MASARYK UNIVERSITY FACULTY OF MEDICINE DEPARTMENT OF PHYSIOLOGY

HALOPERIDOL AFFECTS THE HEART IN TWO ANIMAL MODELS

Ph.D. Thesis in a Field of Physiology and Pathological Physiology

Supervisor Author: Prof. MUDr. Marie Nováková, Ph.D. MUDr. Tibor Stračina

Brno 2019

MASARYKOVA UNIVERZITA LÉKAŘSKÁ FAKULTA FYZIOLOGICKÝ ÚSTAV

HALOPERIDOL OVLIVŇUJE SRDCE VE DVOU ANIMÁLNÍCH MODELECH

Disertační práce v oboru Fyziologie a patologická fyziologie

Školitelka: Autor: Prof. MUDr. Marie Nováková, Ph.D. MUDr. Tibor Stračina

Brno 2019

Abstract

Introduction and Aims. Haloperidol is a potent antipsychotic drug, which is associated with QT interval prolongation. The long QT increases risk of ventricular arrhythmias and sudden cardiac death. The mechanism of haloperidol-induced QT prolongation has not been fully described yet. The thesis was aimed to analyse effects of haloperidol on the QTc interval in two animal models – long-term haloperidol treated guinea pig model and rat neurodevelopmental model of schizophrenia. Methods. To guinea pigs, haloperidol (2 mg/kg) or vehiculum were administered for 21 consecutive days intraperitoneally. To pregnant female rats on gestational day 17, methylazoxymethanol acetate (MAM) or vehiculum were administered intraperitoneally. The offspring of MAM-treated females manifested schizophrenia-like phenotype. In the study, male adult offspring at the age of 30 weeks were used. In order to record electrogram, the heart was isolated and perfused according to Langendorff. After stabilisation, isolated heart was exposed to haloperidol in concentration of 10 nmol/L and subsequent washout. In guinea pig model, QT was measured by automatic detection. The QT/RR coupling was analysed. The QT was corrected according to subject specific model. In rat model of schizophrenia, QT was measured manually and corrected according to Framingham formula. To elucidate the mechanisms of observed effects, gene expression analyses, biochemical and histological analyses and cell line studies were performed. Results. In isolated guinea pig heart, the long-term haloperidol administration lead to decrease of the QT/RR coupling. No significant change of QTc calculated according to subject specific model was detected in stabilisation. Long-term haloperidol administration lead to increase of the expression of sigma 1 receptors and IP3 receptors type 1 and 2 in atria but not in ventricles of guinea pig heart. In the hearts of rat model of schizophrenia, no structural change was detected. In isolated heart of this model, a trend to longer QTc interval (when compared to controls) was detected in stabilization. After acute haloperidol administration, QTc was significantly higher in schizophrenia-like rats than in controls. Conclusion. Based on these results, it may be hypothesised that sigma 1 receptor represents important component in the mechanisms of haloperidol-induced QT prolongation. To study these mechanisms, it is important to identify novel animal models with high translational potential. The rat neurodevelopmental model of schizophrenia seems to be a good candidate.

Keywords guinea pig – haloperidol – isolated heart – QT prolongation – rat – schizophrenia

Abstrakt

Úvod a cíle. Haloperidol je účinné antipsychotikum, často zmiňované v souvislosti s prodloužením QT intervalu. Dlouhé QT zvyšuje riziko vzniku komorových arytmií a náhlé srdeční smrti. Mechanismus haloperidolem indukovaného prodloužení QT dosud nebyl plně objasněn. Tato disertační práce byla zaměřena na analýzu účinků haloperidolu na QTc interval ve dvou animálních modelech – modelu morčete dlouhodobě vystaveného haloperidolu a neuro-vývojovém modelu schizofrenie u potkana. Metody. Morčatům byl intraperitoneálně podáván haloperidol (2 mg/kg) nebo vehikulum po dobu 21 po sobě následujících dní. Samicím potkana byl 17. den březosti podán methylazoxymethanol acetát (MAM) nebo vehikulum. Potomci MAM-exponovaných samic vykazovali schizofrenii podobný fenotyp. V studii byli použiti pouze samci ve věku 30 týdnů. Srdce bylo izolováno, perfundováno podle Langendorffa a byl registrován elektrogram. Po stabilizaci byl na izolované srdce aplikován haloperidol v koncentraci 10 nmol/l a následně byl vymyt. U modelu morčete bylo QT měřeno pomocí automatické detekce. Byl analyzován QT/RR coupling. QT bylo korigováno podle subjekt-specifického modelu. U modelu schizofrenie u potkana bylo QT měřeno manuálně a korigováno podle Framinghamovy rovnice. Dále byly provedeny analýzy genové exprese, biochemické a histologické analýzy a studie na buněčné linii, ve snaze popsat mechanismy pozorovaných vlivů. Výsledky. Dlouhodobé podávání haloperidolu vedlo u izolovaných srdcí morčat ke snížení QT/RR couplingu. U izolovaných srdcí těchto morčat nebyl v stabilizaci nalezen signifikantní rozdíl v QT korigovaném podle subjekt-specifického modelu. U modelu morčete vedlo dlouhodobé podávání haloperidolu ke zvýšení exprese sigma 1 receptoru a IP3 receptorů typu 1 a 2 v srdečních síních, v komorách žádná změna zaznamenána nebyla. V srdcích neuro- vývojového modelu schizofrenie u potkana nebyla nalezena žádná strukturální změna. U izolovaného srdce tohoto modelu byl zjištěn trend k prodloužení QTc intervalu (ve srovnání s kontrolou) ve stabilizaci. Po akutní expozici haloperidolu bylo detekováno výrazně delší QTc u potkanů se schizofrenním fenotypem než u kontrolních zvířat. Závěr. Na základě těchto výsledků lze předpokládat, že sigma 1 receptor představuje důležitou součást v mechanismech haloperidolem navozeného prodloužení QT. Pro studium těchto mechanismů je důležité identifikovat nové zvířecí modely s vysokým translačním potenciálem. Neuro-vývojový model schizofrenie u potkana se zdá být dobrým kandidátem.

Klíčová slova haloperidol – izolované srdce – morče – potkan – prodloužení QT – schizofrenie

I hereby declare that I worked on this thesis independently under the supervision of Prof. MUDr. Marie Nováková, Ph.D. and that I used only sources listed in the bibliography.

………………………………. MUDr. Tibor Stračina

I would like to thank mostly to my supervisor Prof. MUDr. Marie Nováková, Ph.D. for her support and patient guidance in my first steps in laboratory as well as in academia, for her fruitful and beneficial discussions over my work, and for encouraging me in my personal and professional development. I would like to thank to Prof. PharmDr. Petr Babula, Ph.D., the head of the Department of Physiology, for creating a pleasant and stimulating working environment. I would like to thank to Mrs. Branislava Vyoralová and Mgr. Jaroslav Nádeníček for their technical help and support. I would also like to thank to all my students for their inspiring questions and to all my colleagues and collaborators with whom I had the honour to cooperate. Last but not least, I would like to thank to my parents and all my beloved for their love, patience and support during my studies.

This thesis was written at the Masaryk University as a part of the projects number MUNI/A/0957/2013, MUNI/A/1326/2014, MUNI/A/1365/2015, MUNI/A/1355/2016, MUNI/A/1157/2017, and MUNI/A/1255/2018 with the support of the Specific University Research Grant, as provided by the Ministry of Education, Youth and Sports of the Czech Republic.

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Contents

1 Introduction ...... 10 1.1 Association between Schizophrenia and Cardiovascular Diseases ...... 10 1.1.1 Antipsychotic Treatment and Its Adverse Effects ...... 12 1.1.2 Genetic Aspects ...... 14 1.2 Haloperidol: from Clinical Practice back to Basic Research ...... 15 1.2.1 Cardiovascular Adverse Effects of Haloperidol Treatment ...... 17 1.2.2 Haloperidol in Basic Research – with Respect to the Heart ...... 17 1.2.2.1 Sigma Receptors ...... 18 1.2.2.2 Haloperidol and Voltage Gated Ion Channels ...... 19 2 Aims ...... 20 3 Materials and Methods ...... 21 3.1 Chemicals and Drugs ...... 21 3.2 Laboratory Animals ...... 21 3.2.1 Guinea Pig Model of Long-Term Haloperidol Administration ...... 22 3.2.2 Rat Model of Schizophrenia ...... 22 3.3 Isolated Heart Perfused According to Langendorff ...... 22 3.3.1 Evaluation of Preparation Ischemia and Damage of the Isolated Heart throughout the Experiment ...... 23 3.4 Electrogram Analysis ...... 24 3.4.1 R and T Automatic Detection. RR Interval and Heart Rate Measurement. QT Interval Measurement and Correction ...... 24 3.4.2 Incidence of Arrhythmias ...... 25 3.4.3 Analysis of the Relationship between QT and RR Intervals ...... 25 3.4.4 Heart Rate Variability Analysis ...... 25 3.5 Gene Expression Analysis ...... 26 3.5.1 RNA isolation, cDNA preparation ...... 26 3.5.2 Real-time reverse-transcription polymerase chain reaction ...... 26 3.6 Histological and Immunohistological Staining of the Heart Muscle ...... 26 3.7 Cell Line Experiments ...... 27 3.7.1 Analysis of Cell Viability ...... 27 3.7.2 Analysis of Reactive Oxygen Species Production and Lipoperoxidation...... 28

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3.8 Statistical Analysis ...... 28 4 Results ...... 29 4.1 Study A: Long-term Haloperidol Administration Affects Relationship between QT and

RR Intervals and Expression of Sigma 1 and IP3 Receptors ...... 29 4.1.1 Heart rate, QTc interval and incidence of arrhythmias ...... 29 4.1.2 Relationship between QT and RR Intervals ...... 29

4.1.3 Expression of Sigma 1 and IP3 Receptors ...... 33 4.1.4 Preparation Ischemia and Biochemical Analysis ...... 33 4.2 Study B: Prenatal Exposure to MAM Affects Rat Heart and Its Reactivity to Haloperidol ...... 34 4.2.1 Body Weight, Heart Weight, HW/BW Ratio ...... 34 4.2.2 Heart Muscle Structure and Collagen Content ...... 34 4.2.3 Isolated Heart Electrophysiology ...... 34 4.2.3.1 Heart Rate, QTc Interval ...... 34 4.2.3.2 Heart Rate Variability ...... 35 4.2.3.3 Incidence of Arrhythmias ...... 37 4.2.4 Effect of MAM on Viability and Oxidative Status in H9c2 Cells ...... 38 5 Discussion ...... 39 5.1 Study A ...... 40 5.2 Study B ...... 42 6 Conclusion ...... 45 7 References...... 46 8 List of Abbreviations ...... 58 9 List of Figures ...... 59 10 List of Tables ...... 60 11 List of Appendices ...... 61 12 List of Author’s Publications ...... 62 12.1 Thesis-related Publications ...... 62 12.2 Publications Unrelated to the Thesis...... 63 13 Summary of the Thesis Findings ...... 66 14 Souhrn poznatků disertační práce ...... 67

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1 Introduction

Schizophrenia represents one of the major public health concerns. According to International Classification of Diseases (10th revision, in chapter F2),1 it is classified in psychotic disorders. The term schizophrenia was introduced by Eugen Bleuler in 1911.2 More than century of research, especially the last two decades, have brought many important parts into the schizophrenia puzzle. However, not all particular problems are fully resolved. Schizophrenia is a severe mental disease characterised by significant impairment of thinking, perception and emotions. It usually starts in young adulthood. The median global lifetime prevalence of schizophrenia is 4.0/1,000.3 Although schizophrenia is not a very frequent disease, it was listed by the World Health Organization in 2001 as the 8th most burdening disease in the age group 14 – 44 years worldwide according to disability-adjusted life year (DALY) metric.4 The burden of schizophrenia is progressively increasing with substantial economic and social consequences and significant impact on health in all countries of the world.5 In schizophrenia patients, life expectancy is by 15 – 20 years shorter in comparison with the healthy population.6-8 Several reasons for earlier mortality have been identified, including higher risk of suicide and accidents.9 However, the leading cause of excess mortality in schizophrenia patients is increased incidence of cardiovascular diseases.9-11

1.1 Association between Schizophrenia and Cardiovascular Diseases More than 50 years, professionals have uncovered cross-links between mental and cardiovascular diseases. In 1960s, Friedman and Rosenman described association of so-called type A behaviour pattern with coronary artery disease,12 which was supported by the Western Collaborative Group Study and the Framingham study.13 Today, personality type is generally accepted predictor of long-term mortality in patients with coronary artery disease.14,15 Among mental disorders, depression was the first identified as closely related to cardiovascular diseases.16-18 In schizophrenia patients, age and sex standardized incidence of cardiovascular diseases is as high as 18.0/1000 patient-years, which represents nearly three times higher incidence in comparison with non-schizophrenic population.19 Several factors are discussed as the origin of cardiovascular diseases development (Figure 1).20-22 Because of the nature of the disease, schizophrenia patients have tendency to unhealthy lifestyle. Negative symptoms including

| 11 amotivation and anhedonia lead to decreasing physical activity.23 Beside other factors (e.g. neurotransmitter and hormonal changes), physical inactivity contributes to development of obesity and metabolic syndrome in schizophrenia patients.24 Addictions to alcohol, nicotine and other addictive substances, which are very frequent among schizophrenia patients,25,26 may even worsen the situation. Moreover, physical disorders are frequently underdiagnosed and treated insufficiently due to social isolation and negative discrimination of the patients.27-29 Some of the above-mentioned factors are frequently accelerated by adverse effects of antipsychotic treatment.24,30-32

Figure 1: Association between schizophrenia and cardiovascular diseases. Adverse effects of antipsychotics significantly contributes to cardiovascular diseases development. The role of genetic factors has not been fully described yet. DM2 – diabetes mellitus type 2

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1.1.1 Antipsychotic Treatment and Its Adverse Effects Antipsychotics are psychoactive drugs mainly used for treatment of schizophrenia, schizoaffective disorder, acute mania and other psychotic disorders. Some of the drugs are also used in other indications, such as treatment of delirium, posttraumatic stress disorder, excessive vomiting, and chronic pain. Use of antipsychotics increases in most European countries.33 Figure 2 demonstrates such trend in consumption of olanzapine in the Czech Republic. Between years 2011 and 2017, it shows more than 2-fold increase. Antipsychotics are classified into two groups. Typical (first-generation) antipsychotics are available from 1950s34 and are represented (beside others) by haloperidol and chlorpromazine. They are primarily targeted on dopaminergic receptors. Atypical (second-generation) antipsychotic drugs (such as olanzapine, clozapine and risperidone) affect dopaminergic, serotonergic, adrenergic, and other receptor systems – in specific manner for each particular agent.35 Variety of pharmacological profiles allows treating patients with various psychotic symptoms. Although second-generation antipsychotics are usually well tolerated and severe extrapyramidal effects are rare,36 they are no more effective than the first-generation agents.37- 39

14,000,000

12,000,000

10,000,000 DD 8,000,000

Total D 6,000,000

4,000,000

2,000,000

0 2011 2012 2013 2014 2015 2016 2017

HALOPERIDOL OLANZAPINE

Figure 2: Total number of defined daily doses (DDD) of haloperidol and olanzapine prescribed in the Czech Republic between 2011 and 2017. Total number of DDD is given by the product of the number of delivered packages (distributor’s reports according to the Guideline DIS-13 of the State Institute for Drug Control, Czech Republic) and the number of defined daily doses in one package (according to classification of World Health Organization). Consumption of atypical antipsychotic olanzapine (light grey columns) shows increased trend while consumption of typical antipsychotic haloperidol (dark grey columns) remains almost constant in the Czech Republic. Source: State Institute for Drug Control (SÚKL), Czech Republic, March 2018.

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Antipsychotic treatment is associated with wide spectrum of adverse effects. Among the common adverse effects, non-specific effects such as headaches, dizziness and diarrhoea are reported. Neurological adverse effects are represented by extrapyramidal symptoms (such as dystonia, akinesia, akathisia, Parkinsonism, and tremor). Chronic antipsychotic administration may lead to tardive dyskinesia characterised by slow, repetitive, involuntary movements.40 It was supposed that neurological adverse effects are more frequent in the typical antipsychotics. However, clinical trials and recent meta-analysis reported no difference in incidence and severity of extrapyramidal symptoms and tardive dyskinesia between typical and atypical antipsychotics commonly used in clinical practise.38-41 Sedation, anxiety and dysphoria are common in individual agents only.39 All abovementioned adverse effects may stigmatize antipsychotic treated patient and thus may lead to social isolation and negative discrimination. Manifestation of adverse effects may also decrease adherence of the patient to the therapy.42 Although non-specific and neurological adverse effects may negatively affect cardiovascular health, metabolic adverse effects represent significantly higher risk for cardiovascular diseases development. Among antipsychotic drugs, clozapine, olanzapine and chlorpromazine exert the highest liability to metabolic adverse effects.32,38,43 Weight gain is the most frequent metabolic effect of antipsychotics and usually results in central (abdominal) obesity.32 Weight gain potential of antipsychotic drug depends on its pharmacological profile.35,44 It is accelerated by physical inactivity and unhealthy diet, which are both common in patients with schizophrenia (Figure 1).45,46 Antipsychotic treatment is also associated with dyslipidaemia and impaired glucose metabolism. Dyslipidaemia appears strongly related to weight gain and significantly increases risk of cardiovascular mortality.32 Impaired glucose metabolism is represented by hyperglycaemia and/or insulin resistance and potentially leads to type 2 diabetes mellitus. It is particularly associated with second-generation antipsychotics.32,43 More than 50 % increase of type 2 diabetes mellitus was reported in adolescents treated by the second-generation antipsychotics (in comparison with those who did not take the second-generation antipsychotics).47 Abdominal obesity, dyslipidaemia and impaired glucose metabolism together with hypertension are clustered in metabolic syndrome,48 which is associated with more than 2-fold increase in overall cardiovascular mortality.49 Direct cardiovascular adverse effects of antipsychotics are sporadic and include arterial hypertension, QTc interval prolongation and sudden cardiac death.32,43,50 Unfortunately, only limited number of studies is focused on arterial hypertension.32 Excessive increase of arterial blood pressure was reported in patients treated with aripiprazole, a partial agonist of 5-HT1A

| 14 receptor.51 Arterial hypertension is often reported in antipsychotics with a high potential of the metabolic adverse effects (e.g. clozapine, olanzapine).32 Besides other effects, metabolic syndrome increases a risk of atherosclerosis – and thus increases a risk of arterial hypertension.48 On the other hand, clozapine and olanzapine were reported to cause orthostatic hypotension.52,53 Such effect may be partially explained by high affinity of clozapine and 52,53 olanzapine to α1 adrenergic receptor. Orthostatic hypotension was also reported in other antipsychotics.52 For some antipsychotics (especially the first-generation ones), no quantitative data exist about the incidence of orthostatic hypotension. In patients treated with antipsychotics, a risk of sudden cardiac death is increased from twice to three times.50,54-56 A study in a large cohort of patients receiving antipsychotic monotherapy showed that the increase of sudden cardiac death risk depends on antipsychotic dose and it is similar for first- and second-generation agents.57 However, these data are disputable because of possible undocumented as well as over-reported cases of sudden cardiac death in schizophrenia patients.43 Increased sudden cardiac death among schizophrenia patients may be related to antipsychotic drug-induced QTc interval prolongation.43 QT interval on ECG represents duration of ventricular electric revolution (depolarization and repolarization). Prolongation of heart rate corrected QT interval (QTc) is an independent risk factor for sudden cardiac death.58 Many antipsychotic drugs have been reported to prolong QTc in case reports or in randomized studies.59,60 Generally, typical antipsychotic medications leads to a higher risk of QTc prolongation than atypical antipsychotic medications.60,61 Among typical antipsychotics, the highest QTc prolongation potential was identified in phenotiazines, namely thiorizadine.62 Among atypical antipsychotics, the high risk of QTc prolongation was reported in sertindole and ziprasidone.63,64 Besides antipsychotic medication, multiple factors are associated with QTc prolongation. Female sex, electrolyte abnormalities and adverse effects of other drugs increases the risk of QT prolongation development. In patients with inherited mutation in sodium or potassium ion channels (inherited long-QT syndrome), prescription of high-potent QT prolonging antipsychotics should be omitted.

1.1.2 Genetic Aspects Regardless side effects of antipsychotics, impairment of heart function was reported in schizophrenia patients even before the initiation of pharmacotherapy. Japanese authors described significantly longer QT interval in drug-free schizophrenia patients than in age-

| 15 matched healthy subjects.65 They discussed polymorphism in KCNH2 (hERG) potassium channel as possible link between schizophrenia and QT prolongation.65 Although inhibition of the potassium current via hERG channel may clarify a significant QT prolongation,66,67 it seems to be the tip of an iceberg only. It is more probable that association between schizophrenia and cardiovascular diseases is based on more than one gene. Such theory is supported by the results of genome-wide association studies, which identified one hundred and eight genetic loci associated with schizophrenia68 and even more genes associated with various cardiovascular diseases.69,70 Although we recognise tens of overlapping genetic loci associated with both schizophrenia and cardiovascular diseases, there is still a long way to identify a role of all particular genes and their involvement in pathophysiology of cardiovascular diseases in schizophrenia patients.

1.2 Haloperidol: from Clinical Practice back to Basic Research Haloperidol is a typical antipsychotic drug. It can be administered orally, intravenously (haloperidol lactate) or as a depot intramuscular injection (haloperidol decanoate).71 In the Czech Republic, haloperidol is approved for adults for the treatment of schizophrenia, schizoaffective disorder, manic episodes in bipolar disorder, acute agitation, sustained aggression and psychotic symptoms, sever tics (e.g. in case of Tourette’s syndrome), and light and moderate chorea. For paediatric population, it is approved for treatment of schizophrenia and a few other disorders as a drug of the last choice – only in cases where other pharmacotherapeutic agents failed or were not tolerated. During the last decade, consumption of haloperidol in the Czech Republic remains almost stable with more than 3 million DDD/year (between 2011 and 2017; Figure 2). Haloperidol is a butyrophenone derivative. It was first synthesized in 1958.72 The compound underwent clinical trials in the same year and it was introduced to the market in 1959.72 After chlorpromazine (developed in 1950) and reserpine (discovered in 1953), haloperidol was the third antipsychotic drug introduced into the clinical practice. This trio comprised basis of so- called “psychopharmacological revolution”.72 European clinical practice accepted haloperidol with great enthusiasm within few years. Its consumption during first ten years was increased exponentially up to 250 million doses per year (in 1969) in the Europe.72 However, introduction to the market of the North America was intricate. After many clinical and industrial problems, haloperidol was successfully patented in the United States of America in 1969.72 Afterwards, it

| 16 became widely used in entire North America.71 And up today, haloperidol is still the most prescribed typical antipsychotic in the US.71

Table 1: Pharmacological profile of haloperidol

Ki [nmol/l] Target Receptor Human Rat Guinea Pig

73 74 D1 83 (cloned) * 15 (cloned) 75 D2 2 (cloned)* 2 (striatum) 76 D3 10 (cloned) 12 (cloned)* 77 D4 15 (cloned)* 5.1 (cloned)

D5 147 (cloned)* 74 α1 8 (cloned)* 7.3 (cloned) 78 α2 550 (cloned)* 360 (brain) 75 β1,2 >10 000 (cloned)* >10 000 (cortex) 74 5-HT1A 1 202 (cloned)* 2 600 (cloned) 79 5-HT2A 130 (recombinant) 73 (cloned)* 75 80 5-HT3 >10 000 (cloned)* >10 000 (cortex) 15.4 (hippocampus) 75 5-HT4 >10 000 (striatum)

5-HT5 2 247 (cloned)* 74 5-HT6 3 666 (cloned)* 5 000 (cloned) 74 81 5-HT7 378 (cloned)* 260 (cloned) 3 311 (cloned) 82 83 84 σ1 3.7 (cloned) 4 (brain) 4 (brain) 85 85 σ2 6.7 (neuroblastoma) 7.1 (glioma) H1 3 002 (cloned)* 3 630 (brain)78 1 800 (cerebellum)75 H2 1 003 (cloned)*

M1-4 >10 000 (cloned)*

M5 657 (cloned)*

GABAA,B >10 000 (forebrain)* NMDAR >10 000 (cortex)86

Ki values obtained from Ki database of NIMH Psychoactive Drug Screening Program, https://pdspdb.unc.edu/pdspWeb/ (accessed June 8,

2019). All Ki values are expressed in nmol/l (nM). After each Ki value, source of the receptor is defined in the brackets, followed by reference.

