Anhedonia and altered cardiac atrial natriuretic peptide following chronic stressor and endotoxin treatment in mice

Psychoneuroendocrinology (2010) 35, 233—240

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n

Anhedonia and altered cardiac atrial natriuretic peptide following chronic stressor and endotoxin treatment in mice Boubacar Pasto Wann *, Marie-Claude Audet, Julie Gibb, Hymie Anisman Institute of Neuroscience, Department of Psychology, Carleton University, Ottawa, Ontario, Canada K1S 5B6 Received 15 January 2009; received in revised form 24 April 2009; accepted 17 June 2009

KEYWORDS Chronic stress; Lipopolysaccharide; ANP cardiac tissue; Plasma ANP; Cytokines and anhedonia; Depression

Summary Chronic stressors and inflammatory immune activation may contribute to pathophysiological alterations associated with both major depression and cardiovascular disease. The present study, conducted in mice, assessed whether a chronic stressor of moderate severity that induced an anhedonic effect, when coupled with a bacterial endotoxin, lipopolysaccharide (LPS), additively or interactively provoked circulating and heart atrial natriuretic peptide (ANP), a potentially useful diagnostic and prognostic tool in cardiac diseases. As well, given the potential role of inflammatory processes in both depression and cardiovascular disease, we assessed proinflammatory mRNA expression in heart in response to the stressor and the LPS treatments. Male CD-1 mice that had been exposed to a chronic, variable stressor over 4 weeks displayed reduced sucrose consumption, possibly reflecting the anhedonic effects of the stressor. Treatment with LPS (10 mg) provoked increased circulating corticosterone levels in both chronically stressed and non-stressed mice. Moreover, ANP concentrations in plasma and in the left ventricle were increased by both the stressor and the LPS treatments, as were left atrial and ventricular cytokine (interleukin-1b; tumor necrosis factor-a) mRNA expression. Further, these treatments synergistically influenced the rise of plasma ANP. A link may exist between stressor-provoked depressive features (anhedonia) and immune activation, with elevated levels of ANP, a potential marker of cardiovascular disturbance. These findings are consistent with the view that chronic stressors and inflammatory immune activation may represent a common denominator subserving the frequent comorbidity between these illnesses. # 2009 Elsevier Ltd. All rights reserved.

1. Introduction

* Corresponding author at: Institute of Neuroscience, Life Science Research Centre, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6. Tel.: +1 613 520 2600x1694; fax: +1 613 520 4052. E-mail address: [email protected] (B.P. Wann).

Both chronic stressors and activation of the inflammatory immune response may contribute to the pathophysiological processes that increase vulnerability of depressed patients to cardiovascular diseases (Lesperance and Frasure-Smith, 2000). Consistent with this view, major depressive disorder (MDD) has been associated with increased

0306-4530/$ — see front matter # 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2009.06.010

234 levels of inflammatory factors, including pro-inflammatory cytokines (Maes, 1995), and MDD can be induced by administration of cytokines such as interferon-a (Raison et al., 2006), and to some extent by endotoxin administration (Reichenberg et al., 2001). The processes by which immune activation produces such effects are not fully understood, but it seems that direct or indirect actions of cytokines on brain norepinephrine (NE) and serotonin may contribute to MDD (Anisman et al., 2008b; Dantzer et al., 2008). It likewise appears that cardiovascular disturbances in rodents and humans may arise in association with immune activation provoked by a bacterial endotoxin, such as lipopolysaccharide (LPS) (Tavener and Kubes, 2006; Zorn-Pauly et al., 2007). This endotoxin is a strong stimulus that influences immune cells (Blunck et al., 2001), by directly stimulating toll-like receptor 4 (TLR-4) present on neutrophils and monocytes (Tavener et al., 2004), which may infiltrate cardiac tissues and may promote myocardial dysfunction (Grabie et al., 2003; Tavener and Kubes, 2006). This may, in turn, promote the release of atrial natriuretic peptides (ANP and BNP) by the heart, which have been implicated as hormonal markers in cardiac diseases, including heart failure, cardiac hypertrophy and arrhythmia (Goetze et al., 2004; MekontsoDessap and Brochard, 2006). It seems likely that central processes may also contribute to cardiovascular changes. Specifically, the elevated locus coeruleus NE synthesis that occurs in response to stressors and immune activation (Anisman and Merali, 1999) may influence cardiac receptors and increase heart rate, blood pressure and ventricular contraction rate, thereby influencing vulnerability to tachycardia or ventricular fibrillation (Dalack and Roose, 1990). Pro-ANP is the principal storage form of ANP in the granules of atrial cardiomyocytes. It is cleaved to the inactive Nterminal fragment (NT-pro-ANP) and the active hormone ANP, and then released into the bloodstream by exocytosis (Mekontso-Dessap and Brochard, 2006; Michener et al., 1986; Ruskoaho, 1992). It is mainly produced and secreted by the atrium in response to atrial stretch provoked by volume overload (Mekontso-Dessap and Brochard, 2006; Politi et al., 2007; Shanshan et al., 2005; Weber et al., 2006). Physiological effects of this peptide include natriuresis, diuresis, vasorelaxation and modulation of systems that increase extracellular fluid volume and blood pressure (Mekontso-Dessap and Brochard, 2006; Shanshan et al., 2005). Although stressors and LPS may induce unfavorable effects on depressive symptoms and on cardiovascular functioning, they may do so through independent pathways. Alternatively, the treatments may engage common processes (e.g., promotion of monoamine and cytokine alterations) so that they additively or synergistically favor the emergence of behavioral disturbances, such as depression, as well as cardiovascular problems (Anisman et al., 2008a). The present study was conducted to assess whether chronic stressors and immune activation (by LPS) would promote signs of cardiovascular disturbances and elicit depressive-like symptoms, and whether the combination of the stressor and LPS treatments would have additive or synergistic effects in this regard.

