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European Journal of Heart Failure 2007 9(4):329-335; doi:10.1016/j.ejheart.2006.10.014
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© 2007 European Society of Cardiology

{alpha}1-adrenergic stress induces downregulation of Na+/Ca2+ exchanger in myocardial preparations from rabbits at physiological preload

Wolfgang Schillingera,*, Claus Christiansa,1, Samuel Sossallaa, Nils Teucherb, Phuc Nguyen Vana, Harald Köglera, Oliver Zeitza,2 and Gerd Hasenfussa

a Universitaet Goettingen, Herzzentrum, Kardiologie und Pneumologie Goettingen, Germany
b Universitaet Goettingen, Herzzentrum, Thorax-, Herz- und Gefaesschirurgie Goettingen, Germany

* Corresponding author. Georg-August-Universität Göttingen, Herzzentrum Göttingen, Kardiologie und Pneumologie, Robert-Koch-Str. 40, 37099 Göttingen, Germany. Tel.: +49 551 39 6349; fax: +49 551 39 9804. E-mail address: schiwolf{at}med.uni-goettingen.de


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
{alpha}1-adrenergic stimulation and mechanical load are considered crucial for the expression of sarcolemmal Na+/Ca2+ exchanger (NCX1). However, the interaction between these processes is unknown.

We investigated electrically stimulated (1 Hz, 1.75 mmol/L Ca2+) rabbit ventricular trabeculae at physiological preload under stimulation by the selective {alpha}1-agonist phenylephrine (PE, 10 µmol/L). Using quantitative real-time PCR, downregulation of mRNA to 76.5% (p<0.05) was found, while B-type natriuretic peptide (BNP) was increased to 569.5% (p<0.05) compared to control. These changes were abolished in the presence of both the β1-blocker prazosin (13 µmol/L) and the PKC inhibitor GF109203X (1 µmol/L). Furthermore, no changes in NCX mRNA levels under the influence of PE were found in unstretched trabeculae or in unstretched isolated rabbit myocytes (24 h), while BNP was increased in both preparations. In addition, since the {alpha}1-adrenergic effect could be Ca2+-dependent we tested increased extracellular Ca2+ (3.0 mmol/L) in stretched trabeculae and found downregulation of NCX1 to 75.2% (p<0.05).

{alpha}1-stimulation decreases NCX1 mRNA in rabbit myocardium via PKC. This is critically load-dependent and may be mediated by changes in [Ca2+]. In hypertrophy and heart failure, distinct phenotypes with respect to NCX1 expression may result from the interaction between mechanical load and {alpha}1-adrenergic stimulation.

Key Words: Na/Ca exchanger • Hypertrophy • Adrenergic signalling • Mechanical stretch • Rabbits

Received March 25, 2006; Revised July 27, 2006; Accepted October 12, 2006


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Contractile performance of cardiac myocytes is closely dependent on oscillating changes of cytosolic Ca2+ which binds to and thereby activates the contractile apparatus of the cell [1,2]. Evidence suggests that the ability to control intracellular Ca2+ within a narrow range, is disturbed in heart failure, and this may be associated with altered expression and function of sarcolemmal Na+/Ca2+ exchanger (NCX1) [3,4]. The first study investigating NCX1 mRNA and protein levels in myocardial samples from patients with end-stage heart failure found a ~2-fold increase as compared to healthy control subjects [5]. Increased NCX1 expression as well as altered function have also been found in subsequent studies; however, some authors have reported unchanged or decreased expression and function in several animal models (reviewed in [3,4]).

In order to identify stimuli that induce changes in NCX1 expression, several studies have been performed in animal hearts. In rats and cats, upregulation has been shown to occur at the transcriptional level following sustained {alpha}1-adrenergic stimulation in vivo and in vitro depicting a specific role of proliferative signalling pathways [6-9]. In addition, we recently demonstrated that the degree of upregulation significantly correlated with plasma norepinephrine levels of patients with end-stage heart failure [10]. As a consequence, in human heart failure a wide variation of NCX1 expression levels may be found ranging from unchanged NCX1 protein in some hearts up to a 2-3-fold increase in NCX1 protein in other hearts as compared to non-failing controls [10,11]. Mechanical stress has been proposed as a further independent mechanism affecting the expression of NCX1. In rat and feline hearts, it has been recognized that there is a rapid upregulation of NCX1 mRNA in response to pressure overload [12,13].

