Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Elizabeth Hillier; Jason Covone; Matthias G. Friedrich

doi:10.3791/64149

Medicine

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Published: August 17, 2022 doi: 10.3791/64149

Elizabeth Hillier^1,2, Jason Covone¹, Matthias G. Friedrich^1,3

¹Faculty of Medicine and Health Sciences, McGill University, ²Faulty of Medicine and Dentistry, University of Alberta, ³Departments of Medicine and Diagnostic Radiology, McGill University

Summary

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.

Abstract

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.

OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.

As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

Introduction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of MR contrast¹^,²^,³. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation as a marker for vascular function, thereby circumventing the need for any extrinsic, intravenous contrast or pharmacologic stress agents ⁴^,⁵^,⁶.

Breathing maneuvers, including breath-holds and hyperventilation, are highly effective vasoactive measures to alter vasomotion and, because of their safety and simplicity, are ideal for controlled endothelial-dependent vasomotion as part of a diagnostic procedure. Studies have shown an added effectiveness when combining hyperventilation with a subsequent breath-hold⁴^,⁷, as during such a protocol, the vasoconstriction (through the associated decrease of blood carbon dioxide) is followed by vasodilation (increase of blood carbon dioxide); thus, a healthy vascular system transitions through the entire range from vasoconstriction to vasodilation with a strong increase in myocardial blood flow, which in turn increases myocardial oxygenation and, thus, the observable signal intensity in OS-CMR images. The use of cine images for the acquisition also allows for cardiac phase-resolved results with a better signal-to-noise ratio when compared to adenosine infusion⁸.

Breathing maneuvers can replace pharmacological stress agents for inducing vasoactive changes that can be used for assessing coronary vascular function. This not only reduces patient risk, logistical efforts, and associated costs but also helps in providing results that are clinically more meaningful. Pharmacologic stress agents such as adenosine trigger an endothelium-dependent response and, thus, reflect endothelial function itself. Such specific assessment of endothelial function so far was only possible by an intracoronary administration of acetylcholine as an endothelial-dependent vasodilator. This procedure, however, is highly invasive²^,⁹ and, therefore, rarely performed.

Lacking access to direct biomarkers, several diagnostic techniques have used surrogate markers such as tissue uptake of an exogenous contrast agent. They are limited by the need for one or two intravenous access lines, contraindications such as severe kidney disease or atrioventricular block, and the need for the physical presence of staff with training in managing potentially severe side effects¹⁰^,¹¹. The most significant limitation of current imaging of coronary function, however, remains that myocardial perfusion as a surrogate marker does not reflect myocardial tissue oxygenation as the most important downstream consequence of vascular dysfunction².

OS-CMR with vasoactive breathing maneuvers has been utilized to evaluate vascular function in numerous scenarios, including healthy individuals, macrovascular disease in patients with coronary artery disease (CAD), as well as microvascular dysfunction in patients with obstructive sleep apnea (OSA), ischemia with no obstructive coronary artery stenosis (INOCA), after heart transplantation, and heart failure with preserved ejection fraction (HFpEF)⁴^,⁷^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶. In a CAD population, the protocol for the breathing-induced myocardial oxygenation reserve (B-MORE) as derived from OS-CMR was proven to be safe, feasible, and sensitive in identifying an impaired oxygenation response in myocardial territories perfused by a coronary artery with a significant stenosis¹³.

In microvascular dysfunction, OS-CMR demonstrated a delayed myocardial oxygenation response in patients with obstructive sleep apnea, and a blunted B-MORE was found in patients with HFpEF and following heart transplantation¹²^,¹⁴^,¹⁶. In women with INOCA, the breathing maneuver led to an abnormally heterogeneous myocardial oxygenation response, highlighting the advantage of the high spatial resolution of OS-CMR¹⁵. This paper reviews the rationale and methodology for performing OS-CMR with vasoactive breathing maneuvers and discusses its clinical utility in the assessment of vascular pathophysiology in patient populations with microvascular dysfunction, specifically as they relate to endothelial dysfunction.

