Maprotiline treatment differentially influences cardiac β-adrenoreceptors expression under normal and stress conditions

Alterations in cardiac function were observed in antidepressants treated patients and published in several clinical reports. These detected changes could be either a consequence of the treatment or of depression itself, which has already been proved to be a risk factor in heart diseases. In order to determine a possible influence of chronic treatment with norepinephrinergic reuptake inhibitor, maprotiline, on the heart, we investigated gene expression of cardiac β-adrenoceptors both in controls and in animals with signs of depression. The rats were divided into two groups, unstressed controls and those exposed to chronic unpredictable mild stress (CUMS). The groups were further divided into two subgroups, one receiving daily intraperitoneal injections of vehicle (sterile water) and another one maprotiline (10 mg/kg) for four weeks. Tissue samples were collected after the last application. Gene expression of cardiac β1and β2-adrenoceptor was determined using Real-time RT-PCR analysis. Our results show that in control animals expression of both adrenoreceptors was decreased in the right atria after 4 weeks of maprotiline application. Contrary, the same treatment led to a significant increase in expression of cardiac β1adrenoceptor in the stressed rats, with no change in the characteristics of β2-adrenoceptor. Our findings might reflect the that molecular mechanisms are underlying factors involved in the development of cardiovascular diseases linked with antidepressant treatment.


INTRODUCTION
Depression is associated with the autonomic nervous system dysfunction that could have a negative impact on cardiovascular function (Carney et al., 2005).Several studies have demonstrated an exaggerated norepinephrine response in major depression (Lake et al., 1982;Veith et al., 1994;Yehuda et al., 1998;Mausbach et al., 2005).Chronic exposure to elevated levels of catecholamines, released from sympathetic nerve terminals and the adrenal gland, may cause pathologic changes, resulting in alterations in cardiac structure and function (Brum et al., 2002, Lohse et al., 2003).Activation of the closely related β 1 -and β 2 -adrenergic receptors (β 1 -AR and β 2 -AR) by released catecholamines is the primary trigger of molecular changes in the heart.These two subtypes are expressed in cardiac tissue at a ratio of 70:30, and their activation lead to an increase in contractile force and heart rate (Wallukat, 2002).The ratio of β 1 -AR to β 2 -AR subtypes depends on the type of animals and it is modified in pathological conditions (Brodd, Michel, 1999).Both receptors are highly homologous and activate the G protein stimulatory for adenylyl cyclase (Gs),yet they have distinguishable biological effects (Xiang, Kobilka, 2003).Thus, the β 1 -AR plays the dominant role in stimulating heart rate and strength of myocyte contraction, whereas β 2 -AR produces only modest chronotropic effects.Furthermore, chronic stimulation of β 1 -AR produces myocyte hypertrophy and apoptosis, whereas β 2 -AR signaling promotes cell survival (Xiao et al., 2004).
Stress is a key etiological factor in anxiety and major depressive disorders (Caspi et al., 2003).The chronic unpredictable mild stress (CUMS) procedure is an animal model that mimics the role of chronic stress in precipitating depression and induces various long-term physical, behavioural, neurochemical and neuroendocrine alterations that resemble those observed in depressed patients, which are reversed only by chronic antidepressants treatments (Wilner et al., 1992;Mineur et al., 2006;Yalcin et al., 2007).Maprotiline is a antidepressant with an atypical tetracyclic structure, which function is to prevent reuptake by blocking norepinephrine transporter.It is commonly used in elderly patients suffering from depression as longterm medication (Ahles et al., 1984).In the past, there have been only several clinical reports on the rise in heart rate (Hewer et al., 1995) and proarrhythmia (Lentini et al., 2001;Zuchner, 2002) linked to maprotiline.
This raises a question of a possible influence of maprotiline on β-adrenergic receptors gene expression in the heart of these animals.Applying TaqMan RT-PCR we have estimated the influence of long-term treatment with norepinephrinergic reuptake inhibitor, maprotiline, on gene expression of β 1 and β 2 adrenoreceptors, in the right and left atria as well as in the ventricle of unstressed controls and rats exposed to CUMS for 4 weeks.

