The cyclophilin inhibitor alisporivir prevents hepatitis C virus–mediated mitochondrial dysfunction

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Alisporivir (Debio-025) is an analogue of cyclosporine A and represents the prototype of a new class of non-immunosuppressive cyclophilin inhibitors. In vitro and in vivo studies have shown that alisporivir inhibits hepatitis C virus (HCV) replication, and ongoing clinical trials are exploring its therapeutic potential in patients with chronic hepatitis C. Recent data suggest that the antiviral effect is mediated by inhibition of cyclophilin A, which is an essential host factor in the HCV life cycle. However, alisporivir also inhibits mitochondrial permeability transition by binding to cyclophilin D. Because HCV is known to affect mitochondrial function, we explored the effect of alisporivir on HCV protein-mediated mitochondrial dysfunction. Through the use of inducible cell lines, which allow to investigate the effects of HCV polyprotein expression independent from viral RNA replication and which recapitulate the major alterations of mitochondrial bioenergetics observed in infectious cell systems, we show that alisporivir prevents HCV protein-mediated decrease of cell respiration, collapse of mitochondrial membrane potential, overproduction of reactive oxygen species and mitochondrial calcium overload. Strikingly, some of the HCV-mediated mitochondrial dysfunctions could even be rescued by alisporivir. Conclusion: These observations provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C. (HEPATOLOGY 2012)

Hepatitis C virus (HCV)-related liver disease represents a major health burden worldwide.1 Treatment with pegylated interferon-α and ribavirin has limited efficacy and numerous adverse effects.2 While a first generation of directly acting antivirals have entered clinical application, targeting host factors essential for the HCV life cycle represents an attractive alternative therapeutic approach. In this context, non-immunosuppressive analogues of the cyclophilin (Cyp) inhibitor cyclosporine A (CsA) represent a new class of potent anti-HCV agents,3 with efficacy both in vitro as well as in clinical studies in patients with chronic hepatitis C.4-6 Alisporivir (also known as Debio-025 or DEB025) is the prototype and most advanced molecule in this novel class of antivirals. It efficiently inhibits Cyps but, unlike CsA, does not interact with calcineurin, explaining the lack of immunosuppressive effect.7

At least 16 Cyp isoforms are expressed in human cells, and these are involved in diverse cellular processes and pathways, many of which may influence the HCV life cycle.8 The respective roles of Cyp isoforms in the HCV life cycle remains controversial. However, the peptidyl-prolyl cis-trans isomerase activity of cyclophilin A (CypA) is crucial for HCV replication, and its inhibition mediates the antiviral activity of alisporivir.9, 10 CypA may interact with different viral proteins and favor a particular conformation that is required for efficient viral replication and/or could have a role in facilitating the processing of the HCV polyprotein.3

Cyclophilin D (CypD) is a member of the family that has been receiving growing attention because of its role in controlling cell fate.11 It is localized within the mitochondrial matrix and interacts with the mitochondrial permeability transition pore (MPTP), sensitizing its opening by physiological inducers.12, 13 Activation of the MPTP allows the rapid passage of low molecular weight molecules and ions (up to 1.5 kDa) and, when persistent, the release of proapoptotic mitochondrial intermembrane proteins, i.e., proteins located between the outer and inner mitochondrial membranes.12 This last event, whose mechanism has not yet been completely clarified, induces adaptive cellular responses that can lead to mitophagy, apoptosis, or necrotic cell death.14, 15 CsA and its non-immunosuppressive analogs are potent inhibitors of the MPTP, because they bind to CypD and thereby increase its activation threshold.11

Consistent with this notion, alisporivir has been evaluated in models of muscular dystrophy and myopathy and was found to attenuate mitochondria-dependent muscle cell apoptosis/necrosis.16, 17 Moreover, inhibition of mitochondrial permeability transition by alisporivir has been reported to improve functional recovery and to reduce mortality following acute myocardial infarction in mice.18

We have shown that HCV protein expression elicits marked alterations of mitochondria-related activities19, 20 that may cause or be caused by alterations of MPTP and prime proapoptotic setting. Therefore, we tested the hypothesis that the beneficial effect of alisporivir may also depend on its ability to prevent HCV-mediated mitochondrial dysfunction by interfering with the MPTP inducer CypD. To this end, we used an in vitro cell system allowing the inducible expression of the entire HCV polyprotein independent from viral RNA replication21 which is efficiently inhibited by alisporivir. This allowed us to investigate effects of alisporivir on HCV protein-mediated mitochondrial dysfunction.