2. Experimental protocol Male CD-1 mice (N = 80) were obtained from Charles River Canada (St. Constant, Quebec) at about 6—8 weeks of age.

B.P. Wann et al. They were allowed to acclimatize to the laboratory setting for 2 weeks before being used as experimental subjects. Mice were housed individually in a temperature-controlled vivarium with lights on from 08:00 to 20:00 h; food and water were freely available. All procedures were conducted in accordance with the guidelines set out by the Canadian Council on Animal Care and were approved by the Carleton University Animal Care Committee.

2.1. Chronic stressor procedure and LPS treatment Mice were randomly assigned to either a chronic stressor condition or a non-stressed group (N = 40/group). Of these animals all were used for determination of plasma corticosterone. Half the animals were used for ANP determinations and for mRNA expression of ANP and for cytokines. Stressed mice were exposed to a series of different stressors on each day over 4 weeks. During this time stressors were applied twice a day (different stressors in the morning and afternoon). The stressors were presented on a variable and unpredictable schedule. The animals were returned to their home cages between the two stressor sessions of each day. The chronic stressor regimen included the following stressors: restraint in semicircular Plexiglas tubes (4 cm diameter  12 cm long), with tails taped to prevent mice from turning (15 min); exposure to predator odor (rat) by placing the mouse in a cage containing rat soiled bedding (60 min); wet bedding, in which cage bedding was soaked with water (60 min); forced swim in water of 20 8C within a plastic cylinder, 30 cm diameter and 27 cm high (5 min); placement on the open arm of plus maze (5 min); placement in a tight fitting triangular baggie (with a hole for the nose) resulting in complete restraint (15 min), and placing mice in cage with four mice (60 min). These procedures were previously shown to elicit behavioral changes and to altered brain monoamine activity (Anisman et al., 2007; Tannenbaum et al., 2002). Nonstressed mice remained in their home cages, and were not disturbed. On sacrifice day, which occurred on the morning following the last stressor session, the stressed and non-stressed mice were divided into two subgroups and treated with either LPS (10 mg, in a volume of 0.3 ml; from Escherichia coli 026:B6  10,000 EU/mg; Sigma) or saline.

2.2. Sucrose preference test The present study was conducted, in part, to determine whether the effects of LPS would be enhanced given a background of chronic stressor exposure that could potentially be associated with depressive-like symptoms. Thus, it was of interest to establish whether the chronic stressor regimen provoked depressive-like behaviors prior to the introduction of the endotoxin. To this end, a sucrose preference test that has been used as a measure of anhedonia (Redei et al., 2001; Willner et al., 1987) was conducted over 24 h periods on three occasions (prior to the stressor regimen being introduced, and then again 2 and 4 weeks after stressor initiation). In this test, mice had access to two 200 ml bottles that contained either tap water or a 1%

Altered cardiac ANP following chronic stressor and LPS treatment sucrose solution. Intake volume of each was determined on the basis of the bottle weights prior to vs. after the 24 h test period.

2.3. Tissue extraction Cardiac tissues extraction was conducted as previously described (Kim et al., 1999; Tse et al., 2001). Briefly, after sacrifice, left and right atria as well as the ventricles were dissected and stored at 80 8C. Then 1 ml of 1 M acetic acid was added to each tissue sample, which was then transferred to boiling water bath for 10 min. After cooling on ice, samples were homogenized with a sonicator (Model 100, Sonic Dismembrator, and Fisher Scientific), centrifuged at 16,000  g for 15 min and the supernatant frozen at 80 8C until assay. The samples were extracted by Sep-Pak C18 cartridges, which had been pre-washed with 10 ml acetonitrile (CH3CN) and then with 10 ml of 0.1% TFA. The acidified samples were then passed three times through the cartridges. The sample loaded cartridges were washed with 10 ml of 0.1% TFA. This was followed by a 10 ml wash of 10% CH3CN in 0.1% TFA and finally the samples were eluted slowly with 3 ml of 60% CH3CN in 0.1% TFA and lyophilized. The resulting powder samples were stored at 80 8C until assayed. Assays were restricted to the left atrium and ventricle as these portions of the heart tend to be sensitive to pathology than the right (Iyer et al., 2006).