The physiological context of breathing-enhanced oxygenation-sensitive MRI
Under normal physiologic conditions, an increase in oxygen demand is matched by an equivalent increase in oxygen supply through increased blood flow, resulting in no change in local deoxyhemoglobin concentration. In contrast, induced vasodilation leads to "excess" inflow of oxygenated blood without a change in oxygen demand. Consequently, more of the tissue hemoglobin is oxygenated, and thus, there is less deoxyhemoglobin, leading to a relative increase in OS-CMR signal intensity⁴^,¹⁷. If vascular function is compromised, it cannot properly respond to an altered metabolic demand or stimulus to augment myocardial blood flow.

In the setting of a stimulus to elicit vasomotion, such as paced hyperventilation eliciting vasoconstriction or a long breath-hold eliciting carbon dioxide-mediated vasodilation, impaired vasomotor activity would result in a relative increase in local deoxyhemoglobin concentration compared with other regions, and, subsequently, a reduced change in OS-CMR signal intensity. In the setting of inducible ischemia, impaired vascular function would result in increased local demand not met by a local increase in myocardial blood flow even in the absence of epicardial coronary artery stenosis. In OS-CMR images, the net local increase in deoxyhemoglobin concentration leads to a decrease in local signal intensity²^,¹⁸^,¹⁹^,²⁰.

Attenuated vascular smooth muscle relaxation in response to endothelium-dependent and -independent vasodilators (including adenosine) has been demonstrated in patients with coronary microvascular dysfunction²¹^,²²^,²³^,²⁴^,²⁵^,²⁶^,²⁷. Endothelial-independent dysfunction is thought to be due to structural abnormalities from microvascular hypertrophy or surrounding myocardial pathology. In contrast, endothelial dysfunction results in both inadequate vasoconstriction and impaired (endothelium-dependent) vasorelaxation, typically caused by a loss of nitric oxide bioactivity in the vessel wall²¹^,²⁸. Endothelial dysfunction has been implicated in the pathogenesis of a number of cardiovascular diseases, including hypercholesterolemia, hypertension, diabetes, CAD, obstructive sleep apnea, INOCA, and HF²³^,²⁴^,²⁸^,²⁹^,³⁰^,³¹^,³². In fact, endothelial dysfunction is the earliest manifestation of coronary atherosclerosis³³. The imaging of endothelial function has very strong potential, given its role as a significant predictor of adverse cardiovascular events and long-term outcomes, with profound prognostic implications in cardiovascular disease states²³^,²⁹^,³⁰^,³¹^,³⁴^,³⁵.

In contrast to perfusion imaging, the breathing-induced myocardial oxygenation reserve (B-MORE), defined as the relative increase in myocardial oxygenation during a post-hyperventilation breath-hold allows for visualizing the consequences of such a vasoactive trigger on global or regional oxygenation itself²^,³⁶. As an accurate downstream marker of vascular function, B-MORE can, therefore, not only identify vascular dysfunction but also actual inducible ischemia, indicating a more severe local perfusion or oxygenation problem¹⁸^,¹⁹^,³⁷. This is achieved through the ability of OS-CMR to visualize the relative decrease in deoxygenated hemoglobin, which is abundant in the capillary system of the myocardium, which itself represents a significant proportion of myocardial tissue²⁴.

OS-CMR sequence
The magnetic resonance imaging (MRI) sequence used for OS-CMR imaging is a prospectively gated, modified, balanced, steady-state, free precession (bSSFP) sequence acquired in two short-axis slices. This bSSFP sequence is a standard clinical sequence available (and modifiable) on all MRI scanners that perform cardiac MRI, making this technique vendor-agnostic and easily implemented. In a regular bSSFP cine sequence, echo time, repetition time, and flip angle are modified to sensitize the resulting signal intensity to the BOLD effect and, thus, create an oxygenation-sensitive sequence. This approach, a T2-prepared bSSFP readout, has previously been shown to be suitable for acquiring oxygenation-sensitive images with a higher signal-to-noise ratio, higher image quality, and faster scan times when compared to previous gradient echo techniques used for BOLD imaging³⁸. Performing breathing-enhanced OS-CMR with this approach can be applied with very few, mild side effects (Table 1). Of note, more than 90% of participants complete this protocol with sufficiently long breath-hold times⁴^,¹²^,¹³^,¹⁶.

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Protocol

All MRI scans utilizing OS-CMR with vasoactive breathing maneuvers should be performed in compliance with local institutional guidelines. The protocol outlined below has been used in studies approved by several institutional human research ethics committee. Written consent was obtained for all the human participant data and results described in this protocol and manuscript.

