Experimental Models of Cardiovascular Diseases: Methods and Protocols (Methods in Molecular Biology, 2803)

Preface
Contents
Contributors
Part I: In Vitro Models
	Chapter 1: Decellularized Extracellular Matrix for Modeling Cardiac Extracellular Microenvironment
		1 Introduction
		2 Materials
			2.1 Equipment and Glassware
			2.2 Plasticware and Other Disposables
			2.3 Antibiotic and Washing Solutions
			2.4 Decellularization Solutions
		3 Methods
			3.1 Preparation of Cardiac Samples
			3.2 Decellularization
			3.3 Cell Seeding and Tissue Culture
		4 Notes
		References
	Chapter 2: Subtype and Lineage-Mediated Protocol for Standardizing Activin/Nodal and BMP Signaling for hiPSC-Derived Cardiomyo...
		1 Introduction
		2 Materials
			2.1 Maintenance and Culture of Human-Induced Pluripotent Stem Cells (hiPSCs) (Feeder-Free)
			2.2 Stage-Specific Standardization of Activin/Nodal and BMP Signaling During Ventricular Cardiomyocyte Differentiation
			2.3 Cell Dissociation and Preparatory Reagents for ``Day 0´´ of the Cardio Myocyte Differentiation Protocol
			2.4 Cell Dissociation and Preparatory Reagents for ``Day 4´´ Mesoderm and Pre-Cardiac Mesoderm Markers´ Analysis by Celesta Fl...
			2.5 Cardiomyocyte Differentiation
			2.6 Cell Dissociation and Preparatory Reagents for Matured Cardiomyocyte Sorting and Subsequent Collection After Day 25 or 30 ...
			2.7 Evaluation of the hiPSC-Derived Progenitor and Matured FACS-Sorted CMs by Immunostaining Using Confocal Microscopy
			2.8 Characterization of the hiPSCs for Pluripotency Markers by Immunostaining Using Confocal Microscopy
		3 Methods
			3.1 Maintenance and Culture of hiPSCs (Feeder-Free)
				3.1.1 Diluting Matrigel
				3.1.2 Coating Sterile Cell Culture Treated Multiwell Plates (e.g., 6-Well Plates) with Extracellular Matrix (Matrigel)
				3.1.3 Plating hiPSCs from Frozen Vial (Feeder-Free)
				3.1.4 The hiPSCs Expanding/Passaging and Maintenance (See Note 5)
			3.2 Stage-Specific Standardization of Activin/Nodal and BMP Signaling During Ventricular Cardiomyocyte Differentiation (Starti...
				3.2.1 Generation of the EBs (Day 0)
				3.2.2 Day 1 of the Ventricular Cardiomyocyte Differentiation: The Developmental Stage Primitive Streak/Induction of the Germ L...
				3.2.3 Day 3 of the Ventricular Cardiomyocyte Differentiation: The Developmental Stage-Induction of the Cardiac Mesoderm
				3.2.4 Embryoid Bodies´ Dissociation on ``Day 4´´ for Mesoderm (CD56) and Pre-Cardiac Mesoderm (CD13) Markers´ Analysis by Cele...
			3.3 Day 5 to Day 7 of the Ventricular Cardiomyocyte Differentiation (Starting from Day 5 Until Day 7)
			3.4 Day 9 of the Ventricular Cardiomyocyte Differentiation (Starting from Day 9 Until Day 25 or 30) (See Note 19)
			3.5 Characterization of the Differentiated CMs Using Live Stain Markers for Cardiomyocyte (SIRPA) and Fibroblasts (CD90) Witho...
			3.6 Evaluation of the iPSC-Derived Progenitor and Matured FACS-Sorted CMs by Immunocytochemistry (Fig. 4b, c)
				3.6.1 Fixing
				3.6.2 Staining
				3.6.3 Preprocessing for Imaging
		4 Notes
		References
	Chapter 3: Generation of 3D-Multicellular Human iPSC-Heart Organoids for the Noninvasive Assessment of Cardiac Fibrosis
		1 Introduction
			1.1 Pathological Cardiac Fibrosis and Myofibroblast Transformation
			1.2 From Human-Induced Pluripotent Stem Cells to Human Heart Organoids
		2 Materials
			2.1 Cell Culture
		3 Methods
			3.1 Generation of Beating Human Heart Organoids (hHOs) from Human iPS Cells
			3.2 Activation of the TGFβ-Dependent Pro-Fibrotic Response in  hHOs
			3.3 Noninvasive Assessment of Cardiac Fibrosis by Organoid Immunocytochemistry
			3.4 Image Analysis
		4 Notes
		References
	Chapter 4: In Vitro Experimental Approach for Studying Human Pulmonary Artery Smooth Muscle Cells and Endothelial Cells Prolif...