The results obtained provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C.

Abbreviations

AIF, apoptosis-inducing factor; CsA, cyclosporine A; Cyp, cyclophilin; CypA, cyclophilin A; CypD, cyclophilin D; DCF, dichlorofluorescein; EDTA, ethylene diamine tetraacetic acid; ER, endoplasmic reticulum; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; FITC, fluorescein isothiocyanate; HCV, hepatitis C virus; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; LSCM, laser scanning confocal microscopy; MPTP, mitochondrial permeability transition pore; mtCa2+, intramitochondrial calcium; mtΔΨ, mitochondrial membrane potential; PBS, phosphate-buffered saline; ROS, reactive oxygen species; TMRE, tetramethylrhodamine ethyl ester; VDAC, voltage-dependent anion channel.

Materials and Methods

Cell Cultures and Drug Treatment.

UHCV-32 and UHCVcon-57.3 are U-2 OS human osteosarcoma-derived cell lines inducibly expressing the entire open reading frame derived from the HCV H77 prototype and consensus clones, respectively.21 Cell viability was measured by trypan blue exclusion analysis. HCV protein expression in these cells is induced by withdrawal of tetracycline from the culture medium. The effect of tetracycline on the naïve U2 OS cell line was tested measuring mitochondria-related respiration and reactive oxygen species (ROS) production (see below), which remained unchanged (data not shown). Alisporivir (Debio-025, kindly provided by Debiopharm, Lausanne, Switzerland) was prepared in dimethyl sulfoxide at 4 mM and diluted in cell culture medium at the indicated concentrations.

Measurement of Cell Respiration and Citrate Synthase Activity.

Cultured cells were gently detached from the dish by way of trypsinization, washed in phosphate-buffered saline (PBS), harvested by centrifugation, and immediately assessed for O2 consumption by a Clark-type electrode (Hansatech or Oroboros) in a thermostated gas-tight chamber equipped with a stirring device. Typically, 5-7 × 106 viable cells/mL were assayed in 50 mM KPi, 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), and 1 mM ethylene diamine tetraacetic acid (EDTA; pH 7.4) at 37°C; after attainment of a stationary endogenous substrate-sustained respiratory rate, 2 μg/mL of oligomycin and 0.8 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added sequentially within a 10-minute interval. The rates of O2 consumption were corrected for 2 mM KCN-insensitive respiration. Citrate synthase activity was measured spectrophotometrically on total cell lysate as described.22

Laser Scanning Confocal Microscopy Live Cell Imaging of Mitochondrial Membrane Potential, ROS and Intramitochondrial Calcium.

Cells cultured at low density on fibronectin-coated 35-mm glass-bottom dishes were incubated for 20 minutes at 37°C with the following probes (all from Molecular Probes): 2 μM tetramethylrhodamine ethyl ester (TMRE) to monitor mitochondrial membrane potential (mtΔΨ); 10 μM 2,7-dichlorofluorescin diacetate, which is converted to dichlorofluorescein (DCF) by intracellular esterases, for detection of H2O2; and 5 μM X-Rhod-1 AM for mitochondrial Ca2+. Stained cells were washed with PBS and examined with a Nikon TE 2000 microscope (images collected using a 60× objective [1.4 NA]) coupled to a Radiance 2100 dual-laser laser scanning confocal microscopy (LSCM) system (Bio-Rad). TMRE and Rhod-1 red fluorescence was elicited by exiting with the He-Ne laser beam (λex 543 nm) whereas dichlorofluorescein green fluorescence was elicited with the Ar-Kr laser beam (λex 488 nm). Acquisition, storage, and analysis of data were performed with LaserSharp and LaserPix software from Biorad or ImageJ version 1.37 as described by Piccoli et al.19

Immunocytochemistry.

Cells cultured at low density on fibronectin-coated 35-mm glass bottom dishes were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, followed by blocking with 3% bovine serum albumin in PBS and incubated for 1 hour at 20°C with 1:200 diluted mouse monoclonal antibody against cytochrome c (Promega) or 1:100 rabbit polyclonal antibody against voltage-dependent anion channel (VDAC) (Cell Signaling Technology) or 1:100 rabbit polyclonal antibody against apoptosis-inducing factor (AIF) (Chemicon International). After two washes in 3% bovine serum albumin in PBS, the sample was incubated for 1 hour at room temperature with 1:200 fluorescein isothiocyanate (FITC) labeled goat anti-mouse immunoglobulin G or 1:200 rhodamine labelled goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology). The fluorescent signals emitted by the FITC-conjugated antibody (λex, 490 nm; λem, 525 nm) of the labeled cells were analyzed using LSCM as described.19

Subcellular Fractionation.