1. Broad overview

Vary the inclusion and exclusion criteria depending on the study population of interest. Use the following general, common exclusion criteria for an OS-CMR with vasoactive breathing maneuvers protocol: general MRI contraindications (e.g., MRI-incompatible devices such as pacemakers or defibrillators, implanted material, or foreign bodies), consumption of caffeine or vasoactive medication in the 12 h prior to the MRI, and age <18 years.
First, acquire the standard clinical localizer scout and ventricular structure and function images before acquiring the OS acquisitions. Use the bSSFP long-axis cine images to plan the slice positioning of the OS acquisitions.
NOTE: A review of standardized clinical CMR protocols is described elsewhere³⁹.
Baseline breath-hold
1. Acquire the first OS-CMR series as a short, baseline breath-hold acquisition to assess image quality and slice location, check for artifacts, as well as serve as a signal intensity baseline.
2. Perform a short (~10 s), single cardiac cycle acquisition after the participant has been breathing normally. Ensure that the breath-hold is done on end-expiration.
Continuous acquisition with vasoactive breathing maneuver
1. Acquire the second OS-CMR series as a 2 min, continuous acquisition comprised of a short 15 s breath hold and 1 min of paced hyperventilation, followed by a voluntary, maximal breath hold (~45 s). As the continuous acquisition obtains multiple cardiac cycles over 2 min, modify one additional parameter (the number of cardiac cycles acquired by the acquisition) to make this series a repeated-measures acquisition.
  NOTE: The minimum required breath-hold length is 30 s, although a breath-hold of 45-60 s is considered the standard.
2. Convey the instructions for the vasoactive breathing maneuver to the participants in the MRI scanner by manually directing the participant throughout the breathing maneuver through a microphone connected to the MRI speaker system or through a prerecorded .mp3 file (Supplemental File 1) that can be played for the participant through the MRI speaker system.
3. Start the breathing maneuver with a short 15 s, and end the expiration breath hold to acquire one cardiac cycle. Then guide the participant through paced breathing with the use of audible beeps from a metronome at a frequency of 30 breaths/min (one beep indicates breathing in, one beep indicates breathing out). At the 55 s mark of hyperventilation, give a final voice command to "take a deep breath in and then breathe out and hold your breath" to ensure that the breath-hold is performed at an end-expiration level.
  NOTE: The change in blood CO₂ is much more pronounced with the breath-hold at end-expiration (the lung surface is smaller, minimizing the residual diffusion of CO₂ into the alveoli).
Image analysis
1. To measure the B-MORE, consider the first end-systolic image during breath-hold as time 0 s. Compare the global or regional signal intensity values of the end-systolic image acquired closest to the 30 s time point of the breath-hold to the image signal intensity at the 0 s time point. Report BMORE as a percent change in signal intensity at 30 s compared to time 0 s of the breath-hold.

2. Pre-scan procedure

Ensure that every participant passes the MRI safety and compatibility questionnaire of the local institution (MRI General Contraindication form), which should include questions on past medical and surgical history and identify the presence of any implant, device, or metallic foreign body inside or at the surgical site of the participant⁴⁰.
Obtain a pregnancy test, if applicable.
Verify that the patient has abstained from vasoactive medication and caffeine in the 12 h prior to the MRI scan.
Show the participant the instructional breathing maneuver video (Supplemental Video S1).
1. Perform a practice session of 60 s of paced hyperventilation followed by a maximal voluntary breath-hold with every participant outside of the MRI scanning room and provide feedback on the performance of the hyperventilation.
2. Instruct the participants that they can simply resume breathing when they have a strong urge to do so.
  NOTE: See the discussion for points to note and provide feedback on to the participants.

3. MRI acquisition of oxygenation-sensitive sequences

Modify three parameters from the standard bSSFP sequence on the MRI console: increase repetition time (TR), increase echo time (TE), decrease flip angle (FA).
NOTE: The modified values are dependent on THE MRI scanner field strength (Table 2). Increasing TR and TE and decreasing FA results in an increase in T2* or oxygenation sensitivity of the MRI sequence. These modifications will then result in an increase in bandwidth and base resolution of the sequence.
Create two OS series, a baseline (labeled: OS_base) and the continuous acquisition during which the breathing maneuver is performed (labeled: OS_cont_acq). Leave the baseline OS sequence unchanged. In the OS continuous acquisition, increase the repeated measures from 1 to ~15-40 (depending on the scanner type). Increase the number of cardiac cycles (measures) until the acquisition time is ~2.5 min.
NOTE: Two OS-CMR sequences are needed: OS baseline acquisition and OS continuous acquisition with vasoactive breathing maneuvers. The following sections describe these steps.

