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Am J Physiol Heart Circ Physiol 295: H1303–H1310, 2008;
doi:10.1152/ajpheart.01143.2007.
Prolonged administration of a dithiol antioxidant protects against ventricular
remodeling due to ischemia-reperfusion in mice
S. Kelly Ambler,1 Yvonne K. Hodges,1 Gayle M. Jones,1 Carlin S. Long,1,2 and Lawrence D. Horwitz1
1
Division of Cardiology, University of Colorado Denver, Aurora; and 2Denver Health Medical Center, Denver, Colorado
Submitted 2 October 2007; accepted in final form 18 July 2008
echocardiography; fetal gene expression; heart; hypertrophy; ribonuclease protection assay
THE PATHOLOGICAL REMODELING of the heart is a serious adverse
outcome of acute myocardial infarction, despite the widespread
application of reperfusion strategies. Ventricular remodeling,
defined as hypertrophy of the myocytes, fibrosis of the noninfarcted myocardium, and changes in the geometry of the
ventricle, presages the development of congestive heart failure
(4, 29). Ventricular remodeling is also associated with a pattern
of gene expression normally present in the fetal heart but
absent in the adult heart. This abnormal pattern of gene
expression generally accompanies decreased contractile function (22, 28). Changes in ventricular structure, function, and
gene expression have been described in patients with heart
failure (22, 35) and animal models of myocardial infarction (8,
Address for reprint requests and other correspondence: L. D. Horwitz, Div.
of Cardiology, B-130, Academic Office 1, 12631 E. 17th Ave., Rm. 7221A,
PO Box 6511, Aurora, CO 80045 (e-mail: lawrence.horwitz@uchsc.edu).
http://www.ajpheart.org
47). Similar abnormalities in gene expression have been induced in myocardial cell cultures (2, 33).
Oxidative stress has been proposed as an important regulator
of cardiac remodeling (24, 31). Cardiac ischemia-reperfusion
(I/R) triggers a vigorous inflammatory response involving
cytokine release, leukocyte activation, and generation of high
levels of reactive oxygen species (3, 7, 19, 41). Leukocyte
release of reactive oxygen species is very intense immediately
after the onset of reperfusion (6), and there is evidence that
antioxidant therapies given the first few hours of reperfusion
can reduce infarct size (12, 13, 25). However, less robust
increases in oxidative activity, generated from mitochondria or
other sources, may persist for weeks or months (24, 40). A few
studies in genetically altered murine models of permanent
coronary artery occlusion or models of pressure overload have
provided evidence that the long-term generation of oxidative
stress may cause ventricular dysfunction (32, 39, 46). In
addition, in vitro cell culture models support a role for oxidative signaling in the regulation of pathological remodeling at
the cellular level (2, 9, 36). Nevertheless, in two recent human
clinical studies, the administration of the antioxidant vitamin E
was unsuccessful in preventing the development of heart failure (20, 23). Therefore, the importance of oxidative processes
in remodeling or the subsequent development of heart failure
has not been clearly established, particularly following early
reperfusion of myocardial infarctions.
We investigated the long-term protective effects of bucillamine, a potent antioxidant dithiol donor (1, 11, 13, 43) on
ventricular remodeling following myocardial I/R injury in
genetically normal mice. We found that daily treatment with
bucillamine, started during reperfusion and continued for 4 wk,
attenuated ventricular dysfunction and reduced pathological
patterns of myocardial gene expression without altering infarct
size.
MATERIALS AND METHODS
Experimental animals. All procedures were conducted in conformance with the National Institutes of Health’s Guide for the Care and
Use of Laboratory Animals and were approved by the University of
Colorado at Denver and Health Sciences Center Institutional Animal
Care and Use Committee. C57Bl/6J mice (11 to 12 wk old) were
purchased from Jackson Laboratory (Bar Harbor, ME) and allowed to
acclimate for 1 wk before any experimental intervention.