However, little is known about the interaction between {alpha}1-adrenergic stimulation and mechanical load with respect to the regulation of NCX1 expression. We therefore aimed to investigate the influence of {alpha}1-adrenergic stimulation on transcriptional levels of NCX1 in myocardial preparations with and without mechanical load under well-defined in vitro conditions.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animals and materials
Chinchilla bastard rabbits (1.5-2.0 kg) were used. In total, 29 rabbits were used for muscle strip experiments and 11 rabbits for cell culture studies. Tissue from each animal was used for several studies in order to minimise the number of animals used in our institution. All procedures regarding care and use of animals were in accordance with institutional guidelines. Collagenase type II was purchased from Biochrom. M199 cell culture medium, protease type XIV, taurine, D, L-carnitine, creatine, laminin, and 2,3-butanedione monoxime were from Sigma. RNA-later reagent, RNeasy kit and RNase-Free DNase were from Qiagen. All other chemicals indicated were purchased from commercial suppliers with highest purity grade available.

2.2. Muscle strip preparation and registration of mechanical parameters
We have recently shown that mechanical and biochemical characteristics of multicellular muscle strip preparations can be maintained stable over multiday periods allowing the investigation of stimuli that alter gene and consequent protein expression under well-defined physiological conditions [14]. To study changes in gene expression following catecholamine stimulation, rabbits were anesthetized with thiopental sodium (50 mg/kg i.v.) and 1000 IU of heparin was administered to prevent blood coagulation. Hearts were rapidly dissected and washed by retrograde perfusion through the aorta with a Krebs-Henseleit solution containing (in mmol/L) 120 NaCl, 5.0 KCl, 2.0 MgSO4, 1.2 NaH2PO4, 20 NaHCO3, 10 glucose, 0.25 CaCl2, and 20 2,3-butanedione monoxime. During the entire procedure, the solutions were kept at equilibrium with 95% O2-5% CO2, pH 7.4, at 37 °C. Under clean conditions, right ventricular trabeculae were dissected under a stereomicroscope and dimensions were measured at 40x magnification. The dimensions of the preparations were 0.45±0.03 mm in width, 0.39±0.03 mm in thickness, and 4.29±0.20 mm in length. From each heart, two preparations were dissected. The preparations were placed horizontally in an organ bath and were fixed on one side at a basket shaped piece of platinum wire attached to a force transducer (Scientific Instruments, Heidelberg, Germany), and on a pin shaped wire connected to a micrometer device on the other side. The preparations were electrically stimulated with a field stimulation (1 Hz, 3-5 V) by two platinum electrodes. The force transducer signals were amplified and recorded using LabView® based software and were normalized to the dimensions and so specified in mN/mm2.

The organ bath was superfused with Krebs-Henseleit solution, starting with 0.25 mmol/L Ca2+. The Ca2+ concentration was raised stepwise (0.25 mmol/L) up to 1 mmol/L, and then electrical stimulation was started. When muscle preparations started beating, the Ca2+ concentration was increased to 1.75 mmol/L, and after a stabilization phase, the diastolic resting tension was increased until a level of 2-4 mN/mm2 was reached. This reflects a physiological sarcomere length below Lmax (sarcomere length at which developed force is maximum) and produces the best long-time performance of preparations [15]. After another stabilization period, we changed the superfusion solution to M199 medium supplemented with (mmol/L) D, L-carnitine 5.0, creatine 5.0, taurine 5.0, and D, L-glutamine 2.0, with 100 UI/mL penicillin and 0.1 mg/mL streptomycin. Incubation with the test drugs started when the contracting parameters were stable. In the slack experiments, the preparations were not stretched but otherwise identical incubation conditions were applied.

