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 of rodents in studies of cardiac arrhythmias is limited by their small heart size and faster heart rate compared to human or large animal models. Therefore, spontaneous lethal arrhythmias in mice or rats after myocardial infarction are rare². Investigators are forced to focus on indirect secondary changes that might reflect a pro-arrhythmic substrate, such as fibrosis or gene expression, without showing meaningful changes in arrhythmia burden or pro-arrhythmic tendencies. To overcome this limitation, a method that allows a reliable assessment of the susceptibility of mouse and rat hearts to ventricular tachyarrhythmias after genetic modification^6,7 or myocardial infarction² is described in the present protocol. This method combines adrenergic receptor stimulation with programmed electrical stimulation to induce ventricular tachyarrhythmias in isolated, Langendorff-perfused⁸ mouse and rat hearts.

Standard approaches for viral gene transfer in rodent myocardial tissue often involve the exposure of the heart by thoracotomy^9,10,11, which is an invasive procedure and is associated with delayed recovery of the animals after the procedure. This article describes a method of direct intramyocardial injection of virus under ultrasound imaging guidance for the overexpression of transgenes. This less invasive procedure allows for faster animal recovery after viral injection and less tissue injury, as compared to thoracotomy, reduces post-operative pain and inflammation in the animal, and, thus, allows better assessment of the effects of transgenic genes on heart function.

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 from commercial sources (see Table of Materials). All the procedures dealing with animals were performed by staff who have been trained and approved by institutional regulatory committees. Appropriate personal protective equipment was used during all procedures.

1. Viral transgene expression in rodent ventricular tissues

NOTE: Store recombinant adenovirus or AAV that expresses the target gene and the corresponding control virus, such as Ad-GFP (titer of 1 x 10¹⁰ PFU/mL) or AAV9-GFP (titer of 1 x 10¹³ GC/mL) (see Table of Materials), in a −80 °C freezer.

1. On the day of virus injection, thaw the virus on ice. Aspirate the virus expressing the target gene and the control virus into two separate syringes (volume = 50 µL) with 30 G 1/2 needles and keep on ice until use.
   NOTE: Any bubbles in the syringe and needle must be carefully removed.
2. Administer buprenorphine to rats or mice (0.05 mg/kg for rats, 0.1 mg/kg for mice, subcutaneous), 1 h later induce anesthesia using 3% isoflurane, and maintain isoflurane at 2% (in 100% oxygen at a flow rate of 0.5-1.0 L/min) for the subsequent procedure.
3. Gently pinch the animal's body (e.g., the tail) with a pair of forceps, and if the animal does not respond to the pinch with any body movements, proper anesthetization is confirmed.
4. Maintain body temperature at 37 °C using an electric heating pad. Shave the hair in the chest region using a hair clipper.
   NOTE: Care should be taken while using an electric heating pad due to potential uneven heat distribution.
5. Apply ophthalmic ointment to both eyes to prevent the drying of the cornea.
6. Keep the animal in a supine position and use a preclinical imaging system (see Table of Materials) for ultrasound imaging of the animal's heart in the short-axis orientation.
   NOTE: The following steps are performed in a level II biosafety cabinet which provides a sterile environment. Standard safety procedures, including those with safe needle handling, are employed.
7. Sterilize the left lower chest region of the animal with alternating rounds of an iodine-based or chlorhexidine-based scrub and alcohol three times in a circular motion.
8. Under ultrasound imaging guidance, insert the 30 G 1/2 needle of the syringe containing the virus as prepared in step 1.1. into the animal's chest via the left lower chest side of the body.
9. Approach the needle tip into the left ventricular front free wall and slowly inject 10-15 μL of the virus. Verify successful injection in ultrasound images by the enhanced brightness near the tip of the needle.
   NOTE: The amount of virus injected at each site is no more than 15 μL to prevent physical damage to the myocardial tissues.
10. Withdraw the needle from the heart and insert it into other regions of the left ventricle for a second and third injection of the same amount of virus.
    NOTE: The total number of injection sites is determined by the experimental design. If focal transgene expression is required, only one injection is needed; if more diffusive transgene expression is required, multiple injection sites (three to five) are usually needed.
11. After the completion of the injections, return the animal to its cage.
12. Carefully monitor the animal until it has regained sufficient consciousness to maintain sternal recumbency and return it to its housing room.
13. Administer buprenorphine HCl (0.1 mg/kg, twice a day for mice, 0.05 mg/kg, twice a day for rats, subcutaneous) to the animal for the following 2 days. Return animals to the vivarium and monitor daily for any unusual signs of pain or distress, and if found, treat animals per the Institutional animal care committee protocols.