4. OS baseline acquisition

For slice prescription, plan in an end-systolic still frame of a long axis view (two- or four-chamber image). Prescribe two short-axis slices-one at the mid-to-basal and the other at the mid-to-apical ventricular level. See the discussion for points to consider regarding slice location.
Sequence parameter adjustments
1. Adjust the sequence parameters as needed for a given participant. See Table 3 for sequence parameters than can or cannot be changed.
2. Adjust the average gap/spacing between slices based on the size of the participant's heart and ensure proper slice location.
3. Adjust the field of view to avoid wrap artifacts if necessary. Make every effort to keep the field of view between 360 mm and 400 mm.
Shim volume
1. Adjust the shim volume to be tight around the left ventricle in both the long- and short-axis views.
Sequence acquisition
1. Approve the sequence and run it during the end-expiration breath-hold. Ensure that this baseline OS sequence lasts ~10 s, based on the heart rate and MRI scanner.
Image quality check
1. Check both slices of the acquired series-look for any respiratory motion, poor slice location, or the presence of artifacts. Repeat the baseline OS sequence until adequate image quality has been obtained.
For troubleshooting, if the slice location is too basal or too apical, adjust the prescribed slice location to be closer to the mid-ventricular level. If there is an artifact present, follow the steps below:
1. Check the phase encoding direction.
2. Make the field of view larger.
3. Adjust the shim volume around the left ventricle.

5. OS continuous acquisition with vasoactive breathing maneuvers

NOTE: Ensure that every participant has been instructed about the proper performance of the breathing maneuver before they are in the MRI scanner (see section 2).

Sequence planning
1. If possible, copy slice position and adjust volume from the OS baseline image or duplicate the baseline OS sequence and, in repeated measurements, increase from 1 to ~15-40 (or close to 2.5 min acquisition time).
Verify the image and slice positioning, and then capture cycle.
If possible, open the live stream window.
In the control room, plug a device with the breathing maneuver instructions .mp3 file into the auxiliary input or prepare to hold it over the microphone projecting into the MRI scanner. Alternatively, manually guide the participant through the breathing maneuver using a stopwatch for timing and verbally provide instructions through the microphone connected to the MRI speaker system.
Sequence acquisition
1. Simultaneously press play for the OS Continuous Acquisition sequence on the MRI scanner and play for the .mp3 breathing instruction file or start the stopwatch if the participant is being manually instructed.
2. If manually guiding the participant through the breathing maneuvers, instruct them to breathe in and breathe out, then hold their breath for 15 seconds (for the short breath-hold), and start hyperventilating as soon as they hear the metronome beep.
3. Notify the participant at the 40 s mark of hyperventilation (2:40 on the stopwatch).
4. At the 55 s time point of hyperventilation (2:55 on the stopwatch), instruct the participant to "take a deep breath in, breathe out, and hold your breath".
  NOTE: The free breathing and hyperventilation images will have motion artifacts. This is expected. However, there should not be any motion artifacts during the breath-hold. It is critically important that the breath-held images are acquired after exhalation (comfortable end-expiratory position). Only a breath-hold after exhalation leads to the rapid increase of blood CO₂ during the first 30 seconds of the subsequent breath-hold, with the associated change of coronary blood flow and myocardial oxygenation.
5. Monitor the participant's performance of the paced hyperventilation through the control room window or MRI scanner camera to ensure adequate performance of deep breathing. If bellows are used, then monitor the amplitude peaks on the respiratory gating viewer. If hyperventilation is not being adequately performed after initial guidance, abort the acquisition and repeat the OS continuous acquisition sequence.
6. Monitor for any small breaths taken by participants throughout the breath-hold. Do this by monitoring the tracing of a respiration belt on the MRI console or visually through the window/camera.
7. Once the participant starts breathing at the end of the breath-hold, stop the acquisition.
8. After the end of the acquisition, ask the participant if they experienced any adverse effects and allow the participant to breathe normally for 3 min.
Troubleshooting: repeat acquisition
1. If the breathing maneuvers need to be repeated, repeat the baseline OS sequence.
  NOTE: A period of 2-3 min before repeating the acquisition is required to allow the physiology to return to baseline. Previous data have shown that physiology does not return to baseline after 1 min⁴¹.
2. If the image quality of the OS baseline sequence is adequate, repeat the OS continuous acquisition and performance of the breathing maneuvers.

