Macrolides including azithromycin, clarithromycin, and erythromycin are considered as a broad-spectrum class of antibiotics with an expanding role in treating various bacterial infections. Therefore, their efficacy and safety should be carefully investigated [25–27]. Macrolides are generally considered safe antibiotics. However, like all other drugs, this group of antibiotics also has unwanted adverse effects, the most important of which is cardiotoxicity . Several studies have shown a potential association between macrolides and cardiac toxicity including probability of QT interval prolongation, ventricular tachycardia, TdP arrhythmia, and sudden cardiac death [29–31]. According to considerable evidence, there is a relationship between drug-induced QT interval prolongation and blockade of the hERG cardiac potassium channel [32, 33].
Macrolides such as erythromycin and clarithromycin may contribute in QT interval prolongation and TdP arrhythmia by inhibiting the cardiac hERG potassium channels, which mediate the rapid delayed rectifier K + current (IKr) in cardiac myocytes . QT interval represents the duration between ventricular depolarization and repolarization on the ECG [35, 36]. The prolonged QT interval can be due to congenital heart defects or QT-prolonging drugs, involved in extending the time of the cardiac muscle action potential [37–39]. A network of ion channels, including potassium channels, regulates the action potential in the heart cells. The ERG or KV11 is a subfamily of the voltage-gated potassium channels superfamily with three members: KV11.1 (ERG1), KV11.2 (ERG2), and KV11.3 (ERG3) channels. Many studies have been conducted on ERG1 channel, which regulates the cardiac action potential duration. Mutation in the ERG1 channel is associated with increased risks of arrhythmia and sudden cardiac death [40–44].
Previous studies have indicated that dysfunction of hERG channel as an adverse effect of macrolides can lead to life-threatening arrhythmias caused by long QT interval syndrome [31, 42–44].
hERG leads to rapid potassium current, which is an important regulator of repolarization of cardiac potential of action. The most known mutations in hERG lead to reduction in potassium current and finally loss or reduction in the function of potassium fast channels. Previous studies have provided evidence suggest that improper function of the hERG channel leads to both inherited and acquired types of long QT interval syndrome .
Some studies also suggest that increased reactive oxygen species (ROS) production will change the kinetics of hERG potassium conductivity. In a study conducted by Salimi and his colleagues, the isolated heart mitochondria from cardiomyocytes were exposed to erythromycin, azithromycin and clarithromycin. Their results showed that macrolides induced the production of reactive oxygen species, mitochondrial membranes permeability, mitochondrial swelling and, finally, the release of cytochrome C from the mitochondria of cardiac myocytes. According to these results, damage to heart mitochondria is the starting point for the cardiotoxic effect of the macrolides . Although the mechanisms underlying the cardiotoxicity of macrolides has been investigated to some extent, their effects on the expression of potassium channels genes involved in the QT interval prolongation process have not been completely elucidated yet.
In this research, the cardiotoxic effects of three macrolides antibiotics, azithromycin, erythromycin and clarithromycin, have been evaluated at three concentrations of 1, 10 and 25 µg/ml on H9c2 cell line at 48 and 72 hours. The results demonstrate that toxic effects of these antibiotics on H9c2 cells are depend on drug concentration and exposure time. While they showed cytotoxicity effect after 48 hours, the toxicity of the drugs increased 72 hours after the treatment. It is noteworthy that their significant differences in toxicity compared to the control group were occurred at two concentrations of 10 and 25 µg/ml after 72 hours. Among the three antibiotics, azithromycin showed more severe toxic effects than two other antibiotics, and the highest toxicity was observed at 72 hours. It should be noted that all three drugs had inhibitory effects of less than 30% at three concentrations, and not all three concentrations of drugs did show inhibitory effects led to 50% loss in cellular population (IC50).
The results of this study are partly consistent with those of the study conducted by Ray et al. In a cohort study, they examined the increased risk of death associated with short-term cardiac effects of azithromycin, amoxicillin, ciprofloxacin and levofloxacin drugs in a 5-day course of treatment. Their results revealed that patients received the azithromycin had a greater risk of cardiovascular death and death for any reason in comparison to those who did not received the antibiotic, but an increase in the risk of death was not reported for patients received the amoxicillin during this period. Compared to amoxicillin, azithromycin was more associated with the risk of cardiovascular death. The cardiovascular death risk was significantly higher with azithromycin than that of ciprofloxacin, but it showed no significant difference with levofloxacin .
Antzelevitch and colleagues also reported that erythromycin prolongs QT intervals. Using the whole-cell patch clamp techniques on cardiomyocytes isolated from the pig heart, they suggested that erythromycin had a strong effect on inhibiting the rapidly activating component (IKr) but not the slowly activating component (IKs) of the delayed rectifier potassium current (IK). The inward rectifier current (IK1) was also unaffected .
In another study, Ohtani, et al. examined the effect of macrolide antibiotics on cardiac arrhythmias quantitatively. They analyzed the effects of clarithromycin, roxithromycin, and azithromycin on QT interval in terms of pharmacokinetics and pharmacodynamics in comparison with erythromycin in male rats. Their results showed that the rank order of these four antibiotics potencies for QT interval prolongation in rats was as follows: erythromycin > clarithromycin > roxithromycin > azithromycin . The rank order of these antibiotics-induced cardiac arrhythmias is not consistent with the one that we obtained regarding to their effect on cell death and ERG channel gene expression in the H9c2 cell line. This inconsistency may be due to the change of experimental design from animal model in their work to cell line model in our study. On the other hand, azithromycin may enhance the efficiency of the potassium channel by increasing its gene expression and thereby reducing inhibitory effects on the channel, therefore, the toxicity caused by potassium channel inhibition was reduced.
Milberg et al. realized that erythromycin, clarithromycin and azithromycin led to QT interval prolongation, erythromycin and clarithromycin caused TdP and Early after depolarization (EAD) after reducing the potassium concentration. This arrhythmia was not seen in azithromycin case . In addition to their direct effect on the QT interval, erythromycin and clarithromycin have inhibitory effects on the metabolism of some other drugs by inhibiting CYP3A. The incidence of sudden cardiac death was three times more in patients received CYP3A inhibitors in addition to erythromycin compared to those who received erythromycin alone [16, 49].
Han et al. found that mutations in drug-binding sites of the hERG channel could attenuate hERG current obstruction by roxithromycin, but did not significantly modify the disruption of trafficking . According to Hancox et al. study, D85N KCNE1 mutations demonstrated an increase in the sensitivity of IKr/hERG to inhibition with clarithromycin in vitro models .
In the present research, H9c2 cells were treated with azithromycin, erythromycin and clarithromycin. In all three cases, after 48 and 72 hours, the expression of ERG1 gene was enhanced at two concentrations of 10 and 25 µg/ml compared to 1 µg/ml. The effect of azithromycin on ERG expression in all three concentrations and after 48 and 72 hours was higher than that of other two drugs, and this result has been confirmed by other studies . According to the results of MTT assay and considering the study conducted by Salimi et al, it seems that azithromycin has an inhibitory effect on the growth of heart cells through a distinct pathway.
Moreover, our research revealed that the inhibitory effect of clarithromycin is higher than that of other two macrolides. Potassium channels may be more sensitive to clarithromycin and this maybe play a key role in clarithromycin-induced cardiac arrhythmia development.