* - Ki determinations was generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program (NIMH, Bethesda MD, USA).

In comparison with other typical antipsychotics, haloperidol exerts rather narrow spectrum of targeted receptors (Table 1). It is believed that its major therapeutic effects are based on high- affinity antagonism of dopamine D2 receptors and additional antagonistic activity on serotonin

5-HT2A receptors and adrenergic α1 receptors. Besides abovementioned, high affinity was detected to sigma 1 and sigma 2 receptors.

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1.2.1 Cardiovascular Adverse Effects of Haloperidol Treatment Haloperidol is particularly connected with QT interval prolongation.71 QT prolongation in haloperidol treated patients has been repeatedly reported.60,87-89 Long QT increases a risk of potentially fatal ventricular arrhythmias – especially Torsades de Pointes.58 Episodes of Torsades de Pointes has been mostly reported after intravenous haloperidol administration.87,90,91 In response to adverse event reports, the U.S. Food and Drug Administration (FDA) strengthened label warnings for intravenous haloperidol in September 2007. For using intravenous haloperidol in the management of acute delirium, the FDA recommends a continuous electrocardiogram (ECG) monitoring. Some restrictions are also applied in other countries. However, the available data suggest that a total cumulative dose of intravenous haloperidol less than 2 mg can safely be administered without ongoing electrocardiographic monitoring in patients without concomitant risk factors.92 Over all, recent clinical studies and meta-analyses identified increased risk of QT prolongation, Torsade de Pointes and sudden cardiac death in haloperidol treated patients.33,39,58,93 And one case of sudden death was reported even after single oral dose of haloperidol.94

1.2.2 Haloperidol in Basic Research – with Respect to the Heart Haloperidol’s introduction to the clinical practice preceded an understanding of the biological mechanisms underlying its effects. It drove scientists to develop new animal models of schizophrenia. Subsequent research contributed to development of biological psychiatry and modern neurosciences and laid the foundations of hypotheses concerning aetiology of schizophrenia.72 Haloperidol has been used in basic heart research especially because of its affinity to sigma receptors. Effects of the acute and/or chronic haloperidol exposure were previously studied on various experimental models and species, including rat,95 guinea pig95 and rabbit.96 As functions of sigma receptors have been uncovered step-by-step, haloperidol – a clinically relevant sigma ligand – became more pertinent. Haloperidol helps to study putative roles of sigma receptors in pathophysiological processes in the heart. For instance, recent study in animal model of aortic constriction-induced heart failure describes aggravation of cardiac hypertrophy by impairment of mitochondrial calcium signalling after haloperidol treatment.97

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1.2.2.1 Sigma Receptors Haloperidol is a potent sigma receptor antagonist.84,98 Two subtypes of sigma receptors were 99-101 identified – sigma 1 and sigma 2. Haloperidol shows higher affinity to sigma 1 (Ki = 3.7 82,85 nM) than to sigma 2 receptor (Ki = 6.7 nM). Next to other tissues, sigma receptors were identified in the heart.102,103 The minority (25 %) of sigma receptors in the heart is represented by sigma 2 receptors.103 Sigma 2 receptor is poorly understood. Recently, primary structure of sigma 2 receptor was suggested as a TMEM97 membrane protein.104 According to binding studies, high density of sigma 2 receptors is present in the brain,105 lungs, kidneys, liver,106 and in cells with high proliferation rate, including tumour cells.107 Inhibitory effect of sigma 2 agonist on inwardly rectifying potassium channels in the heart was reported.108 However, specific functions of sigma 2 receptors in the heart muscle as well as in other tissues stay unclear. Compared to sigma 2 receptor, sigma 1 receptor is better described. It was identified in rat atrial and ventricular cardiomyocytes109 as well as in intracardiac neurons.110 It was purified and cloned from various tissues, both animal and human ones.111-115 Mei and Pasternak reported that predicted structure of rat sigma 1 receptor is highly homologous with murine (93.3 %), guinea pig (93.7 %) and human (96.0 %) sigma 1 receptor.115 Sigma 1 receptor is widely expressed in neuronal system, where it is involved in numerous physiological functions and pathophysiological processes.116 Among non-neural tissues, high density of the sigma 1 receptor was found in the immune, endocrine, reproductive, and gastrointestinal systems.106,117 Sigma 1 receptor is primarily located in endoplasmic reticulum membrane associated with mitochondria.118 It acts as a pluripotent modulator in the cell. By its chaperone activity and protein-protein interactions, it regulates calcium signalling, enhances the endoplasmic reticulum signalling to nucleus and attenuates response to oxidative stress.116,119,120 Translocation of sigma 1 receptor to plasma membrane and nuclear envelope was reported repeatedly after cellular stress or ligand stimulation.118,121,122 Potential function of sigma 1 receptor in regulation of mitochondrial organization and size in the heart was also reported in sigma 1 receptor knockout mouse model.123 Stimulation of sigma 1 receptors in rats leads to activation of protective Akt-eNOS pathway in the heart and amelioration of pressure overload- induced hypertrophy.124 The relationship between the sigma 1 receptor and hERG potassium channel has been studied intensively. The hERG channels are responsible for the rapid component of delayed rectifier current IKr. Haloperidol was reported to block the hERG channels expressed in Xenopus

| 19 oocytes125 and in human embryonic kidney cells (HEK 293).126,127 In the leukemic K562 cell line, the regulating function of sigma 1 receptors on hERG expression was clarified.128 Sigma 1 receptor binds to hERG channel in endoplasmic reticulum and facilitates hERG maturation and trafficking.128,129

1.2.2.2 Haloperidol and Voltage Gated Ion Channels Because of its reported QT prolonging effect, action of haloperidol on potassium channels has been studied. Potassium channels are the most diverse group of voltage-gated channels.130 Potassium repolarizing current on cardiomyocytes consists of several components. Besides the inhibitory effect on hERG potassium channel (discussed above), haloperidol was reported to inhibit transient outward potassium current (Ito) and significantly decelerate its recovery in rat ventricular cardiomyocytes.131 Direct modulation of potassium channels by sigma 1 receptor was suggested in isolated intracardial neurons.110 In dose-dependent manner, sigma ligands reversibly block delayed outward rectifying potassium current, increase conductance of calcium-sensitive potassium channels and inhibit M-current. Haloperidol does not affect potassium channels only. Haloperidol was reported as mild to moderate blocker of the L-type calcium channel.132 An inhibitory effect of haloperidol on activated sodium current was also reported in human atrial cardiomyocytes and in rat ventricular cardiomyocytes.132,133 Johannessen et al. described modulation of Nav1.5 channels by sigma 1 receptors in cardiomyocytes.134 However, they showed that haloperidol inhibited Nav1.5 channel also on cells with no sigma 1 receptors.134 The sigma 1 receptor has been reported to modulate sodium channels Nav1.2, Nav1.4 and Nav1.5 in non-cardiac cells, as well.135,136

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2 Aims

Haloperidol prescription is restricted in many countries due to its potential QT-prolonging effect. Recent clinical studies indicate stratification of such risk among various groups of patients. However, for identification of risk factors, proper knowledge of the mechanism(s) of haloperidol effect on QT is crucial. Animal models play irreplaceable role in discovering of biological effects of various chemical compounds. Reported effects of haloperidol on electrophysiological parameters in various animal models are rather inconsistent. Novel methodological approaches may shed more light on the long QT phenomenon. Therefore, this thesis was aimed to analyse the effects of haloperidol administration on electrogram in selected animal models. Regarding to describe the mechanisms of observed effects, the thesis was complemented by gene expression analyses, biochemical analyses and cell line studies. Based on the aims, the thesis was divided into two parts:

Study A: Long-term haloperidol treated guinea pig model A1. To evaluate the effect of long-term haloperidol administration on the QT/RR coupling and on QTc calculated according to subject specific model in guinea pig isolated heart. A2. To evaluate the effect of long-term haloperidol administration on the QT/RR coupling and QTc alterations under acute haloperidol administration and washout in guinea pig isolated heart. A3. To evaluate the effect of long-term haloperidol treatment on the expression of sigma 1

receptor and IP3 receptors type 1 and 2 in guinea pig heart.

Study B: Rat neurodevelopmental model of schizophrenia B1. To characterize structural and electrophysiological parameters of the heart in MAM neurodevelopmental model of schizophrenia. B2. To evaluate changes of heart rate, QTc and HRV parameters under acute haloperidol administration. B3. To evaluate general effect of MAM on viability and reactive oxygen species production in H9c2 cell line.

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3 Materials and Methods

In this chapter, only key methodological steps are presented. For more details, see published papers (manuscripts) attached in Appendixes.

3.1 Chemicals and Drugs If not mentioned otherwise, all chemical compounds and drugs were purchased from Sigma- Aldrich (MO, USA) in p.a. or equivalent quality. Haloperidol was prediluted in ethanol (96 %) in concentration of 20 mmol/l (haloperidol stock solution). For intraperitoneal application, haloperidol stock solution was diluted in aqua pro injectione in final concentration of 0.4 mmol/l. Solution for intraperitoneal application was prepared and stored under strict aseptic conditions. For isolated heart perfusion, haloperidol stock solution was diluted in Krebs-Henseleit solution (Table 2) in final concentration of 10 nmol/l. Such solution contained ethanol in concentration of 8.22 nmol/l.

Table 2: Composition of Krebs-Henseleit solution.

Component NaCl NaHCO3 KCl KH2PO4 MgSO4 CaCl2 glucose c (mmol/l) 118.0 27.0 4.8 1.0 1.2 1.2 10.0

3.2 Laboratory Animals All animal experiments were carried out according to the recommendations of the European Community: Guide for the Care and Use of Laboratory Animals and according to experimental protocols approved by the Committee for Ensuring the Welfare of Laboratory Animals at Masaryk University, Faculty of Medicine in accordance with the Act No 242/1992 Sb. (“Zákon na ochranu zvířat proti týrání”), as subsequently amended. Laboratory animals were housed in the Animal Breeding and Experimental Facility, Masaryk University, Faculty of Medicine in the environment with controlled atmospheric pressure, humidity, temperature, and light cycle 12/12 (12 hours light, 12 hours dark; light on at 5:30 am). Animals were kept in groups in standard cages. They were fed with standard diet; water was accessible ad libitum.

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3.2.1 Guinea Pig Model of Long-Term Haloperidol Administration Guinea pigs (unspecified breed, male only, 3 months old) were purchased from Bioveta, Ltd. (Czech Republic). After minimally 10 days of adaptation, animals were randomly divided into two groups: haloperidol-treated (group HAL) and control (group CON). Haloperidol in dose of 2 mg/kg of actual body mass or vehiculum (2 % ethanol in aqua pro injectione) in adequate volume (13.3 ml/kg) were administered by intraperitoneal injection once a day for 21 consecutive days. Maximal volume of single dose did not exceed 10 ml. Weighing and application was always done at the same daytime (around noon). No anaesthetics or analgesics were used before drug administration. To reduce discomfort of animals during the manipulation, gentle handling and quiet approach was applied. Isolated heart experiment or samples collection were performed twenty-four hours after the last dose of haloperidol.

3.2.2 Rat Model of Schizophrenia Neurodevelopmental rat model of schizophrenia was provided by Department of Pharmacology, Faculty of Medicine, Masaryk University. Time-mated female Sprague-Dawley rats were purchased from Charles River (Germany). Briefly, methylazoxymethanol acetate (MAM; Midwest Research Institute, MO, USA) or vehiculum (saline) were administered by intraperitoneal injection to pregnant female rats on gestational day 17.137,138 The offspring were weaned on the postnatal day 22. The offspring of MAM-treated females manifested schizophrenia-like phenotype, which was evaluated by set of standard behavioural tests in adulthood. In the study, male adult offspring at the age of 30 weeks were used.

3.3 Isolated Heart Perfused According to Langendorff Langendorff apparatus allows perfusion of any heart with coronary system explanted from the body.139 The heart is placed in the bath and aorta is cannulated. Through cannula, perfusion solution flows at constant perfusion pressure to the aorta. Pressure of the solution closes aortic valve and solution flows directly to the coronary arteries. Noticeable advantage of the isolated heart model is easy way how to administer strictly defined concentrations of drug directly to coronary system of the heart. Prior the heart preparation, animals were deeply anesthetized by isoflurane (2 %). The heart was rapidly excised from the thorax, placed in a cold (4 °C) Krebs-Henseleit solution (Table 2) and prepared for cannulation. The aorta was cannulated and the heart was perfused at constant perfusion pressure with Krebs-Henseleit solution (Table 2). The solution was prepared before

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each experiment and aerated with 95 % O2 and 5 % CO2. Its temperature was maintained at 37 °C and level of pH was adjusted to 7.4. The isolated heart was stabilized for 20 – 40 minutes. Experimental protocols are summarised in Figure 3. During haloperidol phases, haloperidol was administered to isolated heart diluted in Krebs-Henseleit solution (10 nmol/l; pre-dissolved in ethanol). Six Ag-AgCl electrodes placed on the inner surface of the bath were used for continual recording of electrogram in three orthogonal leads. Besides electrogram, the perfusion pressure and temperature of the perfusion solution were continuously monitored and maintained at 80 mmHg and 37 °C, respectively. All signals were recorded by an acquisition card NI USB- 6229 BNC (National Instruments, TX, USA). Signals were recorded with resolution of 12-bit and sampling frequency of 5 or 10 kHz.

Study A: Guinea pig model S H1 W1 H2 W2

Study B: Rat model S H W

Time [min] 0 20 40 60 80 100 120

Figure 3: Experimental protocols of isolated heart experiments. S – stabilization period; H – haloperidol administration; W – washout

3.3.1 Evaluation of Preparation Ischemia and Damage of the Isolated Heart throughout the Experiment To evaluate the quality of the isolated heart preparation and to detect possible influence of preparation ischemia in guinea pig study (Study A), levels of creatine kinase (CK), lactate dehydrogenase (LD), lactate, and 4-hydroxy-2-nonenal (HNE) were determined in coronary effluent. Samples of coronary effluent were collected at the end of the first minute of stabilization period and at the end of experiment. Biochemical analyses were performed at the Department of Biochemistry, Faculty of Medicine, Masaryk University. Moreover, duration of ischemic period was measured. Preparational ischemic period consists of two parts: shorter warm ischemia (from excision the heart from the thorax to the cooling in cold solution) and longer cold ischemia. Both periods were measured separately.

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3.4 Electrogram Analysis The data processing was performed in off-line mode using MATLAB (version 8.0.2.701, MathWorks, MA, USA). Partial analyses were performed in cooperation with the Department of Biomedical Engineering, Brno University of Technology and in the International Clinical Research Centre St. Anne’s University Hospital in Brno.

3.4.1 R and T Automatic Detection. RR Interval and Heart Rate Measurement. QT Interval Measurement and Correction The positions of R peaks were detected by automatic detection method based on wavelet transformation. In guinea pig electrograms, the ends of T wave were detected by automatic detection method based on the isoline regression. The detection was done for each single beat. Debatable beats were marked as non-detectable. The detection was carefully reviewed by human specialist. In guinea pig electrograms (Study A), RR and QT intervals were analysed by automatic analysis. In rat electrograms (Study B), RR intervals were determined from the window of 10 consecutive beats at the end of each experimental phase. In the same window, QT intervals were measured manually (Figure 4).

QT Voltage [V] [V] Voltage

Time [s] Figure 4: Original record of rat isolated heart electrogram. QT interval length is labelled in the red box (QT).

In guinea pig study (Study A), QT interval in haloperidol and washout phases was corrected using subject specific linear model based on QT/RR coupling140 for nominal RR0 = 400 ms according to formula:

Parameters a1 and a0 were determined푄푇푐 according = 푎1 × 푅푅 to0 individual + 푎0 QT/RR coupling in particular experimental phase.

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In stabilisation phase, modified formula was applied:

Parameter a1 was calculated as푄푇푐 the =average푄푇 + 푎1value × ( of푅푅 the− 푅푅QT/RR0) coupling computed over all phases of the experiment except of the stabilization and RR0 is nominal RR interval (RR0 = 400 ms). In rat study (Study B), QT was corrected according to Framingham formula:141

푄푇푐 = 푄푇 + 0.154 × (1 − 푅푅) 3.4.2 Incidence of Arrhythmias Arrhythmias were classified according to Lambeth Conventions.142 The total number of ventricular premature beats (VPB) and supraventricular premature beats (SVPB) was calculated for each experimental phase separately. The first 10 minutes of stabilisation period were not included in the analysis.

3.4.3 Analysis of the Relationship between QT and RR Intervals The coupling between QT and RR intervals (QT/RR) and their differences (dQT/dRR) were analysed in guinea pig study (Study A) only. QT/RR and dQT/dRR coupling were calculated for all haloperidol and washout phases. The differences in RR interval (dRR) were calculated according to formula:

i The dRRi and the RRi are the values 푑푅푅푖for -th = beat,푅푅푖 −the푅푅푆 RRS is the mean value of the RR at the end of stabilization. Similarly, dQT was calculated. The mean and SD of the filtered courses of RR, QT, dRR, and dQT were calculated.

3.4.4 Heart Rate Variability Analysis Heart rate variability (HRV) was analysed in rat study (Study B). The analysis was performed on interbeat interval sequences. Series of ten-minutes-long sequences were obtained from RR series for each experiment, as follows: 1 x 10 minutes of stabilisation, 4 x 10 minutes of haloperidol phase, 4 x 10 minutes of washout. HRV parameters were computed by HRVAS plugin for MATLAB according to recommendations of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology.143 HRV was analysed in time, geometric, frequency, and non-linear domain. Three parameters were chosen to describe HRV in the isolated heart model: (1) the standard deviation of the NN intervals (SDNN); (2)

| 26 the NN20 parameter, representing the number of interval differences of successive RR intervals greater than 20 ms; and (3) the sample entropy.

3.5 Gene Expression Analysis Gene expression analysis was performed at the Department of Pathological Physiology, Faculty of Medicine, Masaryk University.

3.5.1 RNA isolation, cDNA preparation For RNA isolation, the high pure RNA tissue isolation kit (Roche, Switzerland) was used. Tissue samples were homogenized in 200 µL of lysis buffer. RNA isolation was performed according to manufacturer’s instructions. Isolated RNA was transcribed for cDNA using the transcriptor first strand cDNA synthesis kit (Roche, Switzerland). Synthesised cDNA was diluted in RNase free water up to volume of 100 µL and directly analysed by real-time polymerase chain reaction.

3.5.2 Real-time reverse-transcription polymerase chain reaction Real-time reverse-transcription polymerase chain reaction (PCR) was performed in triplicates using the TaqMan gene expression assay system with the 7500 real-time PCR system (Applied Biosystems Inc., CA, USA). The amplified DNA was analysed by the comparative Ct method using β-actin as an endogenous control. Following primers and probe sets (all from Applied Biosystems Inc., CA, USA) were used: β-actin (Assay ID: Cp03755211_g1), sigma 1 receptor

(Assay ID: Cp03755850_m1), IP3 receptor type 1 (CpLOC100713023), and IP3 receptor type 2 (CpLOC100717278). Real-time PCR was performed under the following amplification conditions: total volume of 20 µL, initial denaturation 95 °C/10 min, then 45 cycles 95 °C/15 s, and 60 °C/1 min. The results were expressed in relation to the reference values. Twofold or higher difference between the groups was accepted as statistically significant.

3.6 Histological and Immunohistological Staining of the Heart Muscle This part of the study was performed in cooperation with the Department of Pathological Physiology and the Department of Biology, Faculty of Medicine, Masaryk University and with the First Department of Pathological Anatomy, St. Anne’s Faculty Hospital and Faculty of Medicine, Masaryk University.

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One sample from each atrium and one from each ventricle were collected. Samples were fixed in buffered paraformaldehyde/glutaraldehyde (4 % paraformaldehyde, 1 % glutaraldehyde in 0.1 M PBS) or formaldehyde, embedded in paraffin and sliced on 3 – 4 µm-thick sections. Sections were deparaffinised using xylene and stained.

In guinea pig study (Study A), immunostaining of sigma 1 receptors and IP3 receptors type 1 was performed in Department of Pathological Physiology, Faculty of Medicine, Masaryk University. Deparaffinised sections were processed for antigen retrieval. Rabbit Anti-inositol 1,4,5-trisphosphate receptor type I (Sigma-Aldrich, MO, USA) and rabbit Anti-OPRS1 (sigma 1 receptor; Abcam, UK) were used as primary antibodies. Goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Invitrogen, MO, USA) was used as secondary antibody. Stained sections were mounted with Vectashield (Vector Laboratories, CA, USA). Fluorescent images were taken using a confocal microscope Zeiss LSM 700 (Zeiss, Germany) using 488 nm laser. In rat study (Study B), haematoxylin and eosin staining was used for evaluation of general morphology. For evaluation of collagen content, van Gieson staining was performed. Microscope Nikon Eclipse Ti-S/L100 (Nikon, Japan) and NIS elements software (Nikon, Japan) was used to process images and to evaluate the resultant pictures.

3.7 Cell Line Experiments For assessment of possible direct effect of MAM on embryonal cardiomyocytes (Study B), the H9c2 cell line (rat cardiomyoblasts, ATCC® CRL-1446™) was used. Cells were cultured in Dulbecco's Modified Eagle's medium supplemented with 1.5 g/L sodium bicarbonate, 10 % foetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a humidified atmosphere of 5 % CO2. Cells were subcultured when reached 80 % confluence. For the experiments, cells were seeded in 24-well plates. After seeding, cells were allowed to grow for 1 day to reach the desired confluence (>75 %). Treatment with MAM in a concentration range from 10 to 1000 μM lasted for 24 hours. Then, cells were washed three times with PBS, harvested and analysed immediately. All experiments were performed in triplicates.

3.7.1 Analysis of Cell Viability Cell viability was assessed using propidium iodide and fluorescein diacetate staining directly on the plates. Dye uptake was visualised by microscope Nikon Eclipse Ti-S/L100 (Nikon, Japan) and NIS elements software (Nikon, Japan) was used to evaluate the resultant pictures.

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Results were verified by flow-cytometric analysis using propidium iodide staining. Flow cytometry was performed at the Department of Biology, Faculty of Medicine, Masaryk University.

3.7.2 Analysis of Reactive Oxygen Species Production and Lipoperoxidation Reactive oxygen species (ROS) production was analysed using CellROX® Green Reagent (ThermoFisher Scientific, MA, USA). The probe was used according to manufacturer’s instructions. Lipoperoxidation was analysed using BODIPY® Lipid Peroxidation Sensor (ThermoFisher Scientific, MA, USA). The probe was used according to manufacturer’s instructions. Microscope Nikon Eclipse Ti-S/L100 (Nikon, Japan) and NIS elements software (Nikon, Japan) was used to process images and to evaluate the resultant pictures.

3.8 Statistical Analysis Basic descriptive statistic was calculated and the results were expressed as mean ± SD. Then, normality of the data was tested. If the data was not normally distributed, the non-parametric tests were applied. To test the differences between experimental groups, Mann-Whitney U test was used. Differences between data from consecutive phases were verified using Wilcoxon signed-rank test. The analysis of electrograms was based on scatter plots and to evaluate the coupling between QT and RR linear regression was used. Spearman's rho was used to assess the correlation between numbers of pathological beats in consecutive experimental phases. For all above tests, P < 0.05 was considered significant.

