Name: viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk
Uploaded: 2022-07-27T14:10:00.000+00:00
Duration: 5 min 15 s
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;MHC-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 &#8722;80 &#176;C freezer.
<ol>
	<li>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 &#181;L) with 30 G 1/2 needles and keep on ice until use. 
	NOTE: Any bubbles in the syringe and needle must be carefully removed.</li>
	<li>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&#160;0.5-1.0 L/min) for the subsequent procedure.</li>
	<li>Gently pinch the animal&#39;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.</li>
	<li>Maintain body temperature at 37 &#176;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.</li>
	<li>Apply ophthalmic ointment to both eyes to prevent the drying of the cornea.</li>
	<li>Keep the animal in a supine position and use a preclinical imaging system (see Table of Materials) for ultrasound imaging of the animal&#39;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.</li>
	<li>Sterilize the left lower chest region of the animal&#160;with alternating rounds of an iodine-based or chlorhexidine-based scrub and alcohol three times in a circular motion.</li>
	<li>Under ultrasound imaging guidance, insert the&#160;30 G 1/2&#160;needle of the syringe containing the virus as prepared in step 1.1. into the animal&#39;s chest via the left lower chest side of the body.</li>
	<li>Approach the needle tip into the left ventricular front free wall and slowly inject 10-15 &#956;L 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 &#956;L to prevent physical damage to the myocardial tissues.</li>
	<li>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.</li>
	<li>After the completion of the injections, return the animal to its cage.</li>
	<li>Carefully monitor the animal until it has regained sufficient consciousness to maintain sternal recumbency and return it to its housing room.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Perform Langendorff perfusion of the rat or mouse heart.
	<ol>
		<li>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).</li>
		<li>Filter the solution with a 0.2 &#956;m filter and bubble with 100% O2 continuously during steps 2.1.6.-2.2.8.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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. 
		&#8203;NOTE: Caution is exercised not to damage the heart during heart collection.</li>
		<li>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)&#160;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.</li>
		<li>Place the cannulated heart in a silicone elastomer-coated9, 10 cm plastic dish with the left ventricle facing up and perfuse the heart9,12 with O2-bubbled Tyrode solution at 37 &#176;C.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Perform adrenergic receptor stimulation and programmed electrical stimulation to induce ventricular tachyarrhythmias.
	<ol>
		<li>After successful aortic cannulation, continue to perfuse the heart with Tyrode solution for 20 min to stabilize the heart preparation.</li>
		<li>Add 1 &#956;M isoproterenol (see Table of Materials) to the Tyrode solution used for heart perfusion in steps 2.2.3.-2.2.8.</li>
		<li>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).</li>
		<li>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&#39;s effective refractory period, ERP)13.</li>
		<li>Monitor any induced ventricular tachyarrhythmias (including ventricular tachycardia and fibrillation) by ECG.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Virani, S. 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. 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=Innate+immune+Nod1/RIP2+signaling+is+essential+for+cardiac+hypertrophy+but+requires+mitochondrial+antiviral+signaling+protein+for+signal+transductions+and+energy+balance.">Innate immune Nod1/RIP2 signaling is essential for cardiac hypertrophy but requires mitochondrial antiviral signaling protein for signal transductions and energy balance.</a> Circulation. 142 (23), 2240-2258 (2020).</li><li>Karunakaran, D., 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=RIPK1+expression+associates+with+inflammation+in+early+atherosclerosis+in+humans+and+can+be+therapeutically+silenced+to+reduce+NF-&#954;B+activation+and+atherogenesis+in+mice.">RIPK1 expression associates with inflammation in early atherosclerosis in humans and can be therapeutically silenced to reduce NF-&#954;B activation and atherogenesis in mice.</a> Circulation. 143 (2), 163-177 (2021).</li><li>Gharibeh, L., 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=GATA6+is+a+regulator+of+sinus+node+development+and+heart+rhythm.">GATA6 is a regulator of sinus node development and heart rhythm.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (1), 2007322118 (2021).</li><li>Lu, A., 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=Direct+and+indirect+suppression+of+Scn5a+gene+expression+mediates+cardiac+Na++channel+inhibition+by+Wnt+signalling.">Direct and indirect suppression of Scn5a gene expression mediates cardiac Na+ channel inhibition by Wnt signalling.</a> Canadian Journal of Cardiology. 36 (4), 564-576 (2020).</li><li>Liang, W., 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=Role+of+phosphoinositide+3-kinase+{alpha},+protein+kinase+C,+and+L-type+Ca2++channels+in+mediating+the+complex+actions+of+angiotensin+II+on+mouse+cardiac+contractility.">Role of phosphoinositide 3-kinase {alpha}, protein kinase C, and L-type Ca2+ channels in mediating the complex actions of angiotensin II on mouse cardiac contractility.</a> Hypertension. 56 (3), 422-429 (2010).</li><li>Kapoor, N., Liang, W., Marban, E., Cho, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+quiescent+cardiomyocytes+to+pacemaker+cells+by+expression+of+Tbx18.">Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18.</a> Nature Biotechnology. 31 (1), 54-62 (2013).</li><li>Kim, N. K., Wolfson, D., Fernandez, N., Shin, M., Cho, H. 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>

