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.

1. Introduction

Inclusion of HIV protease inhibitors (HIV PI)¹ in highly active anti-retroviral therapy substantially reduces the morbidity and mortality of HIV infections. A significant challenge is their adverse effects, which include hyperlipidemia, lipodystrophy, insulin resistance, and increased risks of atherosclerosis and myocardial infarction (for review, see [1]). Arrhythmogenic effects also are reported. HIV PI cause PR and QT_c prolongation and torsade de pointes in patients [2-4] and suppress heterologously expressed hERG currents and I_Kr in ventricular myocytes [2]. Moreover, HIV PI cause endothelial cell damage [5] and decompensated heart failure [6] in experimental settings, and dilated cardiomyopathy and heart block in human neonates [7].

Recent studies indicate that HIV PI augment production of reactive oxygen species (ROS) in the vasculature [8-12], adipocytes [13,14], macrophage-derived foam cells [15], fibroblast [16], and pancreatic β cells [17], suggesting that oxidative stress may play a role in the adverse effects of HIV PI. HIV PI reduce mitochondrial membrane potential, which triggers mitochondrial ROS production [11,15], and the expression of mitochondrial respiratory chain proteins is diminished [16,18]. Moreover, expression of NADPH oxidase subunit mRNAs is augmented [15].

We hypothesize that HIV PI-induced ROS production modulates ion channel function and, thereby, may contribute to untoward effects in heart and perhaps other tissues. One potential target is the outwardly rectifying, volume- and stretch-sensitive Cl⁻ current, I_Cl,swell. We previously demonstrated that activation of ROS production by osmotic swelling, stretch, and downstream signaling proteins, as well as exogenous H₂O₂, elicit I_Cl,swell in ventricular myocytes [19-21]. ROS-induced I_Cl,swell activation also was identified in HeLa and HTC hematoma cell lines [22,23] and primary astrocytes [24]. These studies concluded that ROS production by NADPH oxidase is a required intermediate that elicits I_Cl,swell, and we recently found mitochondrial ROS also is necessary [25].

We tested whether HIV PI elicit I_Cl,swell in cardiomyocytes and identified the source of ROS. Under isosmotic conditions, clinically relevant concentrations of the HIV PI ritonavir (RTV) and lopinavir (LPV) rapidly induced an outwardly-rectifying Cl⁻ current, and RTV decreased action potential duration (APD) by 60 ms, effects that were fully blocked by the I_Cl,swell inhibitor DCPIB. HIV PI-induced I_Cl,swell also was blocked by the ROS scavenger ebselen and by rotenone, which inhibits mitochondrial e⁻ transport. In contrast, the NADPH oxidase inhibitor apocynin had no effect. Furthermore, the HIV PI inhibitors amprenavir (APV) and nelfinavir (NFV) and the HIV integrase inhibitor raltegravir (MK-0518) failed to acutely modulate I_Cl,swell, and APV failed to alter APD. Flow cytometry confirmed that RTV and LPV, but not APV, NFV or MK-0518, augmented ROS production measured in HL-1 myocytes. As found for I_Cl,swell, HIV PI-induced ROS production was inhibited by rotenone but not by suppressing NADPH oxidase activity. Additionally, RTV, but not APV, depolarized mitochondrial membrane potential, ΔΨ_m. These data suggest that RTV and LPV acutely activated I_Cl,swell via mitochondrial ROS production, most likely at complex III, and that ROS arising from NADPH oxidase were not required.

2. Materials and methods

Detailed methods are given in the Online Supplement. Animal studies were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the Declaration of Helsinki, and the Guide for the Care and Use of Animals (National Academy Press, Washington, D.C., 1996).

Ventricular myocytes for electrophysiological studies were freshly isolated from anesthetized New Zealand white rabbits (∼3 kg) by enzymatic digestion. HL-1 cardiomyocytes were used for flow cytometry because they provide a more homogeneous population than freshly isolated myocytes, which unavoidably included damaged cells.

