Effect of escitalopram on cardiomyopathy-induced anxiety in mice

Abstract

The present study was aimed to evaluate the effect of escitalopram on anxiety following doxorubicin (DOX)-induced cardiomyopathy, a rodent model for heart failure (HF), in mice. The study was carried out in Swiss albino mice. DOX was used at a dose of 10 mg/kg intravenously. Escitalopram was administered at the doses of 10 and 20 mg/kg orally for 7 days pre- and 7 days post-DOX. Anxiety was measured on day 8 and on day 14 using elevated plus maze and Vogel’s conflict test. On day 14, serum lactate dehydrogenase (LDH) was estimated. The mice were then killed and their hearts were dissected out for the estimation of malondialdehyde (MDA) and for the transmission electron microscopic (TEM) studies. Our results showed that the DOX administration induced cardiomyopathy in mice. This was evidenced by the increased levels of serum LDH and tissue MDA and was also confirmed by TEM. Escitalopram (20 mg/kg) not only reversed the anxiety-like effects induced by DOX but also DOX-induced increase in LDH and MDA as well as the morphological alterations induced by DOX in TEM studies. Escitalopram, thus, appears to be a good candidate for alleviating anxiety in patients with HF.

Introduction

Psychosocial problems including anxiety and depression in patients with heart failure (HF) serve as a major risk factor for further cardiac events and impede recovery. ^{1 ,2} The effective psychological management in HF has generally focussed on depression, with prevalence rates estimated at 17–85%. ³ There is considerably less literature on anxiety (than on depression) in patients after myocardial infarction (MI) and/or HF, even though both of these are highly comorbid disorders. ⁴ Furthermore, the prevalence of anxiety may be as high as 60% in patients with HF. ⁵ Such behavioural changes in the animal models of MI/HF may be used to represent the aspects of reduced quality of life in patients following HF. ⁶

Doxorubicin (DOX), a broad spectrum quinine-containing anthracycline antibiotic and an extremely effective antitumour drug, is used to treat a variety of neoplastic diseases as well as a range of solid tumours. However, its clinical application is limited by its adverse effects, primarily cardiomyopathy and congestive HF. ⁷ More frequently, DOX causes cardiomyopathy that manifests as congestive HF, and thus DOX is commonly employed as a model of HF in rats. Earlier, we have shown that the DOX also produces cardiomyopathy in a mouse model and thus can be used as a model of HF in mice. ⁸

The drug treatment for anxiety in patients with HF should ensure cardiovascular safety. A drug with dual benefit (both cardiovascular and anxiolytic effects) is desirable. In this study, we evaluated the effect of escitalopram (S-citalopram), a selective serotonin reuptake inhibitor (SSRI), on anxiety and cardiomyopathy in the DOX model of HF in mice. We selected escitalopram for the present study due to the fact that the drug is reported to cause both antidepressant and anxiolytic-like effects, ⁹ and it has a more favourable cardiovascular safety profile when compared with other SSRIs. ¹⁰

Materials and methods

Animals

Swiss strain male albino mice (25–35 g) raised at the Central Animal House Facility of Jamia Hamdard were used. The animals were housed in polypropylene cages (eight per cage) with a natural light–dark cycle. The mice were fed on a standard pellet diet (Amrut rat and mice feed, Pune, India) and water ad libitum. The protocol was approved by the Institutional Animal Ethics committee of Hamdard University (Registration No. 173/CPCSEA; January 28, 2000), New Delhi. Utmost care was taken to ensure that the animals were treated in the most humane and ethically acceptable manner.

