B) and M-mode traces (C and D) from a control
mouse (A and C) and a mouse with large MI (B and D).
Echo test was performed 7 wk after surgery. Infarct size
in the infarcted mouse was 47% of the LV. The akinesis
at the infarcted region (anterior wall) was evident. The
LV inner dimension at diastole (LVIDd) was 3.6 mm for
the sham-operated mouse and 5.7 mm for the infarcted
mouse. Sweep speed ⫽ 100 mm/s.
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Fig. 3. A: maximal rates of increase (dP/dtmax) and decrease in LV
pressure (dP/dtmin) in sham-operated (SH) mice and mice with MI of
moderate (m-IS; ⬍36%) and large infarct size (l-IS; ⬎36%) measured
at 9 wk after surgery. Data are means ⫾ SE; n ⫽ 10–13 per group.
*P ⬍ 0.01 vs. respective SH group; ⫹P ⬍ 0.01 vs. wild-type (WT)
group. TG, transgenic group. B: changes in LV systolic pressure
(LVSP) and dP/dtmax during brief periods of occlusion of the ascending aorta (AO) in infarcted WT (n ⫽ 6) and TG mice (n ⫽ 7) under
open-chest conditions with mechanical ventilation. *P ⬍ 0.05 vs. WT
group.
infarcted mice surviving to the time of study, nine (4
TG, 5 WT) had chronic thrombus in the left atrium. All
but one (32.6%) had IS ⬎36% (38.2–50.5%, average
44 ⫾ 2%).
There was no significant difference among shamoperated and infarcted WT and TG groups in any of the
echocardiographic parameters (Table 2), except that
TG mice had a higher HR. The TG mice with large IS
had significantly lower LVEDP compared with the
respective WT group (P ⬍ 0.05). Although infarcted TG
and WT mice had similar percent reduction in LV
dP/dtmax and dP/dtmin versus respective sham-operated
groups, dP/dt levels were significantly higher than
those in the respective WT groups (P ⬍ 0.01, Fig. 3A).
HR itself is known to influence the measurement of
LV dP/dt (20). The possibility that higher levels of LV
dP/dt in TG mice was due to higher HR was examined
in a separate group of infarcted WT mice (n ⫽ 5) under
conditions of ventilation and rapid atrial pacing by
following the method described by Palakodeti et al.
(20). Increasing HR from 350 ⫾ 10 to 450 to 500
beats/min led to an 18% fall in dP/dtmax (P ⬍ 0.05) from
a basal level of 3,350 ⫾ 250 mmHg/s. A similar change
was also observed for dP/dtmin. This suppression of
dP/dt levels with rapid atrial pacing was immediately
reversed when atrial pacing was stopped. Thus the
difference between WT and TG mice in LV dP/dt cannot be attributed to the difference in HR levels.
The contractile response to acute aortic occlusion
was tested in randomly selected infarcted WT (n ⫽ 6)
and TG mice (n ⫽ 7) with similar IS (38 ⫾ 3% vs. 35 ⫾
3%, P ⫽ NS). Under conditions of open chest and
ventilation, baseline LVSP (P ⫽ 0.069) and dP/dtmax
(P ⬍ 0.05) were higher in TG than in WT mice. LVSP
and dP/dtmax increased in both groups in response to
temporary aortic occlusion. While the peak LVSP was
not significantly different between groups, TG mice
H2460
␤2-AR OVEREXPRESSION AND MYOCARDIAL INFARCTION
had higher levels of dP/dtmax than WT mice did (P ⬍
0.05, Fig. 3B).
Correlation of morphometric and functional parameters. Lung weights correlated well with weights of
heart (r ⫽ 0.704), LV (r ⫽ 0.523), RV (r ⫽ 0.848), and
atria (r ⫽ 0.766; all P ⬍ 0.001). Furthermore, when IS
exceeded a certain level (⬃30%), weights of the heart,
LV, RV, atria, and lungs were increased (Table 1).