A total of 5 × 107 U-2 OS cells were harvested in 250 mM sucrose, 1 mM EDTA, 5 mM HEPES (pH 7.4), 3 mM MgCl2 supplemented with 20 μL/mL of protease inhibitor cocktail (Roche), dounce-homogenized in ice (50 strokes) and centrifuged at 600g for 5 minutes. The supernatant was centrifuged at 12,000g for 15 minutes and the resulting supernatant removed constituting the cytosolic fraction. The pellet was resuspended in the buffer and recentrifuged at 12,000g for 15 minutes; the resulting pellet was resuspended in a minimal volume of the buffer and constituted the mitochondria enriched fraction.

Immunoblotting.

Cells or cellular fractions were lysed in 20 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 2 mM sodium phosphate, and protease inhibitor cocktail, followed by 10% SDS-PAGE and western blotting using monoclonal antibodies 5B-3B1 against HCV NS5B,19, 23 C7-50 against HCV core,19, 23 rabbit anti-cytochrome c (1:400, Abcam), anti-cleaved caspase 3 (1:1000, Cell Signaling Technology), anti-β-subunit of CV (MitoSciences) or anti-β-actin (Sigma) as primary antibodies.

Statistical Analysis.

A two-tailed Student t test was applied to evaluate the statistical significance of differences measured from the datasets reported. P < 0.05 was considered statistically significant.

Results

Alisporivir Prevents HCV Protein-Mediated Collapse of the Respiration-Driven Mitochondrial Membrane Potential.

We have shown that HCV protein expression causes a dose- and time-dependent alteration of the main bioenergetic functions of mitochondria19, 20 with the major dysfunctions observed already 36-48 hours after HCV protein induction.19 To assess a possible involvement of the MPTP in HCV protein-mediated mitochondrial deregulation, we tested the effect of alisporivir, a non-immunosuppressive analog of the MPTP inhibitor CsA.

Opening of the MPTP causes collapse of the respiration-driven mtΔΨ.12 As shown previously19 and confirmed in Fig. 1A, HCV protein expression for 48 hours resulted in a marked reduction of the mtΔΨ, as assessed by the fluorescent probe TMRE. Alisporivir prevented the dissipation of the mtΔΨ in a dose-dependent manner. HCV protein-induced collapse of the mtΔΨ was completely prevented at 0.125 μM, a concentration that has been shown to significantly reduce HCV RNA levels in the HCV replicon system4 (Fig. 1A,B). No significant effects were observed in noninduced control cells treated by low concentrations of alisporivir, whereas a slight hyperpolarization was detected at the highest concentration. Importantly, treatment with 0.125 μM alisporivir did not affect the expression level of HCV proteins after 48 hours of induction (Fig. 1C).

Figure 1.

Dose-dependence of the effect of alisporivir on HCV protein-mediated depolarization of the mitochondrial membrane potential. (A) UHCVcon-57.3 cells were induced by tetracycline withdrawal for 2 days (ON) in the absence or presence of 0.0625, 0.125, 0.25, and 0.5 μM alisporivir. Cells cultured in the presence of tetracycline (that is, those not expressing HCV proteins) served as controls (OFF). The mitochondrial membrane potential (mtΔΨ) in live cells was monitored by LSCM using the specific probe TMRE as described by Piccoli et al.19 as well as in the Materials and Methods section. (B) Quantitative estimation of TMRE fluorescence. Quantification of the emitted fluorescent signal was achieved by averaging the pixel intensity values within the outline of single cells, as a function of each focal plane using ImageJ software (National Institutes of Health, Bethesda, MD) and expressed as arbitrary units. Correction was made for minimal background in cell-free fields. At least 20 cells were randomly selected in each of 8-10 different optical fields under the indicated conditions and statistically analyzed. The values are expressed as the mean ± SEM of four independent experiments for each condition and are reported as the percentage of the pixel intensity per cell of the noninduced untreated sample. #P < 0.01, HCV-induced cells versus noninduced cells. *P < 0.05, **P < 0.005, and ***P < 0.001 as compared with the respective fluorescence of both noninduced cells and HCV-induced cells cultured in the absence of alisporivir. (C) Effect of alisporivir on the HCV protein expression in induced cells. Total protein extracts from cells cultured for 2 days in the absence of tetracycline were analyzed via western blotting with monoclonal antibody C79-50 against the viral core protein as described in the Materials and Methods section. Results shown are representative of four independent experiments.