Surgical generation of myocardial I/R in mice. Mice were anesthetized by an injection of 2% 2,2,2-tribromoethanol (0.66 mg/g ip;
Aldrich Chemical, St. Louis, MO). The mice were then orally intubated with a 20-gauge angiocath and mechanically ventilated with
90% O2-10% room air at a tidal volume of 0.4 ml and a rate of 120
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0363-6135/08 $8.00 Copyright © 2008 the American Physiological Society
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Ambler SK, Hodges YK, Jones GM, Long CS, Horwitz LD. Prolonged administration of a dithiol antioxidant protects against ventricular
remodeling due to ischemia-reperfusion in mice. Am J Physiol Heart Circ
Physiol 295: H1303–H1310, 2008. doi:10.1152/ajpheart.01143.2007.—
The prolonged production of reactive oxygen species due to ischemiareperfusion (I/R) is a potential cause of the pathological remodeling
that frequently precedes heart failure. We tested the ability of a potent
dithiol antioxidant, bucillamine, to protect against the long-term
consequences of I/R injury in a murine model of myocardial infarction. After transiently occluding the left anterior descending coronary
artery for 30 min, saline or bucillamine (10 ␮g/g body wt) was
injected intravenously as a bolus within the first 5 min of reperfusion.
The antioxidant treatment continued with daily subcutaneous injections for 4 wk. There were no differences in infarct sizes between
bucillamine- and saline-treated animals. After 4 wk of reperfusion,
cardiac hypertrophy was decreased by bucillamine treatment (ventricular weight-to-body weight ratios: I/R ⫹ saline, 4.5 ⫾ 0.2 mg/g vs.
I/R ⫹ bucillamine, 4.2 ⫾ 0.1 mg/g; means ⫾ SE; P ⬍ 0.05).
Additionally, the hearts of bucillamine-treated mice had improved
contractile function (echocardiographic measurement of fractional
shortening) relative to saline controls: I/R ⫹ saline, 32 ⫾ 3%, versus
I/R ⫹ bucillamine, 41 ⫾ 4% (P ⬍ 0.05). Finally, I/R-induced injury
in the saline-treated mice was accompanied by a fetal pattern of gene
expression determined by ribonuclease protection assay that was
consistent with pathological cardiac hypertrophy and remodeling
[increased atrial natriuretic peptide, ␤-myosin heavy chain (MHC),
skeletal ␣-actin; decreased sarco(endo)plasmic reticulum Ca2⫹
ATPase 2a, and ␣-MHC-to-␤-MHC ratio]. These changes in gene
expression were significantly attenuated by bucillamine. Therefore,
treatment with a dithiol antioxidant for 4 wk after I/R preserved
ventricular function and prevented the abnormal pattern of gene
expression associated with pathological cardiac remodeling.
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ware. The region at risk and infarct size were determined using the
following equations: Weight of region of interest ⫽ (A1 ⫻ Wt1) ⫹
(A2 ⫻ Wt2) ⫹ (A3 ⫻ Wt3) ⫹ (A4 ⫻ Wt4), where A is area of the
region of interest determined by planimetry from each of the four
heart sections and Wt is the weight of each section. Region of interest
as percentage of the left ventricle ⫽ (Wt of region of interest/Wt of
left ventricle) ⫻ 100%.
Tissue harvest. At the end of the 28-day experimental interval, the
mice were weighed before heart excision. The excised hearts were
rinsed in cardioplegia solution containing (in mM) 140.0 NaCl, 5.4
KCl, 1.0 MgSO4, 1.0 Na2HPO4, 11.0 glucose, 15.0 BES, 1.0 EGTA,
and 30.0 2,3-butanedione monoxime and 0.1% BSA (pH 7.4), and the
atria and major vessels were removed. The combined ventricles were
blotted dry, weighed, and stored in RNALater (Ambion, Austin, TX)
at ⫺20°C. The lung and liver were also excised, trimmed of vascular
tissue, blotted dry, and weighed.
