Cover -- Dedication -- Title page -- Copyright page -- Contents -- List of Contributors -- Preface -- About the Companion Website -- Part 1: Electrophysiology -- 1: Measurement of calcium transient ex vivo -- Introduction -- Preparation and fluorescent indicator loading -- Optical setup -- Calibration -- Analysis and interpretation -- Set-by-step procedure -- References -- 2: Confocal imaging of intracellular calcium cycling in isolated cardiac myocytes -- Introduction -- Materials and methods -- Chamber, electrical stimulation, and superfusate delivery apparatus -- Confocal microscope system -- Cell loading of fluorescent Ca2+ indicators -- Experimental approach -- Data analyses -- Weaknesses and strengths of the method and alternative approaches -- Conclusions -- References -- 3: Generating a large animal model of persistent atrial fibrillation -- Introduction -- Insights from tachypacing-induced AF models -- Protocol -- Generating a tachypacing-induced AF model -- Pacing protocols -- Ventricular rate during AF: to ablate or not to ablate -- Strengths and weaknesses of the model -- Complications and alternative approaches to study AF -- Potential complications -- Alternative AF models -- Conclusion -- Acknowledgement -- References -- 4: Confocal imaging of intracellular calcium cycling in the intact heart -- Introduction -- Measurement of intracellular Ca2+ transients -- Principles and features of confocal microscopy -- Procedure -- Materials and equipment -- Experimental setup -- Limitations, potential pitfalls and alternative approaches -- Additional application: use of confocal imaging to determine efficacy of stem cell integration into host myocardium -- References -- 5: Recording and measurement of action potentials -- Introduction -- Protocol -- Theory of AP recording -- Procedure -- Recording amplifier.

Choices to make before an experiment -- AP recording procedure -- Frequent problems -- AP analysis -- Alternative approaches and extensions -- Weaknesses and strengths -- Conclusion -- References -- 6: Patch-clamp recordings from isolated cardiac myocytes -- Introduction -- Theory of patch-clamp recording -- Cell-attached configuration -- Whole-cell recording configuration -- Inside-out excised patch configuration -- Outside-out excised patch configuration -- Instrumentation for patch-clamp recordings -- Experimental aspects of patch-clamp recordings -- Gigaseal formation -- Whole-cell recordings -- Whole-cell voltage-clamp protocols, data acquisition, and analysis -- Conclusions and future directions -- References -- 7: Optical mapping of the heart -- Introduction -- Protocols -- Experimental set-up -- Tissue preparations (Figure 7.2) -- Staining and image acquisition -- Data processing and analysis -- Representative results -- Alternative approaches -- Weaknesses and strengths of method -- Conclusions -- References -- Part 2: Isolation and Maintenance of Primary Stem Cells -- 8: Isolation of colony-forming endothelial progenitor cells -- Introduction -- Materials -- Laboratory reagents -- Laboratory equipment -- Methods -- MNC isolation from fresh PB or UCB -- EPC-CFA of bulk cell populations in PB, GmPB, BM, or UCB -- Optional-1: EPC-CFA of single CD34+ or CD133+ cells in PB, GmPB, BM, or UCB -- Optional-2: HELIC assay of single CD34+ or CD133+ cells as cell fate assay of HSC -- Application -- Acknowledgements -- Competing interests statement -- References -- 9: Cardiac resident stem cells -- Introduction -- Protocol -- Preparation of basic solutions and culture medium -- Enzymatic digestion of the mouse heart -- Separation of small cells -- Sorting c-kit-positive cells using FACS -- Alternative approaches -- Weaknesses and strength of the method.

Conclusion -- Acknowledgements -- References -- 10: Cardiospheres -- Introduction -- Protocols -- Preparation for explant creation -- Explant creation -- Preparation for cardiosphere formation -- Cardiosphere formation -- Alternative approaches -- Weaknesses and strengths of the method -- Conclusions -- References -- 11: Mesenchymal stem cells -- Introduction -- Protocols -- Isolation of human MSCs from BM aspirates -- Conclusions -- Acknowledgements -- References -- 12: Generation and differentiation of human iPS cells -- Introduction -- Protocol I: Generation of hiPSC from fibroblasts -- Production of the VSV-G Lentivirus (6 days) -- Reprogramming of human fibroblasts (∼14 days) -- Plasmids and media -- Titration of the virus -- Protocol II: differentiation hiPSCs to myocytes -- Future perspectives -- References -- 13: Isolation of neonatal and adult rat cardiomyocytes -- Introduction -- Protocols -- Isolation of neonatal rat ventricular cardiomyocytes -- Isolation of adult rat cardiomyocytes -- Alternative approaches -- Weaknesses and strengths of the method -- Conclusions -- References -- 14: Isolation and culture of vascular smooth muscle cells -- Introduction -- Protocol -- Reagents -- Equipment -- Reagent setup -- Preparation and equipment setup -- Procedure -- Alternative approaches -- Explant method -- Strengths and weaknesses of method -- Conclusion -- References -- 15: Isolation and culture of cardiac endothelial cells -- Introduction -- Protocol for isolation of mouse cardiac endothelial cells -- Materials and preparations -- Procedures -- Important notes -- Alternative approaches -- A novel protocol of endothelial cell isolation -- Conclusions -- Acknowledgements -- References -- 16: Isolation and culture of cardiac fibroblasts -- Introduction -- Cardiac fibroblasts isolation protocol -- Materials and preparations.