Opening of the MPTP may cause leakage of low molecular weight mitochondrial respiratory substrates.24 To test this possibility, we performed measurements of respiratory rates in intact cells by high-resolution oxymetry. As shown in Fig. 2, HCV protein expression for 48 hours resulted in a significant inhibition of the cyanide-sensitive dioxygen consumption under endogenous respiratory setting both in the absence and in the presence of either oligomycin (an ATP-synthase inhibitor) or FCCP (an oxidative phosphorylation uncoupler). Alisporivir completely prevented the inhibition of mitochondrial respiration in induced cells, whereas it did not have any effect in noninduced cells. Notably, basal respiration was decreased in cells harboring HCV proteins, yet oxygen consumption could still be stimulated by FCCP, suggesting that HCV protein expression affected ATP synthesis. This result is consistent with our recent report showing a significant inhibition of the FoF1 ATP-ase activity in HCV protein-expressing cells.23 Consistently, CypD was found to affect synthesis and hydrolysis of ATP by binding ATP synthase with CsA modulating this binding and, thereby, the activity of the ATP synthase.25 This could contribute to the observed rescue of resting respiration by alisporivir.

Figure 2.

Effect of alisporivir on the HCV protein-mediated inhibition of the respiratory activity. Oxygen consumption rates were measured in intact UHCVcon-57.3 cells by high-resolution oxymetry as described in the Materials and Methods section. HCV induction was performed for 2 days without or with 0.125 μM alisporivir. The values are expressed as the mean ± SEM of 3-4 independent experiments for each condition. *P < 0.05 as compared with the respective respiratory activities of both noninduced cells (OFF) and HCV-induced cells cultured in the presence of alisporivir (ON + alisporivir).

Alisporivir Prevents HCV Protein-Mediated Mitochondrial Calcium Overload.

A major trigger of MPTP opening is mitochondrial calcium overload.15 By using the calcium probe Rhod-1, which specifically detects intramitochondrial calcium (mtCa2+), we previously found that HCV protein expression for 48 hours resulted in a significant increase of mtCa2+.19 Inhibitors of the mitochondrial calcium uniporter or of the endoplasmic reticulum (ER) calcium channel efficiently prevented mitochondrial calcium overload.19, 20 Importantly, alisporivir prevented mtCa2+ accumulation in a dose-dependent fashion. As shown in Fig. 3, maximal protective effect was already observed at a concentration of 0.125 μM.

Figure 3.

Dose-dependence of the effect of alisporivir on the HCV protein-mediated intramitochondrial Ca2+ load. UHCVcon-57.3 cells were treated as described in Fig. 1 and intramitochondrial Ca2+ (mtCa2+) was assessed by the specific probe M X-Rhod-1 AM (RHOD-1) as described by Piccoli et al.19 as well as in the Material and Methods section. (A) Representative LSCM analyses. An enlarged detail of the intracellular fluorescent signal in the induced alisporivir-untreated sample is also shown. (B) Quantitative estimation of the RHOD-1 fluorescence. The values are expressed as the mean ± SEM of three independent experiments for each condition. #P < 0.01, HCV-induced cells versus noninduced cells. *P < 0.05, **P < 0.01 as compared with the respective fluorescence of HCV-induced cells cultured in the absence of alisporivir. See Fig. 1 for details.

Alisporivir Prevents HCV Protein-Mediated Production of ROS.

Mounting evidence from experimental and clinical observations indicates that HCV infection is causally linked with alterations of the intracellular redox state and that these may be involved in the pathogenesis of hepatitis C.20 Notably, oxidative stress proved to be a condition favoring MPTP opening.26, 27 As reported previously,19 HCV protein expression resulted in a marked increase of cellular ROS production, as assessed by the hydrogen peroxide-sensitive fluorescent probe dichlorofluorescein (Fig. 4A). Closer analyses by LSCM revealed a bright fluorescence signal in intracellular compartments corresponding to the mitochondrial network. Alisporivir prevented ROS production as a result of HCV protein expression at a concentration as low as 0.125 μM (Fig. 4B).

Figure 4.