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4 Results

4.1 Study A: Long-term Haloperidol Administration Affects Relationship between QT and RR Intervals and Expression of Sigma 1 and IP3 Receptors

4.1.1 Heart rate, QTc interval and incidence of arrhythmias In isolated guinea pig hearts, significant difference in the heart rate was found between control (CON) and haloperidol-treated (HAL) group at the end of stabilisation phase (Table 3). However, no significant difference in QTc was detected between the groups at the end of stabilisation. In both groups, significantly longer QTc was found in both haloperidol phases in comparison with consecutive washout phases (Table 4). Values of RR, QT, dRR, and dQT in H1, W1, H2, and W2 phases are summarized in Table 5. Apparent decrease of total number of ventricular premature beats in phases W1 (compared to H1) was noticeable in both groups (for group CON: 3.9±5.8 in H1 vs. 1.1 ± 1.8 in W1; for group HAL: 4.0 ± 7.9 in H1 vs. 1.4 ± 1.9 in W1). However, no significant differences in total number of ventricular premature beats were detected between the groups as well as between consecutive phases within each group.

4.1.2 Relationship between QT and RR Intervals In QT/RR coupling as well as in dQT/dRR coupling, flatter slopes of fitted lines (lower a1) were found in group HAL then in CON during all phases (Table 6 and 7). In the dQT/dRR coupling, the differences between the groups were more manifest (Table 7, Figure 5).

Table 3: The RR, heart rate, QT, and QTc in guinea pig isolated heart at the end of stabilization phase. Group RR [ms] HR [bpm] QT [ms] QTc [ms] CON 309 ± 31.5 196 ± 20.1 179 ± 25.8 245.0 ± 8.3 HAL 287 ± 42.2 213 ± 33.7 161 ± 32.5 243.0 ± 14.1 P < 0.001 < 0.001 < 0.001 0.253

Values are expressed as mean ± SD. HR – heart rate; QTc – corrected QT interval; Con – control group; HAL – haloperidol-treated group; P – P value (Student’s t-test). The QTc was computed according to formula:

The RR0 is nominal RR interval (RR0 = 400 ms). The pa푄푇푐rameter = 푄푇 a1+ was 푎1 ×calculated (푅푅 − 푅푅 as0) the average value of the QT/RR coupling computed over all phases of the experiment except of stabilization. Adopted from Vesely et al. 2019.144

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Table 4: QTc [ms] in haloperidol and washout phases. H1 W1 H2 W2 CON 250.7 ± 9.7 244.0 ± 8.6 252.1 ± 4.8 242.9 ± 7.4 HAL 252.1 ± 14.1 242.1 ± 8.4 254.8 ± 7.2 249.3 ± 8.0 P 0.171 0.006 < 0.001 < 0.001

The values are expressed as mean ± SD. QT was corrected according to subject specific linear correction based on QT/RR coupling for nominal RR0 = 400 ms:

H1 – the first haloperidol administration; W1 – the first washout;푄푇푐 = H2 푎1 –× the푅푅 second0 + 푎0 haloperidol administration; W2 – the second washout; CON – control group; HAL – haloperidol-treated group; P – P value (Student’s t-test). Adopted from Vesely et al. 2019.144

Table 5: Mean values of RR, QT, dRR, and dQT in guinea pig isolated hearts computed for each individual haloperidol and washout phase. H1 W1 RR [ms] QT [ms] dRR [ms] dQT [ms] RR [ms] QT [ms] dRR [ms] dQT [ms] CON 300.1 173.2 -5.210 -4.097 298.4 171.1 -5.923 -5.023 ±28.6 ±24.4 ±9.745 ±5.184 ±32.8 ±25.2 ±15.106 ±7.502 HAL 280.3 158.0 -0.349 1.067 285.2 159.5 4.192 1.783 ±43.4 ±29.5 ±7.439 ±5.139 ±37.4 ±26.5 ±9.052 ±6.387 P <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

H2 W2 RR [ms] QT [ms] dRR [ms] dQT [ms] RR [ms] QT [ms] dRR [ms] dQT [ms] CON 298.2 169.1 -7.642 -7.184 303.4 169.3 -2.031 -6.752 ±31.6 ±26.1 ±15.559 ±8.171 ±31.6 ±25.2 ±18.554 ±14.098 HAL 279.4 157.5 -3.068 -1.454 283.4 160.1 -1.293 0.698 ±28.8 ±23.7 ±17.061 ±7.530 ±25.1 ±21.5 ±21.268 ±10.141 P <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

The values are expressed as mean ± SD. The dRR was calculated according to formula:

i The dRRi and the RRi are the values for -th beat, the RRS is the푑푅푅푖 mean = 푅푅푖value− of푅푅푆 the RR at the end of stabilization; dQT was calculated similarly. CON – control group; HAL – haloperidol-treated group; P – P value (Student’s t-test); H1 – the first haloperidol administration; W1 – the first washout; H2 – the second haloperidol administration; W2 – the second washout. Adopted from Vesely et al. 2019.144

Table 6: The numeral expression of the QT/RR coupling in guinea pig isolated hearts computed for each individual haloperidol and washout phase.

H1 W1 a1 a0 a1 a0 CON 0.783 ± 0.021 62.392 ± 6.725 0.723 ± 0.015 44.939 ± 4.734 HAL 0.620 ± 0.010 15.323 ± 3.032 0.677 ± 0.006 33.876 ± 1.916 P < 0.001* < 0.001* < 0.001* < 0.001*

H2 W2 a1 a0 a1 a0 CON 0.812 ± 0.009 72.853 ± 2.645 0.762 ± 0.016 61.766 ± 4.755 HAL 0.785 ± 0.008 62.633 ± 2.328 0.799 ± 0.011 65.818 ± 2.970 P < 0.001* < 0.001* < 0.001* < 0.001*

The values are expressed as mean ± SD. The parameter a1 represents the slope of the fitted line:

H1 – the first haloperidol administration; W1 – the first washout;푄푇 = H2 푎1 –× the푅푅 second+ 푎0 haloperidol administration; W2 – the second washout; CON – control group; HAL – haloperidol-treated group; P – P value (Student’s t-test). Adopted from Vesely et al. 2019.144

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Figure 5: The dQT/dRR coupling computed for each individual haloperidol and washout phase. From the left upper corner to the right bottom corner in order H1, W1, H2 and W2. The green colour corresponds to control group (CON), the red to haloperidol-treated group (HAL). Haloperidol-treated group (HAL) exhibited decreased slope of fitted lines (red) in each of the experimental phases. Adopted from Vesely et al. 2019.144

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Table 7: The numeral expression of the dQT/dRR coupling in guinea pig isolated hearts computed for each individual haloperidol and washout phase. H1 W1 a1 a0 a1 a0 CON 0.395 ± 0.017 -2.175 ± 0.193 0.411 ± 0.011 -2.734 ± 0.212 HAL 0.244 ± 0.046 0.918 ± 0.233 0.248 ± 0.033 0.565 ± 0.332 P < 0.001 < 0.001 < 0.001 < 0.001

H2 W2 a1 a0 a1 a0 CON 0.477 ± 0.011 -3.654 ± 0.169 0.670 ± 0.017 -5.331 ± 0.288 HAL 0.246 ± 0.017 -0.992 ± 0.376 0.303 ± 0.015 0.966 ± 0.482 P < 0.001 < 0.001 < 0.001 < 0.001

The values are expressed as mean ± SD. The parameter a1 represents the slope of the fitted line:

H1 – the first haloperidol administration; W1 – the first washout;푑푄푇 H2 = – 푎1 the × second 푑푅푅 haloperidol administration; W2 – the second washout; CON – control group; HAL – haloperidol treated group; P – P value (Student’s t-test). Adopted from Vesely et al. 2019.144

Figure 6: Relative gene expressions of the sigma 1 receptor, IP3 receptor type 1 and type 2 (left), and immunohistochemical staining of the sigma 1 receptors and the IP3 receptors type 1 in the left cardiac atrium (right) of guinea pig heart after repeated haloperidol exposure. Haloperidol significantly increased the gene expression of sigma 1 receptor, IP3 receptor type 1 and type 2 in both atria (LA, RA), but not in ventricles (LV, RV). Each column represents an average of 3 – 10 heart samples. Mean relative mRNA levels in the controls (group C; grey columns) were set as a reference values. Mean relative mRNA levels in haloperidol-treated guinea pigs (group H; black columns) were expressed in relation to the reference values. In immunohistochemical staining (right), slightly increased signal for both receptors was detected in haloperidol-treated guinea pigs (c, d) as compared to controls (a, b). Nuclei were stained by DAPI. Inset (e) shows the negative control, where primary antibody was omitted. Bars represent 50 μm. SigmaR – sigma 1 receptor; InsP3R1 – IP3 receptor type 1; InsP3R2 – IP3 receptor type 2; LA – left atrium; RA – right atrium; LV – left ventricle; RV – right ventricle; o – P < 0.05; a – sigma 1 receptor, control group; b – IP3 receptor type 1, control group; c – sigma 1 receptor, haloperidol-treated group; d – IP3 receptor type 1, haloperidol-treated group. Adopted from Stracina et al. 2015.145

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4.1.3 Expression of Sigma 1 and IP3 Receptors Expression of sigma 1 receptor in cardiac atria was significantly increased in haloperidol- treated guinea pigs (in the left atrium 5.67-times and in the right atrium 2.84-times, respectively;

Figure 6). Expression of the IP3 receptors type 1 and 2 was also elevated (in the left atrium type

1 IP3 receptor 3.01-times and type 2 IP3 receptor 6.81-times, in the right atrium type 1 IP3 receptor 3.23-times and type 2 IP3 receptor 4.08-times, respectively; Figure 6). In ventricles, no significant changes were detected (Figure 6).

Immunohistochemistry demonstrated both sigma 1 receptor and IP3 receptor type 1 in the cytoplasm of atrial and ventricular cardiomyocytes in both groups. Slightly increased signal for both sigma 1 receptor and IP3 receptor type 1 was observed in cardiac atria of the haloperidol- exposed animals, as compared to controls (Figure 6).

4.1.4 Preparation Ischemia and Biochemical Analysis When all experiments were analysed together, mean duration of warm ischemia was 17.5 s and cold ischemic period lasted 155 s. No significant difference in ischemic period duration was found between the groups. In CK, LD and lactate levels, no significant difference was found between haloperidol-treated group (HAL) and controls (CON) in the beginning (sample 1) as well as at the end of perfusion (sample 2). However, the levels of these parameters were significantly higher in sample 1 than in sample 2 in both groups (Table 8). No significant difference in HNE levels was found between groups HAL and CON at the beginning as well as at the end of isolated heart experiment. In addition, no significant differences were detected between sample 1 and 2 within each group.

Table 8: The levels of creatine kinase, lactate dehydrogenase and lactate in the samples of coronary effluent. CON HAL sample 1 sample 2 P sample 1 sample 2 P CK [nkat.min-1] 2.32 0.00 0.012 1.25 0.00 0.028 (1.80; 4.90) (0.00; 0.87) (0.82; 2.50) (0.00; 0.34) LD [nkat.min-1] 37.11 22.93 0.017 32.49 19.98 0.046 (32.23; 39.78) (20.70; 25.33) (26.68; 35.30) (16.94; 24.46) lactate 2.02 0.00 0.028 1.44 0.48 0.028 [μmol.min-1] (0.70; 2.93) (0.00; 1.27) (0.96; 2.54) (0.00; 1.27)

Values are expressed as median (25th; 75th quartile). CON – control group; HAL – haloperidol-treated group; CK – creatine kinase; LD – lactate dehydrogenase; sample 1 – the end of the first minute of stabilization phase; sample 2 – the end of experiment; P – P value (Wilcoxon paired test). Adopted from Vesely et al. 2019.144

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4.2 Study B: Prenatal Exposure to MAM Affects Rat Heart and Its Reactivity to Haloperidol

4.2.1 Body Weight, Heart Weight, HW/BW Ratio Body weight in MAM rats was significantly lower when compared to controls (MAM: 563.2 ± 62.3 g; CON: 608.1 ± 40.0 g; P = 0.041). The same trend was detected in heart weight – heart weight of MAM rats was significantly lower (MAM: 1.43 ± 0.18 g; CON: 1.60 ± 0.16 g; P = 0.045). In HW/BW ratio, no significant difference was observed (MAM: 0.00255 ± 0.00016; CON: 0.00264 ± 0.00025; P = 0.473).

4.2.2 Heart Muscle Structure and Collagen Content No significant differences in structure of myocardium, size of cardiomyocytes and collagen content were observed between the groups. Representative pictures of left ventricle sections in heart of MAM and control rat are presented below (Figure 7).

Figure 7: Representative pictures of histological sections of left ventricle stained using haematoxylin and eosin in MAM rat (right) and control rat (left). No significant difference in the heart muscle structure was detected between the groups. Bar represents 20 µm.

4.2.3 Isolated Heart Electrophysiology 4.2.3.1 Heart Rate, QTc Interval During the whole experiment, insignificantly higher heart rate was detected in the MAM group in comparison with controls (in S: MAM: 290.3 ± 30.5 bpm; CON: 269.5 ± 10.0 bpm). In either

| 35 group, neither haloperidol administration nor washout caused significant heart rate changes when compared to stabilization (Figure 8, left). Nearly significant difference in QTc interval was detected in MAM group when compared to controls in stabilisation (in S: MAM: 221.98 ± 6.41 ms; CON: 216.34 ± 2.60 ms; P = 0.113). Haloperidol administration led to slight increase of QTc in MAM but slight decrease of QTc in CON (in H: MAM: 227.40 ± 8.09 ms; CON: 214.42 ± 3.95 ms; P = 0.002). During washout period, QTc interval was decreased in both groups. In CON group, the decrease was only slight (in W: CON: 212.62 ± 2.78 ms), although the decrease in MAM group was highly significant (in W: MAM: 217.00 ± 6.68 ms; P = 0.026, when compared to QTc in H; Figure 8, right).

Figure 8: Heart rate (right) and QTc interval (left) in rat isolated hearts. Data are displayed as median, the edges of the box indicate 25th and 75th percentiles, circles indicate outliers. MAM – MAM rats (n = 9); CON – controls (n = 6); STAB – stabilisation phase; HALO – haloperidol administration; WASH – washout; * indicates P < 0.05; ** indicates P < 0.01.

4.2.3.2 Heart Rate Variability As a part of electrophysiological data processing, RR intervals were analysed. During haloperidol administration, the RR intervals were gradually prolonged in CON group (by 9% in mean, by 8% in median; Figure 9, right). After 20 minutes of haloperidol administration, RR intervals were stabilized and remained constant for the rest of the experiment. In MAM group, no significant change of RR intervals was detected during the experiment (Figure 9, left). Three parameters were chosen to describe HRV in the isolated heart model. Overall HRV was assessed as the standard deviation of the NN intervals (SDNN). In stabilisation period, no difference was detected in the SDNN between the groups. Haloperidol administration caused gradual decrease of the SDNN in both groups (Figure 10). In CON group, the decrease was more dynamic. During the first 10 minutes (the first quarter) of haloperidol administration, the

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SDNN was reduced to 48% in the CON group (compared to the value in stabilization; Figure 10, right). In MAM group, the same level of the SDNN reduction was reached during the third quarter of haloperidol administration (Figure 10, left). At the end of haloperidol administration, the final reduction of the SDNN was equal in both groups. At the beginning of washout, the SDNN was significantly increased in both groups; however, it did not reach initial values in stabilization. During the washout, the SDNN was decreased again in both groups. In the CON group, higher dynamics of the decrease was detected (Figure 10). In the stabilisation period, the NN20 parameter, representing the number of interval differences of successive RR intervals greater than 20 ms, was slightly higher in the MAM group than in the CON group (Figure 11). In the group CON, almost no extraordinary beat longer than 20 ms was detected throughout the experiment. In the group MAM, the NN20 was decreased at the beginning of haloperidol administration. After that, the NN20 was gradually increased during the haloperidol administration and decreased back during washout (Figure 11, left). Almost during the entire experiment, the sample entropy was higher in the group MAM than in the group CON (Figure 12). The sample entropy is a measure of regularity of successive intervals – higher sample entropy indicates lower regularity and therefore lower predictability of fluctuations in successive RR intervals. The sample entropy was higher in the group MAM than in the group CON. The groups also differed in the trend of change of sample entropy during the haloperidol administration and washout (Figure 12). In the CON group, biphasic trend of sample entropy was detected in both experimental phases – with the peak in the third quarter of each phase (Figure 12, right). In the group MAM, sample entropy was more variable with increasing trend during the haloperidol administration as well as washout (Figure 12, left).

MAM: RR median CON: RR median MAM: SDNN CON: SDNN

Figure 9: RR intervals in rat isolated heart during the Figure 10: The standard deviation of the NN intervals in rat experiment. isolated heart during the experiment. Data are displayed as median, edges of box indicate 25th and 75th Data are displayed as median, the edges of the box indicate 25th percentiles, and circles indicate outliers. MAM – MAM rats th and 75 percentiles, and circles indicate outliers. MAM – MAM (left, n = 9); CON controls (right, n = 6); STAB stabilisation – – rats (n = 9); CON – controls (n = 6); STAB – stabilisation phase; HALO – haloperidol administration; WASH – washout. phase; HALO – haloperidol administration; WASH – washout. | 37

MAM: NN20 CON: NN20 MAM: sample entropy CON: sample entropy

Figure 11: The parameter NN20 in rat isolated heart during Figure 12: The sample entropy in rat isolated heart during the experiment. the experiment. Data are displayed as median, edges of box indicate 25th and 75th Data are displayed as median, edges of box indicate 25th and 75th percentiles, and circles indicate outliers. MAM – MAM rats percentiles, and circles indicate outliers. MAM – MAM rats (left, n = 9); CON – controls (right, n = 6); STAB – stabilisation (left, n = 9); CON – controls (right, n = 6); STAB – stabilisation phase; HALO – haloperidol administration; WASH – washout. phase; HALO – haloperidol administration; WASH – washout.

4.2.3.3 Incidence of Arrhythmias VPB were observed in electrograms of both groups only sporadically, except of two experiments from MAM group, where total number of VPB in washout reached 5 and 48, respectively. SVPB were more frequent. No difference in total number of SVPB was found between MAM group and controls. Number of SVPBs in MAM increases significantly during haloperidol administration and washout in comparison with corresponding previous phases. If both groups are analysed together, more than 1 SVPB were found in 13 %, 67 % and 80 % of total number of experiments in stabilization, haloperidol administration and washout, respectively. More than 10 SVPB were observed in 20 % and 33 % of experiments in the haloperidol administration and washout, respectively. Significant positive correlation (Spearman's rho = 0.74, P < 0.005) was detected between total number of SVPB during haloperidol administration and washout.

100 90 * 80 70 *

Viable cells [%] 60 Figure 13: Viability of H9c2 cell after 24-hours exposure to MAM. 50 Columns represent mean percentage of viable cells (PI-) measured by flow cytometry. Whiskers represent SD. * - significant decrease in comparison with controls (CON; P < 0.05, Mann-Whitney U test)

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4.2.4 Effect of MAM on Viability and Oxidative Status in H9c2 Cells Fluorescence staining revealed high viability of H9c2 cells after 24 hours exposure to low concentrations of MAM (10 nmol/L – 100 μmol/L; Figure 14). The result was proven by flow cytometry, which showed significant decrease of viable cells entirely after exposure to higher concentrations of MAM (500 μmol/L and more; Figure 13). Fluorescent staining revealed slightly increased production of ROS and subsequent lipid peroxidation after exposure to MAM in concentration of 100 μmol/L (Figure 15).

Figure 14: Fluorescein diacetate (FDA)/propidium iodide (PI) staining of H9C2 cells. Staining revealed high cell viability after 24-hours exposure to MAM in low concentrations (10 nM – 100 μM). A – composite of fluorescent and phase contrast images; B – fluorescent images: composite of FDA and PI signal. Bars represent 50 μm.

Figure 15: Reactive oxygen species (ROS) production (A) and lipid peroxidation in H9C2 cells after 24-hours exposure to MAM in low concentrations (10 nM – 100 μM). Staining revealed slightly increased production of ROS and level of lipoperoxidation, especially in concentration of 100 μM. A – CellROX® Green staining: green signal represents oxidized dye bound to DNA in nucleus; B – BODIPY® C11 (Lipid Peroxidation Sensor) staining: signal shift from yellow to green reveals oxidation of the dye by ROS. Bars represent 50 μm.

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5 Discussion

QT interval prolongation is independent risk factor for development of life-threatening arrhythmias potentially leading to sudden cardiac death. QT interval length, as a biomarker, is used by drug developers and regulatory agencies as a measure of drug safety. However, evaluation of QT interval prolongation is rather complicated. The QT interval adapts to changes in heart rate. Therefore, it is difficult to compare the QT interval length recorded at different heart rates. To allow such comparison, a concept of heart rate corrected QTc interval was introduced and many correction formulas have been proposed. Bazzet’s formula is both most frequently used in clinical practice and most criticised.146,147 During the last two decades, subject-specific models for QT correction were discussed as the most convenient – especially in drug-induced QTc prolongation assessment.148,149 Antipsychotic drug haloperidol was repeatedly reported to prolong QTc interval and causes sudden death even after a single dose.58,94,150 Despite restricted haloperidol prescription in some countries and availability of more modern antipsychotic drugs on the market, haloperidol is still widely prescribed in many countries. The incidence of haloperidol cardiac adverse effects is low; however, such side effects may have grave consequences. In drug development and safety assessment, animal models play a crucial role. Cardiac effects of haloperidol were repeatedly studied in various animal models.95-97,109,151 Although many studies have been focused on mechanism of haloperidol cardiac adverse effects, the mosaic is still incomplete. Novel animal models may bring new perspectives on this problem. This thesis was aimed to analyse effects of haloperidol administration on electrogram in two animal models – in long-term haloperidol treated guinea pig model and rat neurodevelopmental model of schizophrenia. Experiments were performed on Langendorff isolated heart. Spontaneously beating Langendorff isolated heart is eligible model for studying direct drug effects on heart electric activity without extracardiac modulation (autonomous nervous system and humoral effects). Heart rate of spontaneously beating isolated heart is affected by rather small changes of experimental conditions (such as temperature and perfusion pressure). To prevent any external influence, experimental conditions in Langendorff apparatus were strictly controlled and precisely monitored. For administration during the isolated heart experiments, haloperidol was diluted in Krebs- Henseleit solution. Concentration of haloperidol was chosen in accordance with previous experimental works.95,151 Concentration of 10 nmol/l corresponds to free plasma concentrations

| 40 in haloperidol-treated patients (10 – 200 nmol/l).96 Because of limited solubility of haloperidol in water, haloperidol was pre-dissolved in ethanol. The final concentration of ethanol in Krebs- Henseleit solution administered to isolated heart was 8.22 nmol/l. To our best knowledge, ethanol in such small concentration has no effect on electrophysiology in guinea pig and rat isolated heart.151-153 One of a few limitations of this model is certain influence of ischemia to which the heart is exposed during preparation.154 Preparation ischemia increases the production of reactive oxygen species in the heart tissue. During increased oxidative stress, HNE is produced by peroxidation of membrane lipids.155 Thus, to evaluate the quality of the isolated heart preparation in the study A and to detect possible influence of preparation ischemia, duration of cold and warm ischemic period was measured and assessment of CK, LD, lactate, and HNE was performed in samples of coronary effluent. As no significant difference was find between the groups (Table 8), we may conclude that the preparation ischemia in our experimental set- up does not affect the reliability of the obtained results.

