Inhibition of Copper-Zinc Superoxide Dismutase Induces
Cell Growth, Hypertrophic Phenotype, and Apoptosis in
Neonatal Rat Cardiac Myocytes In Vitro
Deborah A. Siwik,* John D. Tzortzis,* David R. Pimental, Donny L.-F. Chang, Patrick J. Pagano,
Krishna Singh, Douglas B. Sawyer, Wilson S. Colucci
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Abstract—Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that
inhibition of endogenous antioxidant enzymes can regulate the phenotype of cardiac myocytes. Neonatal rat ventricular
myocytes in vitro were exposed to diethyldithiocarbamic acid (DDC), an inhibitor of cytosolic (Cu, Zn) and extracellular
superoxide dismutase (SOD). DDC inhibited SOD activity and increased intracellular superoxide in a concentrationdependent manner. A low concentration (1 mmol/L) of DDC stimulated myocyte growth, as demonstrated by increases
in protein synthesis, cellular protein, prepro–atrial natriuretic peptide, and c-fos mRNAs and decreased sarcoplasmic
reticulum Ca21ATPase mRNA. These actions were all inhibited by the superoxide scavenger Tiron (4,5-dihydroxy1,3-benzene disulfonic acid). Higher concentrations of DDC (100 mmol/L) stimulated myocyte apoptosis, as evidenced
by DNA laddering, characteristic nuclear morphology, in situ terminal deoxynucleotidyl transferase–mediated nick
end-labeling (TUNEL), and increased bax mRNA expression. DDC-stimulated apoptosis was inhibited by the
SOD/catalase mimetic EUK-8. The growth and apoptotic effects of DDC were mimicked by superoxide generation with
xanthine plus xanthine oxidase. Thus, increased intracellular superoxide resulting from inhibition of SOD causes
activation of a growth program and apoptosis in cardiac myocytes. These findings support a role for oxidative stress in
the pathogenesis of myocardial remodeling and failure. (Circ Res. 1999;85:147-153.)
Key Words: superoxide dismutase n superoxide n myocyte n hypertrophy n apoptosis
S
uperoxide is a “foundation” radical that may lead to the
formation of the reactive oxygen species hydrogen peroxide,1 hydroxyl radical,2 and peroxynitrite.3 Superoxide is
produced intracellularly by electron leakage from mitochondria during oxidative phosphorylation and by activation of
several cellular enzymes, including NADPH oxidases,4 cyclooxygenase, nitric oxide synthase,5 and xanthine oxidase.
Antioxidant enzymes, including superoxide dismutase
(SOD), catalase, and peroxidases protect cells by maintaining
O22 and H2O2 at low levels.6
Studies in animal models suggest that a chronic increase in
oxidative stress in the myocardium, possibly due to impairment of SOD and other antioxidant pathways, could contribute to myocardial remodeling and failure.7,8 Although the
mechanism by which oxidative stress might cause myocardial
remodeling is not clear, oxidative stress has been implicated
as a mediator of both cell death9 and cell growth.10,11
Furthermore, Cheng et al12 showed that mechanical stretch of
papillary muscle increased superoxide and caused apoptosis
and that both effects were inhibited by nitric oxide, leading to
the suggestion that superoxide mediates stretch-induced apoptosis in cardiac myocytes.
We hypothesized that an increase in superoxide due to
inhibition of SOD would affect the growth and survival of
cardiac myocytes. To test this thesis, Cu, Zn SOD was
inhibited in a graded manner with the copper chelator
diethyldithiocarbamic acid (DDC)13 in ventricular myocytes
cultured from neonatal rats.
Materials and Methods
Neonatal Rat Ventricular Myocytes (NRVMs)
NRVMs were prepared as previously described.14 Myocytes were
plated at a density of 300 cells/mm2 on 24-well plates (Costar),
12-mm-diameter coverslips in 24-well plates, or 35- or 100-mm
dishes (Falcon) for 24 hours in DMEM (GIBCO) containing 7%
(vol/vol) heat-inactivated FBS (GIBCO) and 1% (vol/vol) penicillinstreptomycin (GIBCO). The culture medium was changed to serumfree DMEM for 24 hours before exposure to experimental treatments. DDC (Sigma), alone or in combination with the
nonenzymatic superoxide scavenger Tiron (4,5-dihydroxy-1,3benzene disulfonic acid, Sigma)15 or the SOD/catalase mimetic
EUK-8 (Eukarion),16 was added for 24 hours or as indicated. In other
Received October 19, 1998; accepted April 8, 1999.
