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.
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.
Mitral regurgitation (MR) is a common heart valve lesion, diagnosed in 1.7% of the general US population and in 9% of the elderly population greater than 65 years of age1. In this heart valve lesion, improper closure of the mitral valve leaflets in systole, causes regurgitation of blood from the left ventricle into the left atrium. MR can occur due to various etiologies; however, primary lesions of the mitral valve (primary MR) are diagnosed and treated more frequently compared to secondary MR2. Isolated primary MR is often a result of myxomatous degeneration of the mitral valve, resulting in elongation of the leaflets or chordae tendineae, or rupture of some chordae, all of which contribute to the loss of systolic coaptation of the valve.
MR resulting from such valve lesions elevates the blood volume filling the left ventricle in each heartbeat, increasing the end diastolic wall stress and providing a hemodynamic stressor that incites cardiac adaptation and remodeling. Cardiac remodeling in this lesion is often characterized by significant chamber enlargement3,4, mild wall hypertrophy, with preserved contractile function for prolonged periods of time. Since the ejection fraction is often preserved, correction of MR using surgical or transcatheter means is often delayed, until the onset of symptoms such as dyspnea, heart failure, and arrhythmias. However, uncorrected MR is associated with high risks of cardiac adverse events, though currently knowledge regarding the ultrastructural changes underlying these events are unknown.
Animal models of MR provide a valuable model to investigate such ultrastructural changes in the heart, and study longitudinal progression of the disease. Previously, researchers have induced MR in large animals including pigs, dogs, and sheep, by creating an external ventriculo-atrial shunt5, intracardiac chordal rupture6, or leaflet perforation7. While surgical techniques are easier in large animals, these studies have been limited to sub-chronic follow-up in a small sample size, due to the high costs of performing such studies in large animals. Furthermore, molecular analysis of tissue from these models is often challenging due to limited species-specific antibodies and annotated genome libraries for alignment.
Small animal models of MR can provide a suitable alternative to study this valve lesion and its impact on cardiac remodeling. Historically, the rat model of aorto-caval fistula (ACF) of cardiac volume overload has been used. First described in 1973 by Stumpe et al.8, an arterio-venous fistula is surgically created to bypass high pressure arterial blood from the descending aorta into the low pressure inferior vena cava. The high flow rate in the fistula induces a drastic volume overload on both sides of the heart, causing significant right and left ventricular hypertrophy and dysfunction occurring within days of creating the ACF9. Despite its success, ACF does not mimic the hemodynamics of MR, a low-pressure volume overload, which elevates preload but also reduces afterload. Due to such limitations of the ACF model, we sought to develop and characterize a model of MR that better mimics the low-pressure volume overload.
Herein, we describe the protocol for a model of mitral valve leaflet puncture to create severe MR in rats10,11. A hypodermic needle was introduced into the beating rat heart, and advanced into the anterior mitral valve leaflet under real-time echocardiographic guidance. The technique is highly reproducible and a relatively good model that mimics MR as seen in patients. MR severity is controlled by the size of the needle used to perforate the mitral leaflet and severity of MR can be assessed using transesophageal echocardiography (TEE).
Procedures were approved by the Animal Care and Use Program at Emory University under the protocol number EM63Rr, approval date 06/06/2017.
1. Pre-surgical preparation
2. Animal preparation
NOTE: Adult Sprague-Dawley male rats weighing 350-400 g were used in this study. The surgical techniques are amenable to slightly smaller or larger animals, if desired.
3. Left thoracotomy
4. Echo guided MR procedure (Figure 3 & Figure 4)
5. Animal recovery and post-operative care
6. Validation of MR severity with echocardiography (Figure 5)
7. Sham surgery
Feasibility and reproducibility
The proposed MR model is highly reproducible, with a well defined hole in the mitral leaflet achieved in 100% of the rats used in this study. Figure 6A depicts the direction of the needle as it is inserted into the mitral valve. Figure 6B depicts a hole in the mitral valve leaflet from a representative rat explanted at 2 weeks after the procedure.
Survival & Adverse Events
Sixteen rats were induced with MR using the described methods. Severe MR was created in all the rats. One rat died within an hour of creating MR from acute respiratory failure. Therefore, overall survival at 2 weeks after creating MR was 93.75%. Mortality or major cardiac adverse events, such as bleeding, arrhythmias, or stroke were not observed in any animals in the two weeks of observation.