5.1 Study A The main advantage of guinea pig, a model organism used in this study, is that guinea pig cardiac cells express voltage-gated ion channels comparable to those of humans.156 Haloperidol was administered via intraperitoneal route in the dose previously used by Fialova and co- workers.95 Pharmacokinetics of intraperitoneal administration is comparable to oral administration because the primary route of absorption is into portal vein system and passage through liver.157,158 It brings our model closer to clinical situation, since the majority of long- term haloperidol-treated patients use the oral form.61 Two clinical situations were imitated in the isolated heart experiments: (1) administration of haloperidol in isolated hearts of haloperidol-treated guinea pigs (group HAL) simulated acute (intravenous) haloperidol exposure in long-term haloperidol-treated patient; (2) haloperidol administration in isolated hearts of control guinea pigs (group CON) simulated the first haloperidol exposure in untreated patient. It has been shown that acute haloperidol administration decreases the coupling between the QT and RR intervals in both groups (Figure 5). Moreover, flatter dQT/dRR coupling in HAL group over all phases indicates that long-term haloperidol administration alters relation between QT and RR. The flatter relationship between QT and RR reveals a lack of QT adaptation to changing heart rate – QT interval does not adapt to altered heart rate sufficiently.140 Even no

| 41 significant change in incidence of spontaneous arrhythmias was detected in our model, such decreased adaptation of QT to heart rate (RR interval) increases risk of ventricular arrhythmias – especially when the heart rate is accelerated.159,160 For correction of QT to the heart rate, subject-specific correction model was used. It was previously shown that subject-specific linear model is more appropriate for QT correction in guinea pig isolated heart than other frequently used correction methods.161 As linear character of the QT/RR coupling was found in phases of haloperidol administration and washout, linear model of correction was applied. For higher accuracy of correction, calculation was based on parameters of QT/RR coupling in the specific phase of experiment. This approach was applied in all phases, except of stabilisation. In stabilisation phase, non-linear changes of QT/RR coupling are expected. Non-linearity in QT/RR coupling relates to adaptation of isolated heart to experimental conditions. The isolated heart should be considered as stable (and therefore linear character of QR/RR coupling might be expected) in short period at the end of stabilisation phase only. However, the period (not more than 2 minutes of the record) is too short for accurate calculation of linear QT/RR coupling parameters. Therefore, modified formula for QTc correction was used in stabilisation phase. Different QTc correction formulas limit evaluation of the direct effect of acute haloperidol administration on QTc. Nevertheless, the trends in QTc differences between consecutive phases correspond to the generally accepted idea that acute haloperidol administration prolongs QTc – QTc is higher in haloperidol phases than in washouts in both groups (Table 4). However, prolonging effect of long-term haloperidol administration on QTc was not proven. Contrary to expectations, QTc in haloperidol treated group did not differ from control group at the end of stabilization and during the first haloperidol phase. It was even significantly shorter during the first washout (Table 4). It might be explained by different reactivity of QTc in group CON and HAL to acute haloperidol administration (H1) and consecutive washout (W1). Between H1 and W1, the haloperidol treated group (HAL) exerted higher change of QTc than control group (CON). In comparison, the dQT/dRR coupling was significantly flatter in HAL group than in controls in all phases except for stabilisation (Figure 5). Modulation of sigma 1 receptors by haloperidol might at least partially explain changes detected in isolated guinea pig hearts in this study. Haloperidol is a potent sigma 1 ligand (Table 1). Modulatory effect of sigma 1 receptors on potassium channels behaviour was previously reported in various models that.162 In order to demonstrate the putative role of sigma 1 receptors in our model, expression of the sigma 1 receptors was analysed. It was detected that long-term

| 42 haloperidol treatment significantly increased expression of sigma 1 receptors in both cardiac atria (Figure 6). However, no change of sigma 1 receptor expression was detected in cardiac ventricles. It contrasts with previous study in long-term haloperidol treated rat model, in which an increase of sigma 1 receptor´s expression in the cardiac atria as well as ventricles was reported.109 This finding may be attributed to interspecies differences in effect of haloperidol on the heart. In addition, handling of animals during chronic treatment should be taken into consideration. Handling represents a moderate level of stress for animals. Mild stress affects expression of cardiac sigma receptors163 and this reaction may also differ in rat and guinea pig.

Expression of IP3 receptors type 1 and 2, which are known to be coupled with sigma 1 receptors in the heart,164 was also determined. In accordance with the rat model, the increased expression was detected only in cardiac atria of haloperidol treated guinea pigs. Sigma 1 receptor acts as a pluripotent modulator in the cell. By its chaperone activity and protein-protein interactions, it regulates calcium signalling, enhances the endoplasmic reticulum signalling to nucleus and attenuates response to oxidative stress.116,119,120 Moreover, sigma 1 receptor binds to hERG channel in endoplasmic reticulum and facilitates hERG maturation and trafficking.128,129 Inhibition of hERG channel is connected with significant QTc prolongation.66,67 Haloperidol has antagonistic activity in sigma 1 receptor. Absence of increase of sigma 1 expression in cardiac ventricles may contribute to decreased coupling of QT and RR under haloperidol administration.

5.2 Study B According to research evidence, schizophrenia is neurodevelopmental disorder.165 Therefore, neurodevelopmental animal models of schizophrenia are at the forefront of scientists’ interest. As described previously, an injection of the MAM in pregnant female rats at gestational day 17 resulted in long lasting behavioural (i.e. hyper-locomotion, social behaviour impairment and cognitive deficits) and molecular (i.e. dysregulation of dopaminergic and glutamatergic neurotransmission) changes in the offspring.137,166 It was shown that MAM model is highly translatable. The changes of MAM offspring are similar to those observed in schizophrenia patients.166,167 To our best knowledge, electrogram parameters in rat schizophrenia model and their changes under haloperidol administration were evaluated for the first time in this study. It was clearly shown that response of QTc interval to acute haloperidol administration is different in isolated hearts of MAM and control rats (Figure 8). After acute haloperidol administration, QTc was

| 43 significantly higher in MAM rats than in controls. Subsequent washout causes significant decrease of QTc in MAM group. However, significant difference between the groups was kept. Moreover, nearly significant difference in QTc interval was detected in MAM group when compared to controls at the end of stabilisation. In drug-free schizophrenia patients, significantly longer QTc was reported compared to age- matched healthy subjects.65 Fujii and co-authors discussed a role of a variant polymorphism or mutation in hERG (KCNH2 channel) as a possible mechanism common in both pathophysiology of schizophrenia and long QT. Advanced genotyping methods and subsequent analyses help to uncover common genetic factors that contribute to both risk of schizophrenia and cardiovascular diseases.168 The hERG seems to be one of such genes. Association of hERG with QT prolongation was repeatedly proven.66,67,169 During the last decade, hERG (KCNH2) association with high risk of schizophrenia was demonstrated.170-172 Unique, brain specific isoform of hERG was recognised and associated with cognition and neuronal repolarization.170,171 Moreover, by the brain isoform of the hERG, a modulation of treatment response of schizophrenia patients was reported.172 As the isolated heart is detached from all extracardiac influences, the mechanism of altered QT reactivity have to be located in the heart itself. Some changes of electric activity of MAM hearts were uncovered by HRV analysis. In case of heart in situ, HRV is mainly associated with oscillations of autonomous system activity,173 which has modulatory effect on heart functions. In schizophrenia patients, alteration of HRV parameters was described.174 In isolated heart, HRV depicts a variability in intrinsic control and regulatory mechanisms. Minor – however significant – differences were found between the MAM rats and controls in selected HRV parameters (Figure 9 – 12). It indicates alteration of intrinsic control and regulatory mechanisms in the hearts of adult MAM rats. The MAM is a very potent DNA-alkylating agent. It is believed that through selective disturbance of proliferation and migration of neuronal precursor cells undergoing their final mitosis, MAM induces morphological and cytological alterations in rat offspring. This resembles phenomena seen in schizophrenia post-mortem brains.166,167 However, the effects of MAM exposure in embryonic day 17 on heart structure and function have not been previously studied. In this thesis, heart weight and histological structure of MAM hearts were evaluated. Significantly lower heart weight was detected in MAM rats in comparison with controls. However, this difference was caused by significant lower body weight in MAM rats. Such close relationship between body weight and heart weight in mammals was clearly demonstrated.175

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This explanation is supported by analysis of heart weight to body weight ratio, where no significant difference was observed. In histological analyses, no significant differences in structure of myocardium, size of cardiomyocytes and collagen content were observed between the groups (Figure 7). The MAM exposure in the late embryonic stage of development does not cause structural changes in offspring hearts in adulthood. This may be explained by the fact that rat heart is almost fully maturated from embryonic day 16.176 Only minimal macroscopic changes, mostly connected with adaptation of cardiovascular system to postnatal life, were detected.176 At cellular level, formation of highly organized intracellular structure and energy metabolism in cardiomyocytes were described from late embryonic to early postnatal development.177 To support this theory, viability of H9c2 cells after 24 hours exposure to MAM was evaluated. H9c2 is cell line derived from rat cardiomyoblasts. It is a respected cellular model in toxicology studies.178-180 It was demonstrated that MAM decreases viability of H9c2 cells at high concentrations only (Figure 13 and 14). Such concentrations (above 500 μmol/L) are not expected in the hearts of MAM rats during MAM exposition. However, by alkylation, MAM may affect gene transcription181 and consequently electrophysiology of the heart. Although rat heart in embryonic day 17 is almost fully maturated as the structure is concerned, the electrophysiological features as well as intracellular organisation and metabolism of cardiomyocytes undergo substantial changes during the late embryonic development and early postnatal life.177,182 According to experimental data and mathematical models, the most prominent potassium current in the rat heart is inward rectifier K+ current via hERG channel.182 It is the same hERG channel, alteration of which is associated with QT prolongation. However, the precise description of mechanisms of QT alteration in MAM rats needs further investigation.

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6 Conclusion

Haloperidol is a potent typical antipsychotic drug. It is very effective in acute as well as chronic administration protocols. Use of haloperidol is limited by its adverse effects. Besides others, haloperidol was associated with QT prolongation. The long QT increases risk of ventricular arrhythmias and sudden cardiac death. Thus, haloperidol-induced QT prolongation may contribute to early mortality in schizophrenia patients. As schizophrenia is one of the most burdening diseases worldwide with significantly shortened life expectancy, it comes to be very important to study all factors, which may contribute to decreased quality of patient’s life and early mortality. Herein, effects of haloperidol on the QT interval in two animal models were studied. We demonstrated that haloperidol administration decreases the coupling between the QT and RR intervals in long-term haloperidol treated guinea pigs. The loss of coupling between QT and RR intervals points to increased electrical instability, which may lead to increased probability of ventricular arrhythmias, especially at higher heart rates. In MAM model, we demonstrated that schizophrenia-like phenotype leads to QT alteration in rat isolated heart. In schizophrenia- like rats, haloperidol-induced QT prolongation was substantially more manifested. As no change was found in the structure of schizophrenia-like rat heart, it can be assumed that the changed QT reactivity is caused by functional alteration. Based on literature review and results summarised in this thesis, it may be hypothesised that sigma 1 receptor represents important component in the mechanism of haloperidol-induced QT prolongation. Sigma 1 receptor, by its regulatory and chaperon activity, affects calcium handling as well as trafficking of functionally important proteins, such as the hERG channel. For decades, animal models play crucial role in cardiovascular research. To study mechanisms of haloperidol-induced QT prolongation, it is important to identify novel animal models with high translational potential. The rat neurodevelopmental model of schizophrenia seems to be a good candidate.

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170. Huffaker SJ, Chen J, Nicodemus KK, et al. A primate-specific, brain isoform of KCNH2 affects cortical physiology, cognition, neuronal repolarization and risk of schizophrenia. Nat Med. 2009;15(5):509-518. 171. Hashimoto R, Ohi K, Yasuda Y, et al. The KCNH2 gene is associated with neurocognition and the risk of schizophrenia. World J Biol Psychiatry. 2013;14(2):114- 120. 172. Apud JA, Zhang F, Decot H, Bigos KL, Weinberger DR. Genetic variation in KCNH2 associated with expression in the brain of a unique hERG isoform modulates treatment response in patients with schizophrenia. Am J Psychiatry. 2012;169(7):725-734. 173. Shaffer F, Ginsberg JP. An Overview of Heart Rate Variability Metrics and Norms. Front Public Health. 2017;5:258. 174. Montaquila JM, Trachik BJ, Bedwell JS. Heart rate variability and vagal tone in schizophrenia: A review. J Psychiatr Res. 2015;69:57-66. 175. Prothero J. Heart weight as a function of body weight in mammals. Growth. 1979;43(3):139-150. 176. Marcela SG, Cristina RM, Angel PG, et al. Chronological and morphological study of heart development in the rat. Anat Rec (Hoboken). 2012;295(8):1267-1290. 177. Anmann T, Varikmaa M, Timohhina N, et al. Formation of highly organized intracellular structure and energy metabolism in cardiac muscle cells during postnatal development of rat heart. Biochim Biophys Acta. 2014;1837(8):1350-1361. 178. Sathishkumar K, Gao X, Raghavamenon AC, Murthy SN, Kadowitz PJ, Uppu RM. Determination of glutathione, mitochondrial transmembrane potential, and cytotoxicity in H9c2 cardiomyoblasts exposed to reactive oxygen and nitrogen species. Methods Mol Biol. 2010;610:51-61. 179. Korashy HM, Attafi IM, Ansari MA, et al. Molecular mechanisms of cardiotoxicity of gefitinib in vivo and in vitro rat cardiomyocyte: Role of apoptosis and oxidative stress. Toxicol Lett. 2016;252:50-61. 180. Zordoky BN, El-Kadi AO. H9c2 cell line is a valuable in vitro model to study the drug metabolizing enzymes in the heart. J Pharmacol Toxicol Methods. 2007;56(3):317-322. 181. Zhu H, Wang G, Qian J. Transcription factors as readers and effectors of DNA methylation. Nat Rev Genet. 2016;17(9):551-565. 182. Okubo C, Sano HI, Naito Y, Tomita M. Contribution of quantitative changes in individual ionic current systems to the embryonic development of ventricular myocytes: a simulation study. J Physiol Sci. 2013;63(5):355-367.

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8 List of Abbreviations

Akt protein kinase B BDNF brain-derived neurotrophic factor CK creatine kinase DDD defined daily dose hERG human ether-à-go-go-related gene HNE 4-hydroxy-2-nonenal HR heart rate HRV heart rate variability

IP3 inositol 1,4,5-trisphosphate LD lactate dehydrogenase MAM methylazoxymethanol acetate NIMH The National Institute of Mental Health NN20 the number of interval differences of successive RR intervals greater than 20 ms PBS phosphate-buffered saline PCR polymerase chain reaction SDNN standard deviation of the NN intervals SVPB supraventricular premature beats TrkB tropomyosin receptor kinase B VPB ventricular premature beats

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9 List of Figures

Figure 1: Association between schizophrenia and cardiovascular diseases. 11

Figure 2: Total number of defined daily doses (DDD) of haloperidol and olanzapine prescribed in the Czech Republic between 2011 and 2017. 12

Figure 3: Experimental protocols of isolated heart experiments. 23

Figure 4: Original record of rat isolated heart electrogram. 24

Figure 5: The dQT/dRR coupling computed for each individual haloperidol and washout phase. 31

Figure 6: Relative gene expressions of the sigma 1 receptor, IP3 receptor type 1 and type 2 (left), and immunohistochemical staining of the sigma 1 receptors and the IP3 receptors type 1 in the left cardiac atrium (right) of guinea pig heart after repeated haloperidol exposure. 32

Figure 7: Representative pictures of histological sections of left ventricle stained using haematoxylin and eosin in MAM rat (right) and control rat (left). 34

Figure 8: Heart rate (right) and QTc interval (left) in rat isolated hearts. 35

Figure 9: RR intervals in rat isolated heart during the experiment. 36

Figure 10: The standard deviation of the NN intervals in rat isolated heart during the experiment. 36

Figure 11: The parameter NN20 in rat isolated heart during the experiment. 37

Figure 12: The sample entropy in rat isolated heart during the experiment. 37

Figure 13: Viability of H9c2 cell after 24-hours exposure to MAM. 37

Figure 14: Fluorescein diacetate (FDA)/propidium iodide (PI) staining of H9C2 cells. 38

Figure 15: Reactive oxygen species (ROS) production (A) and lipid peroxidation in H9C2 cells after 24-hours exposure to MAM in low concentrations (10 nM – 100 μM). 38

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10 List of Tables

Table 1: Pharmacological profile of haloperidol 16

Table 2: Composition of Krebs-Henseleit solution. 21

Table 3: The RR, heart rate, QT, and QTc in guinea pig isolated heart at the end of stabilization phase. 29

Table 4: QTc [ms] in haloperidol and washout phases. 30

Table 5: Mean values of RR, QT, dRR, and dQT in guinea pig isolated hearts computed for each individual haloperidol and washout phase. 30

Table 6: The numeral expression of the QT/RR coupling in guinea pig isolated hearts computed for each individual haloperidol and washout phase. 30

Table 7: The numeral expression of the dQT/dRR coupling in guinea pig isolated hearts computed for each individual haloperidol and washout phase. 32

Table 8: The levels of creatine kinase, lactate dehydrogenase and lactate in the samples of coronary effluent. 33

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11 List of Appendices

Appendix A: Cardiac sigma receptors - An update. (Review)

Appendix B: Long-term haloperidol treatment prolongs QT interval and increases

expression of sigma 1 and IP3 receptors in guinea pig hearts. (Original article)

Appendix C: Haloperidol affects coupling between QT and RR intervals in guinea pig isolated heart. (Original article)

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12 List of Author’s Publications

All author’s publications indexed in Web of Science (Clarivate Analytics) and/or Scopus (Elsevier B.V) are listed below (updated in June 2019). Conference abstracts are not included. Impact Factor (IF) is stated according to Journal Citation Reports (Clarivate Analytics). “*” indicates corresponding author

12.1 Thesis-related Publications Original and Review Articles in Journals with Impact Factor 1. Raudenska M, Gumulec J, Babula P, Stracina T, Sztalmachova M, Polanska H, Adam V, Kizek R, Novakova M, Masarik M*. Haloperidol Cytotoxicity and Its Relation to Oxidative Stress. Mini-Reviews in Medicinal Chemistry. 2013;13(14):1993-1998. IF 3.186 2. Stracina T, Slaninova I, Polanska H, Axmanova M, Olejnickova V, Konecny P, Masarik M, Krizanova O, Novakova M*. Long-Term Haloperidol Treatment Prolongs QT Interval and Increases Expression of Sigma 1 and IP3 Receptors in Guinea Pig Hearts. Tohoku Journal of Experimental Medicine. 2015;236(3):199-207. IF 1.287 3. Kubickova J, Lencesova L, Csaderova L, Stracina T, Hudecova S, Babula P, Rozborilova E, Novakova M, Krizanova O*. Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through sigma 1R/IP(3)R1 Complex. Cellular and Molecular Neurobiology. 2018;38(1):181-194. IF 3.811 4. Stracina T, Novakova M*. Cardiac Sigma Receptors - An Update. Physiol Res. 2018;67:S561-S576. IF 1.701 5. Vesely P, Stracina T*, Hlavacova M, Halamek J, Kolarova J, Olejnickova V, Mrkvicova V, Paulova H, Novakova M. Haloperidol affects coupling between QT and RR intervals in guinea pig isolated heart. J Pharmacol Sci. 2019;139(1):23-28. IF 2.439

Conference Papers Indexed in Web of Science 6. Stracina T*, Ronzhina M, Stark T, Ruda J, Olsanska E, Vesely P, Micale V, Novakova M. Prolonged QT Interval in Neurodevelopmental Rat Model of Schizophrenia. 2016 Computing in Cardiology Conference (Cinc), Vol 43. 2016;43:1049-1052. 7. Janousek O*, Stracina T, Ronzhina M, Hejc J, Stark T, Ruda J, Micale V, Kolarova J, Novakova M, Provaznik I. The Effect of Haloperidol Administration on Heart Rate

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Variability in Isolated Heart of Schizophrenia-like and Control Rats. In: 2017 Computing in Cardiology. Los Alamitos: IEEE Computer Soc; 2017.

12.2 Publications Unrelated to the Thesis Original and Review Articles in Journals with Impact Factor 8. Gumulec J, Raudenska M, Hlavna M, Stracina T, Sztalmachova M, Tanhauserova V, Pacal L, Ruttkay-Nedecky B, Sochor J, Zitka O, Babula P, Adam V, Kizek R, Novakova M, Masarik M*. Determination of oxidative stress and activities of antioxidant enzymes in guinea pigs treated with haloperidol. Experimental and Therapeutic Medicine. 2013;5(2):479-484. IF 0.941 9. Paulova H*, Stracina T, Jarkovsky J, Novakova M, Taborska E. Hydroxyl radicals' production and ECG parameters during ischemia and reperfusion in rat, guinea pig and rabbit isolated heart. General Physiology and Biophysics. 2013;32(2):221-228. IF 0.875 10. Hlavacova M, Gumulec J, Stracina T, Fojtu M, Raudenska M, Masarik M, Novakova M, Paulova H*. Different doxorubicin formulations affect plasma 4-hydroxy-2-nonenal and gene expression of aldehyde dehydrogenase 3A1 and thioredoxin reductase 2 in rat. Physiol Res. 2015;64 Suppl 5:S653-660. IF 1.643 11. Hudecova S, Markova J, Simko V, Csaderova L, Stracina T, Sirova M, Fojtu M, Svastova E, Gronesova P, Pastorek M, Novakova M, Cholujova D, Kopacek J, Pastorekova S, Sedlak J, Krizanova O*. Sulforaphane-induced apoptosis involves the type 1 IP3 receptor. Oncotarget. 2016;7(38):61403-61418. IF 5.168 12. Fojtu M, Gumulec J, Stracina T, Raudenska M, Skotakova A, Vaculovicova M, Adam V, Babula P, Novakova M, Masarik M*. Reduction of Doxorubicin-Induced Cardiotoxicity Using Nanocarriers: A Review. Current Drug Metabolism. 2017;18(3):237-263. IF 2.655 13. Hlavacova M, Olejnickova V, Ronzhina M, Stracina T, Janousek O, Novakova M, Babula P, Kolarova J, Provaznik I, Paulova H*. Tolerance of isolated rabbit hearts to short ischemic periods is affected by increased LV mass fraction. Physiol Res. 2017;66(4):581-589. IF 1.324 14. Novak J*, Sana J, Stracina T, Novakova M, Slaby O. Doxorubicin and Liposomal Doxorubicin Differentially Affect Expression of miR-208a and let-7g in Rat Ventricles and Atria. Cardiovascular Toxicology. 2017;17(3):355-359. IF 2.989

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15. Ronzhina M*, Olejnickova V, Stracina T, Novakova M, Janousek O, Hejc J, Kolarova J, Hlavacova M, Paulova H. Effect of increased left ventricle mass on ischemia assessment in electrocardiographic signals: rabbit isolated heart study. BMC Cardiovascular Disorders. 2017;17:11.15. IF 1.812

Review Article in Journal without Impact Factor (Indexed in Scopus) 16. Dufek D, Stračina T, Nováková M*. Cardiovascular side effects of antitumour drugs. Onkologie (Czech Republic). 2014;8(6):264-268.

Conference Papers Indexed in Web of Science 17. Vesely P*, Halamek J, Stracina T, Krejcirova L, Novakova M. QT Interval Analysis in Electrograms of Isolated Guinea Pig Hearts Treated with Haloperidol. In: 2013 Computing in Cardiology Conference. New York: IEEE; 2013;40:69-72. 18. Olejnickova V*, Ronzhina M, Paulova H, Hlavacova M, Stracina T, Novakova M. Susceptibility of Isolated Rabbit Hearts with Various Left Ventricular Mass to Short Ischemic Periods. 2014 Computing in Cardiology Conference (Cinc), Vol 41. 2014;41:1097-1100. 19. Fojtu M*, Gumulec J, Sztalmachova M, Raudenska M, Stracina T, Novakova M, Kizek R, Adam V, Hlavacova M, Paulova H, Masarik M. Changes in oxidative stress status after the administration of doxorubicin-loaded nanocarriers. International Journal of Molecular Medicine. 2015;36:S34-S34. IF 2.348 20. Janousek O*, Ronzhina M, Hejc J, Olejnickova V, Stracina T, Fialova K, Novakova M, Provaznik I, Kolarova J. The Effect of Voltage-Sensitive Dye di-4-ANEPPS on Heart Rate Variability in Langendorff-Perfused Isolated Rabbit Heart. In: Murray A, ed. 2015 Computing in Cardiology Conference. New York: IEEE; 2015;42:1049-1052. 21. Hejc J*, Janousek O, Ronzhina M, Stracina T, Olejnickova V, Kolarova J, Novakova M. Response of Ventricular Repolarization Parameters to Preload Changes in the Isolated Working Heart. In: Murray A, ed. 2016 Computing in Cardiology Conference. Vol 43. New York: IEEE; 2016:409-412. 22. Janousek O*, Ronzhina M, Hejc J, Stracina T, Olejnickova V, Novakova M, Provaznik I, Kolarova J. The Effect of Cardiac Filling on Heart Rate Variability in Rabbit Isolated Heart. Paper presented at: 43rd Computing in Cardiology Conference (CinC); Sep 11-14, 2016; Vancouver, Canada. Los Alamitos: IEEE Computer Soc; 2016.

