Stefan Dhein Friedrich Wilhelm Mohr Mario Delmar Practical Methods in Cardiovascular Research Stefan Dhein (Editor) Friedrich Wilhelm Mohr (Editor) Mario Delmar (Editor) Practical Methods in Cardiovascular Research With 33 Tables and 290 Figures Prof. Dr. Stefan Dhein Prof. Dr. Friedrich Wilhelm Mohr Mario Delmar, MD PhD Klinik für Herzchirurgie Klinik für Herzchirurgie Department of Pharmacology Universität Leipzig Universität Leipzig SUNY Upstate Medical University Herzzentrum Herzzentrum 766 Irving Avenue Strümpellstraße 39 Strümpellstraße 39 Syracuse NY 13210 04289 Leipzig 04289 Leipzig USA Germany Germany Library of Congress Control Number:2004105045 ISBN3-540-40763-4 Springer Berlin Heidelberg NewYork This work is subject to copyright. 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Product liability: The publisher cannot guarantee the accuracy of any information about dosage and application thereof contained in this book. In every individual case the user must check such information by consulting the relevant literature. Editor:Dr. Thomas Mager, Heidelberg Development Editor:Natasja Sheriff, Heidelberg Production:Frank Krabbes, Heidelberg Cover Design:deblik, Berlin Page makeup:Hilger VerlagsService, Heidelberg Printing:Krips BV, Meppel, Niederlande Printed on acid-free paper SPIN:10891623 14/3109–543210 0.00 Preface V Preface Cardiovascular disease is among the most common causes of death in Western indus- trialized countries. Although a number of diagnostic and therapeutic possibilities exist today, many problems remain unsolved. This implies the urgent need for future cardiovascular research. The present book gives an overview of a number of available techniques most often used in cardiovascular sciences, especially for investigators coming from other fields of research. The book is divided into three sections. In the first part, the focus is on techniques used for in vivo experimentation. This field has regained great interest, given the availability of knock-out models which now await in-vivo testing. The book describes the most commonly used procedures for anesthesia and analgesia in laboratory ani- mals as well as „classical“ isolated organ techniques, including electrophysiological methods now – again – in common use. In the second part of the book, we move from the organ and tissue level to the cellular domain. Topics covered include electrophysiology, histology and cell culture techniques as well as biochemical methods such as HPLC, protein biochemistry and binding assays. This part closes with a brief chapter on how to register and analyze concentration response curves giving briefly the mathematics and basic analysis tech- niques. In the final part, the focus narrows further into molecular procedures such as surface plasmon resonance, nuclear magnetic resonance, RNA methods and DNA arrays. Finally, a chapter is devoted to the molecular aspects of transgenic mice tech- nology as well as an overview of procedures and regulations applicable to housing and transfer of transgenic animals. The novelty of this book is in the description of practical protocols and trouble- shooting procedures. It is our intention to provide a basic guide for investigators in- terested in applying these techniques in their own laboratories. The book also aims to close the gap between the integrative in-vivo and organ-based methods and the re- ductionist cellular and molecular approaches. We wish all researchers every success with their experiments and that this book may help them start their way toward new frontiers. Stefan Dhein Mario Delmar Friedrich Wilhelm Mohr 0.00 Table of Contents VII Table of Contents In-Vivo Techniques 1 Models of Cardiovascular Diseases 1.1 Anaesthesia (Inhalative or i.v. Narcosis) of Laboratory Animals . . . . . . . . . . . . . . . . 5 Katja Schneider 1.2 Extracorporeal Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Cris W. Ullmann 1.3 Open-chest Models of Acute Myocardial Ischemia and Reperfusion . . . . . . . . . . 37 Kai Zacharowski, Thomas Hohlfeld and Ulrich K.M. Decking 1.4 Models of Chronic Ischemia and Infarction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Nicolas Doll, Heike Aupperle, Fabian Stahl and Thomas Walther 1.5 Models on Left Ventricular Hypertrophy (LVH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Thomas Walther and Volkmar Falk 1.6 Experimental Models of Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Volkmar Falk, Jens Garbade and Thomas Walther 1.7 In-Vivo Models of Arrhythmias: a Canine Model of Sudden Cardiac Death . . . . 