Dose-dependence of the effect of alisporivir on the HCV protein-mediated intracellular production of ROS. UHCVcon-57.3 cells were treated as described in Fig. 1, and ROS were assessed by the hydrogen peroxide-specific probe DCF, as described by Piccoli et al.19 as well as in the Materials and Methods section. (A) Representative LSCM analyses. An enlarged detail of the intracellular fluorescent signal in the induced alisporivir-untreated sample is also shown. (B) Quantitative estimation of the DCF fluorescence. The values are expressed as the mean ± SEM of four independent experiments for each condition. #P < 0.001, HCV-induced cells versus noninduced cells. *P < 0.05, **P < 0.001 as compared with the respective fluorescence of HCV-induced cells and noninduced cells cultured in the absence of alisporivir. See Fig. 1 for details.

Alisporivir Reverts HCV Protein-Mediated Mitochondrial Dysfunction.

The results above clearly demonstrate that alisporivir prevents HCV protein-mediated mitochondrial dysfunction. Next, we asked whether alisporivir may also revert already established mitochondrial dysfunction. To this end, treatment with alisporivir at a concentration of 0.125 μM was initiated 36 hours after the induction of HCV protein expression. As shown in Fig. 5A-C, alisporivir reverted within 12 hours HCV protein-mediated collapse of the mtΔΨ, production of ROS and mitochondrial calcium overload of mtCa2+. Thus, alisporivir cannot only prevent but also revert already established mitochondrial dysfunction in this experimental setting.

Figure 5.

Alisporivir rescues HCV protein-mediated mitochondrial dysfunction. UHCVcon-57.3 cells were cultured for 36 hours in the absence of tetracyline (ON), followed by treatment with either 0.125 μM alisporivir or vehicle for the successive 12 hours (see the experimental scheme at the top of the figure). Cells cultured in the presence of tetracycline (that is, those not expressing HCV proteins) served as controls (OFF). Detection and image quantification of ROS (A), mtCa2+ (B), and mtΔΨ (C) was performed in living cells by fluorescent probe-assisted LSCM analyses, as described in Figs. 4, 3, and 1, respectively. The histograms report quantifications of the fluorescence signals assessed at 36 hours and at 48 hours, as indicated. The values are expressed as the mean ± SEM of three independent experiments for each condition. The statistical comparison between pairs of different values is shown when significant. *P < 0.05. **P < 0.01.

Alisporivir Prevents HCV Protein-Mediated Mitochondrial Dysfunction Outside the Context of Apoptosis.

Opening of the MPTP elicits redistribution of small proapoptotic proteins located in the mitochondrial intermembrane space, such as cytochrome c, to the extramitochondrial compartment.12 As shown in Fig. 6A, inducible expression of the HCV polyprotein resulted in a marked change of the cytochrome c–related immunofluorescence detection. Cytochrome c was concentrated in mitochrondria in U-2 OS cells cultured in the presence of tetracycline (that is, those not expressing HCV proteins). By contrast, induction of HCV protein expression by tetracycline withdrawal over 5 days resulted in blurring and weakening of the fluorescence pattern. Of note, this effect on cytochrome c was observed only after 4-5 days of HCV protein expression but not at earlier time points (Fig. 6C and data not shown). Treatment of the cells with 0.125 μM alisporivir concomitant with the induction of HCV protein expression completely prevented alterations in the cellular appearance/distribution of cytochrome c (Fig. 6A,C). Similar results were obtained with 1 μM CsA (data not shown).

Figure 6.