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23. Novotna P*, Hejc J, Ronzhina M, Janousek O, Stracina T, Olejnickova V, Novakova M, Kolarova J,. Analysis of Hemodynamic Related Changes in High Frequency Content of QRS Complex in Working Isolated Rabbit Heart. In: 2017 Computing in Cardiology. Los Alamitos: IEEE Computer Soc; 2017. 24. Hokynkova A*, Wilhelm Z, Novakova M, Babula P, Stracina T, Paulova H, Hlavacova M, Sedlackova M. Wound healing effects after application of polyunsaturated fatty acids in rat. Ceska a Slovenska Neurologie a Neurochirurgie. 2018;81:S29-S31. IF 0.355

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13 Summary of the Thesis Findings

The thesis was aimed to analyse the effects of haloperidol administration on cardiac electrogram in two animal models. The main findings are as follows:

Finding 1 In guinea pig model, the long-term haloperidol administration leaded to decrease of the QT/RR coupling in isolated heart perfused according to Langendorff. In haloperidol-treated guinea pig isolated heart, no significant change of QTc calculated according to subject specific model was detected in stabilisation. During the second acute haloperidol administration, QTc was significantly higher in haloperidol treated group in comparison with controls.

Finding 2 In guinea pig model, long-term haloperidol administration leaded to increase of the expression of sigma 1 receptors and IP3 receptors type 1 and 2 in cardiac atria contrary to the ventricles where no such change was found.

Finding 3 No structural change was detected in the hearts of rat neurodevelopmental model of schizophrenia. In schizophrenia-like rat isolated heart, a trend to longer QTc interval (when compared to controls) was detected in stabilization.

Finding 4 After acute haloperidol administration, QTc was significantly higher in schizophrenia-like rats than in controls.

Finding 5 In H9c2 cell line, methylazoxymethanol acetate caused significant decrease of viability in concentrations of 500 and 1000 μmol/L. Slight increase of production of ROS and subsequent lipid peroxidation was detected after MAM exposure at the concentration of 100 μmol/L.

Student Supervisor MUDr. Tibor Stračina Prof. MUDr. Marie Nováková, Ph.D.

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14 Souhrn poznatků disertační práce

Tato disertační práce byla zaměřena na studium účinků haloperidolu na elektrogram srdce ve dvou animálních modelech. Hlavní poznatky jsou následující:

Poznatek 1 Dlouhodobé podávání haloperidolu vedlo u morčete ke snížení QT/RR couplingu u izolovaného srdce. U izolovaných srdcí morčat dlouhodobě vystavených haloperidolu nebyl v stabilizaci nalezen signifikantní rozdíl v QTc korigovaném podle pro subjekt specifického modelu. Během druhé akutní expozice haloperidolu byl zaznamenán signifikantně delší QTc u morčat dlouhodobě vystavených haloperidolu v porovnání s kontrolami.

Poznatek 2 V modelu morčete vedlo dlouhodobé podávání haloperidolu ke zvýšení exprese sigma 1 receptoru a IP3 receptorů typu 1 a 2 v srdečných síních, v komorách žádná změna zaznamenána nebyla.

Poznatek 3 V srdcích neuro-vývojového modelu schizofrenie u potkana nebyla nalezena žádná strukturální změna. U izolovaného srdce tohoto modelu schizofrenie byl zjištěn trend k prodloužení QTc intervalu (ve srovnání s kontrolou) ve stabilizaci.

Poznatek 4 U izolovaného srdce modelu schizofrenie u potkana bylo po akutní expozici haloperidolu detekováno výrazně delší QTc (ve srovnání s kontrolou).

Poznatek 5 Methylazoxymethanol acetát způsobil u buněčné linie H9c2 signifikantní pokles viability v koncentracích 500 a 1000 μmol/l. V koncentraci 100 μmol/l byla zjištěna mírně zvýšená produkce ROS a peroxidace lipidů.

Student Školitel MUDr. Tibor Stračina Prof. MUDr. Marie Nováková, Ph.D.

| Appendix

Appendix A

Physiol. Res. 67 (Suppl. 4): S561-S576, 2018

REVIEW

Cardiac Sigma Receptors – An Update

T. STRACINA1, M. NOVAKOVA1

1Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Received March 25, 2018 Accepted September 12, 2018

Summary (Martin et al. 1976). The authors believed that sigma More than four decades passed since sigma receptors were first receptor represents an opioid receptor subtype, which mentioned. Since then, existence of at least two receptor mediates psychomimetic and stimulatory behavioral subtypes and their tissue distributions have been proposed. effects of N-allylnormetazocine (SKF-10047) in chronic Nowadays, it is clear, that sigma receptors are unique ubiquitous spinal dog. Subsequent binding studies in guinea pig and proteins with pluripotent function, which can interact with so rat showed that binding profile of sigma receptor differs many different classes of proteins. As the endoplasmic resident from any other known subtype of opioid receptor as well proteins, they work as molecular chaperones – accompany as other receptor classes (Su 1982, Tam 1983). Therefore, various proteins during their folding, ensure trafficking of the the sigma receptor was defined as novel receptor type maturated proteins between cellular organelles and regulate their (Su 1982). functions. In the heart, sigma receptor type 1 is more dominant. Cardiac sigma 1 receptors regulate response to endoplasmic Two subtypes of sigma receptor reticulum stress, modulates calcium signaling in cardiomyocyte Further research led to differentiation among at and can affect function of voltage-gated ion channels. They least two subtypes of sigma receptors. Based on their contributed in pathophysiology of cardiac hypertrophy, heart diverse ligand selectivity and stereospecificity, association failure and many other cardiovascular disorders. Therefore, with signal transduction mechanism and/or enzyme sigma receptors are potential novel targets for specific treatment function, tissue distribution, subcellular localization, and of cardiovascular diseases. apparent molecular mass, existence of sigma receptor type 1 (sigma 1 receptor) and type 2 (sigma 2 receptor) has Key words been proposed and confirmed (Hellewell and Bowen 1990, Sigma receptor • Heart • Chaperone • Endoplasmic reticulum Quirion et al. 1992, Torrence-Campbell and Bowen 1996). stress The sigma receptor originally described by Su (1982) was recognized as the sigma 1 receptor (Hellewell and Bowen Corresponding author 1990). M. Novakova, Department of Physiology, Faculty of Medicine, Since the molecular structure of the sigma 1 Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. receptor was not known till 1996, various sigma ligands E-mail: [email protected] were employed in the studies of distribution and cellular functions of sigma receptors. Selective ligands A brief history – from an enigmatic binding (e.g. 1,3-di(2-tolyl) guanidine (DTG), SA 4503, site in the brain to ubiquitous receptor and (±)-PPCC, BD 1047) as well as clinically used drugs molecular chaperone which exert affinity to sigma receptors (e.g. haloperidol, fluvoxamine, sertraline, amitriptyline) have played Sigma receptors were first reported in the central crucial role in the research. Based on their different nervous system by Martin and co-workers in 1976 binding profiles, sigma receptors were classified as

PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)  2018 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic Fax +420 241 062 164, e-mail: [email protected], www.biomed.cas.cz/physiolres

S562 Stracina and Novakova Vol. 67

follows (Quirion et al. 1992): sigma 1 receptors exert transmembrane domain for each protomer (Schmidt et al. high affinity to dextromethorphan, (+)-pentazocine, 2016). The cytosolic domain of each of the three (+)-NANM, and carbetapentane; sigma 2 receptors bind protomers contains a -barrel β fold with the ligand- these compounds with low affinity. Haloperidol and binding region at its center. Such structure is substantially DTG, two mostly used sigma ligands in 1980s and 1990s, different from the two-transmembrane domain model show affinity for both sigma subtypes (Kushner and proposed on the basis of biochemical, molecular, in Zukin 1994). Moreover, sigma subtypes exert different silico, and NMR data (Laurini et al. 2011, Laurini et al. stereoselectivity for benzomorphans: sigma 1 receptor 2017). As Laurini et al. (2017) postulated, differences exhibits higher affinity for dextrorotatory benzomorphans may arise from various factors, such as structure and, in contrast, sigma 2 receptor exhibits equal or higher determination methods and experimental conditions used. affinity for the levorotatory benzomorphans (Hellewell Moreover, the protein may adopt different structures and Bowen 1990). under solid and solution states. More studies are needed to prove structural details of sigma 1 receptor protein. Sigma 1 receptor – molecular structure Research on cellular localization(s) and Sigma 1 receptor – ligands function(s) of sigma receptors was significantly facilitated The classification of sigma ligands as agonists by description of molecular structure of the sigma 1 and antagonists is mainly based on animal studies. receptor. The sigma 1 receptor was first purified and Agonists are defined as ligands that induce cloned from guinea pig liver in 1996 (Hanner et al. 1996). hyperlocomotion or other physiological responses The amino acid sequence was structurally unrelated to then through binding to sigma receptor, while antagonists are known mammalian proteins (Hanner et al. 1996). ligands that block or blunt this response (Martin et al. Subsequently, the receptor was cloned from various 1976, Schmidt et al. 2016). Various endogenous tissues, both animal and human ones (Kekuda et al. 1996, substances, such as progesterone, dihydroepi- Seth, Leibach, and Ganapathy 1997, Prasad et al. 1998, androsterone, sphingosine and its derivatives, and Seth et al. 1998, Mei and Pasternak 2001). Mei and N,N-dimethyltryptamine, exert certain affinity to sigma 1 Pasternak (2001) reported that predicted structure of rat receptor (Patterson et al. 1994). Although these sigma 1 receptor is highly homologous with murine substances can bind to sigma 1 receptor under (93.3 %), guinea pig (93.7 %) and human (96.0 %) sigma 1 experimental conditions, up to now none of them has receptor. Many structural models of the receptor were been reported to act as endogenous sigma 1 ligand. Nine postulated (Su and Hayashi 2003, Laurini et al. 2011, years ago, Fontanilla et al. (2009) indicated Schmidt et al. 2016, Laurini et al. 2017). Most of them N,N-dimethyltryptamine as endogenous sigma 1 described sigma 1 receptor as membrane receptor with two regulator. However, relevant doubts were recently cast on transmembrane domains (Laurini et al. 2017). It was this suggestion (Nichols 2018) and the endogenous ligand reported that ligand-binding region of sigma 1 receptor is still seems to be undiscovered. The precise structure of similar to an active site of cupin family proteins, the ligand-binding region may shed light on this problem. oligomeric bacterial and fungal enzymes, and plant seed storage proteins (Hanner et al. 1996, Schmidt et al. 2016). Sigma 1 receptor – localization and function One of them is the yeast sterol C8-C7 isomerase, enzyme The sigma 1 receptor is involved in a wide range essential for ergosterol synthesis and cell proliferation of physiological functions and pathophysiological (Moebius et al. 1997). In spite of structural homology, processes in nervous system, such as neurodegenerative sigma 1 receptor binding region exerts no enzymatic diseases, neuropathic pain, depression and cocaine activity. In addition, Mishra and co-workers reported, that addiction (Su et al. 2016). It has been localized in several sigma 1 receptor can be found either in monomeric or regions of the central nervous system as well as in oligomeric forms in living cells in the presence and/or peripheral nervous system and numerous non-neural absence of various ligands (Mishra et al. 2015). tissues. According to immunohistochemical studies in rat, Recently, evidence of the full crystal structure high density of sigma 1 receptor was found in olfactory was reported (Schmidt et al. 2016). According to X-ray bulb, several hypothalamic nuclei, septum, central grey, data, the solid-state structure of the sigma 1 receptor certain motor nuclei of the hindbrain, and dorsal horn of reveals a trimeric organization with a single spinal cord (Alonso et al. 2000, Bouchard and Quirion 2018 Cardiac Sigma Receptors S563

1997). Among non-neural tissues, high density of the However, different methodological approaches were sigma 1 receptor was found in the immune, endocrine and employed in abovementioned studies. Therefore, many reproductive systems and in the digestive tract (Wolfe et questions concerning sigma 1 receptor localization and al. 1989, Hellewell et al. 1994), as well as in the heart functioning remain unanswered. (Ela et al. 1994, Novakova et al. 1995). Intracellular localization of sigma 1 receptor has Sigma 1 receptor in the heart been intensively studied. Various localizations were reported among different cell types and various stages of Contrary to other tissues, the first report on the cell differentiation. The primary region, where sigma 1 presence of sigma receptors in the heart muscle was receptor is located, is endoplasmic reticulum membrane focused on sigma 2 type (Dumont and Lemaire 1991). associated with mitochondria (Hayashi and Su 2007). Based on specific binding activity of the prototypic sigma Sigma 1 receptor acts here as molecular chaperone. By receptor ligand [3H]-DTG, Dumont and Lemaire interaction with various proteins, it promotes survival of concluded that sigma 2 receptors are present on the rat the cell by regulation of calcium signaling, enhancing the heart membrane preparations. As reported later, the endoplasmic reticulum signaling to nucleus and majority of sigma receptors (75 %) in the rat heart is attenuating response to oxidative stress (Boehning et al. represented by sigma 1 receptors (Novakova et al. 1995). 2003, Mori et al. 2013, Su et al. 2016). Nevertheless, Therefore, numerous studies were focused on cardiac translocation of sigma 1 receptor was reported repeatedly sigma 1 receptor in various experimental models. after sigma ligand stimulation or cellular stress (Hayashi and Su 2007, Mavlyutov and Ruoho 2007). Hayashi and Sigma 1 receptor gene and regulation of its expression Su proved translocation from lipid-enriched sites of the Sigma 1 receptor is coded by SIGMAR1 gene endoplasmic reticulum to the endoplasmic reticulum- (GeneCards®: Human Gene Database). In mouse, it was associated reticular network upon stimulation by recognized on chromosome 4, in rat on chromosome 5. In psychoactive drugs in NG108-15 cells (Hayashi and Su human, the gene is located on the shorter arm of 2003). Johannessen and co-workers reported inhibition of chromosome 9. Polymorphisms and mutations in sodium voltage-gated channel NaV1.5 by sigma 1 ligands SIGMAR1 in human population were identified and their in mouse cardiomyocytes (Johannessen et al. 2009). association with neurodegenerative and psychiatric Voltage-gated sodium channels are resident plasma diseases have been reported (Luty et al. 2010, Al-Saif, membrane channels and such inhibition suggested the Al-Mohanna, and Bohlega 2011, Huang et al. 2011, co-operation of sodium channel with sigma 1 receptor on Mandelli et al. 2017). Up today, there is no evidence of plasma membrane. Translocation to plasma membrane association of SIGMAR1 variants with cardiovascular was proved repeatedly in neuronal tissue and was disorders. previously reviewed (Su et al. 2010). Recently, In human genome, more than 20 regulatory translocation to nuclear envelope was reported after regions for SIGMAR1 gene were identified. Only a few haloperidol treatment in differentiated NG-108 cells regulating mechanisms of SIGMAR1 expression are clear (Kubickova et al. 2018). Mavlyutov and co-authors yet. Nevertheless, numerous factors, which affect determined precise intracellular localization of sigma 1 expression of sigma 1 receptor in heart, have been receptor in retinal neurons using electron microscopy reported in various models. Novakova et al. (2007) found (Mavlyutov et al. 2015). In photoreceptor cells, that sigma 1 receptors in rat heart are upregulated by predominal presence of sigma 1 receptor in the nuclear aging as well as by various stress factors, such as envelope and in the subsurface endoplasmic reticulum immobilization and cold environment. In mice, cisternae was found (Mavlyutov et al. 2015). Authors upregulation was caused by hypoxia (Novakova et al. suggested mechanism of sigma 1 receptor action possibly 2007). Sigma 1 receptors are upregulated also by long- different from that determined in other cell types. term exposure to prototypic sigma ligand haloperidol in It is generally accepted that the sigma 1 receptor heart of rat (Novakova et al. 2010) and guinea pig works as a ubiquitous pluripotent modulator in the (Stracina et al. 2015). Specific regulatory mechanisms of mammalian cells and interacts with many proteins (e.g. these phenomena have not been identified yet. receptors, ion channels, enzymes, chromatin-remodeling The first evidence of a specific transcriptional factors) in various intracellular locations (Su et al. 2016). factor regulating sigma 1 expression was brought by S564 Stracina and Novakova Vol. 67

Mitsuda and coworkers (Mitsuda et al. 2011). They ventricular cardiomyocytes of both rat and guinea pig reported that sigma 1 receptor can be upregulated by (Novakova et al. 2010, Stracina et al. 2015) as well as in escalated endoplasmic reticulum stress by the intracardiac neurons (Zhang and Cuevas 2005). Function PERK/ATF4 pathway. Activation of the pathway leads to of the sigma 1 receptor, as a molecular chaperone, is amelioration of cell death signaling. Recently, key role of based on interaction with various proteins. Due to miRNA-297 in sigma 1 expression regulation during intensive research of sigma receptors in neuronal system, cardiomyocyte hypertrophy was reported (Bao et al. a lot of protein-protein interactions have been identified 2017). Regulation by other microRNAs has been also in neuronal models (Pabba 2013, Su et al. 2016). Based proposed (Su et al. 2016). on direct or indirect evidence, many of them can be applied in cardiomyocytes, too; in others, we can Cellular function of sigma 1 receptor presume that the interactions are analogous. Key protein Sigma 1 receptors are expressed in atrial and interactions are shown in Figure 1.

Fig. 1. The Sigma 1 Receptor Protein Interactions in Cardiomyocyte. Sigma 1 receptors are represented by black flags. They are primarily located on mitochondrion-associated endoplasmic reticulum membrane (MAM). Only key interactions related to the text are presented. All presented interactions were determined experimentally. For more information see corresponding chapters in the text. A – ankyrin B; BiP – binding immunoglobulin protein; Ca2+ – calcium ions; ER – endoplasmic reticulum; GPCRs – G protein-coupled receptors; hERG – human ether-a-go-go-related gene potassium voltage-gated channel; IP3R – inositol 1,4,5-trisphosphate receptor; IRE1α – inositol requiring protein 1α; Kv? – potassium voltage-gated channel ensuring transient outward potassium current; Kv2.1 – Kv2.1 potassium voltage-gated channel; LCC – L-type calcium voltage-gated channel; M – mitochondria; MAM – mitochondrion- associated endoplasmic reticulum membrane; Nav1.5 – Nav1.5 sodium voltage-gated channel; PLC – phospholipase C; RYR – ryanodine receptor; S1R – sigma 1 receptor in trimeric form; XBP1 – spliced X-box binding protein 1.

Interaction with endoplasmic reticulum membrane within the cell. The first studies about effect of sigma proteins – a modulation of calcium handling and dealing ligands on the cardiomyocytes reported increased calcium with endoplasmic reticulum stress influx which led to increased contractility (Ela et al. The endoplasmic reticulum plays an essential 1994, Novakova et al. 1995). It was suggested that role in calcium handling, protein synthesis and folding, sigma 1 receptor directly affects calcium channels or and lipid synthesis in the cell (Glembotski 2012). The modulates potassium channels on plasma membrane (Ela endoplasmic reticulum calcium pool is important for et al. 1994). Subsequent study uncovered that sigma 1 contraction of cardiomyocyte as well as for signaling ligands increase inositol 1,4,5-trisphosphate (IP3) 2018 Cardiac Sigma Receptors S565

production in cardiomyocytes and observed increase of protein response. The rate of general translation is contractility depends on calcium stores in endoplasmic reduced and the expression of endoplasmic reticulum reticulum (Novakova et al. 1998). IP3 activates its resident protein chaperones and protein foldases is receptor, a transmembrane glycoprotein complex increased to restore homeostasis. However, if the primarily located on endoplasmic reticulum membrane. unfolded protein response is unsuccessful, endoplasmic After the activation, IP3 receptor releases calcium from reticulum stress causes cell dysfunction and apoptotic endoplasmic reticulum (Fig. 1). Three types of pathways are activated (Biala and Kirshenbaum 2014). IP3 receptors affect cardiac function to various extent Recently, potential function of sigma 1 receptor in (Kockskämper et al. 2008). IP3 receptors type 1 and 2 regulating normal mitochondrial organization and size in represent important intracellular calcium channels in the heart was reported in sigma 1 receptor knockout cardiomyocytes (Garcia and Boehning 2017). mouse model (Abdullah et al. 2018). An overexpression of IP3 receptor types 1 and 2 were Sigma 1 receptor, as the endoplasmic reticulum reported in rat and guinea pig heart after long-term resident protein, has been found to promote cellular exposure to sigma ligand haloperidol (Novakova et al. survival by regulating specific endoplasmic reticulum 2010, Stracina et al. 2015). stress sensors at the mitochondria-associated membrane The chaperone activity of sigma 1 receptor on region under endoplasmic reticulum stress in various IP3 receptor, first described on Chinese hamster ovary cellular models (Hayashi and Su 2001, Hayashi and cells with transfected receptors (Hayashi and Su 2007), Su 2007, Wu and Bowen 2008, Mitsuda et al. 2011, Mori was proven in cardiomyocytes (Tagashira et al. 2013). et al. 2013). Reported sigma 1 receptor dependent Besides IP3 receptors, an interaction with other important protective signaling pathways significantly differ in intracellular calcium channel, ryanodine receptor type 2, various cell types. Recently, sigma 1 receptor was was discovered in cardiomyocytes. Tagashira et al. reported as an essential component of the unfolded showed that sigma 1 receptors are associated both with protein response pathway eliciting cellular protection in ryanodine receptors type 2 and IP3 receptors type 2 in cardiomyocytes (Alam et al. 2017). Alam et al. (2017) cardiomyocyte endoplasmic reticulum membrane described that sigma 1 receptor regulates C/EBP- (Tagashira et al. 2013). Sigma ligand pentazocine homologous protein expression in association with suppressed ryanodine receptor mediated calcium release activation of the inositol requiring kinase 1α and spliced from endoplasmic reticulum, which consequently led to X-box binding protein 1 (IRE1α/XBP1) pathway (Fig. 1). decreased contraction force. Sigma 1 receptor stimulation The IRE1α/XBP1 pathway is the most conserved branch also promotes mitochondrial calcium transport through of the unfolded protein response in mammals and is IP3 receptor type 2 (Fig. 1) and in turn ATP production important for cardiomyocyte viability and contractile (Tagashira et al. 2013). function (Wang et al. 2014). Cross-talk between endoplasmic reticulum and Numerous implications may be proposed from mitochondrion is important for maintaining homeostasis currently known sigma 1 receptor functions on in both organelles. Impairment of endoplasmic reticulum endoplasmic reticulum membrane. However, there are luminal homoeostasis leads to misfolding or unfolding of a lot of unanswered questions. The answers may bring proteins. An accumulation of unfolded proteins is known some new insights into endoplasmic reticulum and as endoplasmic reticulum stress (Glembotski 2007). mitochondrion coordination and consequently into Protein synthesis and folding are controlled by pathophysiology of many heart diseases. endoplasmic reticulum protein quality control mechanisms and accumulation of unfolded proteins is Interaction with voltage-gated ion channels – sensed by endoplasmic reticulum stress sensors: inositol a modulation of action potential of cardiomyocytes requiring protein 1α (IRE1α), protein kinase RNA-like Generally, three main groups of voltage-gated ER kinase (PERK) and transcriptional factor 6 (ATF6) ion channels are present in working cardiomyocytes: (Liu et al. 2016). In these processes, endoplasmic sodium voltage-gated channels conduct sodium current, reticulum resident proteins take important part. responsible for membrane depolarization; calcium Endoplasmic reticulum stress activates a complex voltage-gated channels conduct calcium current, which signaling pathway to deal with the misfolded and contributes to the plateau phase of action potential; and unfolded proteins, which is referred as the unfolded potassium voltage-gated channels conducting potassium S566 Stracina and Novakova Vol. 67

currents, which ensures returning the depolarized increase conductance of calcium-sensitive potassium membrane to a resting state. There is direct or indirect channels and inhibit M-current. evidence, that sigma 1 receptor modulates all main ion The relationship between the sigma 1 receptor currents in cardiomyocytes. and hERG (human ether-a-go-go-related gene) potassium Inhibitory effect of haloperidol on activated channel has been studied intensively. The hERG channels sodium current was reported in human atrial are responsible for the rapid component of delayed cardiomyocytes and in rat ventricular cardiomyocytes rectifier current IKr. Haloperidol was reported to block the (Crumb et al. 2006, Tarabova et al. 2009). Johannessen et hERG channels expressed in Xenopus oocytes al. (2009) described modulation of Nav1.5 channels by (Suessbrich et al. 1997) and in human embryonic kidney sigma 1 receptors in. They showed that level of Nav1.5 cells (HEK 293) (Martin et al. 2004, Katchman et al. channel inhibition by selective sigma 1 ligands depends 2006). In the leukemic K562 cell line, the regulating on number of sigma 1 receptors on plasma membrane, function of sigma 1 receptors on hERG expression was however non-specific ligands, such as haloperidol, inhibit clarified (Crottès et al. 2011). The sigma 1 receptor

Nav1.5 channel also on cells with no sigma 1 receptors. modulates the hERG current density in the presence of As a possible explanation of above result, direct effect of sigma ligands. However, the direct interaction between non-selective ligands on ion channels as well as sigma 1 receptor and hERG in the plasma membrane is modulation via sigma 2 receptors were proposed not sigma 1 ligand dependent, it is reduced by cholesterol

(Johannessen et al. 2009). Modulation of Nav1.5 channels depletion. Coimmunoprecipitation study proposed that by sigma ligands can be inhibited by progesterone sigma 1 receptors located in lipid rafts (Balasuriya et al. (Johannessen et al. 2011). The sigma 1 receptor has been 2014) potentiate the hERG subunit’s translocation from reported to modulate sodium channels Nav1.2, Nav1.4 and endoplasmic reticulum to Golgi apparatus (Crottès et al.