*Both authors contributed equally to this study.
From the Myocardial Biology Unit, Boston University School of Medicine, and Cardiovascular Division, Boston University Medical Center, Boston,
Mass. Present address of P.J.P is Henry Ford Hospital, Detroit, Mich.
Correspondence to Wilson S. Colucci, MD, Cardiovascular Division, Boston University Medical Center, 88 East Newton St, Boston, MA 02118. E-mail
[email protected]
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
147
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Circulation Research
July 23, 1999
experiments, the combination of xanthine (500 mmol/L; Sigma) plus
xanthine oxidase (0.1 mU/mL; Boehringer Mannheim), referred to as
XXO, was added to cells. Control cells were treated with DMEM
alone.
SOD Activity
SOD activity was measured by the inhibition of pyrogallol autooxidation.17 NRVMs plated on 100-mm dishes were treated for 7
hours with DDC. The cells were trypsinized, centrifuged, and
resuspended in the assay buffer containing 50 mmol/L Tris (pH 8.2)
and 1 mmol/L diethyltriamine pentoacetic acid. The cells were
sonicated and centrifuged at 800g for 5 minutes. Protein concentration in the supernatant was determined by Bradford assay against a
BSA standard (Bio-Rad protein assay dye reagent concentrate).
Pyrogallol (Sigma) auto-oxidation was measured as the rate of
change of the absorbance at 420 nm over 5 minutes of 200 mmol/L
pyrogallol, diluted from acidic stock into assay buffer, in the
presence of 1200 U/mL catalase (Sigma).
Northern Hybridization
NRVMs plated on 100-mm dishes were treated for 24 hours or as
indicated. Total RNA isolations and Northern hybridizations with
32
P-labeled full-length cDNA of rat prepro–atrial natriuretic peptide
(ANP), rabbit sarcoplasmic-endoplasmic reticulum Ca21 ATPase
(SERCA2), rat c-fos, or rat bax20 were performed essentially as
previously described.14 Blots were exposed to a phosphor screen for
2 to 3 hours and quantified with a phosphor imager (GS-363,
Bio-Rad) or exposed to XOMAT-AR film (Kodak, Rochester, NY)
overnight and quantified with an imaging densitometer (GS-700,
Bio-Rad) using Molecular Analyst software (Bio-Rad). mRNA
levels were normalized to 18S rRNA determined by reprobing blots
with a 32P-labeled oligonucleotide complementary to 18S rRNA.
Mitogen-Activated Protein Kinase Activity
NRVMs plated on 100-mm dishes were treated as indicated. The
activities of the extracellular signal–regulated kinases (ERK)-1/
ERK2 (p44/p42 mitogen-activated protein kinase) were measured as
phosphorylation of myelin basic protein by immunoprecipitated
ERK1/ERK2.21
Lucigenin-Enhanced Chemiluminescence
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DDC-enhanced production of superoxide was measured in NRVMs
plated on 35-mm dishes. DDC (1 mmol/L) was added to the medium
for 24 hours before superoxide measurements. The medium was
removed and replaced with physiological buffer (in mmol/L: NaCl
119, HEPES 20, KCl 4.6, MgSO4 1, Na2HPO4 0.15, KH2PO4 0.4,
NaHCO3 5, CaCl2 1.2, and glucose 11.1, pH 7.4). Lucigenin
(100 mmol/L; bis-N-methylacridinium nitrate, Sigma) was added to
the dishes and allowed to equilibrate for 10 minutes at 37°C.
Luminescence, measured with a Turner 20/20 luminometer, was
integrated over 30-second intervals for a total of 5 minutes at room
temperature. Background luminescence was determined in the presence of the nonenzymatic superoxide scavenger Tiron (1 mmol/L).