For the investigation of the {alpha}1-adrenergic pathway the selective {alpha}1-adrenoceptor agonist phenylephrine (PE) and the selective antagonist prazosin (Praz) were used. One preparation each was stimulated in medium supplemented with PE (10 µmol/L), PE+Praz (13 µmol/L), PE+the protein kinase C (PKC) inhibitor, GF109203X (1 µmol/L), or PE+Praz+Ca2+ (3 mmol/L). The control preparation beat in a normal medium under the same conditions but without pharmacological stimulation. After 3, 6, and 10 h, electrical stimulation was stopped and the meat ends, which were irritated by shearing on the loop, were immediately cut away and the muscle strips were shock-frozen in RNA-later reagent by liquid nitrogen and stored at – 80 °C.

2.3. Isolation of ventricular myocytes
Rabbits were heparinized and anesthetized with sodium thiopental (50 mg/kg i.v.). Hearts were rapidly removed, mounted in a modified Langendorff perfusion setup, and perfused with Tyrode I solution containing (in mmol/L): 137 NaCl, 5.4 KCl, 1.2 Na2HPO4, 1.2 MgSO4, 20 HEPES, 15 glucose, 1 CaCl2 aerated with 100% O2 for 5-8 min. Perfusion was then switched to nominally Ca2+-free Tyrode solution for 12-15 min (30 ml/min) and digestion was performed by perfusion for 12-15 min (10 ml/min) with Tyrode-enzyme solution containing 250 U/ml collagenase type II, and 0.04 mg/ml protease type XIV, 0.025 mmol/L Ca2+, 60 mmol/L taurine, 8 mmol/L D, L-glutamic acid, and 2 mmol/L D, L-carnitine. Digestion was stopped by perfusion with 100 mL Tyrode solution containing 50 µmol/L Ca2+, 2% fatty acid-free type V albumin, and 20 mmol/L 2,3-butanedione monoxime. Atria were cut off and the ventricles were immersed in Tyrode solution containing 20 mmol/L 2,3-butanedione monoxime, and 50 µmol/L Ca2+. The heart was cut into chunks and myocytes were released by four rounds of mincing and gentle manual agitation. The myocytes were filtered through sterile nylon gauze (200 µm meshes) and progressively exposed to increasing Ca2+ concentrations in Tyrode solution. The final suspension was laid on top of a 6% albumin/M199 medium (1.75 mmol/L Ca2+) supplemented with 5 mmol/L D, L-carnitine, 5 mmol/L taurine, 5 mmol/L creatine and antibiotics.

2.4. Culture of ventricular myocytes
Myocytes were counted and plated at a density of 1.4 x 103 rod-shaped cells/cm2 onto laminin (10 µg/mL)-coated tissue culture dishes. After 2 h, unattached cells were removed by three wash steps and myocytes were cultured for 24 h. For testing {alpha}1-adrenergic stimulation or Ca2+-dependent effects, the normal M199 medium (1.75 mmol/L Ca2+, no phenylephrine) was tested against medium supplemented with PE (10 µmol/L) or CaCl2 (3.0 mmol/L). After 24 h of culture, cells were washed once with 5 mL PBS, scraped off into 5 mL PBS and centrifuged at 1500 rpm for 5 min at 4 °C. Pellets were frozen in liquid nitrogen and stored at – 80 °C.

2.5. mRNA quantification by real-time PCR
DNA free total RNA was extracted by standard protocol using Qiagen RNeasy kit and RNase-Free DNase Set. First strand cDNA synthesis was carried out with the reverse transcriptase superScript II and random primer (Invitrogen) by a standard Invitrogen protocol. The PCR reactions were performed with a real-time PCR Light Cycler (Roche) as detailed recently [16]. The emission was measured at 530 nm. The primer pairs used and the temperature at which emission was measured are specified in Table 1. RNAs with known concentration were used for quantification standard curves. Expression of NCX1 and brain natriuretic peptide (BNP) was normalized to GaPDH. There was a low degree of variability between independent determinations. Neither in cultured myocytes nor in muscle strip preparations were significant differences in the amount of DNA free total RNA (per mg tissue) detected with respect to different stimulation and control culture conditions as well as compared with freshly frozen tissue. This demonstrates that differences in the expression of particular genes cannot be attributed to differences in preparation stability (data not shown).


