Practical Methods in Cardiovascular Research

1011 Pages·2005·14.28 MB·English

by  Stefan Dhein

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
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Library of Congress Control Number:2004105045
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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
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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
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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
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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
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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
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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