Procedures for isolation of mouse cardiac fibroblasts -- Cell propagation and maintenance -- Alternative approaches -- Isolation of cardiac ventricular fibroblasts from rats -- Conclusions -- References -- 17: Murine bone marrow transplantation model -- Introduction -- Materials -- Procedure -- Animal husbandry -- Alternative approaches -- Weaknesses and strengths of the method -- References -- 18: In vitro differentiation and expansion of vascular endothelial cells derived from mouse embryonic stem cells -- Introduction -- Materials -- Tissue culture -- Methods -- ES-cell culture (see Notes 1 and 2) -- Differentiation of ES-cells to Flk-1+ progenitors -- Purification of Flk-1+ cells -- Expansion/differentiation of Flk-1+ cells -- Notes -- Acknowledgements -- References -- Part 3: Manipulation of the Heart and Vessels in Vivo and ex Vivo -- 19: Coronary ligation -- Introduction -- Protocol -- Alternative approaches -- Weaknesses and strengths of the method -- Conclusions -- Acknowledgments -- References -- 20: Transverse aortic constriction: a model to study heart failure in small animals -- Introduction -- Material -- Procedure -- Preparation of the operative field -- Anesthesia, intubation, and ventilation -- Aortic banding (constriction of transverse aorta) -- Postoperative care -- Notes -- Limitations -- Confirmation of constriction -- Expected outcomes -- Alternative approaches -- Weaknesses and strengths of the method -- Conclusions -- References -- 21: Pharmacological models of hypertrophy and failure -- Overview -- Protocols -- Alternative approaches -- Strengths and weaknesses of the method -- Conclusions -- References -- 22: Hindlimb ischemia -- Introduction -- Protocol -- Induction of anesthesia -- Hair removal prior to surgery -- Surgery preparation -- Femoral artery ligation -- Laser Doppler -- Alternative methods.

Common considerations -- Acknowledgements -- References -- 23: The Langendorff preparation -- Introduction -- Protocol -- Cannulation of the aorta -- Perfusion solution constituents, thermoregulation, and mode of delivery -- Recording of left ventricular pressure, ECG, and coronary flow -- Alternative approaches -- Weaknesses and strengths of the method -- Conclusions -- References -- 24: Myocarditis and other immunological models of cardiac disease -- Introduction -- Protocol -- Induction of EAM -- Notes -- Alternative approaches -- Strength and weaknesses of the method -- Conclusions -- Acknowledgement -- References -- 25: Models of pacing-induced heart failure -- Introduction -- Rapid pacing models - protocols -- Functional and hemodynamic expectations -- Neurohormonal aspects and expectations -- Cellular and biochemical considerations to chronic rapid pacing -- Matrix remodeling and chronic rapid pacing -- Electrophysiology and arrhythmogenesis in chronic rapid pacing -- Chronic rapid pacing-induced heart failure: the issue of reversibility -- Chronic pacing model for therapeutic research and development: technical considerations and limitations -- Summary -- Acknowledgements -- References -- 26: Porcine myocardial ischemia models -- Introduction -- Methods -- Anesthetized pig model with an angioplasty balloon-induced occlusion -- Conscious pig model with an extravascular balloon-induced occlusion -- Conclusion -- Acknowledgements -- References -- 27: Angiogenesis assays -- Introduction -- In vivo Matrigel plug assay -- Preparation of reagents and animals -- Reagents for quantification of angiogenesis -- Equipment -- Matrigel mixtures and injections -- Matrigel harvest and analysis -- Notes -- Mouse corneal micropocket angiogenesis assay -- Preparation of reagents and animals -- Equipment -- Preparation of controlled slow-release pellet.

Surgical procedure.