The authors have no competing financial interests.

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 needle is close to the aortic valve level so that the heart is well-perfused through the coronary arteries. Lastly, bubbles in the perfusion needle need to be removed prior to aortic cannulation&#8212;it is usually helpful to turn on the pump for a few seconds right before cannulation to remove the bubble at the tip of the needle.
The Langendorff-perfused heart preparation has several advantages over the in vivo studies in animals. Firstly, it is possible to accurately control the concentration and duration of drugs applied to the heart (e.g., isoproterenol, as used in this study). Secondly, it allows easy access to the different regions of the heart for either stimulation (e.g., electrical pacing at the apex in this manuscript) or physiological signal recording (e.g., optical mapping of the ventricular tissues after loading with a Ca2+-sensitive or voltage-sensitive dye, which is not described here). In addition, cardiac function, such as ventricular contractility, can be readily measured by inserting a balloon into the left ventricle and connecting the balloon to a pressure transducer8. Thirdly, the intrinsic properties of the heart, such as heart rate and ventricular contractility, can be investigated without the complicating factors in in vivo studies, such as the autonomic nervous system and circulating hormones. However, the limitation of the Langendorff-perfused heart is that it lacks the cross-talk among different organs or tissues in vivo15,16,17 via either circulating factors or the autonomic nervous system, which may be critical players in some studies.
The ex vivo ECG recording of the Langendorff-perfused heart, as described here and in the previous publications2,6,7,9, has the advantage of contactless recording and poses no disturbance to the function of the heart as compared to other approaches such as optical mapping, which requires the loading of Ca2+-sensitive or voltage-sensitive dyes and the use of an excitation-contraction uncoupler to reduce mechanical heart movement (such as blebbistatin)12,18. However, the optical mapping has the advantage of providing more detailed information on the electrical activities of the heart, such as the origin of the heartbeat, the pattern of myocardial activation, and the myocardial conduction velocity.
The method of direct intramyocardial injection of a virus, as described here, can also be used for the delivery of other therapeutic materials19,20,21,22,23,24, such as biomaterials, exons, modified mRNA, and stem cell-derived cardiomyocytes. The ultrasound imaging-guided intramyocardial injection has several advantages over the traditional thoracotomy-based injection approach. Firstly, it is less invasive and allows for faster recovery of the animals after the injection procedure. This reduces procedure-associated effects on the animals (e.g., those caused by post-operative pain and chest tissue inflammation when an invasive thoracotomy is used). Secondly, ultrasound imaging verifies successful virus injection in the heart, which increases the consistency and reproducibility of the results. However, the limitation of the ultrasound-guided virus injection approach is that the locations of the virus injection sites cannot be controlled as precisely as in the thoracotomy-based approach, which allows visual localization of the different heart regions.

