Vol. 279, Issue 6, H3089-H3100, December 2000
Nitroprusside induces cardiomyocyte death: interaction with
hydrogen peroxide
Simon W.
Rabkin and
Jennifer
Y
Kong
Department of Medicine, Division of Cardiology, University of
British Columbia, Vancouver, British Columbia V5Z 3J5, Canada
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ABSTRACT |
We examined
the hypothesis that sodium nitroprusside (SNP) produces cell death in
cardiomyocytes through generation of H2O2. Embryonic chick cardiomyocytes in culture were treated with SNP, and
cell viability was assessed by trypan blue, MTT assay, and fluorescent
activated cell sorting (FACS) analysis. SNP for 24 h
induced a significant (P < 0.001) dose-dependent loss of
cell viability. On MTT assay, the half-maximal effective concentration was 0.53 mM (confidence interval 0.45-0.59 mM).
SNP-treated cardiomyocytes displayed characteristic microscopic
features of apoptosis: reduced cell size, nuclear disintegration, and
membrane bleb formation. FACS analysis demonstrated SNP-induced
apoptosis as well as cell changes consistent with necrosis. The
proportion of cells with nuclear changes of apoptosis, identified by
propidium iodide (PI) staining of permeabilized cells, increased
significantly (P < 0.05) after 0.5 mM SNP for 24 h.
The proportion of apoptotic cells, characterized by dual staining of
intact cardiomyocytes with fluorescein diacetate and PI, was
significantly (P < 0.05) increased after treatment with 0.5 mM SNP for 24 h. SNP metabolism and NO production was suggested by
the significant (P < 0.05) increase in nitrite generation
in the media with 0.5 mM SNP compared with control. SNP-mediated
H2O2 production was implicated in the mechanism
of SNP-induced cell death. First, SNP produced a significant (P
< 0.05) increase in H2O2 detected in the
media after 6 or 24 h of SNP treatment. Second, catalase
completely blocked the reduction of cell viability induced by 0.1 mM
SNP and significantly (P < 0.05) blunted the effect of
0.5 mM SNP. In contrast, the iron chelator deferoxamine did not
alter SNP-induced loss of cell viability. FACS analysis showed that the
combination of low concentrations of H2O2
(10
8 M) that did not alter cell viability augmented
SNP-induced apoptosis. In contrast, the amount of necrotic cell death
was unchanged by the combination of H2O2 and
SNP. H2O2 plus SNP produced a dramatic alteration in cell structure with greater membrane bleb formation, shrunken cells, and more intense cytosolic acridine orange staining and
nuclear fragmentation than either agent alone. These data indicate the
vulnerability of cardiomyocytes to SNP and suggest the involvement of
H2O2 in the pathogenesis of SNP-induced
cardiomyocyte cell death. Establishing apoptosis as a component of the
type of cell death induced by SNP permitted the recognition that
SNP-induced apoptosis was increased by chronic treatment with low
(subtoxic) concentrations of H2O2.
apoptosis
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INTRODUCTION |
SODIUM NITROPRUSSIDE
[Na2(Fe(CN)5NO)] (SNP) is used clinically as
a vasodilator to reduce markedly elevated blood pressure and reduce
left ventricular impedance in the acute management of hypertension. The
potential adverse effects of SNP on cells and tissues have recently
been identified and include SNP-induced cell death in murine
neuroblastoma cells (47), the brain (37), vascular smooth muscle cells (32, 44), and certain tumor
cells (42). However, this cytotoxicity is not demonstrable
in all cells and tissues. SNP does not induce cell death in murine
macrophages (8) or thymocytes (1). In
contrast, a cell-protective function has been attributed to SNP in PC12
cells, sympathetic neuronal cells (13), and endothelial
cells (10) and in ovarian follicles (7). If
the potential cytotoxicity of SNP is dependent on cell type, then it
becomes imperative to determine whether SNP induces cardiomyocyte cell
death, because the loss of cardiomyocytes is part of the underlying
pathogenesis leading to irreversible failure of cardiac function
(23). We therefore sought to determine the potential
toxicity of SNP in cardiomyocytes and the type of cell death that would
be involved. Cell death can be divided mainly into two kinds:
apoptosis, which involves the activation of a genetically programmed
set of enzymes that leads to the cell participating in its own
destruction, or necrosis, a more passive process of cell destruction
that occurs after severe damage to the cell membrane (23, 43,
46).
The underlying mechanisms accounting for SNP-induced cell toxicity are
not completely understood but include several mechanisms related to
free radical-mediated cell injury. First, metabolites of SNP may
undergo redox cycling with oxygen to form superoxide anion
(O2
·) that in turn produces
H2O2 and O2 (36).
Second, the iron moiety of SNP appears to mediate the prooxidative
properties of SNP (37). Third, the metabolism of SNP
releases cyanide, which complexes with the iron of mitochondrial
cytochromes to inhibit ATP production that is critically important for
cell metabolism (25). Fourth, metabolism of SNP releases
nitric oxide (NO) that in turn can regulate myocardial oxygen
metabolism (33). NO also has the potential for
cytotoxicity through the formation of peroxynitrite, although the
extent to which this is relevant in vivo is still open to debate
(2, 28). We sought to determine the extent to which the
putative action of SNP may be mediated through oxidative damage to the cardiomyocyte.
We further sought to explore the potential synergy between SNP and
reactive oxygen species. Reactive oxygen species have been implicated
in myocardial damage in clinically important conditions such as
reperfusion injury and heart failure (for reviews, see Refs.
3, 14). Hydrogen peroxide
(H2O2), one of the reactive oxygen species
released during reperfusion of the ischemic myocardium (38), induces contractile dysfunction, oxidative damage to
membranes, and cell death (4, 5, 9, 21, 38, 40). In
addition to the acute cardiotoxicity of reactive oxygen species, the
concept has recently developed that heart failure may develop because of limited antioxidant reserve of the myocardium (38).
