© 2000 American Heart Association, Inc.
Integrative Physiology |
Direct Evidence for Increased Hydroxyl Radicals Originating From Superoxide in the Failing Myocardium
From Cardiovascular Medicine (T.I., H.T., S.K., N.S., S.H., Y.M., K.E., A.T.), Graduate School of Medical Sciences, and Department of Biophysics, Faculty of Pharmaceutical Sciences (K.I., H.U.), Kyushu University, Fukuoka, Japan.
Correspondence to Hiroyuki Tsutsui, Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, 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 |
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Abstract—Experimental and clinical studies have suggested an increased production of reactive oxygen species (ROS) in the failing myocardium. The present study aimed to obtain direct evidence for increased ROS and to determine the contribution of superoxide anion (·O2-), H2O2, and hydroxy radical (·OH) in failing myocardial tissue. Heart failure was produced in adult mongrel dogs by rapid ventricular pacing at 240 bpm for 4 weeks. To assess the production of ROS directly, freeze-clamped myocardial tissue homogenates were reacted with the nitroxide radical, 4-hydroxy-2,2,6,6,-tetramethyl-piperidine-N-oxyl, and its spin signals were detected by electron spin resonance spectroscopy. The rate of electron spin resonance signal decay, proportional to ·OH level, was significantly increased in heart failure, which was inhibited by the addition of dimethylthiourea (·OH scavenger) into the reaction mixture. Increased ·OH in the failing heart was abolished to the same extent in the presence of desferrioxamine (iron chelator), catalase (H2O2 scavenger), and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; LaMotte) (·O2- scavenger), indicating that ·OH originated from H2O2 and ·O2-. Further, ·O2- produced in normal myocardium in the presence of antimycin A (mitochondrial complex III inhibitor) could reproduce the increase of H2O2 and ·OH seen in the failing tissue. There was a significant positive relation between myocardial ROS level and left ventricular contractile dysfunction. In conclusion, in the failing myocardium, ·OH was produced as a reactive product of ·O2- and H2O2, which might play an important role in left ventricular failure.
Key Words: antioxidant • free radicals • heart failure • myocardial contraction • myocardium
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In patients with heart failure (HF), thiobarbituric acid reactive substances (TBARS) and 8-iso prostaglandin (PG) F2
,
major biochemical consequences of reactive oxygen species (ROS)
generation have been found to be elevated in plasma1 and
pericardial fluid,2 respectively. Animal studies have
shown that antioxidant enzyme activities are decreased and TBARS are
increased in HF myocardium.3 4 5 Even though
these studies suggest that ROS are increased in the failing heart, all
findings are inconclusive and a direct measurement of ROS has not been
performed. Further, it remains unknown which species among 3 major
oxygen metabolites, superoxide anion
(·O2-), hydrogen
peroxide (H2O2), and
hydroxyradical (·OH), are responsible for oxidative stress in
HF. We have recently demonstrated that ·O2- production is increased in the submitochondrial particle fractions isolated from canine failing hearts as a result of rapid ventricular pacing.6 We measured ·O2- by using electron spin resonance (ESR) spectroscopy with 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap, a standard method to detect ROS in the biological tissue. Even though it can quantify ·O2- with high sensitivity and specificity, DMPO adducts are unstable and fall below the detection limits when measured in the multicellular tissue, and thus the fractionation of subcellular components should be required to obtain measurable ESR signals.6 When the subcellular fractions are used, the effects of scavenging enzymes such as superoxide dismutase (SOD), glutathione peroxidase, and catalase, as well as nonenzymatic antioxidants in the cytoplasm, are ignored. Further, because of high specificity of DMPO for ·O2-, DMPO is not applicable for assessing the concomitant formation of other ROS such as H2O2 and ·OH within the tissue. Therefore, it still remains unanswered whether the production of downstream ROS is indeed enhanced in the failing myocardium.