Nav1.5 also in non-cardiac cells (Balasuriya et al. 2012, 2011). Altogether, sigma 1 receptor binds to hERG Gao et al. 2012). channel in endoplasmic reticulum and facilitates hERG Effect on calcium current was reported by Ela et maturation and trafficking. Su et al. (2016) suggested that al. (1994) who described increased influx of calcium into sigma 1 receptor might exert chaperoning activities in the neonatal rat cardiomyocyte after sigma 1 receptor endoplasmic reticulum to facilitate proper protein sorting stimulation. Twelve years after, sigma 1 ligand to their final destinations. However, such relationship of haloperidol was reported as mild to moderate blocker of sigma 1 receptor with other proteins (except of hERG) the L-type calcium channel (Tarabova et al. 2009). It was has not been proven yet. proven that inhibitory effect is independent on channel The effect of many clinically used sigma ligands splice variant (cardiac or vascular). The modulation of on cardiac action potential and electrocardiogram calcium current by sigma 1 receptor was also reported in parameters was observed in various experimental models. non-cardiac cells (Brent et al. 1996, Tchedre et al. 2008). Besides used experimental model and tested ligand, the In retinal ganglion cells, direct interaction of L-type studies usually vary in dosage and route of calcium channel with sigma 1 receptor was proven by administration. In clinical studies, patients are usually coimmunoprecipitation assay (Tchedre et al. 2008). treated by more than one drug and drugs interactions may Potassium channels are the most diverse group make interpretation of obtained results more difficult. of voltage-gated channels (Perney and Kaczmarek 1991). Moreover, sigma 1 ligands may affect various ion Potassium repolarizing current on cardiomyocytes channels as well as various receptor systems in the same consists of several components. In rat ventricular time. Also, direct action of the ligand on ion channel cardiomyocytes, haloperidol was reported as an effective should be taken in consideration. Potential receptor- inhibitor of one of them (Bebarova et al. 2006). independent inhibition of the Kv2.1 channel by sigma Haloperidol inhibits transient outward potassium current ligands was recently reported (Liu et al. 2017). In sum,

(Ito) and significantly decelerates its recovery. Based on the reported effects of sigma 1 ligands on ion channels whole-cell patch-clamp recordings, direct modulation of seem to be inconsistent and most of the specific potassium channels by sigma 1 receptor was suggested in mechanisms of sigma 1 receptor interactions with ion isolated intracardial neurons (Zhang and Cuevas 2005). channels are unclear. In dose-dependent manner, sigma ligands reversibly block delayed outward rectifying potassium channels, 2018 Cardiac Sigma Receptors S567

Other protein interactions – another pieces of incomplete pathway by endogenous oxidative stress may lead to mosaic autophagy of cardiomyocytes (Wang et al. 2018). In It may be expected that sigma 1 receptor addition, exposure to sigma 1 antagonist haloperidol interacts – besides abovementioned – with numerous aggravates hypertrophy by impairment of mitochondrial other proteins in cardiomyocytes. Among others, calcium signaling in cardiomyocytes (Shinoda et al. 2016) interaction with opioid receptors, NMDA receptors and and stimulation of sigma 1 receptors restores abnormal dopamine receptors has been reported in neuronal models mitochondrial calcium mobilization and ATP production (Mei and Pasternak 2002, Navarro et al. 2010, Balasuriya (Tagashira et al. 2013, Tagashira et al. 2014). Recently, et al. 2013). However, action of these receptor systems in miR-297 was proposed as a novel regulator of sigma 1 the heart is not fully understood and only indirect receptor in cardiomyocyte hypertrophy (Bao et al. 2017). evidence of interaction with cardiac sigma 1 receptor has Up-regulation of miR-297 increases the activation of been reported and the mosaic of cardiac sigma 1 receptor XBP1 and ATF4 pathways via targeting sigma 1 receptor, actions stays incomplete. which promotes cardiomyocyte hypertrophy. However, the precise mechanism by which sigma 1 receptor induces this Role of sigma 1 receptor in pathophysiology of action has not been described yet. cardiovascular diseases Our current knowledge about physiological Cardiovascular adverse effects of clinically used sigma 1 functions of cardiac sigma 1 receptors are still incomplete ligands and their pathophysiological roles are largely unknown. Many sigma 1 ligands are used in clinical Only a few studies have been focused on the expression practice for treatment of various diseases (Table 1). Some and function of sigma 1 receptors in cardiovascular of them exert cardiovascular side effects. The spectrum of disorders. Recently, significant roles of sigma 1 receptors the effects varies from frequent mild blood pressure in cardiac hypertrophy and heart failure have been change (hypotension or hypertension) to sporadic life- proposed. Moreover, many of clinically used sigma 1 threating ventricular arrhythmias, which may lead to ligands exert cardiovascular side effects. sudden cardiac death. It is out of scope of this review to pose all the Cardiac hypertrophy and heart failure sigma 1 ligands, in which cardiovascular adverse effects It is well known, that endoplasmic reticulum have been reported. Among all, cardiovascular side stress is important agent in pathophysiology of many effects of the prototypic sigma 1 ligand haloperidol are cardiovascular diseases, such as hypertension, the most controversial: haloperidol-induced QT prolon- atherosclerosis, myocardial infarction, and cardiac gation is comparable to other antipsychotics; however, hypertrophy which ultimately result in heart failure (Liu haloperidol treatment significantly increases a risk of et al. 2016). Cardiac hypertrophy is caused by pressure/ ventricular arrhythmias and sudden cardiac death volume overload or overactivation of neurohumoral (Leonard et al. 2013, Leucht et al. 2013, Wu et al. 2015). systems, such as the renin-angiotensin-aldosterone system. In animal studies, reported effects of haloperidol seem to It is initiated as an adaptive response, but if the process is be consistent. Haloperidol provokes QT interval uncontrolled and prolonged, it may lead to heart failure and prolongation and cardiac arrhythmias, such as torsades eventually to death (Shimizu and Minamino 2016). de pointe. In isolated rat hearts, exposure to haloperidol Moreover, increased myocardium mass may lead to leads to premature ventricular contractions. Incidence of decreased tolerance of heart to ischemia (Hlaváčováet al. arrhythmias was significantly lowered after repeated 2017). haloperidol administration, which suggest desensitization As discussed above, activation of sigma 1 of sigma 1 receptor (Fialova et al. 2009). In rats, receptors leads to amelioration of endoplasmic reticulum increased expression of sigma 1 receptor was detected in stress. Sigma 1 ligands may therefore protect the heart all heart chambers after long-term haloperidol from hypertrophy. Cardioprotective role of the sigma 1 administration (Novakova et al. 2010). QT prolongation receptors was also reported after stimulation by and increase of sigma 1 receptor expression in cardiac dehydroepiandrosterone in ovariectomized rats, which atria were reported in guinea pigs after long-term leads to activation of the Akt-eNOS pathway (Bhuiyan and haloperidol administration (Stracina et al. 2015). Fukunaga 2009). On the other hand, inhibition of Akt Expressions of IP3 receptor type 1 and 2 were also S568 Stracina and Novakova Vol. 67

increased in the atria only. Acute haloperidol exposure Su 2004). Haloperidol can also interact with other receptor causes slowing of intraventricular conduction and systems. Moreover, haloperidol exerts significant lengthening of repolarization in anesthetized guinea pigs cytotoxicity (Raudenska et al. 2013). Altogether with (Mortl et al. 2003). considerable variability in experimental methods, dosage Antipsychotic drug haloperidol is a nonselective and route of administration in the studies, it is difficult to ligand, which acts as a sigma 1 receptor antagonist as well propose a role of sigma 1 receptors in the observed as a dopamine D2 and D3 receptor antagonist (Hayashi and cardiovascular effects of haloperidol.

Table 1. Clinically used drugs with affinity to sigma 1 receptor.

Drug category Sigma 1 ligand References

Analgesics Pentazocine Zhang and Cuevas 2005 Anesthetics Ketamine Robson et al. 2012 Anticonvulsants and Lamotrigine, Phenytoin Cobos et al. 2005 Antiepileptic Agents Antidepressants Citalopram, Escitalopram, Fluoxetine, Hashimoto 2009, Albayrak and Fluvoxamine, Opipramol, Sertraline Hashimoto 2017 Antidiarrheals Loperamide Sánchez-Fernández et al. 2014 Anti-Parkinson and Anti- Amantadine, Donepezil, Memantine Peeters et al. 2004, Albayrak and Dementia Drugs Hashimoto 2017 Antipsychotics Fluphenazine, Gevotroline, Haloperidol, Schuster et al. 1995 Nemonapride, Perphenazine, Remoxipride, Risperidone, Trifluoperazine Antitussive Agents Dextromethorphan, Dimemorphan, Nguyen et al. 2014 Pentoxyverine, Psychostimulants Methylphenidate Zhang et al. 2012 Psychoactive Substances Cocaine, Ibogaine, Methamphetamine, Navarro et al. 2013, Chao et al. 2017 Phencyclidine

Sigma 1 receptor as a cross-link between mental and relationship between heart failure and depression in cardiovascular disorders a mouse model of pressure overload. As a part of link Epidemiological studies have demonstrated between depression and cardiovascular diseases, brain- a close relationship between depression and derived neurotrophic factor (BDNF) was discussed as an cardiovascular diseases (Trebatická et al. 2017). important agent. It was shown that the BDNF-TrkB Cardiovascular diseases can lead to severe depression pathway plays a role in the pathophysiology of (Gottlieb et al. 2004). Among cardiovascular diseases, cardiovascular diseases as well as depression and the strongest association with depression has been interaction between sigma 1 receptor and the BDNF- reported in ischemic heart disease and after myocardial TrkB pathway was discussed (Hashimoto 2013). Bhuyian infarction (Schleifer et al. 1989). Inversely, depression et al. (2013) proposed a hypothesis of sigma 1 receptor can lead to significant increase in the risk of developing mediated protective mechanism in cardiomyocyte, which heart failure and is independently associated with may explain protective effect of selective serotonin increased cardiovascular morbidity and mortality uptake inhibitors on heart failure progression. In mouse (Rumsfeld et al. 2003). model of myocardial infarction, decreased expression of In 2012, Ito et al. suggested that decreased brain brain sigma 1 receptor was reported (Ito et al. 2013). sigma 1 receptor expression contributes to the Brain sigma 1 receptor stimulation improves mental 2018 Cardiac Sigma Receptors S569

disorder and cardiac function in mice after myocardial receptor has not been cloned yet, its exact molecular infarction. Recently, it was reported that heart failure- structure is not known so far. It was suggested that the induced depression in mice is mediated by corticosteroids sigma 2 receptor is identical with the progesterone through reduced sigma 1 receptor expression in the brain receptor membrane component 1 (Xu et al. 2011). (Shinoda et al. 2016). However, Chu et al. (2015) reported that both these Not only depression can affect cardiovascular proteins are different binding sites derived from functions. QT interval prolongation was reported in independent genes. According to binding studies, high drug-free patients suffering from schizophrenia (Fujii et density of sigma 2 receptors are present in cerebellum, al. 2014). Authors suggested a role of potassium channels motor cortex, substantia nigra, hippocampus (Bouchard inhibition in both disorders. QT prolongation was also and Quirion 1997), in lungs, liver, kidneys (Hellewell et found in rat neurodevelopmental model of schizophrenia al. 1994), and in cells with high proliferation rate, such as (Stracina et al. 2016). As the sigma 1 receptors are tumor cells (Xu et al. 2011). Because of overexpression ubiquitous molecular regulators, their contribution to link in many types of tumors, sigma 2 receptors have been between schizophrenia and QT prolongation may be intensively researched in the field of tumor biology, based on their pluripotent chaperone activity. However, cancer diagnostics and treatment (van Waarde et al. this issue needs furthers investigation. 2015). Radiolabeled sigma ligands have been developed and are used for diagnostic imaging using positron Neurodegenerative diseases emission tomography and single photon emission Association of sigma 1 receptor with various computed tomography. Sigma 2 receptors have been also neurodegenerative diseases (e.g. amyotrophic lateral explored as a possible target for anticancer drug delivery. sclerosis, Huntington’s disease, Alzheimer disease, Moreover, cytotoxic and anticancer effects of sigma 2 Parkinson disease) was intensively studied (Cai et al. ligands have been reported (Zeng et al. 2012). 2017). During last decade, disease-causing mutations in Nevertheless, specific functions of sigma 2 receptors in sigma 1 receptor gene (SIGMAR1) were associated with tumor as well as other cells stay unclear. juvenile amyotrophic lateral sclerosis (ALS) and juvenile According to our best knowledge, there is only distal hereditary motor neuropathy in humans (Al-Saif et one relevant study dealing with sigma 2 receptor action in al. 2011, Li et al. 2015). As Mavlyutov and co-authors the heart. Monassier and co-workers reported that reviewed, sigma 1 receptor knockout mouse did not sigma 2 agonist inhibits inwardly rectifying potassium develop ALS (Mavlyutov et al. 2015). However, sigma 1 channels in the heart (Monassier et al. 2007). receptor knockout in mouse SOD-1 model of ALS led to Many studies have reported close and a faster onset of disease and decreased longevity overlapping pharmacological and biochemical properties (Mavlyutov et al. 2013). Recently, sensory and of sigma 1 and sigma 2 receptors. Therefore, sigma 2 autonomic nervous system dysfunction in ALS was receptors might also act as a molecular chaperones (Su et described (Nolano et al. 2017, Vucic 2017). Impaired al. 2016). However, proper description of role of sigma 2 neuronal regulation of cardiac function in ALS patients receptor needs further investigation. was reported previously (Dalla Vecchia et al. 2015). It is not clear, if such impaired regulation may lead to Future perspectives cardiovascular disease progression. In Huntington’s disease, association of neurodegeneration and cardiac Although numerous studies have been focused malfunction was proved (Critchley et al. 2018). However, on the effects of cardiac sigma 1 receptors, many pieces possible role of sigma 1 receptor has not been studied in of the mosaic of their functions are missing. A major step such consequences yet. The proper molecular forward to better understanding of cardiac sigma 1 mechanisms of neuro-cardio link in neurodegenerative receptor function would be the identification of the diseases need to be uncovered. endogenous ligand(s) responsible for the action of sigma 1 receptors under physiological as well as Sigma 2 receptor pathological conditions. This step might be promoted by precise structural model of the receptor. Knowledge of Sigma 2 receptor is one of the most poorly the crystal protein structure opens new possibilities in understood proteins in cell biology today. While sigma 2 design and preparation of novel highly specific sigma 1 S570 Stracina and Novakova Vol. 67

ligands. Such ligands will help in research of new Conflict of Interest therapeutic strategies. In the heart disorders associated There is no conflict of interest. with protein misfolding, sigma 1 receptor-dependent activation of IRE1α-XBP1s pathway may preserve Acknowledgements myocyte viability and contractile function (Alam et al. This review was written at Masaryk University as part of 2017). In cardiac hypertrophy treatment, rescuing the the project “Kardiovaskulární systém napříč obory– od decreased expression of sigma 1 receptor through molekulární diagnostiky po klinická vyšetření” number miR-297 inhibition may be beneficial (Bao et al. 2017). MUNI/A/1157/2017 with the support of the Specific And other perspectives will appear as the further University Research Grant, as provided by the Ministry investigation will shed new light onto the topic. of Education, Youth and Sports of the Czech Republic in the year 2018.

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| Appendix

Appendix B

Tohoku J. Exp. Med., 2015, 236, 199-207Effects of Haloperidol Treatment on Guinea Pig Heart 199

Long-Term Haloperidol Treatment Prolongs QT Interval and

Increases Expression of Sigma 1 and IP3 Receptors in Guinea Pig Hearts

Tibor Stracina,1 Iva Slaninova,2 Hana Polanska,3 Martina Axmanova,3 Veronika Olejnickova,1 Petr Konecny,4 Michal Masarik,3 Olga Krizanova5 and Marie Novakova1,6

1Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 2Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 3Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 4Department of Physiotherapy and Rehabilitation, Faculty of Medicine, Masaryk University, Brno, Czech Republic 5Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia 6International Clinical Research Center, Animal Center, St. Anne’s Faculty Hospital, Brno, Czech Republic

Haloperidol is a neuroleptic drug used for a medication of various psychoses and deliria. Its administration is frequently accompanied by cardiovascular side effects, expressed as QT interval prolongation and occurrence of even lethal arrhythmias. Despite these side effects, haloperidol is still prescribed in Europe in clinical practice. Haloperidol binds to sigma receptors that are coupled with inositol 1,4,5-trisphosphate

(IP3) receptors. Sigma receptors are expressed in various tissues, including heart muscle, and they

modulate potassium channels. Together with IP3 receptors, sigma receptors are also involved in calcium handling in various tissues. Therefore, the present work aimed to study the effects of long-term haloperidol administration on the cardiac function. Haloperidol (2 mg/kg once a day) or vehiculum was administered by intraperitoneal injection to guinea pigs for 21 consecutive days. We measured the responsiveness of the hearts isolated from the haloperidol-treated animals to additional application of haloperidol. Expression of

the sigma 1 receptor and IP3 receptors was studied by real time-PCR and immunohistochemical analyses. Haloperidol treatment caused the significant decrease in the relative heart rate and the prolongation of QT interval of the isolated hearts from the haloperidol-treated animals, compared to the hearts isolated from

control animals. The expression of sigma 1 and IP3 type 1 and type 2 receptors was increased in both atria

of the haloperidol-treated animals but not in ventricles. The modulation of sigma 1 and IP3 receptors may lead to altered calcium handling in cardiomyocytes and thus contribute to changed sensitivity of cardiac cells to arrhythmias.

Keywords: guinea pig; haloperidol; heart; QT prolongation; sigma 1 receptor Tohoku J. Exp. Med., 2015 July, 236 (3), 199-207. © 2015 Tohoku University Medical Press

even sudden cardiac death (Remijnse et al. 2002). Introduction Haloperidol is a non-specific drug with affinity to

Haloperidol is an antipsychotic drug used for a medi- numerous receptors, including dopamine D2 receptors and cation of both acute and chronic psychosis, mainly schizo- serotonin 5HT2 receptors. It is also known as a prototypic phrenia, mania and psychomotor agitation. Oral or paren- ligand of sigma receptors. These receptors were first dis- teral administration according to acute or chronic protocols covered in the central nervous system (Martin et al. 1976). is possible, with respect to patient’s condition and diagno- Later, their presence was proven in various tissues (Su and sis. Rather frequent side effects are observed, neurological, Junien 1994), including the central nervous system (Alonso mental, and last but not least, cardiovascular. The last men- et al. 2000) and numerous peripheral tissues, such as tioned comprise mostly QT interval prolongation; occasion- immune system (Wolfe and De Souza 1993), digestive tract, ally, it is accompanied by arrhythmias, such as Torsade de liver and kidney (Hellewell et al. 1994), endocrine and Pointes, eventually leading to ventricular fibrillation and reproductive systems (Wolfe and De Souza 1993; Kushner

Received February 18, 2015; revised and accepted May 29, 2015. Published online June 19, 2015; doi: 10.1620/tjem.236.199. Correspondence: Marie Novakova, Ph.D., Department of Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. e-mail: [email protected]