Reduction in antioxidant reserve may also be an important determinant
of prognosis in patients with heart failure (22). Thus
myocardial damage may occur acutely due to high bursts of free radical
generation during reperfusion, after ischemia, as well as chronically
because of low grade oxidative stress. High concentrations of
O2
· produce H2O2 that, in
combination with NO, released by SNP, produce peroxynitrite anion
(ONOO
) and hydroxyl radicals that are especially damaging
to tissues (2). The potential adverse consequence of an
interaction between SNP and low concentrations of
H2O2 is a novel hypothesis. Although most
studies focused on factors other than those that mitigate acute
H2O2-induced cardiotoxicity, we explored the
potential of SNP as a factor that might act to accentuate or augment
the effect of low levels of H2O2 to induce
cardiomyocyte injury and cell death.
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MATERIALS AND METHODS |
Cell Cultures
Chick embryonic ventricular cells were cultured from 7-day chick
embryos from white Leghorn eggs using previously described methods
(34). The protocol was approved by the University
committee on use of animals for research. Myocytes were maintained in
culture in medium 818A [73% DBSK (116 mM NaCl, 0.8 mM
MgSO4, 0.9 mM NaH2PO4, 5.5 mM
dextrose, 1.8 mM CaCl2, and 26 mM NaHCO3), 20%
M199, 6% fetal calf serum, 1% antibiotic-antimycotic, 10,000 µg/ml
streptomycin sulfate, 10,000 U/ml penicillin G sodium, and 25 mg/ml
amphotericin B] for 72 h before experimentation. The proportion
of cells showing spontaneous contraction or displaying the
muscle-specific marker myosin on immunohistological examination was in
excess of 90% at this time.
Cell Viability
MTT assay.
The MTT assay, an index of cell viability and cell growth, is based on
the ability of viable cells to reduce MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] from
a yellow water-soluble dye to a dark blue insoluble formazan product
(26). Cardiomyocytes were seeded in multiwell microtiter
plates (Falcon 3072, Becton-Dickinson, Lincoln Park, NJ) at 20,000 cells per well and incubated at 37°C for 72 h. Cardiomyocytes
were then treated with SNP, H2O2, or other
agents and returned to the incubator for 24 h. SNP and
H2O2 were added directly to the media, and
potential inhibitors were added 15 min before either agent. MTT dye was
added to each well for the last 4 h of treatment. The reaction was
stopped with the addition of solubilization reagent (Promega, Madison,
WI), and the optical density was determined at 570 nm on a multiwell
plate reader (Bio-Rad model 3550, Bio-Rad, Mississauga, Canada).
Background absorbance of medium in the absence of cells was subtracted.
All samples were assayed in duplicate, and the mean for each experiment was calculated. The relationship of the absorbance of MTT to cell number was verified in separate experiments in which 1,200 to 37,000 cardiomyocytes were added to different wells of a microtiter plate.
There was a significant (P < 0.01) linear relationship between cell number and absorbance with a correlation coefficient of
0.98.
Trypan blue exclusion.
Cardiomyocytes were grown on coverslips in dishes for 72 h in
culture before treatment with SNP. After the addition of SNP to the
media, dishes were returned to the incubator for 24 h. The
coverslips were removed, stained with 0.4% trypan blue, and then
examined microscopically in a hemocytometer to determine the total
number of cells and the number of dead cells, i.e., those retaining
trypan blue. Four representative high-power fields (hpf) of each
slide, ~300 cells, were assessed, and the mean was calculated per coverslip.
Microscopy
To examine cell morphology, cardiomyocytes grown on coverslips
were treated with H2O2 or SNP or the
combination. The medium was removed, and cells were stained acridine
orange (100 µg/ml). Cells were examined microscopically with a Zeiss
(Standard 16) microscope using a fluorescent light source as previously
described (35).
H2O2 Assay
The ability of SNP to generate H2O2 was
assessed by the ability of H2O2 to
oxidize Fe2+ to Fe3+ that binds to and alters
an indicator dye (xylenol orange), which is measured
spectrophotometrically. Samples are reported as absorbance at 560 nm
minus a blank, which consists of the sample plus 1 vol of 2.5 M
H2SO4:100 vol of sorbitol (100 mM) plus xylenol
orange (125 µM) in water read at 560 nm. This is done to adjust for
the presence of endogenous iron in the cells. Cardiomyocytes were grown
in petri dishes for 72 h, after which they were treated with 0.5 mM SNP for 6 or 24 h. The media was removed and assayed using a
quantitative assay kit for H2O2 from OXIS
(Portland, OR). To eliminate possible effects of SNP, media blanks,
i.e., media plus SNP without cells, were also measured and subtracted
from the samples with cells. The absence of an effect of catalase
directly on this assay was verified by the addition of catalase to the reaction mixture with an aliquot of media after exposure of cells to
SNP and SNP plus H2O2 (10
7 M).
The sensitivity of the assay is reflected in the
H2O2 standard curve that was linear in the
range of 0.1-20 µM.
Nitrite Assay
For measurement of nitrite (NOx), cardiomyocytes
that had been in petri dishes for 72 h after culture were treated
with SNP for 24 h, after which the medium was removed. Medium, 100 µl, was added to equal volumes of the Greiss reagent [0.1%
N-(1 naphthyl) ethylenediamine dihydrochloride, 1%
sulfanilamide, and 5% phosphoric acid]. The mixture was kept at
20°C for 10 min, and the absorbance was measured at 570 nm.
NOx concentration was determined from a standard curve
calibrated with NaNO2.