Ribonuclease protection assay. Tissue RNA was purified using
TRIzol reagent (Invitrogen, Carlsbad, CA). Blinded analysis of myocardial mRNAs was performed by ribonuclease protection assay using
a cassette of mouse cardiac riboprobes as described previously (14).
Briefly, 10 ␮g aliquots of RNA were hybridized with [32P]-labeled
anti-sense probes for 1) cardiac specific genes, including murine ␣and ␤-myosin heavy chain (MHC), sarco(endo)plasmic reticulum
Ca2⫹ ATPase 2a (SERCA2a), atrial natriuretic peptide (ANP), skeletal ␣-actin, and GAPDH (internal control) or 2) cytokine genes,
including interleukin-1␤ (IL-1␤), IL-6, tumor necrosis factor-␣ (TNF-␣),
and GAPDH (BD Biosciences, San Jose, CA). Unhybridized RNA was
digested with RNase, and the protected fragments were separated by polyacrylamide gel electrophoresis. The detection and quantitation of the individual protected fragments were accomplished by Phosphorimager densitometry
(Molecular Dynamics, Sunnyvale, CA). The densitometry values for each
mRNA species were normalized to the GAPDH mRNA signal from the
same sample to correct for variations in RNA loading.
Experimental groups. The mice to be analyzed after 4 wk of
reperfusion were randomly assigned to one of four experimental
groups: 1) a sham-operated group not exposed to I/R and injected
daily for 4 wk with control saline solution (Sham ⫹ saline), 2) a group
exposed to I/R and treated with daily injections of saline for 4 wk
(I/R ⫹ saline), 3) a sham-operated group not exposed to I/R and
injected daily with bucillamine for 4 wk (Sham ⫹ bucillamine), and
4) a group exposed to I/R and treated with daily injections of
bucillamine for 4 wk (I/R ⫹ bucillamine).
Mice to be analyzed for infarct size after euthanasia at 48 h were
randomly assigned to two groups: 1) a group assigned to I/R that
received bucillamine as described above for 2 days of I/R (I/R ⫹
bucillamine) and 2) a group assigned to I/R that received saline as
described above for 2 days of I/R (I/R ⫹ saline).
Data analysis. All data were expressed as means ⫾ SE. Statistical
comparisons between treatment groups were performed by two-way
ANOVA with Bonferroni’s post test (GraphPad Software, San Diego,
CA). A P ⬍ 0.05 was considered statistically significant.
RESULTS
Infarct size. Mice receiving bucillamine or saline (control)
were euthanized after 48 h of reperfusion for the determination
of infarct size. Individual values of infarct size as a percentage
of the left ventricle weight are plotted in Fig. 1. The area-atrisk measurements were 37 ⫾ 5% (saline) versus 36 ⫾ 6%
(bucillamine). The infarct size measurements were 14 ⫾ 6%
(saline) versus 15 ⫾ 8% (bucillamine). There were no significant differences or trends between the two groups.
The 4-wk experimental model. There were no apparent
adverse reactions to bucillamine during the 4-wk treatment
period in any of the treated mice. The gain in body weight was
similar and did not differ statistically among the experimental
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breaths/min (model CIV-101, Columbus Instruments, Columbus,
OH). The heart was accessed via a parasternal thoracotomy at the
fourth intercostal space and a 7-0 silk suture passed under the left
anterior descending coronary artery (LAD) at the point where it
emerged from under the left atrial flap. Myocardial ischemia was
achieved by occluding the LAD against a 22-gauge J-shaped stainless
steel probe and verified by visually noting the regional akinesis and
blanching of the left ventricle. The chest was closed in layers, with the
long end of the probe remaining outside the chest wall, allowing the
animal to be removed from the ventilator. After 30 min of ischemia,
reperfusion was initiated by carefully pulling the probe out from under
the ligature and then removing it from the chest cavity. Other
investigators have shown that a 30-min ischemic interval in wild-type
mice results in infarction of ⬃50% of the area at risk (45). Following
the surgical procedure, the mouse was allowed to recover on a
warmed surface, with supplemental oxygen delivered through a nose
cone. Sham-operated animals underwent all procedures described,
except the actual occlusion of the LAD. I/R was verified by three-lead
electrocardiograms, which were obtained preoperatively, at the end of
the ischemic interval and immediately after the initiation of reperfusion. Mice fully recovered from the surgical procedure were returned
to standard animal housing conditions. Postsurgical pain was controlled with buprenorphine injections (2 ␮g/g sc, bid) for the first 48
hr following surgery and acetaminophen (2 mg/ml, ad libitum in the
drinking water) for 7 days.
