NIH grant HL070241 to P.D.

All methods and procedures described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Tulane University School of Medicine.
1. Tools and instruments for AAB model creation
<ol>
	<li>Obtain disinfectants, such as 70% isopropyl alcohol and povidone-iodine.</li>
	<li>Obtain ketamine and xylazine for anesthesia and buprenorphine for analgesia.</li>
	<li>Obtain a heating pad and heavy absorbency disposable underpad with the dimensions of 18 inches x 30 inches.</li>
	<li>Obtain a 100% cotton twine roll, a tape and a hair clipper.</li>
	<li>Obtain a 20 cm x 25 cm plastic board, thickness range between 3-5 mm.</li>
	<li>Obtain a Z-LITE fiber optic illuminator.</li>
	<li>Obtain a mechanical ventilator for small animals (e.g., SAR-830/AP).</li>
	<li>Obtain 2-0 and 3-0 Vicryl taper sutures and nylon 3-0 monofilament suture, sterile gauze pads and sterile extra large cotton tips and sterile gloves.</li>
	<li>Obtain 16 G angiocath for intubation.</li>
	<li>Purchase the following surgical tools.
	<ol>
		<li>Obtain a Weck stainless steel Hemoclip ligation and stainless-steel ligating clips.</li>
		<li>Obtain hardened fine iris scissors.</li>
		<li>Obtain Adson forceps.</li>
		<li>Obtain two curved Graefe forceps.</li>
		<li>Obtain a Halsted-Mosquito Hemostats-straight forceps.</li>
		<li>Obtain a Mayo-Hegar needle holder.</li>
		<li>Obtain an Alm chest retractor with blunt teeth.</li>
	</ol>
	</li>
	<li>Utilize and obtain an autoclave and a bead sterilizer.</li>
</ol>
2. Ascending aortic banding surgical procedure
<ol>
	<li>Anesthetize the animal with an intraperitoneal injection of a mix of 75-100 mg/kg Ketamine and 10 mg/kg Xylazine. 
	NOTE: Allow a few minutes for the animal to be completely sedated and flaccid. If the anesthetic dose is not sufficient and the animal is still moving in the cage, re-inject the animal with the same anesthetic dose after allowing enough time, around 5-10 minutes between subsequent injections. Most animals require 1-2 injections to achieve deep sedation and anesthesia.</li>
	<li>Shave the hair on the surgical site located at the right lateral thoracic area under the right armpit.</li>
	<li>Stabilize the animal by gently taping all four limbs to the plastic board. Then perform endotracheal intubation with a 16 G angiocath. After the animal is successfully intubated, initiate mechanical ventilation with tidal volumes of 2 mL at 50 cycles/min and FiO2 of 21%. Look for the symmetrical rise in chest wall with each breath.</li>
	<li>Turn the animal slowly to lie on its left lateral side, and then bend the tail in a U-shape manner and stabilize it by gently taping it to the plastic board. Then go ahead and disinfect the shaved area with topical application of povidone-iodine.&#160;</li>
	<li>Infiltrate the skin at the incision site with 50/50 mix by volume of 1-2% Lidocaine/0.25-0.5 % Bupivacaine as preemptive analgesia before making the incision.</li>
	<li>Perform a right horizontal skin incision, 1-2 centimeters long, in the right axillary area 1 cm below the right armpit. Then, dissect the thoracic muscular layer until reaching the thoracic rib cage. Make a 1 cm thoracotomy between the 2nd and 3rd rib cage.
	<ol>
		<li>While dissecting the muscular layer of the chest, be careful and avoid injury of the right axillary artery, which runs underneath the right armpit. 
		NOTE: Thoracotomy performed between the 1st and the 2nd rib carries the risk of banding the right brachiocephalic artery instead of the ascending aorta. Thoracotomy between the 3rd and the fourth rib makes it hard to visualize and band the ascending aorta, as the operator will be looking at the right atrium. 
		NOTE: Avoid extending the thoracotomy too medially towards the sternum to avoid dissecting and injuring the right internal mammary artery.</li>
	</ol>
	</li>
	<li>Dissect the two lobes of the thymus gland gently and push them apart on the side. Then identify the ascending aorta and isolate it from the superior vena cava by blunt dissection via a curved Graefe forceps. 
	NOTE: Significant manipulation of the thymus gland will render it swollen and makes it hard to visualize the ascending aorta.
	<ol>
		<li>Dissect the superior vena cava from the aorta with extra caution to avoid injury or rupture of the superior vena cava, which is fatal. This may be the trickiest part of the procedure and is expected to happen from time to time even in most experienced hands, but often with beginners and learners.</li>
	</ol>
	</li>
	<li>Lift gently the ascending aorta with a curved Graefe forceps and place the vascular clip around the ascending aorta.
	<ol>
		<li>Adjust the vascular hemoclip ligation tool via a plastic pre-cut 7&#34; piece to obtain a vascular clip of the desired internal area of 1.5 mm2 or 2 mm2, depending on which HF model is desired.</li>
	</ol>
	</li>
	<li>Suture the thorax via a Vicryl 2-0 monofilament suture. Then suture the muscular layer of the chest via a 3-0 Vicryl taper suture. Then suture the skin incision via a Nylon 3-0 monofilament suture.</li>
	<li>Administer a combination of the following drugs after completion of the surgery for 48-72 hrs to serve as analgesia in the post-operative period: 1) Buprenorphine 0.01-0.05 mg/kg subcutaneously every 8-12h, 2) Meloxicam 2 mg/kg subcutaneously every 12h, and 3) Morphine 2.5 mg/kg subcutaneously every 2-4h as needed for severe pain. 
	NOTE: Leave the animal to recover on a heating pad under regular monitoring. Once the animal shows signs of recovery from anesthesia (able to breath spontaneously - without evidence of gasping or use of accessory muscles for more than two minutes - and has good reflexes, red and warm extremities), extubate the animal and return it to the cage.</li>
</ol>
3. Echocardiography
<ol>
	<li>Sedate the animal with intraperitoneal injection of 80-100 mg/kg ketamine. Ensure adequate sedation for proper acquisition of good quality echo images. 
	NOTE: The use of isoflurane as an anesthetic is discouraged for its cardiodepressor effect, especially in the setting of severe pressure overload and might give a false impression of LV dilatation and systolic dysfunction that resolves once animal is off anesthetic.
	<ol>
		<li>Be cautious and administer half or even one third of the dose of ketamine in animals that look dyspneic and tachypneic with suspicion that they have developed the HFrEF phenotype.</li>
	</ol>
	</li>
	<li>Shave the hair of the chest, anteriorly, in the completely sedated animal.</li>
	<li>Lay the animal on its back and stabilize it to the plastic board.</li>
	<li>Acquire 2D parasternal long axis and 2D parasternal short axis view clips at the level of the papillary muscle. Also, obtain M-mode images from the short parasternal axis view at the level of the papillary muscle to measure LV septal and posterior wall thickness in diastole as well as LV end-diastolic and end-systolic diameter.
	<ol>
		<li>Acquire images or clips at a heart rate of 370 - 420 beats per minute to ensure proper assessment of LV size and function. Acquisition of images at lower heart rates will lead to a false impression of depressed LV function and LV dilatation. 
		NOTE: Acquisition of foreshortened 2D long parasternal axis view images/clips lead to false measurements. For quality control purposes, make sure that the LV apex and the aorto-mitral angle are visualized within the same plane cut.</li>
		<li>Acquire 2D short parasternal axis view images/clips at the level of the mid papillary muscle. This will serve as a reference to obtain reliable serial and subsequent LV measurements while following the animals over time throughout the study period.</li>
	</ol>
	</li>
	<li>Obtain M-mode images in long parasternal axis view at the level of the aortic valve to assess the relative aortic to left atrium (LA) diameter at end systole. 
	NOTE: Animals with the MOD and HFrEF phenotypes should show evidence of LA dilatation with LA/Ao ratio being &#8805;1.25 and &#60;1.5 in MOD HF phenotype and &#8805;1.5 in the HFrEF phenotype10.</li>
</ol>