IS correlated positively with LVIDd, LVIDs, and
LVEDP and negatively with FS, LVSP, dP/dtmax, and
dP/dtmin (all P ⬍ 0.01; Fig. 5). FS modestly correlated
with dP/dtmax in either WT (r ⫽ 0.421) or TG mice (r ⫽
0.369; both P ⬍ 0.05). The extent of LV dilatation,
estimated from the LVEDd correlated negatively with
FS (r ⫽ ⫺0.781, P ⬍ 0.0001). LVEDP correlated significantly with weights of the RV ( r ⫽0.466), atria (r ⫽
0.408), and lungs (r ⫽ 0.526; all P ⬍ 0.01).
DISCUSSION
In this study, the development of HF in WT mice
with MI was evidenced by pulmonary congestion, pleu-
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Fig. 4. Cardiac functional responses to increasing doses of dobutamine in WT mice subjected to sham operation or MI for 9 wk. The
increase in LV dP/dtmax (A) and LVSP (B) was depressed in both
infarcted groups. The increase in heart rate was also blunted in the
l-IS group (C). Data are means ⫾ SE; n ⫽ 9–10 per group. *P ⬍ 0.05
vs. SH group by ANOVA.
ral effusion, LV dilatation, elevation in LVEDP, and
reduction in both LV contractility (dP/dt) and FS. The
blunted inotropic and chronotropic responses to dobutamine indicate downregulation of the ␤-adrenergic system in this model. Similarly to that in other species,
including humans (21, 22), IS in mice is a major determinant of the extent of cardiac dysfunction, morphometric abnormalities, incidence of pathological events,
and mortality. These data elucidate the features of the
murine model of MI.
We observed that, 9 wk after MI, TG mice with large
IS had lower LVEDP and a lower incidence of pleural
effusion compared with the WT group. Although dP/dt
fell significantly in TG mice to the extent comparable to
that in the infarcted WT mice, dP/dt levels were significantly higher in infarcted TG mice than in respective
WT mice irrespective of IS. Furthermore, the peak
levels of dP/dtmax caused by a brief occlusion of the
ascending aorta were also significantly higher in TG
than in WT mice with infarct. These findings suggest
that the noninfarcted myocardium in TG mice maintained the phenotype of an enhanced inotropy. Therefore, these data demonstrate that overexpressing
␤2-AR in the heart provides inotropic support to the
infarcted and failing ventricle.
It might be expected that markedly enhanced ␤2adrenergic activity under conditions of MI would increase the risk of arrhythmias. In the present study,
MI did not lead to excessive death in TG mice, with
mortality slightly lower than that in WT mice. There
was no evidence to indicate arrhythmic death in either
WT or TG mice, although arrhythmias were recorded
occasionally during the functional experiments. Thus
overexpression of ␤2-AR at a high level did not cause
adverse consequences in mice following MI.
We recently observed facilitated onset of HF and
higher mortality due to critical HF in ␤2-AR transgenic
mice following thoracic aortic constriction (7). The different outcomes from HF models of pressure overload
and MI raise the possibility that the effect of ␤2-AR
overexpression is not only dependent on the receptor
number, as suggested by recent studies (6), but also on
the etiology of HF. Interestingly, it is known that the
extent of ␤-adrenergic downregulation and desensitization is more severe in pressure-overloaded heart
than in ischemic disease (5). The mechanism responsible for such differences remains unclear, and potential
differences in some key molecules, such as ␤ARK1 and
Gi protein, require further investigation.
In infarcted TG and WT mice, the percent reduction
in LV dP/dt from the respective sham-operated control
levels was comparable. However, the absolute levels of
dP/dtmax and dP/dtmin were significantly higher in infarcted TG than in WT groups. This finding indicates
that, in TG mice, the noninfarcted myocardium maintained its hyperdynamic phenotype supported by the
genetically overexpressed ␤2-AR. Interestingly, although infarcted TG mice had LV dP/dt levels similar
to those of sham-operated WT mice, the extent of LV
dilatation measured by echocardiography did not significantly improve compared with that of the infarcted
␤2-AR OVEREXPRESSION AND MYOCARDIAL INFARCTION
H2461
WT groups. The average levels of FS in TG groups were
20–30% higher than those in WT groups, but such
differences were statistically not significant. It is not
clear why cardiac ␤2-AR overexpression differentially
influences LV contractility and echocardiographic measures. We found similar FS levels in sham-operated
WT and TG groups, although LV contractility was
much higher in the latter. Unchanged echo indexes
were also previously observed in mice with cardiac
overexpression of adenylyl cyclase or ␤2-AR, although
the basal LV dP/dt was significantly higher compared
with that of WT littermate controls (9, 27).
