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

Summary

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.

Abstract

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.

Introduction

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 autophagy^2,3,4,5,6. 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 models^7,8,9. The phenotypic variability encountered after PO in rat, Sprague-Dawley strain, is described elsewhere^10,11. 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 process^10,12. 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 PO^10,11 and in infarct size^13,14 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.

Protocol

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

1. Obtain disinfectants, such as 70% isopropyl alcohol and povidone-iodine.
2. Obtain ketamine and xylazine for anesthesia and buprenorphine for analgesia.
3. Obtain a heating pad and heavy absorbency disposable underpad with the dimensions of 18 inches x 30 inches.
4. Obtain a 100% cotton twine roll, a tape and a hair clipper.
5. Obtain a 20 cm x 25 cm plastic board, thickness range between 3-5 mm.
6. Obtain a Z-LITE fiber optic illuminator.
7. Obtain a mechanical ventilator for small animals (e.g., SAR-830/AP).
8. 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.
9. Obtain 16 G angiocath for intubation.
10. Purchase the following surgical tools.
    1. Obtain a Weck stainless steel Hemoclip ligation and stainless-steel ligating clips.
    2. Obtain hardened fine iris scissors.
    3. Obtain Adson forceps.
    4. Obtain two curved Graefe forceps.
    5. Obtain a Halsted-Mosquito Hemostats-straight forceps.
    6. Obtain a Mayo-Hegar needle holder.
    7. Obtain an Alm chest retractor with blunt teeth.
11. Utilize and obtain an autoclave and a bead sterilizer.

2. Ascending aortic banding surgical procedure

1. 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.
2. Shave the hair on the surgical site located at the right lateral thoracic area under the right armpit.
3. 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.
4. 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. 
5. 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.
6. 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 2^nd and 3^rd rib cage.
   1. 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 1^st and the 2^nd rib carries the risk of banding the right brachiocephalic artery instead of the ascending aorta. Thoracotomy between the 3^rd 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.
7. 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.
   1. 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.
8. Lift gently the ascending aorta with a curved Graefe forceps and place the vascular clip around the ascending aorta.
   1. Adjust the vascular hemoclip ligation tool via a plastic pre-cut 7" 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.
9. 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.
10. 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.

3. Echocardiography

1. 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.
   1. 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.
2. Shave the hair of the chest, anteriorly, in the completely sedated animal.
3. Lay the animal on its back and stabilize it to the plastic board.
4. 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.
   1. 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.
   2. 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.
5. 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 ≥1.25 and <1.5 in MOD HF phenotype and ≥1.5 in the HFrEF phenotype¹⁰.

Results

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, respective...

Discussion

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 ec...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adson forceps	F.S.T.	11019-12	surgical tool
Alm chest retractor with blunt teeth	ROBOZ	RS-6510	surgical tool
Graefe forceps, curved	F.S.T.	11152-10	surgical tool
Halsted-Mosquito Hemostats, straight	F.S.T.	13010-12	surgical tool
Hardened fine iris scissors, straight	Fine Science Tools F.S.T.	14090-11	surgical tool
hemoclip traditional-stainless steel ligating clips	Weck	523735	surgical tool
Mayo-Hegar needle holder	F.S.T.	12004-18	surgical tool
mechanical ventilator	CWE inc	SAR-830/AP	mechanical ventilator for small animals
Weck stainless steel Hemoclip ligation	Weck	533140	surgical tool