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	<li>McMurray, J. J., Petrie, M. C., Murdoch, D. R., Davie, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+epidemiology+of+heart+failure:+public+and+private+health+burden.">Clinical epidemiology of heart failure: public and private health burden.</a> European Heart Journal. 19 (Suppl P), P9-P16 (1998).</li><li>Berk, B. C., Fujiwara, K., Lehoux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+remodeling+in+hypertensive+heart+disease.">ECM remodeling in hypertensive heart disease.</a> Journal of Clinical Investigation. 117 (3), 568-575 (2007).</li><li>Frey, N., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+hypertrophy:+the+good,+the+bad,+and+the+ugly.">Cardiac hypertrophy: the good, the bad, and the ugly.</a> Annual Review of Physiology. 65, 45-79 (2003).</li><li>Hill, J. A., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+plasticity.">Cardiac plasticity.</a> New England Journal of Medicine. 358 (13), 1370-1380 (2008).</li><li>Kehat, I., Molkentin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathways+underlying+cardiac+remodeling+during+pathophysiological+stimulation.">Molecular pathways underlying cardiac remodeling during pathophysiological stimulation.</a> Circulation. 122 (25), 2727-2735 (2010).</li><li>Rothermel, B. A., Hill, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+load-induced+heart+disease.">Autophagy in load-induced heart disease.</a> Circulation Research. 103 (12), 1363-1369 (2008).</li><li>Barrick, C. 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=Parent-of-origin+effects+on+cardiac+response+to+pressure+overload+in+mice.">Parent-of-origin effects on cardiac response to pressure overload in mice.</a> American Journal of Physiology-Heart and Circulatory Physiology. 297 (3), H1003-H1009 (2009).</li><li>Barrick, C. J., Rojas, M., Schoonhoven, R., Smyth, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+response+to+pressure+overload+in+129S1/SvImJ+and+C57BL/6J+mice:+temporal-+and+background-dependent+development+of+concentric+left+ventricular+hypertrophy.">Cardiac response to pressure overload in 129S1/SvImJ and C57BL/6J mice: temporal- and background-dependent development of concentric left ventricular hypertrophy.</a> American Journal of Physiology-Heart and Circulatory Physiology. 292 (5), H2119-H2130 (2007).</li><li>Lygate, C. 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=Serial+high+resolution+3D-MRI+after+aortic+banding+in+mice:+band+internalization+is+a+source+of+variability+in+the+hypertrophic+response.">Serial high resolution 3D-MRI after aortic banding in mice: band internalization is a source of variability in the hypertrophic response.</a> Basic Research in Cardiology. 101 (1), 8-16 (2006).</li><li>Chaanine, A. H., Hajjar, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Differential+Progression+of+Left+Ventricular+Remodeling+in+a+Rat+Model+of+Pressure+Overload+Induced+Heart+Failure.+Does+Clip+Size+Matter?.">Characterization of the Differential Progression of Left Ventricular Remodeling in a Rat Model of Pressure Overload Induced Heart Failure. Does Clip Size Matter?.</a> Methods in Molecular Biology (Clifton, N.J.). 1816, 195-206 (2018).</li><li>Chaanine, A. H., 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=Mitochondrial+Integrity+and+Function+in+the+Progression+of+Early+Pressure+Overload-Induced+Left+Ventricular+Remodeling.">Mitochondrial Integrity and Function in the Progression of Early Pressure Overload-Induced Left Ventricular Remodeling.</a> Journal of the American Heart Association. 6 (6), (2017).</li><li>Chaanine, A. H., 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=Potential+role+of+BNIP3+in+cardiac+remodeling,+myocardial+stiffness,+and+endoplasmic+reticulum:+mitochondrial+calcium+homeostasis+in+diastolic+and+systolic+heart+failure.">Potential role of BNIP3 in cardiac remodeling, myocardial stiffness, and endoplasmic reticulum: mitochondrial calcium homeostasis in diastolic and systolic heart failure.</a> Circulation: Heart Failure. 6 (3), 572-583 (2013).</li><li>Takagawa, 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=Myocardial+infarct+size+measurement+in+the+mouse+chronic+infarction+model:+comparison+of+area-+and+length-based+approaches.">Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.</a> Journal of Applied Physiology (Bethesda, Md. : 1985). 102 (6), 2104-2111 (2007).</li><li>Vietta, G. 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All authors report no conflict of interest.