111 George E. Billman 1.8 In-Vivo Models of Atrial Fibrillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Ulrich Schotten, Yuri Blaauw and Maurits Allessie In-Vitro Techniques 2 Isolated Organs 2.1 The Langendorff Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Stefan Dhein 2.2 The Working Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Marc W. Merx and Jürgen Schrader 2.3 Isolated Papillary Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Stefan Dhein 2.4 Isolated Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Rudolf Schubert 0 VIII Table of Contents 3 Electrophysiological Techniques 3.1 Optical Techniques for the Recording of Action Potentials . . . . . . . . . . . . . . . . . . . . 215 Helmut A. Tritthart 3.2 Recording Action Potentials Using Voltage-Sensitive Dyes . . . . . . . . . . . . . . . . . . . 233 Vladimir G. Fast 3.3 Epicardial Mapping in Isolated Hearts – Use in Safety Pharmacology, Analysis of Torsade de Pointes Arrhythmia and Ischemia-Reperfusion-Related Arrhythmia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Stefan Dhein 3.4 Voltage-Clamp and Patch-Clamp Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 Hans Reiner Polder, Martin Weskamp, Klaus Linz, Rainer Meyer 3.5 L-Type Calcium Channel Recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 Uta C. Hoppe, Mathias C. Brandt, Guido Michels and Michael Lindner 3.6 Recording Cardiac Potassium Currents with the Whole-Cell Voltage Clamp Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Erich Wettwer and Ursula Ravens 3.7 Recording the Pacemaker Current If . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Uta C. Hoppe, Fikret Er and Mathias C. Brandt 3.8 Recording Gap Junction Currents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Ardawan J. Rastan and Stefan Dhein 3.9 Recording Monophasic Action Potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Paulus Kirchhof, Larissa Fabritz and Michael R. Franz 3.10 Heart on a Chip – Extracellular Multielectrode Recordings from Cardiac Myocytes in Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Ulrich Egert and Thomas Meyer 4 Histological Techniques 4.1 Immunohistochemistry for Structural and Functional Analysis in Cardiovascular Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457 Wilhelm Bloch, Yüksel Korkmaz and Dirk Steinritz 4.2 Classical Histological Staining Procedures in Cardiovascular Research . . . . . . . . 485 Wilhelm Bloch and Yüksel Korkmaz 4.3 Practical Use of in situ Hybridisation and RT in situ PCR in Cardiovascular Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 Yüksel Korkmaz, Dirk Steinritz and Wilhelm Bloch 4.4 Practical Use of Transmission Electron Microscopy for Analysis of Structures and Molecules in Cardiovascular Research . . . . . . . . . . . . . . . . . . . . . . 525 Wilhelm Bloch, Christian Hoffmann, Eveline Janssen and Yüksel Korkmaz 4.5 DAF Technique for Real-Time NO Imaging in the Human Myocardium . . . . . . . 546 Dirk Steinritz and Wilhelm Bloch 0.00 Table of Contents IX 5 Cell Culture Techniques 5.1 Isolation and Culture of Adult Ventricular Cardiomyocytes . . . . . . . . . . . . . . . . . . . 557 Klaus-Dieter Schlüter and Hans Michael Piper 5.2 Culture of Neonatal Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568 Aida Salameh and Stefan Dhein 5.3 Culture of Embryoid Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Cornelia Gissel, Dirk Nierhoff, Bernd Fleischmann, Jürgen Hescheler and Agapios Sachinidis 5.4 Culturing and Differentiation of Embryonic and Adult Stem Cells for Heart Research and Transplantation Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592 Marcel AG van der Heyden and Henk Rozemuller 5.5 Preparation of Endothelial Cells from Micro- and Macrovascular Origin . . . . . . 610 Saskia C. Peters, Anna Reis and Thomas Noll 5.6 In Vitro Cultivation of Vascular Smooth Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 630 Daniel G. Sedding and Ruediger C. Braun-Dullaeus 5.7 Engineering Heart Tissue for In Vitro and In Vivo Studies . . . . . . . . . . . . . . . . . . . . . 640 Wolfram-Hubertus Zimmermann, Ivan Melnychenko, Michael Didié, Ali El-Armouche and Thomas Eschenhagen 6 Biochemical and Analytical Techniques 6.1 High-Performance Liquid Chromatography (HPLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . 661 Veronika R. Meyer 6.2 Protein Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686 Aida Salameh, Stefan Lehr, Jürgen Weiss and Peter Rösen 6.3 Receptor and Binding Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723 Peter Hein, Martin C. Michel, Kirsten Leineweber, Thomas Wieland, Nina Wettschureck and Stefan Offermanns 6.4 Recording and Analysing Concentration-Response Curves . . . . . . . . . . . . . . . . . . . 