Flow Cytometry (FACS Analysis)
Several different cell preparation and staining methods were
used followed by fluorescent-activated cell sorting (FACS) analysis. Cardiomyocytes at 72 h of culture were exposed to
H2O2, SNP, or both. After 24 h, the
reaction was stopped by the removal of media followed by brief exposure
to trypsin (0.01% in DMS8) to suspend the adherent cells.
Trypsinization was stopped by dilution with 818A media containing 6%
fetal calf serum. The suspended cardiomyocytes were gently spun (1,000 rpm for 5 min) and washed with PBS. Cardiomyocytes were incubated with
fluorescein diacetate (FDA), 1 µg/ml, for 30 min at 37°C in a dark
environment. FDA is a substrate for esterases whose product is the
flurochrome fluorescein that becomes entrapped in live cells but
escapes from necrotic cells (16). Cells were then
resuspended in 0.5 µg/ml propidium iodide (PI) for 30 min at room
temperature. Cells were washed twice with PBS and then resuspended in
PBS for FACS analysis. Cardiomyocytes, ~10,000, were aspirated into
the FACS machine (flow cytometer, model Epics XL MCL, Coulter
Electronics, Burlington, Canada) and examined for fluorescence on
separate channels for FDA (FL1) and PI (FL3).
To examine nuclear characteristics based on PI staining after
cardiomyocytes were exposed to drugs, the reaction was stopped by the
removal of media from the cells followed by brief exposure to trypsin
(0.01% in DMS8) to suspend the adherent culture. Trypsinization was
halted by dilution with 818A media, which contains 6% fetal calf
serum. The suspended cardiomyocytes were gently spun down and washed
with PBS. Cells were then permeabilized with 70% ethanol for 30 min at
room temperature. Cells were then gently spun down, and the ethanol was
removed. The resulting permeabilized cells were then stained with PI
staining mix (0.1% Triton X-100, 0.1 mM EDTA, 0.5 mg/ml RNAse A, and
50 µg/ml PI) to visualize the nuclei.
Materials
All cell culture media components were from GIBCO-BRL Life
Sciences (Burlington, Ontario, Canada). Catalase, deferoxamine, and
acridine orange were from Sigma Chemicals (St. Louis, MO). Trypan blue
was from BDH Chemicals. SNP was from Hoffman LaRoche (Mississauga,
Ontario, Canada). MTT assay kit was from Promega. All
chemicals were purchased from Fischer Scientific (Ottawa, Ontario,
Canada). Chemicals for flow cytometry were from VWR (Mississauga, Ontario, Canada).
Data Analysis
The data are presented as the means ± SE. Hypothesis
testing used one-way ANOVA. Examination of a pair of groups used
Kruskal-Wallis multiple-comparison test. Calculation of
EC50 used nonlinear least-squares fit of the concentration
response relationship. The null hypothesis was rejected if the
probability of a type I error was <5% (P < 0.05).
 |
RESULTS |
SNP treatment of cardiomyocytes produced a dose-dependent
reduction in cell survival. Cardiomyocytes treated with SNP for 24 h displayed a significant (P < 0.001) dose-dependent
increase in the proportion of dead cells determined by the trypan blue assay (Fig. 1). The proportion of dead to
live cells per hpf increased from 0.25 ± 0.06 (means ± SE)
in control to 0.96 ± 0.33 for 0.1 mM SNP to 1.5 ± 0.2 for
0.5 mM SNP and to 4.9 ± 0.7 for 1.0 mM SNP. Each concentration of
SNP was significantly (P < 0.01) greater than control.
The percentage of dead cells per hpf increased significantly (P < 0.01) from control of 20.3 ± 2.2 to
48.9 ± 10.0%, 57.3 ± 6.8, and 81.8 ± 11% for,
respectively, 0.1, 0.5, and 1.0 mM SNP. Evaluation of cell survival by
the MTT assay demonstrated a significant (P < 0.0001)
dose-dependent reduction in MTT absorbance in cardiomyocytes treated
with SNP for 24 h (Fig. 2). On the
basis of the ability of viable cells to reduce MTT and the direct
relationship between cardiomyocyte cell number and MTT absorbance, SNP
induced a significant loss of cell viability (see inset,
Fig. 2). The EC50 was 0.53 mM (confidence intervals
0.45-0.59 mM).

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Fig. 1.
Cardiomyocyte viability after treatment with sodium
nitroprusside (SNP) as assayed by trypan blue exclusion. Cells were
treated with 0.1 mM (n = 4), 0.5 mM (n = 4), or 1.0 (n = 4) SNP or control (n = 14) for 24 h. The proportion of dead to alive cells per high
power field (hpf) was calculated. Four representative hpfs are counted
with a total of ~300 cells, and the mean is calculated for each
coverslip. The percentage of dead cells of the entire population per
hpf was calculated and is shown in the inset. The results
are presented as the means ± SE, and the data with SNP are
compared with control (**P < 0.01).
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Fig. 2.
Cardiomyocyte viability after treatment with SNP as
assayed by the MTT assay. Cardiomyocytes were seeded in multiwell
microtiter at 20,000 cells per well. After 72 h, cells were
treated with SNP for 24 h. MTT dye was added to each well for the
last 4 h of treatment. The reaction was stopped, and the
absorbance (optical density) was determined at 570 nm. Background
absorbance of medium in the absence of cells was subtracted. The
results are presented as the means ± SE for 0.01 mM
(n = 7), 0.1 mM (n = 30), 0.5 mM (n = 40), or 1.0 mM (n = 17) SNP and control (n = 40) for 24 h. Hypothesis
testing compared SNP and control cells (*P < 0.05;
**P < 0.01). The inset shows loss of cell
viability as % control.
