Surgically Induced Cardiac Volume Overload by Aortic Regurgitation in Mouse

Summary

This protocol presents a practical guide on the surgery for creation of aortic regurgitation (AR) in the mouse. Assessment of the AR mouse by echocardiography and invasive hemodynamic measurement recapitulates its clinically relevant characteristics of volume overload-induced eccentric hypertrophy, suggesting its promising application in the study of cardiac hypertrophy.

Abstract

Aortic regurgitation (AR) is a common valvular heart disease that exerts volume overload on the heart and represents a global public health problem. Although mice are widely applied to shed light on the mechanisms of cardiovascular disease, mouse models of AR, especially those induced by surgery, are still paucity. Here, a mouse model of AR was described in detail which is surgically induced by disruption of the aortic valves under high-resolution echocardiography. In accordance with regurgitated blood flow, AR mouse hearts present a distinctive and clinically relevant volume overload phenotype, which is characterized by eccentric hypertrophy and cardiac dysfunction, as evidenced by echocardiographic and invasive hemodynamic evaluation. Our proposal, in a reliable and reproducible manner, provides a practical guide on the establishment and assessment of a mouse model of AR for future studies on molecular mechanisms and therapeutic targets of volume overload cardiomyopathy.

Introduction

In the presence of increased volume overload (preload) or pressure overload (afterload), the heart undergoes enlargement, a condition termed hypertrophy. Although cardiac hypertrophy is a compensatory response to maintain perfusion of peripheral organs before cardiac failure, it is also an independent risk factor for major cardiovascular events^1,2. Volume overload is one of the important manifestations of increased mechanical stress. Volume overload occurs during cardiac diastole and induces eccentric cardiac hypertrophy, which is not only commonly seen in valvular diseases, such as aortic regurgitation and mitral regurgitation, but also in end-stage hypertensive heart disease, myocardial infarction, dilated cardiomyopathy, and excessive exercise. In addition, in clinical practice, some drugs that can better reduce the myocardial hypertrophy induced by pressure overload have unsatisfactory effects in the treatment of myocardial hypertrophy induced by volume overload¹. It is therefore of great significance to discover the mechanism and intervention methods of eccentric cardiac remodeling caused by volume overload. However, such research on volume overload has been significantly hampered for a long time, which can be, in large part, attributed to the lack of small animal models that can be easily operated, efficiently quantified, and stably replicated³.

As for small animal species, mice have become the mainstream model animal for cardiovascular disease research due to their short life cycle, convenient operation, clear genome, and ease of genetic modification⁴. In terms of model categories, compared to genetic modification models and drug-treated models, surgical models have obvious unique advantages. The surgical model can avoid excessive and laborious mouse breeding and gene identification that are necessary for the genetic modification model and can also avoid the non-specific effects on extracardiac tissues and organs that are difficult to control in drug-treated models. The mouse model of aortocaval shunt has been documented to induce cardiac volume overload in previous literature⁵. However, aortocaval shunt accounts for a small fraction of cardiac eccentric hypertrophy in the clinic and causes biventricular overload⁵, making it of little translational significance to be used in left ventricular eccentrical hypertrophy study. Nevertheless, valvular heart disease represents a major public health problem worldwide; it is estimated that around 15% of the population >75 years of age has significant valvular disorder⁶. Although aortic regurgitation (AR) occupies a portion of valvular heart disease, it distinctively causes eccentric left ventricular (LV) hypertrophy due to an increase in volume overload by regurgitant blood flow^7,8. Considering the right common carotid artery (RCCA) provides a route to reach the location of aortic valves, it is conceptually intriguing to disrupt aortic valves via the RCCA to cause regurgitant blood flow in mice. Inspired by the techniques of creating oscillating aortic flow⁹, a mouse model of aortic regurgitation (AR) was recently established in our lab to surgically induce volume overload⁷. This AR mouse demonstrates obvious LV eccentric hypertrophy, which is a clinically transformative approach and demonstrates a great translational potential for studying the overloaded heart phenotype and its underlying mechanism. Here, a detailed step-by-step procedure was described to perform AR surgery in mice, recapitulated by high frequency echocardiography and invasive hemodynamics to ensure the success of the surgery (Figure 1).

Protocol

This protocol has received ethical approval from the Animal Care and Use Committee of Zhongshan Hospital, Fudan University, and follows the recommendations of Guide for the Care and Use of Laboratory Animals (No. 85-23, revised 2011; National Institutes of Health, Bethesda, MD, USA). NOTE: Animal experiments were performed on male C57BL/6J mice >10 weeks of age. The surgeon in this protocol should be skillful in the manipulation of murine echocardiography, before he/she performs the AR ope…

Representative Results

To guarantee successful AR, we validated regurgitant blood flow using color Doppler and pulse wave Doppler echocardiography. In mice with AR, the color Doppler spectrum of the aortic arch showed regurgitant flow (red) immediately post-operation, which was absent in sham mice (no flow in diastole; Figure 3A). Consistently, the pulse wave Doppler demonstrated robustly elevated regurgitant flow in AR mice (Figure 3B,C). With a further confirmation …

Discussion

The surgical induction of AR in the mouse is a technically challenging, new technique but has significant translational relevance. To master the technique, a surgeon should at least be familiar in advance with murine cervical and cardiac anatomy, mouse handling, and echocardiography. Skillful operation in invasive hemodynamic measurement is a plus. For successful AR operation, special care should be taken on several critical steps.

Cutting open the RCCA is the most crucial step. The hole on th…

Materials

Copper plate	JD.com Inc.	Customized	20 X 15 cm or bigger is prefeered
Curved Tying forceps	66 Vision Tech	53324A	to stretch and isolate muscle, tissue, and vessel
Heating pad	JD.com Inc.	Changzhi 55	warm the copper plate and mouse by the way
Long-handed Curved Tying Forceps	MECHENIC	TS-15	to stretch vessel
Metal Wire (stainless steel)	JD.com Inc.	0.18 mm in diametter	work with a plastic catheter to puncture aortic valves
Needle Holder	Shanghai Jinzhong	131110	suture of skin
Plastic Catheter	Anilab software & instruments	PE-0402	work with a metal wire to puncture aortic valves
Pressure Catheter	Millar Instruments	SPR 835	1.4F in size
Pressure Data Acquisition Device and Analog/Digital Converter	AD Instruments	Labchart 5	connected with pressure catherter
Scissor	Suzhou Shiqiang	Stronger 13Cr	to cut skin
Smallpinch Scissors	Shanghai Jinzhong	YBE030	to cut vessel
Stereomicroscope	Olympus Corporation	SMZ845	for incision and intubation of vessel
Straight Tying forceps	66 Vision Tech	53320A	to stretch and isolate muscle, tissue, and vessel
Thumbforceps	Suzhou Shiqiang	5307B	to clamp and stretch skin and muscle
Ultrasound Gel	PARKER	Aquasonic-100	to transfer ultrasound signal
Ultrasound Imaging System	VisualSonics	2100	includes B-mode, M-model, color Doppler and pulse wave Dopper
Vaporizer	RWD Life Science	R540	for anesthesia