Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Summary

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Abstract

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

Introduction

Patients with acquired and congenital cardiovascular diseases, including pulmonary hypertension (PH), are at risk of right ventricular (RV) dysfunction and failure¹. RV adaptation as a result of increased pressure load is characterized by concentric hypertrophy in early stages and progressive dilatation in end-stage disease. Furthermore, it is associated with disorders in metabolism and the extracellular matrix, processes of inflammation and, eventually, RV failure^2,3,4,5,6. Animal models have been developed to explore the underlying processes of the progression towards RV failure. However, optimization of models and adequate assessment of RV function and dimensions has been challenging. For noninvasive assessment of RV function and dimensions, cardiac magnetic resonance (CMR) imaging is the golden standard. This technique creates images of the beating heart by using a strong magnetic field and radiofrequency waves. CMR is available for humans, and for animals such as laboratory rodents. As the latter require higher spatial resolution due to the smaller size of the heart, the magnetic field required to provide adequate images must be higher, compared to humans.

Multiple models mimicking RV pressure overload are available, including models of PH^{7,8,9,10,11,12,13,14,15,16,17} and models of proximal RV pressure load^{2,3,10,18,19,20,21,22,23}. The choice of either a model of PH or a model of proximal RV pressure load depends on the research question: the effect of an intervention on the pulmonary vasculature and therefore possibly RV afterload modulation (i.e., PH models), or the direct effect on the RV (i.e., proximal RV pressure load models). Several methods for experimental induction of PH are available, including use of monocrotaline (MCT)^{12,13,14,16,22,24,25,26}, MCT combined with an aortocaval shunt⁹, chronic hypoxia^7,27,28,29, and the combination of a vascular endothelial growth factor receptor antagonist, Sugen 5416, with chronic hypoxia^8,10,30,31. Such models represent progressive pulmonary models of proximal RV pressure load and are not targeted at the pulmonary vasculature but induce a constant afterload by constriction of the pulmonary artery, with an accompanying increase of RV afterload^2,3. This can be performed by a suture-banding (pulmonary artery banding, PAB) or a vascular clip around the pulmonary artery. PAB has been performed in several animal species, and cardiac dimensions and function have been studied in various ways, such as histology, transthoracic echocardiography (including speckle tracking), and heart catheterization^{2,32,33,34,35,36,37,38,39,40}. PAB in small rodents, such as mice, is challenging. This is because subtle differences between the tightness of artery constriction have marked results on the degree of RV pressure load and subsequent functional status and survival. When the constriction is very tight, the animal will die during or shortly after operation, whereas the desired phenotype will not be achieved when the constriction is not tight enough. However, the use of mice has advantages compared to other animals, because of the excellent genetic modification possibilities (i.e., transgenic or knockout models) and fast breeding. This is of added value in the study of diseases and in exploring the contribution of molecular and (epi-) genetic factors.

Animal study designs are shifting towards the investigation of temporal changes during disease^2,3,8,13,21. For such studies, noninvasive modalities are necessary, because serial assessments can be performed. Alternatives to CMR in the assessment of cardiac remodeling could be (1) tissue characterization using histopathology, with multiple animals being sacrificed at different time points, (2) invasive functional assessment by pressure-volume analysis, or (3) echocardiography, which allows the researcher to identify cardiac hypertrophy or dilatation noninvasively within the same animal serially. CMR has two major advantages in assessment of the RV: (1) CMR is a noninvasive modality, enabling serial measurements in one animal, hereby contributing to reducing animal numbers needed for studies, and (2) CMR does not rely on a particular geometric shape and visualizes three-dimensionally. CMR-derived RV volumes and function measurements have been shown to be accurate and are considered to be the noninvasive golden standard in different cardiac entities in humans^42,43,44,45, but had not yet been translated to a CMR protocol for mice with RV pressure overload.

Many models of PAB are described in the literature, but with high heterogeneity in methods of assessing hemodynamic effects and RV function and adaptation. This protocol outlines the procedure of PAB in mice with validation of the model by measuring the PAB gradient by echocardiography and evaluating cardiac dimensions and function with CMR. While a protocol of CMR in animals subjected to PAB has been published for rats, this combination has not been described for mice until now. While rats are most commonly used for PH models^{8,12,13,14,15,16,22,24,25,26,27,28,29,30,31,46}, mice are most often used for transgenic or knock-out studies and thereby contribute to our understanding of mechanisms in pressure-loaded RV failure. This protocol could form the basis for future studies to unravel signaling pathways involved in the transition towards RV failure.

Protocol

All experiments and animal care are conducted according to the Dutch Animal Experimental Act and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The Animal Experiments Committee of the University of Groningen, the Netherlands, approved the current experimental protocol (permit number: 2014-041/3005). 1. Housing and acclimatization Use 20–30 g wild type C57 black 6 (C57BL/6) mice (institutional breeding line de…

Representative Results

Mortality rate of the PAB surgical procedure is around 10%. The presented results show characteristics of mice in the sham (n = 5) and PAB (n = 8) groups. As shown in Figure 3, PAB gradient values significantly increased compared to sham animals at 2 and 6 weeks after PAB. This increase of loading caused RV dilatation expressed as increased RV, EDV, and RV ESV (Figure 4A,B). RV dysfunction occurred as RV EF decreased (Figure…

Discussion

This protocol provides a reproducible method for PAB in mice and the subsequent assessment of cardiac remodeling and functional adaptation using CMR.

PAB differs from other models of increased RV pressure load because it involves absolute and static increase of afterload without the presence of other triggers. RV pressure load in models of hypoxia, monocrotaline, shunt, or a combination of these inducers are based on remodeling of the pulmonary vasculature. This remodeling is driven by endothe…

Materials

14.0 MHz i13L-echocardiography transducer	GE Healthcare, Waukesha, WI, USA
20G cannula
23G needle
9.4T magnetic resonance scanner with 1,500 mT/m gradient set	Bruker BioSpin, Ellingen, Germany
Anesthesia induction chamber
Blunt 25G needle
Buprenorphine
Chloride-hexidine
CMR post-processing software	Medis Medical Imaging Systems, Leiden, The Netherlands		Qmass version 7.6
Data visualisation and statistical software	GraphPad Prism Inc, La Jolla, CA, USA		software version 7.02
Echocardiography machine	GE Healthcare, Waukesha, WI, USA		Vivid Dimension 7
Eye ointment
Heat mat
Incubator (37°C)
Isoflurane
Isoflurane evaporator
Miniventilator for rodents	Hugo Sachs	model 687
monofilament polypropylene 5-0 sutures
monofilament polypropylene 6-0 sutures
Needle and syringe for subcutaneous injections
Pediatric electrocardiogram-stickers
pure polyglycolic acid 5-0 sutures
Sterile surgical instruments
Ventilation mask