6. Image analysis

Import the OS-CMR image data sets into a DICOM viewer with image contouring capabilities, best with functionality to automatically analyze oxygenation-sensitive signal intensity changes.
Markers and their measurement
1. Acquire a baseline image during a short, baseline breath-hold acquisition before hyperventilation (rest). Compare the first image of the breath-hold (representing the end of the "stress" stimulus) to the baseline image.
  NOTE: Hyperventilation is a vasoconstrictive stimulus that reduces myocardial oxygenation (Healthy: %ΔSI ≈−5% to −10%¹³).
2. Obtain many images (and cardiac cycles) during the breath-hold. Use the first image of the breath-hold as the baseline and compare all following images to this image.
  NOTE: Breath-hold is a vasodilating stimulus that increases myocardial oxygenation (%ΔSI ≈ +5%-15%¹²^,¹³^,¹⁴^,¹⁶).
3. Myocardial contours
  For manual analysis: cardiac phase selection
  1. As the breath-hold can contain greater than 400 images, analyze only a single phase of each cardiac cycle. As a result, focus the analysis on the end-systolic images of each cardiac cycle.
  2. Identify the end-systolic images of each cardiac cycle.
  3. Draw the epicardial and endocardial contours around the myocardium.
  4. Window the image to look for artifacts, which will appear as either dark (susceptibility) or bright areas due to poor gating in the myocardium.
    NOTE: Avoid including pixels with partial volume effects from the left and right ventricular blood pools. Most contouring errors occur from the endocardial contour, including pixels with partial volume effects and resultant artificially elevated signal intensity from the left ventricular blood pool. To avoid this, draw the endocardial contour one full pixel inside the myocardium. Similarly, ensure that the epicardial contour is one full pixel inside the myocardium to avoid partial volume effects from the right ventricular blood pool, epicardial fat, or air-lung interface.
  5. Copy and paste the endocardial and epicardial contours from the first end-systolic image of the cardiac cycle to the end-systolic image of the next cardiac cycle. Adjust the contours as needed.
    For automated analysis:
    NOTE: With automated contouring capabilities, if desired, all phases of the cardiac cycle can be contoured and analyzed.
  6. Check all images to ensure accurate contouring.
    NOTE: Some commercially available automated contouring capabilities have been trained on data sets that were contoured for volumetric analysis. These contours are prone to partial volume effects as they are meant to border the blood pool and myocardium. OS-CMR contours must be fully inside of the myocardium.

7. Segmentation for regional analysis

To obtain regional information, identify the anteroseptal and inferoseptal insertion of the right ventricle to divide the myocardium into American Heart Association (AHA) segmentation⁴².

8. Calculating B-MORE

Express B-MORE as a percent change in signal intensity from baseline to vasodilation (see equation 1):
(1)
Calculate the global B-MORE as the global mean myocardial signal intensity of the end-systolic images at 30 s compared to 0 s of the breath-hold⁸ (see equation 2):
(2)

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Representative Results

Interpreting B-MORE
In previously published studies utilizing OS-CMR with vasoactive breathing maneuvers, the global or regional B-MORE was calculated by comparing the first end-systolic image of the breath-hold to the end-systolic image closest to 15 s, 30 s, 45 s, etc. of the breath-hold. The end-systolic phase of the cardiac cycle was chosen for several reasons. The end-systolic image is the most consistent phase identified among and between readers: it contains the greatest number of pixels in the myocardium, it normally occurs at roughly the same trigger time independent of participant heart rate, and it is always present in the acquisition (whereas end-diastole may not be shown in prospectively gated images throughout a breath-hold where the heart rate may change).