784 Stefan Dhein 6.5 Measurement of NO and Endothelial Function in Cardiovascular Research . . . 799 Renate Roesen, Anke Rosenkranz, Dirk Taubert and Reinhard Berkels 6.6 Biomechanical Stimulation of Vascular Cells In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . 813 Henning Morawietz and Andreas Schubert 6.7 Measurement of Function and Regulation of Adrenergic Receptors . . . . . . . . . . 829 Marc Brede, Melanie Philipp and Lutz Hein 6.8 Measurement of Function and Regulation of Muscarinic Acetylcholine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848 Björn Kaiser and Chris J. van Koppen 6.9 Cytometry in Cardiovascular Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 863 Attila Tárnok 0 X Table of Contents Molecular Biology in Cardiovascular Research 7 Molecular Biology Techniques 7.1 Real-Time PCR Analysis of Cardiac Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891 Dalton A. Foster and Steven M. Taffet 7.2 Use of cDNA Arrays to Explore Gene Expression in Genetically Manipulated Mice and Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 907 Dumitru A. Iacobas, Sandra Iacobas and David C. Spray 7.3 Using Antibody Arrays to Detect Protein-Protein Interactions . . . . . . . . . . . . . . . . 916 Heather S. Duffy, Ionela Iacobas, David C. Spray and Anthony W. Ashton 7.4 Surface Plasmon Resonance as a Method to Study the Kinetics and Amplitude of Protein-Protein Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 936 Brian D. Lang, Mario Delmar and Wanda Coombs 7.5 How to Solve a Protein Structure by Nuclear Magnetic Resonance – The Connexin43 Carboxyl Terminal Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 948 Paul L. Sorgen 7.6 Managing a Transgenic Mouse Colony: A Guide for the Novice . . . . . . . . . . . . . . . 959 Robert H. Quinn and Karen L. Vikstrom Appendix Colour Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 979 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 997 0.00 List of Abbreviations XI List of Abbreviations AP action potential COMT catechol-O-methyltransferase 1D one-dimensional COS cells kidney cells from the monkey 2D two-dimensional Cercopithecus aethiops 3D three-Dimensional CPB cardiopulmonary bypass 5-HT serotonin CS cell signaling αAR α-Adrenoceptor CSD cell cycle-shape-differentiation 1 1 AADC aromatic L-amino acid decarboxylase CV superior caval vein ABC avidin-biotin peroxidase complex Cx43 connexin43 AC adenylyl cyclase CY cytoskeletal Ach acetylcholine ACT activated clotting time DA diacetate AEC 3-amino-9-ethylcarbazole DAB diaminobenzidine tetrahydro- AF atrial fibrillation chloride AMI acute myocardial infarction DAFs diaminofluoresceins Ao Aorta DAF-T triazole-fluoresceins APHIS Agriculture Animal and Plant Health Daun daunorubicin Inspection Service DβH dopamine β-hydroxylase Ara-C cytosine-β-D-arabinofuranoside dd double-distilled ARI activation-recovery-interval DDSA dodecenyl succinic anhydride AT-1 angiotensin type 1 receptor DEPC diethylpyrocarbonate AT-2 angiotensin type 2 receptor DMEM Dulbecco’s modified Eagle medium DMEM-F12 Dulbecco’s Modified Medium B0 the static magnetic field DMP tri(dimethylaminomethyl)phenol β3-AR β3-adrenoceptors DMSO dimethylsulfoxide BCIP 5-bromo-4-chloro-3- Dox doxorubicin indolylphosphate dP/dt maximum rate of left ventricular max BCL basic cycle length pressure development BDM 2,3-butanedione-monoxime dP/dt maximum relaxation velocity min BM bone marrow dP/dt minimum rate of pressure min BrdU 5-bromo-2-desoxyuridine development BSA bovine serum albumin DSEVC discontinuous single electrode BSA biotin-streptavidin voltage clamp amplifier BSS balanced salt saline DSS Dahl salt sensitive rat BTP breakthrough-points DTT dithiothreitol C capacitance EBNA-LP Epstein-Barr virus nuclear antigen- CABG coronary artery bypass grafting leader protein CARB carbachol EBs embryoid bodies CARD catalyzed reporter deposition EC embryonal carcinoma CCD charge-coupled device EC endothelial cells CDC Centers for Disease Control ECG electrocardiogram CF coronary flow ECL HRP enhanced chemiluminescence CHO Chinese hamster ovary cells horseradish peroxidase CK creatine kinase EDC 1-Ethyl 3-3-dimethylaminopropyl CK/CKMB cCreatine kinase/cardiospecific CK carbodiimide CK-MB cardiospecific CK EDP end-diastolic pressure CN cellulose nitrate EDTA ethylenediaminetetraacetate CNBD cyclic nucleotide-binding domain EGF epidermal growth factor CNS crystallography & NMR systems EGFP enhanced green fluorescent protein CO cardiac output EnMet Energy and Metabolism