2.1. Electrophysiology

Recordings were made at room temperature with fire-polished pipettes (2–3 MΩ). Myocytes were suprafused at ∼2 ml/min, junction potentials were corrected, and a 3-M KCl-agar bridge served as ground. Seal resistances were 5–30 GΩ, and myocytes were dialyzed for 10 min before data collection. Currents were recorded with an Axoclamp 200B and Digidata 1322A under pClamp 9 and were low-pass filtered. Successive 500-ms steps were made at 1-s intervals from −60 mV, and current-voltage (I–V) relationships were plotted from the late current. APD at 50 and 90% repolarization (APD₅₀, APD₉₀) was determined in myocytes stimulated at 1 Hz. At least 30 action potentials were recorded to verify stability, and the last 10 were averaged. Bath and pipette solutions for measuring I_Cl,swell were designed to isolate anion currents, and those for action potentials contained physiologic anions and cations. Solution composition, preparation of reagents, and the structure of HIV PI and integrase inhibitors are given in the Online Supplement.

2.2. ROS and ΔΨ_m measurement by flow cytometry

ROS production was detected in HL-1 myocytes loaded with C-H₂DCFDA-AM (Invitrogen), which is hydrolyzed by intracellular esterases and oxidized to fluorescent carboxy-DCF (excitation 488 nm; emission 525 nm), primarily by H₂O₂. Confluent cells were incubated with C-H₂DCFDA-AM (2.5 or 5 μM) for 30 min (37°C), washed with DPBS, and isolated by brief trypsinization. JC-1 (Invitrogen) was used to detect ΔΨ_m. HL-1 myocytes were incubate with 1 μM JC-1 for 90 min (37°C) and washed. ΔΨ_m depolarization increases monomer (green) and decreases J-aggregate (orange-red) fluorescence (excitation: 488 nm; emission 525 and 575 nm). C-H₂DCFDA-AM and JC-1-loaded cells were assayed separately (EPICS XL; Beckman Coulter).

2.3. Statistics

Data are reported as mean ± SEM; n denotes number of cells (electrophysiology) or experiments (flow cytometry). One-way Repeated Measures ANOVA and the Holm-Sidak method were used (P <0.05; SigmaStat 3.11). For clarity, percent block (± SEM) is sometimes reported and was calculated using each myocyte as its own control. Geometric means of fluorescence data were not normally distributed and were analyzed using Kruskal-Wallis One-way ANOVA on Ranks and Dunn's method. Non-linear fitting was done in SigmaPlot 10.1.

3. Results

3.1. Ritonavir activates I_Cl,swell

Exposure of ventricular myocytes to 15 μM RTV for 20 min activated a Cl⁻ current that outwardly rectified. Families of membrane currents (Fig. 1A) and corresponding I–V relationships (Fig. 1B) are shown. This outwardly rectifying current is likely to be I_Cl,swell because it was abolished by DCPIB, a selective I_Cl,swell blocker [26], in the presence of RTV. RTV increased the current at +60 mV by 1.70 ± 0.16 pA/pF, from 0.42 ± 0.04 pA/pF under control conditions to 2.12 ± 0.15 pA/pF (n = 19, P <0.001) (Fig. 1C). Adding DCPIB (10 μM; 6–12 min) with RTV inhibited 95 ± 3% (n = 4, P <0.002) of RTV-induced current, and current after block was not different than control (n = 4, P = 0.71).

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).

Activation of RTV-induced current at +60 mV was sigmoidal; latency for activation was ∼3–7 min, and steady state was reached in 15–20 min. The time course for fractional activation, Y, was well-described by: Y = 1/[1 + exp(-(t-t_1/2)/τ)], where t_1/2, the time for half-activation, was 12.0 ± 0.9 min, and τ, the time constant, was 1.8 ± 0.2 min (n = 4) (Fig. 1D). Thus, despite variable latency, activation occurred with a consistent time constant once it began.

Although RTV-induced I_Cl,swell was readily blocked by DCPIB, stimulation of current was not easily reversed. The RTV-induced current was 1.16 ± 0.09 pA/pF and remained at 1.20 ± 0.02 pA/pF despite ∼25 min of washout in control bath solution in 2 cells.

