Manual of Research Techniques in Cardiovascular Medicine

While some aspects of clinical and scientific research are common across medicine, research in cardiovascular diseases requires knowledge of an increasing array of procedures, techniques, models and measurements that are highly specialized and unique to this area of investigation. Edited and written by senior investigators, many of whom have been involved in pioneering studies in cardiovascular disease, this new book provides today’s cardiology research professionals with practical how–to guidance on designing, conducting, measuring and reporting their research. Manual of Research Techniques in Cardiovascular Medicine : • Reviews the must–know techniques, methods, analyses, models and measurements of cardiovascular research in a practical, “how–to–do–it” approach, resulting in clear–headed guidance to even the most complicated techniques and methods. • Covers each technique or method beginning with an explanation of when and why it should be selected followed by step–by–step instructions that include why each step is important; what can go wrong; how to trouble shoot and correct errors, and how to analyze and report results. • Helps ensure research success, but also, critically, enables readers to understand why and how some experiments fail. • Contains illustrative figures to reinforce the key components/steps of each method covered. • Includes a companion website with video clips to support the discussion of particularly challenging techniques, for example animal surgical procedures. Manual of Research Techniques in Cardiovascular Medicine is an invaluable resource for investigators at all levels, from technicians, students, fellows and other healthcare professionals who are approaching research for the first time, to seasoned investigators in cardiovascular disease, pharmacology, and related areas who need expert guidance on emerging techniques or unfamiliar models or methods.

