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European Journal of Heart Failure 2008 10(1):39-46; doi:10.1016/j.ejheart.2007.11.002
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© 2008 European Society of Cardiology

The volume-sensitive chloride channel inhibitors prevent both contractile dysfunction and apoptosis induced by doxorubicin through PI3kinase, Akt and Erk 1/2

Alexandra d'Anglemont de Tassignya,b,c, Alain Berdeauxa,b,c,d, Rachid Souktanib, Patrick Henrye and Bijan Ghaleha,b,c,d,*

a INSERM U841 Equipe 3, Créteil, F-94010, France
b Université Paris 12, Faculté de Médecine, Institut Mondor de Médecine Moléculaire (IFR 10) Créteil, F-94010, France
c Ecole Nationale Vétérinaire d'Alfort Maisons-Alfort, F-94700, France
d Fédération de Cardiologie, Groupe Hospitalier Albert Chenevier - Henri Mondor Créteil, F-94010, France
e Service de Cardiologie, Hôpital Lariboisièe and INSERM U572 75010 Paris, France

* Corresponding author. Laboratoire de Pharmacologie, INSERM U841 Eq 3, Faculté de Médecine de Créteil, 8, rue Général Sarrail, 94010 CRETEIL Cedex, France. Tel.: +33 1 49 81 35 93; fax: +33 1 49 98 36 61. E-mail address: bijan.ghaleh{at}creteil.inserm.fr (B. Ghaleh).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Contractile dysfunction and cardiomyopathies secondary to apoptotic cell death are limiting factors for treating cancer with doxorubicin. Inhibition of volume-sensitive chloride currents (ICl,vol) has been reported to blunt doxorubicin-induced apoptosis in cardiomyocytes. To investigate cellular contractility during acute induction of apoptosis by doxorubicin and to determine whether ICl,vol inhibitors are able to prevent the subsequent contractile dysfunction, electrically paced ventricular myocytes freshly isolated from adult rabbits were acutely exposed to doxorubicin in the presence and absence of ICl,vol inhibitors IAA-94 or DIDS. Doxorubicin induced increases in both annexin V labelling and caspase-3 activity and decreases in cell volume. Alteration in cardiac contractility was observed after doxorubicin exposure. Both IAA-94 and DIDS abolished the doxorubicin-induced decreases in peak shortening and cell volume as well as the increases in caspase-3 activity and annexin V labelling. These protective effects of ICl,vol inhibitors were abolished by previous inhibition of PI3kinase, Akt and Erk 1/2. Thus, ICl,vol inhibitors prevent doxorubicin-induced apoptosis and subsequent contractile dysfunction through PI3kinase/Akt and Erk 1/2. Inhibition of ICl,vol may represent a new pharmacological strategy for developing cytoprotective drugs against apoptotic cell death and contractile dysfunction.

Key Words: Volume-sensitive chloride channels • Cardiomyocyte • Contractility • Apoptosis • Doxorubicin

Received February 19, 2007; Revised October 17, 2007; Accepted November 12, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Doxorubicin, a quinone-containing anthracycline, is widely used as a chemotherapeutic agent for the treatment of a large spectrum of human neoplasic diseases but its administration in humans is limited by the risk of severe cardiotoxicity [1]. This results from an impaired contractile function and subsequent development of cardiomyopathies secondary to apoptotic cell death [2-4] with ceramide accumulation, mitochondrial dysfunction, cytochrome c release [2,5-7] and generation of reactive oxygen species [8].

Our group has previously demonstrated that doxorubicin activates a current electrophysiologically characterized as a volume-sensitive chloride channel (ICl,vol) [9]. This current is responsible for a rapid and intense cell shrinkage which is known to play a key role during apoptosis [9-13]. Interestingly, ICl,vol blockade inhibits doxorubicin-induced apoptosis [9] but to date, the mechanisms involved in this cardioprotective effect remain to be investigated. In addition, it is unknown whether blockade of apoptosis by ICl,vol inhibitors would also necessarily result in the preservation of contractile function under doxorubicin.