Severity of mitral regurgitation
Table 1 summarizes the hemodynamic profile of the left heart at baseline and at 2 weeks after inducing MR. A paired t-test was used to determine statistical significance between baseline and MR severity at 2 week, with a statistical significance defined as p < 0.05. An MR jet was vivid at two weeks after the surgery, with an average area of 21.15 ± 8.11 mm2 (p < 0.0001 compared to baseline) and a mean velocity time integral of 39.72 ± 7.52 cm. Normalized MR fraction at 2 weeks was 41.91 ± 8.3%, which is considered severe according to the guidelines of the American Society of Echocardiography. The severity of MR was adequate to induce pulmonary flow reversal, with a decrease in S/D ratio from 0.91 ± 0.17 at baseline to -0.69 ± 0.65 at 2 weeks (p < 0.0001).
Cardiac chamber remodeling
Figure 7 shows morphological changes in a representative heart after severe MR for 2 weeks, compared to a heart from a rat that underwent sham surgery. After two weeks post-surgery, the heart from the rat with MR was spherical and severely dilated, with a 29.65% increase in end diastolic volume (baseline EDV: 462.49 ± 39.62 µL; and post-2 week MR EDV: 599.79 ± 58.59 µL, p < 0.0001). End systolic volume increased by 10.06%, from 153.90 ± 18.78 µL at baseline, to 169.36 ± 24.64 µL (p = 0.01) at 2 weeks after MR induction. Hypercontractility of the heart was observed in the first two weeks as expected, due to afterload reduction, as evident from an elevated ejection fraction (66.77 ± 2.02% at baseline to 71.82 ± 2.31% at 2 weeks (p < 0.0001)). Exposure to MR for two weeks, increased the left atrial area by 99.59% (p < 0.0001).
Figure 1: Intubation technique. (A) A 16 G angiocath with a guidewire used for endotracheal intubation in this rat model; (B) Image of the pharyngeal view using an otoscope, and the target region to insert the endotracheal tube; (C) Final configuration of the endotracheal tube; (D) Attachment of the endotracheal tube to the mechanical ventilator. Please click here to view a larger version of this figure.
Figure 2: Transthoracic and transesophageal imaging. Transthoracic imaging: (A1) Setup for transthoracic imaging of the rat, depicting the angle of the imaging probe; (A2) Parasternal long axis view of the heart; (A3) Short axis view of the heart. Transesophageal imaging: (B1) 8 Fr intracardiac echo probe with probe inserted into the esophagus while the animal is intubated; (B2) High esophageal views of the left heart, depicting the left atrium, mitral valve and left ventricle. Please click here to view a larger version of this figure.
Figure 3: Surgical procedure. (A) Surgical layout showing left thoracotomy at 5th intercostal space, and ICE catheter into the esophagus of the rat for image guidance, and a 23 G needle inserted into the LV apex where thee purse-string suture is placed. (B) Surgical view during transesophageal echo guided leaflet perforation. (C) Echocardiographic image of the needle insertion into the left ventricle in diastole. (D) Echocardiographic image of the needle insertion into the left ventricle in systole. (E) Echocardiographic image of the needle pierced through the anterior leaflet. Please click here to view a larger version of this figure.
Figure 4: Imaging of the procedure. (A) Baseline echo 2 chamber view prior to creating MR; (B) 23 G needle, visualized on echo during beating heart, advanced into the left atrium through the anterior mitral valve leaflet; (C) Color Doppler imaging showing MR jet seen in systole. Please click here to view a larger version of this figure.
Figure 5: Representative echo images to validate MR severity at 2 weeks post-surgery. (A) Left atrial area traced in white and MR jet area traced in red; (B) MR VTI trace in red; (C) Pulmonary flow showing systolic reversal. Please click here to view a larger version of this figure.
Figure 6: Needle puncture. (A) Orientation of needle puncture on an ex vivo heart. Needle punctured through apex of the LV at an angle, a longitudinal section of the LV with the needle directed towards the mitral valve leaflet, and the needle punctured through the mitral valve leaflet into atrial space. (B) Representative explant photograph depicting a hole in the anterior mitral leaflet. Please click here to view a larger version of this figure.
