© 1999 American Heart Association, Inc.
Cellular Biology |
Mitochondrial Electron Transport Complex I Is a Potential Source of Oxygen Free Radicals in the Failing Myocardium
From the Research Institute of Angiocardiology and Cardiovascular Clinic (T.I., H.T., S.K., K.A., K.E., A.T.) and Department of Clinical Chemistry and Laboratory Medicine (H.U.), Kyushu University School of Medicine, Fukuoka, Japan; Department of Biophysics (D.K.), Kyushu University, Fukuoka, Japan; Department of Neurology (N.H.), Juntendo University School of Medicine, Tokyo, Japan; and Laboratory of Food and Biodynamics (K.U.), Nagoya University School of Bioagricultural Sciences, Nagoya, Japan.
Correspondence to Hiroyuki Tsutsui, MD, PhD, Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. E-mail prehiro{at}cardiol.med.kyushu-u.ac.jp
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Abstract—Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5,5'-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH–ubiquinone oxidoreductase (complex I) and succinate–ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P<0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274±13 versus 136±9 nmol · min-1 · mg-1 protein, P<0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium.
Key Words: antioxidant • free radical • heart failure • myocardial contraction • reactive oxygen species
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Introduction |
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Congestive heart failure (HF) is an important cause of morbidity and mortality in patients with various heart diseases. Despite extensive studies, the fundamental mechanisms responsible for the development and progression of left ventricular (LV) failure have not yet been fully elucidated. Reactive oxygen species (ROS) such as superoxide anions (·O2-) and hydroxy radicals (·OH) cause the oxidation of membrane phospholipids, proteins, and DNAs, and they have been implicated in a wide range of pathological conditions including ischemia-reperfusion injury, neurodegenerative diseases, and aging. Under physiological conditions, their toxic effects are prevented by such scavenging enzymes as superoxide dismutase (SOD), glutathione peroxidase, and catalase as well as by other nonenzymatic antioxidants. However, when the production of ROS becomes excessive, oxidative stress might have a harmful effect on the functional integrity of biological tissue. Oxygen free radicals have been shown to cause contractile failure and structural damage in the myocardium.1 2 However, their significance has been demonstrated to limited subsets of cardiac diseases, which include ischemic heart disease3 and adriamycin-induced cardiac toxicity.4
Recent investigations have suggested the generation of ROS to increase
in chronic HF. Lipid peroxides and 8-iso-prostaglandin
F2
, which are the major biochemical
consequences of ROS generation, have been shown to be elevated in
plasma and pericardial fluid of patients with HF and also positively
correlated to the severity of HF.5 6 7 In addition, a
decrease of myocardial antioxidant reserve has been shown in animal
models of HF.8 9 10 However, these experimental and
clinical findings have provided only indirect evidence of ROS
generation in failing myocardial tissue. Direct measurements of ROS
using electron spin resonance (ESR) spectroscopy11 are
therefore needed for direct quantitation within biological tissues.
The cellular sources and mechanisms for the increased ROS in HF also remain to be elucidated. Within the heart, possible cellular sources of ROS generation include cardiac myocytes, endothelial cells, and neutrophils. The contribution of neutrophils to ROS generation was found to be minor in an animal model of HF owing to rapid ventricular pacing, because the infiltration of inflammatory cells within the myocardium is not prominent. Within cardiac myocytes, oxygen free radicals can be produced by several mechanisms including the mitochondrial electron transport and xanthine dehydrogenase/xanthine oxidase.3 Because of the very low xanthine oxidase activity in some species12 and an extreme abundance of mitochondria in cardiac myocytes, mitochondrial electron transport could thus be a major subcellular source of ROS in the failing myocardium.13 14 Therefore, the ROS production was directly monitored in the mitochondria isolated from canine failing hearts induced by rapid ventricular pacing by means of ESR spectroscopy in the presence of a spin trap.11 The magnitude and mechanisms of ROS generation in the failing hearts were also compared with the findings in similar preparations from normal control hearts.