The PI3kinase/Akt signalling pathway is probably the best-characterized and the most prominent pathway with regard to the transmission of anti-apoptotic signals in cell survival. The mitogen-activated protein kinases (MAPKs), which include Erk 1/2 are known to play fundamental roles in survival, proliferation and apoptosis. Importantly, the pharmacological or molecular modulation of MAPK signalling has been determined to influence apoptotic responses of anti-cancer drugs. The involvement of these molecules in most signalling pathways of cardioprotective strategies constitutes a potential mechanism for the protective effect of ICl,vol inhibitors.

In this context, the aims of the present study were (i) to investigate the contractility of isolated cardiomyocytes from adult rabbits during acute induction of apoptosis by doxorubicin, (ii) to determine whether IAA-94 and DIDS, two ICl,vol inhibitors, are able to prevent the subsequent contractile dysfunction induced by doxorubicin, and (iii) to investigate the signalling pathways involved in this cardioprotection.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by NIH (NIH publication No. 85-23, revised 1996).

2.1. Isolation of rabbit ventricular myocytes
Single ventricular cells were obtained from male New Zealand rabbit hearts as previously described [14]. Animals (2-2.5 kg) were anesthetized with a solution of pentobarbital (50 mg/kg) and received heparin (200 UI/kg). The hearts were excised and immediately perfused on a nonrecirculating home-made perfusion system with oxygenated (95% O2-5% CO2) calcium-free and isotonic tyrode solution (in mM: NaCl 135, KCl 5.4, Na2PO4 0.33, MgCl2 1.0, HEPES 10, with pH adjusted to 7.4 with NaOH 1 N at 37 °C, 280-300 mOsmol/kg H2O). Then, all hearts were perfused in recirculating mode with the same calcium-free tyrode solution (coronary flow rate, 10-15 mL/min) supplemented with 1 mg/mL of collagenase type II (Sigma, Saint Quentin Fallavier, France) and 0.28 mg/mL of protease type XIV (Sigma, Saint Quentin Fallavier, France). Finally, all hearts were perfused in nonrecirculating mode with the same tyrode solution supplemented with 0.3 mM CaCl2 for 10 min. The left ventricle was removed and further dissected into small pieces, the cellular dissociation being achieved by gentle mechanical agitation. Extracellular calcium was added incrementally up to 1.0 mM. Isolated myocytes were maintained in a serum free medium for up to 90 min prior to experimentation.

All cells studied were rod-shaped, had clear cross-striations and lacked any visible vesicles on their surfaces under observations with an optical microscope. The cells used for measurement of contractile function and determination of cell surface must remain rod-shaped during the whole protocol. Every cell which became rounded during the 6 h of experiment was rejected. We estimate that 10% of cardiomyocytes became rounded during these experiments.

2.2. Annexin V labelling
Annexin V labelling of phosphatidylserine was performed using the MiniMacs cell isolation kit (Miltenyi Biotec, Bergisch, Gladbach, Germany). Phosphatidylserine-exposing cells were magnetically labelled with annexin V microbeads and passed through a column placed in a magnetic field. The magnetically labelled phosphatidylserine-exposing cells were retained on the column while the unlabelled ones (necrotic and nonapoptotic cells) were run through. After removal of the column from the magnetic field, the magnetically retained phosphatidylserine-exposing cells were eluted as a positively selected cell fraction and counted with a Malassez cell, the percentage of apoptotic cells was reported in relation to the initial number of cells.

2.3. Caspase-3 activity measurement
Cells were lysed and the supernatant was used for measurement of caspase-3 activity using the AK-005 kit (Biomol Research Laboratories, Plymouth Meeting, PA, USA). The fluorogenic substrate for caspase-3 (DEVD) was labelled with the fluorochrome 7-amino-4-methyl coumarin (AMC) which produces a yellow-green fluorescence detectable by exposure to UV light at 360/460 nm for 210 min. AMC was released from the substrates upon cleavage by caspase-3, the enzyme activity being expressed in fmol/min.

2.4. Measurement of myocyte contractility
Myocytes were transferred to a warmed (37 °C) and continuously perfused cell chamber located on the stage of an inverted microscope (Nikon). The chamber was perfused with physiological buffer containing (in mM): NaCl 140; KCl 5.4; CaCl2 1; MgCl2 0.8; HEPES 10 and glucose 5.6 (pH=7.4; 290 mOsmol/kg H2O).

