The antiarrhythmic drug amiodarone has microbicidal activity against fungi, bacteria and protozoa. In Saccharomyces cerevisiae, amiodarone triggers an immediate burst of cytosolic Ca2+, followed by cell death markers. Ca2+ transients are a common response to many forms of environmental insults and toxic compounds, including osmotic and pH shock, endoplasmic reticulum stress, and high levels of mating pheromone. Downstream signaling events involving calmodulin, calcineurin and the transcription factor Crz1 are critical in mediating cell survival in response to stress. In this study we asked whether amiodarone induced Ca2+ influx was beneficial, toxic or a bystander effect unrelated to the fungicidal effect of the drug. We show that downregulation of Ca2+ channel activity in stationary phase cells correlates with increased resistance to amiodarone. In actively growing cells, extracellular Ca2+ modulated the size and shape of the Ca2+ transient and directly influenced amiodarone toxicity. Paradoxically, protection was achieved both by removal of external Ca2+ or by adding high levels of CaCl2 (10 mM) to block the drug induced Ca2+ burst. Our results support a model in which the fungicidal activity of amiodarone is mediated by Ca2+ stress, and highlight the pathway of Ca2+ mediated cell death as a promising target for antifungal drug development.
Amiodarone [2-butyl-3-benzofuranyl-4-[2-(diethylamio)-ethoxy]-3,5-diiodophenyl-ketone hydrochloride; Fig. 1ainset] is listed as a Class III antiarrhythmic drug that prevents atrial fibrillation and ventricular tachycardia (Doggrell, 2001; Varbiro et al., 2003; Stevenson & Tedrow, 2007). It acts by blocking K+ channels and prolonging the action potential and duration of refractory periods, leading to reduced membrane excitability in all myocardial tissues. However, the therapeutic basis of action is complex, and amiodarone has also been shown to block calcium and sodium channels, β- and α-adrenergic receptors and ion exchangers (Hageluken et al., 1995; Kodama et al., 1997). Although it has few adverse cardiovascular effects, high doses of amiodarone can damage noncardiac tissues, most prominently the lungs and thyroid glands (Auer et al., 2002). Nevertheless, amiodarone remains an effective and reasonably safe agent for the restoration of sinus rhythm and has been widely prescribed for the last 30 years.
Recently, amiodarone was shown to exhibit potent fungicidal activity against Saccharomyces cerevisiae and a range of pathogenic fungi, including Candida albicans, Cryptococcus neoformans, Fusarium oxysporum and Aspergillus nidulans (Courchesne, 2002). For example, 5 μM amiodarone completely blocked proliferation of C. neoformans. This was shown to be due to a fungicidal, rather than fungistatic effect. It appeared that Ca2+ played a role in the cytotoxic effect of amiodarone because addition of 10 mM CaCl2 protected against growth inhibition of S. cerevisiae, whereas Ca2+ chelators [0.5–2 mM 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA)] exacerbated amiodarone toxicity (Gupta et al., 2003). Amiodarone was shown to elicit an immediate influx of Ca2+ that was monitored by a rise in the luminescence of aequorin-coelenterazine photoprotein (Courchesne & Ozturk, 2003; Gupta et al., 2003), increased uptake of 45Ca2+ (Yadav et al., 2007), and nuclear translocation of the Ca2+-regulated transcription factor Crz1-GFP (Zhang & Rao, 2007). Mutants lacking key regulators of calcium homeostasis, including the secretory pathway Ca2+ pump Pmr1, the vacuolar H+-ATPase, and Ca2+/calmodulin-activated protein phosphatase calcineurin, were shown to be hypersensitive to amiodarone, corroborating an important role for Ca2+ in the cellular mechanism of amiodarone toxicity (Gupta et al., 2003; Yadav et al., 2007; Zhang & Rao, 2007).