From a physiological perspective, the 0 s and 30 s time points of the breath-hold were specifically chosen for the following reasons. Time 0 s (or the first end-systolic image of the breath-hold) is an assessment of signal intensity after a period of "stress" (60 s of hyperventilation) and, therefore, the point of maximal vasoconstriction. Translated to signal intensity, this represents decreased myocardial blood flow with no increase in demand, resulting in a local increase in deoxyhemoglobin concentration and a decreased signal intensity when compared to baseline. Throughout the breath-hold, the signal intensity increases with carbon dioxide-mediated vasodilation, effectively increasing myocardial blood flow in the context of no increase in local demand. At the ~15 s time point of the breath-hold, the signal intensity curve begins to plateau⁴^,⁸. Therefore, the theoretical minimum breath-hold required for OS-CMR analysis is 15 s (or two cardiac cycles acquired to assess the difference between two data points). However, the 30 s time point of the breath-hold has been demonstrated to be more robust and is, therefore, considered the true minimum required breath-hold length.

After the calculation of global B-MORE (comparison of 30 s to 0 s of breath-hold), these data can be displayed visually and quantitatively. Quantitatively, global B-MORE values have been compared between healthy volunteers and patients with OSAS, CAD, INOCA, and HFpEF, as well as post heart transplant¹²^,¹³^,¹⁴^,¹⁵^,¹⁶ (Table 4). Visually, pixelwise color overlay maps can be generated to augment quantitative measurements in the assessment of myocardial oxygenation (Figure 1).

Figure 1: Myocardial oxygenation reserve visualized with a signal intensity map to assess global and/or regional tissue oxygenation obtained with OS-CMR and vasoactive breathing maneuvers. (A) Global myocardial oxygenation is maintained in a healthy volunteer; (B) a decrease in regional myocardial oxygenation in a patient with a left anterior descending stenosis (100% occlusion on quantitative coronary angiography); (C) a global reduction in myocardial oxygenation in a patient with heart failure. The color bar provides a visual representation of myocardial oxygenation, with black/ blue representing impaired and green representing a healthy myocardial oxygenation response. Abbreviations: OS-CMR = oxygenation-sensitive cardiac magnetic resonance imaging; LAD = left anterior descending. Please click here to view a larger version of this figure.

Figure 2: Visual representation of a full OS-CMR scan with vasoactive breathing maneuvers. (A) The standard acquisitions of a cardiac magnetic resonance imaging scan, including localizers, short-axis and long-axis cine function images, and tissue characterization images (such as T1 and/or T2 mapping). (B) The performance, physiologic effects, acquisition, and changes in MRI signal intensity throughout the vasoactive breathing maneuver. Abbreviations: OS-CMR = oxygenation-sensitive cardiac magnetic resonance imaging; DeoxyHb = deoxyhemoglobin. Please click here to view a larger version of this figure.

Procedures	Risks	Reasons	Frequency	Severity	Persistence
Cardiac MRI	Headache, nausea	Magnetic Field	Common (10%)	Light to severe	Reversible
Cardiac MRI	Anxiety, claustrophobia	Limited space	Rare (<5%)	Light to severe	Reversible
Breathing Maneuvers	Tingly fingers	Hyperventilation	Common (20%)	Light	Reversible (<60 s)
	Dizziness, headache	Hyperventilation	Common (10%)	Light	Reversible (<60 s)
	Dry Mouth	Hyperventilation	Rare (<5%)	Light	Reversible (<60 s)

Table 1: Reported side effects of undergoing a cardiac magnetic resonance imaging scan and performance of vasoactive breathing maneuvers. The reported data were collected from studies performed at the McGill University Health Centre in over 300 participants (unpublished data collected at the Research Institute of the McGill University Health Centre).

2.1 SIEMENS

		3T Siemens modified SSFP (OS)	1.5T Siemens Modified SSFP (OS)
TR	Repetition Time (ms)	41.4	39
TE	Echo Time (ms)	1.7	1.63
FA	Flip Angle (deg)	35	35
	Voxel Size (mm)	2.0 x 2.0 x 10.0	2.0 x 2.0 x 10.0
	Bandwidth (Hz/Px)	1302	1302

2.2 GE HEALTHCARE

		3T GE Healthcare modified SSFP (OS)	1.5T GE Healthcare modified SSFP (OS)
TR	Repetition Time (ms)	2.8	3.22
TE	Echo Time (ms)	0.97	1.2
FA	Flip Angle (deg)	35	35
	Voxel Size (mm)	2.0 X 2.0 X 10.0	1.6 x 1.6 x 10.0
	Bandwidth (Hz/Px)	1302	1302