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Materials and Methods |
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Animal Model of HF
HF was produced in adult mongrel dogs (15 to 25 kg body weight) by rapid ventricular pacing at 240 bpm for 4 weeks, in accordance with methods described previously.15 All operative procedures were carried out under full surgical anesthesia with sodium pentobarbital (25 mg/kg IV). Two-dimensional and M-mode echocardiograms were recorded by ultrasonography. LV short-axis (cross-sectional) views were recorded at the papillary muscle level, and the internal LV dimensions were measured. The LV ejection fraction (%) was calculated using the formula [(LV End-Diastolic Dimension)3-(LV End-Systolic Dimension)3]/[(LV End-Diastolic Dimension)3]x100. After making echocardiographic recordings, the animals were generally anesthetized, intubated, and ventilated with a respirator on a heating pad to maintain body temperature at 37°C. A catheter was inserted into the aortic arch via the left carotid artery to measure the systemic arterial pressure. After a thoracotomy was performed, an externally calibrated 7F catheter-tipped pressure transducer was inserted into the LV through the left atrium for the measurement of LV pressure. At the time of the terminal study, the animals were killed with a lethal dose of
-chloralose. All LV
myocardial samples were immediately excised, freeze-clamped, and stored
in liquid nitrogen for the subsequent ESR measurements. All procedures and animal care were approved by the Committee on Ethics of Animal Experiments, Faculty of Medicine, Kyushu University, and were conducted according to the Guidelines for Animal Experiments of Faculty of Medicine, Kyushu University.
Measurement of Thiobarbituric Acid Reactive Substances
(TBARS)
Lipid peroxidation is a major biochemical consequence of ROS
attack on biological tissue. We therefore determined the degree of
lipid peroxidation in the myocardial tissue through biochemical assay
of TBARS.16 LV myocardial tissue was
homogenized (10% wt/vol) in 1.15% KCl solution (pH 7.4).
The homogenate was mixed with 0.4% sodium dodecyl
sulfate, 7.5% acetic acid adjusted to pH 3.5 with NaOH, and 0.3%
thiobarbituric acid. Butylated hydroxytoluene (0.01%) was added to the
assay mixture to prevent autoxidation of the sample. The mixture was
kept at 5°C for 60 minutes and was heated at 100°C for 60 minutes.
After cooling, the mixture was extracted with distilled water and
n-butanol:pyridine (15:1, vol/vol) and centrifuged
at 1600g for 10 minutes. The absorbance of the organic phase
was measured at 532 nm. The amount of TBARS was determined by the
absorbance with the molecular extinction coefficient of 156 000 and
expressed as µmol/g wet weight.
Immunohistochemistry of 4-Hydroxy-2-Nonenal (HNE)–Modified
Protein
To assess the cellular localization of lipid peroxidation by
histochemical analysis, sections of LV myocardium
obtained from the free wall at the papillary muscle level were
immunolabeled with an antibody raised against HNE-modified protein, an
aldehydic byproduct of lipid peroxidation.17 18
Paraffin-embedded tissue sections (3 µm thick) were
deparaffinized with xylene and refixed with Bouin's solution for 20
minutes and immersed in 70%, 90%, and 100% ethanol to remove picric
acid. To inhibit endogenous peroxidase, the sections were
incubated with 0.3% H2O2
in methanol for 30 minutes. After rinsing in 0.01 mol/L PBS, the
sections were incubated with normal goat serum (diluted to 1:10) to
inhibit nonspecific binding of antibodies. The sections were further
incubated with polyclonal antiserum raised against an HNE-modified
histidyl peptide (Gly3-His-Gly3) conjugated with keyhole-limpet
hemocyanin, which had been confirmed to be specific in our previous
studies.17 18 After rinsing with 0.01 mol/L PBS, the
sections were incubated with biotin-labeled goat anti-rabbit IgG
antiserum (diluted 1:100; DAKO A/S) for 60 minutes and then with
avidin-biotin complex (Vectastain ABC kit; 1:100) for 60 minutes. After
rinsing, the sections were finally incubated with 0.02%
3,3'-diaminobenzidine and 0.03% hydrogen peroxide in deionized water
for 6 to 9 minutes. As a negative control, the sections were also
incubated with normal rabbit serum.
A morphometric analysis of HNE-positive myocardial area was performed with tissue sections stained with HNE. Briefly, each section was photographed under a microscope and magnified (final magnification, x200). Three to four fields were randomly selected from one or two coronal sections in each animal. As a result, the HNE-positive areas were measured at approximately five to seven fields for each animal. Within each field, myocardial segments that stained positively with anti-HNE antibody were identified and were manually traced by using a digitizing pad with a computer to calculate the traced area.