Superoxide levels are reported as Tiron-inhibited arbitrary units per
minute.18
Cell Number and Membrane Integrity
Nitroblue Tetrazolium (NBT) Reduction
DNA Laddering
NRVMs plated on 100-mm dishes were treated with DDC for 24
hours, and the medium was replaced with physiological buffer with
NBT (100 mmol/L; Sigma) for 90 minutes. Cells were collected and
centrifuged for 10 minutes at 12 000g, and the resulting pellet was
resuspended in pyridine (100 mmol/L; Sigma). Formazan, the product of the reaction of superoxide with NBT, was extracted by heating
the samples at 80°C for 90 minutes and was measured by absorbance
at 540 nm. The quantity of formazan was calculated as NBT
reduction5A z V4(T z n z e z l), where A is the absorbance value at
540 nm, V is the volume of solution, T is the time period of NBT
incubation, n is the number of cells, e is the extinction coefficient of
formazan (0.72 mmol/L21 z mm21), and l is the length of the light
path. Nonspecific NBT reduction was determined by addition of
EUK-8 (100 mmol/L) at the time of DDC treatment. Superoxide
levels are reported as EUK-8 –inhibited NBT reduction. Tiron was
not used in these experiments because it interferes with the solubility
of NBT.19
Protein Synthesis
NRVMs were plated on 24-well plates, and [3H]leucine incorporation was determined as previously described.14 To account for
possible changes in cell number with experimental treatment, cell
number was determined in parallel plates as described below, and
[3H]leucine incorporation is reported as dpm/1000 cells.
Protein Content
Total protein content was determined in 24-well plates by Bradford
assay against a BSA standard. Cell number was determined in
parallel plates as described below, and protein values are reported as
mg protein/1000 cells.
Cell number was determined in 24-well plates treated with DDC or
XXO, with or without Tiron for 24 hours. An aliquot of the medium
(including floating cells) was counted with a hemacytometer
(Hausser). The number of adherent cells was determined by
trypsinization (GIBCO) and subsequent counting with a hemacytometer. Cell membrane integrity was determined in NRVMs plated on
100-mm dishes treated with DDC or XXO, with or without EUK-8,
for 24 hours. The medium, containing floating cells, was removed
and collected. Adherent cells were trypsinized and pooled with the
corresponding floating cells. The cells were pelleted at 730g for 5
minutes and resuspended in PBS containing 0.04% (wt/vol) trypan
blue (Sigma). An aliquot of the cell suspension, containing both
adherent and floating cells, was counted with a hemacytometer, and
the percentage of cells excluding dye was calculated.
NRVMs plated on 100-mm dishes were treated for 24 hours as
indicated. The medium, containing floating cells, was removed and
collected. Adherent cells were trypsinized and pooled with the
corresponding floating cells. The cells were pelleted at 730g for 5
minutes, washed once with PBS, and then lysed on ice in buffer
containing 10 mmol/L EDTA (pH 8.0), 10 mmol/L Tris-HCl (pH
7.4), and 0.5% (v/v) Triton X-100 for 30 minutes. Lysed cells were
centrifuged at 16 000g for 20 minutes, and the supernatants were
treated with 0.4 mg/mL RNase A for 30 minutes at 37°C, followed
by 0.4 mg/mL proteinase K for 30 minutes at 37°C. The DNA was
precipitated overnight in 1 mol/L NaCl and 50% isopropanol. The
DNA was pelleted at 16 000g for 20 minutes, air-dried, and dissolved in 10 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L EDTA (pH
8.0). The entire DNA sample for each treatment was electrophoresed
on a 1.5% agarose gel with 40 mmol/L Tris-acetate and 2 mmol/L
EDTA (pH 8.5). The DNA ladders were visualized with ethidium
bromide and UV light, and DNA band sizes were estimated using
DNA size markers (PCR Markers, Promega).
In Situ Terminal Deoxynucleotidyl
Transferase–Mediated Nick End-Labeling of DNA
Strand Breaks (TUNEL)
NRVMs plated on glass coverslips in 24-well dishes were treated for
24 hours as indicated. Cells were fixed in 3.7% formaldehyde for 30
minutes at room temperature and then permeabilized in 0.1%
(vol/vol) Triton X-100 and 0.1% (wt/vol) sodium citrate for 30
minutes at 4°C. Coverslips were exposed to the TUNEL reaction
mixture containing terminal deoxynucleotide transferase and fluorescein-labeled dUTP (In Situ Cell Death Detection Kit—Fluorescein, Boehringer Mannheim) for 1 hour in a humidified 37°C
incubator, washed, and counterstained with Hoechst 33342 for 10
Siwik et al
Effect of Oxidative Stress on Myocyte Phenotype
149
minutes at room temperature. Coverslips were mounted onto glass
slides and visualized with an epifluorescent microscope. At least 100
total nuclei (Hoechst stained) were counted from each coverslip, and
the number of TUNEL-positive cells for each field was determined.
Slides were scored in a blinded fashion.
Statistical Analysis
All data are presented as mean6SEM. Statistical analysis used the
Student t test or 1-way ANOVA with Bonferroni correction, as
appropriate. A P value #0.05 was considered significant.
