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^4,5,6. Coronary microvascular dysfunction involving both functional and structural abnormalities underlies subendocardial ischemia, diffuse interstitial fibrosis, and left ventricular dysfunction in HFpEF^7,8. Extensive research has been conducted on the pathological role of CMD in cardiovascular diseases^9,10. A clinical study showed that microvascular dysfunction (evidenced by an impaired CFR <2.0) is directly associated with worse left ventricular diastolic function and may be a promising therapeutic target in HFpEF¹¹. Echocardiographic assessment in the prevalence of microvascular dysfunction in heart failure with preserved ejection fraction (PROMIS-HFpEF) study revealed that 75% of patients with HFpEF had CMD, based on CFR measurements in the left coronary artery¹². Furthermore, the abnormal CMD correlated with an increased risk of adverse outcomes, including significantly increased mortality hazard^13,14. Consequently, employing CMD as a key biomarker in animal models greatly facilitates the investigation of HFpEF pathophysiology and assists in validating the successful replication of the disease phenotype.

Currently, methods for assessing coronary microvascular function, such as positron emission tomography (PET), cardiovascular magnetic resonance (CMR), fractional flow reserve (FFR), and index of microcirculatory resistance (IMR) are difficult to apply in rodent studies due to the small size of the animals and the limited availability of dedicated imaging hardware. Coronary flow reserve (CFR) is recommended by international guidelines as a tool for interrogating coronary physiology^15,16. Moreover, CFR serves as a reliable marker of cardiac ischemia, a gauge of coronary vascular dysfunction, and a valuable prognostic indicator of cardiovascular risk¹⁷. Doppler echocardiography for CFR assessment is a validated and reproducible method for evaluating global coronary vascular function in preclinical rodent models. Although prior studies have reported the use of CFR in mouse/rat models of myocardial ischemia-reperfusion injury^18,19, it has never been applied in animal models of HFpEF. Moreover, the CFR assessment method described in this study, which rapidly locates the LCA using color Doppler echocardiography, can be extended to a range of animal models, including those of diabetic cardiomyopathy, hypertensive heart disease, and chronic inflammatory or autoimmune disorders. In the present study, given the absence of epicardial coronary stenosis in the murine model, the reduction in coronary flow reserve (CFR) primarily reflects coronary microvascular dysfunction rather than combined epicardial and microvascular contributions. Thus, CFR serves as a reliable surrogate marker for CMD in this HFpEF model.

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, the mice were anesthetized with 1.5% isoflurane and euthanized by cervical dislocation. The reagents and equipment used are listed in the Table of Materials.

1. Establishment of the HFpEF mouse model

1. Randomly allocate mice into two groups: a control (CTRL) group and an HFpEF group, with eight mice in each.
2. Employ an established protocol^{21,22,23,24,25} to induce the HFpEF mouse model as follows:
   1. Feed six- to eight-week-old mice a high-fat diet (HFD, 60% fat) and provide 0.5 g/L N-nitro-L-arginine methyl ester (L-NAME) dissolved in drinking water for 8 weeks.
      NOTE: Prepare L-NAME drinking water in brown bottles to avoid light exposure and refresh every 2–3 days.
3. Feed age-matched mice a standard normal diet and provide regular drinking water for the same duration as the control group.

2. Assessing coronary flow reserve by echocardiography

1. Precede echocardiographic assessment by anesthetizing the mouse via inhalation of 1.5% vaporized isoflurane administered at 1.5–2.0 L/min with 100% O₂ flow in an anesthesia chamber.
   CAUTION: Isoflurane is a volatile anesthetic. Ensure adequate ventilation during operation. Wear nitrile gloves when adding liquid isoflurane to the vaporizer to avoid skin contact.
2. Confirm absence of reaction to skin pinching and toe or tail stimulation, then position the animal supine on the imaging platform.
3. Switch the gas anesthesia machine from chamber to mask delivery and maintain a constant 1.5% anesthetic concentration throughout the procedure.
4. Acquire physiological data (ECG and respiration) by applying conductive gel to the copper sheet (ECG electrodes) of the imaging platform and taping the four paws onto them.
   NOTE: Maintain isoflurane at 1.5% for maintenance and keep core body temperature close to 37 °C using the built-in warming platform. Maintain a target breathing rate of 100–140 breaths per minute to ensure physiological stability during imaging.
5. Remove chest hair by applying depilatory cream with a cotton-tipped applicator.
6. Wipe away excess cream and loose fur with water-soaked gauze to prepare for imaging.
   NOTE: Completely remove residual depilatory cream to avoid skin irritation or toxicity.
7. Apply sterile lubricating ophthalmic ointment to both eyes to prevent scleral drying.
8. Apply ultrasound transmission gel to the anterior heart area and position the imaging platform at approximately a 30°–45° angle to the horizontal.
   NOTE: Avoid air bubbles in the ultrasound gel.
9. Place the high-frequency ultrasound transducer slowly on the parasternal line and align it parallel to the thorax to obtain the adjusted parasternal long-axis (PLAX) view of the left ventricle (Figure 1A).
10. Angle the probe to ensure light contact with the chest wall, avoiding compression.
11. Press the Bright Mode button to switch to brightness mode imaging.
12. Adjust the angle of the probe and imaging platform to locate the left coronary artery (LCA).
    NOTE: Identify the left ventricle (LV), left atrium (LA), left ventricular anterior wall (LVAW), left ventricular posterior wall (LVPW), interventricular septum (IVS), aorta (AO), pulmonary artery (PA), pulmonary vein (PV), mitral valve (MV), and papillary muscle within the acoustic window.
13. Activate color doppler mode by pressing the Color Doppler button on the touchscreen. In real-time display, flow away from the aortic valve (toward the probe) appears red.
14. Adjust the probe angle and position to visualize the complete LCA from the aortic sinus to the distal branch site while minimizing interference from pulmonary vein flow.
15. Activate pulse-wave doppler (PW) mode. Position the PW measurement window parallel to the LCA blood flow direction (Figure 1B,C).
    NOTE: Maintain the insonation angle below 60° and apply real-time angle correction by aligning the Doppler cursor parallel to coronary flow.
16. Select Save Clip to capture the baseline LCA flow velocity waveform.
17. Increase the isoflurane concentration to 3% and observe the gradual increase in coronary flow velocity.
18. After 45 s of continuous exposure to 3% isoflurane, select Save Clip to capture hyperemic coronary flow velocity.
    NOTE: Continuously monitor LCA blood flow. If the PW sample volume drifts due to respiratory or cardiac motion, pause acquisition, return to Color Doppler mode to re-identify the LCA, reposition the PW gate, and resume PW mode.
19. Upon completion, discontinue isoflurane inhalation and remove ultrasound gel. Place the mouse on a heating pad until it recovers fully, then return it to its cage.

3. Statistical analysis

1. Average measurements from three consecutive cardiac cycles traced in the analysis software to obtain baseline and peak LCA flow velocities. Calculate CFR as the ratio of hyperemic peak diastolic coronary flow velocity to baseline peak diastolic coronary flow velocity (CFV).
2. Implement a double-blind design between the experimenter and data analyst. Assess normality using the Shapiro–Wilk test. Analyze normally distributed data using a two-tailed unpaired Student’s t-test. Analyze non-normally distributed data using the Mann–Whitney U test.

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