2. Assessment of cardiac arrhythmia susceptibility

NOTE: At 4-6 days after adenovirus injection and 1-2 weeks after AAV injection, assess the susceptibility of the animal hearts to cardiac arrhythmias following steps 2.1.-2.2.

1. Perform Langendorff perfusion of the rat or mouse heart.
   1. Prepare Tyrode solution by adding the following to 1 L of water: NaCl, 7.9 g (final = 135 mM); KCl, 0.37 g (5.0 mM); CaCl₂, 0.27 g (1.8 mM); MgCl₂, 0.24 g (1.2 mM); HEPES, 2.38 g (10 mM); and glucose, 1.8 g (10 mM). Adjust the pH to 7.40 with NaOH (see Table of Materials).
   2. Filter the solution with a 0.2 μm filter and bubble with 100% O₂ continuously during steps 2.1.6.-2.2.8.
   3. Administer heparin to the rat or mouse (500 U/kg, intraperitoneal injection) and 10 min later anesthetize the animal with 3% isoflurane. Ensure adequate anesthetization with a gentle pinch on the body.
   4. Use a scalpel to make a 1-1.5 cm vertical incision in the mid-clavicular line for a left thoracotomy and expose the heart.
   5. Collect the heart by cutting with a pair of scissors at the aortic arch level and immediately put the heart into ice-cold Tyrode solution.
      ​NOTE: Caution is exercised not to damage the heart during heart collection.
   6. Cannulate the aorta of the heart with a blunt needle (18 G for rats; 23 G for mice) connected to a modified Langendorff perfusion system (see Table of Materials) in the constant flow rate mode. Adjust the flow rate to keep the perfusion pressure at 70-80 mmHg.
      NOTE: Any bubbles in the perfusion needle must be removed before aortic cannulation. Using a dissecting microscope facilitates the cannulation.
   7. Place the cannulated heart in a silicone elastomer-coated⁹, 10 cm plastic dish with the left ventricle facing up and perfuse the heart^9,12 with O₂-bubbled Tyrode solution at 37 °C.
   8. Verify the correct aortic cannulation at the beginning of perfusion by observing the washout of blood from the heart during the first two to three heartbeats and the changing of the heart color from red to pale.
   9. Place the electrodes of a small animal ECG system (see Table of Materials) around the heart by inserting them into the silicone elastomer coating in the dish (Figure 1). Record ECG using compatible software.
2. Perform adrenergic receptor stimulation and programmed electrical stimulation to induce ventricular tachyarrhythmias.
   1. After successful aortic cannulation, continue to perfuse the heart with Tyrode solution for 20 min to stabilize the heart preparation.
   2. Add 1 μM isoproterenol (see Table of Materials) to the Tyrode solution used for heart perfusion in steps 2.2.3.-2.2.8.
   3. After 10 min of isoproterenol perfusion, stimulate the heart at the apex with two platinum electrodes connected to an electrical stimulator (see Table of Materials).
   4. Start with the stimulation procedure (Figure 2), which includes 10 consecutive stimuli (S1, 5 V, 100 ms intervals) that are followed by an extra stimulus (S2) with an initial interval of 80 ms. Repeatedly reduce the S2 interval by 2 ms each time until the heartbeat can no longer be captured (i.e., reaching the heart's effective refractory period, ERP)¹³.
   5. Monitor any induced ventricular tachyarrhythmias (including ventricular tachycardia and fibrillation) by ECG.
   6. If no arrhythmias are induced by the above procedure, add another extra stimulus (S3) after S2 with the same initial (80 ms) and decrement (by 2 ms) intervals until the ERP is reached.
   7. If ventricular tachyarrhythmias are still not induced, add one more extra stimulus (S4) after S3 with the same initial and decrement intervals until the ERP is reached.
   8. If ventricular tachyarrhythmias are still not induced (as expected in control healthy hearts), stop the electrical stimulation protocol and consider the heart to be non-inducible.

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 occurs ...

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

List of materials used in this article

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