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Because the iron moiety of SNP mediates some of the cytotoxicity of SNP
(37), cardiomyocytes were treated with the iron chelator
deferoxamine, and the impact of SNP-mediated cell death was
ascertained. A SNP concentration of 0.5 mM was chosen because it is
close to the EC50 and it significantly reduces cell
viability. Deferoxamine, 0.001 µM, which had no cytotoxicity, did not
alter SNP-induced loss of cell viability (Fig.
3). A higher concentration of
deferoxamine, 0.01 µM, which slightly reduced cardiomyocyte viability
alone, in combination with SNP did not prevent SNP-induced cell death.

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Fig. 3.
The effect of deferoxamine (Def) on
cardiomyocyte viability after treatment with SNP as assayed by the MTT
assay. Cardiomyocytes were seeded in multiwell microtiter at 20,000 cells per well. After 72 h, cells were treated with SNP with or
without Def for 24 h. MTT dye was added to each well for the last
4 h of treatment, the reaction was stopped, and the absorbance
(optical density) was determined at 570 nm. Background absorbance of
medium in the absence of cells was subtracted. The results are
presented as the means ± SE for 0.001 µM
(n = 4) or 0.01 µM Def (n = 11),
0.05 mM SNP (n = 40), 0.5 mM SNP + 0.001 µM
(n = 4) or 0.01 µM Def (n = 7), and
control (n = 40) for 24 h. The
inset shows the loss of cell viability as a percentage of
control. Hypothesis testing compared each data set and control
(*P < 0.05 or **P < 0.01). There were
no differences between SNP + Desf and SNP alone.
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Because metabolites of SNP may undergo redox cycling with oxygen to
produce H2O2 (36), we sought to
determine whether catalase that degrades H2O2
would antagonize the action of SNP (Fig.
4). Cardiomyocytes were pretreated with
catalase beginning 2 h before SNP. Catalase, 1.0 mg/ml, completely
blocked the reduction of cell viability induced by 0.1 mM SNP and
significantly (P < 0.05) blunted the effect of 0.5 mM
SNP. We then sought confirmatory evidence that SNP induced
H2O2 production in these cells. Measurement of
H2O2 release into the media revealed that SNP
produced a significant (P < 0.05) increase in
H2O2 after 6 or 24 h of SNP treatment that was, respectively, 3.5 ± 0.2- and 4.3 ± 0.7-fold greater
than control (Fig. 5). In contrast, in
the absence of cells, there was no significant change in
H2O2 production, as
H2O2 was 1.0 ± 0.1- and 1.0 ± 0.2-fold times control for, respectively, 0.5 mM SNP for 24 h
(n = 4) and 0.5 mM SNP plus 10
7 M
H2O2 (n = 3).

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Fig. 4.
The effect of catalase (Cat) on cardiomyocyte viability
after treatment with SNP as assayed by the MTT assay. Cardiomyocytes
were seeded in multiwell microtiter at 20,000 cells per well. After
72 h, cells were treated with SNP with or without Cat for 24 h. MTT dye was added to each well for the last 4 h of treatment,
the reaction was stopped, and the absorbance (optical density) was
determined at 570 nm. Background absorbance of medium in the absence of
cells was subtracted. The results are presented as the means ± SE
for 0.1 mM SNP (n = 30), SNP + 0.1 mg/ml Cat
(n = 6), SNP + 1.0 mg/ml Cat (n = 7),
0.5 mM SNP (n = 40), SNP + 0.1 mg/ml Cat
(n = 5), SNP + 1.0 mg/ml Cat (n = 10),
and control (n = 40) for 24 h. The
inset shows the loss of cell viability as a percentage of
control. Hypothesis testing compared each data set to control
(*P < 0.05 or **P < 0.01) or SNP
( P < 0.05).
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Fig. 5.
Ability of SNP to generate H2O2.
Cardiomyocytes were grown in petri dishes for 72 h, after which
they were treated with 0.5 mM SNP for 6 or 24 h. The medium was
removed and assayed at 590 nm using a quantitative assay kit for
H2O2. A standard curve for absorbance of
H2O2 permitted calculation of
H2O2 in the medium. The effect of endogenous
iron was eliminated by subtracting a blank with reagents, and, to
eliminate the effect of SNP itself, media plus SNP without cells was
also measured and subtracted from the samples of cells treated with
SNP. Top, data after 6 and 24 h of SNP treatment;
bottom, data compared with control cells. The data are
presented as the means ± SE (*P < 0.05;
**P < 0.01).
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Because H2O2 production is thought to be
mediated by NO release from SNP (33) and NO release
indicates SNP metabolism (29), we sought to obtain an
indication of the amount of NO released during SNP treatment.
NOx concentration in the media was determined after
treatment of cardiomyocytes with SNP for 24 h. There was a
significant (P < 0.05) increase in NOx
generation concentration with SNP treatment (Fig.
6). SNP, 0.5 mM, produced a 4.6 ± 0.5-fold increase in NOx concentration compared with
control. Cyanide can also be released by SNP metabolism. Although it is
difficult to know the extent of SNP metabolism in cell culture and
cyanide liberation, considering that 1 mM SNP produced an increase in NOx in the 1 µM range, we examined the effect of cyanide
on cell viability of cyanide ~1 µM. We did not find any increase in
cell death assessed by the MTT assay after exposure of cardiomyocytes to cyanide (KCN) of 1 × 10
6 M for 24 h [
2.5 ± 3.1% (n = 8)], whereas
10
5 M produced a small 13.3 ± 4.4%
(n = 13) reduction in cell viability.

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Fig. 6.
Nitric oxide (NO) release in SNP-treated
cardiomyocytes. Cardiomyocytes that had been in petri dishes
for 72 h after culture were treated with SNP for 24 h, after
which the medium was removed. Medium, 100 µl, was added to equal
volumes of the Greiss reagent, and the absorbance was measured at 570 nm. SNP, 0.1 (n = 4), 0.5 (n = 7), and
1.0 mM (n = 4), and control (n = 7) are
shown. The inset shows the change relative to control. The
data are presented as the means ± SE (**P < 0.01 from control).
