Name: viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk
Uploaded: 2022-07-27T14:10:00
Description: Watch this Scientific Journal Video about Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk at JoVE.com

This work was supported by the Canadian Institutes of Health Research (CIHR) Project Grants (PJT-148918 and PJT-180533, to WL), the CIHR Early Career Investigator Award (AR8-162705, to WL), the Heart and Stroke Foundation of Canada (HSFC) McDonald Scholarship and New Investigator Award (S-17-LI-0866, to WL), Student Scholarships (to JW and YX), and a Postdoctoral Fellowship (to AL) from the University of Ottawa Cardiac Endowment Funds at the Heart Institute. The authors thank Mr. Richard Seymour for his technical support. Figure 2 was created with Biorender.com with approved licenses.

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. Ctnnb1flox/flox and &#945;M...

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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&#8212;ideally, the tip of the...

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 modification6,7 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 thoracotomy9,10,11, 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&#160;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&#160;transgenic genes on heart function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G 1/2 PrecisionGlide Needle</td>
			<td>Becton Dickinson (BD)</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>adeno-associated virus (AAV9-GFP)</td>
			<td>Vector Biolabs</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>adenovirus (Ad-GFP)</td>
			<td>Vector Biolabs</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>adenovirus (Ad-Wnt3a)</td>
			<td>Vector Biolabs</td>
			<td>ADV-276318</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet (Level II)</td>
			<td>Microzone Corporation</td>
			<td>N/A</td>
			<td>Model #: BK-2-4</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Vetergesic</td>
			<td>DIN 02342510</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>102378</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher Chemical</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>WAHL Clipper Corporation</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Sigma-Aldrich</td>
			<td>20701</td>
			<td>705 LT, volume 50 &#956;L</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Life Brand</td>
			<td>E12107</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fresenius Kabi</td>
			<td>DIN 02264315</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fresenius Kabi Ltd.</td>
			<td>M60303</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>1351005</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart 8 software</td>
			<td>ADInstruments Inc.</td>
			<td>Version 8.1.5</td>
			<td>for ECG recording</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice (Ctnnb1flox/flox)</td>
			<td>Jackson Labs</td>
			<td>4152</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice (&#945;MHC-MerCreMer)</td>
			<td>Jackson Labs</td>
			<td>5650</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>S9i</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>MS400 transducer</td>
			<td>VisualSonic Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Systane</td>
			<td>DIN 02444062</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure meter</td>
			<td>NETECH</td>
			<td>DigiMano 1000</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>Cole-Parmer</td>
			<td>UZ-77924-65</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Rat (Sprague-Dawley, male)</td>
			<td>Charles River</td>
			<td>400</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10007-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>Down Inc.</td>
			<td>Sylgard 184</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Small animal ECG system</td>
			<td>ADInstruments Inc.</td>
			<td>N/A</td>
			<td>Powerlab 8/35 and Animal Bio Amp</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>567530</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>IonOptix</td>
			<td>MyoPacer EP</td>
			<td></td>
		</tr>
		<tr>
			<td>VEVO3100 Preclinical Imaging System</td>
			<td>VisualSonic Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;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&#160;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.

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&#160;which oc...

Watch this Scientific Journal Video about Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk at JoVE.com

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

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.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

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