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Figure 2. Inhibition of SOD increases protein synthesis and protein content. Myocytes were incubated with DDC (0.01 to
100 mmol/L), the superoxide scavenger Tiron (100 mmol/L), or
both, for 24 hours. A, Protein synthesis as assessed by
[3H]leucine incorporation. B, Net cellular protein content as measured by the Bradford method. Both [3H]leucine incorporation
and protein content were normalized to cell number determined
in parallel plates. A and B, Data are mean6SEM from 3 to 5
experiments. *P#0.05 vs control; #P#0.05 vs DDC.
Results
DDC Inhibits SOD and Increases Superoxide
Levels in a Concentration-Related Manner
The addition of DDC13,22 to cultures for 7 hours inhibited
SOD activity in a concentration-dependent manner, reducing
total cellular activity by 33610% and 6365% at 1 mmol/L
and 1 mmol/L, respectively (Figure 1A). Exposure to DDC
(1 mmol/L, 24 hours) increased superoxide levels by
162613% as measured by lucigenin-enhanced chemiluminescence (Figure 1B) and by 4365% as measured by NBT
reduction (Figure 1C), which confirms that inhibition of Cu,
Zn SOD causes a modest increase in superoxide levels.
Figure 1. DDC inhibits SOD activity and increases basal superoxide levels in neonatal rat ventricular myocytes. A, Myocytes
were treated with DDC (1 mmol/L or 1 mmol/L) for 7 hours, and
cytosolic proteins were prepared and tested for the ability to
inhibit pyrogallol auto-oxidation as measured by absorbance at
420 nm. B and C, Myocytes were treated for 24 hours with DDC
(1 mmol/L), and the increment in quenchable superoxide was
measured by lucigenin-enhanced chemiluminescence (B) or
NBT reduction (C). A through C, Data are mean6SEM from 3
experiments. *P#0.05 vs control.
Inhibition of SOD Stimulates Myocyte Growth
Exposure to DDC for 24 hours increased protein synthesis, as
measured by [3H]leucine incorporation, in a concentrationdependent manner with a maximum increase of 109634% at
1 mmol/L (Figure 2A). Concomitant addition of the superoxide scavenger Tiron15 (100 mmol/L) with DDC (1 mmol/L)
abolished the effect of DDC. DDC (24 hours) likewise
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Figure 3. Inhibition of SOD stimulates growth-related pathways
involving c-fos and ERK1/ERK2. A, Time course for the induction of c-fos mRNA after exposure to DDC (1 mmol/L). B, Effect
of DDC (1 mmol/L) on ERK1/ERK2 activity measured as phosphorylation of myelin basic protein by immunoprecipitated
ERK1/ERK2.
increased cellular protein content in a concentration-dependent
manner, and this effect was abolished by Tiron (Figure 2B).
DDC (1 mmol/L) caused induction of c-fos mRNA (Figure 3A)
and activation of ERK1/ERK2 (Figure 3B).
The effects of SOD inhibition were mimicked by addition
of XXO for 24 hours, which caused increases in [3H]leucine
incorporation (88622%; n57; P50.002) and protein content
(9269%; n57; P,0.001) that were abolished by Tiron.
Effect of SOD Inhibition on Myocyte Phenotype
DDC (1 mmol/L, 24 hours) increased ANP mRNA by
194658% and decreased SERCA2 mRNA by 3465% (Figure 4). Both effects of DDC were inhibited by Tiron
(100 mmol/L).
Effect of SOD Inhibition on Cell Membrane
Integrity and Apoptosis
Exposure to DDC for 24 hours at concentrations up to
1 mmol/L had no effect on the membrane integrity of
adherent or floating myocytes, as evidenced by the ability to
exclude trypan blue dye (control cells, 8566%; DDC-treated
cells, 8765%; n53; P5NS), which indicated that DDC did
not cause cell necrosis. At DDC concentrations .1 mmol/L,
there was a decrease in the number of adherent cells (data not
shown). XXO had no effect on membrane integrity (control
cells, 8566%; XXO-treated cells, 80612%; n53; P5NS).
DDC (100 mmol/L, 24 hours) increased DNA laddering
(Figure 5), and this effect was inhibited by concomitant
treatment with the SOD/catalase mimetic EUK-8 (10 mmol/L).16
Likewise, DDC (100 mmol/L, 24 hours) caused an apparent
increase in the fraction of cells with nuclear condensation and
fragmentation as visualized with the fluorescent DNA dye
Hoechst 33342 (Figure 6A).