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><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 &amp;mu;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 (&lt;em&gt;Ctnnb1&lt;/em&gt;&lt;sup&gt;flox/flox&lt;/sup&gt;)</td><td>Jackson Labs</td><td>4152</td><td></td></tr><tr><td>Mice (&amp;alpha;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 occurs in many arrhythmogenic conditions such as myocardial infarction and heart failure. During the programmed electrical stimulation, the successful pacing of the heart is verified by (1) the 1:1 capture of the heartbeat during the consecutive S1 stimuli and (2) the prolonged (or wider) QRS complex during S1 pacing (Figure 3 and Figure 4). The latter is because the heart is being paced at the apex and the slower conduction of the activation in the ventricular tissues increases the QRS complex duration.
As demonstrated in the published data2,7 (Figure 3 and Figure 4), these combined adrenergic and electrical stimulations induced no ventricular tachyarrhythmias (defined as at least three consecutive premature ventricular complexes, PVCs) in healthy mouse hearts (sham-operated wild-type hearts in Figure 3) or in control rat hearts (control Ad-GFP-injected rat hearts in Figure 4). By contrast, the same protocol induced ventricular tachyarrhythmias in 77% of wild-type mouse hearts after myocardial infarction (Figure 3B) and in three out of four rat hearts after intramyocardial injection of Ad-Wnt3a (Figure 4). This demonstrates the high fidelity of this ventricular arrhythmia-inducing approach.
Successful intramyocardial transgene expression after virus injection can be verified by upregulated mRNA and protein levels of the transgene in the myocardial tissues identified by real-time quantitative RT-PCR, western blot, or immunohistochemistry. If the virus expresses a fluorescent reporter gene, such as GFP, successful viral transduction can also be verified in isolated, live, single cardiomyocytes by their expression of GFP9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64073/64073fig01.jpg" /> 
Figure 1: Langendorff perfusion of an isolated rat heart. The heart is collected from the animal, and the aorta is cannulated with a blunt 18 G needle. The needle is connected to a 10 mL syringe filled with 37 &#176;C Tyrode solution at a pressure of 70-80 mmHg. The heart is placed in a silicone elastomer-coated, 10 cm plastic dish with the left ventricle facing up. Electrodes (red, green, and black colors) of a small animal ECG system are placed around the heart by inserting them into the silicone elastomer coating in the dish. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64073/64073fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64073/64073fig02.jpg" /> 
Figure 2: Adrenergic and electrical stimulations of the Langendorff-perfused heart. (A) Rat or mouse heart is perfused on a Langendorff system as shown in Figure 1, and 1 &#181;M isoproterenol is added to the perfusing Tyrode solution to stimulate the &#946;1 adrenergic receptors of the heart. The heart is then stimulated at the apex by two platinum electrodes connected to a stimulator. (B) Representation of the programmed electrical stimulation. For details, see step 2.2. The figure is modified with permission from Wang et al.2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64073/64073fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64073/64073fig03.jpg" /> 
Figure 3: Ventricular tachyarrhythmias induced in mouse hearts after myocardial infarction. (A) Representative ex vivo ECG (Lead II) showing programmed electrical stimulation (PES)-induced ventricular tachycardia (VT, defined as three or more consecutive premature ventricular complexes, PVCs) in a wild-type (WT) mouse heart (at 8 weeks after myocardial infarction, MI) when stimulated with one extra stimulus (S2) but only one single PVC in a cardiac-specific &#946;-catenin knockout (KO) mouse heart (at 8 weeks after MI) when stimulated with three extra stimuli (S4). Isoproterenol (1 &#181;M) was included in the perfusing Tyrode solution during the PES stimulation. Cardiac-specific &#946;-catenin (Ctnnb1) knockout mice were generated by cross-breeding Ctnnb1flox/flox mice (with exons 2 to 6 floxed) with &#945;MHC-MerCreMer mice. At the age of 8-12 weeks, Ctnnb1flox/flox;&#945;MHC-MerCreMer+/&#8722; mice (KO) and littermate Ctnnb1flox/flox;&#945;MHC-MerCreMer&#8722;/&#8722; mice (used as control wild-type mice) received daily subcutaneous injections of tamoxifen (20 mg/kg/day) for 5 consecutive days before they were assigned to either the MI or sham group. (B) Summary of PES-induced PVCs and VTs in WT and KO hearts at 1 week or 8 weeks after MI. An arrhythmia score was assigned to each heart according to the criteria in the left table. At 8 weeks after MI, VT was successfully induced in 77% of WT hearts but only in 18% of KO hearts. Data were analyzed by two-way ANOVA and a Bonferroni post-hoc comparison. The error bars indicate standard error (SE). The figure is reproduced with permission from Wang et al.2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64073/64073fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64073/64073fig04.jpg" /> 
Figure 4: Ventricular tachyarrhythmias induced in rat hearts after intramyocardial injection of a virus expressing Wnt3a. (A) PES induced ventricular arrhythmias in hearts of rats (200-250 g, male, Sprague-Dawley) at day 4-6 after intramyocardial injection of an adenovirus expressing Wnt3a (Ad-Wnt3a, bottom) but not in hearts injected with control adenovirus expressing GFP (Ad-GFP, top). Hearts were isolated and perfused on a Langendorff system. Isoproterenol (1 &#181;M) was included in the perfusing Tyrode solution during the PES stimulation. Ex vivo ECG was continuously recorded during PES stimulation. Note that 11 consecutive S1 stimuli (blue color) were used in this experiment. (B) Summary of studies in Panel (A): Ventricular tachycardia (VT) was induced by PES in three out of four hearts with Ad-Wnt3a injection but in none of the four hearts with control Ad-GFP. The figure is reproduced with permission from Lu et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64073/64073fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