Figure 7: Gross morphology of whole hearts of a sham operated control rat (A) and a rat which underwent MR surgery (B) 2 weeks post-surgery. The rat with severe MR has significant left ventricular dilation and chamber enlargement compared to the sham operated control. Please click here to view a larger version of this figure.
Baseline (n = 15) | 2wk MR (n = 15) | p-value | |
Left atrial area (mm2) | 25.03 ± 8.70 | 49.95 ± 14.78 | p < 0.0001 |
MR jet area (mm2) | 0 | 21.15 ± 8.11 | p < 0.0001 |
MR fraction (%) | 0 | 41.91 ± 8.30 | p < 0.0001 |
MR VTI (cm) | 0 | 39.72 ± 7.52 | p < 0.0001 |
S wave (m/s) | 0.39 ± 0.07 | -0.51 ± 0.41 | p < 0.0001 |
D wave (m/s) | 0.44 ± 0.04 | 0.70 ± 0.17 | p < 0.0001 |
S/D wave ratio | 0.91 ± 0.17 | -0.69 ± 0.65 | p < 0.0001 |
Table 1: Mitral regurgitation characteristics.
A reproducible rodent model of severe MR with good survival (93.75% survival after surgery) and without significant post-operative complications is reported. Real-time imaging with transesophageal echocardiography and introduction of a needle into the beating heart to puncture the mitral leaflet are feasible and can be taught. Severe MR was produced with the 23 G needle size in this study, which can be varied as desired using a smaller or larger needle. MR induced in this model creates a low-pressure volume overload on the left ventricle, which is a better representation of clinically observed mitral valve lesions. Severe left atrial and left ventricular dilatation are observed within two weeks after MR onset in this model, but without contractile dysfunction measured by ejection fraction. Analogous to such a situation are patients with primary MR, who remain asymptomatic without heart failure for prolonged periods, despite progressive dilatation of their left sided cardiac chambers.
This MR model of volume overload differs in several ways from the widely used aorto-caval fistula model of volume overload. Procedural ease of ACF, which requires a simple laparotomy without the need for intubation and mechanical ventilation, has encouraged its adoption by the scientific community12. Despite its clear procedural advantages, arterio-venous fistulae shunt a large volume of blood into the vena cava, which overloads the venous reservoir, and also the right ventricle. Elevated central venous pressure from venous congestion may induce hepatic congestion and suboptimal renal filtration, which may cause hepatic fibrosis or activation of the renin-angiotensin-aldosterone (RAAS) system. The confounding effect of the RAAS system on ventricular-arterial coupling is known, and thus the ACF model fails to present a true volume overload on the left ventricle as seen in the setting of mitral regurgitation. When compared to the mitral valve defect model, lack of afterload reduction further diverges this model from the clinical situation of MR. Altogether, a significant different hemodynamic stress on the LV in the ACF model, introduces rapid changes with pronounced hypertrophy, dilation, and dysfunction that were not observed in our model13.
Beyond the novelty of introducing MR with a needle stick, our model has multiple applications in answering clinically important questions. Patients with primary MR that emerges from a mitral valve lesion often are asymptomatic for long periods and receive correction of their MR only at the onset of pulmonary or cardiac failure symptoms. Recent clinical data indicates that such delayed correction of MR does not enable functional recovery of the left ventricle, despite relief of fatigue and symptoms14. In a recent study using this rodent model, we demonstrated that MR introduces rapid and early remodeling of the cardiac extracellular matrix, which is a precursor of structural changes in the left ventricle10. Such mechanistic insights that provide a physiological basis for mitral valve intervention can be developed using this model. Combined with cardiac imaging, it is possible to develop biomarkers that represent these early left ventricular changes to guide the timing of intervention. Additionally, this model of MR can be combined with ventricular cardiomyopathies such as ischemic, non-ischemic and other etiologies, to understand the effect of MR on remodeling of diseased left ventricles. For example, secondary MR, a frequent occurrence in myopathic ventricles after an infarction or with chronic ischemia, is a lesion that is clinically challenging to manage. Whether MR is a bystander in this disease state and a product of LV dysfunction, or if it actively contributes to cardiac remodeling are controversial. We recently extended this model of MR to investigate if post-infarction hearts with MR differ in their cardiac remodeling potential compared to those without MR11, elucidating potential mechanisms involved in worsening heart failure in patients with MR. This model provides the flexibility to investigating the impact of early onset versus late onset of MR on cardiac remodeling to failure, which could have a significant clinical impact in guiding interventions.