Myocyte contraction was induced once per second (1 Hz) by platinum field electrodes placed in the cell chamber that were attached to a stimulator (Grass S88K, Grass Instruments, Trappes, France). Cell images were continuously acquired through a x20 objective lens and transmitted to a 240 sample/s charge coupled device (CCD) video camera (Myocam, Ionoptix, Milton, MA, USA). The output from the CCD camera was displayed on a video monitor.

Myocytes were selected for study according to the following criteria [15]: a rod-shaped appearance with clear striations and no membrane blebs, no spontaneous contractions when stimulated in 1 mM Ca2+, steady diastolic length and contractile amplitude at basal stimulation rates. Sarcomere length was measured using a video motion edge detector (DSI 200, Ionoptix, Milton, MA, USA) and data were acquired at 240 samples/s. The camera images were converted to sarcomere length measurements by the video sarcomere detector and were analyzed by the data-acquisition system. Percent shortening and rate of shortening were then calculated.

2.5. Measurement of cell volume changes
After their dissociation, ventricular cardiomyocytes were placed on coverslips, with a density of {approx}1.8x105 cells/coverslip, each placed in 35-mm Petri dishes containing Dulbecco's modified Eagle's medium supplemented with 0.01 mM L-glutamine, 0.25 mg/L ampicillin and 1 mg/L kanamycin. Cardiomyocytes were then incubated at 37 °C in a humidified 5% CO2-95% air mixed for 2 h. Each coverslip containing attached cardiomyocytes was placed on an inverted microscope (Leica, France). Only viable, rod-shaped cardiomyocytes were used as their own control. A video camera was used to take digital pictures of myocytes (optical: x200, numeric: x2) every 5 min throughout the entire experimental protocol for subsequent analysis. Each picture was then analyzed to calculate myocyte surface (Image J, USA) used as an index of changes in cell volume. This method has been previously found to be very well correlated to cell volume changes [16].

2.6. Experimental protocol
Isolated cardiomyocytes were continuously exposed to 1 µM doxorubicin [17,18]. Annexin V labelling and measurements of caspase-3 activity were performed after 3 h and 8 h of exposure to doxorubicin, respectively. Contractility and cell volume variations of cardiomyocytes exposed to doxorubicin were recorded every hour for 6 h. After treatment, cells were compared to control cardiomyocytes which were not exposed to doxorubicin.

To determine the effects of ICl,vol channels on doxorubicin-induced apoptosis and contractile dysfunction, cardiomyocytes exposed to doxorubicin were treated concomitantly with the ICl,vol inhibitors indanyloxyacetic acid 94 (IAA-94, 10 µM) [19] or 4-4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS, 100 µM) [20]. In additional experiments to determine whether it is possible to reverse the doxorubicin-induced dysfunction, IAA-94 (10 µM) was added 4 h after the onset of exposure to doxorubicin.

To examine the cellular mechanisms involved in the protective effects of ICl,vol inhibitors, the cardiomyocytes were concomitantly treated with doxorubicin, ICl,vol inhibitors and selective inhibitors of PI3kinase (wortmannin, 100 nM and LY294002, 20 µM), Akt (Akt inhibitor IV, 50 µM) and Erk 1/2 (PD098059, 10 µM).

2.7. Data analysis
Data are reported as mean±SEM. The results for annexin V labelling and caspase-3 activity, were analyzed using a one-way ANOVA procedure. When a significant difference was found, specific differences between groups were identified using a PLSD Fisher test. In order to reduce the number of comparisons for higher statistical power, only individual comparisons vs. control were performed. Results for peak shortening and apoptotic volume decrease were analyzed using two-way ANOVA for repeated measures with treatment effect, time effect and interaction time*treatment. As all the interactions tested in this study were significant, all these ANOVAs were followed by PLSD Fisher in order to detect individual differences. Significant differences were determined as p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
As shown in Table 1, the average sarcomere length of cells used in this study was not significantly different among groups.