199 200 T. Stracina et al. and Zukin 1994), and also the heart muscle (Dumont and and type 2 in guinea pigs repeatedly exposed to haloperidol Lemaire 1991; Novakova et al. 1995). Three subtypes of was also studied. sigma receptor are distinguished. In the heart muscle, sigma 1 and sigma 2 receptors are expressed (Ela et al. Materials and Methods 1994; Novakova et al. 1995). Their stimulation leads to a The study was performed on 30 guinea pigs (unspecified breed, release of Ca2+ from the intracellular store into the cytosol male only, 3 months old). Animals were housed in Laboratory (Novakova et al. 1995, 1998; Maurice and Su 2009). In Animal Breeding and Experimental Facility, Faculty of Medicine, 2001, Hayashi and Su (2001) reported the existence of the Masaryk University, Brno, Czech Republic. All animal experiments trimeric structure consisting of sigma 1 receptor, inositol were carried out according to the recommendations of the European Community Guide for the Care and Use of Laboratory Animals and 1,4,5-trisphosphate (IP3) receptor and ankyrin isomer 220 on the membrane of endoplasmic reticulum in cultured according to the experimental protocol approved by the Committee rodent cells. Dissociation of this triplet and translocation of on the Protection of Animals, Faculty of Medicine, Masaryk either a dimer sigma 1 receptor/ankyrin (in case of sigma University and by the Committee of Ministry of Agriculture of the Czech Republic. receptor agonist binding) or sigma 1 receptor alone (after Animals were divided into two groups: haloperidol-exposed antagonist binding) has been described in the same model (group H; 20 animals; average body mass 320.9 ± 48.8 g) and control (Hayashi and Su 2003). Due to the effect on calcium cur- (group C; 10 animals; average body mass 322.4 ± 54.6 g). rent in cardiomyocytes (Tarabova et al. 2009) and an impact Haloperidol (Sigma Aldrich, USA; 2 mg/kg once a day by intraperito- on the regulation of intracellular calcium store, sigma neal injection) or vehiculum (13.3 ml of 2% alcohol solution per 1 kg receptors are suggested to play a role in modulation of car- of actual body mass) was administered for 21 consecutive days. The diac functions. Moreover, activation of sigma 2 receptors dose of haloperidol was chosen according to the previous studies results in inhibition of inward rectified potassium channels (Inoue et al. 2000; Fialova et al. 2009). The proper dose of haloperi- (Monassier et al. 2007). Haloperidol acts as a potent sigma dol or vehiculum was calculated daily for each animal according to 1 antagonist and a sigma 2 agonist (Colabufo et al. 2004; its actual body mass. Weighing and application was done always at Cobos et al. 2007). the same daytime (around noon). No anaesthetic or analgesic agents Haloperidol is not the only sigma ligand used as an were used in this part of experiment. Gentle handling and quiet efficient drug in clinical practice. Many other neuropsychi- approach was applied to reduce discomfort of animals during the atric drugs such as fluvoxamine and donepezil are sigma 1 manipulation. agonists, and opipramol and sertraline are sigma 1 antago- nists. Bhuiyan and co-workers (2010) suggested that selec- Isolated heart experiments Twenty-four hours after the last haloperidol dose, guinea pigs tive serotonin reuptake inhibitors, such as fluvoxamine, were deeply anaesthetised by isoflurane (2%). The heart was rapidly have a cardioprotective effect in case of pressure-overload- removed from the thorax, placed in a cold (4°C) Krebs-Henseleit induced dysfunction of the rat heart by upregulating sigma solution and prepared for cannulation. The aorta was cannulated and 1 receptor expression and stimulating sigma 1 receptor- the heart was perfused according to Langendorff at constant perfusion mediated Akt-eNOS signalling. Recently, sigma 1 recep- pressure (80 mmHg) with Krebs-Henseleit solution (NaCl, 118 mM; tors were reported as a target for cardioprotection (Bhuiyan NaHCO3, 24 mM; KCl, 4,2 mM; KH2PO4, 1.2 mM; MgCl2, 1.2 mM; and Fukunaga 2011). On the other hand, administration of CaCl2, 1.25 mM; glucose, 5.5 mM) aerated with 95% O2 and 5% CO2. sigma 1 antagonist results in negative cardiovascular The temperature was maintained constant (37°C) throughout the effects. A recent study described coronary artery athero- experiment. Experimental protocol consisted of four 20-min lasting sclerosis after sertraline treatment in female premeno pausal consecutive phases: stabilization, the first haloperidol exposure (H1), primates (Shively et al. 2015). washout with Krebs-Henseleit solution, and the second haloperidol We reported that repeated exposure to haloperidol, a exposure (H2). Haloperidol was administered diluted in Krebs- sigma receptor ligand, increased expression of sigma 1 Henseleit solution at the concentration of 10 nmol/l. This concentra- tion was chosen in order to approximate clinical situation; free plasma receptor and IP3 receptor type 1 and type 2 in rat atria (Novakova et al. 2010). In ventricles, only expression of levels of haloperidol in patients are usually between 10-200 nmol/l the sigma 1 receptor was increased. In another study, (Flanagan 1998). Throughout the entire experiment, electrogram as three orthogonal leads was continually recorded by touch-free method silencing of both, the type 1 and type 2 of IP receptors 3 (Fialova et al. 2009). resulted in a decrease of sigma 1 receptor’s expression The recorded signals were subsequently analysed: ten succes- (Novakova et al. 2007). This fact suggests the mutual sive RR intervals were averaged at the end of the 5th, 10th, 15th, 20th, direct or indirect interaction(s) of abovementioned recep- 25th, and 30th minute of each phase and the heart rate was calculated. tors. In the same way, QT interval was measured. QT interval correction Despite possible side effects, haloperidol is still pre- according to Bazzet formula (QTc) was performed. Manual detection scribed in Europe in clinical practice. Therefore, the pres- of arrhythmias was done: each arrhythmia was classified and the time ent work aimed to study the effects of long-term haloperi- of its appearance was noted. dol treatment on guinea pig hearts and the responsiveness of these hearts to the additional doses of haloperidol. Expression of sigma 1 and IP3 receptors

Expression of the sigma 1 receptor and IP3 receptors type 1 Twenty-four hours after the last haloperidol dose, guinea pigs Effects of Haloperidol Treatment on Guinea Pig Heart 201 were deeply anaesthetised by isoflurane (2%). The heart was rapidly Statistical analysis removed from the thorax, placed in a cold (4°C) Krebs-Henseleit Mean relative expressions of the genes in group C were set as a solution and washed from the blood. Small samples of myocardium reference values. Values of expression in the group H were expressed from free wall of right and left atria and both ventricles were obtained in relation to the reference values. Twofold or higher/lower value in and placed into RNA later (Roche, Switzerland) or formaldehyde group H than in group C was accepted as statistically significant. (Sigma, USA) for stabilization until further processing. The expres- Results of the heart rate and QTc were expressed as means ± SEM. sion of IP3 and sigma 1 receptors in heart tissues were proved using The heart rate in H1, washout and H2 phases were expressed in per- RT-PCR and immunohistochemistry. cent of the heart rate value in the 30th minute of the stabilisation period of the respective experiment. Standard parametric and non- RNA isolation, cDNA preparation parametric descriptive statistics (mean, median, range) were done for For RNA isolation High pure RNA Tissue isolation kit (Roche, each phase. The differences between consecutive phases of the Switzerland) was used. Tissue samples or cell samples were homog- experiment were analysed by Student’s paired t-test. Differences enized in 200 µL of lysis buffer. Subsequently, lysates were trans- between individual phases of each experiment and its stabilisation ferred into the column and RNA isolation was carried out according phase were analysed by one-sample t-test. Differences between two to manufacturer’s instructions. Isolated RNA was used for cDNA experimental groups were analysed by Student’s unpaired t-test. P synthesis. Total RNA (600 ng) was transcribed using Transcriptor values were calculated, two-side p < 0.05 was considered significant. first strand cDNA synthesis kit (Roche, Switzerland) according to Analyses were performed using GraphPad Prism® 5 (version 5.01, manufacturer’s instructions. Prepared cDNA (20 µL) was diluted GraphPad Software, Inc., San Diego, CA). with RNase-free water to 100 µL and directly analyzed by real-time polymerase chain reaction. Results Animals were randomly divided into two groups: hal- Real-time reverse-transcription polymerase chain reaction operidol-exposed (group H; 20 animals; average body mass Real-time reverse-transcription polymerase chain reaction 320.9 ± 48.8 g) and controls (group C; 10 animals; average (PCR) was performed in triplicates using the TaqMan gene expres- body mass 322.4 ± 54.6 g). At the end of stabilization, no sion assay system with the 7500 real-time PCR system (Applied significant difference in the heart rate of isolated hearts was Biosystems, CA, USA). The amplified DNA was analyzed by the observed (group H 197.70 ± 13.19 beats/min, and group C comparative Ct method using β-actin as an endogenous control. The 195.00 ± 29.00 beats/min, respectively). primers and probe set for β-actin (Assay ID: Cp03755211_g1; Applied Biosystems), sigma 1 receptor (Assay ID: Cp03755850_m1, Significant decrease in relative heart rate was observed in group H from the beginning of phase H1 to the end of Applied Biosystems), IP3R1 (CpLOC100713023, Applied experiment; heart rate in group C was basically stable (p < Biosystems), and IP3R2 (CpLOC100717278, Applied Biosystems) were used. Real-time PCR was performed under the following ampli- 0.05; Fig. 1). In group H, the mean values of heart rate fication conditions: total volume of 20 µL, initial denaturation showed decreasing tendency during the H1 phase and this 95°C/10 min, then 45 cycles 95°C/15 sec, 60°C/1 min. decrease was statistically significant (p < 0.05, compared to the end of stabilization) from the 5th minute of H1 phase. Immunohistochemical staining At the end of H1, the mean heart rate in group H was 91.8% Immunostaining of sigma 1 and type 1 IP3 receptors was perfor- ± 1.1% and its values did not further change. Also, mean med on 3-4 µm-thick formaldehyde fixed, paraffin-embedded tissue value of QTc in group H was significantly higher than in sections, which were deparrafinized using xylen, rehydrated by -dec group C (362 ± 4 ms in group H vs. 329 ± 15 ms in group reasing ethanol concentration washes, and then processed for antigen C, p < 0.05). This difference between the groups was pre- retrieval. Antigen retrieval was performed by heating the slides in 10 served and mostly significant (p < 0.05) during the experi- mM citrate buffer (pH 6) at 95-99°C for 40 min. Samples were then ment (Fig. 2). pre-incubated in PBS (137 mmol/l NaCl, 2.7 mmol/l KCl, 1 mmol/l Mean QTc showed increasing tendency during the H1 KH2PO4, and 6.5 mmol/l Na2HPO4; pH 7.4) with 0.1% Triton X100 phase, being statistically significant in group H (p < 0.05) (Sigma, USA) and 10% normal goat serum (Sigma, USA) for 1 h at th room temperature. After washing in PBS the samples were incubated from the 15 minute (compared to the end of stabilization). with primary antibodies at 4°C overnight. Anti-Inositol 1,4,5- At the end of H1 phase, mean QTc in group H was 384 ± 6 Triphosphate Receptor (Type I) (Sigma, USA) and Anti-OPRS1 ab ms. During washout, insignificant decrease was observed. 53852 (sigma-receptor; Abcam) both produced in rabbit and diluted In H2 phase, mean QTc values increased again, but this at 1% bovine serum albumin (BSA) at PBS were used as primary change was already insignificant. In group C, the same, but antibodies. After washing in PBS, secondary antibody (goat anti insignificant trends were observed. rabbit IgG conjugated to Alexa Fluor 488; Invitrogen) was added and Incidence of arrhythmias was low in both groups. samples were incubated for 1 hour at room temperature in the dark. Sporadic supraventricular premature beats were the only Slides were then washed as described above and stained in DAPI (1 detected type of arrhythmia. No ventricular electrical dis- μg/ml) for 15 min, rinsed again in PBS, then mounted with antifade turbance was observed. Vectashield (Vector Laboratories, USA). Negative controls were Expression of sigma 1 receptor in the heart atria was obtained by overnight incubation in 1% BSA instead of primary anti- significantly increased in group H (in left atrium 5.67-times body. Fluorescent images were taken using a confocal microscope and in right atrium 2.84-times, respectively; Fig. 3a, b). Zeiss LSM 700 (Zeiss, Germany) using 488 nm laser. Expression of the IP3 receptors type 1 and 2 was also ele- 202 T. Stracina et al.

Fig. 1. Changes of relative heart rate in isolated hearts. Fig. 2. Changes of QTc in isolated hearts. Changes of the relative heart rate in isolated hearts of Changes of QTc in isolated hearts of haloperidol-exposed haloperidol-exposed guinea pigs (squares; n = 10) and guinea pigs (squares; n = 10) and controls (circles; n = 3). controls (circles; n = 3). Haloperidol (10 nM) was Haloperidol (10 nM) was administered in two phases of administered in two phases of experiment (H1, H2), first experiment (H1, H2), first exposure was followed by exposure was followed by washout period. Significant washout period. QTc was significantly prolonged in decrease in relative heart rate in group H caused by the group H as compared to controls. Results are expressed first haloperidol administration as compared to controls as mean ± SEM. Statistical significance: *p < 0.05 com- was observed; this effect was not washable. Results are pared to the 30th minute of stabilisation (one-sample t- expressed as mean ± SEM. Statistical significance: *p < test); op < 0.05 compared to group C (Student’s unpaired 0.05 compared to the 30th minute of stabilisation (one- t-test). sample t-test); op < 0.05 compared to group C (Student’s unpaired t-test).

vated (in left atrium type 1 IP3 receptor 3.01-times and type suitable model for studying drug effects on electrocardio- 2 IP3 receptor 6.81-times, and in right atrium IP3 receptor 1 gram, since guinea pig cardiac cells exhibit specific ion 3.23-times and IP3 receptor 2 4.08-times, respectively; Fig. channels quite comparable to those of humans (Busch et al. 3a, b). In ventricles, no significant changes were detected 1994). Furthermore, outcomes of our previous studies indi- (Fig. 3c, d). cate that long-term exposure to haloperidol affects electro- Immunohistochemistry demonstrated both sigma 1 and gram parameters of isolated heart in rat and guinea pig and type 1 IP3 receptors in the cytoplasm of cells of atria and also expression of sigma receptors in the rat heart (Fialova ventricles in both control and haloperidol-exposed groups. et al. 2009; Novakova et al. 2010). Compared to the control, slightly increased signal for both In this work it was clearly shown that three weeks receptors was observed in the cytoplasm of these cells in exposure of guinea pigs to haloperidol resulted in signifi- the haloperidol-exposed animals (Fig. 4c, d). cant decrease in the relative mean heart rate after the addi- tional haloperidol administration to isolated heart, although Discussion basal HR of these hearts did not differ. However, further Although haloperidol administration often resulted in washout of this bolus and another bolus did not cause any various cardiovascular side effects, in many countries it is consecutive change in this parameter. This pattern resem- still prescribed as an efficient antipsychotic agent. Clinical bles changes in the relative mean heart rate of isolated rat aspects of acute treatment with haloperidol and its impact hearts of animals exposed to haloperidol for three weeks on cardiovascular system, primarily QT interval prolonga- (Fialova et al. 2009; Novakova et al. 2010). tion and associated arrhythmias, were studied repeatedly QT interval reflects the duration of the ventricular (Metzger and Friedman 1993; Hennessy et al. 2002; Beach action potential and depends on ventricular repolarization. et al. 2013). Effects of the acute and/or chronic exposure to Ventricular repolarization is ensured mainly by various out- haloperidol have been also studied on various biomodels ward potassium currents. Two main delayed rectifying cur- and species, including rat (Fialova et al. 2009), guinea pig rents operate to achieve repolarization, a rapid [IKr] and a (Testai et al. 2007; Fialova et al. 2009), and rabbit (Dhein et slow [IKs] current. When these currents are reduced, repo- al. 2008). larization is prolonged, the ventricular action potentials In this study, effects of chronic exposure to haloperidol broaden and the duration of the QT interval increases. were studied in guinea pig hearts. Guinea pig represents a Interference with the IKr current is the most common Effects of Haloperidol Treatment on Guinea Pig Heart 203

Fig. 3. Changes in relative gene expression.

Changes in relative gene expressions of the sigma 1 receptor, IP3 receptor type 1 and type 2 in guinea pig hearts after repeated haloperidol exposure. The column graphs present the mean relative mRNA levels of sigma 1 receptor (Sig-

maR), IP3 receptor type 1 (InsP3R1) and type 2 (InsP3R2) in left atrium (LA; a), right atrium (RA; b), left ventricle (LV; c), and right ventricle (RV; d) in guinea pig hearts. Haloperidol significantly increased the gene expression of sigma 1 receptor, IP3R1 and IP3R2 in both atria (a, b), but not in ventricles (c, d). Each column is displayed as mean ± SEM and represents an average of 3-10 heart samples. Mean relative mRNA levels in the controls (group C; hatched columns) were set as a reference values. Mean relative mRNA levels in haloperidol-exposed guinea pigs (group H; black col- umns) were expressed in relation to the reference values. Statistical significance:op < 0.05 in group H as compared to group C. mechanism of QT prolongation (reviewed by Witchel and tually sudden cardiac death. Hancox 2000). In humans, IKr is conducted by human In this work, acute exposure to haloperidol increased ether-a-go-go related gene (hERG) potassium channels QTc duration in guinea pig isolated hearts from both (Sanguinetti et al. 1995). Heterologous hERG was blocked groups, although in group C this increase was insignificant. by haloperidol when expressed in Xenopus oocytes Haloperidol concentration of 10 nmol/l used in our experi-

(Suessbrich et al. 1997). Monassier et al. (2007) showed ments on isolated hearts is close to IC50 for hERG blocking that haloperidol blocks human recombinant hERG potas- (Redfern et al. 2003; Katchman et al. 2006). Therefore, one sium channels in COS-7 monkey kidney cells via stimula- of the mechanisms of haloperidol QT prolongation in tion of sigma 2 receptors. On the other hand, haloperidol guinea pigs might be connected with decreasing activity of has affinity for dopamine 2 D receptors, which are located in IKr. guinea pig heart (Gomez et al. 2002). Dopamine D2 ago- Our previous work (Novakova et al. 2010) showed that nists may increase QT interval duration (Gomez et al. the rate-corrected QT interval in isolated hearts from halo-

2002). However, haloperidol acts as a dopamine D2 antago- peridol-exposed rats was shorter than in control animals. nist and the dopaminergic effect on the heart rate and QT Fialova et al. (2009) described a wide range of arrhythmias interval duration is disputable. As far as we know, the in isolated hearts of control rats, including Torsade de direct effect of haloperidol on the heart activity mediated Pointes and sustained ventricular fibrillation. In isolated via dopaminergic receptors has not been studied yet. QT hearts of haloperidol-exposed rats, an absence of haloperi- interval changes are related to the appearance of arrhyth- dol-induced arrhythmias after additional bolus of haloperi- mias; therefore, monitoring of the QT interval is important dol was described (Fialova et al. 2009). Such inter-species in the identification of warning signs that precede serious differences may be attributed to different equipment of ven- rhythm disturbances, such as Torsade de Pointes and even- tricular cardiac cells with specific ion channels. Guinea pig 204 T. Stracina et al.

Fig. 4. Immunohistochemical staining of the sigma 1 receptors and the IP3 receptors type 1. Immunohistochemical staining of the sigma 1 receptors and the IP3 receptors type 1 in the left cardiac atrium in controls and haloperidol-exposed guinea pigs. (a) Sigma 1 receptors stained in controls, (b) IP3R1 stained in controls, (c) sigma 1 receptors stained in haloperidol-exposed animals, (d) IP3R1 stained in haloperidol-exposed animals. In haloperidol- exposed guinea pigs, slightly increased signal for both receptors (c, d) was visible. Nuclei were stained by DAPI. Inset (e) shows the negative control, where primary antibody was omitted. Scale bar represents 50 μm. ventricular cardiomyocytes do not develop transient out- receptors type 1 and 2, which are coupled with sigma 1 ward potassium current, whereas rat ventricular myocytes receptors (Novakova et al. 1998), was studied. The study do. Furthermore, delayed rectifier potassium current of- rel did not focus on IP3 receptors type 3 since there is no evi- atively high amplitude found in guinea pig cardiac cells, but dence of the presence of IP3 receptor type 3 in guinea pig is negligible or even absent in rat (Varró et al. 1993). heart. According to available data IP3 receptor type 3 is Except for direct effects on potassium channels, there expressed in ferret, rat, human and mouse cardiac cells, is another explanation how haloperidol might affect the including conductive system (reviewed in Kockskamper et action potential duration and consequently QT interval al. 2008). Moreover, type 3 IP3 receptor has a lesser impact duration in ventricular cardiac cells. Haloperidol is a proto- on regulation of heart function than the other two subtypes typic ligand of sigma receptors. Concentration of haloperi- in all mentioned species (Kockskamper et al. 2008). An dol used in isolated heart experiments is close to Kd value increase of sigma 1 receptor’s expression in the heart atria of the haloperidol binding on cardiac sigma receptors of haloperidol-exposed animals, compared to control ones (Novakova et al. 1995). In the heart muscle, both sigma 1 was observed. On contrary to the rat hearts (Novakova et and sigma 2 receptors are expressed (Ela et al. 1994; al. 2010), in guinea pig ventricles no changes of sigma 1 Novakova et al. 1995). Modulation of sigma 1 receptors receptor expression were observed. This difference might by haloperidol might at least partially explain changes in account for different species. Moreover, daily manipulation electrophysiological parameters that were detected in iso- of animals during chronic treatment represents a moderate lated guinea pig hearts in this study since it has been level of stress for animals. Mild stress modulates expres- reported in various models that sigma receptors modulate sion of cardiac sigma receptors (Novakova et al. 2007) and potassium channels behaviour (e.g. McKay and Kaczmarek this reaction may differ in rat and guinea pig. Hand by 2002). In order to elucidate the putative role of sigma hand with sigma 1 receptor overexpression, expression of receptors in our experimental setup, expression of the sigma the IP3 receptors type 1 and 2 were increased in atria only.

1 receptors was determined. Moreover, expression of IP3 The same effect of long-lasting haloperidol administration Effects of Haloperidol Treatment on Guinea Pig Heart 205 on IP3 receptor’s expressions was reported previously in rat istration—on IKr current, on co-localization of sigma 1 and hearts (Novakova et al. 2010). IP3 receptors, and last but not least on cytosolic calcium Immunohistochemical staining detected both sigma 1 availability in the cardiac cells. and IP3 type 1 receptors in the same compartment of cardio- myocytes, namely in the cytoplasm. Hayashi and Su (2001, Acknowledgments 2003) described functional coupling of sigma 1 receptor The study was performed at Masaryk University as part of with IP3 receptor and ankyrin 220 in a rodent cell line, the project “Cardiovascular system from the cell to patient’s NG-108. However, in our study, co-localization of sigma 1 bed” MUNI/A/1326/2014 with the support of the Specific University Research Grant, as provided by the Ministry of and IP3 type 1 receptors was not studied. Previous studies Education, Youth and Sports of the Czech Republic in the year revealed that stimulation of sigma receptors lead to a 2015. The financial support from the European Regional Devel- release of Ca2+ from the intracellular store into the cytosol opment Fund Project FNUSA-ICRC No. CZ.1.05/1.1.00/02.0123 (Novakova et al. 1995, 1998; Maurice and Su 2009). The is also highly acknowledged. The authors wish to thank prof. direct effect of haloperidol on calcium current in isolated Pavel Braveny for critical reading of the manuscript, prof. Petr Dobsak for fruitful discussion above the manuscript, and Mrs. rat cardiomyocytes was also described (Tarabova et al. Branislava Vyoralova for excellent technical assistance during 2009). Therefore we have proposed that haloperidol treat- the experiments. ment affects cytosolic calcium availability in the cardiac cells and as a consequence changes arrhythmogenic thresh- Conflict of Interest old. The authors declare no conflict of interest. Question arises about the functional consequences of altered expression of sigma 1 and IP3 receptors in the heart. References Numerous local actions, where the sigma 1 receptor plays a Agelink, M.W., Malessa, R., Kamcili, E., Zeit, T., Lemmer, W., key role, were reported, but its general function remains Bertling, R. & Klieser, E. (1998) Cardiovascular autonomic unclear. IP3 receptors type 1 and 2 are known as the intra- reactivity in schizophrenics under neuroleptic treatment: A cellular calcium channels participating in calcium release potential predictor of short-term outcome? Neuropsychobi- ology, 38, 19-24. from the endoplasmic reticulum (Lencesova and Krizanova Alonso, G., Phan, V., Guillemain, I., Saunier, M., Legrand, A., 2012). Novakova and co-workers (2010) found that pro- Anoal, M. & Maurice, T. (2000) Immunocytochemical local- longed exposure to haloperidol significantly increased ization of the sigma(1) receptor in the adult rat central nervous mRNA levels of sigma 1 receptors in both rat atria and ven- system. Neuroscience, 97, 155-170. Beach, S.R., Celano, C.M., Noseworthy, P.A., Januzzi, J.L. & tricles. Sigma 1 receptor mRNA was increased also in iso- Huffman, J.C. (2013) QTc prolongation, torsades de pointes, lated cardiomyocytes. Haloperidol affected the expression and psychotropic medications. Psychosomatics, 54, 1-13. of IP3 receptors type 1 and 2 in cardiac atria, but not in car- Bhuiyan, M.S. & Fukunaga, K. (2011) Targeting sigma-1 receptor signaling by endogenous ligands for cardioprotection. Expert diac ventricles. In the heart, higher levels of IP3 receptors type 1 in cardiac ganglia and IP receptors type 2 in cardio- Opin. Ther. Targets, 15, 145-155. 3 Bhuiyan, M.S., Tagahsira, H., Sioda, N. & Fukunaga, K. (2010) myocytes were described (Krizanova et al. 2008). Targeting sigma-1 receptor with fluvoxamine ameliorates Haloperidol causes changes in both electrical activity and pressure-overload-induced hypertrophy and dysfunctions. mechanical performance of the heart. Decrease in heart Expert Opin. Ther. Targets, 14, 1009-1022. rate, increase in atrioventricular effective refractory period, Busch, A.E., Malloy, K., Groh, W.J., Varnum, M.D., Adelman, J.P. & Maylie, J. (1994) The novel class III antiarrhythmics the atrioventricular conduction time, and increase in left NE-10064 and NE-10133 inhibit IsK channels expressed in ventricular developed pressure in isolated rat hearts were Xenopus oocytes and IKs in guinea pig cardiac myocytes. reported (Medlin et al. 1996). Approving results were Biochem. 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Witchel, H.J. & Hancox, J.C. (2000) Familial and acquired long qt Wolfe, S.A. Jr. & De Souza, E.B. (1993) Sigma and phencyclidine syndrome and the cardiac rapid delayed rectifier potassium receptors in the brain-endocrine-immune axis. NIDA Res. current. Clin. Exp. Pharmacol. Physiol., 27, 753-766. Monogr., 133, 95-123. | Appendix

