ORIGINAL ARTICLE

High-dose Norepinephrine Induces Disruption of

Myocardial Extracellular Matrix and
Left Ventricular Dilatation and Dysfunction in
a Novel Feline Model
Yung-Tsung Chiu1, Ching-Chang Cheng1, Nai-Nu Lin1, Yi-Wen Hung1, Ying-Tsung Chen2,
Shih-Lan Hsu1, Ching-Shiang Chi3, Yun-Ching Fu3,4*
1
Department of Education and Research, 2Cardiovascular Center, and 3Department of Pediatrics,
Taichung Veterans General Hospital, Taichung, and 4Department of Pediatrics,
National Yang-Ming University School of Medicine, Taipei, Taiwan, R.O.C.

Background: Intravenous norepinephrine (NE) at a dose of 1–6 μg/kg/minute can induce increased extracellular matrix
(ECM) and hypertrophic cardiomyopathy. This study aimed to investigate the effects of a higher dose of NE on cardiac
remodeling.
Methods: After intraperitoneal urethane-chloralose anesthesia, 7 cats (3.03 ± 0.58 kg) received intravenous infusion of
NE 30 μg/kg/minute for 3 hours. Aortic blood pressure and heart rate (HR) were measured by polygraphy at 0, 5, 15, 30,
60, 90, 120, and 180 minutes. Left ventricular size and ejection fraction (EF) were measured by M-mode echocardio-
graphy before and after NE administration. Histopathology was performed by hematoxylin-eosin, silver impregnation, and
Sirius red staining. Activity of matrix metalloproteinases (MMP) in the left ventricle was measured by zymography.
Results: Mean blood pressure (mmHg) increased from 139 ± 20 to 198 ± 19, 187 ± 23, and 166 ± 16 at 15, 30, and 60
minutes, respectively, during NE infusion. HR (beats/minute) decreased from 214 ± 10 to 158 ± 28 at 15 minutes and
then recovered gradually. The left ventricles showed significant dilatation (end-diastolic diameter: from 1.20 ± 0.18 to
1.58 ± 0.23 cm, p = 0.003; end-systolic diameter: from 0.62 ± 0.23 to 1.35 ± 0.29 cm, p = 0.002) and hypokinesia
(EF: from 86.2 ± 5.2 to 33.1 ± 16.5%, p < 0.001). Histopathology revealed that left ventricular myocytes were elongated,
wavy, and fragmented, while collagen fibers were overstretched, straightened, and disrupted. MMP-9 activity was signifi-
cantly elevated (p = 0.003 vs. control), while MMP-2 activity was unchanged.
Conclusion: High-dose NE increases MMP-9 activity and causes ECM disruption, left ventricular dilatation and dysfunc-
tion. [J Chin Med Assoc 2006;69(8):343–350]

Key Words: extracellular matrix, heart failure, matrix metalloproteinases, norepinephrine

Introduction myocardium and governs tissue stiffness.1,2 It has

Collagens are the major extracellular matrix (ECM)
proteins found in the heart.1,2 The fibrillar collagens
of the heart surround and interconnect myocytes and
muscle fibers to enable muscle fiber and myocyte
alignment, which imparts mechanical support to the
become increasingly evident that the myocardial
ECM is not a static structure but, rather, a dynamic
entity that may play a functional role in myocardial
adaptation to pathologic stress and thereby facilitate
remodeling in different kinds of cardiomyopathy.3–7
Hypertrophic cardiomyopathy is characterized by an

*Correspondence to: Dr Yun-Ching Fu, Department of Pediatrics, Taichung Veterans General Hospital, 160,
Section 3, Chung-Kang Road, Taichung 407, Taiwan, R.O.C.
E-mail: ivanfu@vghtc.gov.tw Received: September 29, 2005
● Accepted: June 19, 2006
●