Recent studies have shown that the formation of ROS can be monitored by using ESR combined with the nitroxide radical 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxy-TEMPO) as a spin probe.7 8 9 Nitroxide radical is a stable chemical and thus can be easily detected by ESR. Biological reductants can reduce nitroxides into corresponding nonparamagnetic hydroxylamine compounds, which are ESR silent.7 8 Therefore, the reduction of nitroxides into nonparamagnetic products can provide a new dimension in monitoring redox metabolism within the biological tissue.10 11 12
The present study aimed to address the question whether ·OH production is increased in the HF myocardium. For this purpose, we measured the rate of hydroxy-TEMPO ESR signal decay in the presence of myocardial tissue and compared its rate between control and HF. To delineate the origin of ·OH generated in HF, the effects of ROS scavengers and iron chelator on ·OH production were examined.
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Materials and Methods |
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ESR Experiments
ESR measurements were performed at room temperature using an X-band (9.45-GHz) ESR spectrometer (JES-RE-1X; Jeol).
In Vitro Validation Studies
To interpret the rate of ESR signal decay accurately and to
confirm its efficiency in the quantification of ROS, we performed in
vitro validation studies on the metabolism of hydroxy-TEMPO
in the presence of the exogenous ROS generating system before applying
this method to the failing heart. ·OH was generated in vitro by
the combination of H2O2 (1
to 5 mmol/L) plus Fe3+-nitriotriacetate
(NTA; 20 µmol/L), and
·O2- was generated in
vitro by xanthine (500 µmol/L) plus xanthine oxidase (20 mU/mL).
All ESR samples were prepared using PBS (150 mol/L NaCl, 3 mmol/L
KCl, and 5 mmol/L phosphate; pH 7.4), mixed quickly, and then
drawn into glass tubes. The reaction mixture was immediately reacted
with hydroxy-TEMPO (0.1 mmol/L) in PBS, and its ESR spectra were
recorded for up to 10 minutes at intervals of 10 to 15 seconds. The
rate of signal decay was measured in the presence of exogenous
·OH or ·O2-.
Animal Model of HF
HF was produced in adult mongrel dogs by rapid
ventricular pacing according to methods described
previously.6 All procedures and animal care were approved
by the Committee on Ethics of Animal Experiments, Kyushu University,
and conducted according to animal care guidelines of the American
Physiological Society.
Quantification of ROS in Myocardial Tissue
At the time of the study, the animals were euthanized by a
lethal dose of pentobarbital. Freeze-clamped left
ventricular (LV) myocardial samples were
homogenized in 50 mmol/L sodium phosphate buffer (pH
7.4) containing protease inhibitors. The
homogenate was immediately reacted with hydroxy-TEMPO
(0.1 mmol/L), and its ESR spectra were recorded as described
above. The formation of ·OH was confirmed by adding
dimethylthiourea (DMTU; 50 mmol/L) into the
reaction mixture. The contribution of
·O2- and
H2O2 was examined in the
presence of 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; LaMotte)
(100 µmol/L) and catalase (50 U/mL) as a competitive reagent,
respectively.
Statistical Analysis
Data are expressed as mean±SEM. To compare the data between
control and HF, a Student unpaired t test was used. For
multiple comparisons, 1-way ANOVA was used to evaluate mean differences
in conjunction with a post hoc test with the Scheffé correction.
Correlation was examined by linear regression analysis using
the least square method. Multiple regression analysis was also
performed to analyze the relation between the signal decay and
LV contractile function. All tests were considered statistically
significant at P<0.05.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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In Vitro Validation Studies
ESR signals of hydroxy-TEMPO were observed as 3 sharp lines, and the peak signal intensity was stable with time. In the presence of ·OH generated by a combination of H2O2 plus Fe3+-NTA, ESR signals reduced more rapidly (Figure 1A
). There was a linear relation
in the semilogarithmic plot of peak signal intensity versus time,
indicating the first-order kinetics of the signal decay (Figure 1B). The rate of signal decay was proportional to the
concentrations of H2O2 in
the reaction mixture and thus the amount of ·OH (Figure 1B and 1C). Increase in the rate of signal decay was completely
abolished by the addition of DMTU, specific ·OH scavenger, into
the reaction mixture, confirming that increased decay rate indeed
resulted from the generation of ·OH. In contrast,
·O2- elicited by
xanthine plus xanthine oxidase did not attenuate hydroxy-TEMPO signal
(Figure 2). Further addition of SOD,
which can lead to the production of
H2O2, did not affect the
rate of signal decay. In contrast, further addition of SOD and
Fe3+ into the reaction mixture, which can produce
·OH via the Fenton reaction, increased the decay of
hydroxy-TEMPO signal. Thus, hydroxy-TEMPO can be reduced into
nonparamagnetic hydroxylamine compound only by ·OH, not by
H2O2 or
·O2-, in our
experimental conditions.