Following PO related to AAB in rat, the LV undergoes concentric remodeling by increasing LV wall thickness, known as concentric LVH, as a compensatory mechanism to counteract for the increase in LV wall stress. Increase in LV wall thickness becomes noticeable during the first week following AAB and reaches its maximum thickness at 2-3 weeks post-AAB. During this time period, activation of maladaptive signal transduction pathways lead to progressive enlargement of the LV with increases in LV volumes, a process known as eccentric hypertrophy or remodeling. It is expected that the HF phenotype in rat develops around 8 weeks following AAB in most of the animals with few of them developing HF at week 12 following AAB. Two HF phenotypes ensue depending on the severity of AAB. The MOD phenotype is obtained via the creation of ascending aortic banding (AAB) with a vascular clip of 2 mm2 in internal diameter, whilst, the creation of the HFrEF phenotype requires AAB with a tighter vascular clip of 1.5 mm2 in internal diameter. It is important to perform echocardiography at 2-3 weeks following ascending aortic banding to verify the presence of severe concentric LVH. Severe LVH is defined as LV septal and posterior wall thickness &#8805;1.5 times normal (0.19 cm), and usually ranges between 0.27 - 0.3 cm. Animals that do not develop severe LVH at week 3 following AAB, will be deemed as having unsuccessful AAB and should not be followed thereafter. Those that have developed severe LVH at week 3 following AAB, will undergo echocardiography at week 8 following AAB to assess for the development of the desired HF phenotype. It is not rare to encounter animals that had severe LVH at week 3 following AAB to have regression or resolution of LVH at week 8 following AAB, for reasons that we will address in the latter section of the discussion. Animals with severe LVH and concentric remodeling without or with mild eccentric remodeling at week 8 following AAB, therefore the CR and MILD phenotypes, respectively, are unlikely to develop further eccentric remodeling even if they are followed for an extended month or two. Those that are in between the MILD and MOD phenotype, may develop the MOD HF phenotype if they are followed for one more month.
The PO rat model can be frustrating due to the associated high mortality and failure rates10, despite use of a standardized vascular clip size and surgical technique, which also adds to the research expense, due to the large number of animals that need to undergo AAB in order to achieve the desired target number (n), and the length of time that the animals need to be followed before they develop the desired HF phenotype. Failure to develop severe LVH is related to either unsuccessful banding or banding of the right brachiocephalic artery instead of the aorta, which is not uncommon. Regression and/or resolution of severe LVH in subsequent follow up assessments is related to aneurysm formation and peri-band aortic remodeling that leads to loss in the severity of PO9. It remains unclear why animals with severe LVH and PO develop phenotypic variability in regard to eccentric remodeling despite having the same clip size, sex and strain. It is recommended to visualize the ascending aorta to screen for peri-band aortic remodeling and aneurysmal formation. Animals that develop ascending aortic aneurysm &#8805;1 cm in diameter should be euthanized, as this will cause dyspnea and distress to the animal due to impingement on surrounding structures. Also, it is recommended to check for turbulent flow across the band by color Doppler, but unfortunately precise estimation of pressure gradient across the band by continuous Doppler is not feasible due to the inability to align the continuous Doppler with the blood flow direction in the ascending aorta.
The MOD is an attractive HF phenotype and constitutes an evolution of the traditionally created HFrEF phenotype as it allows for targeting of signal transduction pathways that play role in myocardial remodeling early on in the disease process, especially when it pertains to perturbations in mitochondrial dynamics and function, myocardial metabolism and calcium cycling and extracellular matrix remodeling and myocardial stiffness; features that are highly evident in the MOD HF phenotype11. Also, the early postoperative mortality (defined as mortality in the first 7 days post-AAB) is lower with the clip size of 2 mm2, for the creation of MOD phenotype, than the clip size of 1.5 mm2, for the creation of HFrEF phenotype10, (5% vs 21%, P = 0.009 using Fisher&#39;s exact test). However, the success rate between the two clip sizes, for the creation of MOD and HFrEF phenotypes, is not statistically significant10, (20% vs 13%, P = 0.56 using Fisher&#39;s exact test). Moreover, the aortic banding by vascular clip is advantageous over the aortic banding by tightening a nylon suture against a 27 G needle, a technique often used to constrict the transverse aorta in mice, because there is less variation in clip size and less trauma to the aorta compared to the suture technique.
The PO model of HF is advantageous over the myocardial infarction (MI) model of HF because the PO-induced circumferential and meridional wall stress is homogeneously distributed across all segments of the myocardium. However, both models suffer from variability in the severity of PO10,11 and in infarct size13,14 along with intense inflammation and scarring at the infarct site15 as well as adhesion to the chest wall and surrounding tissues observed in the MI model of HF. Moreover, the rat PO induced HF model is challenging to create as it is associated with high mortality and failure rates10, with only 20% of the operated rats developing the MOD HF phenotype10. When compared to the spontaneously hypertensive rat (SHR) model, the PO-induced HF model is a better model to study pathways related to myocardial remodeling. The increase in afterload and myocardial wall stress in systole is much higher in the PO-induced HF model than the SHR model. It takes about two years for the SHR to develop systolic HF and the mechanism of systolic HF is not entirely known and is confounded by aging16. The SHR model and other models of hypertension, such as the DOCA salt model, are more frequently used to investigate mechanisms and therapies related to hypertension and possibly diastolic dysfunction16.
In conclusion, the MOD HF phenotype is an attractive model to study signal transduction pathways in the context of myocardial remodeling and can be utilized for application and testing of potential therapeutic strategies, before validation of their efficacy in large animal models and in human heart failure.