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  		Table 1 Sarcomere length

 
3.1. Effect of doxorubicin on myocyte contractility and apoptosis
Doxorubicin induced a time-dependent decrease of sarcomere shortening as compared to control (e.g. at 4 h and 6 h, –53±8% vs. –19±2% and –66±7% vs. –21±3% for doxorubicin, n=5 and control, n=7, p<0.05, respectively). Similar results were obtained with maximal velocity of shortening as shown in Table 2. The averaged initial value for Vmax in control conditions was 3.14±0.38 µm/s (n=5). This effect started to be significant after 4 h of exposure to doxorubicin (–53±8%, n=5, vs. –19±2%, n=7, from baseline for doxorubicin and control, respectively, p<0.05). The averaged initial value for peak cell shortening was 15.7±0.4% (n=7) in control conditions. Concomitantly, doxorubicin induced significant increases in annexin V labelling (263±8% after 3 h, n=5) and caspase-3 activity (80±6 fmol/min vs. 11±11 fmol/min for doxorubicin vs. control after 8 h, respectively, n=5). The averaged value for the effect of doxorubicin on annexin V labelling in percentage of total number cells was 84.9±3.9% (n=5).


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  		Table 2 Time-dependent evolution of maximal velocity of sarcomere shortening

 
3.2. Effect of ICl,vol inhibitors on doxorubicin-induced contractile dysfunction and apoptosis
As illustrated in Fig. 1a, the ICl,vol inhibitors IAA-94 and DIDS abolished the doxorubicin-induced reduction in peak shortening of cardiomyocytes. Similar results were observed with maximal velocity of shortening (Table 2). In addition, the increases in annexin V labelling and caspase-3 activity induced by doxorubicin were blocked by IAA-94 (Fig. 1b and c, respectively). Similarly, the decrease in cell surface induced by doxorubicin was blocked by IAA-94 (e.g. at 4 h and 6 h, –39±6% vs. 0±1% and –42±5% vs. –3±1% for doxorubicin, n=5 and doxorubicin with IAA-94, n=5, p<0.05, respectively). The protective effect of IAA-94 was however lost when this ICl,vol inhibitor was added 4 h after doxorubicin exposure. Indeed, cell surface and contractile function of cardiomyocytes treated with doxorubicin were not maintained to a control level when IAA-94 was added 4 h after doxorubicin exposure (–36±1% in cell surface and –65±2% in peak shortening at 6 h).


Figure 01
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  		Fig. 1 The ICl,vol inhibitors IAA-94 and DIDS abolished both contractile dysfunction and apoptosis induced by doxorubicin (Doxo). (a) IAA-94 and DIDS time-dependently inhibited the doxorubicin-induced contractile dysfunction in ventricular cardiomyocytes. (b) Percent change in annexin V labelling was assessed 3 h after exposure to doxorubicin in the presence and in the absence of ICl,vol inhibitor. (c) Caspase-3 activity was measured 8 h after exposure to doxorubicin with and without ICl,vol inhibitor. Mean±SEM, n=5-7 cells per group, *: p<0.05 vs. control.

 
3.3. Role of PI3kinase/Akt in the protective effect of ICl,vol inhibitors
As shown in Fig. 2a and b, LY294002 and wortmannin abolished the protective effect of IAA-94 on doxorubicin-induced cell volume decrease and contractile dysfunction. Similar results were observed with maximal velocity of shortening (Table 2). Concomitantly, the protective effects of IAA-94 on doxorubicin-induced increase in annexin V labelling and caspase-3 activity were abolished by wortmannin and LY294002 (Fig. 3a and b). Finally, LY294002 and wortmannin per se did not induce any effect on doxorubicin-induced caspase-3 activity and annexin V labelling (Fig. 3a and b).


Figure 02
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  		Fig. 2 Role of PI3kinase in the protective effect of ICl,vol inhibitors. (a) PI3kinase inhibitor wortmannin (100 nM) abolished the protective effect of IAA-94 on doxorubicin-induced surface decrease in ventricular cardiomyocytes. (b) PI3kinase inhibitors wortmannin (100 nM) and LY294002 (20 µM) abolished the protective effect of IAA-94 on doxorubicin-induced contractile dysfunction in ventricular cardiomyocytes. Mean±SEM, n=5 cells per group, *: p<0.05 vs. control. Doxo: doxorubicin.