2.4. Plasma corticosterone Before treatments (baseline) and at 2 and 4 weeks following the initiation of the stressor schedule, tail blood samples were collected on filter paper standardized to absorb blood in a homogenous manner so that uniform punches from any section of the sample would yield the same quantity of blood (Worthman and Stallings, 1997). The filter papers were stored at 80 8C until assayed. Tail blood was collected in the morning between 08:00 and 09:00 h before the stressor session commenced. The length of time to collect blood ranged from 1 to 2 min, although preparing the animal for this procedure could take a longer time and hence represents a stressor that might account for the relatively high corticosterone levels in these mice. As well, after sacrifice by rapid decapitation (90 min after LPS or saline treatment, at 10:00—12:00 h), trunk blood was collected (over 15—20 s) in tubes containing 10 mg of EDTA, centrifuged for 20 min at 2000  g and stored at 80 8C until assayed using a radioimmunoassay kit according to the manufacturer’s protocol (ICN Biomedicals, CA, USA). Plasma corticosterone concentrations were determined in duplicate, in a single run to preclude inter-assay variability; the intra-assay variability was less than 10%.

2.5. ANP radioimmunoassay protocol ANP levels in plasma and in cardiac tissue were determined by radioimmunoassay according to the manufacturer’s protocol (Phoenix Pharmaceuticals, Inc.). After reconstitution with the RIA buffer (with 150 ml), the standard peptide (1 ml of RIA buffer), the rabbit anti-peptide serum (13 ml of RIA buffer), the positive control (1 ml of RIA buffer) and samples

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with RIA buffer, dilutions of the standard were prepared as indicated in the experimental protocol from Phoenix Pharmaceuticals and radioactivity determined using a gamma counter.

2.6. Determination of ANP, IL-1b and TNF-a mRNA expression Total RNA was extracted from frozen samples with Trizol according to the manufacturer’s protocol (Invitrogen; Burlington, Ontario, Canada). The total RNA was then reverse transcribed using Superscript II reverse transcriptase (Invitrogen; Burlington, Ontario, Canada). Aliquots of this reaction were then used in simultaneous QPCR reactions. For QPCR, SYBR green detection was used according to the manufacturer’s protocol (Stratagene Brillant QPCR kit). A stratagene MX-4000 real-time thermocycler was used to collect the data. All PCR primer pairs used generated amplicons between 100 and 200 bp. Amplicon identity was checked by restriction analysis. Primer efficiency was measured from the slope relation between absolute copy number or RNA quantity and the cycle threshold using the MX-4000 software. All primer pairs had a minimum of 90% efficiency. GAPDH was used for normalization because this gene showed to be a stable expressed housekeeping gene (Gutkowska et al., 2007). In additional experiment assessing the effects of LPS on brain cytokines, we found that GAPDH and B-actin yielded the same effects. GAPDH, forward: AAA TGG TGA AGG TCG GTG TG, reverse: GAA TTT GCC GTG AGT GGA GT. ANP, forward: CCA GAG TGG ACT AGG CTG CAA C, reverse: AAT GTG ACC AAG CTG CG TGA C. IL-1b, forward: TGT CTG AAG CAG CTATGG CAA C, reverse: CTG CCT GAA GCT CTT GTT GAT G. TNF-a, forward: CTC AGC CTC TTC TCA TTC CTG C, reverse: GGC CAT AGA ACT GAT GAG AGG G.

3. Statistical analyses Corticosterone, ANP in the blood and the tissues, and ANP and cytokine mRNA expression were assessed using a two factor (stressor  LPS treatment) analysis of variance (ANOVA). For the mRNA analyses, the nCt values were converted to fold changes using the 2( Delta Delta C(T)) method (Livak and Schmittgen, 2001). Sucrose preference was analyzed as a mixed measures ANOVA with stressor as a between group variable, and time as a within group measure. Follow-up comparisons were conducted using the least significant difference (LSD) to maintain the alpha at 0.05.

4. Results 4.1. Sucrose preference test Sucrose consumption varied as a function of the stressor condition  time interaction, F(2,156) = 6,57, p < 0.005. The follow-up comparisons confirmed that the preference for the sucrose solution after 2 weeks did not differ between the stressed and control mice. However, during the fourth week the sucrose consumption was significantly reduced in stressed mice relative to their non-stressed counterparts (Fig. 1).