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To further examine the relationship between SNP and
H2O2, it was first necessary to determine the
concentration-effect relationship between chronic treatment with
H2O2 and cell viability (Fig.
7). Cardiomyocytes were treated with
a range of H2O2 concentrations for 24 h.
H2O2 produced a significant (P < 0.001) dose-dependent reduction in MTT absorbance and reduction in
cell viability (Fig. 5) The EC50, the
H2O2 concentration at which there was a 50%
increase in cell death, was 4.2 × 10
4 M (confidence
interval 1.2-8.4 × 10
4 M). The nature of the
concentration-effect relationship was such that there was a small
nonsignificant reduction in cell survival, <10%, at
H2O2 concentrations of 10
6 M or
less. To examine for a potential interaction of SNP with a low
concentration of H2O2 that induced minimal cell
death, 10
6 M H2O2 was selected
(Fig. 8). H2O2,
10
6 M, increased cell death 9.9 ± 7.3% compared
with control; 0.1 mM SNP increased cell death 20.2 ± 5.9%; and
the combination of both agents increased cell death 33.9 ± 11.1%, similar to the expected arithmetic sum of these concentrations
of H2O2 and SNP. Higher concentrations of 0.5 mM SNP increased cell death by 56.1 ± 4.9%. The combination of
this concentration of SNP plus 10
6 M
H2O2 increased cell death by 59.1 ± 6.5%, which is similar to the arithmetic sum of SNP plus
H2O2. To examine the impact of catalase on the
combination of SNP, the cardiomyocytes were pretreated with catalase
beginning 2 h before SNP plus H2O2
treatment (Fig. 9). Catalase antagonized
the reduction in cell viability produced by the combination of
H2O2 and 0.1 mM SNP and significantly (P < 0.05) blunted the effect of the combination of
H2O2 and 0.5 mM SNP.

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Fig. 7.
The effect of H2O2 on
cardiomyocyte viability as assayed by the MTT assay. Cardiomyocytes
were seeded in multiwell microtitre at 20,000 cells per well. After
72 h, cells were treated with H2O2 for
24 h. MTT dye was added to each well for the last 4 h of
treatment, the reaction was stopped, and the absorbance (optical
density) was determined at 570 nm. Background absorbance of medium in
the absence of cells was subtracted. The results are presented as the
means ± SE for 10 8 M (n = 12), 10 6 M (n = 30), 10 5
M (n = 15), 10 4 M (n = 8), and 10 3 M (n = 4)
H2O2, and control (n = 40) for
24 h. The inset shows the loss of cell viability as a
percentage of control. Hypothesis testing compared each data set and
control (**P < 0.05).
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Fig. 8.
The effect of the combination of H2O2 and
SNP on cardiomyocyte viability as assayed by the MTT assay.
Cardiomyocytes were seeded in multiwell microtiter at 20,000 cells per
well. After 72 h, cells were treated with SNP for 24 h. MTT
dye was added to each well for the last 4 h of treatment, the
reaction was stopped, and the absorbance (optical density) was
determined at 570 nm. Background absorbance of medium in the absence of
cells was subtracted. The results are presented as the means ± SE
for control (n = 40), 10 6 M
H2O2 (n = 30) (shown on both
sides of the graph), 0.1 mM SNP (n = 4), SNP plus
H2O2 (n = 13) (shown on the
left side of the graph), 0.5 SNP (n = 40),
and 0.5 mM SNP plus H2O2 (n = 23) (shown on the right side of the graph) for 24 h.
The inset shows the loss of cell viability as a percentage
of control. Hypothesis testing compared data set and control
(*P < 0.05; **P < 0.01).
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Fig. 9.
The effect of catalase on the combination of
H2O2 and SNP on cardiomyocyte viability.
Cardiomyocytes were seeded in multiwell microtiter at 20,000 cells per
well. After 72 h, cells were treated with SNP plus
H2O2 for 24 h with and without
pretreatment with catalase. MTT dye was added to each well for the last
4 h of treatment, the reaction was stopped, and the absorbance
(optical density) was determined at 570 nm. Background absorbance of
medium in the absence of cells was subtracted. The results are
presented as the means ± SE for 10 6 M
H2O2 (n = 30), 0.01 mM SNP
(n = 4), SNP plus H2O2
(n = 13), 0.5 SNP (n = 40), and 0.5 mM
SNP plus H2O2 (n = 23), and
control (n = 40) for 24 h. The inset
shows the loss of cell viability as a percentage of control. Hypothesis
testing compared data set and control (*P < 0.05;
**P < 0.01) or compared with SNP ( P
<0.05).
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Microscopic examination of cardiomyocytes was next undertaken to
explore the structural nature of SNP-induced cardiotoxicity and the
potential interaction with H2O2. Cardiomyocytes
were examined after staining with acridine orange, which preferentially
stains nuclei and lysosomal structures. SNP, 0.5 mM, produced a
reduction in cell size accompanied by nuclear fragmentation and
membrane bleb formation (Fig. 10). Low
concentrations of H2O2 produced little morphological alteration of cell structure, with the most notable being
reduction in cell size and slight nucleolar fragmentation. H2O2 plus SNP produced a dramatic alteration in
cell structure with membrane bleb formation, shrunken cells, and
nuclear fragmentation. There was more intense cytosolic staining that
reflects, in part, lysosomal changes that occur with reduced pH in
these structures.

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Fig. 10.
The cell morphology of cardiomyocytes after SNP and
H2O2. Cardiomyocytes were grown in culture on
coverslips for 72 h and then treated with 0.5 mM SNP,
10 8 M H2O2, and the combination.