3.2. Mitochondrial ROS elicit RTV-induced I_Cl,swell

NADPH oxidase and mitochondria both generate O₂^−• that is readily dismutated to H₂O₂ spontaneously and by superoxide dismutase. Previous studies found that NADPH oxidase- dependent and exogenous H₂O₂ activate I_Cl,swell in ventricular myocytes [19-21,25] and other cells [22-24]. The role of H₂O₂ in the RTV-induced activation of I_Cl,swell was tested with ebselen, a membrane-permeant, glutathione peroxidase mimetic that scavenges H₂O₂. After RTV elicited I_Cl,swell, adding ebselen (15 μM, 12–20 min) in the presence of RTV blocked 105 ± 3% (n = 5, P <0.001) of RTV-induced current (Fig. 2A-C).

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).

Next we considered the source of ROS. NADPH oxidase is a required for stretch and swelling to elicit I_Cl,swell [19-21,25]. The role of NADPH oxidase in RTV-induced I_Cl,swell activation was evaluated with apocynin, a selective inhibitor that prevents translocation of p47^phox to the membrane and assembly of NADPH oxidase [27]. Apocynin (500 μM, 20 min) failed to inhibit RTV-induced current (n = 4, P = 0.98) (Fig. 3A, B). In contrast, we previously showed that apocynin effectively suppresses I_Cl,swell elicited by swelling of cardiomyocytes [21].

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).

Besides NADPH oxidase, mitochondria are an important source of ROS in cardiomyocytes. Complex III leaks O₂^−• to the intermembrane space from which ROS accesses the cytoplasm [28,29]. To test the role of mitochondrial ROS production, we used rotenone, a highly selective complex I inhibitor that prevents e⁻ transport to and ROS production by complex III. Rotenone (10 μM, 26–32 min) inhibited 100 ± 3% (n = 5, P <0.001) of RTV-induced I_Cl,swell-activation (Fig. 3C, D). Rotenone also suppresses the activation of I_Cl,swell by antimycin A [25], which directly stimulates complex III ROS production.

3.3. Lopinavir activates I_Cl,swell via mitochondrial ROS

RTV was not the only HIV PI that activated I_Cl,swell. As shown in Fig. 4A and B, LPV (15 μM, 20 min) increased current at +60 mV by 1.69 ± 0.14 pA/pF, from 0.81 ± 0.06 to 2.50 ± 0.16 pA/pF (n = 18, P <0.001). The magnitude of current evoked by LPV and RTV, 1.70 pA/pF (Fig. 1), were indistinguishable.

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).

Experiments identifying the LPV-induced current as I_Cl,swell and demonstrating that mitochondrial ROS production, rather than NADPH oxidase, was responsible are summarized in Fig. 4C. First, the I_Cl,swell inhibitor DCPIB (10 μM, 6–12 min) blocked the LPV-induced current at +60 mV (115 ± 10%; n = 5, P <0.001) in the presence of LPV, suggesting the current was I_Cl,swell. Second, the H₂O₂ scavenger ebselen (15 μM, 12–20 min) suppressed (95 ± 5%; n = 5, P <0.001) LPV-induced current, demonstrating the involvement of ROS. Third, the NADPH oxidase inhibitor apocynin (500 μM, 20 min) failed to significant suppress LPV-induced I_Cl,swell (8 ± 5%; n = 4, P = 0.46), implying that ROS production by NADPH oxidase was not required. Fourth, blocking mitochondrial e⁻ transport with rotenone (10 μM, 26–32 min) decreased LPV-induced I_Cl,swell by 82 ± 4% (n = 4, P <0.01). These data recapitulate the results with RTV (Fig. 1–3) and suggest that LPV and RTV modulate I_Cl,swell by the same ROS-dependent mechanism.

3.4. Nelfinavir, amprenavir and raltegravir, an integrase inhibitor, did not block I_Cl,swell

Although both RTV and LPV activated I_Cl,swell, neither NFV (15 μM, 30 min; n = 5, P = 0.55) nor APV (15 μM, 30 min; n = 6, P = 0.11), two other HIV PI, significantly altered I_Cl,swell within the period tested (Fig. 5). This failure to activate I_Cl,swell was not due to the absence of the channels in the particular cells examined; hypoosmotic swelling in 0.7T elicited I_Cl,swell in the same cells. In the NFV experiments, current at +60 mV increased from 0.89 ± 0.11 pA/pF in control to 1.44 ± 0.08 pA/pF in 0.7T (n = 5, P <0.001), and activation by 0.7T was confirmed in 2 of 2 cells challenged with APV and 0.7T (P <0.001). The HIV integrase inhibitor raltegravir (MK; 15 μM, 30 min; n = 5, P = 0.86) also failed to stimulate I_Cl,swell, whereas 0.7T elicited I_Cl,swell in 2 of 2 cells exposed to MK and 0.7T (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.