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Figure 1 Sucrose preference (means  SEM) for non-stressed mice and for stressed mice at baseline and after 2 and 4 weeks of treatment. *p < 0.05.

B.P. Wann et al. stressed mice, administration of LPS markedly increased the plasma ANP levels relative to mice that received neither or only one of the treatments (see Fig. 3). In addition to the plasma ANP changes, the ANP mRNA expression in the atrium was significantly higher in the LPS than in saline-treated mice, F(1,29) = 4.29, p < 0.05 (Fig. 3C). Although the interaction between the stressor  LPS treatments was not significant, follow-up tests of the simple effects were conducted based on a priori hypotheses and the fact that this interaction accounted for a large portion of the variance (h2 = 0.17). These comparisons indicated that in the absence of the stressor LPS provoked only a small non-significant increase of ANP mRNA expression. In contrast, among stressed mice, LPS engendered a relatively strong and statistically significant elevation of ANP expression. Finally, the ANP concentration in ventricular tissue were significantly elevated in the stressed relative to the non-stressed mice, F(1,29) = 4.36, p < 0.05 (Fig. 3), but the interaction with the LPS treatment did not reach statistical significance.

4.4. IL-1b and TNF-a mRNA expressions 4.2. Corticosterone levels Plasma corticosterone levels over the 4-week stressor period varied as a function of stress treatment  time interaction F(2,156) = 5.95, p < 0.005. The follow-up tests indicated a significant increased of plasma corticosterone level in the stressed mice during Week 4 ( p < 0.05) compared to the control mice at this time, as well as in comparison to baseline (Fig. 2). In addition, analysis of corticosterone levels sacrifice was increased in the LPS compared to the saline-treated mice, F(1,72) = 57.18, p < 0.005. However, this was the case irrespective of the stressor treatment mice had previously received (Fig. 2).

The mRNA expression for IL-1b and TNF-a in the atrium were significantly elevated by the LPS treatment, F(1,35) = 20.67, 59.05, p < 0.001, respectively. Moreover, in the ventricle, mRNA expression of IL-1b and TNF-a was elevated in the LPS treated mice relative to controls, F(1,31) = 6.88, 33.17, p < 0.05. The effects of the stressor treatment were less remarkable, although mRNA expression of TNF-a in the left ventricle was increased in the stressed mice F(1,33) = 4.67, p < 0.05. The LPS  stressor interaction was not statistically significant (Fig. 4). Thus, it appeared that TNF-a increased within the ventricle increased additively in response to the LPS and stressor treatments.

4.3. Plasma and ventricular ANP levels and atrial mRNA expression

5. Discussion

Plasma ANP concentrations varied as a function of the stressor condition  LPS interaction, F(1,34) = 22.46, p < 0.001. Neither the chronic stressor nor the LPS treatments alone affected plasma ANP levels. However, among chronically

Inflammatory factors and stressors have been implicated in both depressive and cardiovascular illness (Reichenberg et al., 2001; Wright et al., 2005), and have been shown to additively or synergistically influence several behavioral outcomes (e.g., sickness related to LPS), norepinephrine

Figure 2 (A) Plasma corticosterone levels (means  SEM) at baseline and after 2 and 4 weeks of stressor exposure, in the absence of LPS treatment. (B) Plasma corticosterone levels after LPS-treatment or saline treatment among mice that had been exposed to the chronic stressor (or that had not been stressed (n = 16—20/group). *p < 0.05.

Altered cardiac ANP following chronic stressor and LPS treatment

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Figure 3 Effects of LPS and saline among chronically stressed and non-stressed mice on (A) plasma ANP (means  SEM), (B) left ventricular tissue, as well as (C) ANP mRNA expression (fold changes) in the left atrium. *p < 0.05.

release in brain, as well as inflammatory processes (Anisman et al., 2003; Johnson et al., 2003). The present study assessed whether a chronic stressor regimen, that elicits anhedonic-like effects, would increase plasma and heart ANP, as well as ANP mRNA expression and that of pro-inflammatory cytokines in the heart. Consistent with reports that a chronic stressor regimen engendered anhedonia (Redei et al., 2001; Willner et al., 1987), the chronic variable stressor regimen (involving stressors of moderate severity) reduced sucrose consumption. As frequently observed (Willner et al., 1987), this outcome required a relatively sustained chronic stressor regimen, as the reduced consumption was not apparent following 2 weeks of treatment, but was evident after 4 weeks (twice daily) of stressor exposure. Moreover, in line with earlier studies assessing the impact of chronic stressors (Ayensu et al., 1995; Grippo et al., 2005; Silberman et al., 2002), as well as studies showing elevated corticosterone levels in MDD (Holsboer, 2000), the anhedonic effect was accompanied by an increase of plasma corticosterone. Consistent with earlier findings (Anisman et al., 2008b; Dantzer et al., 2008), LPS increased circulating corticosterone levels; however, unlike the augmented effects of this treatment evident immediately after exposure to a social stressor (Gibb et al., 2008), in the present study, in which LPS was administered 24 h afterward, there was no indication of synergy between the treatments. Yet, as corticosterone changes were only assessed at a single time point following