In control myocytes, 662% of nuclei were TUNELpositive. Treatment with DDC (100 mmol/L, 24 hours)
increased the percentage of TUNEL-positive nuclei 3.862.2–
fold (Figures 6B and 7). EUK-8 (10 mmol/L) alone had no
effect on the number of TUNEL-positive nuclei but prevented
completely the DDC-stimulated increase (Figure 7). DDC
Figure 4. Effect of DDC on the expression of ANP and SERCA2
mRNAs. A, Representative Northern hybridization in myocytes
treated with DDC (1 mmol/L), Tiron (100 mmol/L), or both for 24
hours. B and C, Mean changes in ANP and SERCA2 mRNA levels from 4 to 12 experiments. Data are normalized to 18S rRNA
and are mean6SEM. *P#0.05 vs control; #P#0.05 vs DDC
alone.
concentrations .1 mmol/L caused cell detachment, which
may be a stimulus for apoptosis known as anoikis.23 To avoid
the possibility that apoptosis was due to detachment, per se,
TUNEL staining was assessed only in the adherent cells.
XXO likewise increased DNA laddering (data not shown)
and increased the number of TUNEL-positive nuclei
4.961.2–fold (n53; P50.014).
DDC (24 hours) increased the expression of bax mRNA in
a concentration-dependent manner (Figure 8). The level of
bax mRNA was not affected at DDC concentrations of
1 mmol/L or less but was increased at 10 mmol/L and
maximal at 100 mmol/L. XXO likewise increased bax
52613% (n55; P,0.001).
Discussion
The major new finding of this study is that a sustained
subnecrotic increase in superoxide level caused by partial
inhibition of SOD has profound effects on the growth,
Siwik et al
Effect of Oxidative Stress on Myocyte Phenotype
151
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Figure 5. Inhibition of SOD stimulates DNA laddering. Myocytes
were incubated with DDC (100 mmol/L), the SOD/catalasemimetic EUK-8 (10 mmol/L), or both for 24 hours. Low molecular
weight DNA was extracted and visualized by gel electrophoresis
and ethidium bromide staining. The figure is representative of 4
similar experiments.
phenotype, and death of cardiac myocytes. These effects were
related to the concentration of DDC. At low concentrations
(1 mmol/L), DDC stimulated cell growth, induced a fetal gene
program, and activated growth-signaling pathways involving
c-fos and ERK1/ERK2. At a higher concentration (100 mmol/L),
DDC stimulated apoptosis and increased expression of bax
mRNA. DDC-stimulated growth and apoptosis were mimicked by superoxide generation with XXO and prevented by
a superoxide scavenger or an SOD/catalase-mimetic, which
supports the conclusion that the observed effects were due to
increased superoxide levels caused by SOD inhibition.
Oxidative Stress Stimulates Myocyte Growth
The growth effect of DDC is comparable in magnitude with
that observed with several other stimuli such as norepinephrine,24 interleukin-1b,14 or endothelin25 in neonatal rat cardiac
myocytes. Coincident with cell growth, there was increased
expression of ANP mRNA, which is typical of myocardial
hypertrophy,26 and decreased expression of SERCA2 mRNA,
which may be observed with myocardial hypertrophy.26
Oxidative stress is emerging as a growth signal in other cell
types. In vascular smooth muscle cells, the hypertrophic
effect of angiotensin depends on increased production of
superoxide anion via NADH oxidase.10 A similar role was
suggested in fibroblasts, in which increases in superoxide
anion and oxidative stress have been implicated in mediating
the growth effects of stimuli acting through ras.11
Figure 6. Effects of DDC (100 mmol/L, 24 hours) on nuclear
morphology, as assessed by staining with Hoechst 33342 (A),
and DNA fragmentation, as assessed by TUNEL staining (B).