Heart failure (HF) is a prevalent disease and is associated with high morbidity and mortality1. Rodent pressure-overload (PO) models of HF, produced by ascending or transverse aortic banding, are commonly used to explore molecular mechanisms leading to HF and to test potential novel therapeutic targets in HF. They also mimic changes seen in human HF secondary to prolonged systemic hypertension or severe aortic stenosis. Following PO, the left ventricular (LV) wall gradually increases in thickness, a process known as concentric LV hypertrophy (LVH), to compensate and adapt for the increase in LV wall stress. However, this is associated with the activation of a number of maladaptive signaling pathways, which lead to derangements in calcium cycling and homeostasis, metabolic and extracellular matrix remodeling and changes in gene expression as well as enhanced apoptosis and autophagy2,3,4,5,6. These molecular changes constitute the trigger for the initiation and propagation of myocardial remodeling and transition into a decompensated HF phenotype.
Despite the use of inbred rodent strains and standardization of clip size and surgical technique, there is tremendous phenotypic variability in LV chamber structure and function in aortic banding models7,8,9. The phenotypic variability encountered after PO in rat, Sprague-Dawley strain, is described elsewhere10,11. Of those, two HF phenotypes are encountered with evidence of myocardial remodeling and activation of signal transduction pathways leading to a state of heightened oxidative stress. This is associated with metabolic remodeling, altered gene expression and changes in posttranslational modification of proteins, altogether playing a role in the remodeling process10,12. The first is a phenotype of moderate remodeling and early systolic dysfunction (MOD) and the second is a phenotype of overt systolic HF (HFrEF).
The PO model of HF is advantageous over the myocardial infarction (MI) model of HF because the PO-induced circumferential and meridional wall stresses are homogeneously distributed across all segments of the myocardium. However, both models suffer from variability in the severity of PO10,11 and in infarct size13,14 along with intense inflammation and scarring at the infarct site15 as well as adhesion to the chest wall and surrounding tissues, which are observed in the MI model of HF. Moreover, the rat PO induced HF model is challenging to create as it is associated with high mortality and failure rates10, with only 20% of the operated rats developing the MOD HF phenotype10.
The MOD is an attractive HF phenotype and constitutes an evolution of the traditionally created HFrEF phenotype as it allows for early targeting of signal transduction pathways that play a role in myocardial remodeling, especially when it pertains to perturbations in mitochondrial dynamics and function, myocardial metabolism, calcium cycling and extracellular matrix remodeling. These pathophysiological processes are highly evident in the MOD HF phenotype11. In this manuscript, we describe how to create the MOD and HFrEF phenotypes and we address pitfalls while performing the ascending aortic banding (AAB) procedure. We also elaborate on how to best characterize by echocardiography the two HF phenotypes, MOD and HFrEF, and how to differentiate them from other phenotypes that fail to develop severe PO or that develop severe PO and concentric remodeling but without significant eccentric remodeling.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adson forceps</td>
			<td>F.S.T.</td>
			<td>11019-12</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Alm chest retractor with blunt teeth</td>
			<td>ROBOZ</td>
			<td>RS-6510</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Graefe forceps, curved</td>
			<td>F.S.T.</td>
			<td>11152-10</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito Hemostats, straight</td>
			<td>F.S.T.</td>
			<td>13010-12</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Hardened fine iris scissors, straight</td>
			<td>Fine Science Tools F.S.T.</td>
			<td>14090-11</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>hemoclip traditional-stainless steel ligating clips</td>
			<td>Weck</td>
			<td>523435</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Mayo-Hegar needle holder</td>
			<td>F.S.T.</td>
			<td>12004-18</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>mechanical ventilator</td>
			<td>CWE inc</td>
			<td>SAR-830/AP</td>
			<td>mechanical ventilator for small animals</td>
		</tr>
		<tr>
			<td>Weck stainless steel Hemoclip ligation</td>
			<td>Weck</td>
			<td>533140</td>
			<td>surgical tool</td>
		</tr>
	</tbody>
</table>

a rat model of pressure overload induced moderate remodeling and systolic dysfunction as opposed to overt systolic heart failure