Effect of alisporivir on the intracellular localization of cytochrome c in cells expressing HCV proteins. UHCVcon-57.3 cells were induced (ON) by tetracycline withdrawal for 2 or 5 days in the absence or presence of 0.125 μM alisporivir. Cells cultured in the presence of tetracycline (that is, those not expressing HCV proteins [OFF]) served as controls. (A) Representative LSCM analyses of the effect of alisporivir on the immunocytochemical fluorescence detection of cytochrome c in cells cultured in the absence of tetracycline for 5 days. Cytochrome c was visualized by an FITC-conjugated secondary antibody as described in the Materials and Methods section. (B) Representative LSCM analyses of the immunofluorescence of the VDAC and the AIF in cells cultured for 5 days with/without tetracycline. Both VDAC and AIF were visualized by rhodamine (R)-conjugated secondary antibodies. (C) Statistical analysis of the cytochrome c-, VDAC-, and AIF-related immunofluorescence signal. The conditions are as described in (A) and (B), with additional analyses performed after 2 days of HCV protein induction in the case of the cytochrome c. Digitalized images were analyzed using ImageJ software and the mean pixel intensity per cell together with the respective standard deviation expressed in the gray-scale values (0-256). At least 20 different cells were randomly selected in each of 5-6 different optical fields. The histogram shows for all the indicated conditions the mean values ± SEM of the pixel intensity per cell expressed as percentage of the noninduced alisporivir-untreated sample and the standard deviation/(pixel intensity per cell) of 3-4 independent experiments. *P < 0.05. **P < 0.01. (D) Functional evaluation of the mitochondrial mass assessed by the citrate synthase activity. UHCVcon-57.3 cells, cultured under the indicated conditions (concentration of alisporivir, 0.125 μM), were harvested, ultrasound lysed, and assayed spectrophotometrically as described by Barrientos.22 Citrate synthase activity is expressed as the rate of CoASH release, and the values shown are expressed as the mean ± SEM from three independent sample preparations under each condition. (E) Effect of HCV protein expression and alisporivir treatment on the cellular cytochrome c content. Total protein extracts from lysates of UHCVcon-57.3 cells, cultured for 2 or 5 days under the indicated conditions (0.125 μM alisporivir), were western blotted with antibodies against cytochrome c as described in the Materials and Methods section. Immunoblotting of the viral NS5B and core proteins in cells cultured for 5 days in the absence of tetracycline is also shown. Results shown are representative of 3-4 different sample preparations. (F) Immunoblotting against cytochrome c in cytosolic (C) and mitochondria-enriched fractions (M). Control and HCV-induced U-2 OS cells (5 days)) were homogenized and fractionated by differential centrifugation as described in the Materials and Methods section. Subunit β of complex V (CV) and β-actin were used as markers of the cytosolic and mitochondrial fractions, respectively. Representative of three independent preparations yielding similar results.

Measurement of the standard deviation of the pixel intensity normalized to the mean fluorescence intensity per cell (a direct index of signal heterogeneity28) resulted in comparable values between induced and noninduced cells (Fig. 6C, gray bars) thus indicating that, irrespective of the loss of the fluorescent signal, the intracellular distribution of cytochrome c was mostly unchanged in HCV protein-expressing cells. Measurement of citrate synthase activity, a mitochondrial mass marker, ruled out changes of the mitochondrial content in HCV-induced cells (Fig. 6D). Moreover, western blotting of cytochrome c in total cell lysate resulted in a comparable protein content irrespective of HCV protein induction or alisporivir treatment, thereby excluding the possibility of an HCV-mediated proteolytic degradation of cytochrome c (Fig. 6E). In addition, Fig. 6E shows that under prolonged condition of induction, alisporivir did not significantly affect the expression of HCV proteins. Intriguingly, immunofluorescence imaging of the outer mitochochondrial marker VDAC and of another intermembrane proapoptotic protein (AIF) resulted in a decrease of the fluorescence signal following 5 days of HCV protein induction, similar to what was observed for cytochrome c (Fig. 6B), but without effect of their heterogeneity index (Fig. 6C). Finally, immunoblotting of cytochrome c in subcellular fractions showed conclusively that cytochrome c was not released from mitochondria into the cytoplasm following 5 days of HCV-induction (Fig. 6F).

The impact of HCV protein expression on apoptosis was assessed by evaluating the activation of the marker caspase 3 via western blotting. Fig. 7A shows no appreciable cleavage of caspase 3 (diagnostic of apoptosis induction12) up to 5 days after HCV protein induction. This observation supported the effect of HCV protein expression on cell growth and viability evaluated by cell density analyses and trypan blue exclusion assay, demonstrating no significant changes both in the cell growth rate and in the relative amount of cell death (<5%) irrespective of HCV protein expression or alisporivir treatment (Fig. 7B).

Figure 7.

Effect of HCV protein expression and of alisporivir treatment on apoptosis and cell growth. (A) Analysis of the cleavage state of the apoptotic marker caspase 3. Total protein extracts from lysates of UHCVcon-57.3 cells cultured for 2 or 5 days under the indicated conditions (±0.125 μM alisporivir) were analyzed via western blotting with antibodies against the uncleaved and cleaved form of caspase 3, as described in the Materials and Methods section. Cells treated with 1 μM staurosporin for 12 hours served as a positive control. The results shown are representative of three different sample preparations under each condition. (B) Evaluation of cell growth. UHCVcon-57.3 cells were seeded at the initial density of 1.5 × 105 cells in 60-mm culture dish and trypan blue negative (TP) and positive cells (TP+) counted daily for 5 days. The values are expressed as the mean ± SEM of three independent experiments. DOI, days of induction.