Arrows identify condensed or fragmented nuclei (A) or TUNELpositive cells (B). The figure is representative of 3 similar experiments. Bar5100 mm.
by changes in nuclear morphology and increases in DNA
laddering and in situ TUNEL staining. It should be noted that
although DNA laddering is specific for apoptosis, it is not
quantitative, and conversely, that TUNEL staining is quantitative but not specific for apoptosis.27
Oxidative Stress Stimulates Apoptosis
Oxidative stress can cause necrotic cell death characterized
by a loss of membrane integrity.22 However, DDC concentrations up to 1 mmol/L did not impair cell membrane
integrity, which suggests that apoptosis occurred at a level of
oxidative stress that was subnecrotic. Apoptosis was assessed
Figure 7. Inhibition of SOD increases TUNEL-positive myocytes. Cells were treated for 24 hours with DDC (100 mmol/L),
EUK-8 (10 mmol/L), or both and stained as per Figure 6. Shown
is the mean percentage of Hoechst 33342-stained nuclei that
are TUNEL-positive from 3 to 4 experiments. *P#0.05 vs control; #P#0.05 vs DDC alone. Data are mean6SEM.
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July 23, 1999
high concentrations of H2O2 can activate ERK1/ERK2 in
neonatal rat cardiac myocytes.35 Both Euk-8, which has SOD
and catalase activities, and Tiron, which scavenges superoxide, would be expected to decrease H2O2 levels.
Implications
These observations were made in vitro in myocytes from
neonatal rats, and therefore, they may not reflect events in
adult myocytes in vivo. Nevertheless, the effects demonstrated here by inhibition of SOD suggest that oxidative stress
may be an important mediator of myocyte growth, functional
phenotype, and apoptosis. Oxidative stress may be increased
in the myocardium of patients with heart failure.36 Therefore,
our findings suggest that a decrease in SOD activity could
contribute to pathologic myocardial remodeling in humans
and, conversely, that antioxidants might attenuate the development of myocardial failure.
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Acknowledgments
Figure 8. DDC stimulates bax mRNA in a concentrationdependent manner. A, Representative Northern hybridization. B,
Mean6SEM from 5 experiments, normalized to 18S rRNA.
*P#0.05 vs control.
Prior observations have implicated superoxide as a mechanism of cardiac myocyte apoptosis. Cheng et al12 showed
that stretch causes myocyte apoptosis associated with increased levels of reactive oxygen species and that scavenging
superoxide with a nitric oxide donor reduced the extent of
apoptosis. Sawyer et al28 implicated superoxide as a mechanism for anthracycline-stimulated myocyte apoptosis. Likewise, Dhalla et al8 showed that vitamin E is cardioprotective
in pressure overload–induced hypertrophy, which appears to
involve myocyte apoptosis.29
Mechanism of Action of DDC
DDC inactivates Cu, Zn SOD, and extracellular SOD by
chelating the copper ion at the active sites13 and has been
shown to inhibit SOD activity in rat cardiac myocytes.22 Of
note, chronic dietary deficiency of copper causes a characteristic cardiomyopathy that is associated with myocyte
hypertrophy, ventricular dilation,30 and decreased Cu, Zn
SOD activity31 and is ameliorated by antioxidants.32 Dithiocarbamates such as DDC may have other actions relevant to
the redox state of a cell and its response to oxidative stress.
As thiols, they can auto-oxidize to form superoxide and other
reactive oxygen species,33 and alternatively, at high concentrations they may act as reducing agents.34 Dithiocarbamates
may also inhibit the activation of nuclear factor-kB.34 However, the ability of the superoxide scavenger Tiron15 and the
SOD/catalase mimetic EUK-816 to inhibit DDC-stimulated
myocyte growth and apoptosis strongly supports a mechanistic role for superoxide anion in these experiments.
It is unclear whether DDC-stimulated hypertrophy and
apoptosis are mediated by superoxide anion or other downstream reactive oxygen species, such as H2O2, produced by
the spontaneous dismutation of superoxide anion. Relatively
This work was supported by NIH grants HL42539, HL52320, and
HL61639 (to W.S.C.); HL57947 (to K.S.); HL03878 (to D.B.S.);
HL55425 (to P.J.P.); and HL07224 (to D.A.S.); and by a Grant-inAid from the American Heart Association (to P.J.P.); a Grant-in-Aid
from the American Heart Association, Massachusetts Affiliate (to
D.B.S.); and a Merit Grant from the Department of Veterans Affairs
(to K.S.).
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Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Inhibition of Copper-Zinc Superoxide Dismutase Induces Cell Growth, Hypertrophic
Phenotype, and Apoptosis in Neonatal Rat Cardiac Myocytes In Vitro
Deborah A. Siwik, John D. Tzortzis, David R. Pimental, Donny L.-F. Chang, Patrick J. Pagano,
Krishna Singh, Douglas B. Sawyer and Wilson S. Colucci
Circ Res. 1999;85:147-153
doi: 10.1161/01.RES.85.2.147
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