In S. cerevisiae, cytosolic Ca2+ entry is considered to be critical for survival under a variety of cell stresses including prolonged exposure to pheromone (Iida et al., 1990), hypertonic shock (Matsumoto et al., 2002), unfolded proteins (Bonilla et al., 2002), and alkaline pH (Viladevall et al., 2004). Such elevations are thought to trigger protective responses that are crucial to cell survival. For example, brief exposure to hypertonic stress was shown to trigger a Ca2+ transient that preconditioned cells to growth in high salt conditions (Matsumoto et al., 2002). Paradoxically, calcium is a double-edged sword, such that prolonged or uncontrolled elevation of cytosolic Ca2+ can lead to cell death. High levels of amiodarone (80 μM) were shown to elicit mitochondrial fragmentation and subsequent cell death, similar to the effect of pheromone and the Ca2+ ionophore A23187 (Pozniakovsky et al., 2005) and thereby implicating Ca2+ toxicity as fungicidal. However, a causative role for Ca2+ influx in amiodarone mediated cell death has not been established.
The goal of this study was to determine whether Ca2+ influx is protective, toxic or merely a bystander effect in the fungicidal activity of amiodarone. We manipulated the metabolic status of cells and composition of culture medium to modulate Ca2+ channel activity and correlated Ca2+ influx to cell viability. Our findings demonstrate that Ca2+ stress mediates yeast cell death in response to amiodarone and highlight the potential of this pathway as a target for antifungal development.
Materials and methods
Media, strains and reagents
Yeast strains were grown at 30 °C in standard synthetic complete (SC) medium (Bio101 Inc., Vista, CA) or YPD. Where specified, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) was added from a stock solution of 200 mM in water, buffered to pH 8.8 with KOH. Stock solutions of amiodarone (Sigma, St Louis, MO) were 5 mM in dimethyl sulfoxide. Cycloheximide (Sigma) was added from a 10 mg mL−1 stock in dimethyl sulfoxide. Saccharomyces cerevisiae wild-type strain BY4742 (MATa, his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) was purchased from Invitrogen (Carlsbad, CA). Plasmid pEVP11-Aeq89 has been described (Matsumoto et al., 2002).
The procedure was modified from Gupta (2003) as follows: 1 OD600 nm of overnight cultures carrying pEVP11-Aeq-89 were spun down and resuspended in 50 μL SC media containing 0.75 μg of coelenterazine (Invitrogen, Carlsbad, CA) and incubated in the dark at 30 °C for 1 h. Following incubation, the cells were washed twice with 2% glucose and resuspended to 0.5 OD600 nm mL−1 in SC media. Luminescence was quantified on a white opaque 96-well plate using a BMG FLUOStar OPTIMA™ plate reader (BMG Labtechnologies Durham, NC). Total luminescence (Lmax) was determined following lysis with 4% Triton X-100 (Fisher Scientific) in 2 M CaCl2. Free Ca2+ concentrations (in μM) were calculated as described in Gupta (2003). All assays performed on a microplate had 150 μL of culture well−1.
2-Chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide (FUN-1) assay
WT cells (1 OD600 nm) were preloaded with 4 μM FUN-1 for 1 h at 30 °C in SC medium (Millard et al., 1997). Following incubation, cells were washed twice with 2% glucose and resuspended to 0.5 OD600 nm mL−1 in SC media. Green fluorescence was quantified on a black, clear-bottomed 96-well plate using a BMG FLUOstar Optima microtiter plate reader (Excitation, 485 nm; Emission, 520 nm). Data were normalized for cell density, measured as OD600 nm units, and are averages of triplicates; figures are representative of at least two experiments. Data were organized, plotted and analyzed using bmg fluostar optima version 1.20 software (BMG Labtechnologies Durham, NC) and microsoft excel (Microsoft Corp., Redmond, WA) software.