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Animal Characteristics
Chronic rapid ventricular pacing caused an 
89%
increase in end-systolic LV dimension and a 51% decrease in LV
ejection fraction by echocardiography. For the HF
dogs, LV peak positive dP/dt was depressed, and LV
end-diastolic pressure was increased, compared with control
values (Table).
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ROS Production in HF Myocardium
To determine whether ·OH production is enhanced in
the failing myocardium, myocardial tissue
homogenates were reacted with hydroxy-TEMPO, and its ESR
spectra were recorded. The intensity of ESR signals reduced more
rapidly in HF hearts compared with control (Figure 3A). There was also a linear relation in
the semilogarithmic plot of peak signal intensity versus time (Figure 3B). The rate of signal decay, calculated from the slope of this
line, was significantly (P<0.01) higher in HF (0.047±0.002
versus 0.024±0.002 minutes; Figure 3C). In the absence of
myocardial tissue, ESR signals did not decline during the time of
study. DMTU (50 mmol/L) abolished the increase in the rate of
signal decay seen in HF (Figure 3C), confirming an enhanced
generation of ·OH.
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P<0.01 for difference from HF only (no
scavenger).
Desferrioxamine (0.5 mmol/L), an iron chelator, also inhibited the
increase of signal decay in HF (Figure 3C
), indicating that the
highly reactive ·OH radical is generated via interaction of
·O2- and
H2O2 through the
iron-catalyzed Harber-Weiss reaction
(·O2-+H2O2
·OH+OH-+·O2-)
and/or interaction of H2O2
and iron through the Fenton reaction
(H2O2+Fe2+·OH+OH-+Fe3+).
Catalase (50 U/mL) also attenuated the increased rate of signal decay
in HF (Figure 3C), which implies the existence of a component of
H2O2 in the
production of ·OH. Further, Tiron, a nonenzymatic
·O2- scavenger, and
catalase were similarly effective in attenuating the increase of the
signal decay (Figure 3C), which suggested that
·O2- contributed to the
production of H2O2.
Importantly, for control, catalase (50 U/mL) and DMTU (50 mmol/L)
had no such effects on the rate of signal decay. Taken together,
·O2- is primarily
produced in the failing heart and is converted into
H2O2 and further to
·OH via iron-catalyzed reaction.
ROS Production Under Functional Block of Mitochondria in
Normal Myocardium
To further confirm that
H2O2 and ·OH can be
secondarily produced when
·O2- is present
within the myocardium, ROS production was assessed
in the presence of antimycin A (200 µmol/L). Antimycin A has
been shown to selectively block the mitochondrial electron transport at
the site of complex III and to enhance the production of
·O2-.6 ESR
signals of hydroxy-TEMPO in the presence of normal myocardial
homogenates were also observed as 3 sharp lines (Figure 4A). At baseline conditions, the peak
signal intensity decreased slowly with time. In the presence of
antimycin A (200 µmol/L), ESR signal decay was significantly
accelerated (Figure 4A and 4B). Similarly to HF hearts, this
increase was inhibited by DMTU (100 mmol/mL), desferrioxamine
(0.5 mmol/L), and catalase (100 U/mL), indicating that
endogenously generated
·O2- is converted to
H2O2 and further to
·OH in the myocardial tissue. These results further demonstrate
that the formation of
·O2- can mimic the
production of downstream ROS seen in HF.