As with any experimental model, there are some advantages and limitations that should be considered when applying results from animals to humans. The clear advantage of this model is the reproducible severity of MR, which aids in understanding cardiac chamber remodeling in clinically diagnosed conditions such as primary MR from chordal rupture. The increase in cardiac chamber volumes observed in this model and extracellular matrix remodeling observed in the myocardium represent the changes observed previously in larger animals and humans with primary MR14,15. The limitation of this leaflet perforation model is that MR develops acutely, representing only a subset of patients with primary MR from acute chordal rupture. Notwithstanding the limitations, acute onset of MR accounts for a significantly large patient population that undergo mitral valve interventions, and this model is very relevant to such a situation. Another limitation of this model is that MR is not reversible or repairable, which does not enable studies on the effect or timing of intervention on cardiac remodeling.
The authors have nothing to disclose.
This work was funded by grant 19PRE34380625 and 14SDG20380081 from the American Heart Association to D. Corporan and M. Padala respectively, grants HL135145, HL133667, and HL140325 from the National Institutes of Health to M. Padala, and infrastructure funding from the Carlyle Fraser Heart Center at Emory University Hospital Midtown to M. Padala.
23G needle | Mckesson | 16-N231 | |
25G needle, 5/8 inch | McKesson | 1031797 | |
4-0 vicryl | Ethicon | J496H | |
6-0 prolene | Ethicon | 8307H | |
70% ethanol | McKesson | 350600 | |
ACE Light Source | Schott | A20500 | |
ACUSON AcuNav Ultrasound probe | Biosense Webster | 10135936 | 8Fr Intracardiac echo probe |
ACUSON PRIME Ultrasound System | Siemens | SC2000 | |
Betadine | McKesson | 1073829 | |
Blunted microdissecting scissors | Roboz | RS5990 | |
Buprenorphine | Patterson Veterinary | 99628 | |
Carprofen | Patterson Veterinary | 7847425 | |
Chest tube (16G angiocath) | Terumo | SR-OX1651CA | |
Disposable Surgical drapes | Med-Vet | SMS40 | |
Electric Razor | Oster | 78400-XXX | |
Gentamycin | Patterson Veterinary | 78057791 | |
Heat lamp with table clamp | Braintree Scientific | HL-1 120V | |
Hemostatic forceps, curved | Roboz | RS7341 | |
Hemostatic forceps, straight | Roboz | RS7110 | |
Induction chamber | Braintree Scientific | EZ-1785 | |
Injection Plug, Cap, Luer Lock | Exel | 26539 | |
Isoflurane | Patterson Veterinary | 6679401725 | |
Mechanical ventilator | Harvard Apparatus | Inspira ASV | |
Microdissecting forceps | Roboz | RS5135 | |
Microdissecting spring scissors | Roboz | RS5603 | |
Needle holder | Roboz | RS6417 | |
No. 15 surgical blade | McKesson | 1642 | |
Non-woven sponges | McKesson | 446036 | |
Otoscope | Welch Allyn | 23862 | |
Oxygen | Airgas Healthcare | UN1072 | |
Pulse Oximeter | Nonin Medical | 2500A VET | |
Retractor, Blunt 4×4 | Roboz | RS6524 | |
Rodent Surgical Monitor | Indus Instruments | 113970 | The integrated platform allows for monitoring of vital signs and surgical warming |
Scale | Salter Brecknell | LPS 150 | |
Scalpel Handle | Roboz | RS9843 | |
Silk suture 3-0 | McKesson | 220263 | |
Small Animal Anesthesia System | Ohio Medical | AKDL03882 | |
Sterile saline (0.9%) | Baxter | 281322 | |
Sugical Mask | McKesson | 188696 | |
Surgical cap | McKesson | 852952 | |
Surgical gloves | McKesson | 854486 | |
Syringe 10mL | McKesson | 1031801 | |
Syringe 1mL | McKesson | 1031817 | |
Ultra-high frequency probe | Fujifilm Visualsonics | MS250 | |
Ultrasound gel | McKesson | 150690 | |
VEVO Ultrasound System | Fujifilm Visualsonics | VEVO 2100 |