Appendix C

Journal of Pharmacological Sciences 139 (2019) 23e28

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Full Paper Haloperidol affects coupling between QT and RR intervals in guinea pig isolated heart

* Petr Vesely a, b, Tibor Stracina c, , Miroslava Hlavacova d, Josef Halamek e, Jana Kolarova b, Veronika Olejnickova c, Veronika Mrkvicova f, g, Hana Paulova d, Marie Novakova a, c a International Clinical Research Center, St. Anne's Faculty Hospital, Brno, Czech Republic b Department of Biomedical Engineering, Brno University of Technology, Brno, Czech Republic c Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic d Department of Biochemistry, Faculty of Medicine, Masaryk University, Brno, Czech Republic e Institute of Scientific Instruments, Academy of Sciences, Brno, Czech Republic f Department of Sports Medicine and Rehabilitation (KFDR), St. Anne's Faculty Hospital, Masaryk University, Brno, Czech Republic g Department of Public Health, Faculty of Medicine, Masaryk University, Brno, Czech Republic article info abstract

Article history: Prolonged QT interval is an independent risk factor for development of ventricular arrhythmias. Halo- Received 22 June 2018 peridol is one of the drugs inducing QT prolongation. Previous studies showed that haloperidol affects Received in revised form not only QT duration but also heart rate (RR interval). The present work focused on relationship between 8 November 2018 QT and RR and its changes under acute and chronic haloperidol administration. The study included 14 Accepted 12 November 2018 male guinea pigs divided into control and haloperidol-treated group. After 21-days administration of Available online 24 November 2018 haloperidol or vehiculum, electrograms in isolated hearts were recorded. QT/RR and dQT/dRR coupling were calculated. Chronic haloperidol administration significantly decreases the coupling between QT and Keywords: fi Drug-induced QT prolongation RR. Acute haloperidol exposure signi cantly decreases the dQT/dRR coupling in both treated and un- Guinea pig treated guinea pig hearts. Flatter QT/RR relationship reveals a lack of QT adaptation to increased heart Haloperidol rate. It should be emphasized that in such situation ECG recording will not show significant QT pro- Isolated heart longation evaluated according to clinical rules. However, if QT interval does not adapt to increased heart QT/RR coupling rate sufficiently, the risk of ventricular arrhythmias may be increased despite practically normal QT in- terval length. The results are supported by findings in biochemical analyses, which proved eligibility of the used model. © 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

1. Introduction The QT prolongation in haloperidol-treated patients is not as frequently diagnosed as in patients under sertindole or risperi- QT lengthening represents an independent risk factor for done.3 On the other hand, life-threatening arrhythmias and sudden development of arrhythmias, such as Torsades de Pointes, eventu- cardiac death have been reported even after a single haloperidol e ally leading to sudden cardiac death.1 A link between QT prolon- dose.4 6 gation and certain antipsychotic drugs, such as clozapine, Haloperidol is a derivative of butyrophenone used for treatment risperidone, sertindole, and haloperidol has been previously re- of mental disorders. Its sedative and antiemetic effects are also ported.2 From all above mentioned, haloperidol is the least toler- utilized in post-operative and palliative care. Haloperidol is a non- ated antipsychotic substance.3 The main reason is a high incidence specific drug with known affinity to wide range of cerebral and of extrapyramidal side effects and weight gain during the therapy. extracerebral receptors, including dopamine D2 receptors, seroto- nin 5HT2 receptors, alpha adrenergic receptors, histamine H1 re- 7e9 ceptors, muscarinic M1 receptors, and sigma receptors. * Corresponding author. Department of Physiology, Faculty of Medicine, Masaryk Clinical aspects of haloperidol treatment and its impact on QT University, Kamenice 5, 625 00 Brno, Czech Republic. prolongation were studied repeatedly.10,11 Effects of the acute and/ E-mail address: [email protected] (T. Stracina). or chronic exposure to haloperidol were also studied on various Peer review under responsibility of Japanese Pharmacological Society. https://doi.org/10.1016/j.jphs.2018.11.004 1347-8613/© 2018 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 24 P. Vesely et al. / Journal of Pharmacological Sciences 139 (2019) 23e28 experimental models and species, including rat,12 guinea pig12,13 After the first minute of perfusion in stabilization period, coro- and rabbit.14 In previous studies on rat and guinea pig isolated nary effluent was collected (sample 1). Another sample (sample 2) hearts, significant changes of both RR and QT intervals were was obtained at the end of experiment (the last minute of W2). observed after acute as well as chronic haloperidol administra- 12,13 tion. Haloperidol has been clearly linked to higher risk of ven- 2.2. Electrogram analysis tricular arrhythmias,11 despite the relatively mild QT prolongation. 15 Moreover, haloperidol exerts significant cytotoxicity and it is The beat-to-beat analysis was performed using MATLAB known as potentially cardiotoxic drug.16 (version 8.0.2.701, MathWorks, MA, USA). The positions of R peak In animal studies under well-controlled conditions, reported and the end of T wave were subsequently detected. The R detection effects of haloperidol seem to be consistent. However, most of was based on wavelet transform. The simple wavelet db1 with scale haloperidol treated-patients exert normal QTc. Nevertheless, they 32 was chosen from MATLAB wavelet library. RT intervals were are under increased risk of ventricular arrhythmias.11 Such situa- detected in one electrogram channel per experiment only; the 17 tion is similar to congenital long QT syndrome (cLQTS). In some channel with the best shape of T wave was used. For detection of types of cLQTS, patients exert normal QTc at rest; however, adap- the T wave end, the isoline regression method was used. The tation of QT to heart rate changes is deteriorated. It remains unclear, detection was done for each single beat. Debatable beats were if chronic haloperidol treatment may affect relationship between marked as non-detectable. The courses of all studied parameters heart rate and QT. were expressed as beat-dependent and were filtered and deci- The present study was focused on the effect of chronic halo- mated by a median filter with the length of 100 beats. This peridol administration on the response of guinea pig isolated heart approach enabled elimination of quick changes that were probably to acute haloperidol perfusion. QTc according to subject specific not caused by the haloperidol administration. model, relationship between QT and RR intervals and their changes Mean RR, heart rate and QT were computed from the window under acute haloperidol administration were assessed after beat- approximately 2 min long at the end of stabilization in each to-beat analysis. Moreover, several biochemical parameters were experiment. The coupling between QT and RR intervals (QT/RR) and determined in order to evaluate the model eligibility. their differences (dQT/dRR) were analysed in each phase of experiment (except of stabilization) for both experimental groups. 2. Material and methods The differences in QT and RR intervals between each analysed in- terval and the mean one were computed: The study was carried out according to the recommendations of the European Community Guide for the Care and Use of Laboratory dRRi ¼ RRi À RRS Animals and according to experimental protocol approved by the Committee for Ensuring the Welfare of Laboratory Animals at where dRRi and RRi are the values for ith beat, the RRS is the mean Masaryk University, Faculty of Medicine. value of the RR at the end of stabilization. Similarly, dQT was The study was performed on 14 guinea pigs (unspecified breed, calculated. The mean and SD of the filtered courses of RR, QT, dRR, male only, 3 months old). Animals were housed in Laboratory of and dQT were computed. Animal Breeding and Experimental Facility, Faculty of Medicine, QT was corrected to the heart rate according to subject specific 18,21 Masaryk University, Brno, Czech Republic under controlled condi- linear correction model based on QT/RR coupling. For more tions with light cycle 12/12 light/dark. details, see Supplementary materials. Animals were randomly divided into two groups: haloperidol- Premature ventricular beats were detected manually and clas- 19 treated (group T; 6 animals; average body mass 364.2 ± 51.3 g) sified according to Lambeth conventions. Distribution of prema- and control (group C; 8 animals; average body mass 381.3 ± 21.7 g). ture ventricular beats was analysed within each experimental Haloperidol (Sigma Aldrich, USA) in dose of 2 mg/kg or vehiculum phase. Total numbers of premature ventricular beats were (2% ethanol in aqua pro injectione) in identical volume were compared in each phase between the groups as well as in consec- administered by intraperitoneal injection once a day for 21 utive phases within each group. consecutive days. The dose of haloperidol was chosen according to previous studies.12,13 2.3. Biochemical analysis

2.1. Isolated heart experiments Levels of two cardiac enzymes (creatine kinase e CK and lactate dehydrogenase e LD) and lactate were measured immediately in Twenty-four hours after the last dose, guinea pigs were deeply the samples of coronary effluent using commercial kits (Erba- anaesthetized by isoflurane (2%). The heart was rapidly excised and Lachema and BioVendor, Czech Republic) according to the manu- perfused according to Langendorff with Krebs-Henseleit solution as facturer's instructions. Determination of 4-hydroxy-2-nonenal 20 described previously.13 The isolated heart was allowed to stabilize (HNE) was performed as described previously. All chemicals used for 40 min. Immediately after stabilization (S), experimental pro- in biochemical analyses of HNE were of the p.a. or HPLC grade, tocol continued with four 20-min-lasting consecutive phases: the purchased from SigmaeAldrich (St. Louis, MO, USA). The standard first haloperidol exposure (H1), washout (W1), the second halo- of HNE was obtained from Cayman Chemical (Ann Arbor, MI, USA). peridol exposure (H2), and the second washout (W2). Haloperidol was administered diluted in Krebs-Henseleit solution at the con- 2.4. Statistical analysis centration of 10 nM. Six Ag-AgCl electrodes placed in the inner surface of the Langendorff apparatus bath were used for continual The analysis of electrograms was based on scatter plots and recording of electrogram in three orthogonal leads. The perfusion linear regression was used to evaluate the coupling between the pressure and temperature of the perfusion solution were continu- parameters. ANOVA was used to analyse statistical significance and ously monitored and maintained at 80 mmHg and 37 C, respec- bootstrap methodology with repetition 100 times was used to tively. All signals were recorded by an acquisition card NI USB-6229 analyse distribution of regression parameters. For other parame- BNC (National Instruments, TX, USA; sampling frequency: 10 kHz, ters, Wilcoxon paired test for comparison of paired samples and resolution: 12-bit). ManneWhitney U test for groups' comparison were used. The data P. Vesely et al. / Journal of Pharmacological Sciences 139 (2019) 23e28 25 are presented as mean ± standard deviation (SD); differences were these parameters were significantly higher in sample 1 than in considered significant when P < 0.05. sample 2 in both groups (Table S1 in Supplementary materials). Concerning HNE, no significant difference was found between groups C and T at the beginning as well as at the end of isolated 3. Results heart experiment. Also, no significant differences were detected between sample 1 and 2 within each group. 3.1. Electrogram analysis

Significant difference in heart rate was found between groups C 4. Discussion and T at the end of S phase (Table 1). However, there is no signifi- cant difference in QTc between the groups at the end of S phase. The experimental setting in the study allows to compare re- Values of RR, QT, dRR, and dQT in H1, W1, H2, and W2 are sum- sponses to acute haloperidol administration in two situations: marized in Table 2. In QT/RR coupling as well as in dQT/dRR Haloperidol administration in isolated hearts of haloperidol- coupling, flatter slopes of fitted lines (lower a1) were found in treated guinea pigs (group T) simulated the effect of acute (intra- group T then in C during all phases (Tables 4 and 5). The differences venous) haloperidol exposure in long-term haloperidol-treated. between the groups are more significant in the dQT/dRR coupling Haloperidol administration in isolated hearts of control guinea pigs (Fig. 1). In both groups, longer QTc was found in both haloperidol (group C) simulated effect of the first haloperidol exposure in un- phases in comparison with consecutive washout phases (Table 3). treated patient. Apparent decrease of total number of ventricular premature The significant difference in dQT/dRR coupling between group T beats in phases W1 (compared to H1) was noticeable in both groups and C clearly shows that chronic haloperidol administration affects (for group C: 3.9 ± 5.8 in H1 vs. 1.1 ± 1.8 in W1; for group T: 4.0 ± 7.9 relationship between heart rate and QT (Fig. 1). The flatter rela- in H1 vs. 1.4 ± 1.9 in W1). However, no significant differences in tionship between QT and RR reveals a lack of QT adaptation to total number of ventricular premature beats were detected be- changed heart rate.18 tween the groups as well as between consecutive phases within Chronic haloperidol pre-treatment did not prolong QTc in each group. guinea pig isolated heart (Table 1). It is in contrast to previous studies.13 The different finding may be caused by different method of analysis and QT correction used in the present study. Evaluation 3.2. Biochemical analysis of the effect of acute haloperidol on QTc is limited due to different models used for QT correction in phase S and H1 (see In levels of CK, LD and lactate, no significant difference was Supplementary materials). Nevertheless, the trends in QTc differ- found between the groups C and T in the beginning (sample 1) as ences between consecutive phases correspond to the general well as at the end of perfusion (sample 2). However, the levels of meaning, that acute haloperidol administration prolongs QTc (Table 3) e QTc is higher in haloperidol phases than in washouts in Table 1 both groups. However, effect of long-term haloperidol adminis- The RR, heart rate and QT computed from the last 2 min of stabilization phase. tration is not visible. Contrary to expectations, QTc in haloperidol Group Phase of the experiment treated group does not differ from control group during the H1 phase and it is even significantly shorter during the W1. It might be The end of stabilization (S) due to different reactivity of QTc in group C and T to acute halo- RR [ms] HR [beats/min] QT [ms] QTc [ms] peridol administration (H1). Between H1 and W1, group T exerts C 309 ± 31.5 196 ± 20.1 179 ± 25.8 245.0 ± 8.3 higher change of QTc than control group. In comparison, the dQT/ T 287 ± 42.2 213 ± 33.7 161 ± 32.5 243.0 ± 14.1 dRR coupling is significantly flatter in haloperidol treated group < < < P 0.001* 0.001* 0.001* 0.253 than in controls during all H1, W1, H2, and W2 phases (Fig. 1). As Values are expressed as mean ± SD. HR e heart rate; QTc e corrected QT interval; C stated above, this clearly shows that evaluation of QTc (even ac- e control group; T e haloperidol treated group; P e P value; * e significant differ- cording to subject-specific model) may be insufficient in uncover- ence between the groups C and T. The QTc was computed according to formula * ing the effects of long-term haloperidol administration. QTc ¼ QT þ a1 (RR À RRC) where the RRC is nominal RR interval (RRC ¼ 400 ms). The parameter a1 was calculated as the average value of the QT/RR coupling It has been hypothesized that an impaired adaptation of QT computed over all phases of the experiment except of the stabilization. interval to abrupt changes in the heart rate is the primary process

Table 2 Mean values of RR, QT, dRR, and dQT computed for each individual haloperidol and washout phase.

Phase of the experiment

H1 W1

RR [ms] QT [ms] dRR [ms] dQT [ms] RR [ms] QT [ms] dRR [ms] dQT [ms]

C 300.1 ± 28.6 173.2 ± 24.4 À5.210 ± 9.745 À4.097 ± 5.184 298.4 ± 32.8 171.1 ± 25.2 À5.923 ± 15.106 À5.023 ± 7.502 T 280.3 ± 43.4 158.0 ± 29.5 À0.349 ± 7.439 1.067 ± 5.139 285.2 ± 37.4 159.5 ± 26.5 4.192 ± 9.052 1.783 ± 6.387 P <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001*

H2 W2

RR [ms] QT [ms] dRR [ms] dQT [ms] RR [ms] QT [ms] dRR [ms] dQT [ms]

C 298.2 ± 31.6 169.1 ± 26.1 À7.642 ± 15.559 À7.184 ± 8.171 303.4 ± 31.6 169.3 ± 25.2 À2.031 ± 18.554 À6.752 ± 14.098 T 279.4 ± 28.8 157.5 ± 23.7 À3.068 ± 17.061 À1.454 ± 7.530 283.4 ± 25.1 160.1 ± 21.5 À1.293 ± 21.268 0.698 ± 10.141 P <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001* <0.001*

The values are expressed as mean ± SD. dRR was calculated according to formula: dRRi ¼ RRi À RRS, where dRRi and RRi are the values for ith beat, the RRS is the mean value of the RR at the end of stabilization; dQT was calculated similarly. C e control group; T e haloperidol treated group; P e P value; * e significant difference between the groups C and T; H1 e the first haloperidol administration; W1 e the first washout; H2 e the second haloperidol administration; W2 e the second washout. 26 P. Vesely et al. / Journal of Pharmacological Sciences 139 (2019) 23e28

Table 3 such situations, RR intervals will initially shorten, which is followed QTc in haloperidol and washout phases. by QT intervals shortening. However, the shortening of the latter * QTc ¼ a1 RR0 þ a0 [ms] does not follow RR shortening swiftly enough, which may lead to

Phase of the experiment ventricular arrhythmias. Nevertheless, no significant increase of ventricular premature H1 W1 H2 W2 beats was detected during haloperidol exposure in guinea pig iso- C 250.7 ± 9.7 244.0 ± 8.6 252.1 ± 4.8 242.9 ± 7.4 lated hearts. This finding is in accordance with previous study in ± ± ± ± T 252.1 14.1 242.1 8.4 254.8 7.2 249.3 8.0 guinea pig isolated hearts.12 Possible explanation is that isolated P 0.171 0.006* <0.001* <0.001* heart is protected from abrupt changes of the heart rate (RR in- The values are expressed as mean ± SD. QT was corrected according to subject tervals) both by strictly controlled experimental conditions and by specific linear correction based on QT/RR coupling for nominal RR0 ¼ 400 ms: * autonomous denervation due to heart explantation. QTc ¼ a1 RR0 þ a0. H1 e the first haloperidol administration; W1 e the first washout; H2 e the second haloperidol administration; W2 e the second washout; C To explain decreased coupling between QT and RR, several e control group; T e haloperidol treated group; P e P value; * e significant differ- mechanisms may be hypothesised. QT interval reflects duration of ence between the groups C and T. the ventricular action potential and depends on ventricular repo- larization. The ventricular repolarization is ensured mainly by po- tassium currents. When these currents are reduced, repolarization Table 4 The numeral expression of the QT/RR coupling computed for each individual halo- is longer, the ventricular action potentials broaden, and the QT peridol and washout phase. interval prolongs. One of the repolarizing potassium currents is the

* rapid delayed rectifier current (IKr). Alteration of the IKr current is QT/RR (QT ¼ a1 RR þ a0) the most common mechanism of QT prolongation.23 In humans, IKr Phase of the experiment is conducted by human ether-a-go-go related gene (hERG) potas- H1 W1 sium channels.24 Guinea pig heart expresses potassium channels 25 a1 a0 a1 a0 quite comparable to human. Haloperidol concentration of 10 nM used in our experiments on isolated hearts is close to IC50 for hERG C 0.783 ± 0.021 62.392 ± 6.725 0.723 ± 0.015 44.939 ± 4.734 26,27 T 0.620 ± 0.010 15.323 ± 3.032 0.677 ± 0.006 33.876 ± 1.916 blocking. Therefore, one of the mechanisms of haloperidol ef- P <0.001* <0.001* <0.001* <0.001* fect on QT interval might be related to decreasing activity of IKr. Next to its direct effects on ionic channels, haloperidol binds to H2 W2 various receptor systems, with the highest affinities to dopamine a1 a0 a1 a0 and sigma receptors. Dopamine D2 receptors are expressed also in ± ± ± ± C 0.812 0.009 72.853 2.645 0.762 0.016 61.766 4.755 guinea pig heart and it has been shown that D2 agonists may in- T 0.785 ± 0.008 62.633 ± 2.328 0.799 ± 0.011 65.818 ± 2.970 28 crease QT interval duration. However, haloperidol acts as D2 P <0.001* <0.001* <0.001* <0.001* antagonist and the dopaminergic effect on the heart rate and QT The values are expressed as mean ± SD. The parameter a1 represents the slope of the interval duration is disputable. Haloperidol is also a prototypic fitted line. H1 e the first haloperidol administration; W1 e the first washout; H2 e ligand of sigma receptors. In the heart muscle, both sigma 1 and the second haloperidol administration; W2 e the second washout; C e control group; T e haloperidol treated group; P e P value; * e significant difference between sigma 2 receptors are expressed, with majority of sigma 1 sub- 29 the groups C and T. type. Concentration of haloperidol used in this study is close to Kd value of the haloperidol binding on cardiac sigma 1 receptors.29 Previous studies have proven that expression of sigma 1 receptors Table 5 in the guinea pig and rat atria increased after repeated exposure to The numeral expression of the dQT/dRR coupling computed for each individual haloperidol.13,30 Modulation of sigma 1 receptors may partially haloperidol and washout phase. elucidate the QT/RR coupling changes observed in this study since * dQT/dRR (dQT ¼ a1 dRR þ a0) it has been reported that sigma receptors modulate potassium 9,31 Phase of the experiment channels behaviour. It was reported that haloperidol adminis- tration leads to impairment of mitochondrial calcium mobilization H1 W1 and ATP depletion via influencing of sigma 1 receptors in cultured a1 a0 a1 a0 rat cardiomyocytes.32 Moreover, it was shown that stimulation of C 0.395 ± 0.017 À2.175 ± 0.193 0.411 ± 0.011 À2.734 ± 0.212 sigma 2 receptors by haloperidol blocks human recombinant hERG T 0.244 ± 0.046 0.918 ± 0.233 0.248 ± 0.033 0.565 ± 0.332 potassium channels in COS-7 cells.33 < < < < P 0.001* 0.001* 0.001* 0.001* Findings of the present study may partly clarify certain con- H2 W2 troversy in the reported effects of haloperidol in clinical practice: a1 a0 a1 a0 haloperidol-induced QT prolongation is comparable to other anti- psychotics; however, haloperidol treatment significantly increases C 0.477 ± 0.011 À3.654 ± 0.169 0.670 ± 0.017 À5.331 ± 0.288 3,34 T 0.246 ± 0.017 À0.992 ± 0.376 0.303 ± 0.015 0.966 ± 0.482 a risk of ventricular arrhythmias and sudden cardiac death. P <0.001* <0.001* <0.001* <0.001* Limitations of such studies, such as comorbidities and medica- 35,36 The values are expressed as mean ± SD. The parameter a1 represents the slope of the tions must be considered. Nevertheless, the impaired adapta- fitted line. tion of QT interval to abrupt changes in the heart rate might be one C e control group; T e haloperidol treated group; P (anova1) e P value; * e sig- of possible explanations of haloperidol-induced ventricular fi e fi ni cant difference between the groups C and T; H1 the rst haloperidol admin- arrhythmias. e fi e istration; W1 the rst washout; H2 the second haloperidol administration; W2 fi e the second washout. In summary, the study proves for the rst time that chronic haloperidol administration significantly decreases the coupling between QT and RR intervals (dQT/dRR) in the guinea pig model. 17,22 behind the drug-induced long QT syndrome. In such situation, Acute haloperidol exposure significantly decreases the dQT/dRR resting ECG recording does not show significant QT prolongation. coupling in both treated and untreated guinea pig hearts. The Changed coupling will be unmasked only in situations when RR flatter dQT/dRR coupling reveals impaired QT adaptation to heart interval is shortened, such as during physical exercise or stress. In rate changes. The impaired adaptation of QT interval to abrupt P. Vesely et al. / Journal of Pharmacological Sciences 139 (2019) 23e28 27

Fig. 1. The dQT/dRR coupling computed for each individual haloperidol and washout phase. From the left upper corner to the right bottom corner in order H1, W1, H2 and W2. The green colour corresponds to control group (C), the red to haloperidol-treated group (T). Haloperidol-treated group exhibits decreased slope of fitted lines (red) in each of the experimental phases. For numeral expression, see Table 5. changes in the heart rate might be one of possible explanations of Grant, as provided by the Ministry of Education, Youth and Sports of haloperidol-induced ventricular arrhythmias.17 It is necessary to the Czech Republic in the year 2018. emphasize that changed coupling will be unmasked only in situa- tion when RR interval is changed and that the resting ECG recording may or may not show significant QT prolongation. Appendix A. Supplementary data

Supplementary data related to this article can be found at Conflicts of interest https://doi.org/10.1016/j.jphs.2018.11.004.

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