2.3 PHILIPS

		3T Philips modified SSFP (OS)	1.5T Philips modified SSFP (OS)
TR	Repetition Time (ms)	3.41	3.3
TE	Echo Time (ms)	1.71	1.64
FA	Flip Angle (deg)	35	55
	Voxel Size (mm)	1.98 X 1.98 X 10.0	1.19x1.19x10.0
	Bandwidth (Hz/Px)	571.1	571.1

Table 2: Parameter differences between of the modified SSFP (BOLD) sequence at 3 Tesla and 1.5 Tesla for 2.1 Siemens, 2.2 General Electric Healthcare, and 2.3 Philips scanners. Abbreviations: GE = General Electric; SSFP = steady-state, free precession; OS = oxygen-sensitive.

Modifiable		Non-Modifiable
Field of View (mm)	360-400	Slice Thickness (mm)	10
Gap (%)	0-200	Flip Angle	35
Acquisition Time (s/measurement)	8	Segments	12 – 16
Measurements	1 (baseline) or 15+ (continuous acquisition)	ECG	Triggered /Prospective
Acquisition Window	No set limitations	TE (ms)	1.7
		TR (ms)	40.68 (3.4)
		Bandwidth (Hertz/Pixel)	1302

Table 3: Modifiable and non-modifiable OS-CMR sequence parameters during image acquisition. Abbreviations: OS-CMR = oxygenation-sensitive cardiac magnetic resonance imaging; ECG = electrocardiography; TE = echo time; TR = repetition time.

Disease State	Healthy Controls		Patient Populations		p-value*
	Age	B-MORE	Age	B-MORE
OSAS	49±12 (n=36)	9.8±6.7	60±12 (n=29)	4.3±7.6	0.01
CAD	27±4 (n=10)	11.3±6.1	64±11 (n=26)	2.1±4.4	<0.001
INOCA	52±4 (n=20)	4.97±4.2	54±6 (n=20)	5.0±6.82	0.75
Post Heart transplant	47±8 (n=25)	6.4±6.0	59±11 (n=46)	2.6±4.6	0.01
HFpEF	56±5 (n=12)	9.1±5.3	61±11 (n=29)	1.7±3.9	<0.001

Table 4: Global breathing-induced myocardial oxygenation reserve (B-MORE) values from previously published studies utilizing OS-CMR with vasoactive breathing maneuvers¹²^,¹³^,¹⁴^,¹⁵^,¹⁶. B-MORE values are represented as mean ± standard deviation. *p-value for B-MORE comparison. Abbreviations: B-MORE = breathing-induced myocardial oxygenation reserve; CAD = coronary artery disease; HFpEF = heart failure with preserved ejection fraction; INOCA = ischemia with no obstructive coronary artery stenosis; OSAS = obstructive sleep apnea syndrome.

Supplemental File 1: Prerecorded .mp3 file guiding the patient through the vasoactive breathing maneuver. Please click here to download this File.

Supplemental Video S1: Instructional breathing maneuver video. Please click here to download this Video.

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Discussion

x`The addition of an OS-CMR acquisition with standardized, vasoactive breathing maneuvers to an already established research or clinical MRI protocol adds little time to the overall scan. With this short addition, information about underlying macro- and microvascular function can be obtained (Figure 2). An important consequence of endothelial dysfunction is the inability of the vasculature to respond to physiologic stimuli, as initially demonstrated through abnormal flow-mediated relaxation in the heart⁴³. OS-CMR with endothelial-dependent vasoactive breathing maneuvers allows for direct monitoring of myocardial oxygenation during vasoactive breathing maneuvers and circumvents the need for exogenous contrast agents and pharmacologic vasoactive stress agents. Hyperventilation and apnea trigger a reproducible, measurable vascular response through the endothelium and may, therefore, provide a more physiologic model than other methods for assessing microvascular function.

Findings from studies utilizing OS-CMR with vasoactive breathing maneuvers have provided important contributions to the understanding of the underlying pathophysiology in patients with ischemic conditions without explanatory coronary artery stenosis, specifically INOCA, HFpEF, and inflammation (e.g., after cardiac transplantation). The potential addition of OS-CMR with standardized vasoactive breathing maneuvers to identify patients presenting with ischemic chest pain as having explanatory microvascular dysfunction or to the clinical work-up of HFpEF and cardiac transplant patients would significantly improve clinical decision-making in these patient populations⁴⁴.