Cells were stained with acridine orange and examined with a fluorescent
microscope at ×1,600. Representative photomicrographs are shown of
control cardiomyocytes (A), SNP (B),
10 8 M H2O2 (C), and
the combination (D).
|
|
To determine the nature of the cardiomyocyte death, cells were treated
with SNP and subjected to FACS analysis using two different approaches.
The first approach examined for the presence of apoptotic cells focused
on the nuclear changes of apoptosis. Cells were first permeabilized,
allowing PI to enter the cell and stain the nucleus. The intensity of
PI staining is indicative of the amount of DNA. Cells undergoing
apoptotic cell death are characterized by low DNA content or very low
PI nuclear staining (30). A representative experiment
shows the cell population distribution according to the degree of PI
staining (FL3) (Fig. 11A).
Of the 10,000 cells analyzed, only a very small proportion showed PI
(staining) fluorescence around 10. In contrast, the proportion of the
population with this degree of low staining (hypoploidy) was increased
in SNP-treated cells. Indeed, 0.5 mM SNP produced a significant
(P < 0.001) and 2.1 ± 0.2-fold increase in
apoptosis compared with control (Fig. 11B).

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Fig. 11.
A: DNA content frequency histograms of
cardiomyocytes treated with SNP determined by FACS analysis.
Cardiomyocytes were treated with SNP 0.5 mM for 24 h.
Approximately 10,000 cells permeabilized with ethanol and stained with
propidium iodide (PI) were aspirated into the flow cytometer and
examined for fluorescence. Representative studies are shown for control
(top) and SNP-treated (bottom) cardiomyocytes.
Apoptotic cells (Apo) are identified by low PI staining (FL3).
B: apoptotic population defined by low DNA content (PI
staining) is shown in control and SNP-treated
cardiomyocytes. The proportion of the population with
apoptotic nuclei per 100 cells is shown at top and the ratio
of apoptotic nuclei in SNP-treated cells compared with control is shown
at bottom. Data analysis was performed comparing SNP with
control (*P < 0.05; **P < 0.01).
|
|
With a low H2O2 concentration,
10
8 M, that did not induce cell death, cardiomyocytes
were treated with the combination of H2O2 and
0.5 mM SNP and were examined by FACS (Fig.
12). H2O2 plus
SNP increased the proportion of the cell population with low PI
staining indicative of apoptosis. SNP, 0.5 mM, produced a 2.1 ± 0.2-fold increase in the apoptotic population, whereas the combination of SNP plus 10
8 M H2O2 produced a
3.0 ± 0.5-fold increase in the population of cells with apoptotic
nuclei compared with control cells untreated with either SNP or
H2O2. To further examine the relationship
between SNP and H2O2, a second FACS technique,
dual staining with FDA and PI, was used. The FDA dye readily enters the
cell in the uncharged form. FDA is a substrate for esterases, an enzyme
ubiquitous in most cells, which converts FDA into a charged,
fluorescent form. If the cell is alive with an intact membrane, FDA
will remain trapped within the cell. Necrotic cells have damaged plasma
membranes, and FDA readily leaks out of the cell, whereas apoptotic
cells have intact plasma membranes. In the late stages of apoptosis, there is loss of membrane integrity so the necrotic population is a
mixture of necrotic and apoptotic cells. PI is a viability exclusion
dye because PI does not enter viable cells but enters dead cells, where
it primarily stains nuclei. Hence, early necrotic cells will exhibit
poor membrane integrity and death (low FDA and high PI), whereas early
apoptotic cells exhibit good membrane integrity with little death (low
FDA and low PI). In the later stages of cell death, cellular debris is
present and is not recognized by either stain. A representative dot
plot exhibits three distinct populations: alive, apoptotic, and
necrotic cells defined by flow cytometry analysis of cardiomyocytes
dually stained with FDA and PI (Fig.
13). Low concentrations of
H2O2, 10
8 M, did not change the
proportion of apoptotic or necrotic cells, whereas 0.5 M SNP increased
the population of apoptotic and necrotic cells. The combination of
H2O2 plus SNP produced a marked increase in the
cell population of apoptotic cells. SNP produced a significant (P < 0.05) and 4.1 ± 0.8-fold increase in the
apoptotic population and a significant (P < 0.001) and
3.5 ± 0.6 increase in the necrotic population (Fig.
14). The combination of
H2O2 plus SNP produced a 5.7 ± 1.5-fold
increase in the apoptotic population compared with control. In contrast
to apoptosis, there was no increase in the necrotic population with the
combination of H2O2 plus SNP compared with SNP
alone, i.e., respectively 3.6 ± 0.5- versus 3.5 ± 0.6-fold
greater than control.

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Fig. 12.
The effect of SNP on nuclear evidence of apoptosis.
Top: proportion of cells with apoptotic nuclei per 100 cells; bottom: apoptotic nuclei relative to control.
Cardiomyocytes were treated with 0.5 mM SNP (n = 5),
10 8 M H2O2 (n = 5), and the combination (n = 6), and control cells
(n = 6), that had been permeabilized with ethanol,
stained with PI, were aspirated into the flow cytometer. The proportion
of the 10,000 cells with low DNA content (PI staining)
defining the apoptotic population was calculated. The data for control
and SNP are repeated from Fig. 9 for ease of comparison. The data are
presented as the means ± SE with data analysis compared with
control (*P < 0.05; **P < 0.01) or SNP
( P < 0.05).
|
|

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Fig. 13.
Cell population characteristics were assessed using flow
cytometry. Cardiomyocytes were dually stained with fluorescein
diacetate (FDA) (y-axis) and propidium iodide
(x-axis). The defined apoptotic (A) population [low
fluorescein diacetate (FDA), low PI] was observed in cardiomyocytes
(circled population) and a necrotic (N) population defined as low FDA
with higher PI. Four representative studies of 10,000 cells each
showing control, 0.5 mM SNP, 10 8 M
H2O2, and 10 8 M
H2O2 plus SNP.
|
|

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Fig. 14.