Dose dependence of Ca2+ burst and metabolic inactivation elicited by amiodarone
Amiodarone is a cationic amphiphilic drug (Fig. 1a inset) that is known to interact strongly with the lipid bilayer and alter membrane fluidity in model and native membranes (Antunes-Madeira et al., 1995, 2002; Rosa et al., 2000). Addition of amiodarone to yeast cells triggered the opening of plasma membrane Ca2+ channels that can be monitored by a rise in luminescence from the Ca2+ activated photoprotein aequorin-coelenterazine (Courchesne & Ozturk, 2003; Gupta et al., 2003). Here, we show that both the kinetics and amplitude of the Ca2+ signal depend on drug concentration (Fig. 1a): higher doses of amiodarone elicited both sharper and higher peaks, consistent with a concerted recruitment of more Ca2+ channels. Estimated peak values for free Ca2+, calibrated from total luminescence, increased from 2.2 μM in response to 20 μM amiodarone to 6.2 μM (40 μM amiodarone) and 12.7 μM (80 μM amiodarone). In comparison, hypertonic shock (1 M NaCl) transiently elevated cytosolic Ca2+ to c. 3 μM (Matsumoto et al., 2002) and secreted toxins from the pathogenic fungus Trichoderma elicited peak Ca2+ levels of >7 μM in soybean cells (Navazio et al., 2007). Concomitantly, amiodarone triggered a dose-dependent loss of metabolic activity, monitored by a rise in green fluorescence of the vital stain FUN-1 (Millard et al., 1997) as seen in Fig. 1b. These results show that amiodarone toxicity correlates with the kinetics and amplitude of the Ca2+ burst.
Loss of Ca2+ channel activity correlates with resistance to amiodarone in stationary phase cells
We found that the fungicidal activity of amiodarone was dependent on the metabolic status of the cells. Following overnight growth of the culture to stationary phase, yeast cells were largely resistant to amiodarone toxicity, even after transfer to fresh SC medium for 0.5 h (Fig. 2a). However, amiodarone sensitivity was regained following continued growth in fresh medium to exponential phase (Fig. 2a; 5 h). Investigation of amiodarone induced aequorin-coelenterazine luminescence revealed that stationary phase cultures that were resistant to drug toxicity also showed low levels of drug induced Ca2+ influx (Fig. 2b). In contrast, amiodarone was able to elicit large dose-dependent Ca2+ bursts in the exponential phase, drug sensitive culture (Fig. 2c). This suggested that the expression and/or activity of Ca2+ influx channels were dependent on growth phase, being down-regulated in stationary phase cells. Upon transfer of stationary phase cells to fresh medium, we observed a time-dependent increase in the size of the Ca2+ burst (Fig. 2d; 40 μM amiodarone), consistent with increasing numbers of active channels. Addition of cycloheximide completely blocked the time-dependent reappearance of Ca2+ burst, indicating a requirement for new protein synthesis (Fig. 2e). Glucose, possibly as the energy source, was necessary for the recovery of Ca2+ influx; cultures transferred to SC medium lacking glucose failed to show full activation of Ca2+ channels. However, glucose alone, in the absence of media, was insufficient for this effect (Fig. 2f). Taken together, the data show that Ca2+ channel activity is dependent on metabolic state and suggest that stationary phase cells may be more tolerant to amiodarone toxicity because of the downregulation of Ca2+ channels.
Glucose removal from growing cells reduces both calcium burst and amiodarone toxicity
It was possible that amiodarone resistance in stationary phase yeast may have arisen due to altered metabolic response to the drug rather than a consequence of altered Ca2+ influx. Therefore, we assessed the effect of glucose removal from exponentially growing yeast. Upon removal of glucose from the medium, there was an immediate, although modest, decrease in Ca2+ influx (Fig. 3a). Remarkably, this was accompanied by a reproducible increase in cell viability in the same culture (Fig. 3b). After treatment with amiodarone and normalization for cell density as described in Fig. 3b, cultures containing glucose were found to have significant reduction in CFU relative to cultures without glucose (43±14% from four independent trials). These results suggest that even small changes in Ca2+ influx could influence amiodarone toxicity, irrespective of the metabolic status of the cells.