J Chin Med Assoc • August 2006 • Vol 69 • No 8 343

© 2006 Elsevier. All rights reserved.
 Y.T. Chiu, et al
increase in ECM, ventricular thickening, and impaired
diastolic function.4 However, dilated cardiomyopathy
is characterized by a decrease in ECM, ventricular dilata-
tion, and impaired systolic function.4–7
Collagen degradation is an important step in the
remodeling of ECM.1 Collagens are degraded extra-
cellularly by a family of matrix metalloproteinases
(MMPs) capable of enzymatically digesting a wide
variety of ECM.1,8–17 So far, more than 20 different
subtypes of MMP are known.8 Among them, matrix
gelatinases (MMP-2 and MMP-9) may be involved
in the disorganization of the contractile apparatus in
dilated cardiomyopathy.15 Nishikawa et al16 demon-
strated that MMP-9 rather than MMP-2 is involved
in left ventricular dilatation in systolic heart failure.
Catecholamines are important regulators of myo-
cardial contractility and metabolism.18 However,
excessive release or administration of catecholamines
exceeding physiologic doses may result in reversible
or even irreversible cardiac damage.18,19 Catechola-
mine cardiotoxicity or cardiomyopathy is found in
patients who have received large amounts of pressor
agents and in those with pheochromocytoma or cer-
tain brain lesions.18–26 Previous animal models of
catecholamine cardiomyopathy used relatively lower
doses (1–6 μg/kg/minute) and longer (90 minutes–14
days) durations of norepinephrine (NE) administra-
tion, which caused hypertrophic remodeling of the
ventricle.8–10,27–29 However, whether a higher dose of
NE treatment has a different effect on the ECM and
cardiac remodeling remains unclear. Our present study
presents a novel feline model of catecholamine cardio-
myopathy using a relatively high dose of NE (30 μg/
kg/minute), which can increase MMP-9 activity and
cause disruption of the ECM, and left ventricular
dilatation and dysfunction within 3 hours.
Methods
Animal and experimental procedure
The investigation conformed to the Guide for the
Care and Use of Laboratory Animals published by the
US National Institutes of Health (NIH Publication
No. 85-23, revised 1996) and was approved by the
Institutional Animal Care and Use Committee of
Taichung Veterans General Hospital. After intraperi-
toneal injection of alpha-chloralose 40 mg/kg and
urethane 400 mg/kg for 1 hour, each cat was cannu-
lated in the right femoral artery and vein. The arterial
line was connected to a polygraph system for continuous
monitoring of blood pressure and heart rate (HR).30
NE 30 μg/kg/minute was administered intravenously
344
for 3 hours. M-mode echocardiography (12 MHz
probe in Philips Sono 5500, USA) assessed the left
ventricular diameter at end-diastole and end-systole
and ejection fraction (EF) (Teichholz method pro-
vided by the manufacturer) before and after NE
treatment.31 After completing the experiment, each
cat was killed immediately by intravenous potassium
chloride injection and the heart was removed. Some
of the left ventricular tissues were stored in –70°C liq-
uid nitrogen for the determination of MMP activity,
and the others were fixed in 10% buffer formalin for
histopathology.
MMP activity measured by zymography
MMP activity was assayed by SDS-PAGE zymography
using gelatin as the substrate.32 For gelatin-containing
zymograms, equal volumes (10 μL) of samples nor-
malized for protein concentration were subjected to
electrophoresis, without boiling or reduction, through
a 10% polyacrylamide gel with gelatin (0.5 mg/mL).
After electrophoresis, the gel was incubated for 1 hour
at 25°C in 2.5% Triton X-100 solution, washed twice
for 20 minutes each with water and then incubated
overnight at 37°C in 0.05 M Tris–HCl buffer, pH
8.0, containing 5 mM CaCl2. The gels were fixed with
40% methanol and 7% acetic acid, stained with 0.25%
Coomassie brilliant blue R250 and then destained
with 10% methanol and 7% acetic acid. Enzyme
activity attributed to MMP-2 and MMP-9 is visual-
ized in the gelatin-containing zymograms as clear
bands against a blue background. Standards for the
active forms of recombinant human MMP-2 and
MMP-9 were included in the gels for comparison and
identification.
Histopathologic examination
After gross examination of the heart, the left ven-
tricular tissues were fixed in 10% buffer formalin and
embedded in paraffin.33 The 6 μm sections were stained
with hematoxylin-eosin (H-E) and picro-Sirius,34 while
25 μm sections were stained with silver impregnation.4
Statistical analysis
All data were expressed as mean ± SD. Repeated
measured analysis of variance (ANOVA) test was used
for statistical comparisons between the values of mean
blood pressure and HR. Paired t test was used for
comparison between the values obtained after NE
infusion and the baseline value for left ventricular size
and EF. Student t test was used for the comparison of
MMP activity between NE and control groups. The
mean values of the 2 groups were considered signifi-
cantly different if p < 0.05.
J Chin Med Assoc • August 2006 • Vol 69 • No 8
 NE induces disruption of extracellular matrix