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Relation Between ROS and Contractile Dysfunction
To determine whether the myocardial level of ROS is correlated to
the severity of HF, LV contractile function indices were plotted
against the rate of signal decay measured using the same hearts. This
analysis was possible because myocyte contractile function
varied widely within a group of HF animals. There was a significant
inverse relation between the rate of signal decay and LV ejection
fraction (r=0.869, n=19 dogs; P<0.01; Figure 5A) and positive correlation between the
rate of decay and LV end-diastolic pressure
(r=0.901, n=18 dogs; P<0.01; Figure 5B).
A significant correlation was also found even in the analysis
of the HF group only (r=0.684, n=10 dogs, P<0.01
for LV ejection fraction, and r=0.724, n=9 dogs,
P<0.01 for LV end-diastolic pressure).
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, n=9) and HF (•, n=10) dogs. For linear regression data,
the correlation coefficient was calculated using a least-squares linear
fit analysis.A multiple linear regression analysis was performed to analyze the relation between the rate of signal decay, LV ejection fraction, and LV end-diastolic pressure. There was a significant positive correlation between the rate of signal decay and LV end-diastolic pressure (P<0.05), but not with ejection fraction. Further, there was a significant inverse relation between LV ejection fraction and LV end-diastolic pressure (P<0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using an ESR technique, the present study provided direct evidence that ·OH formation was enhanced in the failing myocardium. Importantly, ·OH originated from ·O2- via H2O2 within the tissue. Mitochondrial ·O2- generation could lead to ·OH production in the normal heart, which further supports the conclusion that ·OH is produced from ·O2- and H2O2 in HF. Further, oxidative stress was associated with a parallel decrease in LV contractile performance.
Measurement of ROS by ESR
It is difficult to quantify the amount of ROS in the intact
biological system, because they are unstable and rapidly react with
unoxidized adjacent molecules, and thus their half-life is very short.
ESR combined with a spin trap such as DMPO is a reliable, direct method
for measuring ROS.13 14 However, the application of this
method to the biological sample is limited because of the low stability
of DMPO adducts within the tissue. We thus used ESR spectroscopy with
hydroxy-TEMPO in the present study to quantify ROS. Nitroxide
radical is a stable chemical and can be easily detected by
ESR.7 8 9 Biological reductants can reduce nitroxides into
corresponding nonparamagnetic hydroxylamine compounds, which are ESR
silent.7 Therefore, the decay of nitroxides can provide a
direct method to assess the generation of ROS within biological tissues
in vitro,15 as well as in vivo.10 12 This
method provides several advantages in the assessment of ROS generation.
First, it can allow a direct quantitative assessment of ROS, which is
not possible with other techniques to measure byproducts of ROS
such as TBARS and 8-iso-PGF2. Our in vitro validation studies have
confirmed that the increase in the rate of signal decay is proportional
to the amount of ·OH. Second, this method does not require
complicated procedures to prepare the subcellular fractions, which may
alter the status of ROS. It can measure the net amount of ROS in the
presence of antioxidants and/or reductants within the whole tissue.
Therefore, this method is considered to reproduce the in vivo setting
more closely than other techniques.
We have to acknowledge 2 crucial limitations of ESR measurement with
nitroxide radical. First, given the highly transient nature of ROS, ESR
measurement procedures themselves may modify the status of ROS by
destroying antioxidant defenses and/or activating the oxidant
production system. However, the contribution of artifactual
generation of ROS in our measurement is considered to be minimal, given
that the rate of signal decay was not altered by ROS scavengers in
normal myocardium. Second, the reduction of hydroxy-TEMPO
might not be specific for ROS in living tissue, because
hydroxy-TEMPO can lose its paramagnetism not only by direct
interaction with ·OH but also by various mechanisms, including
the reductants in the cytosol, 1-electron reduction due to enzymatic
processes, and mitochondrial electron flow.11 However, the
contribution of other mechanisms in the reduction of hydroxy-TEMPO
could be neglected in our quantification, because we confirmed that
increased rate of signal decay in HF was inhibited in the presence of
specific ·OH scavenger within the reaction mixture (Figure 3).