the LPS treatment, it is uncertain whether additive or synergistic effects of the treatments might have been detected at other intervals, or whether the combined effects of the treatments were more protracted than those elicited by either treatment alone. As described earlier, chronic stressors negatively influenced hemodynamic markers of cardiovascular disturbances (Grippo et al., 2002; Inagaki et al., 2004; Shigemi et al., 1991), reflected by elevated circulating ANP levels. In this regard, plasma ANP levels were particularly elevated in mice that had been chronically stressed and then treated with LPS. In fact, when administered alone, neither the chronic stressor nor the endotoxin, affected plasma ANP, but the concentrations of this peptide were markedly increased among chronically stressed mice that had been treated with LPS. It was likewise observed that ANP mRNA expression in the left atrium was greater among mice that had been exposed to the stressor and then challenged with LPS, than among mice that received the stressor but not subsequently challenged with LPS. Inasmuch as high plasma levels and upregulation of ANP have been associated with cardiac diseases, including congestive heart failure, cardiac hypertrophy and arrhythmia (Mockel et al., 2005; O’Donoghue and Januzzi, 2005; Sullivan et al., 2005; Takemura et al., 1998), the high levels of circulating ANP in the left ventricle might be a harbinger or marker for stressor-provoked cardiovascular dysfunction. The upregulation of ANP in plasma and in the left atrium (reflected by increased ANP mRNA in the latter instance) is

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B.P. Wann et al.

Figure 4 Effects of LPS and saline among chronically stressed and non-stressed mice on mRNA expression of IL-1b (data expressed as fold changes; means  SEM) in the (A) left atrium and (C) left ventricle, and TNF-a mRNA expression in the (B) left atrium, and (D) left ventricle. *p < 0.05.

suggestive of alterations of cardiomyocytes in the atrium, where ANP is primarily synthesized. However, ANP is also produced, albeit to a lesser extent, in the ventricles, and thus a role for ventricular synthesis of ANP in governing the response to stressors cannot be dismissed. It is noteworthy though, that the effects of the treatments on atrial and ventricular ANP could be dissociated from one another. In particular, unlike the ventricular ANP increase that occurred in response to the stressor, atrial ANP mRNA expression was apparent in response to the LPS treatment. Whether these differential effects of LPS and stressors on atrial vs. ventricular ANP reflect different processes (e.g., the site of ANP synthesis being primarily in the atrium) is uncertain. However, the possibility cannot be dismissed that stressors and LPS induce ultrastructural changes in cardiomyocytes. It will be recalled that depression and cardiovascular illness are frequently comorbid (Lesperance and FrasureSmith, 2000; Musselman et al., 1998), and it was reported that MDD was associated with an increase of plasma Nterminal pro-BNP (NT-pro-BNP), a hormonal marker of heart disease that possesses the same biological effects as BNP (Politi et al., 2007). To our knowledge, similar effects have not previously been reported with respect to ANP, but the present findings suggest a link between a chronic stressor that was associated with depressive features (anhedonia) and increased ANP levels in the plasma and cardiac tissue. As indicated earlier, elevated mRNA expression of ANP in the atrium was also evident in response to the bacterial endo-

toxin, just as increased plasma concentration of ANP was found in patients with septic shock (Witthaut et al., 2003). These findings are consistent with the view that inflammatory immune activation may represent a common denominator for these pathologies and might account for their comorbidity, although the present findings do not speak to a causal role for ANP in this regard. The processes responsible for promoting the ANP changes have yet to be determined. Nevertheless, in humans that had experienced a chronic stressor, monocytes displayed a functional resistance to glucocorticoids, even in the absence of elevated corticoid levels. The down-regulated response to corticoids favors activation of pro-inflammatory functioning, which under chronic conditions may promote cardiovascular dysfunction (Miller et al., 2008). Circulating IL-1b and TNF-a were shown to be involved in impaired cardiac contractility (Hofmann et al., 2007), and depression has been associated with increased levels of circulating cytokines (Maes, 1995). The connection between inflammatory processes and heart disease related to immunogenic and stressor challenges is supported by our finding that LPS treatment strongly increased the mRNA expression of IL-1b and TNF-a in the left atrium and the left ventricle. Moreover, increased mRNA expression of TNF-a was evident in the left ventricle in response to the stressor. At the same time, however, synergy between the LPS and stressor treatments was not apparent with respect to cytokine mRNA expression, as observed with respect to ANP mRNA changes or the cytokine—stress synergy evident in plasma. It seems