Bucillamine treatment. Powdered bucillamine (⬎99% purity) was
obtained from Keystone Biomedical (Los Angeles, CA). Stock solutions of bucillamine (5 mg/ml) were made in normal saline, pH
adjusted to ⬃7.4 with equimolar NaOH, and filter sterilized. Within 5
min of reperfusion being initiated, an intravenous bolus of bucillamine
(10 ␮g/g) was administered via tail-vein injection. The mice were
subsequently treated with daily injections of bucillamine (10 ␮g/g sc),
rotating the injection sites. Control mice received saline injections.
Echocardiography. Cardiac function was assessed in the University
of Colorado at Denver Small Animal Hemodynamic Core Facility by
two-dimensional transthoracic echocardiography (echo). The mice
were sedated with intraperitoneal injections of fentanyl (34 ng/g) plus
droperidol (1.7 ␮g/g) to maintain heart rates consistently above 550
beats/min. Echoes were obtained with an HP Sonos 5500 echocardiograph machine using a 15-MHz linear array intraoperative probe
(Philips Ultrasound, Andover, MA). Parasternal short-axis views,
long-axis views, and M-modes (at the level of the short axis) were
routinely obtained. Echo images were obtained on the mice 4 – 6 days
before surgical intervention (baseline) and then 4 wk following
surgery just before death. All analyses were performed off-line by an
individual blinded as to treatment status.
Infarct size. Region at risk and infarct size were determined in mice
that underwent I/R surgery as described in Surgical generation of
myocardial I/R in mice, except that a slip knot was tied in the suture
used to occlude the LAD. Forty-eight hours after reperfusion began,
the mice were anesthetized and heparinized, and the hearts were
excised. The slip knot was then pulled taut to reocclude the LAD. The
aorta was cannulated and the heart perfused with 10 ml of cardioplegia solution containing (in mM) 140.0 NaCl, 15.0 KCl, 1.0 MgSO4,
1.0 Na2HPO4, 11.0 glucose, 15.0 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 10.0 EGTA, and 30.0 2,3-butanedione
monoxime and 0.10% BSA and 10 U/ml heparin. Regions of the heart
still receiving blood flow during LAD occlusion were identified by
perfusion with 5 ml of 2% Evans blue. After the heart was removed
from the cannula, the atria and right ventricle were trimmed away
before transversely slicing the left ventricle into four sections. Infarcted myocardium was identified by incubating the heart slices with
1% triphenyltetrazolium chloride at 37°C for 15 min. Each slice was
weighed and then imaged with a Nikon SMZ800 stereoscope
equipped with a Cool-Snap CCD camera. Perfused (dark blue),
nonperfused but noninfarcted (brick red), and infarcted (white) myocardial regions were quantitated by planimetry using ImagePro soft-
OXIDATIVE INJURY AND VENTRICULAR REMODELING
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Following sham operations, contractile function, measured
as fractional shortening (FS), remained constant over the 4-wk
interval in both the saline- and bucillamine-treated mice (Table
1). However, I/R injury resulted in a large and highly significant (67%, P ⬍ 0.01) decrease in FS in the I/R ⫹ saline mice
compared with the Sham ⫹ saline mice (Fig. 4). The decrease
in FS caused by I/R was significantly attenuated in the I/R ⫹
bucillamine group (P ⬍ 0.05 compared with the I/R ⫹ saline
groups (Fig. 2A). All mice exposed to I/R had visible infarcts
postmortem.