Animals and study design
Adult Wistar rat males weighing 280-320 g at the onset of experiments and maintained in a temperature--controlled room (21±1.0o C) and 12 h/12 h light/dark cycle, were used.Care was taken to minimise the pain and discomfort of the animals in accordance with the Guide for Care and Use of Laboratory Animals of the National Institute of Health, Bethesda, MD, U.S.A. Research investigations were approved by Ethical Committee of the "Vinca" Institute, Belgrade (Application No. 02/11).The rats were randomly divided into control (unstressed) and group subjected to CUMS according to the method by Grippo et al. (2006).These two groups were further divided into two subgroups each, and the animals were receiving daily injections of vehicle (sterile water) or maprotiline (10 mg/kg) by i.p. route.Exposure to CUMS and the vehicle, i.e. drug administration started on the same day and were continued for 4 weeks.Maprotiline (Sigma-Aldrich Chemie, Germany) solutions in sterile water, sonicated for approximately 10 min were prepared ex tempore.Upon completion of 4 weeks, the animals exposed to CUMS and the corresponding controls were decapitated, the right and left cardiac atria and ventricles rapidly dissected, frozen in liquid nitrogen and stored at −70 o C until analysed.

RNA isolation and cDNA synthesis
Total RNAs were isolated using TRIZOL reagent (Invitrogen, CA, U.S. A.). Reverse transcription was performed using Ready-To-Go You-Prime First-Strand Bead (AP, Biotech) and pd (N)6 primer according to manufacturer's protocol.

Real-time RT-PCR
TaqMan PCR assays were carried out using Assayon-Demand Gene Expression Products (Applied Biosystems, USA) for β 1 -AR (Rn 00824536_s1) and β 2 -AR (Rn 00560650_s1).The reactions were performed in a 25 µL reaction mixture containing 1x TaqMan Universal Master Mix with AmpErase UNG, 1x Assay Mix (Applied Biosystems) and cDNA template (10 ng of RNA converted to cDNA).PCR reactions were performed in the ABI Prism 7000 Sequence Detection System at 50 •C for 2 min, 95 •C for 10 min, followed by 40 cycles at 95 o C for 15 s and 60 o C for 1 min.A reference, endogenous control, was included in each analysis to correct the differences in the inter-assay amplification efficiency and all transcripts were normalised to cyclophyline A (ID:Rn 00690933) expression.Quantification was done using the 2 -ΔΔCt method according to Livak and Schmittgen (2001).

Statistical analysis
The results are reported as means ± S.E.M. Significance of the differences between the groups in gene expression levels of the examined β-AR were estimated by Two-way ANOVA test, followed by the Tukey post hoc test.Statistical significance was accepted at p < 0.05.

RESULTS
In our present study, we investigated the alterations in relative gene expression of β 1 -and β 2 -AR in the right and left atria and ventricles after chronic maprotiline application in rats exposed to CUMS for 4 weeks in comparison with unstressed controls.Results indicate that stress procedure induced a considerable effect (F (1, 20) = 17.87, p<0.001) on β 1 -AR gene expression in the left atria.Chronic stress increased β 1 -AR mRNA levels by 75% (p <0.01) in the left atria.On the other hand, antidepressant treatment affected β 1 -AR gene expression (F (1, 20) = 45.21,p<0.001) in the right atria.
In addition, maprotiline effect on the unstressed animals was quite different from that on the stressed ones.Thus, this antidepressant led to an increase in β 1 -AR mRNA levels by 224% (p<0.001) in the right atria of CUMS rats, but decreased it by 47% (p<0.001) in controls.Analysis of data also displayed a significant interaction (F (1, 20) = 9.56, p<0.01) between effects of chronic stress and antidepressant treatment on β 1 -AR mRNA levels in the left ventricle.In animals subjected to CUMS for 4 weeks and parallelly treated with maprotilin, β 1 -AR mRNA levels were increased by 76% (p<0.01) in the left ventricle (Fig. 1).