Extracellular Ca2+ modulates amiodarone toxicity
Addition of the Ca2+ chelating agent EGTA resulted in a decrease in the amplitude of the amiodarone induced Ca2+ burst, as expected, consistent with a removal of free extracellular calcium by the chelator (Fig. 4a). At 6.8 mM added EGTA, Ca2+ influx was completely abolished. We assessed the viability of cells immediately after the Ca2+ burst (Fig. 4b): increasing amounts of chelator conferred increasing protection against amiodarone induced cell death, with maximal protection observed at 6.8 mM EGTA, similar to the untreated control. We also noted that increasing amounts of EGTA resulted in a progressive delay in the time to peak, suggesting that extracellular Ca2+ influenced channel opening (Fig. 4a). To evaluate this more carefully, we added different amounts of CaCl2 to EGTA buffered medium and assessed amiodarone induced Ca2+ bursts together with cell viability. As described in Fig. 4, addition of 6.8 mM EGTA to exponentially grown cells in SC medium completely abolished Ca2+ influx and conferred protection against cell death in response to 40 μM amiodarone (Fig. 5a and b). Introduction of 1 mM CaCl2 reinstated both the Ca2+ influx peak and amiodarone sensitivity to levels similar to control (no EGTA; Fig. 5a and b). Surprisingly, higher levels of added CaCl2 actually decreased Ca2+ influx; Fig. 5a shows that addition of 10 mM CaCl2 completely inhibited influx to levels achieved with EGTA alone. Consistent with inhibition of Ca2+ influx, 10 mM CaCl2 also protected yeast cells against amiodarone toxicity (Fig. 5b). Taken together, these results support a model in which the fungicidal activity of amiodarone is mediated by Ca2+ influx.
Ca2+ influx is a common cellular consequence of exposure to many forms of environmental stress and cytotoxic drugs. Hyper- or hypo-osmotic stress, cell wall damage, endoplasmic reticulum stress and prolonged exposure to mating pheromone all trigger the opening of calcium channels resulting in the elevation of cytosolic free Ca2+ from submicromolar baseline levels. At the plasma membrane, the Cch1/Mid1 complex has been implicated in Ca2+ entry following exposure to pheromones or tunicamycin (Bonilla et al., 2002). At the vacuolar membrane, the TRP-like channel Yvc1 mediates Ca2+ release in response to hypertonic shock (Denis & Cyert, 2002). Following cytoplasmic Ca2+ influx, homeostasis is accomplished by an array of ATP-dependent pumps (Pmr1, Pmc1, V-ATPase) and exchangers (Vcx1) that work to sequester Ca2+ into stores or transport it out of the cell (reviewed by Ton & Rao, 2004). In S. cerevisiae, the absence of key transporters, particularly the Vma H+-pump that provides a driving force for secondary transport, and the secretory pathway Ca2+-ATPase Pmr1, results in hypersensitivity to numerous stresses and toxic drugs, including amiodarone, that are accompanied by a rise in intracellular Ca2+ (Yadav et al., 2007). The stress or drug induced influx of Ca2+ activates signaling pathways, most prominently calmodulin–calcineurin–Crz1, that mediates a cellular response which includes cell wall synthesis, cell cycle arrest and upregulation of ion homeostasis factors (Matsumoto et al., 2002; Yoshimoto et al., 2002; Miyakawa & Mizunuma, 2007; Zhang & Rao, 2007). Although not essential for viability under normal growth conditions, disruption of the calcineurin signaling pathway is lethal to both model and pathogenic fungi under stress: calcineurin mutants or cells treated with the calcineurin inhibitors cyclosporine A or FK506 rapidly lose viability in the face of cell stress, antifungal agents, and other toxic drugs (Kraus & Heitman, 2003; Steinbach et al., 2007; Zhang & Rao, 2007).