Twenty-one of the 22 mice originally receiving injections
survived the 4-wk treatment interval. One mouse that had
undergone I/R injury and had received saline injections died
from cardiac rupture 5 days after surgery. The final animal
numbers per group were as follows: Sham ⫹ saline (n ⫽ 6),
I/R ⫹ saline (n ⫽ 5), Sham ⫹ bucillamine (n ⫽ 4), and I/R ⫹
bucillamine (n ⫽ 6).
There were no statistical differences in lung weights or liver
weights between the different groups (data not shown).
Cardiac mass. Figure 2B shows the cardiac mass, as defined
by the heart weight-to-body weight ratio, in the four groups of
mice. There was a significant increase in cardiac mass (13%,
P ⬍ 0.05) in the I/R ⫹ saline group compared with the
Sham ⫹ saline group 4 wk after I/R exposure. Bucillamine had
no effect on cardiac mass in the sham-operated mice. The
increase in heart weight-to-body weight ratio in response to I/R
was attenuated in the I/R ⫹ bucillamine group and was
statistically indistinguishable from the Sham ⫹ bucillamine
group. Thus bucillamine provided a long-term protective effect
against the cardiac hypertrophic response to injury following
I/R injury.
Cardiac function. Figure 3 shows M-mode echo tracings
from individual mice representative of each experimental
group. The echo measurement data compiled for each experimental group are shown in Table 1. I/R injury caused a
statistically significant increase in the left ventricular endsystolic diameter in the I/R ⫹ saline group compared with the
Sham ⫹ saline group. The end-diastolic diameter tended to
increase in the I/R ⫹ saline group compared with the Sham ⫹
saline group, but the results did not reach statistical significance. There were no differences in ventricular dimensions
between the saline- and bucillamine-treated sham-operated
mice. Neither end-diastolic diameter nor end-systolic diameter
was statistically different between the I/R ⫹ bucillamine group
and either of the two Sham groups. No significant differences
were seen in septal or posterior wall thicknesses in any group,
although there was a trend toward an increase in posterior wall
thickness in the I/R ⫹ saline group compared with the Sham ⫹
saline group.
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Fig. 1. Infarct sizes in bucillamine-treated and control mice. Infarct size as a
percentage of the left ventricle was measured after 48 h of reperfusion as
described in MATERIALS AND METHODS. Individual values for each mouse are
represented by circles, and the mean value for each group is designated by a
horizontal line.
Fig. 2. Bucillamine protects against cardiac hypertrophy following ischemiareperfusion (I/R) injury. A: body mass. Each mouse was weighed before
surgery, weekly during the treatment interval, and just before death. Group
averages are shown for the weights just before surgery (presurgery) and at the
end of the experiment interval [28-day (28d) postsurgery]. B: cardiac mass (n,
number of mice/group). Twenty-eight days after surgical induction of I/R
injury, hearts were excised and trimmed of atria and major blood vessels. The
ventricles were blotted dry before weighing. Ventricular weights were normalized to body weights for each mouse (heart weight-to-body weight ratio,
in mg/g).
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Fig. 3. Representative echocardiographic M-mode
tracings. Echocardiograms were obtained as detailed
in MATERIALS AND METHODS. M-mode echoes representative for each experimental group are shown.
prevented by bucillamine treatment (Fig. 5E). Finally,
SERCA2a expression significantly decreased following I/R
injury in the saline-treated mice, and this decrease was attenuated by bucillamine (Fig. 5F).
Thus, 4 wk after I/R injury, the pathological pattern of gene
expression (increased ␤-MHC, ANP, and skeletal ␣-actin; and
decreased ␣-MHC, ␣-MHC-to-␤-MHC ratio, and SERCA2a
mRNA) appeared in saline-treated mice but was attenuated by
bucillamine.
Cytokine gene expression. Since redox activity early in
ischemia may stimulate cytokine activation, we tested whether
the protection of cardiac function by bucillamine was mediated
by prolonged alterations in myocardial cytokine gene expression. No statistically significance or trend toward differences in
the cardiac expression of IL-1␤, IL-6, or TNF-␣ was seen
among the experimental groups (data not shown).
