Assessment of Coronary Flow Reserve in a Mouse Model of Heart Failure with Preserved Ejection Fraction Using Doppler Echocardiography

Summary

This protocol describes a rapid, non-invasive approach to assess coronary microvascular dysfunction in a mouse model of heart failure with preserved ejection fraction (HFpEF). Coronary flow reserve (CFR) is determined by Doppler ultrasound-guided localization of the left coronary artery, calculated as the ratio of hyperemic to baseline coronary flow velocity.

Abstract

Heart failure with preserved ejection fraction (HFpEF) constitutes a considerable global health burden and a major clinical challenge due to the absence of effective therapies, and the underlying pathophysiology remains elusive. Coronary microvascular dysfunction (CMD), which is highly prevalent in patients with HFpEF, triggers myocardial ischemia that impairs both left ventricular diastolic and systolic function. Given that microvascular dysfunction is a central feature of HFpEF, CMD is now considered an important factor in its pathogenesis. Coronary flow reserve (CFR), defined as the ratio of maximal hyperemic to resting blood flow velocity, is a highly valuable marker of myocardial ischemia. Furthermore, it serves as a comprehensive indicator of coronary vasomotor dysfunction, measuring the combined hemodynamic effects of both epicardial and microvascular coronary arteries on myocardial perfusion. This protocol focused on assessing the changes in CFR in a mouse model of HFpEF using Doppler echocardiography. In this study, control mice showed a greater than twofold increase in peak coronary blood flow velocity with isoflurane-induced vasodilation compared to resting values, whereas this response was significantly attenuated in HFpEF mice. In HFpEF, a reduced CFR predicts adverse outcomes and reflects underlying microvascular dysfunction, positioning it as a key tool for studying disease progression and guiding patient selection.

Introduction

Heart failure (HF) with preserved ejection fraction (HFpEF) has been the dominant form of HF and is associated with poor prognosis. However, there is no proven treatment to reduce its related morbidity and mortality^1,2. The presence of multiple risk factors, including advanced age, hypertension, diabetes, dyslipidemia, and obesity, underlies the complexity and heterogeneity in HFpEF³. In recent years, accumulating evidence has shown that coronary microvascular dysfunction (CMD) is an independent and direct risk factor for HFpEF, rather than a comorbidity

Protocol

All procedures complied with the protocols approved by the Institutional Ethics Committee of Nanjing Drum Tower Hospital (Approval No. 20011141) and adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines²⁰. In this study, the HFpEF model was established in six- to eight-week-old male C57BL/6J mice (BW, ~25 g). Throughout the experimental duration, mice were housed in a specific-pathogen-free (SPF) room under controlled conditions of temperature (23 ± 1 °C), humidity (65%–70%), and a 12 h light/dark cycle. All animals had ad libitum access to food and water. After the study, t....

Results

In this study, 16 mice (HFpEF, n = 8; CTRL, n = 8) were used. The left coronary artery was identified under color Doppler guidance in the parasternal long-axis (PLAX) view, and coronary flow velocity was measured using pulsed-wave (PW) Doppler. Following the acquisition of sufficient and reproducible images from each mouse at both 1.5% (baseline) and 3% (hyperemic) isoflurane concentrations²⁶ CFR was determined by averaging measurements from at least 3 consecutive cardiac cycles; representative re.......

Discussion

This study presents a protocol that uses a non-invasive, straightforward, and reproducible method to assess macro- and microvascular dysfunction in HFpEF animal models. Doppler echocardiography for assessing Coronary Flow Reserve is recognized by the European Society of Cardiology as a validated and reproducible method¹⁵. Under normal physiological conditions in C57BL/6J mice, the coronary flow reserve is expected to be ≥2.0. In this study, compared to the CTRL group, HFpEF mice demonst.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Animal physiology monitor	Fujifilm	VEVO 3100	Monitor heart rate,respiration rate and body temperature
C57 mice	GemPharmatech Co., Ltd
Camera	Canon,Japan	EOS R50	Record the positions of the imaging platform and the ultrasound probe
Depilatory cream	Veet,French		Hair removal
Gauze	Curity	CAR-6339-PK	Sterile
Isoflurane	REWARD	R510-22-10	Anesthesia
N-nitro-L-arginine methyl ester (L-NAME)	Sigma-Aldrich	N5751	HFpEF mice model introduction
Research Diets, 60% fat	Research Diets, Inc.	D12492	High-fat diet
Surgical thermostatic heating pad	Globalebio, China	GE0-20W	Temperature control
Ultrasound Gel	Parker Laboratories	W60698L	Ultrasound transmission gel
Vevo 3100 preclinical imaging platforn	Fujifilm	VEVO 3100	Echocardiography
VevoLAB software	Fujifilm	VevoLAB 3.2.6	Echocardiography data analysis