 


Figure 03
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  		Fig. 3 PI3kinase inhibitors wortmannin (100 nM) and LY294002 (20 µM) abolished the protective effect of IAA-94 on doxorubicin-induced apoptosis in ventricular cardiomyocytes. (a) Percent change in annexin V labelling was assessed 3 h after exposure to doxorubicin. (b) Caspase-3 activity was measured 8 h after exposure to doxorubicin. Mean±SEM, n=5 cells per group, *: p<0.05 vs. control.

 
Akt inhibitor IV abolished the effect of IAA-94 on doxorubicin-induced contractile dysfunction (Fig. 4a and Table 2), cell volume decrease (Fig. 4b) and caspase-3 activity (92±9 and 114±9 vs. 21±8 and 27±10 fmol/min for doxorubicin and doxorubicin with both IAA-94 and Akt inhibitor IV vs. control and doxorubicin with IAA-94, respectively, p<0.05). Akt inhibitor IV per se did not induce any effect on doxorubicin-induced caspase-3 activity (16±5 fmol/min).


Figure 04
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  		Fig. 4 Role of Akt and Erk 1/2 in the protective effect of ICl,vol inhibitors. (a) Akt inhibitor IV (50 µM) abolished the protective effect of IAA-94 on doxorubicin-induced contractile dysfunction in ventricular cardiomyocytes. (b) Akt inhibitor IV (50 µM) abolished the protective effect of IAA-94 on doxorubicin-induced surface decrease in ventricular cardiomyocytes. (c) PD098059 (10 µM) abolished the protective effect of IAA-94 on doxorubicin-induced contractile dysfunction in ventricular cardiomyocytes. (d) PD098059 (10 µM) abolished the protective effect of IAA-94 on doxorubicin-induced surface decrease in ventricular cardiomyocytes. Mean±SEM, n=5 cells per group, *: p<0.05 vs. control. Doxo: doxorubicin, Akti: Akt inhibitor IV.

 
3.4. Role of Erk 1/2 in the protective effect of ICl,vol inhibitors
PD098059 abolished the effect of IAA-94 on doxorubicin-induced contractile dysfunction (Fig. 4c), cell volume decrease (Fig. 4d) and caspase-3 activity (92±9 and 107±9 vs. 21±8 and 27±10 fmol/min for doxorubicin and doxorubicin with both IAA-94 and PD098059 vs. control and doxorubicin with IAA-94, respectively, p<0.05). Similar results were observed with maximal velocity of shortening (Table 2). PD098059 per se did not induce any effect on doxorubicin-induced contractile dysfunction (Fig. 4c and Table 2) and caspase-3 activity (20±7 fmol/min).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, acute exposure of adult isolated cardiomyocytes to doxorubicin induced a time-dependent contractile dysfunction. This phenomenon occurred along with the induction of apoptosis assessed by cell volume decrease, annexin V labelling and caspase-3 activation. We demonstrated for the first time that ICl,vol inhibition prevents doxorubicin-induced cardiomyocyte contractile dysfunction suggesting that apoptosis and more precisely apoptotic volume decrease could be a target to modulate contractile function. Indeed, this protective effect was concomitant with a protection against apoptosis as shown by the measurements of cell volume decrease, caspase-3 activity and annexin V labelling. The mechanism of this protection involves PI3kinase, Akt and Erk 1/2.

A major hallmark of apoptosis is normotonic cell shrinkage so-called apoptotic volume decrease [9,12]. This phenomenon is known to be coupled with chloride anion release through ICl,vol channels [9,12,13] and is a pre-requisite for the induction of apoptosis [9,12]. Indeed, this decrease in cell volume is allowed by concomitant effluxes of water and KCl [11,21,22] and precedes cytochrome c release, caspase-3 activation and DNA laddering. In this setting, Maeno et al. [12] reported that prevention of apoptotic volume decrease by IAA-94 inhibits subsequent apoptosis, demonstrating the crucial role of ICl,vol in the apoptotic process. Accordingly in the present study, DIDS and IAA-94, two ICl,vol inhibitors, abolished apoptotic volume decrease and subsequent apoptosis.