DISCUSSION
It is well established that early reperfusion of the infarcted
myocardium results in the generation of high levels of oxidative activity. Considerable attention has been paid to possible
reduction in infarct size by the acute administration of antioxidants or other anti-inflammatory measures during the early
stages of reperfusion (12, 13, 25). However, there has also been
evidence of the prolonged elevation of oxidative activity for
weeks or months following I/R (10, 24, 40), and little is known
about its importance in the long-term recovery from acute
myocardial infarction. This study demonstrates that a thiol
Table 1. Cardiac dimensions and functional parameters
Treatment Groups
n
LVDd, mm
LVDs, mm
IVSd, mm
PWd, mm
Presurgery
28-day postsurgery
Saline
Sham
I/R
Bucillamine
Sham
I/R
21
2.9⫾0.1
6
5
4
6
FS, %
HR, beats/min
1.3⫾0.1
0.96⫾0.02
0.94⫾0.03
56⫾2
639⫾9
3.2⫾0.2
3.8⫾0.3†
1.5⫾0.2
2.5⫾0.3*†
1.02⫾0.04
0.95⫾0.03
0.87⫾0.07
0.99⫾0.06
55⫾3
32⫾3*†
677⫾4
679⫾8
2.9⫾0.2
3.3⫾0.2
1.4⫾0.1
2.0⫾0.2
1.04⫾0.05
1.02⫾0.06
0.95⫾0.06
0.87⫾0.04
51⫾2
41⫾4
668⫾20
668⫾7
Values are means ⫾ SE for each animal group; n, number of animals in each group. Left ventricular dimensions were obtained from short-axis
two-dimensional-guided M-mode echoes. Measurements were obtained over 3 separate contractile cycles and then averaged to obtain mean values for each
animal before obtaining the group means. Left ventricular internal diameter (LVD) was measured at end diastole (d) and end systole (s). Interventricular septal
(IVS) and posterior wall (PW) thicknesses were measured at end diastole (d). Heart rate (HR) was calculated from diastole-to-diastole intervals. Percent fractional
shortening (FS) for each animal was calculated as FS ⫽ 关(LVDd ⫺ LVDs)/LVDd兴 ⫻ 100%. *P ⬍ 0.01, ischemia-reperfusion (I/R) ⫹ saline vs. Sham ⫹ saline;
†P ⬍ 0.05, I/R ⫹ saline vs. Sham ⫹ bucillamine.
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group). Therefore, long-term treatment with bucillamine preserves cardiac contractile function following I/R injury.
Fetal gene expression. Expression of fetal isoforms of several
genes has been observed in pathological cardiac hypertrophy (8,
22, 33, 35, 47). For example, the expression of the natriuretic
peptides, ANP and brain natriuretic peptide, the skeletal isoform
of ␣-actin, and the ␤-isoform of MHC are increased. In contrast,
the expression of the SERCA2a gene decreases in pathological
hypertrophy. The expression of the ␣-isoform of MHC is also
decreased, resulting in a further decrease in the ␣-MHC-to-␤MHC ratio. We compared the myocardial expression of these
genes in saline- and myocardial-treated mice 4 wk after I/R
injury.
Figure 5 shows the effects of I/R injury, without or with
bucillamine treatment, on the expression of the fetal isoforms
of specific cardiac genes. The data were expressed as the
abundance of each mRNA species relative to the sham-operated controls. I/R injury resulted in a significant increase in
␤-MHC expression in the saline-treated mice. This increase
was prevented by long-term bucillamine treatment (Fig. 5A).
I/R injury in the saline-treated mice also resulted in increases
in ANP and skeletal ␣-actin expression (Fig. 5, B and C).
Again, bucillamine prevented the increases in the expression of
these genes.
As shown in Fig 5D, I/R tended to decrease the expression
of ␣-MHC in both the saline- and bucillamine-treated mice,
although these results did not reach statistical significance.