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Cell Splitting and Plating
			2.3 Cell Freezing
			2.4 Cell Proliferation ELISA
			2.5 Transwell Migration Assay
		3 Methods
			3.1 Thawing and Plating hPASMC/hPAEC
			3.2 Splitting and Plating Cells
			3.3 Cell Freezing
			3.4 Cell Proliferation
			3.5 Cell Migration Assay
		4 Notes
		References
Part II: Ex Vivo Models
	Chapter 5: Culturing Cardiac Tissue Slices Under Continuous Physiological Mechanical Stretches
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Preparation of Heart Slices
			2.3 CTCM Device and Equipment
		3 Methods
			3.1 Incubator Setup
			3.2 LabChart Setup and Pressure Probe Calibration (See Fig. 1)
			3.3 Heart Tissue Slicing (See Note 5)
			3.4 Assembling CTCM Device (See Note 13; Fig. 2)
			3.5 Adhering Tissue Slices to Support Rings and Inserting into CTCM (See Fig. 3)
			3.6 Connecting the CTCM Devices to the Pneumatic Driver and C-Pace
			3.7 Media Change and Tissue Collection (See Notes 29 and 30)
		4 Notes
		References
	Chapter 6: Quantitative Assessment of Mitochondrial Morphology and Electrophysiological Function in the Diabetic Heart
		1 Introduction
		2 Materials
			2.1 Mouse Strains
			2.2 Heart Extraction and Cannulation for High-Resolution Optical Action Potential (AP) Mapping
			2.3 High-Resolution Cardiac AP Optical Mapping
			2.4 Left Ventricular Samples Preparation for Electron Microscopy (EM) Imaging
			2.5 EM Images Analysis
		3 Methods
			3.1 Retrograde-Perfusion via the Aorta for High-Resolution AP Optical Mapping
			3.2 High-Resolution Cardiac AP Optical Mapping
			3.3 Left Ventricular Samples Preparation for EM Imaging
			3.4 Analysis of Mitochondrial Morphology
		4 Notes
		References
	Chapter 7: Ex Vivo Working Porcine Heart Model
		1 Introduction
			1.1 Working Heart Model Overview
			1.2 Working Heart Model Objectives
			1.3 Establishing Cardiac Afterload
			1.4 Actively Controlled Afterload
		2 Materials
			2.1 Measurement
			2.2 Sedation and Anesthesia
			2.3 Ex Vivo Working Heart Setup
		3 Methods
			3.1 Preparation of the Ex Vivo Working Heart Setup
			3.2 Preparation of the Pig Heart
			3.3 Ex Vivo Working Heart
		4 Notes
		References
Part III: Small Animal Models
	Chapter 8: Conventional Method of Cardiac Ischemia/Reperfusion Injury in Mice
		1 Introduction
		2 Materials
			2.1 Animal
			2.2 Site Preparation
			2.3 Surgery
			2.4 Echocardiography
		3 Methods
			3.1 Presurgical Preparation
			3.2 Preparation of Mice (Anesthesia, Intubation, and Ventilation)
			3.3 LAD Ligation and the Direct Myocardial Injection of Therapeutic Agent
			3.4 Reperfusion
			3.5 Evaluating the Systolic Function of the Heart
		4 Notes
		References
	Chapter 9: Rat Model of Isoproterenol-Induced Myocardial Injury
		1 Introduction
			1.1 Unveiling Cardiac Consequences of Isoprotrenol (ISO) Administration
			1.2 Mechanism of ISO-Induced Myocardial Injury
		2 Materials
			2.1 Reagents
			2.2 Surgical Tool Kit
			2.3 Animal Selection and Housing
			2.4 Anesthesia and Anesthetic Systems
			2.5 ECG Analysis
			2.6 Echocardiography
			2.7 Isolated Heart Langendorff Technique
			2.8 Whole Heart Homogenate Preparation
			2.9 Triphenyl Tetrazolium Chloride (TTC) Powder
			2.10 Common Instrumentations
		3 Methods
			3.1 Animal Preparation Before Conducting the Experiment
			3.2 ISO Preparation Based Upon Chronicity and Severity of Myocardial Injury
			3.3 Experimental Grouping of Rats
			3.4 Isoproterenol Administration
			3.5 Monitoring
			3.6 Electrocardiography (ECG) Analysis
			3.7 Echocardiography Analysis
			3.8 Blood Collection and Plasma Separation
			3.9 Sacrifice and Tissue Collection
			3.10 Ex Vivo Cardiac Functional Performance
			3.11 Hypertrophy Assessment
			3.12 Evaluation of Infarct Size and Tissue Viability
		4 Notes
		References
	Chapter 10: Pop-Out Myocardial Infarction Induction in Mice
		1 Introduction
		2 Materials
			2.1 Sterilization Supplies
			2.2 Surgical Room Setup
			2.3 Surgical Tools
			2.4 Analgesia and Stress Management
		3 Methods
			3.1 Preoperative Preparation and Surgical Area Setup
			3.2 Intubation
			3.3 Thoracotomy
			3.4 Post-Op  Care
		4 Notes
		References
	Chapter 11: Rat Models of Cardiorenal Syndrome and Methods for Functional Assessment
		1 Introduction
		2 Materials