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  		Table 1 Primer pairs used in real-time PCR and temperature at which emission at 530 nm was measured

 
2.6. Statistical analysis
Data are presented as mean±SEM. Differences in mRNA levels were tested for significance by paired t-test.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Dose-response relationship of PE and isometric force
In order to ensure that the PE dose used in our experiments stimulated the {alpha}1-adrenergic cascade sufficiently, we performed preliminary experiments in electrically stimulated, isolated ventricular trabeculae. A clear dose-dependent increase in force was found that peaked at a concentration of 10 µmol/L (37.4±5.6 mN/mm2 vs. 15.5±2.6 mN/mm2 at baseline without PE). This dose was used in our experiments. Previous studies have used 10 and 20 µmol/L PE for investigating alterations in NCX1 expression in adult [6] and neonatal [7] rat myocytes, respectively.

3.2. Electrically stimulated rabbit ventricular trabeculae
Trabeculae were electrically stimulated and gradually stretched to physiological preload. Then, the continuously beating trabeculae were incubated in 10 µmol/L PE for 3, 6, and 10 h. After 3 and 6 h, mRNA levels of NCX1 did not significantly change compared to trabeculae beating in medium containing no PE (not shown). However, after 10-h incubation in PE there was a significant decrease in NCX1 mRNA to 76.5% (0.048±0.006 vs. 0.036±0.005, p<0.05) and a significant increase in BNP mRNA to 569.5% (0.008±0.002 vs. 0.045±0.010, p<0.05) relative to GaPDH and compared to control. The PE-dependent effect on NCX1 expression was absent in trabeculae that were beating slack, i.e. that had not been stretched before the PE incubation period (Fig. 1).


Figure 01
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  		Fig. 1 PE effect in trabeculae with and without physiological load. Effect of 10-h incubation with 10 µmol/L PE (hatched bars) on mRNA levels of NCX and BNP relative to GaPDH (therefore the ratio has no units) in electrically stimulated, isometrically contracting rabbit ventricular trabeculae that were gradually stretched to physiological preload (A) and in trabeculae that were devoid of stretch and beat slack (B) as compared to control (Contr., same conditions, no PE, open bars). *p<0.05; n=6 each from 6 different rabbits.

 
Fig. 2 demonstrates that the PE effect on transcriptional levels of NCX1 and BNP could be completely blocked by the selective {alpha}1-blocker prazosin. Similar results were obtained when the intracellular downstream pathway of the {alpha}1-adrenergic receptor was blocked by the PKC inhibitor GF109203X. Under these conditions, PE did not induce significant changes in NCX1 or BNP mRNA levels (Fig. 2). Furthermore, elevation of extracellular Ca2+ (3.0 mmol/L compared to 1.75 mmol/L) induced a significant decrease in NCX1 mRNA levels to 75.2% (0.076±0.008 vs. 0.057±0.006, p<0.05) and an increase in BNP mRNA levels to 291.8% (0.015±0.006 vs. 0.045±0.013, p<0.06) (Fig. 3).


Figure 02
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  		Fig. 2 {alpha}1-adrenergic blockade and PKC-inhibition in trabeculae with physiological load. Absence of a PE effect (hatched bars) on NCX and BNP mRNA levels in stretched rabbit ventricular trabeculae in the presence of (A) prazosin (Praz) or (B) the PKC inhibitor GF109203X (GF) after 10-h incubation. Contr.=control medium without PE, prazosin and GF109203X (open bars), n=6 each from 6 different rabbits.

 


Figure 03
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  		Fig. 3 Ca2+ effect in trabeculae with physiological load. Effect of elevated extracellular Ca2+ (3 mmol/L, hatched bars) on mRNA levels of NCX and BNP relative to GaPDH (therefore the ratio has no units) in stretched rabbit ventricular trabeculae as compared to control (1.75 mmol/L, open bars). *p<0.05; n=6 each from 6 different rabbits.