However, I/R caused a statistically significant decrease in the
␣-MHC-to-␤-MHC ratio in the saline-treated mice, which was
OXIDATIVE INJURY AND VENTRICULAR REMODELING
donor antioxidant administered daily for 1 mo after I/R markedly attenuates ventricular remodeling in genetically normal
mice. In previous studies of transgenic animals, either antioxidant capacity was impaired or there was exaggerated oxidative
activity before, during, and after the initiation of ischemia.
Bucillamine [N-(2-mercapto-2-methylpropionyl)-L-cysteine]
is a member of a group of low molecular weight, cysteinederived thiol donors that includes N-acetylcysteine (NAC) and
N-2-mercaptopropionyl glycine (MPG). These compounds
readily enter cells through the cysteine transport pathway and
exert their antioxidant effect by maintaining the endogenous
glutaredoxin and thioredoxin systems in a reduced state by
transfer of thiol groups (1, 43). Bucillamine contains two
donatable thiol groups, making it a considerably more potent
antioxidant than NAC or MPG, which each contain only one
thiol group (11, 13, 43). Based on the proven effectiveness of
bucillamine in counteracting oxidative stress (1, 11, 13, 26, 38,
43), we tested whether bucillamine would reduce adverse
remodeling following I/R. In our investigator blinded and
sham-surgery controlled study, a long-term treatment with
bucillamine attenuated the increase in cardiac mass, loss of
contractile function, and pathological gene expression observed in saline-treated mice 4 wk after I/R injury.
A few previous studies have suggested the potential of
antioxidant therapy to prevent long-term remodeling processes
due to oxidative stress. Yamamoto et al. (46) reported that
transgenic mice overexpressing a dominant negative form of
thioredoxin, an endogenous antioxidant, developed cardiac
hypertrophy in the absence of exogenous stress. This hypertrophy was prevented by 4 wk of treatment with MPG. Dimethylthiourea, which has antioxidant effects but also blocks
sodium/calcium exchange by an unrelated mechanism (48),
improved ventricular function when given after coronary ligation in mice (15). Treatment with the antioxidant flavonoid,
7-monohydroxyethylrutoside, before ischemia partially preserved cardiac contractile responses after I/R in mice (5),
although these authors did not examine cardiac function beyond 2 wk. The knockout of the myeloperoxidase gene in mice
protects against the loss of cardiac function 24 days after I/R,
without altering infarct size (42). Adenoviral transfer of heme
oxygenase-1 to rat myocardium prevented I/R-induced cardiac
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fibrosis and ventricular remodeling for up to 3 mo (18). Our
study extends these results to show that a sustained delivery of
a thiol donor antioxidant after the onset of reperfusion in
normal mice attenuates long-term I/R-induced cardiac hypertrophy, loss of cardiac contractile function, and pathological
patterns of gene expression. Oxidative activity immediately at
the onset of reperfusion may primarily affect cytokine release
and acute inflammatory responses (27), whereas later oxidative
activity may have a greater influence on genes that control
myocyte function and size (2, 9). Thus the benefits of antioxidant therapy following I/R may extend beyond the acute injury
phase and continue during the chronic phase of myocardial
remodeling.
Although oxidative stress is a determinant of infarct size in
the acute response to I/R injury (12, 13, 25), there is not
necessarily a consistent correlation between infarct size and the
subsequent development of ventricular remodeling. As alluded
to above, Vasilyev et al. (42) found that mice deficient in
myeloperoxidase (an enzyme that catalyzes the generation of
reactive oxygen species in leukocytes) had improved cardiac
function 24 days after I/R compared with wild-type mice,
despite equivalent infarct sizes. Additionally, in an ischemia
model without reperfusion, Shiomi et al. (32) demonstrated
that an overexpression of glutathione peroxidase in transgenic
mice prevented ventricular remodeling and heart failure, independently of infarct size. Our data are in agreement with these
published studies showing an improvement in cardiac function
following antioxidant treatment, despite equivalent infarct
sizes in treated and control animals. Thus it is likely that the
development of ventricular remodeling is not solely related to
the amount of damage sustained during or immediately following ischemia but, rather, is strongly influenced by the evolving
response to injury in the surviving myocardium.
