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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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics-2020+update:+A+report+from+the+American+Heart+Association.">Heart disease and stroke statistics-2020 update: A report from the American Heart Association.</a> Circulation. 141 (9), 139 (2020).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte-specific+deletion+of+&#946;-catenin+protects+mouse+hearts+from+ventricular+arrhythmias+after+myocardial+infarction.">Cardiomyocyte-specific deletion of &#946;-catenin protects mouse hearts from ventricular arrhythmias after myocardial infarction.</a> Scientific Reports. 11 (1), 17722 (2021).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+exercise+training+on+the+FNDC5/BDNF+pathway+in+spontaneously+hypertensive+rats.">Effect of exercise training on the FNDC5/BDNF pathway in spontaneously hypertensive rats.</a> Physiological Reports. 7 (24), 14323 (2019).</li><li>Lin, H. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+complete+atrioventricular+block+recapitulates+clinical+indices+of+bradycardia+and+provides+a+platform+to+test+disease-modifying+therapies.">A rat model of complete atrioventricular block recapitulates clinical indices of bradycardia and provides a platform to test disease-modifying therapies.</a> Scientific Reports. 9 (1), 6930 (2019).</li><li>Cingolani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+to+inhibit+the+calcium+channel+beta+subunit:+Physiological+consequences+and+pathophysiological+effects+in+models+of+cardiac+hypertrophy.">Gene therapy to inhibit the calcium channel beta subunit: Physiological consequences and pathophysiological effects in models of cardiac hypertrophy.</a> Circulation Research. 101 (2), 166-175 (2007).</li><li>Ionta, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHOX2+overexpression+favors+differentiation+of+embryonic+stem+cells+into+cardiac+pacemaker+cells,+improving+biological+pacing+ability.">SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability.</a> Stem Cell Reports. 4 (1), 129-142 (2015).</li><li>Guss, S. B., Kastor, J. A., Josephson, M. E., Schare, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ventricular+refractoriness.+Effects+of+cycle+length,+pacing+site+and+atropine.">Human ventricular refractoriness. Effects of cycle length, pacing site and atropine.</a> Circulation. 53 (3), 450-455 (1976).</li><li>Segel, L. D., Ensunsa, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Albumin+improves+stability+and+longevity+of+perfluorochemical-perfused+hearts.">Albumin improves stability and longevity of perfluorochemical-perfused hearts.</a> The American Journal of Physiology. 254, 1105-1112 (1988).</li><li>Hong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NLRP3+inflammasome+as+a+potential+treatment+in+ischemic+stroke+concomitant+with+diabetes.">NLRP3 inflammasome as a potential treatment in ischemic stroke concomitant with diabetes.</a> Journal of Neuroinflammation. 16 (1), 121 (2019).</li><li>Lin, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-NLRP3+inflammasome+activation+exacerbates+cardiac+dysfunction+after+ischemic+stroke+in+a+mouse+model+of+diabetes.">Macrophage-NLRP3 inflammasome activation exacerbates cardiac dysfunction after ischemic stroke in a mouse model of diabetes.</a> Neuroscience Bulletin. 36 (9), 1035-1045 (2020).</li><li>Lin, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral-cardiac+syndrome+and+diabetes:+Cardiac+damage+after+ischemic+stroke+in+diabetic+state.">Cerebral-cardiac syndrome and diabetes: Cardiac damage after ischemic stroke in diabetic state.</a> Frontiers in Immunology. 12, 737170 (2021).</li><li>Brack, K. E., Narang, R., Winter, J., Ng, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+uncoupler+blebbistatin+is+associated+with+significant+electrophysiological+effects+in+the+isolated+rabbit+heart.">The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart.</a> Experimental Physiology. 98 (5), 1009-1027 (2013).</li><li>Allison, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroconductive+nanoengineered+biomimetic+hybrid+fibers+for+cardiac+tissue+engineering.">Electroconductive nanoengineered biomimetic hybrid fibers for cardiac tissue engineering.</a> Journal of Materials Chemistry. B. 5 (13), 2402-2406 (2017).</li><li>Hamel, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+human+cardiac+myocytes+for+medical+research:+Promises+and+challenges.">De novo human cardiac myocytes for medical research: Promises and challenges.</a> Stem Cells International. 2017, 4528941 (2017).</li><li>Liang, W., Lu, A., Davis, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell-based+treatment+of+acquired+heart+block:+The+battle+for+tomorrow+has+begun.">Induced pluripotent stem cell-based treatment of acquired heart block: The battle for tomorrow has begun.</a> Circulation. Arrhythmia and Electrophysiology. 10 (5), 005331 (2017).</li><li>McLaughlin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+human+recombinant+collagen+matrices+limit+adverse+remodeling+and+improve+cardiac+function+after+myocardial+infarction.">Injectable human recombinant collagen matrices limit adverse remodeling and improve cardiac function after myocardial infarction.</a> Nature Communications. 10 (1), 4866 (2019).</li><li>Villanueva, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyoxalase+1+prevents+chronic+hyperglycemia+induced+heart-explant+derived+cell+dysfunction.">Glyoxalase 1 prevents chronic hyperglycemia induced heart-explant derived cell dysfunction.</a> Theranostics. 9 (19), 5720-5730 (2019).</li><li>Kanda, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deterministic+paracrine+repair+of+injured+myocardium+using+microfluidic-based+cocooning+of+heart+explant-derived+cells.">Deterministic paracrine repair of injured myocardium using microfluidic-based cocooning of heart explant-derived cells.</a> Biomaterials. 247, 120010 (2020).</li></ol>

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 ...