Preparation of Cardiac Subcellular Fractions
The frozen LV tissues were homogenized at 4°C for
1 minute in 6 volumes of buffer consisting of 10 mmol/L HEPES-NaOH
(pH 7.4) and 250 mmol/L sucrose with a Polytron
homogenizer. The homogenate was
centrifuged at 4°C and 700g for 10 minutes to
remove any nuclear and myofibrillar debris, and the resultant
supernatant was centrifuged at 7000g for 10 minutes
to separate any cardiac subcellular fractions. To isolate the
mitochondria, the pellet was resuspended at 4°C in a buffer
consisting of 10 mmol/L HEPES-NaOH (pH 7.4), 1 mmol/L EDTA,
and 250 mmol/L sucrose (HES) and was washed three times with HES
buffer. The frozen and thawed mitochondrial fraction was suspended in a
buffer consisting of 10 mmol/L HEPES-NaOH (pH 7.4) and 1
mmol/L EDTA and kept at 4°C for 30 minutes. Submitochondrial
particles were prepared by sonicating the mitochondria. The sonicated
mitochondrial suspension was centrifuged at 21 000g
for 10 minutes. The submitochondrial particle pellet was washed three
times with HES buffer and stored at -80°C. The postmitochondrial
supernatant was centrifuged for 1 hour at 170 000g
at 4°C, and the final supernatant was used as the cytosolic fraction.
The resultant pellet was resuspended at 4°C in HES buffer and was
washed three times to obtain the microsomal fraction. These
submitochondrial particle, cytosolic, and microsomal fractions were
used for the ESR measurement of
·O2-.
The cardiac SOD levels were determined in the submitochondrial particle, microsomal, and cytosolic fractions using the xanthine:xanthine oxidase:cytochrome c assay according to methods described previously.19 The protein concentration of each fraction was adjusted to obtain approximately a 50% inhibition of the rate of cytochrome c reduction produced by the xanthine:xanthine oxidase system. No SOD activity could be detected in the submitochondrial particle fractions from either control or HF hearts. There was no significant difference in the SOD activity of each fraction between the control and HF.
Special care was taken to minimize any artifactual generation of radical signals, in accordance with methods described by Zweier et al.20
ESR Measurement of ROS
To demonstrate ROS in the mitochondria obtained from control and
HF hearts, the submitochondrial particle fractions were reacted with
the substrate and a spin trapping agent,
5,5'-dimethyl-1-pyrroline-N-oxide (DMPO; LABOTEC, Ltd), and
processed for ESR spectroscopy. Immediately after starting the reaction
(< 45 seconds), ESR spectra were recorded at an ambient
temperature (25°C) with an ESR spectrometer (JES-RE-1X; JEOL)
operating at X-band (4.95 GHz). The microwave power was 10 mW, the
field modulation width was 0.063 mT, and the magnetic field range was
swept at a scan rate of 5 mT/min. The quantitation of the DMPO signal
intensity was performed by comparing the amplitude of the observed
signal to the standard Mn2+/MgO marker.
Two segments of the respiratory chain are primarily responsible for ROS
generation in the mitochondria, the NADH–ubiquinone reductase in
complex I and the ubiquinol–cytochrome c reductase in
complex III (Figure 1
).21 22 23 NADH
(200 µmol/L) was used as a substrate for complex I and succinate
(10 mmol/L) for complex II (succinate–ubiquinone
oxidoreductase).
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To determine the amount of ·O2- production in the microsomal and cytosolic fraction, each fraction obtained from control and HF hearts was reacted with NADH (200 µmol/L), and ESR spectra of DMPO were recorded in accordance with the same methods as those performed in the submitochondrial fractions.
Assay of Mitochondrial Complex I Activity
To delineate the basis for the block of electron transfer at
complex I, its enzymatic activity was measured. To measure the complex
I activity, the submitochondrial particle fractions were assayed for a
reduction of ubiquinone analog, decylubiquinone, using a
spectrophotometer, in accordance with the method of Trounce et
al24 with some modifications. Enzyme activity was
expressed in nmol · min-1 ·
mg-1 protein.
Statistical Analysis
All data are expressed as mean±SEM. To compare the data between
control and HF, Student's unpaired t test was used. For
multiple comparisons, one-way ANOVA was used in conjunction with the
post hoc test using Scheffé's correction. All tests were
considered to be statistically significant at P<0.05.
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Results |
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Contractile Dysfunction in HF Model
Rapid pacing caused an approximately 125% increase in end-systolic LV dimension and a 60% decrease in LV ejection fraction (Table 1
). For HF dogs, LV peak
positive dP/dt was depressed, and LV end-diastolic pressure
was elevated in comparison to the control values. As a result, HF dogs
showed similar hemodynamic characteristics to those of
humans with dilated
cardiomyopathy.15 25
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Lipid Peroxidation of Failing Myocytes
TBARS significantly increased in HF animals (Figure 2A). Most importantly, an
immunohistochemical analysis of HNE-modified protein revealed
the lipid peroxides to be positively stained in myocytes from HF dogs,
whereas no labeling was observed in the control myocardium
(Figure 2B). The myocardial area stained positively with HNE was
18±6% in HF hearts, whereas it was 2±0.2% in control
(P<0.01).