The proportion of apoptotic and necrotic cells as defined by dual
staining with FDA and PI. Cardiomyocytes treated with 0.5 mM SNP
(n = 5), 10 8 M
H2O2 (n = 5), the combination
(n = 6), and control cells (n = 6) were
stained with FDA and PI and aspirated into the flow cytometer. The
proportion of the population of 10,000 cells that were classified as
apoptotic are shown at top and as necrotic at
bottom, with the ratio of treatment to control displayed at
right. The data are presented as the means ± SE with
data analysis compared with control (**P < 0.01) or SNP
( P < 0.05).
|
|
 |
DISCUSSION |
This study, to our knowledge, is the first detailed report of the
ability of nitroprusside to induce cell death in cardiomyocytes. We
demonstrated the cardiotoxicity of SNP by finding that SNP damaged
cellular functions that are integral markers of cell viability, specifically loss of plasma membrane integrity, as assessed by the
inability to exclude trypan blue or PI from entering the cell and the
failure to keep a fluorescent probe from leaving the cell. These data
were further supported by SNP-induced damage to mitochondrial function,
as assessed by the lack of mitochondrial dehydrogenase to reduce MTT
(18, 26). The adverse impact of SNP on cardiomyocyte cell
viability is consistent with the findings in murine neuroblastoma N1E-115 cells (47), the brain (37), and
vascular smooth muscle cells (32, 44). In other cell
types, SNP does not induce cell death (1, 7, 8, 10, 13).
Although the toxicity of SNP may be specific for only certain cell
types, it is noteworthy that our finding that 1.0 mM SNP produced an
87% increase in cell death in cardiomyocytes is similar to the 95%
reported by Yamada et al. (47) in neuroblastoma cells with
the same SNP concentration.
Although several factors have been implicated to explain SNP-induced
cell death, the precise mechanism has been uncertain (2, 27, 36,
37). Our data provide a compelling argument for the involvement
of free radicals in SNP-induced cardiotoxicity. We and others have
documented SNP-induced H2O2 production in cell culture (47) as SNP metabolism yields metabolites that
undergo redox cycling with oxygen to produce O2
·
(36). The O2
· formed during this cycle
readily produces H2O2 (36).
Treatment with SNP required 6 h before loss of cell viability was
recognized (47), consistent with the time lag needed to
produce a metabolite such as H2O2. We also
demonstrated NO production from SNP in cardiomyocytes. Although NO can
be released spontaneously from SNP (19), in the absence of
cells, SNP releases trivial amounts of NO (19, 20). NO is
released from SNP following metabolism involving one electron reduction
(36). NO induces mitochondria H2O2
production through an effect on cytochrome oxidase explained by
Ponderoso et al. (33) and demonstrated to occur in the
isolated heart (33). The potential of SNP toxicity
directly from NO is possible, but other NO donors such
S-nitrosoglutathione and
S-nitroso-N-acetyl-penicillamine afforded
protection against reactive oxygen species-induced toxicity in some
tissues and situations (44). The role of
H2O2 in SNP-mediated cardiac toxicity is
supported by our finding that catalase completely prevented SNP-induced
cell death at low SNP concentrations and significantly blunted it at
high SNP concentrations. Although we cannot exclude the possibility
that the preparation of catalase may have small amounts of superoxide
dismutase, the dominant effect should be that of catalase. Our findings
with catalase are supported by the data that catalase has the capacity
to totally inactivate H2O2 (5) and
exogenous catalase that can enter or become associated with myocytes
inhibits H2O2-induced cell death in this
cardiomyocyte type (17).
In contrast to the protective effects of catalase, we found no evidence
to support the contention that the iron moiety of SNP mediates cell
toxicity in cardiomyocytes compared with the findings in other
cell types (27, 36). The iron chelator deferoxamine did
not alter SNP-induced cardiomyocyte cell death. Although there are data
that deferoxamine can blunt H2O2-induced
cardiomyocyte cell damage, as reflected in loss of lactate
dehydrogenase activity, deferoxamine does not significantly
eliminate H2O2-mediated cardiomyocyte injury
(5). An alternative possibility is that any potential beneficial effects of deferoxamine on SNP-mediated cardiotoxicity is
offset by the deleterious effects of deferioxamine reacting with
hydroxyl radicals to produce a nitroxide free radical
(31). Another potential mechanism for SNP-induced
cardiotoxicity is the release of cyanide from the SNP molecule
(25). However, cyanide itself does not readily induce cell
death, as we found in these cardiomyocytes and others have found in
different cells culture (29, 47). Further cyanide
antidotes and trapping agents do not ameliorate but rather increase
SNP-induced cytotoxicity and lipid peroxidation in isolated hepatocytes
(29). Although a role for cyanide in SNP-induced
cardiotoxicity has not been excluded, the ability of catalase to
effectively reverse SNP-induced cell death at some SNP concentrations
strongly implicates a major role for H2O2 in
the pathogenesis of SNP-induced cardiomyocyte cell death. Furthermore,
cyanide does not induce the release of H2O2
(12).
SNP metabolism occurs rapidly. Within minutes of its intravenous
administration in animals and humans, SNP is metabolized (39), making calculation of serum concentration and volume
of distribution difficult. SNP metabolism also occurs in cell culture (29, 47) as contact with the sulfhydryl groups in the cell wall immediately initiates SNP degradation (39). Although
the rates of SNP metabolism in cell culture appears to be slower than those in humans (29), the rapid metabolism presents
problems in extrapolating the data from cell culture to intact animals and humans. High concentrations of SNP were used in the present study.