TG mice overexpressing ␤2-AR have markedly enhanced inotropy and chronotropy (19, 29) without deleterious changes in function and histology up to 8 mo of
age, except for a mild increase in collagen content in
the LV myocardium (7). In contrast, a TG line overexpressing ␤1-AR by 15-fold exhibits early mortality,
cardiac hypertrophy, and failure (8, 23). The reasons
for the differences in these TG lines remain to be
elucidated and may be related to some important differences between ␤1-AR and ␤2-AR in downstream
coupling and signaling, such as coupling to Gi proteins
by ␤2-AR but not by ␤1-AR and the compartmentalization of ␤2-AR-activated AC/cAMP signaling (2, 15a, 29).
␤2-AR overexpression (⬃200-fold) in mice with disruption of muscle lim protein (MLP⫺/⫺) leads to worsening of HF and higher mortality (24). In another HF
model caused by overexpression of Gq␣, overexpressing
␤2-AR at a high level worsened the outcome, whereas a
low level overexpression (⬃30-fold) reversed ventricular dysfunction and suppressed cardiac hypertrophy
(6). Expressing a ␤ARK inhibitor enhanced ventricular
contractility in normal mice (1, 15) and rescued the
cardiomyopathic phenotype in MLP⫺/⫺ mice (24) but
not in Gq␣-overexpressing mice (6). Overexpressing
adenylyl cyclase in Gq␣ mice by crossbreeding improved ventricular function (26). These studies and the
findings from the present study indicate that enhanced
␤-adrenergic activity is not always associated with
adverse consequences.
Rapid pacing per se is able to induce the development of HF in large animals (28). It is therefore possible that higher HR in TG mice may confound the
findings of the present study. The ␤2-AR TG strain is
unique for the high HR level with the ionic mechanism
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Fig. 5. Correlation between infarct size and functional parameters in sham-operated control and
infarcted mice. The data were combined if there
was no significant different between WT and TG
groups. E, WT mice; 䊐, TG mice. LVEDP, LV
end-diastolic pressure; LVIDs, LV inner dimension at systole; FS, fractional shortening; LVEDd,
LV external dimension at diastole.
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␤2-AR OVEREXPRESSION AND MYOCARDIAL INFARCTION
We thank Dr. Robert J. Lefkowitz for supplying the transgenic
line. We thank the staff at the Biological Research Unit, Elodie
Percy, Binghui Wang, Rodney Dilley, and Brian Jones, for help in
animal breeding, transgene screening, and image analysis.
This work was supported by the National Health and Medical
Research Council of Australia and a grant from the Merck Sharp &
Dohme Research Fund (Australia). X.-M. Gao is the recipient of a
scholarship from the Australia/China/Indonesia/Singapore Heart
Foundation via Prof Y. M. Lim, Director, Singapore National Heart
Center.
REFERENCES
1. Akhter SA, Eckhart AD, Rockman HA, Shotwell K,
Lefkowitz RJ, and Koch WJ. In vivo inhibition of elevated
myocardial ␤-adrenergic receptor kinase activity in hybrid transgenic mice restores normal ␤-adrenergic signaling and function.
Circulation 100: 648–653, 1999.
2. An RH, Heath BM, Higgins JP, Koch WJ, Lefkowitz RJ,
and Kass RS. ␤2-Adrenergic receptor overexpression in the
developing mouse heart: evidence for targeted modulation of ion
channels. J Physiol (Lond) 516: 19–30, 1999.
3. Asai K, Yang GP, Geng YJ, Takagi G, Bishop S, Ishikawa Y,
Shannon RP, Wagner TE, Vatner DE, Homcy CJ, and
Vatner SF. ␤-Adrenergic receptor blockade arrests myocardial
damage and preserves cardiac function in the transgenic Gs␣
mouse. J Clin Invest 104: 551–558, 1999.