Taken together, these results show that prolonged HCV protein expression causes an alisporivir-sensitive decrease of the antigenicity of cytochrome c which, however, retained its mitochondrial localization. Consistently, no evidence of significant induction of apoptosis was observed in HCV protein-expressing cells.

Discussion

In this study, we investigated the effect of the non-immunosuppressive CsA analogue alisporivir on HCV-mediated mitochondrial dysfunction. Well-characterized cell lines inducibly expressing the entire HCV polyprotein were chosen as an in vitro model, allowing to study the effects of alisporivir on mitochondrial physiology independent from its antiviral effect.21 In a recent model, proposed by us, the earliest event leading to mitochondrial dysfunction is the entry of Ca2+ into mitochondria19 (see also Li et al.29 and Dionisio et al.30). This event was suggested to take place at mitochondrial-ER contact sites and is likely due to ER stress induced by HCV proteins.31, 32 Increased steady-state levels of mtCa2+ induce further alterations comprising production of nitric oxide, inhibition of the respiratory chain and generation of ROS, thereby creating the conditions for a state of oxidative stress. Both Ca2+ and ROS are inducers of the MPTP, enhancing its opening probability.13, 14, 26, 27 Transient activation of the MPTP is thought to regulate the homeostasis of mtCa2+ levels and of the mtΔΨ.33 However, conditions leading to a persistent opening of the MPTP cause a complete collapse of the mtΔΨ and release of low molecular weight metabolites as well as coenzymes, with consecutive impairment of energy production by the oxidative phosphorylation system.28, 33, 34 Finally, continuous activation of the MPTP causes the release of proapoptotic factors residing within the mitochondrial intermembrane space. Depending on the prevailing conditions, this may lead to selective removal of damaged organelles, programmed cell death, or necrosis.14, 15 Enhanced hepatocyte apoptosis has been demonstrated in chronic hepatitis C.35 Nevertheless, HCV infection persists in the majority of patients. The consequences of apoptosis in chronic hepatitis C are not well understood. Proapoptotic and antiapoptotic effects have been described in vitro for some HCV proteins, in particular for core and NS5A.36 However, it is unknown which viral proteins affect apoptosis in a natural HCV infection in vivo. Insufficient apoptosis, with failure to remove cells carrying genetic alterations, and increased proliferation in the context of persistent inflammation, may promote the development of hepatocellular carcinoma. However, chronic apoptotic stimulation may also contribute to cancer development because of the high rate of regeneration invoked in the tissue, which enhances the risk of mitotic errors. Therefore, therapeutic strategies aimed at inhibiting apoptosis may be beneficial in chronic hepatitis C, and phase 2 trials are ongoing to explore the effect of a pancaspase inhibitor in chronic hepatitis C.37

The main mechanism of action of CsA and alisporivir is based on the inhibition of CypA, the peptidyl-prolyl cis-trans isomerase activity of which has been identified as being essential for HCV replication.9, 10 However, these agents may have additional beneficial effects. Indeed, despite the well-known effect of CsA in counteracting CypD-mediated activation of the MPTP in a variety of cell systems,16, 17 there is, to our knowledge, no specific study linking the homeostasis of the MPTP to a beneficial therapeutic effect of CsA and its analogs in the treatment of chronic hepatitis C. However, it has been reported that a suboptimal dose of alisporivir given for 4 weeks as monotherapy decreased ALT levels in previous nonresponder patients in the absence of a significant decrease in viral load.38 Although speculative, this may indicate a cytoprotective effect of alisporivir that is independent of its antiviral activity.

Here, we show that alisporivir preserved in cells expressing HCV proteins the mitochondrial membrane potential and respiratory activity. The simplest explanation for the protective effect of alisporivir may relate to its desensitizing action on the MPTP.