3.5. APD

If activation of outwardly rectifying I_Cl,swell by HIV PI is functionally significant, these agents should decrease APD in physiological solutions (Fig. 6). RTV significantly decreased APD₅₀ and APD₉₀ by 37 ± 7 and 60 ± 9 ms, respectively (15 μM, 15 min; n = 4), whereas APV did not alter APD (15 μM, 15 min; n = 3). Moreover, the RTV-induced reduction of APD was reversed by the I_Cl,swell blocker DCPIB (10 μM, 10 min) in the presence of RTV. These responses are consistent with the effects of RTV, APV, and DCPIB on I_Cl,swell and illustrate the importance of I_Cl,swell in modulating APD. Nevertheless, RTV also reduced overshoot and plateau voltage, actions insensitive to suppression of I_Cl,swell by DCPIB. This suggests I_Cl,swell was not the only channel modulated by RTV, but the details were not further studied.

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.

3.6. Mitochondrial ROS production and ΔΨ_m

To more directly assess the proposed mitochondrial ROS-dependent mechanisms of I_Cl,swell activation, the effect of HIV PI on ROS production in HL-1 cardiomyocytes was determined by flow cytometry with C-H₂DCFDA-AM, which primarily detects H₂O₂. Fig. 7A,B display log fluorescence histograms from a single experiment. Geometric means of histograms normalized by background fluorescence are depicted in Fig. 7C. Measurements of ROS production were fully consistent with the notion that I_Cl,swell is a ROS sensor. Both RTV and LPV (15 μM, 30 min) significantly increased ROS production 4.2-fold from background levels. This was due to mitochondrial ROS production and did not depend on NADPH oxidase. RTV- and LPV-induced ROS production was suppressed by pretreatment (30 min) with rotenone (10 μM) but was unaffected by pretreatment with the NADPH oxidase blocker gp91ds-tat (500 nM). Moreover, the HIV PI inhibitors NFV and APV and the integrase inhibitor MK failed to elicit ROS production (15 μM, 60 min). In comparison, fluorescence elicited by H₂O₂ (100 μM, 15 min) was increased 5.6-fold above background (Fig. 7A,C). Negligible responses were obtained from myocytes in the absence of fluorophore (−F) with or without addition of LPV or RTV (Fig. 7A), indicating neither myocytes nor HIV PI themselves contributed to the signal attributed to ROS. We previously demonstrated that gp91ds-tat (500 nM) fully inhibits ROS production elicited by endothelin-1, which stimulates NADPH oxidase [25].

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.

ΔΨ_m also was assessed in HL-1 myocytes with the potential-sensitive dye JC-1. Fig. 8 shows that RTV (15 μM, 60 min, n = 5) significantly depolarized ΔΨ_m, as reflected by the increased green:orange-red fluorescence ratio over time. After 30 min, a time when ROS production and I_Cl,swell were augmented, the JC-1 fluorescence ratio was 2.1-fold (median) greater than control. On the other hand, APV (15 μM, 60 min, n = 4) elicited only a 20% increase at 30 min when ROS production and I_Cl,swell were unaffected. This suggests I_Cl,swell activation was triggered by significant ΔΨ_m depolarization. As a positive control, mitochondrial respiration was uncoupled with DNP (0.3 mM, 30 min, n = 3); this evoked a 4.5-fold increase in JC-1 fluorescence, whereas ΔΨ_m was unchanged over 60 min in control media (n = 3).

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.