 
3.3. Isolated, cultured unstretched rabbit myocytes
Isolated, cultured rabbit myocytes devoid of stretch were incubated in the presence of 10 µmol/L PE (1.75 mmol/L Ca2+). After 24 h, transcriptional levels of NCX1 and BNP were determined by real-time PCR (Fig. 4). In accordance with the results in unstretched trabeculae, no significant changes with respect to NCX1 mRNA levels (0.051±0.012 vs. 0.051±0.010, n.s.) were found whereas BNP levels were significantly upregulated to 257.7% (0.011±0.002 vs. 0.028±0.003, p<0.05).


Figure 04
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  		Fig. 4 PE effect in isolated unstretched myocytes. Effect 10 µmol/L PE (hatched bars) on mRNA levels of NCX and BNP relative to GaPDH (therefore the ratio has no units) after 24-h incubation of isolated, cultured adult rabbit cardiomyocytes as compared to control (Contr., no PE, otherwise identical conditions, open bars). n=11 each from 11 different myocyte isolations.

 
We also tested the effect of elevation of the extracellular Ca2+ concentration. No significant changes were found in NCX1 (0.041±0.003 vs. 0.042±0.004, n.s.) or BNP (0.008±0.003 vs. 0.007±0.003, n.s.) mRNA levels in rabbit myocytes incubated in control medium containing 1.75 mmol/L Ca2+ compared to control medium containing 3.0 mmol/L Ca2+.


   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrates that 1) in ventricular trabeculae from rabbit hearts that had been stretched to physiological preload, sustained PE stimulation promoted downregulation of NCX1 mRNA. 2) {alpha}1-adrenergic stimulation induced no change in NCX1 mRNA in unloaded rabbit trabeculae and myocytes. 3) The PE-dependent effect in physiologically stretched trabeculae was abolished after blockade of either the {alpha}1-adrenoceptor or PKC. 4) Elevation of the extracellular Ca2+ concentration resulted in decreased NCX1 mRNA only in stretched rabbit trabeculae while no change was found in unloaded myocytes.

Under combined mechanical load and {alpha}1-adrenergic stress, we demonstrated a significant effect on NCX1 expression at the transcriptional level in multicellular myocardial preparations from rabbits and somewhat unexpectedly NCX1 transcription was downregulated. This was mediated by the {alpha}1-subtype of the adrenoceptor and depended on intracellular downstream signalling via PKC. Previously, other authors have suggested that {alpha}1-adrenergic pathways induce hypertrophy independent from load [17]. In a study of adult unstretched rat cardiomyocytes, sustained stimulation with the selective {alpha}1-agonist PE was shown to induce an increased expression of NCX1 at the mRNA and protein level [6]. In the present work, in contrast to the findings in trabeculae with physiological preload, no change in NCX1 expression was found in unstretched trabeculae that underwent an otherwise identical PE stimulation protocol. In accordance with these findings and contrasting with previous data in isolated rat myocytes [6], PE did not induce changes in the expression of NCX1 in isolated unstretched rabbit myocytes. However, BNP, a sensitive cardiac load indicator and a clinically reliable index of heart failure [18], was upregulated under PE stimulation in stretched and unstretched preparations, which demonstrates correct functioning of intracellular downstream signalling in our experiments. Moreover, in rat myocytes we were able to stimulate an increase in NCX1 mRNA using the same culture system in accordance with the previous studies (data not shown).

The role of mechanical stretch in hypertrophy has been extensively investigated and is considered important for the induction of an early gene program and protein synthesis. Regarding NCX1 expression, Kent and co-workers demonstrated a rapid increase in NCX1 mRNA in response to pressure overload in feline hearts [12]. Little is known about how biomechanical stress is sensed by the cardiomyocytes and transduced into pro-hypertrophic intracellular signals. Several authors have suggested pathways that involve the formation of auto- and/or paracrine factors subsequent to mechanical stress such as endothelin-1, norepinephrine or angiotensin II, the receptors of which are coupled to G-proteins responsible for submitting the intracellular signal transduction cascade [17,19]. Other authors have suggested pathways that are completely independent from humoral or neuronal factors, but involve cellular mechanoreceptors like β1-integrins [20] or proteins from the sarcomeric Z-disc [21]. The exact points of cross-talk between {alpha}1-adrenergic and stretch-dependent pathways in the present study remain speculative because this was beyond the scope of the study. However, recent data from adenoviral co-transfection studies provide evidence that MAP kinases play an important role in regulating the expression of NCX1 in adult rat cardiocytes suggesting a specific role of ERK1/2 in mediating downstream signalling from both integrins and {alpha}1-adrenergic stimulation (reviewed in [9]).