The analysis of gene expression in heart tissue from human
patients has identified a pattern of abnormal myocardial gene
expression that occurs with cardiac remodeling (22, 35). Similar changes are observed in rodent models (8, 47), although the
magnitude of the responses of individual genes differs among
species. Although it is not necessarily the case that all the
changes in genetic expression have direct functional consequences, decreases in SERCA2a expression and the ␣-MHCto-␤-MHC ratio are associated with the loss of myocyte contractility (22, 28). Furthermore, sustained decreases in
SERCA2a protein levels are thought to contribute to the loss of
cardiac function during the progression to heart failure (28).
Therefore, the attenuation of I/R-induced decreases in SERCA
and ␣-MHC-to-␤-MHC ratio by antioxidant therapy, as seen in
this study, has potential usefulness for the prevention of heart
failure. Indeed, in human clinical studies, the improvement of
cardiac function through ␤-adrenergic blockade (21) or left
ventricular assist devices (44) correlated with a reversal of the
pathological pattern of gene expression.
A bucillamine-induced attenuation of the expression of the
pathological pattern of gene expression may occur through the
prevention of abnormal oxidative signaling. Oxidative stress
induces pathological gene expression in isolated cardiac myocytes (2). In addition, SERCA2a mRNA levels are reduced by
oxidative stress in the acute phase of I/R injury (37). Thus
antioxidant therapy may modulate abnormal signaling pathways that alter myocardial gene transcription following I/R
injury. Finally, the prevention of adverse remodeling by buci-
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Fig. 4. Bucillamine protects against the loss of cardiac contractile function
following I/R injury. Fractional shortening (FS) was measured as described in
Table 1. The change in FS over the experimental interval in each individual
animal was calculated as (FSpre ⫺ FS28d)/FSpre ⫻ 100%, where FSpre is FS
before surgery and FS28d is FS 28 days after surgery. The individual values
were averaged to determine the means ⫾ SE for each experimental group.
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Fig. 5. Bucillamine attenuates the expression of the pathological fetal gene program
following I/R injury. Ventricular tissue was
analyzed by ribonuclease protection assay
for expression of pathological marker gene
mRNAs as described in MATERIALS AND
METHODS. Individual values for each mRNA
species were normalized to the mean sham
value (saline or bucillamine, as appropriate)
for the same mRNA. Each panel indicated
the individual mRNA species measured, as
follows: ␤-myosin heavy chain (␤-MHC; A);
atrial natriuretic peptide (ANP; B); skeletal
␣-actin (C); ␣-MHC (D); ␣-MHC-to-␤MHC ratio (E); and sarco(endo)plasmic reticulum Ca2⫹ ATPase 2a (SERCA2a; F).
llamine could indirectly result in a more physiological pattern
of gene expression due to a decreased stress environment in the
heart.
Despite evidence in animal models that increased levels of
oxidant stress are associated with cardiac remodeling and
development of heart failure, vitamin E, a lipid-soluble antiAJP-Heart Circ Physiol • VOL
oxidant, did not reduce the incidence of heart failure either in
patients with established atherosclerotic disease (20) or with
recent myocardial infarction (23). Vitamin E largely exerts its
antioxidant effects by preventing lipid peroxidation. However,
lipid peroxidation is only one of the mechanisms by which
adverse effects of oxidative stress may occur. The prevention
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OXIDATIVE INJURY AND VENTRICULAR REMODELING
ACKNOWLEDGMENTS
We gratefully acknowledge Nancy Sherman and Ursula Jiron for daily
mouse care, Dr. Ping Yue for performing the echo measurements, and Dr. R.
Dale Brown for insightful discussions and critical review of the manuscript.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-55291 (to L. D. Horwitz) and HL-66399 and HL-79160 (to C. S.
Long).
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