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Mitochondria as the Source of ROS Production
At baseline conditions, only small ESR signals appeared for the
submitochondrial particle fractions obtained from the normal heart in
the presence of NADH or succinate as a substrate (Figure 3). When submitochondrial particle
fractions were treated with rotenone (200 µmol/L), which
selectively blocks the electron transport at the distal site of complex
I, in the presence of NADH, prominent ESR signals of DMPO–superoxide
adduct, DMPO-OOH, were observed with characteristic hyperfine
splittings that yielded 12 resolved peaks (Figure 3A). A
preliminary dose-response assay of rotenone concentration versus
DMPO-OOH signal magnitude showed a maximal effect of rotenone to be
achieved at a concentration of 200 µmol/L. We verified that the
observed DMPO signals reflected the presence of
·O2- by a comparison of
the signals with the identical hyperfine splittings elicited in the
presence of pure ·O2-
generated from the reaction of hypoxanthine (500 µmol/L) and
xanthine oxidase (100 mU) in vitro (Figure 3C). In addition, the
DMPO signals were completely attenuated both in the presence of SOD
(100 U/mL) and by the combination of SOD plus catalase (500 U/mL)
(Table 2). These results indicate that
the functional block of complex I is capable of producing
·O2-, which is
consistent with the findings of previous
studies.21 22 In addition, antimycin A (200 mmol/L),
an inhibitor of complex III, further increased the DMPO-OOH
signals in normal mitochondria, which thus indicated the additive
nature of the two sites (complex I and complex III) in mitochondrial
·O2- production
(Figure 3A and Table 2). DMPO-OOH signals elicited by
NADH and antimycin A were also attenuated in the presence of SOD or SOD
plus catalase (Table 2), which indicated that the observed
signals originated from
·O2-.
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To further confirm the generation of
·O2- at complex III,
normal submitochondrial particles were treated with antimycin A
(200 mmol/L) in the presence of succinate (10 mmol/L).
Antimycin A also increased the DMPO-OOH signals (Fig-ure
3B and Table 2
), thus indicating that
·O2- can also be
produced from the cytochrome b-c1 segment
of complex III. The DMPO signals elicited by succinate and antimycin A
were abolished in the presence of SOD or SOD plus catalase (Table 2). This finding is consistent with a previous study
that showed ·O2- to be
produced by a reduced component of complex III when antimycin A is
present.23 Taken together, these results suggest
that both complex I and complex III are important sources of ROS in
cardiac myocytes.
ROS Production in HF Mitochondria
To determine whether
·O2- production
is enhanced in mitochondria isolated from HF and to identify its
production site, submitochondrial particles obtained from HF
hearts were reacted with NADH (200 µmol/L), and the ESR spectra
of DMPO were recorded. Only small signals appeared for normal
control heart, whereas HF exerted a prominent ESR signal of DMPO-OOH,
thus indicating the generation of
·O2- (Figure 4A). The magnitude of
·O2- was 2.8-fold
(P<0.01) higher in HF than in control mitochondria (Figure 5). For the HF mitochondria studied, each
sample demonstrated DMPO-OOH spectra. Repeated experiments on the same
heart exerted the same spectra (repeated measurements of DMPO-OOH
magnitude: 100±5% of initial value, P=NS). These ESR
signals were attenuated in the presence of SOD (100 U/mL) alone or SOD
(100 U/mL) plus catalase (500 U/mL), thus suggesting that most ESR
signals derived from ·O2 (Figure 5).
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P<0.05 for difference from the HF
value in the presence of NADH.
To examine the production of
·O2- at complex III, the
submitochondrial particles were incubated with succinate (10
mmol/L). In contrast to NADH as a substrate, ESR demonstrated very few
DMPO signals in the HF heart (Figure 4B). The production
of ·O2- in normal
mitochondria treated with complex I inhibitor, rotenone, in
the presence of NADH (Figure 3A) was comparable to the baseline
·O2- production
in the HF mitochondria (Figure 4A). An important finding was
that ·O2-
production was initiated by the addition of NADH but not of
succinate, thus indicating that the electron transfer function at
complex I was primarily responsible for such production in
HF.