Our study demonstrates the existence of a close relationship between apoptosis, changes in cell volume and contractile dysfunction. It is likely that cell volume decrease via ICl,vol activation is a requisite for caspase-3 activation. According to previous studies [12,13], physiological concentrations of potassium and chloride ions inhibit the apoptotic machinery, including caspase-3 activation. A significant decrease in the overall intracellular ionic strength due to volume changes is required to activate various caspases and nucleases which are known to be involved in apoptosis [12,23]. In this context, we have shown that doxorubicin induces cell volume decrease via ICl,vol and caspase-3 activation suggesting the trigger role of apoptotic volume decrease in the caspase-3 activation. Indeed, the inhibition of cell volume decrease by IAA-94 did not elicit caspase-3 activation. Although it was not directly demonstrated in this study, we can suppose that caspase-3 activation may end at alterations in myofibrillar structure of the sarcomere, calcium pumps or channels. Indeed, caspase-3 activation is involved in the contractile dysfunction observed in various models of myocardial injury [24-26]. Furthermore, the loss of cell volume may also operate in this phenomenon by altering gene transcription, phosphorylation of various proteins, and/or allowing physical disruption of normal protein-protein interactions leading to contractile dysfunction. Accordingly, the present study suggests that ICl,vol inhibitors can reverse the doxorubicin-induced contractile dysfunction only if they are added before this intracellular ionic deficit responsible for caspase activation. If these ICl,vol inhibitors are added after this event, contractile dysfunction becomes irreversible. In this context, apoptotic volume decrease could be a target to modulate contractile function rather than to be a simple functional consequence of apoptosis.

PI3kinase is a key enzyme for a number of intracellular functions that induce cell proliferation, growth cell motility, contractility and cell survival [27]. The present results demonstrate that PI3kinase is involved in the protective mechanism of ICl,vol blockade as inhibition of PI3kinase with LY294002 and wortmannin abolished the protective effect of IAA-94 against doxorubicin-induced contractile dysfunction. This is consistent with the previously demonstrated link between PI3kinase activation and modulation of cell volume through ICl,vol channels in physiological conditions [28,29]. The present study also demonstrates a relationship between Akt and ICl,vol channels. Indeed, the Akt inhibitor IV was able to prevent the protective effect of IAA-94 on doxorubicin-induced contractile dysfunction. It should be stressed that the literature regarding the relationship between Akt and activation of ion channels is limited [30,31]. Nevertheless, our results are consistent with previous reports demonstrating the involvement of the PI3kinase/Akt signalling pathway in cardioprotective strategies.

Mitogen-activated protein kinases which include Erk 1/2 are also known to play a fundamental role in cell survival, proliferation and apoptosis [32]. In this setting, a link between Erk 1/2 and modulation of ICl,vol channels has been demonstrated in physiological conditions [33]. In the present study, PD098059, an Erk 1/2 inhibitor, abolished the protective effect of IAA-94 against doxorubicin-induced contractile dysfunction. Inhibition of PI3kinase/Akt or Erk 1/2 similarly inhibited the protective effects of ICl,vol inhibitors against doxorubicin-induced apoptosis and contractile dysfunction. This may suggest that: (i) the two signalling pathways share a common mechanism for inducing protection by ICl,vol inhibition and/or (ii) the two pathways are redundant suggesting a cross-talk [34].

In conclusion, this study demonstrates that acute exposure to doxorubicin in adult isolated cardiomyocytes is responsible for a contractile dysfunction that occurs along with the induction of apoptosis. ICl,vol inhibitors are able to prevent apoptosis and the subsequent contractile dysfunction through PI3kinase/Akt and Erk 1/2. Inhibition of ICl,vol may represent a new pharmacological strategy for developing cytoprotective drugs against apoptotic cell death and contractile dysfunction. However, further research is necessary to understand how cell volume decrease is able to activate PI3kinase/Akt and Erk 1/2


   Acknowledgements
 
Alexandra d'Anglemont de Tassigny was a recipient from the Ligue Contre le Cancer. The authors wish to thank Alain Bizé for his technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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