Although DCPIB distinguishes between I_Cl,swell and a number of plasmalemmal anion and cation channels [26], its mechanism and site of action are unknown. One possibility is that DCPIB blocks mitochondrial inner membrane Cl⁻ channel, thereby restoring ΔΨ_m and decreasing ROS production. Rather than enhancing ΔΨ_m, DCPIB (10 μM, 10 min) increased JC-1 fluorescence in HL-1 myocytes 8.7 ± 0.9-fold (n = 6), indicating it strongly depolarized ΔΨ_m. In contrast, a low DCPIB concentration (0.1 μM, 10 min) that fails to suppress I_Cl,swell increased JC-1 fluorescence by only ∼20% (n = 2). These data suggest mitochondrial actions of DCPIB may, at least in part, explain its block I_Cl,swell.

4. Discussion

We demonstrated that certain HIV PI activate the volume-sensitive Cl⁻ current, I_Cl,swell, in ventricular myocytes under isosmotic conditions. At clinically relevant concentrations, RTV and LPV acutely induced an outwardly-rectifying Cl⁻ current, RTV decreased APD₉₀ by 60 ms, and both effects were fully abolished by the I_Cl,swell-blocker DCPIB. Two other HIV PI, APV and NFV, and the HIV integrase inhibitor raltegravir (MK-0518) did not effect I_Cl,swell, and APV also did not alter APD.

Activation of I_Cl,swell by RTV and LPV was ROS-dependent. H₂O₂ is a required intermediate in the signaling cascade regulating I_Cl,swell, and exogenous H₂O₂ activates I_Cl,swell in rabbit ventricular myocytes [19-21,25], adenocarcinoma HeLa cells, hepatoma HTC cells, and cultured primary astrocytes [22-24]. We found RTV- and LPV-induced I_Cl,swell was fully suppressed by the H₂O₂ scavenger ebselen. RTV and LPV augmented ROS production, as detected by C-H₂DCFDA-AM, in HL-1 myocytes, whereas APV and NFV failed to generate significant ROS after 1 h. Dichlorofluoresceins, such as C-H₂DCFDA-AM, are nominally H₂O₂-selective, but ONOO^−•, HOCl, and peroxides also can contribute to its fluorescence [30]. Measurements of ΔΨ_m with JC-1 in HL-1 myocytes suggest that RTV elicited mitochondrial ROS production, at least in part, by depolarizing ΔΨ_m. ROS and ΔΨ_m measurements were made in HL-1 myocytes rather than acutely dissociated adult myocytes to avoid inclusion of cells damaged during isolation. Other studies confirm that I_Cl,swell is activated by a number of the same mechanisms in HL-1 cells as in adult myocytes [31].

The two major sources of H₂O₂ in cardiomyocytes are sarcolemmal NADPH oxidase and mitochondria. These systems generate O₂^−•, which is readily dismutated to H₂O₂ spontaneously and by superoxide dismutase. Previously we showed that NADPH oxidase is essential for the activation of I_Cl,swell by β1-integrin stretch and osmotic swelling and that I_Cl,swell can be blocked by selectively inhibiting NADPH oxidase with apocynin or gp91ds-tat [19-21,25]. However, apocynin failed to suppress the RTV-and LPV-induced currents, and pretreatment with gp91ds-tat failed to suppress C-H₂DCFDA-AM fluorescence. These data are consistent with the notion that activation of I_Cl,swell by HIV PI was independent of NADPH oxidase.

Complex I and Complex III are the major mitochondrial redox centers that leak single e⁻ to molecular O₂ to generate O₂^−• [28,29]. Normally O₂^−• generated by complex I is exclusively released into the matrix of intact mitochondria and degraded by the matrix anti-oxidant system [32]. By contrast, Complex III releases O₂^−• to both sides of the mitochondrial inner membrane and accounts for extra-mitochondrial release of O₂^−• [28,32]. We propose that HIV PI elicit I_Cl,swell via mitochondrial ROS production by Complex III. RTV- and LPV-induced I_Cl,swell were completely abolished by rotenone, a selective Complex I inhibitor that limits downstream e⁻ flux to Complex III and, therefore, inhibits mitochondrial ROS generation [28]. In addition, a 1-hr pretreatment with rotenone fully abrogated RTV- and LPV-induced H₂O₂ production measured directly by flow cytometry. Although we favor H₂O₂ as the active species, we cannot rule out the possibility that the activation of I_Cl,swell by HIV PI involves other ROS derived from O₂^−• or H₂O₂.