·O2- production
by complex I in the submitochondrial particles from HF hearts was
maximally enhanced and there was no significant
"rotenone"-recruitable reserve (Figure 4 and Table 2). We next examined whether the inhibition of complex III in HF
mitochondria could further increase the
·O2- production
as shown in normal mitochondria. Antimycin A further increased the
DMPO-OOH signals in the HF mitochondria in the presence of either NADH
or succinate as a substrate (Figure 4 and Table 2), thus
indicating that the additive nature of complex I and complex III in the
·O2- production
is also present in HF. Most importantly, the combination of Figure 3 and Figure 4 suggests that
·O2- is produced via a
functional block at complex I in HF.
The magnitude of DMPO-OOH signals obtained from the microsomal
fractions was found to be comparable between control and HF (Table 3). However, the signals could not be
detected in the cytosolic fractions.
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Complex I Enzymatic Activity
Complex I activity significantly decreased in HF
myocardium in comparison to control values (274±13 versus
136±9 nmol · min-1 ·
mg-1 protein, P<0.01).
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Discussion |
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The present study using ESR spectroscopy provided the first direct evidence for the increased generation of ·O2- in the mitochondria isolated from the failing heart. The decrease in complex I enzymatic activity and the resultant impairment of electron transfer lead to the deleterious production of ROS in the intracellular space, which could result in the myocyte injury in HF.
Mitochondria produce ROS through one electron carrier in the
respiratory chain. Under physiological conditions,
small quantities of ROS are formed during mitochondrial respiration,
which, however, can be detoxified by the endogenous
scavenging mechanisms of myocytes. In line with the findings of
previous studies,26 the inhibition of electron transport
at the sites of complex I and complex III in the normal
submitochondrial particles resulted in a significant production
of ·O2- (Figure 3). The most important findings of the present study were
that HF mitochondria produce more
·O2- than normal
mitochondria in the presence of NADH but not succinate (Figure 4) and that complex I is the predominant source of such
·O2- production
(Figures 3 and 4, Table 2). Furthermore, HF
mitochondria were also found to be associated with a decrease in the
complex I activity. Although the present study could not prove the
mechanisms for this decreased activity, it has provided compelling
evidence that the defects in electron transfer function lead to
mitochondrial ROS production. In aging and neurodegenerative
diseases such as Parkinson disease, the mitochondria are the
predominant source of ROS.27 In this respect, our results
demonstrated a similar pathophysiological link
between mitochondrial dysfunction and oxidative stress in failing
hearts.
Cardiac myocytes are the likely targets of ROS attack in the failing
heart (Figure 2B). It is conceivable that free radicals cause
damage at or near the site of their formation, given that they are a
highly reactive and short-lived species. Therefore, as major sources of
ROS production, mitochondria also could be major targets of ROS
attack and thus be particularly susceptible to its
attack,28 which further impairs the function of the
respiratory chain (T.I., unpublished data, 1998) and accelerates
·O2- production
within the mitochondria. Mitochondrial ROS production may
underlie the mutations and/or deletions of mitochondrial DNA, which
subsequently should lead to a further impairment of the mitochondrial
function.29 The high susceptibility of mitochondrial DNA
to mutation and oxidative damage is likely a reflection of this
localized production of ROS by electron transport
oxidation.30
To determine whether the decreased antioxidant capacity may further aggravate the ROS accumulation in HF, SOD was quantified in the myocardial tissues. However, there was no significant difference in the SOD content between control and HF (H.T., unpublished data, 1998), which thus indicates that oxidative stress in HF is primarily due to the enhancement of mitochondrial prooxidant generation rather than the decline in antioxidant defenses. It should be acknowledged that other potential sources of ROS generation within the heart including vascular endothelial cells (via xanthine oxidase and/or NADPH oxidase) and activated leukocytes (via NADPH oxidase)3 could not be completely ruled out in the present study. Although mitochondrial electron transport plays an important role in the ROS production in HF, other mechanisms also might be involved. The activation of neurohumoral factors commonly seen in HF, including catecholamines and cardiac sympathetic tone, renin-angiotensin system, and nitric oxide, can contribute to the generation of ROS.31 32 33 34
The present study demonstrated that the production of ROS and ROS-induced lipid peroxidation are enhanced in HF heart. Oxygen radicals ·OH and ·O2- have been demonstrated to impair myocardial contractile function in vivo and in vitro.2 35 Further, lipid peroxides, byproducts of ROS generation, could also cause myocardial contractile defects and ultimately lead to structural damage.36 Although the pathophysiology of intense cellular damage resulting from the radical exposure commonly seen under ischemia and reperfusion could not be equated to the cardiac damage in HF, ROS production exceeding cellular antioxidant defense capabilities can have potentially lethal consequences for cardiac myocytes over longer periods. Recently, oxygen radicals have been suggested to be involved in apoptotic cell death.37 38 As a result, apoptosis might play an important role in the pathogenesis of HF.39
Although the ESR technique is a direct method to detect ROS within
biological tissue, several crucial steps must be performed carefully.