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation of signal transduction pathways, to counteract, in the short-term, for the cardiac myocyte loss and or the increase in wall stress. However, prolonged activation of these pathways becomes detrimental leading to the initiation and propagation of cardiac remodeling leading to changes in left ventricular geometry and increases in left ventricular volumes; a phenotype seen in patients with systolic heart failure (HF). Here, we describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction (MOD) by ascending aortic banding (AAB) via a vascular clip with an internal area of 2 mm2. The surgery is performed in 200 g Sprague-Dawley rats. The MOD HF phenotype develops at 8-12 weeks after AAB and is characterized noninvasively by means of echocardiography. Previous work suggests the activation of signal transduction pathways and altered gene expression and post-translational modification of proteins in the MOD HF phenotype that mimic those seen in human systolic HF; therefore, making the MOD HF phenotype a suitable model for translational research to identify and test potential therapeutic anti-remodeling targets in HF. The advantages of the MOD HF phenotype compared to the overt systolic HF phenotype is that it allows for the identification of molecular targets involved in the early remodeling process and the early application of therapeutic interventions. The limitation of the MOD HF phenotype is that it may not mimic the spectrum of diseases leading to systolic HF in human. Moreover, it is a challenging phenotype to create, as the AAB surgery is associated with high mortality and failure rates with only 20% of operated rats developing the desired HF phenotype.

