Sodium, potassium adenosine triphosphatase as a potential target of the anti-tuberculosis agents, clofazimine and bedaquiline

Abstract

ABSTRACT BACKGROUND: Tuberculosis (TB) is a disease caused by the acid-fast bacterium, Mycobacterium tuberculosis (M. tuberculosis). It is currently the leading cause of morbidity and mortality worldwide due to a single infectious agent. One of the main contributing factors to the burden of the disease is an alarming increase in the number of drug-resistant tuberculosis (DR-TB) cases. Recently, an efficient chemotherapeutic regimen, containing two second-line drugs, namely clofazimine (CFZ) and bedaquiline (BDQ), has been introduced. The use of these antibiotics has been associated with prolongation of the cardiac QT interval (this is measurement of the heart rate shown on the electrocardiogram), which leads to fatal cardiac arrhythmias. However, the mechanism by which these agents cause cardiac arrhythmia remains unknown. In this context, CFZ has been reported to inhibit the activity of the sodium, potassium-adenosine triphosphatase (Na+,K+-ATPase) enzyme in T lymphocytes, while the effects of BDQ on Na+,K+-ATPase have, to date, been unexplored. Importantly, however, both CFZ and BDQ target K+ channels in human mammalian cells including those of the mononuclear leukocytes (MNLs) and cardiac muscle cells (cardiomyocytes: CMs). Despite this, the effect of these two antibiotics on Na+,K+-ATPase of cardiomyocytes has not been described.

AIM AND OBJECTIVES: The aim of the current study was to evaluate the effects of CFZ and BDQ on the activity of Na+,K+-ATPase of MNLs and rat cardiomyocytes (RCMs), by determining the activity of Na+,K+-ATPase via inorganic phosphate (Pi) concentration measurements, quantification of intracellular ATP levels and cellular viability.

METHODS: The MNLs were isolated from healthy adult volunteers while the RCMs were obtained commercially. The MNLs and RCMs were treated with varying concentrations of CFZ and BDQ individually and in combinations (0.15 - 5 mg/L). Thereafter, Pi concentrations (using a Na+,K+-ATPase activity assay), ATP levels (using a colorimetric ATP assay) and number of viable cells (measured flow cytometrically) were determined.

RESULTS: All antibiotic assays demonstrated inhibition in Na+,K+-ATPase activity in a dose-response related manner. Clofazimine was found to have the greatest inhibitory effect on the Na+,K+-ATPase activity in MNLs followed by BDQ while the effect was attenuated when the two antibiotics were used in combination. However, in RCMs, the greatest inhibitory effect of the antibiotics was demonstrated by combinations of the two antibiotics, followed by CFZ and BDQ alone, which however, demonstrated a comparable effect. In both cell lines, the inhibitory effect on Na+,K+-ATPase activity by the antibiotics was associated with an increase in ATP concentrations. Additionally, these effects were followed by a decline in cellular viability in the case of MNLs while a slight increase in cellular viability was observed in the RCMs for all antibiotic treatments. However, there was a decrease in cellular viability of the RCMs when the antibiotics were used in combination at their highest concentration of 5 mg/L.

CONCLUSION: The results of the current study illustrate the implication of the Na+,K+-ATPase in the beneficial effects of CFZ and BDQ on the immunomodulatory roles of the MNLs while, importantly, demonstrating the potentially detrimental effects of these antibiotics, especially BDQ and, more notably, when the two antibiotics are used in combination, on the viability of the cardiomyocytes via targeting the Na+,K+-ATPase pump. This may contribute to the risk on individuals administered these drugs of developing cardiac arrhythmia.