Oxidative Stress in HF
The present study has provided a definitive and direct
demonstration of enhanced generation of ROS in the failing
myocardium. These results are concordant with our recent
study using the subcellular fractions that
·O2- production
was increased in HF mitochondria.6 They are also in
agreement with the studies by others in which TBARS and 8-iso-PGF2
are increased in HF.1 2
·O2- is a primary
radical that can lead to the formation of other ROS, such as
H2O2 and ·OH. The
present study has demonstrated that
·O2- leads to the
formation of H2O2 and
·OH in the failing myocardium (Figures 3 and 4). ·OH arises from electron exchange between
·O2- and
H2O2 via the Harber-Weiss
reaction. In addition, ·OH is also generated by the reduction of
H2O2 in the presence of
endogenous iron by means of the Fenton reaction. The
generation of ·OH implies a
pathophysiological significance of ROS in HF, given
that ·OH radicals have been suggested to be the predominant
oxidant species causing cellular injury.16
The present study has demonstrated that
·O2- produced in the
presence of antimycin A can lead to the further generation of
downstream ROS, including
H2O2 and ·OH, within
the myocardium (Figure 4). Our recent study
indicated that ·O2-
production was enhanced in the mitochondria from the failing
heart.6 Taken together, it is conceivable that
·O2- originated from the
mitochondrial complex I could lead to the production of
H2O2 and ·OH in
HF.
Previous studies by Haywood et al17 and by us18 have demonstrated enhanced production of NO in the failing heart, which originated from inducible NO synthase. ·O2- and NO can react to the powerful prooxidant, peroxynitrite, which can form hydroxyl radicals. Peroxynitrite can increase the rate of hydroxy-TEMPO signal decay and might be involved in the development of HF.
Both ·OH and ·O2-
can diminish myocardial contractile function19 and also
cause lipid peroxidation of membrane phospholipids, which ultimately
leads to myocyte structural damage.20 Recently, ROS have
been suggested to be involved in apoptosis,21 22
which might play an important role in the pathogenesis of HF. Moreover,
ROS can cause endothelial dysfunction23
and induce arrhythmia, both of which may contribute to the
progression of HF. Therefore, oxidant stress may play an important role
in myocardial failure. This is supported by a close correlation between
oxidative stress and contractile dysfunction in this study (Figure 5). Nonetheless, we have to admit that our findings do not prove
a causal relation. Indeed, given the importance of ROS in the
pathophysiology of HF, it is crucial to determine the effects of
radical scavengers on the reversibility or prevention of HF, and an
establishment of a cause-and-effect relationship is a major focus of
our future research. In our preliminary studies using a mouse model of
myocardial infarction and failure, chronic
intraperitoneal administration of DMTU (50 mg/kg
per day) could attenuate the production of ·OH in the
myocardium and also LV remodeling process (cavity
dilatation), as well as contractile failure without affecting the size
of myocardial infarction (our unpublished data, 1999). These
results may suggest that oxidative stress could be an important cause
in the initiation and progression of HF.
Conclusions
The present study provided definite and direct evidence for an
increased production of ·OH originating from
·O2- and
H2O2 in the failing
myocardium. Oxidative stress was in parallel to LV
contractile dysfunction. The enhanced production of ROS might
play 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 of Japan (Grants 07266220, 08258221, and 09670724).
Received September 16, 1999; accepted November 3, 1999.
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C.-s. Liang, W. Mao, C. Iwai, S. Fukuoka, and S. Y. Stevens Cardiac sympathetic neuroprotective effect of desipramine in tachycardia-induced cardiomyopathy Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H995 - H1003. [Abstract] [Full Text] [PDF]
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S. Ichihara, A. Noda, K. Nagata, K. Obata, J. Xu, G. Ichihara, S. Oikawa, S. Kawanishi, Y. Yamada, and M. Yokota Pravastatin increases survival and suppresses an increase in myocardial matrix metalloproteinase activity in a rat model of heart failure Cardiovasc Res, February 15, 2006; 69(3): 726 - 735. [Abstract] [Full Text] [PDF]
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