Results 24 mmHg, p = 0.013) due to heart failure. HR (beats/

Blood pressure and HR
Figure 1 shows the time course of hemodynamic data.
Mean aortic blood pressure increased from 139 ± 20
to 198 ± 19, 187 ± 23, and 166 ± 16 mmHg at 15,
30, and 60 minutes, respectively, during NE infusion
(p < 0.001, p = 0.011, and p = 0.005, respectively). It
was lower than baseline data at 180 minutes (120 ±
minute) decreased from 214 ± 10 to 158 ± 28 at 15
minutes and then recovered gradually.
Cardiac size and function
After a 3-hour treatment with NE, the left ventricles
showed significant dilatation (end-diastolic diameter:
from 1.20 ± 0.18 to 1.58 ± 0.23 cm, p = 0.003; end-
systolic diameter: from 0.62 ± 0.23 to 1.35 ± 0.29 cm,

250 300
* †
250 †
200 ‡
†
‡

HR (beats/min)
† 200
MBP (mmHg)

150
150
100
100

50
50

0 0
0 15 30 60 90 120 150 180 0 15 30 60 90 120 150 180
Time (min) Time (min)

Figure 1. Time course of mean blood pressure (MBP) and heart rate (HR) during norepinephrine treatment. Values are mean ± SD of
7 cats. *p < 0.001; †p < 0.05; ‡p < 0.01 vs. baseline values.

A O B O

> <
LV
LV

Figure 2. Two-dimensional echocardiography in the short-axis view shows significant dilatation of the left ventricle. (A) Before and
(B) after electrical stimulation.

J Chin Med Assoc • August 2006 • Vol 69 • No 8 345

 Y.T. Chiu, et al

Before NE
†
2.0 After NE * 100
1.8 *
1.6 80

Ejection fraction (%)

1.4
Diameter (cm)

1.2 60
1.0
0.8 40
0.6
0.4 20
0.2
0.0 0
LVIDs LVIDd Before NE After NE

Figure 3. The internal diameter of the left ventricle (LVID) at end-diastole (LVIDd) and end-systole (LVIDs) significantly increased, while
the ejection fraction significantly decreased 180 minutes after beginning norepinephrine (NE) treatment. Values are mean ± SD of 7
cats. *p < 0.01; †p < 0.001 vs. baseline values.

A B
80,000
Control *
NE
Control NE 60,000
M 1 2 3 4 5 6 7
Intensity

130 kD 40,000
MMP-9

73 kD 20,000
MMP-2
54 kD

0
MMP-2 MMP-9

Figure 4. (A) Representative zymogram shows clear proteolytic bands of matrix metalloproteinase (MMP-9 and MMP-2). (B) Quantitative
analysis demonstrated that the increase in MMP-9 zymographic activity was more pronounced in the norepinephrine (NE) group (n = 7)
than in the control group (n = 6) (p = 0.003). There was no difference in MMP-2 zymographic activity between NE and control groups
(p = 0.192). *p < 0.01 vs. control.

p = 0.002) concomitant with hypokinesia (EF: from 4B). There was no difference in MMP-2 zymographic
86.2 ± 5.2% to 33.1 ± 16.5%, p < 0.001) (Figures 2 activity between the NE and control groups (21,590 ±
and 3). 10,237 vs. 15,555 ± 2,874, p = 0.192).

MMP activity Histopathologic findings

A representative zymogram for left ventricular MMP
activity is shown in Figure 4A. Clear proteolytic bands
were revealed on gelatin zymography gels correspon-
ding to both gelatinase MMP-9 and MMP-2. Quantita-
tive analysis demonstrated that the increase in MMP-9
zymographic activity was more pronounced in the
NE group (n = 7) than in the control group (n = 6)
(50,436 ± 16,273 vs. 24,738 ± 2,565, p = 0.003) (Figure
Gross examination of NE-treated hearts revealed
remarkable dilatation of both ventricles indicated by
rounding of the apex (Figure 5A). Myocardial hem-
orrhage was also noted, especially at the base of the
papillary muscles (Figures 5A and B). In H-E stain,
cardiomyocyte damage was characterized by myofib-
rillar weaving (Figure 5C), elongation, and disrup-
tion (myocytolysis, Figure 5D). Normal collagens of

346 J Chin Med Assoc • August 2006 • Vol 69 • No 8

 NE induces disruption of extracellular matrix

A B
1 cm 1 cm

C D

Figure 5. Gross examination of norepinephrine-induced hearts showed: (A) remarkable dilatation of both ventricles indicated by round-
ing of the apex; (B) myocardial hemorrhage was also noted, especially at the base of the papillary muscles. In hematoxylin-eosin stain,
cardiomyocyte damage was characterized by: (C) myofibrillar weaving and (D) disruption (myocytolysis).