			2.1 MI Surgery
			2.2 Surgical Instruments for MI Autoclaved Prior to Surgery
			2.3 Assessing Cardiac Function
				2.3.1 Echocardiography
				2.3.2 Cardiac Catheterization for Pressure-Volume Loops
			2.4 STNx Surgery
			2.5 Surgical Instruments for STNx Autoclaved Prior to Surgery
			2.6 Assessing Renal Function
				2.6.1 Creatinine and Proteinuria
				2.6.2 Glomerular Filtration Rate
		3 Methods
			3.1 MI Surgery: Permanent Ligation of the Left Coronary Artery
				3.1.1 Anesthesia
				3.1.2 Intubation
				3.1.3 Thoracotomy
				3.1.4 Ligation of the LCA and Chest Closure
			3.2 STNx Surgery: 5/6 Subtotal Nephrectomy
				3.2.1 Preoperative  Care
				3.2.2 Anesthesia
				3.2.3 STNx Surgery
				3.2.4 Wound Closure and Recovery
				3.2.5 Postoperative  Care
			3.3 Assessment of Cardiac Function
				3.3.1 Echocardiography (Echo)
				3.3.2 Pressure-Volume Loops (PV Loops)
			3.4 Assessment for Renal Function
				3.4.1 Collection of Blood and Urine for Creatinine and Proteinuria Measurements
				3.4.2 GFR Measurements
			3.5 Tissue Collection
		4 Notes
		References
	Chapter 12: The Sugen/Hypoxia Rat Model for Pulmonary Hypertension and Right Heart Failure
		1 Introduction
		2 Materials
			2.1 Animal and Housing
			2.2 Reagents and Equipment
		3 Methods
			3.1 Experimental Model
			3.2 Hemodynamic Measurement for PA and RV Function
				3.2.1 Preparation
				3.2.2 Hemodynamic Measurements
		4 Notes
		References
	Chapter 13: Unilateral Lung Removal in Combination with Monocrotaline or SU5416 in Rodents: A Reliable Model to Mimic the Path...
		1 Introduction
		2 Materials
			2.1 Animal Preparation and Anesthesia
			2.2 PH Model Establishment
			2.3 PH Functional Assessment
		3 Methods
			3.1 Anesthesia, Intubation, and Ventilation
			3.2 Preparation of Sterile Environment
			3.3 Unilateral Lung Removal
			3.4 Postoperative Recovery
			3.5 Monocrotaline Injection 1 Week After Unilateral Lung Removal
			3.6 Sugen (SU5416) Injection 1 Week After Unilateral Lung Removal
			3.7 PH Model Evaluation
				3.7.1 Pulmonary Artery Pressure Measurement at Terminal Time Point
				3.7.2 Lung Tissue Histology
		4 Notes
		References
Part IV: Large Animal Models
	Chapter 14: Swine Model of Myocardial Infarction Induced by Ischemia-Reperfusion and Embolization
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Drugs
		3 Methods
			3.1 Preparation and Anesthesia
			3.2 Baseline Cardiac Assessment with Echocardiography (See Note 8)
			3.3 Induction of Myocardial Infarction
		4 Notes
		References
	Chapter 15: Porcine Model of Hypertrophy-Independent Left Ventricular Stiffening via Repetitive Pressure Overload
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Sterile Placement of Indwelling Jugular Vein Catheter
			2.3 Transient Pressure Overload via Phenylephrine Infusion
			2.4 Physiologic Studies
		3 Methods
			3.1 Construction of Modified Angiocatheter
			3.2 Sterile Placement of Indwelling Jugular Vein Catheter
			3.3 Repetitive Pressure Overload via Phenylephrine Infusion
			3.4 Physiologic Studies
		4 Notes
		References
Chapter 16: Swine Model of Coronary Artery Dissection
	1 Introduction
	2 Materials
	3 Methods
		3.1 CAD with Wire Injury
		3.2 CAD with Guiding Catheter Injury
	4 Notes
	References
Chapter 17: Hypoxia-Induced Piglet Model of Cardiac Arrest with Assisted Resuscitation by Extracorporeal Membrane Oxygenation
	1 Introduction
	2 Materials
		2.1 Animal Preparation and Anesthesia
		2.2 Hypoxia Model Establishment
		2.3 Assisted Resuscitation with ECMO After Cardiac Arrest
	3 Methods
		3.1 Anesthesia, Intubation, and Ventilation
		3.2 Creation of Hypoxia-Induced Cardiac Arrest
		3.3 Assisted Resuscitation with ECMO
	4 Notes
	References
Chapter 18: A Dynamic Sheep Model to Induce Pulmonary Hypertension and Right Ventricular Failure
	1 Introduction
	2 Materials
		2.1 Medications and Fluids
		2.2 Surgical Instruments and Equipment
		2.3 Testing and Monitoring Equipment
	3 Methods
		3.1 Animal Preparation Prior to Surgery
		3.2 Day of Surgery
		3.3 Day of Surgery, Operating Suite
		3.4 Operative Procedure
		3.5 Postoperative Recovery
		3.6 Chronic PA Banding (Postoperative Weeks: 1-10 Weeks)
		3.7 Conduct Weekly Exercise Sessions
		3.8 Invasive Interval Model Analysis, and Reoperative Surgery
	4 Notes
	References
Index