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. | 8 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