Direct Evidence for Increased Hydroxyl Radicals Originating From Superoxide in the Failing Myocardium
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.
In patients with heart failure (HF), thiobarbituric acid reactive substances (TBARS) and 8-iso prostaglandin (PG) F2α, 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.
Materials and Methods
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.
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.
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.
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⇓).
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.
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.
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).
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).
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-PGF2α. 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-PGF2α 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.
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.
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.
- © 2000 American Heart Association, Inc.
Belch JJ, Bridges AB, Scott N, Chopra M. Oxygen free radicals and congestive heart failure. Br Heart J. 1991;65:245–248.
Mallat Z, Philip I, Lebret M, Chatel D, Maclouf J, Tedgui A. Elevated levels of 8-iso-prostaglandin F2α in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation progression to heart failure. Circulation. 1998;97:1536–1539.
Dhalla AK, Singal PK. Antioxidant changes in hypertrophied and failing guinea pig hearts. Am J Physiol. 1994;266:H1280–H1285.
Hill MF, Singal PK. Right and left myocardial antioxidant responses during heart failure subsequent to myocardial infarction. Circulation. 1997;96:2414–2420.
Ide T, Tsutsui H, Kinugawa S, Utsumi H, Kang D, Hattori N, Uchida K, Arimura K, Egashira K, Takeshita A. Mitochondrial electron transport complex I is the potential source of oxygen free radicals in the failing myocardium. Circ Res. 1999;85:357–363.
Dikalov S, Skatchkov M, Bassenge E. Spin trapping of superoxide radicals and peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine and the stability of corresponding nitroxyl radicals towards biological reductants. Biochem Biophys Res Commun. 1997;231:701–704.
Utsumi H, Takeshita K, Miura Y, Masuda S, Hamada A. In vivo EPR measurement of radical reaction in whole mice–influence of inspired oxygen and ischemia-reperfusion injury on nitroxide reduction. Free Radic Res Commun. 1993;19:S219–S225.
Zweier JL, Flaherty JT, Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci U S A. 1987;84:1404–1407.
Zweier JL, Kuppusamy P. Electron paramagnetic resonance measurements of free radicals in the intact beating heart: a technique for detection and characterization of free radicals in whole biological tissues. Proc Natl Acad Sci U S A. 1988;85:5703–5707.
Bolli R, Jeroudi MO, Patel BS, Aruoma OI, Halliwell B, Lai EK, McCay PB. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion: evidence that myocardial “stunning” is a manifestation of reperfusion injury. Circ Res. 1989;65:607–622.
Haywood GA, Tsao PS, von der Leyen HE, Mann MJ, Keeling PJ, Trindade PT, Lewis NP, Byrne CD, Rickenbacher PR, Bishopric NH, Cooke JP, McKenna WJ, Fowler MB. Expression of inducible nitric oxide synthase in human heart failure. Circulation. 1996;93:1087–1094.
Yamamoto S, Tsutsui H, Tagawa H, Saito K, Takahashi M, Tada H, Yamamoto M, Katoh M, Egashira K, Takeshita A. Role of myocyte nitric oxide in β-adrenergic hyporesponsiveness in heart failure. Circulation. 1997;95:1111–1114.
Josephson RA, Silverman HS, Lakatta EG, Stern MD, Zweier JL. Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes. J Biol Chem. 1991;266:2354–2361.
Ide T, Tsutsui H, Kinugawa S, Utsumi H, Takeshita A. Amiodarone protects cardiac myocytes against oxidative injury by its free radical scavenging action. Circulation. 1999;100:690–692.
Cheng W, Li B, Kajstura J, Li P, Wolin MS, Sonnenblick EH, Hintze TH, Olivetti G, Anversa P. Stretch-induced programmed myocyte cell death. J Clin Invest. 1995;96:2247–2259.
Siwik DA, Tzortzis JD, Pimental DR, Chang DL, Pagano PJ, Singh K, Sawyer DB, Colucci WS. Inhibition of copper-zinc superoxide dismutase induces cell growth, hypertrophic phenotype, and apoptosis in neonatal rat cardiac myocytes in vitro. Circ Res. 1999;85:147–153.
Stewart DJ, Pohl U, Bassenge E. Free radicals inhibit endothelium-dependent dilation in the coronary resistance bed. Am J Physiol. 1988;255:H765–H769.