This protocol allows the user to achieve efficient transgene expression in the ventricular myocardium, facilitating gene expression studies to examine cardiac arrhythmias and function in the ventricular tissue. The technique for viral transgene expression is minimally invasive for the animal, causing less damage and reduced recovery time than conventional approaches that involve a thoracotomy. The technique for inducing arrhythmias in small rodent hearts is useful for assessing the susceptibility to arrhythmias in small animal models of heart disease.Demonstrating the procedure will be Doctor Alice Lu, a post-doctoral fellow from the lab. To begin, sterilize the left lower chest region of the anesthetized mouse with alternating rounds of an iodine-based or chlorhexidine-based scrub, in alcohol, three times in a circular motion. Under ultrasound imaging guidance, insert the needle of the syringe containing the virus into the animal's chest.Approach the needle tip into the left ventricular front free wall and slowly inject 10 to 15 microliters of the virus. Verify successful injection in ultrasound images by the enhanced brightness near the tip of the needle. Withdraw the needle from the heart and insert into other regions of the left ventricle for a second and third injection of the same amount of virus.To perform Langendorff perfusion of the mouse heart, place the cannulated heart in a silicone-elastomer-coated, 10-centimeter plastic dish, with the left ventricle facing up. Cannulate the aorta of the heart with a blunt needle connected to a modified Langendorff profusion system in the constant flow rate mode. Perfuse the heart with oxygen-bubbled Tyrode solution at 37 degrees Celsius.At the beginning of perfusion, verify the correct aortic cannulation by observing the washout of blood from the heart during the first two to three heartbeats and changing the heart color from red to pale. Adjust the flow rate to keep the perfusion pressure at 70 to 80 millimeters of mercury. After confirmation, place the electrodes of a small animal ECG system around the heart by inserting them into the silicone-elastomer coating in the dish.Then, record the ECG using compatible software. Next, perform adrenergic receptor stimulation and perfuse the heart with isoproterenol and Tyrode solution. After 10 minutes, perform programmed electrical stimulation to induce ventricular tachyarrhythmias by stimulating the heart at the apex with two platinum electrodes connected to an electrical stimulator.Start the stimulation procedure with initial 10 consecutive stimuli, S1, followed by an extra stimulus, S2, with an initial interval of 80 milliseconds. Then, repeatedly reduce the S2 interval by two milliseconds each time until the heartbeat can no longer be captured or the heart's effective refractory period, or ERP, is reached. Monitor any induced ventricular tachyarrhythmias, including ventricular tachycardia and fibrillation, by ECG.If no arrhythmias are induced. add another extra stimulus, S3, after S2, with the same parameters until the ERP is reached. If ventricular tachyarrhythmias are still not induced, stop the electrical stimulation and consider the heart non-inducible.During the programmed electrical stimulation, the successful pacing of the heart is verified by the one-to-one capture of the heartbeat during the consecutive S1 stimuli and the prolonged QRS complex during S1 pacing. These combined adrenergic and electrical stimulations induced no ventricular tachyarrhythmias in healthy, sham-operated wild-type mouse hearts, or in control adenovirus-GFP-injected rat hearts. In contrast, the same protocol induced ventricular tachyarrhythmias in 77%of wild-type mouse hearts after myocardial infarction and in three out of four rats after intramyocardial injection of Ad-Wnt3a.While inserting the needle, ensure that the needle tip is embedded within the free wall and not inside the ventricle. Also, securing and cannulating the aorta of the heart is the most critical step. Following these procedures, hearts can be used for standard histological studies or molecular biology assays.Alternatively, live cells can be isolated in order to perform single-cell electrophysiology studies.

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JoVE Journal

Biology

1.5K visualizações.  University of Ottawa Heart Institute. Este protocolo permite ao usuário alcançar uma expressão transgênica eficiente no miocárdio ventricular, facilitando os estudos de expressão gênica para examinar arritmias cardíacas e função no tecido ventricular. A técnica de expressão transgênica viral é minimamente invasiva para o animal, causando menos danos e tempo de recuperação reduzido do que as abordagens convencionais que envolvem toracotomia. A técnica de indução de arritmias em corações de pequenos roedores é...