Experimental procedure

Escitalopram (C-PRAM ; Unichem Laboratories Ltd, Mumbai, India) was administered at the doses of 10 and 20 mg/kg orally (p.o.) 7 days pre- and post-DOX. DOX (ADRIM; Dabur Pharmaceuticals Ltd, Ghaziabad, India) was used at a dose of 10 mg/kg intravenously (i.v.), which is being the dose that produced cardiotoxicity in 100% of toxic animals without mortality. This dose was standardized in our laboratory. ⁸ Animals were randomly divided into five groups having eight animals in each group: Group I comprised animals treated with normal saline (1 ml/kg); group II comprised animals treated with DOX only (10 mg/kg on day 7); group III and IV comprised animals treated with escitalopram (10 and 20 mg/kg, respectively) 7 days pre- and 7 days post-DOX (10 mg/kg); group V comprised animals treated with escitalopram per se (20 mg/kg).

Assessment of anxiety

Anxiety was measured on day 8 (before starting the posttreatment) and on day 14 using the following methods.

Elevated plus maze

Briefly, in the method of elevated plus maze (EPM), the mice were placed in the central square of the maze, and the total time spent by the animal in the open or enclosed arm and the number of open or closed arm entries were recorded during a 5-min observation period. Confinement to closed arm was associated with more anxiety-related behaviour as per the method of Pellow et al. ¹¹

Vogel’s conflict test

Vogel’s conflict test (VCT) was performed using an anxiometer (Coulbourne, Whitehall, Pennsylvania, USA). In this method, animals deprived of water for 18 h were put into separate chambers, and they were allowed to drink water freely for 40 min (the habituation procedure). A few days later, the same animals, again deprived of water for 18 h prior to the test were put into the chambers 30 min after the administration of the drug or their vehicle. The mice were allowed to access water from the spout in the chamber, and the number of licks of the spout was counted simultaneously in the chamber for 30 min. At every 20th lick, a mild shock (0.1 mA) through the grid was given, and the number of licks during the 30-min period was recorded as per the method of Umezu. ¹²

Biochemical and ultrastructural studies

On day 14, blood was collected from the tail vein of mice, and the level of lactate dehydrogenase (LDH) was estimated in the serum following the methods of Bergmeyer ¹³ and Lum and Gambino. ¹⁴ The mice were then killed and their hearts were dissected out, and the level of malondialdehyde (MDA) was measured following the method described in a previous study. ¹⁵ The protein content was determined following the method of Lowry et al. ¹⁶ Transmission electron microscopy was performed using the electron microscope (Philips, CM-10).

Statistical analysis

The results were analysed by analysis of variance followed by Dunnett’s t test. Statistical analyses were performed using Graphpad Prism 3.0 (San Diego, California, USA). p < 0.05 were considered as statistically significant.

Results

Elevated plus maze

DOX (10 mg/kg i.v.) reduced the number of open arm entries and time spent in open arms after pretreatment (i.e. on day 8). However, on day 14, the number of open arm entries were reduced significantly (p < 0.05) indicating anxiety. Escitalopram (10 and 20 mg/kg p.o.) significantly reversed the reduction in the number of open arm entries and the time spent in open arms after pre- and post- (i.e. on day 8 and 14) DOX treatment, where the dose of 20 mg/kg p.o. being more effective. However, ESC (20 mg/kg p.o.) per se did not exhibit significant anxiolytic-like effects when compared with normal control (Figure 1(a) and (b)).

Figure 1.

Effect of escitalopram on the number of open arm entries (a) and the time spent on open arms (b) in elevated plus maze on day 8 (pretreatment) and day 14 (posttreatment) following DOX in mice. DOX was administered intravenously, while ESC was given orally for 14 days (7 days pre- and 7 days post-DOX). Each group has a total number of eight animals. All the values were expressed as mean ± SEM. *p < 0.05 versus group I (normal); ^## p < 0.01, ^# p < 0.05 versus group II (toxic control). DOX: doxorubicin; ESC: escitalopram.

Vogel’s conflict test

DOX (10 mg/kg i.v.) significantly reduced the number of licks and shocks in Vogel’s conflict test (p < 0.01). Escitalopram (10 and 20 mg/kg p.o.) significantly reversed the reduction in the number of licks and shocks after pre- and post-DOX, where the dose of escitalopram (20 mg/kg p.o.) being more effective and statistically significant. ESC (20 mg/kg p.o.) per se did not show significant anxiolytic-like effect when compared with normal control (Figure 2(a) and (b)).