Since load has been shown to alter Ca2+ gradients across the sarcolemma, and {alpha}1-adrenoceptor stimulation results in increased cytoplasmic Ca2+ [2] we hypothesized that changes in intracellular Ca2+ might be crucial for inducing altered NCX1 expression. This hypothesis was further supported by the observation that veratridine, a Na+ and Ca2+ influx stimulator in excitable cells induced an upregulation of NCX1 in adult feline myocytes similar to mechanical load [13]. Moreover, enhanced NCX1 expression and function have been found in neonatal rat myocytes when cytosolic Ca2+ was increased by ouabain treatment [22]. One potential focal regulator of cardiomyocyte hypertrophy in response to altered intracellular Ca2+ is the calmodulin-activated serine/threonine protein phosphatase calcineurin. Once activated by increases in Ca2+, calcineurin mediates the hypertrophic response through its downstream transcriptional effector nuclear factor of activated T cells (NFAT) [23]. In the present study, Ca2+-dependent downregulation of NCX1 was found in stretched ventricular rabbit trabeculae, similar to the effect of {alpha}1-adrenergic stimulation. In contrast, in unstretched, isolated rabbit myocytes, no Ca2+-dependent alterations in NCX1 transcript levels were found.

Our finding that NCX1 expression following {alpha}1-adrenergic stimulation at physiological load was downregulated may be species related. In vivo studies in rats investigating {alpha}1-stimulation by means of an osmotic minipump have shown increased expression of NCX1 [8]. Furthermore, our group has demonstrated a significant correlation between plasma norepinephrine levels and NCX1 protein levels in myocardium from end-stage heart failure patients [10]. It is well known that the same stimulus may induce divergent responses in different species, e.g. in dogs, sustained tachycardia pacing induced NCX1 upregulation at the protein level [24] whereas in rabbits it provoked NCX1 downregulation at the mRNA level by 36% [25], but did not amount to a significant response at the protein level [26]. In a direct comparative study, rats had the highest cardiac {alpha}1-adrenoceptor density among seven species, including man, guinea pig, mouse, rabbit, and pig, which exceeded that of all other species by at least 5 times. These data indicate that the high {alpha}1-adrenoceptor density in rat ventricles may be a particular feature of that species and necessitates great care in extrapolation of rat data to other species [27]. Our findings may also be related to the model. In several rabbit models of heart failure NCX1 overexpression has also been reported, e.g. heart failure subsequent to myocardial infarction or to combined pressure-volume overload [3,4,28]. It may therefore be speculated that additional factors interfere with neurohumoral activation and mechanical load in these models resulting in particular phenotypes with respect to NCX1 expression. We want to stress that we measured transcriptional levels because we aimed to address short term regulation of the NCX gene. From a functional point of view, protein levels may be more interesting. However, it is questionable whether a reduction in NCX protein levels could be measured in ex vivo culture systems in adult cardiac tissue, because the protein half-life of NCX has been reported to be 33 h [29].

Taken together, in the present model regulation of NCX1 expression by {alpha}1-adrenergic stimulation is critically dependent on load and might rely on intracellular Ca2+ concentrations. As a result, NCX1 is downregulated. Thus, NCX1 expression is the consequence of the activation of distinct hypertrophic pathways. These findings should be considered when extrapolating data from experimental studies to the in vivo situation of cardiac hypertrophy or failure.


   Acknowledgements
 
This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich Transregio 2 to Gerd Hasenfuss. We gratefully acknowledge the expert technical assistance of Michael Kothe and Gudrun Müller.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
1 Current address: Klinikum Leverkusen, Klinik fuer Kinder und Jugendliche, Leverkusen Germany. Back

2 Current address: Universitaetsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde, Hamburg, Germany. Back


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

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