First, given the highly transient nature of ROS, the ESR measurement
procedures themselves may modify the status of ROS by destroying the
antioxidant defenses and/or activating the ROS production
system. Therefore, we took special care to minimize the artifactual
generation of ROS and to preserve the in situ status of ROS by using
freeze-clamped myocardial tissues, rigorously maintaining the sample
under anaerobic conditions in the liquid
nitrogen.20 More importantly, a differential effect of the
experimental procedures on HF tissue seems unlikely. Second, although
DMPO has been the most versatile and commonly used spin trap for
measuring ·O2-, the
half-life of DMPO–superoxide adduct, DMPO-OOH, is as short as 50
seconds in aqueous media, and it spontaneously decomposes to form the
DMPO–OH adduct,40 which was identical to that formed from
trapping the ·OH radical. To overcome these limitations, ESR
spectra were recorded immediately after (< 45 seconds) the
addition of DMPO to the sample. We confirmed that the DMPO signals
detected in HF mitochondria are specific for
·O2- by use of SOD
(Figure 5), and identical DMPO-OOH signals were observed in the
presence of pure ·O2-
generated by the interaction of hypoxanthine and xanthine oxidase in
vitro (Figure 3). Furthermore, we obtained the same results by
using
5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide,40
a more sensitive and stable spin trap than DMPO (T.I.,
unpublished data, 1998).
Our findings do not prove a causal relationship. Indeed, given the importance of ROS in the pathophysiology of HF, the effects of radical scavengers on the reversibility or prevention of HF and the establishment of a cause-and-effect relationship will need to be determined in future studies.
HF is associated with alterations of various signal molecules in the myocardium, including the renin-angiotensin and sympathetic nervous systems, peptide growth factors, cytokines, nitric oxide, and ROS. All of these have the potential to exert profound effects on the phenotype of the myocardium. There are probably multiple levels of autocrine and paracrine interactions among these mediators resulting from both positive and negative feedback loops; therefore, studies in isolated tissues may not replicate the in vivo environment.41 It must be stated that a purely reductionistic approach might overlook the critical interactions among the various signal molecules in the intact heart. Although a more focused experimental approach is essential for determining the cellular and subcellular mechanisms stimulating these systems in HF, it will be important to search for a better understanding of the integrated regulation of various mediators in HF in future studies.
The present study provides, for the first time, direct evidence for the increased production of ·O2- at the complex I site in the mitochondria isolated from failing cardiac myocytes. Although the focus of this study was the heart, these mechanisms of ROS production probably have a common foundation with other pathological conditions in other organs such as the brain, liver, and kidney. These results provide a direct link among ROS generation, mitochondrial dysfunction, and contractile defects in HF and suggest that oxidative stress plays an important role in the pathogenesis of HF.
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Acknowledgments |
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This work was supported in part by grants from the Ministry of Education, Science, and Culture (Nos. 07266220, 08258221, and 09670724).
Received February 15, 1999; accepted June 25, 1999.