Altered cardiac ANP following chronic stressor and LPS treatment that the parallel between the effects of stressors and endotoxin challenges on ANP and cytokine variations in plasma and heart were not perfect. Nonetheless, the stressor and endotoxin treatments clearly affected these processes, providing at least prima facie evidence in favor of a relationship in the processes governing depressive illness and markers of cardiovascular disturbances. In addition to the aforementioned limitations of the present findings, it needs to be underscored that ANP may be a marker of heart disease, but in the present investigation no specific heart ailment was assessed. Thus, it remains to be established whether the observed ANP changes would, in fact, predict the subsequent development of cardiopathology. Finally, in the present investigation only a single stressor regimen was employed and biological changes were measured at only a single post-stressor interval. To more fully determine the course of the illness, and the longer-term ramifications of a chronic stressor, more detailed parametric analyses will be necessary. Although the focus of this report has been on ANP and cytokines, the influence of other factors provoked by stressors and endotoxin challenges cannot be dismissed. By example, aldosterone was not determined in the present study, but it is possible that plasma ANP released could block the physiological action of this hormone. In this regard, aldosterone promotes sodium and water reabsorption and potassium release. This elevates blood volume and blood pressure and may thus influence ANP expression (Lumbers, 1999). Likewise, by virtue of effects on epinephrine (Packer, 2001), LPS plus stressor exposure could potentially influence cardiac ANP through sympathetic nervous system mechanisms. Summarizing, the present findings indicated that a chronic, intermittent, unpredictable stressor increased left ventricular ANP levels, which may reflect a marker for potential cardiac disturbances. The LPS treatment increased atrial and ventricular IL-1b and TNF-a mRNA expression, whereas the stressor had limited effects in this regard. Significantly, the combination of the stressor and LPS treatments markedly augmented changes of plasma ANP and that of ANP mRNA expression in the left atrium. In fact, in the absence of the stressor, circulating ANP was unaffected by the LPS administration. As the stressor procedure itself provoked an anhedonic effect, which might reflect depressive-like symptom, these findings are consistent with the perspective that chronic stressors and inflammatory immune activation might contribute to the frequent comorbidity between depression and cardiovascular illness.

Role of funding sources The grant organizations did not have any impact in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Conflict of interest The authors have no commercial or other relations pertaining to the present findings.

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Acknowledgments This work was supported by Canadian Institute of Health Research (CIHR). HA holds a Canada Research Chair in Neuroscience. M-CA is supported by a fellowship from FRSQ and from the office of VP Research and International, Carleton University.

References Anisman, H., Gibb, J., Hayley, S., 2008a. Influence of continuous infusion of interleukin-1beta on depression-related processes in mice: corticosterone, circulating cytokines, brain monoamines, and cytokine mRNA expression. Psychopharmacology (Berl.) 199, 231—244. Anisman, H., Merali, Z., 1999. Anhedonic and anxiogenic effects of cytokine exposure. In: Dantzer, R., Wollman, E.E., Yirmiya, R. (Eds.), Cytokines, Stress and Depression. Kluwer Academic/Plenum Publishers, New York. Anisman, H., Merali, Z., Hayley, S., 2003. Sensitization associated with stressors and cytokine treatments. Brain Behav. Immun. 17, 86—93. Anisman, H., Merali, Z., Hayley, S., 2008b. Neurotransmitter, peptide and cytokine processes in relation to depressive disorder: comorbidity between depression and neurodegenerative disorders. Prog. Neurobiol. 85, 1—74. Anisman, H., Prakash, P., Merali, Z., Poulter, M.O., 2007. Corticotropin releasing hormone receptor alterations elicited by acute and chronic unpredictable stressor challenges in stressor-susceptible and resilient strains of mice. Behav. Brain Res. 181, 180— 190. Ayensu, W.K., Pucilowski, O., Mason, G.A., Overstreet, D.H., Rezvani, A.H., Janowsky, D.S., 1995. Effects of chronic mild stress on serum complement activity, saccharin preference, and corticosterone levels in Flinders lines of rats. Physiol. Behav. 57, 165—169. Blunck, R., Scheel, O., Muller, M., Brandenburg, K., Seitzer, U., Seydel, U., 2001. New insights into endotoxin-induced activation of macrophages: involvement of a K+ channel in transmembrane signaling. J. Immunol. 166, 1009—1015. Dalack, G., Roose, S., 1990. Perspectives on the relationship between cardiovascular disease and affective disorder. J. Clin. Psychiatry 51, 4—11. Dantzer, R., O’Connor, J.C., Freund, G.G., Johnson, R.W., Kelley, K.W., 2008. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat. Rev. Neurosci. 9, 46—56. Gibb, J., Hayley, S., Gandhi, R., Poulter, M.O., Anisman, H., 2008. Synergistic and additive actions of a psychosocial stressor and endotoxin challenge: circulating and brain cytokines, plasma corticosterone and behavioral changes in mice. Brain Behav. Immun. 22, 573—589. Goetze, J.P., Videbaek, R., Boesgaard, S., Aldershvile, J., Rehfeld, J.F., Carlsen, J., 2004. Pro-brain natriuretic peptide as marker of cardiovascular or pulmonary causes of dyspnea in patients with terminal parenchymal lung disease. J. Heart Lung Transplant. 23, 80—87. Grabie, N., Hsieh, D.T., Buono, C., Westrich, J.R., Allen, J.A., Pang, H., Stavrakis, G., Lichtman, A.H., 2003. Neutrophils sustain pathogenic CD8+ T cell responses in the heart. Am. J. Pathol. 163, 2413—2420. Grippo, A.J., Francis, J., Beltz, T.G., Felder, R.B., Johnson, A.K., 2005. Neuroendocrine and cytokine profile of chronic mild stressinduced anhedonia. Physiol. Behav. 84, 697—706. Grippo, A.J., Moffitt, J.A., Johnson, A.K., 2002. Cardiovascular alterations and autonomic imbalance in an experimental model of depression. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R1333—R1341.

