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 H(2)O(2) 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-H(2)DCFDA-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.

Fig. 1

Ritonavir (RTV) activated volume-sensitive Cl⁻ current (I_Cl,swell) under isosmotic conditions; RTV-induced currents outwardly rectified and were blocked by DCPIB. (A) Families of currents (-100 to + 60 mV, 500 ms) in control (Ctrl), after RTV exposure (15 μM, 20 min), and after addition of DCPIB (10 μM, 6 min) in presence of RTV (+DCPIB). (B) I-V relationships from A. (C) Current densities at +60 mV (n = 4; Ctrl vs. RTV, P <0.002; RTV vs. +DCPIB, P <0.002; Ctrl vs. +DCPIB, P = 0.71). (D) Kinetics of activation of I_Cl,swell by RTV (n = 4).

Fig. 2

RTV-induced activation of I_Cl,swell was reversed by ebselen, a H₂O₂ scavenger. (A) Currents in control (Ctrl), after RTV exposure (15 μM, 20 min), and after addition of ebselen (15 μM, 15 min) in presence of RTV. (B) I-V relationships from A. (C) Current densities at +60 mV (n = 5; Ctrl vs. RTV, P <0.001; RTV vs. +Ebselen, P <0.001; Ctrl vs. +Ebselen, P = 0.81).

Fig. 3

RTV elicited I_Cl,swell via ROS from mitochondria but not NADPH oxidase. (A, B) Apocynin (Apo; 500 μM, 20 min), a specific NADPH oxidase inhibitor, failed to suppress RTV (15 μM, 20 min)-induced I_Cl,swell (n = 4; Ctrl vs. RTV, P <0.001; RTV vs. +Apo, P = 0.98; Ctrl vs. +Apo, P <0.001). (C, D) In contrast, rotenone (Rot; 10 μM, 30 min), which suppresses mitochondrial ROS production, fully inhibited RTV-induced I_Cl,swell (n = 5; Ctrl vs. RTV, P <0.001; RTV vs. +Rot, P <0.001; Ctrl vs. +Rot, P = 0.89).

Fig. 4

Lopinavir (LPV) activated I_Cl,swell via mitochondrial ROS production but not NADPH oxidase. (A, B) LPV (15 μM, 25 min) elicited outwardly rectifying Cl⁻ current that was completely blocked by DCPIB. (C) Current densities at +60 mV showing block of LPV-induced current by DCPIB (n = 5, Ctrl vs. LPV, P <0.001; LPV vs. +DCPIB, P <0.001; Ctrl vs. +DCPIB, P = 0.55), ebselen (n = 5, Ctrl vs. LPV, P <0.001; LPV vs. +Ebselen, P <0.001; Ctrl vs. +Ebselen, P = 0.72), and rotenone (n = 4, Ctrl vs. LPV, P <0.003; LPV vs. +Rot, P <0.006; Ctrl vs. +Rot, P = 0.45). In contrast, LPV-induced current was unaffected by apocynin (n = 4, Ctrl vs. LPV, P <0.001; LPV vs. +Apo, P = 0.79; Ctrl vs. +Apo, P <0.001).

Fig. 5

The HIV PI nelfinavir (NFV; 5 μM, 30 min; n = 5) and amprenavir (APV; 15 μM, 30 min, n = 6) and the HIV integrase inhibitor raltegravir (MK; 15 μM, 30 min, n = 5) failed to activate I_Cl,swell acutely. By contrast, I_Cl,swell was elicited by osmotic swelling in 0.7T bath solution in the continued presence of HIV protease or integrase inhibitors (for drug + 0.7T, n = 5, 2 and 2, respectively). Currents were normalized to Ctrl for each experimental group.

Fig. 6

Effects of RTV and APV on ventricular action potentials and normalized APD₅₀ and APD₉₀. (A,B) RTV (15 μM, 15 min; n = 4) significantly decreased APD₅₀ and APD₉₀, and abbreviation of APD was reversed by DCPIB (10 μM, 10 min). *, P < 0.05 vs Ctrl; †, vs 15 min RTV; ns, vs Ctrl. (C,D) APV (15 μM, 15 min; n = 3) did not significantly alter APD; ns, vs Ctrl.

Fig. 7

RTV- and LPV-induced mitochondrial ROS production in HL-1 myocytes. (A) Log fluorescence (FL) histograms by flow cytometry. Negative controls in absence of probe (−F) without (−RTV) or with (+RTV) RTV (15 μM, 30 min). In fluorophore-loaded myocytes, background fluorescence (Bkgnd) and response to H₂O₂ (100 μM, 15 min) reflect ROS. (B) RTV elicited ROS production, and pretreatment with rotenone (10 μM, 30 min; Rot+RTV) suppressed ROS to background levels; A and B, from same experiment. (C) Geometric means of histograms normalized by background fluorescence (FL/FL_O). Both RTV and LPV (15 μM, 30 min; n = 5, for each) significantly increased ROS production, which was fully suppressed by pretreatment with Rot (10 μM, 30 min; n = 5, for each) but not by pretreatment with gp91ds-tat (500 nM, 30 min; n = 3, for each). In contrast, NFV (n = 4), APV (n = 3), and MK (n = 3; 15 μM, 60 min, for each) failed to augment ROS production. H₂O₂ (100 μM, 15 min; n = 9) was positive control. Box plots show 25, 50 and 75%ile, and whiskers show 5 and 95%ile; dotted line, background fluorescence.

Fig. 8

Time-dependence of RTV-induced ΔΨ_m depolarization in HL-1 myocytes. Ratios of JC-1 fluorescence (FL1/FL2) normalized by control fluorescence ratio are plotted for RTV (15 μM; n = 5), APV (15 μM; n = 4), and a time control (Ctrl; n = 3). Treatment with DNP (0.3 mM, 20 min) served as positive control. Increased FL1/FL2 corresponds to depolarization of ΔΨ_m.