4. Böhm M, Deutsch HJ, Hartmann D, Rosee KL, and Stablein A. Improvement of postreceptor events by metoprolol
treatment in patients with chronic heart failure. J Am Coll
Cardiol 30: 992–996, 1997.
5. Bristow MR. Mechanism of action of ␤-blocking agents in heart
failure. Am J Cardiol 80: 26L–40L, 1997.
6. Dorn, GW II, Tepe NM, Lorenz JN, Koch WJ, and Liggett
SB. Low- and high-level transgenic expression of ␤2-adrenergic
receptors differentially affect cardiac hypertrophy and function
in G␣q-overexpressing mice. Proc Natl Acad Sci USA 96: 6400–
6405, 1999.
7. Du XJ, Autelitano DJ, Wang BH, Dart AM, and Woodcock
EA. ␤2-Adrenergic receptor overexpression exacerbates develop-
ment of heart failure following aortic stenosis. Circulation 101:
71–77, 2000.
8. Engelhardt S, Hein L, Wiesmann F, and Lohse MJ. Progressive hypertrophy and heart failure in ␤1-adrenergic receptor
transgenic mice. Proc Natl Acad Sci USA 96: 7059–7064, 1999.
9. Gao MH, Lai NC, Roth DM, Zhou J, Zhu J, Anzai T, Dalton
N, and Hammond HK. Adenylylcyclase increases responsiveness to catecholamine stimulation in transgenic mice. Circulation 99: 1618–1622, 1999.
10. Gao XM, Dart AM, Dewar E, Jennings GL, and Du XJ.
Serial echocardiographic assessment of left ventricular dimensions and function after myocardial infarction in mice. Cardiovasc Res 45: 330–338, 2000.
11. Gilbert EM, Abraham WT, Olsen S, Hattler B, White M,
Mealy P, Larrabee P, and Bristow MR. Comparative hemodynamic, left ventricular functional, and antiadrenergic effects
of chronic treatment with metoprolol versus carvedilol in the
failing heart. Circulation 94: 2817–2825, 1996.
12. Heibrunn SM, Shah P, Bristow MR, Valantine HA, Ginsburg R, and Fowler MB. Increased ␤-receptor density and
improved hemodynamic response to catecholamine stimulation
during long-term metoprolol therapy in heart failure from dilated cardiomyopathy. Circulation 79: 483–490, 1989.
13. Iaccarino G, Tomhave ED, Lefkowitz RJ, and Koch WJ.
Reciprocal in vivo regulation of myocardial G protein-coupled
receptor kinase expreession by ␤-adrenergic receptor stimulation and blockade. Circulation 98: 1783–1789, 1998.
14. Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP,
Kudej RK, Wight DC, Wagner TE, Ishikawa Y, Homc CJ,
and Vatner SF. Adverse effects of chronic endogenous sympathetic drive induced by cardiac Gs␣ overexpression. Circ Res 78:
517–524, 1996.
15. Koch WJ, Rockman HA, Samama P, Hamilton R, Bond RA,
Milano CA, and Lefkowitz RJ. Cardiac function in mice overexpressing the ␤-adrenergic receptor kinase or a ␤ARK inhibitor.
Science 268: 1350–1353, 1995.
15a.Kuschel M, Zhou YY, Cheng H, Zhang SJ, Chen Y, Lakatta
EG, and Xiao RP. Gi protein-mediated functional compartmentalization of cardiac ␤2-adrenergic signaling. J Biol Chem 274:
22048–22052, 1999.
16. Liang CS, Frantz RP, Suematsu M, Sakamoto S, Sullebarger JT, Fan TM, and Guthinger L. Chronic ␤-adrenoceptor blockade prevents the development of ␤-adrenergic subsensitivity in experimental right-sided congestive heart failure in
dogs. Circulation 84: 254–266, 1991.
17. Maurice JP, Hata JA, Shah AS, White DC, McDonald PH,
Dolber PC, Wilson KH, Lefkowitz RJ, Glower DD, and
Koch WJ. Enhancement of cardiac function after adenoviralmediated in vivo intracoronary ␤2-adrenergic receptor gene delivery. J Clin Invest 104: 21–29, 1999.