A mitochondrial ROS-dependent mechanism for the activation of I_Cl,swell is consistent with previous work showing that HIV PI reduce mitochondrial membrane potential and increases leak of e⁻ from the transport chain to O₂ to produce O₂^−• [11,15]. It also is consistent with our previous studies showing that blocking mitochondrial ROS production inhibits activation of I_Cl,swell by endothelin-1 [31]. We did not evaluate whether mitochondrial ROS-induced ROS release [33] contributed to the response to HIV PI.

Despite the failure of APV and NFV to modulate I_Cl,swell in 30 min or elicit ROS production after 1 h, it remains possible that these HIV PI also would activate cardiac I_Cl,swell with chronic administration. APV and NFV have been shown to elicit ROS production in endothelial cells [9,34,35], vascular smooth muscle cells [10], macrophages [14], adipocytes [13,36], fibroblasts [16], and pancreatic β cells [17]. Prolonged exposures to HIV PI often employed to detect oxidative stress cannot be replicated in patch clamp studies using each myocyte as its own control.

DCPIB is regarded as a highly selective I_Cl,swell blocker because it suppresses I_Cl,swell without affecting several other plasmalemma Cl⁻, Na⁺, Ca²⁺ and K⁺ channels [26], but evidence that it binds directly to I_Cl,swell channels is lacking. Studies with JC-1 indicated that DCPIB very strongly depolarized ΔΨ_m. This raises the possibility that DCPIB uncouples mitochondrial e⁻ transport and blocks ROS production, which may in part explain how DCPIB inhibits I_Cl,swell. Nevertheless, DCPIB also suppresses I_Cl,swell evoked by 100-μM exogenous H₂O₂ [32], suggesting a mitochondria-independent effect. Although DCPIB is useful to distinguish between ion channels, fully understanding its actions on mitochondrial function and the mechanism of I_Cl,swell blockade requires additional investigation.

4.1. Potential Implications

Adverse cardiovascular effects of HIV PI include coronary artery disease and myocardial infarction [37,38], and certain HIV PI are associated with QT interval prolongation and torsade de pointes [2,4]. Anson et al. [2] demonstrated that RTV, LPV, NFV, and saquinavir substantially suppress HERG currents expressed in HEK293 cells and the corresponding endogenous current, I_Kr, in mouse neonatal myocytes. Block of HERG favors APD prolongation, early afterdepolarizations, and torsade de pointes [39]. Because I_Cl,swell produces an outward current near the plateau potential, its activation favors reduction of APD. Consequently, HIV PI-induced I_Cl,swell would antagonize APD prolongation due to block of HERG and thereby reduce the risk of drug-induced long QT syndrome and torsade de pointes. On the other hand, APD abbreviation will favor reentrant arrhythmias, such as atrial fibrillation, but an association between HIV PI and reentrant arrhythmias has not been documented. Although the present acute studies on isolated cardiomyocytes from normal animals raise important several questions, they are not sufficient to establish the role of I_Cl,swell in HIV-infected patients who are treated with HIV PI as part of multidrug highly active anti-retroviral therapy.

Although several mechanisms underlying the untoward effects of HIV PI have been the focus of research, the potential role of ion channels in these outcomes largely has been ignored. Because I_Cl,swell is present in a number of tissues affected by HIV PI, such as macrophages, hepatocytes, endothelial cells, and pancreatic β cells, it is reasonable to consider whether I_Cl,swell activation might contribute to the untoward effects of HIV PI. HIV PI-induced ROS production also may regulate other redox sensitive ion channels and transporters that are widely distributed. Na channels [40], L-type Ca²⁺ channels, transient outward K⁺ channels, inwardly rectifying K⁺ channels, ATP-sensitive K⁺ channels, ryanodine receptors, inositol-1,4,5- trisphosphate receptors, sarcoplasmic reticulum and plasma membrane Ca²⁺ ATPases, and the Na⁺–Ca²⁺ exchanger [41] are influenced by ROS. Whether HIV PI modulate these channels and transporters and whether they contribute to the adverse effects of HIV PI in heart and other systems remains to be elucidated.