HIV Protease Inhibitors Activate Volume-Sensitive Chloride Current in Ventricular Myocytes by Generating Mitochondrial Reactive Oxygen Species

Abstract

HIV protease inhibitors (HIV PI) have been successfully used to reduce morbidity and mortality of HIV infection. However, their long-term use causes significant side effects including cardiac arrhythmias. Previously we showed that outwardly-rectifying, volume-sensitive Cl current (ICl,swell) is regulated by signaling pathways that elicit production of reactive oxygen species (ROS). Because certain HIV PI recently were reported to augment ROS production, we tested whether HIV PI stimulate ICl,swell in rabbit ventricular myocytes. Under isosmotic conditions, ritonavir (15 μM, 20 min) and lopinavir (15 μM, 25 min) induced outwardly-rectifying Cl currents (1.5 ± 0.3 pA/pF and 1.9 ± 0.3 pA/pF at +60 mV, respectively) that were fully inhibited by the highly selective ICl,swell-blocker DCPIB (10 μM). In contrast, amprenavir (15 μM, 30 min) and nelfinavir (15 μM, 30 min) did not modulate ICl,swell, and raltegravir (MK-0518, 15 μM, 30 min), an HIV integrase inhibitor, also was ineffective. Two major sources of ROS in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. The specific NADPH oxidase inhibitor apocynin (500 μM) failed to inhibit the ritonavir- or lopinavir-induced currents, although we previously found apocynin blocks ICl,swell activation upon osmotic swelling and stretch. In contrast, rotenone (10 μM, 30 min), a mitochondrial complex I inhibitor that limits electron flux to and ROS production by complex III, blocked 102 ± 4% of ritonavir- and 82 ± 12% of lopinavir-induced ICl,swell. Furthermore, the membrane-permeant, glutathione peroxidase mimetic ebselen (15 μM, 15 min) suppressed ICl,swell elicited by ritonavir (102 ± 3%) and lopinavir (93 ± 6%). These results suggest that ritonavir and lopinavir activate ICl,swell via mitochondrial ROS production by complex III. Activation of ICl,swell by certain HIV PI may contribute to their untoward effects in heart and potentially other tissues.