Characterization of the HF phenotypes, that develop 8-12 weeks following AAB, could be easily performed via echocardiography. Representative M-mode images of Sham, Week 3 post-AAB, MOD and HFrEF phenotypes are presented in Figure 1A. Figure 1B and Figure 1C are showing the vascular clip size for the creation of the MOD HF phenotype and HFrEF phenotype, respectively. The LV end-diastolic (LVEDV) and end-systolic (LVESV) volumes could be calculated by using the formulae of the area length method: V=5/6&#215;A&#215;L, where V is the volume in ml; A is the cross sectional area of the LV cavity in cm2, obtained from the short parasternal axis view at the level of the mid-papillary muscle in diastole (Ad) and in systole (As); and L is the length of the LV cavity in cm, measured from the long parasternal axis view as the distance from the endocardial LV apex to the mitral-aortic junction in diastole (Ld) and in systole (Ls). Representative 2D long parasternal axis and short parasternal axis echocardiography images, with illustration on how to measure Ld, Ls, Ad and As, in Sham and MOD HF phenotype are presented in Figure 2. The LVEDV in the MOD HF phenotype usually ranges between 600 - 700 &#181;L, with very few animals having LVEDV greater than 700 &#181;L and up to 1000 &#181;L; whilst, the LVESV in the MOD phenotype ranges between 120 - 160 &#181;L (Table 1). From the 2D short parasternal axis view echocardiography images presented in Figure 2, one could appreciate the degree of LVH in the MOD phenotype compared to the sham. Representative pressure-volume loop tracings of the Sham, Week 3 post-AAB, MOD and HFrEF phenotypes are presented in Figure 3. The LV maximum pressure is at least 200 mmHg, even at week 3 post-AAB, and increases further at week 8 post-AAB due to the mismatch between the growth of the animal and aorta and the fixed created stenosis in the ascending aorta. Note that the animals at week 3 post-AAB are fully compensated with shift of the LVEDV and LVESV to the left compared to the sham. With progressive eccentric hypertrophy and remodeling, there is a shift in LVEDV and LVESV to the right in the MOD and HFrEF phenotypes compared to week 3 post-AAB. One could appreciate also the significant increase in LVESV in the MOD phenotype and the profound increase in LVESV in the HFrEF phenotype, which reflects the significant and profound decreases in stroke volume and LVEF in the MOD and HFrEF phenotypes, respectively, compared to week 3 post-AAB. Moreover, one could appreciate the significant increase in LVEF at week 3 post-AAB and the significant decrease in LVEF in the HFrEF phenotype compared to the sham.
The rat PO induced HF model is associated with high mortality and failure rates. Only about 20% of the rats that undergo AAB, with a vascular clip of 2 mm2 in internal diameter, will transition to develop the MOD HF phenotype. Representative M-mode images of the failed phenotypes are presented in Figure 4. Figure 4A is showing representative M-mode images of animals that did not develop LVH at week 8 post-AAB, and had completely lost the PO with complete regression of LVH (sham-like) or had variable degree of LVH and PO at week 8 post-AAB causing a mild-moderate LVH phenotype. The second failed phenotype group is presented in Figure 4B showing representative M-mode images of animals with severe PO (LV maximum pressure &#62;200 mmHg) and severe LVH who remained compensated with no evidence of eccentric remodeling, concentric remodeling (CR) group, or with a mild (MILD group) eccentric remodeling. Echocardiography and hemodynamic data of the sham, failed, and successful/desired phenotypes are presented in Figure 5 and Table 1. Note the progressive increases in heart weight and LV weight as the animals transition from a compensated phenotype to a more eccentric and remodeled phenotype. Also, there is an exponential increase in LVESV and decrease in LVEF as the animals transition from a compensated concentric remodeling to a decompensated eccentrically remodeled phenotype. Of particular interest is that both the MOD and HFrEF HF phenotypes have a similar degree of myocardial stiffness as measured by the stiffness-coefficient &#946; of the end-diastolic pressure volume relationship (EDPVR (mmHg/&#181;L)) compared to all the other phenotypes, whereas there is a gradual decrease in LV efficiency as the animals transition to a more eccentrically remodeled phenotype. LV efficiency is calculated from the end-systolic pressure volume relationship (ESPVR) divided by the arterial elastance (EA). Despite that there is no significant statistical difference in ESPVR and ESPVR/EA between the MOD and HFrEF phenotypes and the sham group, this is falsely the case as the MOD and HFrEF phenotypes have a significantly higher LV end-systolic pressure compared to the sham, making the ESPVR slope falsely steeper with shift in V0 to the right compared to the sham. Moreover, when the MOD and HFrEF phenotypes are compared to the compensated and concentrically remodeled phenotypes, which have the same degree of PO, then one could appreciate the significant and progressive increase in LVESV and drop in ESPVR and ESPVR/EA with progressive eccentric remodeling, as observed in the MOD and HFrEF phenotypes compared to the CR and MILD phenotypes (Figure 5 and Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60954/60954fig1.jpg" /> 
Figure 1: Representative heart failure phenotypes at week 8 following ascending aortic banding. (A) Representative M-mode images of sham animals, animals three weeks following ascending aortic banding (AAB) and eight weeks following AAB. Figure 1A has been modified from Chaanine et al., American Journal of Physiology-Heart and Circulatory physiology, 2016. (B) Vascular clip size for the creation of severe left ventricular hypertrophy (LVH) with moderate eccentric remodeling (MOD). (C) Vascular clip size for the creation of severe LVH with overt systolic heart failure (HFrEF). Figures 1B and 1C has been obtained and modified from Chaanine et al., Methods in Molecular Biology, 2018. <a href="https://www.jove.com/files/ftp_upload/60954/60954fig1large.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/60954/60954fig2.jpg" /> 
Figure 2: Calculation of left ventricular volumes by echocardiography using the area-length method. Representative 2D long parasternal and 2D short parasternal axis view echocardiography images to measure left ventricular (LV) cavity length in diastole (Ld) and in systole (Ls) and LV cavity cross sectional area in diastole (Ad) and in systole (As) in order to calculate LV volumes at end of diastole and systole. <a href="https://www.jove.com/files/ftp_upload/60954/60954fig2large.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/60954/60954fig3.jpg" /> 