240 Gutkowska, J., Paquette, A., Wang, D., Lavoie, J.M., Jankowski, M., 2007. Effect of exercise training on cardiac oxytocin and natriuretic peptide systems in ovariectomized rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R267—R275. Hofmann, U., Heuer, S., Meder, K., Boehler, J., Lange, V., Quaschning, T., Ertl, G., Bonz, A., 2007. The proinflammatory cytokines TNF-alpha and IL-1 beta impair economy of contraction in human myocardium. Cytokine 39, 157—162. Holsboer, F., 2000. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23, 477—501. Inagaki, H., Kuwahara, M., Tsubone, H., 2004. Effects of psychological stress on autonomic control of heart in rats. Exp. Anim. 53, 373—378. Iyer, V., Edelman, E., Lilly, L., 2006. Pathophysiology of heart disease: a collaborative project of medical students and faculty. In: Lilly, L. (Ed.), Pathophysiology of Heart Disease. Lippincott Williams & Wilkins, p. 473. Johnson, J.D., O’Connor, K.A., Hansen, M.K., Watkins, L.R., Maier, S.F., 2003. Effects of prior stress on LPS-induced cytokine and sickness responses. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R422—R432. Kim, E.M., Grace, M.K., Welch, C.C., Billington, C.J., Levine, A.S., 1999. STZ-induced diabetes decreases and insulin normalizes POMC mRNA in arcuate nucleus and pituitary in rats. Am. J. Physiol. 276, R1320—R1326. Lesperance, F., Frasure-Smith, N., 2000. Depression in patients with cardiac disease: a practical review. J. Psychosom. Res. 48, 379—391. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2( Delta Delta C(T)) method. Methods 25, 402—408. Lumbers, E.R., 1999. Angiotensin and aldosterone. Regul. Pept. 80, 91—100. Maes, M., 1995. Evidence for an immune response in major depression: areview and hypothesis. Prog. Neuropsychopharmacol. Biol. Psychiatry 19, 11—38. Mekontso-Dessap, A., Brochard, L., 2006. BNP in intensive care unit: what prospect for the patient under mechanical ventilation. Re ´animation 15, 48—54. Michener, M.L., Gierse, J.K., Seetharam, R., Fok, K.F., Olins, P.O., Mai, M.S., Needleman, P., 1986. Proteolytic processing of atriopeptin prohormone. Mol. Pharmacol. 30, 552—557. Miller, G.E., Chen, E., Sze, J., Marin, T., Arevalo, J., Doll, M., Ma, R., Cole, S.W., 2008. A functional genomic fingerprint of chronic stress in humans: blunted glucocorticoid and increased NF-kappaB signaling. Biol. Psychiatry 64, 266—272. Mockel, M., Muller, R., Vollert, J.O., Muller, C., Carl, A., Peetz, D., Post, F., Kohse, J.K., Lackner, K.J., 2005. Role of N-terminal proB-type natriuretic peptide in risk stratification in patients presenting in the emergency room. Clin. Chem. 51, 1624—1631. Musselman, D.L., Evans, D.L., Nemeroff, C.B., 1998. The relationship of depression to cardiovascular disease: epidemiology, biology, and treatment. Arch. Gen. Psychiatry 55, 580—592. O’Donoghue, M., Januzzi Jr., J.L., 2005. N-terminal proBNP: a novel biomarker for the diagnosis, risk stratification and management of congestive heart failure. Expert Rev. Cardiovasc. Ther. 3, 487—496. Packer, M., 2001. Current role of beta-adrenergic blockers in the management of chronic heart failure. Am. J. Med. 110, 81—94. Politi, P., Minoretti, P., Piaggi, N., Brondino, N., Emanuele, E., 2007. Elevated plasma N-terminal ProBNP levels in unmedicated patients with major depressive disorder. Neurosci. Lett. 417, 322—325. Raison, C.L., Capuron, L., Miller, A.H., 2006. Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol. 27, 24—31.