myocardial ECM are perimysial coil-like fibers, which
are parallel to the cardiomyocytes in picro-Sirius
(Figures 6A and C) and silver impregnation staining
(Figure 6E). In NE-treated hearts, they became over-
stretched, progressively straightened, and ultimately
disrupted (Figures 6B, D and F).
Discussion
The main finding in this study was that a high dose
of NE of 30 μg/kg/minute could increase MMP-9
but not MMP-2 expression, which was associated
with the disruption of ECM and left ventricular
dilatation and dysfunction. MMPs are responsible for
ECM degradation and remodeling, so previous stud-
ies concluded that the activation of MMPs plays a cru-
cial role in left ventricular dilatation, and MMPs are
therapeutic targets for the prevention of left ventricular
remodeling.3,11,16 However, whether all activated
MMPs contribute equally to left ventricular remodel-
ing is not known. Nishikawa et al16 demonstrated that
activation of MMP-2 occurred in association with pro-
gressive myocardial fibrosis and was independent of
ventricular dilatation. In contrast, although MMP-9
was somewhat activated in diastolic heart failure, its
activity was more enhanced in systolic heart failure
with left ventricular dilatation.16 Moreover, immuno-
histochemical study and in situ zymography demon-
strated that the region with increased gelatin lysis
corresponded to that with increased MMP-9 expres-
sion.16 The above findings suggest that MMP-9 is
likely to contribute to left ventricular dilatation irre-
spective of underlying cardiovascular diseases. Li et al17
reported downregulation of MMP-9 in patients with
dilated cardiomyopathy following support with left
ventricular assist devices. This suggests that an increased
left ventricular filling pressure and, hence, an elevated

J Chin Med Assoc • August 2006 • Vol 69 • No 8 347

 Y.T. Chiu, et al

Control Norepinephrine

A B

C D

E F
100 μm 100 μm

Figure 6. Normal collagens of myocardial extracellular matrix are coil-like fibers, which are parallel to the cardiomyocytes in picro-Sirius
(A, C) and silver impregnation staining (E). In norepinephrine-treated hearts, they became overstretched, progressively straightened,
and ultimately disrupted (B, D, F).

left ventricular wall stress are responsible for the disruption of ECM and left ventricular dilatation and
enhanced expression of MMP-9.17 Our results showed dysfunction, whereas MMP-2 was unchanged. This
that a high dose of NE of 30 μg/kg/minute could cardiac remodeling is different from that induced by a
increase MMP-9 expression, which resulted in the lower dose of NE. Previous studies demonstrated that

348 J Chin Med Assoc • August 2006 • Vol 69 • No 8

 NE induces disruption of extracellular matrix
NE at a dose of 1–6 μg/kg/minute induced hyper-
trophic remodeling of the heart, especially the left ven-
tricle.8–10,27–29 These findings indicate that a differential
remodeling response to NE is dose-dependent. A low
dose of NE increases MMP-2 expression, which is a
sign of the remodeling process accompanied by cardiac
fibrosis. However, a high dose of NE increases MMP-9
expression, which facilitates disruption of ECM and
results in ventricular dilatation.
In our model, an increase in blood pressure
occurred abruptly after NE administration and per-
sisted for about 60 minutes (Figure 1). However, it
was initially accompanied by a decrease and not an
increase in HR. There are some reasons for this. First,
hypertension can activate baroreflex and parasympa-
thetic activity. Second, high-dose NE can damage not
only the myocytes and ECM, but also the pacemaker
cells in the heart. We observed arrhythmia, including
ventricular premature beat, sinus bradycardia and
sinus arrhythmia in the initial 30 minutes. After that,
HR recovered at 60 minutes and became elevated at
120 minutes. The latter tachycardia might be due to
sympathetic compensation because of heart failure.
Many studies have revealed that the myocardial
damage in catecholamine cardiotoxicity is coagulative
myocytolysis, also known as myofibrillar degeneration
or contraction band necrosis, which is characterized
by sarcoplasmic coagulation, granulation, vacuoliza-
tion, and myofibrillar disruption.20,27,35 In addition to
its well-known effect on myocytes, NE can alter the
ECM and regulate cardiac remodeling. Our observa-
tions showed that the deformed collagen fibers in the
ECM caused by the huge dose of NE contributed to
the dilatation and dysfunction of the left ventricle.
In conclusion, an extremely high dose of NE
increases MMP-9 activity and causes disruption of the
ECM and left ventricular dilatation and dysfunction.
The inhibition of MMP-9 activity might be a thera-
peutic target for the prevention of NE-induced cardiac
remodeling.
Acknowledgments
This work was supported by a grant from Taichung
Veterans General Hospital (TCVGH-946511D).
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