HIV protease inhibitors elicit volume-sensitive Cl⁻ current in cardiac myocytes via mitochondrial ROS

Abstract 

HIV protease inhibitors (HIV PI) reduce morbidity and mortality of HIV infection but cause multiple untoward effects. Because certain HIV PI evoke production of reactive oxygen species (ROS) and volume-sensitive Cl⁻ current (I_Cl,swell) is activated by ROS, we tested whether HIV PI stimulate I_Cl,swell in ventricular myocytes. Ritonavir and lopinavir elicited outwardly rectifying Cl⁻ currents under isosmotic conditions that were abolished by the selective I_Cl,swell-blocker DCPIB. In contrast, amprenavir, nelfinavir, and raltegravir, an integrase inhibitor, did not modulate I_Cl,swell acutely. Ritonavir also reduced action potential duration, but amprenavir did not. I_Cl,swell activation was attributed to ROS because ebselen, an H₂O₂ scavenger, suppressed ritonavir- and lopinavir-induced I_Cl,swell. Major ROS sources in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin failed to block ritonavir- or lopinavir-induced currents, although it blocks I_Cl,swell elicited by osmotic swelling or stretch. In contrast, rotenone, a mitochondrial e⁻ transport inhibitor, suppressed both ritonavir- and lopinavir-induced I_Cl,swell. ROS production was measured in HL-1 cardiomyocytes with C-H₂DCFDA-AM and mitochondrial membrane potential (ΔΨ_m) with JC-1. Flow cytometry confirmed that ritonavir and lopinavir but not amprenavir, nelfinavir, or raltegravir augmented ROS production, and HIV PI-induced ROS production was suppressed by rotenone but not NADPH oxidase blockade. Moreover, ritonavir, but not amprenavir, depolarized ΔΨ_m. These data suggest ritonavir and lopinavir activated I_Cl,swell via mitochondrial ROS production that was independent of NADPH oxidase. ROS-dependent modulation of I_Cl,swell and other ion channels by HIV PI may contribute to some of their actions in heart and perhaps other tissues.

Abbreviations: APD, action potential duration, DNP, 2,4-dinitrophenol, HIV PI, HIV protease inhibitors, ROS, reactive oxygen species, I_Cl,swell, volume-sensitive Cl⁻ current, ΔΨ_m, mitochondrial membrane potential, RTV, ritonavir, LPV, lopinavir, APV, amprenavir, NFV, nelfinavir, MK-0518, raltegravir

Keywords: I_Cl,swell, VRAC, HIV protease inhibitors, Reactive oxygen species, Mitochondria, Mitochondrial membrane potential, NADPH oxidase, Swelling, Cell volume