Interestingly and quite unexpectedly, alisporivir was also able to counteract HCV protein-mediated enhancement of ROS production and mtCa2+ overload. These observations suggest that inhibition of the MPTP per se has a protective effect against oxidative stress and deregulation of calcium homeostasis. Indeed, although it is well-established that pro-oxidant conditions increase MPTP opening, it is also known that activation of the MPTP may lead to enhanced mitochondrial ROS production. This is likely due to the efflux/depletion of low molecular weight antioxidants (such as glutathione)39 and/or reducing substrates.24 Consistently, alteration of the oxidative state was shown to affect the activity of both ER and mitochondrial calcium channel/transporters.26, 34

Taken together, our observations on HCV protein-mediated mitochondrial dysfunction invoke a positive feedback pathogenetic loop. As illustrated in Fig. 8, this initiates with an increased flux of Ca2+ from the ER into mitochondria, proceeds by enhanced ROS production, thereby inducing MPTP opening. Activation of the MPTP in turn promotes further alteration of the redox state, which affects ER-mitochondria Ca2+ homeostasis and so on. Such a progressive self-nourishing mechanism of HCV-mediated mitochondrial dysfunction implies that the observed alterations cannot only be prevented but also rescued at least to some extent after they have been established. Indeed, we show in this study that alisporivir was able not only to prevent but also to revert mitochondrial dysfunction induced by HCV protein expression.

Figure 8.

Proposed pathogenetic mechanism of HCV infection. The metabolic changes elicited by HCV protein-mediated mitochondrial dysfunction are drawn schematically. Impairment of oxidative phosphorylation (OXPHOS) results in inhibition of fatty acid β-oxidation and in an adaptive up-regulation of glycolysis via stabilization of hypoxia-inducible factor (HIF-1). The combination of these metabolic shifts along with other factors (described in the Discussion) will cause enhanced lipogenesis and accumulation of triglycerides (TG) in the form of lipid droplets, which constitute a functional platform for virion assembly. The potential involvement of the HCV proteins core, NS2, NS5A, and NS5B, is also shown, as is the proposed site of action of alisporivir with CypD and CypA. See Discussion for further details. FA, fatty acid; RC, respiratory chain.

However, in spite of the HCV protein-mediated dysregulation of mitochondrial function, no overt evidence of increased apoptotic cell death was observed. The lower antigenicity of cytochrome c as well as of AIF, observed only after longer induction of HCV protein expression, was not diagnostic of a proapoptotic setting. Indeed, both intermembrane proteins largely retained their mitochondrial localization. The faded detection of cytochrome c, observed by immunofluorescence, was likely due to generalized protein modifications related to the unbalanced nitro-oxidative state.40 Our conclusion is supported by the observation that the outer mitochondrial membrane VDAC also displayed a similar immunogenic behavior in HCV protein-expressing cells and that immunoblotting of cytochrome c in subcellular fractions did not change upon HCV induction.

The involvement of the MPTP in the HCV-mediated alterations of the mitochondrial physiology but in the absence of a proapoptotic setting is not counterintuitive in keeping the notion that the MPTP oscillates between the closed and open configurations (flickering) and that severe alteration of outer mitochondrial membrane permeability occurs only when the MPTP is kept open permanently by activating effectors or conditions.

The possible impact of mitochondrial dysfunction on cell metabolism and virus-host interactions is further illustrated in Fig. 8. Loss of the mtΔΨ and reduction of respiratory chain efficiency impairs the driving force for aerobic ATP synthesis by the oxidative phosphorylation system. The infected cell adapts by shifting its metabolism toward glycolysis.41

This occurs by up-regulation of the prosurvival hypoxia-inducible factor under normoxic conditions, as recently shown by us and others.23, 42 Moreover, HCV infection leads to reprogramming of lipid metabolism, consisting of decreased β-oxidation of fatty acids (requiring functional mitochondria) and enhanced lipogenesis.41, 43 Enhanced cellular lipid storage in the form of lipid droplets provides a functional and structural platform required for HCV assembly.44 Therefore, mounting evidence supports a scenario in which earliest alterations of mitochondrial homeostasis caused by HCV proteins prime the host cell toward adaptive responses beneficial to the viral life cycle. In this context, the therapeutic efficacy of Cyp inhibitors such as alisporivir can be conceivably rationalized in terms of its capability to block at pharmacological concentrations both CypD and CypA which, according to our model (Fig. 8), are involved upstream and downstream, respectively, in the HCV-mediated pathogenetic mechanism.

In conclusion, our results provide new insights into the pathogenesis of HCV-related liver disease, highlighting the role of the MPTP in amplifying initial insults caused by HCV proteins on the mitochondrial calcium and redox homeostasis. Moreover, it is shown that the use of an inhibitor of the MPTP, alisporivir, prevents and substantially reverts, at least in vitro, HCV protein-mediated mitochondrial dysfunction. This unveils a thus far neglected additional pharmacological effect of alisporivir that may contribute to its therapeutic potential in chronic hepatitis C.

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