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A. Warnholtz and T. Munzel The failing human heart: Another battlefield for the NAD(P)H oxidase? J. Am. Coll. Cardiol., June 18, 2003; 41(12): 2172 - 2174. [Full Text] [PDF]
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N. Suematsu, H. Tsutsui, J. Wen, D. Kang, M. Ikeuchi, T. Ide, S. Hayashidani, T. Shiomi, T. Kubota, N. Hamasaki, et al. Oxidative Stress Mediates Tumor Necrosis Factor-{alpha}-Induced Mitochondrial DNA Damage and Dysfunction in Cardiac Myocytes Circulation, March 18, 2003; 107(10): 1418 - 1423. [Abstract] [Full Text] [PDF]
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 X. Qi, A. S. Lewin, W. W. Hauswirth, and J. Guy Optic Neuropathy Induced by Reductions in Mitochondrial Superoxide Dismutase Invest. Ophthalmol. Vis. Sci., March 1, 2003; 44(3): 1088 - 1096. [Abstract] [Full Text] [PDF]
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M. R. Sayen, A. B. Gustafsson, M. A. Sussman, J. D. Molkentin, and R. A. Gottlieb Calcineurin transgenic mice have mitochondrial dysfunction and elevated superoxide production Am J Physiol Cell Physiol, February 1, 2003; 284(2): C562 - C570. [Abstract] [Full Text] [PDF]
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Y. Machida, T. Kubota, N. Kawamura, H. Funakoshi, T. Ide, H. Utsumi, Y. Y. Li, A. M. Feldman, H. Tsutsui, H. Shimokawa, et al. Overexpression of tumor necrosis factor-alpha increases production of hydroxyl radical in murine myocardium Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H449 - H455. [Abstract] [Full Text] [PDF]
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D. T. Lucas, P. Aryal, L. I. Szweda, W. J. Koch, and L. A. Leinwand Alterations in mitochondrial function in a mouse model of hypertrophic cardiomyopathy Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H575 - H583. [Abstract] [Full Text] [PDF]
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R. J. Scheubel, M. Tostlebe, A. Simm, S. Rohrbach, R. Prondzinsky, F. N. Gellerich, R.-E. Silber, and J. Holtz Dysfunction of mitochondrial respiratory chain complex I in human failing myocardium is not due to disturbed mitochondrial gene expression J. Am. Coll. Cardiol., December 18, 2002; 40(12): 2174 - 2181. [Abstract] [Full Text] [PDF]
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S. Srivastava, B. Chandrasekar, A. Bhatnagar, and S. D. Prabhu Lipid peroxidation-derived aldehydes and oxidative stress in the failing heart: role of aldose reductase Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2612 - H2619. [Abstract] [Full Text] [PDF]
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P. J Hanley, J. Ray, U. Brandt, and J. Daut Halothane, isoflurane and sevoflurane inhibit NADH: ubiquinone oxidoreductase (complex I) of cardiac mitochondria J. Physiol., November 1, 2002; 544(3): 687 - 693. [Abstract] [Full Text] [PDF]
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Y. Sun, J. Zhang, L. Lu, S. S. Chen, M. T. Quinn, and K. T. Weber Aldosterone-Induced Inflammation in the Rat Heart : Role of Oxidative Stress Am. J. Pathol., November 1, 2002; 161(5): 1773 - 1781. [Abstract] [Full Text] [PDF]
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P.a. Pacher, L. Liaudet, J. G. Mabley, K. Komjati, and C. Szabo Pharmacologic inhibition of poly(adenosine diphosphate-ribose) polymerase may represent a novel therapeutic approach in chronic heart failure J. Am. Coll. Cardiol., September 4, 2002; 40(5): 1006 - 1016. [Abstract] [Full Text] [PDF]
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P. J Hanley, M. Mickel, M. Loffler, U. Brandt, and J. Daut KATP channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart J. Physiol., August 1, 2002; 542(3): 735 - 741. [Abstract] [Full Text] [PDF]
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E. Novalija, S. G. Varadarajan, A. K. S. Camara, J. An, Q. Chen, M. L. Riess, N. Hogg, and D. F. Stowe Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated hearts Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H44 - H52. [Abstract] [Full Text] [PDF]
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K. Nakamura, K. Kusano, Y. Nakamura, M. Kakishita, K. Ohta, S. Nagase, M. Yamamoto, K. Miyaji, H. Saito, H. Morita, et al. Carvedilol Decreases Elevated Oxidative Stress in Human Failing Myocardium Circulation, June 18, 2002; 105(24): 2867 - 2871. [Abstract] [Full Text] [PDF]
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W. F. Saavedra, N. Paolocci, M. E. St. John, M. W. Skaf, G. C. Stewart, J.-S. Xie, R. W. Harrison, J. Zeichner, D. Mudrick, E. Marban, et al. Imbalance Between Xanthine Oxidase and Nitric Oxide Synthase Signaling Pathways Underlies Mechanoenergetic Uncoupling in the Failing Heart Circ. Res., February 22, 2002; 90(3): 297 - 304. [Abstract] [Full Text] [PDF]
