CARDIOVASCULAR EFFECTS OF ORAL CALCIUM SENSITIZERS ON EXPERIMENTAL HEART FAILURE Marjut Louhelainen Institute of Biomedicine Pharmacology University of Helsinki Finland Academic Dissertation Helsinki 2010 To be presented, with the permission of the Medical Faculty of the University of Helsinki for public examination in Lecture Hall 2, Biomedicum Helsinki, Haartmaninkatu 8, on the 29 of January 2010 at 12 noon. Supervisor Professor Eero Mervaala, MD, PhD Institute of Biomedicine Pharmacology University of Helsinki Finland Reviewers Docent Jaana Rysä, PhD Institute of Biomedicine Department of pharmacology and toxicology University of Oulu Finland Docent Jyri Lommi, MD, PhD Division of Cardiology Department of Medicine Helsinki University Central Hospital Finland Opponent Professor Raimo Tuominen, MD, Ph.D Faculty of Pharmacy University of Helsinki Finland ISBN 978-952-92-6765-1 (paperback) ISBN 978-952-10-6016-8 (PDF) http://ethesis.helsinki.fi Helsinki University Print Helsinki 2009 2 To Jani and Martta 3 Table of contents List of original publications ...................................................................................................................... 6 Main abbreviations ...................................................................................................................................... 7 Abstract............................................................................................................................................................ 8 1 Introduction ............................................................................................................................................ 9 2 Review of the literature ....................................................................................................................11 2.1 Heart failure..................................................................................................................................11 2.1.1 Epidemiology and prevalence..........................................................................................................11 2.1.2 Clinical symptoms and diagnosis....................................................................................................11 2.1.3 Treatment..................................................................................................................................................12 2.1.4 Mechanisms of heart failure..............................................................................................................15 2.1.4.1 Single gene mutations and genetic polymorphisms ....................................................................... 15 2.1.4.2 Renin‐angiotensin‐aldosterone‐system............................................................................................... 16 2.1.4.3 Increased sympathetic signalling............................................................................................................ 16 2.1.4.4 Ca2+ handling and regulation of excitation‐contraction coupling............................................. 17 2.1.4.5 Cardiac hypertrophy..................................................................................................................................... 18 2.1.4.5.1 Mechanisms involved in heart failure‐related cardiac hypertrophy ............................. 19 2.1.4.6 Oxidative stress............................................................................................................................................... 21 2.1.4.7 Apoptosis........................................................................................................................................................... 23 2.1.4.8 Dysfunctional energy utilization ............................................................................................................. 24 2.1.4.9 Cardiomyocyte senescence........................................................................................................................ 25 2.2 Experimental models of heart failure ..................................................................................26 2.3 Levosimendan ..............................................................................................................................28 2.3.1 Mechanism of action.............................................................................................................................28 2.3.2 Pharmacokinetics of levosimendan...............................................................................................29 2.3.3 Pharmacodynamics of levosimendan ...........................................................................................29 2.3.3.1 Clinical use of levosimendan..................................................................................................................... 29 2.3.3.2 Adverse effects................................................................................................................................................ 30 2.3.3.3 Clinical studies on levosimendan............................................................................................................ 31 2.3.3.4 Clinical studies on oral levosimendan .................................................................................................. 35 2.3.3.5 Experimental studies on levosimendan............................................................................................... 35 2.3.4 The active metabolite OR‐1896.......................................................................................................36 2.3.4.1 Mechanism of action of OR‐1896............................................................................................................ 36 3 Aims of the study .................................................................................................................................37 4 Materials and methods......................................................................................................................38 4.1 Experimental models and animal welfare..........................................................................38 4.1.1 Dahl/Rapp salt sensitive (SS) rat....................................................................................................38 4.1.2 Diabetic Goto Kakizaki (GK) rat.......................................................................................................38 4.1.3 Animal welfare........................................................................................................................................39 4.2 Study design ..................................................................................................................................39 4.3 Experimental myocardial infarction ....................................................................................40 4.4 Levosimendan and OR­1896 dosage.....................................................................................40 4.5 Blood pressure measurement and sample preparation................................................41 4.6 Echocardiography .......................................................................................................................41 4.7 Infarct size, collagen volume fraction and cardiomyocyte cross­sectional area...41 4.8 Myocardial morphology............................................................................................................42 4.9 Immunohistochemistry.............................................................................................................42 4.10 TUNEL apoptosis assay ...........................................................................................................42 4.11 Apoptosis microarray .............................................................................................................43 4.12 Western blotting .......................................................................................................................43 4.13 Electrophoretic mobility shift assay of nuclear FOXO3a transcription factor.....43 4.14 Quantitative real­time RT­PCR.............................................................................................44 4 4.15 Biochemical analyses ..............................................................................................................46 4.16 Statistical analyses ...................................................................................................................46 5 Results.....................................................................................................................................................47 5.1 Survival (All studies)..................................................................................................................47 5.2 Cardiac functions and blood pressure (All studies) ........................................................47 5.3 Hypertrophy (All studies), cardiomyocyte cross­sectional area (Study III and IV)... ...........................................................................................................................................................48 5.4 Cardiomyocyte morphology (Studies I and II), infarct size, fibrosis (Studies III and IV).......................................................................................................................................................49 5.5 Calcium handling proteins (Studies I and III)....................................................................50