However, the SNP concentrations were the same or even less than other
studies of the cytotoxicity of SNP in other cell types in culture
(29, 47). Clinically, SNP is administered up to 800 µg/min so patients may receive >1 g in 24 h (39).
The volume of distribution of SNP is not known, but it is likely large
enough to considerably reduce the effective SNP concentration. The
sensitivity of the human myocardium to SNP and its metabolites are
additional factors that would influence the potential for SNP-induced
cardiotoxicity in humans.
Apoptotic cell death was demonstrated in cardiomyocytes after SNP
treatment. Cardiomyocyte cell structure was altered by SNP treatment
with cardiomyocytes displaying the characteristic features of
apoptosis, namely, reduction in cell size, membrane bleb formation, and
nuclear disintegration (46). The DNA staining
characteristics of cardiomyocyte nuclei evaluated by flow cytometry
demonstrated SNP-induced alterations of nuclear structure
characteristic of apoptosis (30). Evaluation of membrane
permeability by dual staining with FDA and PI (16) also
established that SNP treatment produced a significant increase in
apoptosis. The degree of apoptotic cell death was between twofold when
assessed by nuclear PI staining and fourfold when assessed by dual
staining with PI and FDA. The differences in the magnitude of apoptosis
reflect the different methodologies as the former technique examines
the nuclear properties of permeabilized cells and demonstrates
hypoploidy or low DNA content due to DNA fragmentation, a late event in
the process of apoptosis. The dual-staining approach with FDA and PI
evaluates membrane integrity and esterase activity of intact
cardiomyocytes, an early event in apoptosis. SNP was also found to
damage cellular membrane integrity in a manner consistent with necrotic
cell death; however, in the late stage of apoptosis there is also a
loss of membrane integrity as the cell dies (43). Our
demonstration of SNP-induced apoptosis in cardiomyocytes is consistent
with the findings in other cell types (32, 42, 44, 47). In addition, we were able to determine the magnitude of the effect of SNP
on cell viability and apoptosis and demonstrated that SNP-induced membrane alterations of the type associated with necrotic cell death
was the predominant change produced by SNP.
A major contribution of the present study is the demonstration that low
concentrations of H2O2 at levels that do not
alter cardiomyocyte viability produce a significant enhancement of
SNP-induced apoptosis. The concentration-effect relationship between
H2O2 and cardiomyocyte viability was
established and was consistent with the findings of Byler et al.
(5), who used the same cell type and duration of
H2O2 treatment but only a single high
H2O2 concentration. We chose low concentrations
of H2O2 that produced minimal or no significant
damage to cardiomyocytes; however, when combined with SNP, there was a
significant increase in apoptotic cell death. Interestingly, the amount
of necrotic cell death was not altered by the combination of SNP and
H2O2. As the later mode of cell death
predominated in SNP-induced cardiotoxicity, the potential synergy
between SNP and H2O2 on apoptotic cell death could not be identified using a total assessment of cellular death as
measured by MTT reduction. Recognizing the need for caution in using a
single-parameter measurement as an indicator of apoptosis (16), we used two different flow cytometry approaches.
FDA/PI staining of intact cells and PI staining of nuclei of
permeabilized cells provided concordant data of a synergistic effect of
SNP and H2O2. Cellular structural alterations
reflective of apoptosis (46), as determined by fluorescent
microscopy, were also accentuated when H2O2 was
combined with SNP. Taken together, these data indicate that the
combination of SNP plus H2O2 is especially
toxic to cardiomyocytes, and the effect is essentially restricted to
apoptotic cell death, especially in the early phase of apoptosis. The
adverse effect of the combination of nitroprusside and
H2O2 has been recognized by
H2O2 enhancement of the tumoricidal activity of
NO in Fu5 cells (20), cytotoxicity in V79 Chinese hamster
lung fibroblasts (45), and DNA fragmentation and cell
lysis in murine lymphoma cells (15). In contrast to these
studies that used lethal concentrations of
H2O2, our data is novel in the use of low,
nontoxic concentrations of H2O2 as well as
primary cells in culture rather than tumor cells or a cell line.
Extrapolation of the data must take into account the embryonic and
avian nature of the cardiomyocytes examined. However, this model has
several advantages, especially the ability to study the morphological
aspects of apoptosis in the entire cell. Second, these cells are
ideally suited for the study of chronic exposure to agents because they
are stable for days in culture and our studies used prolonged, 24-h
exposure to SNP and H2O2. Another positive
feature of using this cell type is that elements of the apoptotic
pathway such as the bcl-2 gene are abundant in the chick heart and show a high sequence homology to human bcl-2
(11). When the caveats associated with extrapolation from
cell culture data to patients are recognized, the data suggest the need
for caution in the use of SNP in situations associated with increases in myocardial H2O2, such as myocardial ischemia
and reperfusion or heart failure (3, 14, 38). The clinical
implications of the use SNP in situations with enhanced free radical
generation may warrant investigation from the perspective of enhanced
apoptotic cell death.
In summary, our results present novel findings for cardiomyocyte injury
induced by SNP. We established that the mode of SNP-induced cardiomyocyte cell death includes apoptosis. Because of the nature of
the nitroprusside molecule, we focused on free radical generation as a
potential cause of SNP-induced cardiotoxicity and implicated H2O2 by demonstrating SNP-induced
H2O2 generation and reversal of SNP-induced
loss of cell viability by catalase. The toxicity of
H2O2 to cardiac cell is consistent with many
studies (3, 4, 5). We extended those results by
demonstrating that under certain circumstances even low concentrations
of H2O2 have the potential for toxicity.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: S. Rabkin, Univ. of British Columbia, D410-2733 Heather St., Vancouver, BC, Canada V5Z 3J5 (E-mail: rabkin{at}interchange.ubc.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 April 1999; accepted in final form 14 July 2000.
 |
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