The purpose of this study was to understand whether amiodarone-induced Ca2+ influx was beneficial, deleterious or unrelated to cell viability. On the one hand, a Ca2+ transient can be viewed as a signaling event, occurring downstream from a stress sensor, and allowing the cell to respond to its environment. Thus, the delayed influx of Ca2+, reported to occur after an hour of tunicamycin treatment (Bonilla et al., 2002), has been interpreted as a protective mechanism that is necessary for viability. In this case, deletion of the plasma membrane Cch1/Mid1 channel complex resulted in decreased viability in tunicamycin treated cultures. Alternatively, the opening of Ca2+ channels may simply be a physical consequence of membrane perturbation (such as mechanical or electrical). Examples might include membrane shrinking or swelling in response to osmotic changes (Denis & Cyert, 2002), or partitioning of a drug such as amiodarone into the bilayer. In such cases, calcineurin mediated signaling events would represent the cellular response to counter Ca2+ stress. If the Ca2+ stress is small, the protective response effectively preconditions cells to further insults, as was found to occur following brief exposure to high salt (Matsumoto et al., 2002). However, the Ca2+ stress can overwhelm the protective response and lead to cell death.
In this study, we show that high levels of Ca2+ entry elicited by the amphiphilic drug amiodarone correlate with cell death. Furthermore, varying the size and temporal kinetics of Ca2+ transients by altering metabolic status or extracellular Ca2+ levels, resulted in concomitant changes in amiodarone toxicity. First, we confirmed that the dose dependence of amiodarone induced Ca2+ transients correlated with loss of viability, measured by the vital stain FUN-1 or by drop tests. Second, we found that drug sensitivity was dependent on the metabolic status of the cells. Stationary phase cells were more resistant to amiodarone, up to half hour following dilution into fresh medium, and amiodarone failed to induce significant Ca2+ transients in these cultures. Over time, amiodarone sensitivity increased with concomitant increases in the amiodarone induced Ca2+ burst, strongly suggesting that cell death was downstream of Ca2+ influx. This increase in Ca2+ influx required new protein synthesis, as it was inhibited by cycloheximide. Presumably, one or more Ca2+ channel proteins are downregulated in stationary phase cells, although we cannot exclude that membrane lipid composition or other components upstream of calcium channel opening may be altered as well. The Cch1/Mid1 channels appear not to be involved in amiodarone mediated toxicity, since both single and double gene deletions showed no loss of amiodarone-induced Ca2+ transients (data not shown; see also, Gupta et al., 2003). This is consistent with the findings of Loukin (2007) that there are functionally redundant Ca2+ entry pathways in S. cerevisiae that remain to be identified. The immediate decrease in Ca2+ influx upon glucose removal from actively growing cells may be related to the reported drop in plasma membrane potential in the absence of glucose. It has been established that H+ pump activity of Pma1 is immediately downregulated in low glucose medium (Serrano, 1983; Lecchi et al., 2007). These findings raise the interesting possibility that opening of calcium channels may be triggered by changes in plasma membrane potential and warrant further investigation.
Third, we show that addition of the Ca2+ chelator EGTA to the culture medium minutes before drug addition decreased the Ca2+ burst and concomitantly increased cell viability, assessed immediately after the Ca2+ burst (Fig. 4). This contrasts with the exacerbating effect of EGTA (2 mM) on sensitivity to amiodarone, which was monitored over 24 h of growth as reported earlier (Gupta et al., 2003). Calcium starvation over this period of time is harmful to cells, and likely to induce a compensatory opening of calcium channels so that the drug actually has a slightly more harmful effect under these conditions. The ability of high extracellular Ca2+ to block drug-mediated Ca2+ entry (Fig. 5) explains the paradoxical protective effect of high CaCl2 on growth toxicity of amiodarone, reported earlier (Courchesne, 2002; Gupta et al., 2003). The protective effect of high Ca2+ may be mediated by unknown membrane effects that mitigate drug toxicity or integration, by rapid desensitization via Ca2+ block of the channel pore, or by activation of calcineurin and other effectors. Taken together, our findings clearly show that the calcium burst is closely coupled to the fungicidal effect of amiodarone, although the drug may affect more than one cellular pathway. Our findings point to the importance of Ca2+ stress in mediating the cytotoxic effects of multiple environmental challenges and drugs and highlight the potential of key regulators of Ca2+ homeostasis and signaling as drug targets.
This work was supported by a Public Health Service grant from the National Institutes of Allergy and Infectious Disease (R01AI065983).