DISCUSSION
Antidepressant prescriptions are often associated with an increased risk of heart disease (Rosenberg et al., 2010).Tata et al. (2005) reported that this risk is more likely associated to factors relating to depression itself than to specific adverse drug effects.Therefore, we examined whether the effect of maprotiline treatment on gene expression of β 1 and β 2 -adrenoceptor in the heart of animals with depressive symptoms, which were induced by the 4 weeks exposure to CUMS, differed from that in unstressed rats.In control animals expression of both adrenoreceptors was decreased in right atria after four weeks of maprotiline application.Down-regulation of the β-adrenergic receptors appears to be a common effect of most tricyclic antidepres- sants (Duman et al., 1997;Deupree et al., 2007).This may be related to adaptive processes regarded as a secondary effect to the increased concentration of norepinephrine in the synaptic cleft.Similarly, prolonged agonist exposure decreases both β 1 -AR binding activity and β 1 -AR mRNA levels (Dunigan et al., 2002).Port et al.(1992) identifed a 35 kDa β-AR mRNA-binding protein (β-ARB protein) which is involved in agonist-induced destabilization of β 2 -AR mRNA.They have shown that the amount of β-ARB protein correlates inversely with the decrease in β 2 -AR mRNA during the treatment with agonists in hamster heart cells.Antidepressants might also affect β-adrenergic receptors transcription that is independent of the inhibition of norepinephrine uptake.An intact Sp1 site is required for the full activity of the β 1 -AR promoter in heart cells.The conservation of this binding site across mammalian β 1 -AR genes suggests that this element is crucial for the β 1 -AR gene expression regulation (Bahouth et al., 2002).It has been observed that chronic desipramine produced a significant reduction in Sp1-binding activity (Frechilla et al., 1998).In our experiments, as a result of exposure to CUMS, a significant increase in β 1 -and β 2 -adrenoceptor mRNA levels was recorded in left atria whereas β 2 -ARs mRNA levels were increase only in left ventricles.According to Ueyama and colleagues (2003) activation of β-ARs is the primary trigger of emotional stress-induced molecular changes in the heart.Glucocorticoids are known to affect expression of these receptors through GRE sequences in promotor region (Malbon, Hadcock, 1988).In these animals, as previously shown, plasma corticosterone levels were elevated (Dronjak et al., 2007). Similarly, Misliveček et al. (2003) noticed that after hydrocortisone treatment density of both β 1 -AR and β 2 -AR was increased.In contrast to control group of animals, maprotiline treatment led to a significant increase in the expression of β 1 -AR but remained without effect on β 2 -AR expression in stressed rats.Chronic antidepressant treatment, which enhances norepinephrine synaptic transmission, was shown to increase phosphorylation of CREB and CRE-mediated gene expression in several brain regions (Thome et al., 2000;Abdel-Razaq et al., 2007).Tseng and co-workers (1998) demonstrated that cAMP mediates the induction of β 1 -AR gene expression by interacting with CRE within the promoter region.It has been also observed that glucocorticoids, which are present during chronic stress, interacting with transcription factor CREB, can prevent downregulation of β 2 -AR number and mRNA expression (Mak et al., 1995;Adcock et al., 1996).According to Dangel and co-workers (1996), glucocorticoids also induce changes in the β 2 -AR RNA stability by reducing the amount of β-ARB protein.In addition, antidepressants could also regulate mRNA stability.The studies conducted by Headley and coworkers (2004) demonstrated that stimulation of mitogenactivated protein kinases-MAPKs (JNK, p38) resulted in marked stabilization of β-AR mRNA.Budziszewska et al. (2010) observed that chronic treatment with antidepressant drugs attenuated the stress-induced decreased levels of MAPKs in the brain of rats subjected to prenatal stress, but had no effect on its concentration in control animals.Longer half-life may explain the higher levels of β 1 -AR mRNA in stressed treated animals, in comparison to placebo group.The results of the mentioned authors can explain the different gene expression of these receptors in normal and stressed conditions.Norepinephrine-promoted destabilization and stress-induced transcription seem to be underlying factors for the interplay of the two opposing pathways controlling receptor mRNA levels.Further experiments on transcriptional activation and mRNA stability will be required to unravel the complexity of stress-and antidepressant-dependent regulation of beta-adrenoceptor gene expression.In chronically stressed individuals, treated with maprotiline, the increased expression of this receptor might be a prerequisite in the development of cardiovascular disease given that it initiates irreversible damage to cardiac tissue.

FIGURE 1 -
FIGURE 1 -The effects of chronic maprotiline treatment on β 1 -adrenoceptors mRNA levels in left and right cardiac atrium and ventricle of unstressed and CUMS rats.The values are means ± S.E.M. of 7 rats.Statistical significance: ## p< 0.01; ### p< 0.001 CUMS vs. control (Tukey-test); **p< 0.01; ***p< 0.001 maprotiline vs. vehicle (Tukey-test).The final result was expressed as fold change relative to the calibrator and normalized to cyclophyline A.

FIGURE 2 -
FIGURE 2 -The effects of chronic maprotiline treatment on β 2adrenoceptors mRNA levels in left and right cardiac atrium and ventricle of unstressed and CUMS rats.The values are means ± S.E.M.of 7 rats.Statistical significance: # p<0.05; ## p<0.01 CUMS vs. control (Tukey-test); *p< 0.05 maprotiline vs. vehicle (Tukey-test).The final result was expressed as fold change relative to the calibrator and normalized to cyclophyline A.