List of Contributors ix Preface xix About the Companion Website xx Part 1 Electrophysiology 1 Measurement of calcium transient ex vivo 3 Kenneth R. Laurita and Bradley N. Plummer 2 Confocal imaging of intracellular calcium cycling in isolated cardiac myocytes 12 Søren Grubb, J. Andrew Wasserstrom, and Gary L. Aistrup 3 Generating a large animal model of persistent atrial fi brillation 20 Raphaël P. Martins and José Jalife 4 Confocal imaging of intracellular calcium cycling in the intact heart 32 Neha Singh, Manvinder Kumar, James E. Kelly, Gary L. Aistrup, and J. Andrew Wasserstrom 5 Recording and measurement of action potentials 41 Kenneth S. Ginsburg and Donald M. Bers 6 Patch–clamp recordings from isolated cardiac myocytes 50 Kai–Chien Yang, Wei Wang, and Jeanne M. Nerbonne 7 Optical mapping of the heart 60 Di Lang, Sarah Gutbrod, Jacob Laughner, and Igor R. Efi mov Part 2 Isolation and Maintenance of Primary Stem Cells 8 Isolation of colony–forming endothelial progenitor cells 71 Haruchika Masuda and Takayuki Asahara 9 Cardiac resident stem cells 87 João Ferreira–Martins, Fumihiro Sanada, and Marcello Rota 10 Cardiospheres 95 Rachel Ruckdeschel Smith 11 Mesenchymal stem cells 104 Jose S. Da Silva and Joshua M. Hare 12 Generation and differentiation of human iPS cells 110 Sebastian Diecke, Lei Ye, Sophia Zhang, and Jianyi Zhang 13 Isolation of neonatal and adult rat cardiomyocytes 117 Md. Abdur Razzaque and Jeffrey Robbins 14 Isolation and culture of vascular smooth muscle cells 125 Milton Hamblin, Lin Chang, and Y. Eugene Chen 15 Isolation and culture of cardiac endothelial cells 131 Asish K. Ghosh, Joseph W. Covington, and Douglas E. Vaughan 16 Isolation and culture of cardiac fi broblasts 140 Asish K. Ghosh, Joseph W. Covington and Douglas E. Vaughan 17 Murine bone marrow transplantation model 146 Prasanna Krishnamurthy, Suresh Kumar Verma, and Raj Kishore 18 In vitro differentiation and expansion of vascular endothelial cells derived from mouse embryonic stem cells 149 Anees Fatima, Carey Nassano–Miller, and Tsutomu Kume Part 3 Manipulation of the Heart and Vessels in Vivo and ex Vivo 19 Coronary ligation 157 Alexander R. Mackie and Hossein Ardehali 20 Transverse aortic constriction: a model to study heart failure in small animals 164 Suresh Kumar Verma, Prasanna Krishnamurthy, and Raj Kishore 21 Pharmacological models of hypertrophy and failure 170 Angela C. deAlmeida, Tariq Hamid, and Sumanth D. Prabhu 22 Hindlimb ischemia 177 Jerry C. Lee, Ngan F. Huang, and John P. Cooke 23 The Langendorff preparation 187 Hugh Clements–Jewery and Michael J. Curtis 24 Myocarditis and other immunological models of cardiac disease 197 Daniela C¡ iháková and Noel R. Rose 25 Models of pacing–induced heart failure 203 James A. Shuman, Rupak Mukherjee, and Francis G. Spinale 26 Porcine myocardial ischemia models 215 Xian–Liang Tang and Roberto Bolli 27 Angiogenesis assays 225 Susmita Sahoo and Douglas W. Losordo 28 Immunohistochemical analysis of cardiac tissue 232 Barbara Ogórek, Donato Cappetta, and Jan Kajstura 29 A murine model of cardiac arrest by exsanguination 237 Guangming Cheng, Yiru Guo, Harold K. Elias, Carrie M. Quinn, Arash Davani, Yanjuan Yang, Magdy Girgis, Roberto Bolli, and Buddhadeb Dawn Part 4 Small Animal Imaging 30 Blood pressure, telemetry, and vascular measurements in the rodent model 245 Robert S. Danziger 31 The setting: imaging conscious, sedated, or anesthetized rodents 250 Gene H. Kim and Roberto M. Lang 32 Echocardiography: standard techniques (M–mode, two–dimensional imaging, and Doppler) 254 Gene H. Kim, Lauren Beussink–Nelson, Sanjiv J. Shah, and Roberto M. Lang 33 Echocardiography: advanced techniques (tissue Doppler, speckle tracking, and three–dimensional imaging) 275 Lauren Beussink–Nelson, Gene H. Kim, Roberto M. Lang, and Sanjiv J. Shah 34 In vivo tomographic cardiac imaging: positron emission tomography and magnetic resonance imaging 287 Bruno C. Huber, Patricia K. Nguyen, and Joseph C. Wu 35 In vivo hemodynamics 295 Alexander R. Mackie, Kyle K. Henderson, Sol Misener, and Hossein Ardehali Part 5 Metabolism, Mitochondria, and Cell Death 36 Fractionation of cardiomyocytes and isolation of mitochondria 307 Christopher P. Baines 37 Assessment of glucose and fatty acid metabolism ex vivo 313 Darrell D. Belke and E. Dale Abel 38 Quantification and characterization of atherosclerotic lesions in mice 320 Abhinav Agarwal, Millicent G. Winner, Srinivas D. Sithu, and Sanjay Srivastava 39 Assessment of cell death in the heart 332 Russell S. Whelan, Klitos Konstantinidis, and Richard N. Kitsis 40 Assessment of mitochondrial function in isolated cells 343 Amy K. Rines and Hossein Ardehali 41 Multinuclear NMR spectroscopy of myocardial energetics and substrate utilization in isolated perfused mouse hearts 351 Stephen C. Kolwicz, Jr. and Rong Tian 42 Measurement of reactive oxygen species in cardiovascular disease 359 Mahmood Khan, Fatemat Hassan, Sashwati Roy, and Chandan K. Sen 43 Assessing autophagy 371 Roberta A. Gottlieb 44 Assessment of cardiomyocyte size 378 A. Martin Gerdes and Alessandro Pingitore Part 6 Manipulation of Gene Expression in Vitro and in Vivo 45 Generation of Cre–loxP mouse models for conditional knockout and overexpression of genes in various heart cells 389 Marisa Z. Jackson and Warren G. Tourtellotte 46 Modulation of myocardial genes via use of adenoviral vectors and RNA interference approaches 397 Qianhong Li 47 Overexpression and downregulation of proteins in vitro 407 Marina Bayeva and Hossein Ardehali 48 In vivo microRNA studies 416 Eva van Rooij Part 7 Model Systems 49 Vascular and cardiac studies in zebrafi sh 425 Hans–Georg Simon, Molly Ahrens, and Brandon Holtrup 50 Vascular and cardiac studies in Drosophila 432 Lin Yu, Joseph P. Daniels, and Matthew J. Wolf Index 441

Edited by Hossein Ardehali MD, PhD, FAHA , Associate Professor of Medicine, Molecular Pharmacology and Biological Chemistry Feinberg Cardiovascular Research Institute Northwestern University Feinberg School of Medicine, Chicago, IL USA Roberto Bolli MD Professor of Medicine Chief, Division of Cardiovascular Medicine Director, Institute of Molecular Cardiology University of Louisville Louisville, KY USA Douglas W. Losordo MD Vice President New Therapeutic Development Baxter Healthcare Corporation Deerfield, IL USA