Figure 2.

(a), (b): Effect of escitalopram on the number of licks and shocks received by mice in Vogel conflict test on day 8 (pretreatment) and day 14 (posttreatment) following DOX. DOX was administered intravenously, while ESC was given orally for 14 days (7 days pre- and 7 days post-DOX). Each group has a total number of eight animals. All the values were expressed as mean ± SEM. *p < 0.05 versus group I (normal control); ^## p < 0.01, ^# p < 0.05 versus group II (toxic control). DOX: doxorubicin; ESC: escitalopram.

LDH and MDA

DOX (10mg/kg i.v.) significantly raised the levels of serum LDH (p < 0.01) and tissue lipid peroxidation marker MDA (p < 0.01). Escitalopram at both the doses significantly (p < 0.01) reversed the serum LDH level, while at 20 mg/kg p.o., it also reversed the raised MDA levels (Figure 3(a) and (b)).

Figure 3.

(a), (b): Effect of escitalopram on LDH and tissue MDA levels in DOX-induced cardiomyopathy in mice. DOX was administered intravenously, while ESC was given orally for 14 days (7 days pre- and 7 days post-DOX). Each group has a total number of eight animals. All the values were expressed as mean ± SEM. **p < 0.01, *p < 0.05 versus group I (normal control); ^# p < 0.05, ^## p < 0.01 versus group II (toxic control). DOX: doxorubicin; ESC: escitalopram; LDH: lactate dehydrogenase; MDA: malondialdehyde.

Ultrastructural changes

Significant morphological changes including cytoplasmic vacuolisation (V), loss of myofibrils and nuclear chromatin migration were observed in DOX group (Figure 4(c) and (d)) when compared with normal control (Figure 4(a) and (b)). DOX + ESC-treated group and ESC per se group showed normal mitochondria and nucleus with normal membrane and fewer condensed pieces of coarse chromatin clumping (Figure 4(e) to (i)).

Figure 4.

Electron micrograph of mice myocardium of normal control group showed normal mitochondria (M), myofibrils (MF) and nucleus (N) (magnification of (a) and (b) is 0.89 × 10,000 and 0.71 × 10,000, respectively). DOX-treated groups showing myofibrils with vacuoles and chromatin migration at nuclear membrane (magnification of (c) and (d) is 0.56 × 10,000 and 0.56 × 10,000, respectively), DOX + ESC (10 + 10 mg/kg)-treated groups showed normal mitochondria, myofibril integrity and nuclear membrane with little chromatin migration at nuclear membrane (magnification of (e) and (f) is 0.44 × 10,000 and 0.56 × 10,000, respectively), DOX + ESC (10 + 20 mg/kg)-treated groups showed normal nucleus, mitochondria and normal myofibrils (magnification of (g) and (h) is 0.56 × 10,000 and 0.71 × 10,000, respectively), ESC per se-treated groups showed normal mitochondria and integrity of myofibrils (magnification of (i) and (j) is 0.71 × 10,000 and 0.89 × 10,000, respectively).

Discussion

The present study evaluated the effects of escitalopram on DOX-induced anxiety and cardiomyopathy in mice. DOX induced anxiety-like effects in two commonly used methods to evaluate anxiety (EPM and VCT) in experimental animals. After receiving DOX, mice exhibited a reduction in the number of open arm entries, time spent in open arms and anxiety-like behaviour in open arms such as freezing, immobility, defecation in EPM and a reduction in the number of licks and shocks in VCT. Statistically significant effects on anxiety were observed only after 7 days post-DOX. Present findings are consistent with our previous observations following DOX administration in mice. ⁸