Figure 3: Pressure-volume loop tracings were obtained via a 1.9 F rat pressure-volume catheter using the open chest and left ventricular apical puncture approach. Representative pressure-volume loop tracings in Sham, week 3 following AAB, MOD and HFrEF phenotypes at week 8 following AAB. Figure has been modified from Chaanine et al., Circulation: Heart Failure, 2013. <a href="https://www.jove.com/files/ftp_upload/60954/60954fig3large.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/60954/60954fig4.jpg" /> 
Figure 4: Encountered phenotypes at week 8 following AAB with failure to develop the desired heart failure phenotype(s). (A) Representative M-mode images of animals that lost the pressure overload (PO) and did not develop LVH (Sham-like) and those with variable PO and LVH (mild-moderate LVH) phenotypes. (B) Representative M-mode images of animals that developed severe PO, LVH and concentric remodeling (CR), but without (CR) or with mild (MILD) eccentric remodeling phenotypes. Figure 4B has been modified from Chaanine et al., Journal of American Heart Association, 2017. <a href="https://www.jove.com/files/ftp_upload/60954/60954fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60954/60954fig5.jpg" /> 
Figure 5: Echocardiography and pressure-volume loop parameters in the different phenotypes. Data are presented as individual values (dots) with median (horizontal line) in the different phenotypes at week 8 post-AAB. Statistical analysis results of the presented data in the different phenotypes are shown in table 1. LVESV: left ventricular end-systolic volume, LVEF: left ventricular ejection fraction, EDPVR: end-diastolic pressure volume relationship, ESPVR: end-systolic pressure volume relationship, EA: arterial elastance. <a href="https://www.jove.com/files/ftp_upload/60954/60954fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Sham (n=5)</td>
			<td>Sham-like (n=5)</td>
			<td>Mild-mod LVH (n=8)</td>
			<td>CR (n=11)</td>
			<td>MILD (n=14)</td>
			<td>MOD (n=14)</td>
			<td>HFrEF (n=5)</td>
		</tr>
		<tr>
			<td>Body weight (g)</td>
			<td>594 &#177; 37</td>
			<td>466 &#177; 66</td>
			<td>464 &#177; 22</td>
			<td>497 &#177; 43</td>
			<td>530 &#177; 59</td>
			<td>478 &#177; 39</td>
			<td>546 &#177; 18</td>
		</tr>
		<tr>
			<td>HW (mg)</td>
			<td>1269 &#177; 124.5</td>
			<td>1328 &#177; 119</td>
			<td>1614 &#177; 177</td>
			<td>1645 &#177; 191a</td>
			<td>1821 &#177; 169a,b</td>
			<td>2106 &#177; 292a,b,c,d,e</td>
			<td>2897 &#177; 182a,b,c,d,e,f</td>
		</tr>
		<tr>
			<td>LVW (mg)</td>
			<td>897 &#177; 94</td>
			<td>968 &#177; 91</td>
			<td>1161 &#177; 144</td>
			<td>1222 &#177; 152a</td>
			<td>1372 &#177; 135a,b</td>
			<td>1580 &#177; 219a,b,c,d,e</td>
			<td>1726 &#177; 82a,b,c,d,e</td>
		</tr>
		<tr>
			<td>RVW (mg)</td>
			<td>218&#177; 22</td>
			<td>218 &#177; 23</td>
			<td>266 &#177; 24</td>
			<td>239 &#177; 26</td>
			<td>249 &#177; 26</td>
			<td>283 &#177; 42a,b</td>
			<td>565 &#177; 76a,b,c,d,e,f</td>
		</tr>
		<tr>
			<td>IVSd (cm)</td>
			<td>0.19 &#177; 0.01</td>
			<td>0.21 &#177; 0.01</td>
			<td>0.23 &#177; 0.01a</td>
			<td>0.29 &#177; 0.01a,b,c</td>
			<td>0.28 &#177; 0.02a,b,c</td>
			<td>0.28 &#177; 0.01a,b,c</td>
			<td>0.28 &#177; 0.02a,b,c</td>
		</tr>
		<tr>
			<td>LVPWd (cm)</td>
			<td>0.20 &#177; 0.01</td>
			<td>0.21 &#177; 0.02</td>
			<td>0.24 &#177; 0.01a,b</td>
			<td>0.29 &#177; 0.02a,b,c</td>
			<td>0.28 &#177; 0.02a,b,c</td>
			<td>0.28 &#177; 0.01a,b,c</td>
			<td>0.30 &#177; 0.02a,b,c</td>
		</tr>
		<tr>
			<td>LVEDV (&#956;l)</td>
			<td>560.5 &#177; 25.8</td>
			<td>570 &#177; 32</td>
			<td>668 &#177; 143</td>
			<td>442 &#177; 42,c</td>
			<td>583 &#177; 45d</td>
			<td>697 &#177; 129d,e</td>
			<td>881.5 &#177; 55.7a,b,c,d,e,f</td>
		</tr>
		<tr>
			<td>LVESV (&#956;l)</td>
			<td>105.9 &#177; 8.9</td>
			<td>93 &#177; 15</td>
			<td>111 &#177; 20</td>
			<td>59 &#177; 7a,b,c</td>
			<td>85.3 &#177; 10.6d</td>
			<td>139.7 &#177; 22.5a,b,c,d,e</td>
			<td>319.2 &#177; 51.5a,b,c,d,e,f</td>
		</tr>
		<tr>
			<td>LVEF (%)</td>
			<td>81.1 &#177; 1.2</td>
			<td>83.7 &#177; 2.9</td>
			<td>83.1 &#177; 2.5</td>
			<td>86.5 &#177; 2.2a,c</td>
			<td>85.4 &#177; 1.7a</td>
			<td>79.8 &#177; 1.9b,c,d,e</td>
			<td>64.1 &#177; 3.6a,b,c,d,e,f</td>
		</tr>
		<tr>
			<td>LVPmax (mmHg)</td>
			<td>121 &#177; 19</td>
			<td>126 &#177; 23</td>
			<td>186 &#177; 23a,b</td>
			<td>218 &#177; 18a,b</td>
			<td>221 &#177; 22a,b,c</td>
			<td>*234 &#177; 25a,b,c</td>
			<td>262 &#177; 16a,b,c,d,e</td>
		</tr>
		<tr>
			<td>EDPVR (mmHg/&#956;l)</td>
			<td>0.018 &#177; 0.005</td>
			<td>0.017 &#177; 0.004</td>
			<td>0.041 &#177; 0.013</td>
			<td>0.043 &#177; 0.017</td>
			<td>0.039 &#177; 0.015</td>
			<td>*0.068 &#177; 0.025a,b,c,d,e</td>
			<td>0.079 &#177; 0.017a,b,c,d,e</td>
		</tr>
		<tr>
			<td>ESPVR/EA</td>
			<td>1.57 &#177; 0.67</td>
			<td>1.96 &#177; 0.61</td>
			<td>2.63 &#177; 1.52</td>
			<td>3.35 &#177; 1.23a</td>
			<td>2.62 &#177; 0.55</td>
			<td>*1.63 &#177; 0.41d</td>
			<td>0.82 &#177; 0.24c,d,e</td>
		</tr>
		<tr>
			<td colspan="8">Data are presented as mean &#177; standard deviation. Statistical analysis was performed using One-way ANOVA. P &#60; 0.05 was considered significant.</td>
		</tr>
		<tr>
			<td colspan="8">aP &#60; 0.05 vs Sham</td>
		</tr>
		<tr>
			<td colspan="8">bP &#60; 0.05 vs Sham-like</td>
		</tr>
		<tr>
			<td colspan="8">cP &#60; 0.05 vs Mild-modearte LVH</td>
		</tr>
		<tr>
			<td colspan="8">dP &#60; 0.05 vs CR</td>
		</tr>
		<tr>
			<td colspan="8">eP &#60; 0.05 vs MILD</td>
		</tr>
		<tr>
			<td colspan="8">fP &#60; 0.05 vs MOD</td>
		</tr>
		<tr>
			<td colspan="8">*n=6</td>
		</tr>
		<tr>
			<td colspan="8">Abbreviations: HW: heart weight, LVW: left ventricular weight, RVW: right ventricular weight, IVSd: septal wall thickness in diastole, LVPWd: left ventricular posterior wall thickness in diastole. LVEDV: left ventricular end-diastolic volume, LVESV: left ventricular end-systolic volume, LVEF: left ventricular ejection fraction, LVPmax: left ventricular maximal pressure, EDPVR: end-diastolic pressure volume relationship, ESPVR: end-systolic pressure volume relationship, EA: arterial elastance.</td>
		</tr>
	</tbody>
</table>
Table 1: Echocardiography and pressure-volume parameters in Sham, Sham-like, Mild-moderate LVH, CR, MILD, MOD and HFrEF phenotypes.