B.P. Wann et al. Redei, E., Ahmadiyeh, N., Baum, A., Sasso, D., Slone, J., Solberg, L., Will, C., Volenec, A., 2001. Novel animal models of affective disorders. Semin. Clin. Neuropsychiatry 6, 43—67. Reichenberg, A., Yirmiya, R., Schuld, A., Kraus, T., Haack, M., Morag, A., Pollmacher, T., 2001. Cytokine-associated emotional and cognitive disturbances in humans. Arch. Gen. Psychiatry 58, 445—452. Ruskoaho, H., 1992. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol. Rev. 44, 479—602. Shanshan, P., Yan, Z., Aiyun, L., Chen, P., 2005. Effect of exercise on gene expression of atrial natriuretic peptide receptor of kidney. Life Sci. 76, 1921—1928. Shigemi, K., Morimoto, T., Itoh, T., Natsuyama, T., Hashimoto, S., Tanaka, Y., 1991. Regulation of vascular compliance and stress relaxation by the sympathetic nervous system. Jpn. J. Physiol. 41, 577—588. Silberman, D.M., Wald, M., Genaro, A.M., 2002. Effects of chronic mild stress on lymphocyte proliferative response. Participation of serum thyroid hormones and corticosterone. Int. Immunopharmacol. 2, 487—497. Sullivan, D.R., West, M., Jeremy, R., 2005. Utility of brain natriuretic peptide (BNP) measurement in cardiovascular disease. Heart Lung Circ. 14, 78—84. Takemura, G., Takatsu, Y., Doyama, K., Itoh, H., Saito, Y., Koshiji, M., Ando, F., Fujiwara, T., Nakao, K., Fujiwara, H., 1998. Expression of atrial and brain natriuretic peptides and their genes in hearts of patients with cardiac amyloidosis. J. Am. Coll. Cardiol. 31, 754— 765. Tannenbaum, B., Tannenbaum, G.S., Sudom, K., Anisman, H., 2002. Neurochemical and behavioral alterations elicited by a chronic intermittent stressor regimen: implications for allostatic load. Brain Res. 953, 82—92. Tavener, S.A., Kubes, P., 2006. Cellular and molecular mechanisms underlying LPS-associated myocyte impairment. Am. J. Physiol. Heart Circ. Physiol. 290, H800—H806. Tavener, S.A., Long, E.M., Robbins, S.M., McRae, K.M., Van Remmen, H., Kubes, P., 2004. Immune cell Toll-like receptor 4 is required for cardiac myocyte impairment during endotoxemia. Circ. Res. 95, 700—707. Tse, M.Y., Watson, J.D., Sarda, I.R., Flynn, T.G., Pang, S.C., 2001. Expression of B-type natriuretic peptide in atrial natriuretic peptide gene disrupted mice. Mol. Cell. Biochem. 219, 99—105. Weber, M., Mitrovic, V., Hamm, C., 2006. B-type natriuretic peptide and N-terminal pro-B-type natriuretic peptide–—diagnostic role in stable coronary artery disease. Exp. Clin. Cardol. 11, 99—101. Willner, P., Towell, A., Sampson, D., Sophokleous, S., Muscat, R., 1987. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology 93, 358—364. Witthaut, R., Busch, C., Fraunberger, P., Walli, A., Seidel, D., Pilz, G., Stuttmann, R., Speichermann, N., Verner, L., Werdan, K., 2003. Plasma atrial natriuretic peptide and brain natriuretic peptide are increased in septic shock: impact of interleukin-6 and sepsis-associated left ventricular dysfunction. Intensive Care Med. 29, 1696—1702. Worthman, C.M., Stallings, J.F., 1997. Hormone measures in fingerprick blood spot samples: new field methods for reproductive endocrinology. Am. J. Phys. Anthropol. 104, 1—21. Wright, C.E., Ebrecht, M., Mitchell, R., Anggiansah, A., Weinman, J., 2005. The effect of psychological stress on symptom severity and perception in patients with gastro-oesophageal reflux. J. Psychosom. Res. 59, 415—424. Zorn-Pauly, K., Pelzmann, B., Lang, P., Machler, H., Schmidt, H., Ebelt, H., Werdan, K., Koidl, B., Muller-Werdan, U., 2007. Endotoxin impairs the human pacemaker current If. Shock 28, 655— 661.