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J. Shite, F. Qin, W. Mao, H. Kawai, S. Y. Stevens, and C.-s. Liang Antioxidant vitamins attenuate oxidative stress and cardiac dysfunction in tachycardia-induced cardiomyopathy J. Am. Coll. Cardiol., November 15, 2001; 38(6): 1734 - 1740. [Abstract] [Full Text] [PDF]
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J. Shiraishi, T. Tatsumi, N. Keira, K. Akashi, A. Mano, S. Yamanaka, S. Matoba, J. Asayama, T. Yaoi, S. Fushiki, et al. Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis Am J Physiol Heart Circ Physiol, October 1, 2001; 281(4): H1637 - H1647. [Abstract] [Full Text] [PDF]
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J. M. Hare Oxidative Stress and Apoptosis in Heart Failure Progression Circ. Res., August 3, 2001; 89(3): 198 - 200. [Full Text] [PDF]
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H. Tsutsui, T. Ide, S. Hayashidani, N. Suematsu, T. Shiomi, J. Wen, K.-i. Nakamura, K. Ichikawa, H. Utsumi, and A. Takeshita Enhanced Generation of Reactive Oxygen Species in the Limb Skeletal Muscles From a Murine Infarct Model of Heart Failure Circulation, July 10, 2001; 104(2): 134 - 136. [Abstract] [Full Text] [PDF]
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T. Ide, H. Tsutsui, S. Hayashidani, D. Kang, N. Suematsu, K.-i. Nakamura, H. Utsumi, N. Hamasaki, and A. Takeshita Mitochondrial DNA Damage and Dysfunction Associated With Oxidative Stress in Failing Hearts After Myocardial Infarction Circ. Res., March 16, 2001; 88(5): 529 - 535. [Abstract] [Full Text] [PDF]
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T. Ukai, C.-P. Cheng, H. Tachibana, A. Igawa, Z.-S. Zhang, H.-J. Cheng, and W. C. Little Allopurinol Enhances the Contractile Response to Dobutamine and Exercise in Dogs With Pacing-Induced Heart Failure Circulation, February 6, 2001; 103(5): 750 - 755. [Abstract] [Full Text] [PDF]
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H. Tsutsui, T. Ide, S. Hayashidani, N. Suematsu, H. Utsumi, R. Nakamura, K. Egashira, and A. Takeshita Greater susceptibility of failing cardiac myocytes to oxygen free radical-mediated injury Cardiovasc Res, January 1, 2001; 49(1): 103 - 109. [Abstract] [Full Text] [PDF]
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G. S. Filippatos, B. D. Uhal, H. Tsutsui, T. Ide, S. Kinugawa, A. Takeshita, and H. Utsumi Effects of Amiodarone on Heart Cells Response Circulation, November 14, 2000; 102 (20): e170 - e170. [Full Text] [PDF]
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S. Kinugawa, H. Tsutsui, S. Hayashidani, T. Ide, N. Suematsu, S. Satoh, H. Utsumi, and A. Takeshita Treatment With Dimethylthiourea Prevents Left Ventricular Remodeling and Failure After Experimental Myocardial Infarction in Mice : Role of Oxidative Stress Circ. Res., September 1, 2000; 87(5): 392 - 398. [Abstract] [Full Text] [PDF]
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D. B. Sawyer and W. S. Colucci Mitochondrial Oxidative Stress in Heart Failure : "Oxygen Wastage" Revisited Circ. Res., February 4, 2000; 86(2): 119 - 120. [Full Text] [PDF]
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T. Ide, H. Tsutsui, S. Kinugawa, N. Suematsu, S. Hayashidani, K. Ichikawa, H. Utsumi, Y. Machida, K. Egashira, and A. Takeshita Direct Evidence for Increased Hydroxyl Radicals Originating From Superoxide in the Failing Myocardium Circ. Res., February 4, 2000; 86(2): 152 - 157. [Abstract] [Full Text] [PDF]
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W. F. Saavedra, N. Paolocci, M. E. St. John, M. W. Skaf, G. C. Stewart, J.-S. Xie, R. W. Harrison, J. Zeichner, D. Mudrick, E. Marban, et al. Imbalance Between Xanthine Oxidase and Nitric Oxide Synthase Signaling Pathways Underlies Mechanoenergetic Uncoupling in the Failing Heart Circ. Res., February 22, 2002; 90(3): 297 - 304. [Abstract] [Full Text] [PDF]
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G. D. Thomas, W. Zhang, and R. G. Victor Impaired Modulation of Sympathetic Vasoconstriction in Contracting Skeletal Muscle of Rats With Chronic Myocardial Infarctions : Role of Oxidative Stress Circ. Res., April 27, 2001; 88(8): 816 - 823. [Abstract] [Full Text] [PDF]
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D. R. Pimentel, J. K. Amin, L. Xiao, T. Miller, J. Viereck, J. Oliver-Krasinski, R. Baliga, J. Wang, D. A. Siwik, K. Singh, et al. Reactive Oxygen Species Mediate Amplitude-Dependent Hypertrophic and Apoptotic Responses to Mechanical Stretch in Cardiac Myocytes Circ. Res., August 31, 2001; 89(5): 453 - 460. [Abstract] [Full Text] [PDF]
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