18. MERIT-HF Study Group. Effect of metoprolol CR/XL in
chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure. Lancet 353: 2001–2007,
1999.
19. Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn
TR, Chien KR, Johnson TD, Bond RA, and Lefkowitz RJ.
Enhanced myocardial function in transgenic mice overexpressing the ␤2-adrenergic receptor. Science 264: 582–586, 1994.
20. Palakodeti V, Oh S, Oh BH, Mao L, Hongo M, Peterson KL,
and Ross J Jr. Force-frequency effect is a powerful determinant
of myocardial contractility in the mouse. Am J Physiol Heart
Circ Physiol 273: H1283–H1290, 1997.
21. Pfeffer MA, Pfeffer JM, Fishbein MC, Fletcher PJ, Spadaro J, Kloner RA, and Braunwald E. Myocardial infarct size
and ventricular function in rats. Circ Res 44: 503–512, 1979.
22. Pfeffer MA and Braunwald E. Ventricular remodeling after
myocardial infarction. Experimental observations and clinical
implications. Circulation 81: 1161–1172, 1990.
23. Port JD, Weinberger HD, Bisognano JD, Knudson OA,
Bohlmeyer TJ, Pende A, and Bristow MR. Echocardiographic and histopathologic characterization of young and old
transgenic mice overexpressing the human ␤1-adrenergic receptor (Abstract). J Am Coll Cardiol 31, Suppl A: 177A, 1998.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on June 17, 2017
undefined. Patch-clamp studies have shown that there
are no significant changes in slow delayed rectifier K⫹
current but that the density of L-type Ca2⫹ current is
increased because of modulation by the activated AC/
cAMP system (2, 29). Higher HR would increase energy
expenditure and shorten the diastolic filling time. It is
likely that, in TG mice with genetically induced cardiomyopathy or pressure overload, the higher energy demand and restricted usage of Frank-Starling mechanism, due to shortened diastolic filling time, compromise
the cardiac function and that this contributes to the
adverse outcome (7, 24). It is also possible that a higher
energy demand partially offsets the beneficial effect, observed in the present study, of ␤2-AR overexpression in
TG mice with MI.
Our results demonstrate a preserved ventricular
contractility in ␤2-AR TG mice following chronic MI.
This finding implies that an improved ␤2-adrenergic
activity by ␤2-AR overexpression in the infarcted and
failing heart could provide inotropic support to the
overall ventricular contractility and is in keeping with
the view for ␤2-AR expression or transfection as a
potential approach for HF gene therapy (6, 17, 24).
However, considering the diverse effects of ␤2-AR overexpression observed in other HF models (6, 7, 24), it is
likely that the effect of transgenic overexpression of
␤2-AR on HF is dependent on the etiology of HF.
␤2-AR OVEREXPRESSION AND MYOCARDIAL INFARCTION
24. Rockman HA, Chien KR, Choi DJ, Iaccarino G, Hunter JJ,
Ross J Jr, Lefkowitz RJ, and Koch WJ. Expression of a
␤-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc Natl Acad
Sci USA 95: 7000–7005, 1998.
25. Rockman HA, Choi D, Akhter SA, Jaber M, Giros B, Lefkowitz RJ, Caron MG, and Koch WJ. Control of myocardial contractile function by the level of ␤-adrenergic receptor kinase 1 in
gene-targeted mice. J Biol Chem 273: 18180–18184, 1998.
26. Roth DM, Gao MH, Lai NC, Drumm J, Dalton N, Zhou JY,
Zhu J, Entrikin D, and Hammond HK. Cardiac-directed
adenylyl cyclase expression improves heart function in murine
cardiomyopathy. Circulation 99: 3099–3102, 1999.
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27. 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.
28. Vatner DE, Sato N, Galper JB, and Vatner SF. Physiological
and biochemical evidence for coordinate increases in muscarinic
receptors and Gi during pacing-induced heart failure. Circulation 94: 102–107, 1996.
29. Xiao RP, Avdonin P, Zhou YY, Cheng H, Akhter SA,
Eschenhagen T, Lefkowitz RJ, Koch WJ, and Lakatta
EG. Coupling of ␤2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res 84:
43–52, 1999.
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