Activation of sympathetic nervous system and hypothalamic pituitary adrenal axis are the physiological responses that occur in conjunction with anxiety. ⁵ The sympathoadrenal hyperactivity, the exaggerated platelet reactivity and the alterations in the platelet serotonin system are hazardous in such patients. Thus, effective management with anxiolytic drugs that act by lowering serotonergic activation may exert beneficial effects in sympathoadrenal hyperactivity and exaggerated platelet reactivity in patients with cardiovascular disease. In our study, pre- and posttreatment of escitalopram (10 and 20 mg/kg p.o.) could reverse DOX-induced behavioural changes in EPM and VCT, the dose of 20 mg/kg being more effective. The anxiolytic effects of escitalopram have been shown by other workers in a number of other rodent models of anxiety such as foot-shock-induced ultrasonic vocalization in rats, exploratory behaviour in black and white box model in both mice and rats and escape behaviour induced by the stimulation of dorsal periaqueductal grey matter. ¹⁷ Anxiety-like behaviour in the EPM is due to a chronic decrease in serotonergic activity leading to the upregulation of 5 HT_1A receptors. ¹⁸ Thus, the antianxiety effects of escitalopram in EPM could be attributed to the increased levels of serotonin, which by acting on presynaptic 5 HT_1A receptor, causing hypoactivity of serotonergic system, where anxiety is being associated with hyperactivity of serotonergic neurons. ^{19 ,20}

Postinfarct treatment is aimed mainly at improving life expectancy, whereas less attention is paid to psychosocial issues including anxiety. Since anxiety is considered to be a major risk factor for further cardiac events, it is important to manage anxiety with drugs having a favourable effect on the cardiovascular system. We induced cardiomyopathy in mice by a single i.v. injection of DOX (10 mg/kg). There was a significant increase in the serum LDH enzyme. It is possible that the rapid swelling of sarcolemmal bulb and injury to the myocardium caused the cytosolic enzyme (LDH) to leak out from injured heart muscle cells into the blood stream. ^21,22 The electron microscopic studies of mice myocardium further confirmed the myocardial damage by exhibiting cell shrinkage, condensation of chromatin and its migration at the nuclear membrane, extensive vacuolisation and distortion of myofibrils in the myocardium of DOX-treated group. DOX-induced cardiomyopathy is considered to be multifactorial mainly due to the increased generation of free radicals, lipid peroxidation and decreased myocardial endogenous antioxidants. ²³ Recently, it has also been reported that chronic cardiotoxicity of DOX is due to apoptosis (i.e. programmed cell death) of cardiomyocytes. ²⁴ In our study, cardiotoxicity was evidenced by an increase in lipid peroxidation measured as MDA in the-DOX treated group. Escitalopram (10 and 20 mg/kg, p.o.) when given for 14 days (7 days each for pre- and posttreatment) reduced the raised levels of LDH and MDA as well as restored the myocardial cells integrity as evidenced by electron microscopic studies. Our results are consistent with the antioxidant effects of escitalopram as reported earlier, although not in the heart. ²⁵ Although the exact mechanism of cardioprotective effects of escitalopram observed in the present study is not clear, it could be related to the antiplatelet effects of escitalopram. Serotonin is normally released by activated platelets, causing enhanced platelet aggregation, which may further contribute to the pathogenesis of acute MI. ²⁶ SSRIs with high affinity for serotonin transporters attenuate platelet activation by depleting serotonin storage, ²⁷ and have been shown to decrease platelet activity in patients with coronary artery disease. ²⁸ The antiplatelet activity of SSRIs, most of which have higher affinity for serotonin transporter, has been implicated to lower the risk of MI, ²⁹ where escitalopram is being the most selective SSRI inhibiting the serotonin transporter. ⁹

Conclusions

The anxiety-like behaviour of a cardiotoxic mouse was improved by escitalopram. Furthermore, the drug exhibited potential for cardioprotective effects against DOX-induced cardiomyopathy in mice. However, additional investigations are required to ascertain the effects in other rodent models of HF and anxiety and also to determine if other serotonergic antidepressants have similar effects.