viral-transgene-expression-rodent-hearts-assessment-cardiac

Nanyang Technological University

Research

JoVE Journal

Biology

1.5K Views.  University of Ottawa Heart Institute. Dit protocol stelt de gebruiker in staat om efficiënte transgene expressie in het ventriculaire myocardium te bereiken, waardoor genexpressiestudies worden vergemakkelijkt om hartritmestoornissen en functie in het ventriculaire weefsel te onderzoeken. De techniek voor virale transgene expressie is minimaal invasief voor het dier en veroorzaakt minder schade en verminderde hersteltijd dan conventionele benaderingen waarbij een thoracotomie betrokken is. De techniek voor het induceren van arit...

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

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Genetics

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Medicine

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Electroporation-Based Transgenesis of Cardiac Cells in Mouse Hearts

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

Author Spotlight: Cardiac Cell Transgenesis for Rapid Gene Screening

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

Developmental Biology

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes following physical activity, stress, or catecholamine challenge, which can deteriorate into potentially fatal ventricular fibrillation. The mouse heart is a widespread species for modeling inherited cardiac arrhythmic diseases, including CPVT. Simultaneous optical mapping of transmembrane potential (Vm) and calcium transients (CaT) from Langendorff-perfused mouse hearts has the potential to elucidate mechanisms underlying arrhythmogenesis. Compared with the cellular level investigation, the optical mapping technique can test some electrophysiological parameters, such as the determination of activation, conduction velocity, action potential duration, and CaT duration. This paper presents the instrumentation setup and experimental procedure for high-throughput optical mapping of CaT and Vm in murine wild-type and heterozygous RyR2-R2474S/+ hearts, combined with programmed electrical pacing before and during the isoproterenol challenge. This approach has demonstrated a feasible and reliable method for mechanistically studying CPVT disease in an ex vivo mouse heart preparation.

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

This protocol introduces dual-dye optical mapping of mouse hearts obtained from wild-type and knock-in animals affected by catecholaminergic polymorphic ventricular tachycardia, including electrophysiological measurements of transmembrane voltage and intracellular Ca2+ transients with high temporal and spatial resolution.

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video