Amiodarone Protects Cardiac Myocytes Against Oxidative Injury by its Free Radical Scavenging Action
Background—Oxidative stress plays an important role in the pathophysiology of ischemic heart disease and heart failure, and antioxidants might be beneficial in the treatment of these patients. This study was performed to determine the scavenging effects of amiodarone on oxygen free radicals and its protective effects against oxygen radical-mediated injury in cardiac myocytes.
Methods and Results—The formation of the radical spin adduct with hydroxy radical (·OH) in the presence of H2O2 (10 mmol/L) and Fe3+-nitrilotriacetate (20 μmol/L) was monitored by electron paramagnetic resonance spectroscopy combined with a spin trapping agent, 5,5-dimethyl pyrroline-N-oxide (DMPO). Amiodarone decreased the intensity of the DMPO-OH signals in a dose-dependent manner (0.1 to 100 μmol/L), whereas other antiarrhythmia drugs such as disopyramide and atenolol had no such effects. Furthermore, amiodarone (10 μmol/L) protected intact adult canine cardiac myocytes against ·OH-mediated myocyte injury, as assessed by the degree of morphological change from rod shape to the irreversible hypercontracture state during the exposure of cells to H2O2 and Fe3+ in vitro.
Conclusions—Amiodarone can protect cardiac myocytes against oxidative stress-mediated injury by directly scavenging oxygen free radicals. Antioxidant action of amiodarone might potentially contribute to the beneficial effects of this drug in the treatment of patients with ischemic heart disease and congestive heart failure.
Recent studies have demonstrated that amiodarone improves the clinical status and left ventricular function in patients with heart failure (HF).1 2 It is noteworthy that amiodarone may reduce total mortality and arrhythmic/sudden death in HF patients or survivors of myocardial infarction.3 This is in clear contrast to other antiarrhythmia drugs, which have actually increased mortality, as shown by carefully evaluated large randomized trials of high risk patients.3 4 These significant clinical benefits have made amiodarone the antiarrhythmic agent of choice in the current treatment of HF patients.5
Oxidative stress has been shown to play an important role in the pathophysiology of ischemic heart disease6 and recently in congestive HF.7 8 Oxygen radicals can produce deleterious effects on the myocardium, including contractile dysfunction and structural damage.9 In addition, they can damage vascular endothelial cells. Therefore, oxygen radical-mediated myocardial injury may be involved in the initiation and progression of HF. In addition, it has been suggested that the beneficial effects of carvedilol on HF may be attributable, at least in part, to its antioxidant action.10 11 Recent studies have suggested that amiodarone exhibits inhibitory effects against oxygen radical-mediated lipid peroxidation of rat liver mitochondria.12 Antioxidant activity, if present, may provide additional cardiovascular effects of this drug. However, to our knowledge, no study has examined the radical scavenging action of amiodarone and its protective effects against oxygen radical-mediated cardiac injury in myocytes.
The present study was undertaken to determine whether amiodarone is a free radical scavenger by using in vitro electron paramagnetic resonance (EPR) spectroscopy with spin trap. We also sought to determine whether it is protective against exogenously generated oxygen radical-mediated injury in isolated intact cardiac myocytes. We used isolated myocyte preparations to avoid the confounding systemic effects of amiodarone and to examine its direct effects on myocytes.
Direct EPR Measurements of Oxygen Radicals
The procedures for EPR with spin trapping agents to directly detect oxygen free radicals were described previously by Mak et al.13 Briefly, hydroxy free radical (·OH) was generated from H2O2 (10 mmol/L) in the presence of an iron redox chelate, Fe3+-nitrilotriacetate (NTA; 20 μmol/L).9 A spin trap, 5,5′-dimethyl-1-pyroline-N-oxide (DMPO; 80 mmol/L), was reacted with ·OH in the presence or absence of amiodarone or other test drugs. The formation of the radical spin adduct, DMPO-OH, was monitored with a spectrometer (JES-RE-1X, JEOL Ltd) operating at X-band (9.43 GHz) and a microwave power of 5.0 mW. A range of external magnetic field of 20 mT was swept at a scan rate of 10 mT/min. The amplitude of EPR spin signal, which is proportional to the free radical formation, was used to estimate the scavenging effects of drugs. Quantification of the DMPO signal intensity was performed by comparing the amplitude of the observed signal to a standard Mn2+/MgO marker.
·OH-Mediated Cardiac Myocyte Injury
Cardiac myocytes were isolated from the canine left ventricular free wall as described previously.14 Isolated cells were placed in a chamber on the stage of an inverted microscope (Olympus) and superfused with the oxygenated Krebs buffer (pH 7.4, 35°C). Amiodarone and other test drugs were preincubated for 15 minutes before the addition of H2O2 (10 mmol/L)-Fe3+-NTA (20 μmoll/L). The image of the cell was acquisited via CCD camera and recorded continuously on a video tape during the experiment. The length of rod shaped cells (15 to 30 cells per experiment), determined along its longitudinal axis, was measured.
Data are expressed as mean±SEM. An ANOVA with repeated measures was used to compare the time-dependent changes of cell length after the exposure of myocytes to ·OH between control and HF. Differences were considered statistically significant at P<0.05.