Watch this Scientific Journal Video about A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure at JoVE.com

A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

In response to an injury, such as myocardial infarction, prolonged hypertension or a cardiotoxic agent, the heart initially adapts through the activation ...

a-rat-model-pressure-overload-induced-moderate-remodeling-systolic

Nanyang Technological University

Research

JoVE Journal

Medicine

6.4K Views.  Tulane University. We describe the creation of a rat model of pressure overload induced moderate remodeling and early systolic dysfunction where signal transduction pathways involved in the initiation of the remodeling process are activated. This animal model will aid in identifying molecular targets for applying early therapeutic anti-remodeling strategies for heart failure.

Video: A Rat Model of Pressure Overload Induced Moderate Remodeling and Systolic Dysfunction as Opposed to Overt Systolic Heart Failure

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 ...

Ascending Aortic Constriction in Rats for Creation of Pressure Overload Cardiac Hypertrophy Model

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart failure. Here, we describe a detailed surgical procedure for creating pressure overload and cardiac hypertrophy in rats by constriction of the ascending aorta using a small metallic clip. After anesthesia, the trachea is intubated by inserting a cannula through a half way incision made between two cartilage rings of trachea. Then a skin incision is made at the level of the second intercostal space on the left chest wall and muscle layers are cleared to locate the ascending portion of aorta. The ascending aorta is constricted to 50&#8211;60% of its original diameter by application of a small sized titanium clip. Following aortic constriction, the second and third ribs are approximated with prolene sutures. The tracheal cannula is removed once spontaneous breathing was re-established. The animal is allowed to recover on the heating pad by gradually lowering anesthesia. The intensity of pressure overload created by constriction of the ascending aorta is determined by recording the pressure gradient using trans-thoracic two dimensional Doppler-echocardiography. Overall this protocol is useful to study the remodeling events and contractile properties of the heart during the gradual onset and progression from compensated cardiac hypertrophy to heart failure stage.

We describe a stepwise procedure for creating pressure overload and left ventricular hypertrophy in Wistar rats by constriction of the ascending aorta using a small metallic clip. This model is extensively used for studying remodeling changes during cardiac hypertrophy and for identifying and evaluating strategies for regression of such changes.

Ascending aortic constriction is the most common and successful surgical model for creating pressure overload induced cardiac hypertrophy and heart ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to suboptimal clinical results. We set out to establish a chronic ovine model of FTR and right heart failure to investigate the mechanisms of FTR. Twenty adult male sheep (6-12 months old, 62 &#177; 7 kg) underwent a left thoracotomy and baseline echocardiography. A pulmonary artery band (PAB) was placed and cinched around the main pulmonary artery (PA) to at least double the systolic pulmonary artery pressure (SPAP), inducing right ventricular (RV) pressure overload and signs of RV dilatation. PAB acutely increased the SPAP from 21 &#177; 2 mmHg to 62 &#177; 2 mmHg. The animals were followed for 8 weeks, symptoms of heart failure were treated with diuretics, and surveillance echocardiography was used to assess for pleural and abdominal fluid collection. Three animals died during the follow-up period due to stroke, hemorrhage, and acute heart failure. After 2 months, a median sternotomy and epicardial echocardiography were performed. Of the surviving 17 animals, 3 developed mild tricuspid regurgitation, 3 developed moderate tricuspid regurgitation, and 11 developed severe tricuspid regurgitation. Eight weeks of pulmonary artery banding resulted in a stable chronic ovine model of right ventricular dysfunction and significant FTR. This large animal platform can be used to further investigate the structural and molecular basis of RV failure and functional tricuspid regurgitation.

Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments.

The pathophysiology of severe functional tricuspid regurgitation (FTR) associated with right ventricular dysfunction is poorly understood, leading to ...

Therapeutic Potential of Tuina Massage for Knee Osteoarthritis

Tuina Intervention in Sodium Monoiodoacetate Injection-Induced Rat Model of Knee Osteoarthritis

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable structural damage of the joint. Investigations into the link between articular cartilage, muscles, synovium, and other tissues surrounding the knee joint in KOA are of great importance. Currently, managing KOA includes lifestyle modifications, exercise, medication, and surgical interventions; however, the elucidation of the intricate mechanisms underlying KOA-related pain is still lacking. Consequently, KOA pain remains a key clinical challenge and a therapeutic priority. Tuina has been found to have a regulatory effect on the motor, immune, and endocrine systems, prompting the exploration of whether Tuina could alleviate KOA symptoms, caused by the upregulation of inflammatory factors, and further, if the inflammatory factors in skeletal muscle can augment the progression of KOA.
We randomized 32 male Sprague Dawley (SD) rats (180-220 g) into four groups of eight animals each: antiPD-L1+Tuina (group A), model (group B), Tuina (group C), and sham surgery (group D). For groups A, B, and C, we injected 25 &#181;L of sodium monoiodoacetate (MIA) solution (4 mg MIA diluted in 25 &#181;L of sterile saline solution) into the right knee joint cavity, and for group D, the same amount of sterile physiological saline was injected. All the groups were evaluated using the least to most stressful tests (paw mechanical withdrawal threshold, paw withdrawal thermal latency, swelling of the right knee joint, Lequesne MG score, skin temperature) before injection and 2, 9, and 16 days after injection.

Author Spotlight: Treating Knee Osteoarthritis with Tuina - A New Perspective 

This protocol describes the methods of Tuina intervention in sodium monoiodoacetate injection-induced rat model of knee osteoarthritis (KOA), which provides a reference for the application of Tuina in KOA animal models. This protocol also studies the effective mechanism of Tuina for KOA, and the results will help promote its application.

Knee osteoarthritis (KOA), a common degenerative joint disorder, is characterized by chronic pain and disability, which can progress to irreparable ...