Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Summary

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

Abstract

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

Introduction

Cardiovascular disease is the leading cause of death worldwide, claiming the lives of 18 million people in 2017 alone¹. Rodents, especially mice and rats, have become the most commonly used model in cardiovascular research due to the ease of handling and the availability of various transgenic overexpression or knockout lines. Rodent models have been fundamental for understanding the disease mechanisms and for identifying potential new therapeutic targets in myocardial infarction², hypertension³, heart failure⁴, and atherosclerosis⁵. However, the use ....

Protocol

All the methods and procedures described were approved by the animal research ethical review board at the University of Ottawa and the biosafety review committee at the University of Ottawa Heart Institute. The developed safety protocols include that all the procedures dealing with recombinant adenovirus or adeno-associated virus (AAV) were performed in a level II biosafety cabinet. All the items in contact with the virus were thoroughly decontaminated after the experiment. Ctnnb1^flox/flox and αMHC-MerCreMer mice (8-12 weeks old, of either sex) and Sprague-Dawley rats (200-250g, male) were used for the present study. The animals were obtained ....

Results

When perfused following the protocol described here (Figure 1), an isolated rat or mouse heart beats rhythmically and stably for at least 4 h. If the experimental design requires a longer period of heart perfusion, it is helpful to add albumin into the perfusion solution to reduce the occurrence of myocardial edema after long-time perfusion¹⁴. The inclusion of isoproterenol in the perfusion solution mimics the activation of the sympathetic nervous system which oc.......

Discussion

Several steps are critical for the success of the Langendorff-perfused, isolated heart preparation. Firstly, it is important to avoid any damage to the heart during heart collection (e.g., due to accidental squeezing or cutting with the scissors). Secondly, it is critical to put the collected heart into cold Tyrode solution as soon as possible because this will stop the heartbeat and reduce the oxygen consumption of the heart. Thirdly, the needle insertion into the aorta must not be too deep—ideally, the tip of the.......

Materials

Name	Company	Catalog Number	Comments
30 G 1/2 PrecisionGlide Needle	Becton Dickinson (BD)	305106
adeno-associated virus (AAV9-GFP)	Vector Biolabs	7007
adenovirus (Ad-GFP)	Vector Biolabs	1060
adenovirus (Ad-Wnt3a)	Vector Biolabs	ADV-276318
Biosafety cabinet (Level II)	Microzone Corporation	N/A	Model #: BK-2-4
Buprenorphine	Vetergesic	DIN 02342510
Calcium Chloride	Sigma-Aldrich	102378
D-Glucose	Fisher Chemical	D16-1
Hair clipper	WAHL Clipper Corporation	78001
Hamilton syringe	Sigma-Aldrich	20701	705 LT, volume 50 μL
Heating pad	Life Brand	E12107
Heparin	Fresenius Kabi	DIN 02264315
HEPES	Sigma-Aldrich	H4034
Isoflurane	Fresenius Kabi Ltd.	M60303
Isoproterenol hydrochloride	Sigma-Aldrich	1351005
LabChart 8 software	ADInstruments Inc.	Version 8.1.5	for ECG recording
Magnesium chloride hexahydrate	Sigma-Aldrich	M2393
Mice (Ctnnb1flox/flox)	Jackson Labs	4152
Mice (αMHC-MerCreMer)	Jackson Labs	5650
Microscope	Leica	S9i	for Langendorff system
MS400 transducer	VisualSonic Inc.	N/A
Ophthalmic ointment	Systane	DIN 02444062
Potassium Chloride (KCl)	Sigma-Aldrich	P9541
Pressure meter	NETECH	DigiMano 1000	for Langendorff system
Pump	Cole-Parmer	UZ-77924-65	for Langendorff system
Rat (Sprague-Dawley, male)	Charles River	400
Scalpel blades	Fine Science Tools	10010-00
Scalpel handle	Fine Science Tools	10007-12
Silicone elastomer	Down Inc.	Sylgard 184	for Langendorff system
Small animal ECG system	ADInstruments Inc.	N/A	Powerlab 8/35 and Animal Bio Amp
Sodium Chloride	Sigma-Aldrich	S7653
Sodium Hydroxide	Sigma-Aldrich	567530
Stimulator	IonOptix	MyoPacer EP
VEVO3100 Preclinical Imaging System	VisualSonic Inc.	N/A