·OH Scavenging Action of Amiodarone
The formation of the spin adduct DMPO-OH was evidenced by the appearance of 1:2:2:1 EPR hyperfine splitting pattern characteristic for ·OH (Figure 1A⇓). As expected, catalase (H2O2 scavenger; 5 U/mL) or mannitol (·OH scavenger; 100 mmol/L) showed significant attenuation of EPR spin signals, indicating that EPR signals indeed originated from ·OH. Amiodarone inhibited the DMPO-OH signal height in a concentration-dependent manner (Figure 1B⇓). Under the same conditions, disopyramide (10 μmol/L) and atenolol (10 μmol/L) had no such effects on the EPR signals. These results indicate that amiodarone is capable of directly scavenging ·OH generated from the H2O2+Fe3+-NTA system.
Protective Effects of Amiodarone against ·OH-Mediated Cardiac Myocyte Injury
Myocytes had no morphological changes in the control buffer without H2O2 or Fe3+-NTA during the time of the study for 25 minutes. The addition of either H2O2 or Fe3+-NTA did not induce any morphological changes. The combined addition of H2O2 and Fe3+-NTA induced myocyte hypercontracture after 10 minutes of exposure; thereafter, myocytes shortened to a square hypercontracture state after 20 minutes (Figure 2⇓), which was irreversible even after replacing the bathing media into normal buffer (n=8 preparations). ·OH-induced hypercontracture was significantly inhibited in the presence of catalase (50 U/mL) or mannitol (100 mmol/L), indicating that hypercontracture was indeed mediated by the generation of ·OH radical.
Amiodarone (10 μmol/L; n=8 preparations) exerted significant (P<0.05) protection against the loss of viability (Figure 2⇑). In contrast, disopyramide (10 μmol/L) and atenolol (10 μmol/L) had no such effects.
The present study indicated, for the first time, that amiodarone could directly quench ·OH radical in vitro and exert a protective effect against ·OH radical-mediated cardiac myocyte injury. These effects were not observed in other antiarrhythmia drugs such as disopyramide and atenolol.
EPR studies demonstrated that the signal height of DMPO-OH, proportional to the amount of ·OH, was decreased in the presence of amiodarone, which indicated that this compound effectively suppressed the formation of the DMPO-OH adduct. Therefore, amiodarone has an ability to directly scavenge ·OH radicals. This is in accordance with the recent observation that this drug can inhibit lipid peroxidation in rat liver mitochondrial membranes challenged by an iron-dependent oxygen radical generating system.12
The exposure of isolated adult cardiac myocytes to exogenously generated ·OH radicals resulted in time-dependent morphological changes, as evidenced by the irreversible hypercontracture, which has been known as an in vitro model of oxidant stress-induced myocyte injury.9 Amiodarone showed a partial but significant inhibition of oxygen radical-mediated hypercontracture in cardiac myocytes. The protective effects of amiodarone could result from its direct scavenging action on ·OH before reaching the cellular sites of injury because cell damage was prevented at a concentration similar to that for inhibition of DMPO-OH adduct formation. Further, even though a direct comparison may not be appropriate, the magnitude of the effects of amiodarone on cellular damage shown in Figure 2⇑ is commensurate with that on ·OH formation shown in Figure 1⇑. However, amiodarone may also interact with the membrane lipids and interrupt the free radical chain reactions, which could contribute to its protection against ·OH-induced lipid peroxidation and cellular injury. The present study did not intend to identify the structural requirements for the antioxidant capacity of amiodarone, and thus the mechanisms by which it scavenges oxygen radicals remain to be clarified.
The effective concentration of amiodarone for cardioprotective effects in this study (10 μmol/L) appears to be several times higher than its plasma concentration in patients given this drug (0.5 to 2.5 μg/mL or 0.7 to 3.5 μmol/L).15 However, this drug is a highly lipophilic compound,16 suggesting a high affinity of this drug to the plasma membranes.17 Therefore, it is conceivable that an effective tissue concentration level of amiodarone for exerting the cardioprotective action may be attainable when administered in vivo.
We have shown that amiodarone is an antioxidant and is unique among antiarrhythmia drugs in this respect. Recently, oxidative stress has been implicated in the progression of HF as evidenced by increased oxygen radical generation in failing hearts.7 8 Vitamin E, an endogenous antioxidant, has been shown to preserve myocardial structure and function in an animal model of HF.18 We thus speculate that antioxidant effects of amiodarone might play an important role in the reversibility or prevention of HF. However, the clinical significance of antioxidant action of amiodarone has not been established in this study.
In conclusion, amiodarone protects cardiac myocytes against oxidative stress-mediated injury by scavenging oxygen free radicals. In view of increasing evidence that oxygen radical-mediated myocardial injury is implicated in the pathogenesis of HF,7 8 the antioxidant effects of amiodarone, along with its antiarrhythmic effects, would potentially increase its therapeutic value in the treatment of patients with HF.
Supported in part by grants from the Ministry of Education, Science, and Culture (Nos. 07266220, 08258221, and 09670724).
- Received May 20, 1999.
- Revision received June 21, 1999.
- Accepted June 28, 1999.
- Copyright © 1999 by American Heart Association
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