When performing OS-CMR with vasoactive breathing maneuvers, there are some aspects to look for in the participants' performance of the breathing maneuvers and in the images. Commonly, participants try to keep up with the pace of the metronome (30 breaths/min) and are not breathing in deeply. It is more important to breathe deeply than it is to keep up the pace of 30 breaths/min (e.g., "belly breathing" is more effective than shallow chest breathing). In healthy participants, the heart rate is expected to increase by ~20 beats/min during hyperventilation. Patient participants tend to have a heart rate increase of 5-10 beats/min⁴⁵. Some participants may be tempted to take a small breath in to increase the time of the breath-hold. Therefore, patients should be informed that the test will lose its diagnostic accuracy if the protocol is not carefully followed and that any small breath will end the test.

If a slice location is too basal (close to the valvular plane), the outflow tracts may not allow for differentiating LV from RV or may be in the left ventricular outflow tract as a result of through-plane motion and would impact the ability to analyze the images. If a slice is too apical, the images may not be perpendicular to the ventricular wall and, therefore, may contain blood or paracardiac tissue and impair the evaluation. Additionally, if a slice is too apical, there are significantly less pixels of true myocardium, increasing the risk of including pixels with partial volume effects in the analysis.

Global impairment of myocardial oxygenation
OS-CMR with vasoactive breathing maneuvers has previously demonstrated an impaired global myocardial oxygenation reserve in patients with OSA and HFpEF and cardiac transplant recipients¹²^,¹⁴^,¹⁶. The finding of a global reduction in B-MORE in patients with HFpEF conflicts with the findings of a previous study demonstrating impaired myocardial perfusion but maintained myocardial oxygenation in patients with non-ischemic HF⁴⁶. However, the previous studies used adenosine, an endothelial-independent vasodilator, as the stress agent. Therefore, endothelial-dependent microvascular dysfunction and the potential impact on myocardial oxygenation were not investigated. The presence or absence of endothelial dysfunction in patients with chronic heart failure has important clinical implications, as the severity of endothelial dysfunction may not only determine the clinical presentation but also have prognostic value with respect to future hospitalization, cardiac transplantation, or death³⁴^,⁴⁷.

The presence of a marked global reduction in B-MORE in cardiac transplant patients both with and without cardiac allograft vasculopathy as compared to healthy controls is an important finding for shedding light on underlying pathophysiology and the timing and reduction of invasive follow-up testing and has prognostic implications. The reduction in B-MORE in cardiac transplant patients with and without cardiac allograft vasculopathy is likely a result of diminished coronary vasoreactivity. This explanation is additionally supported by the association of further B-MORE impairment with the severity of cardiac allograft vasculopathy¹⁴. As annual screening for microvascular dysfunction with invasive coronary angiography is recommended in patients post cardiac transplantation⁴⁸, the ability of OS-CMR with vasoactive breathing maneuvers to identify and monitor the severity of microvascular dysfunction in this patient population may provide an alternative non-invasive and needle-free screening methodology.

Regional impairment of myocardial oxygenation
In many centers, 50%-70% of patients who undergo invasive coronary angiography do not have significant obstructive coronary artery stenosis, calling for a non-invasive imaging technique to both identify INOCA and provide prognostic information on cardiovascular outcomes in this not well-understood patient population. The clinical evaluation of patients with INOCA has historically applied coronary reactivity testing, including measuring the index of microcirculatory resistance during invasive coronary angiography²⁵^,²⁶. However, this method is limited by its invasiveness, lack of reproducibility, and cost. Additionally, invasive angiography does not assess the level of the critical downstream pathophysiologic effect, i.e., the effect on myocardial oxygenation. Recently, OS-CMR with vasoactive breathing maneuvers in women with INOCA demonstrated intriguing findings. While there was no impairment in the global B-MORE when compared to age-matched healthy controls, the coronary vascular responsiveness, as defined by a change in myocardial oxygenation, showed a heterogeneous pattern of impaired oxygenation response when compared to that of age-matched healthy subjects¹⁵.

The observed regional variations in endothelial function and myocardial oxygenation in patients with chest pain and INOCA provide an important insight into the physiology of microvascular dysfunction in this patient population. Regional variations in myocardial oxygenation could potentially be mediated through local abnormalities in endothelial-derived relaxing factors, abnormal neural stimuli to the coronary microcirculation resulting in regionality of flow and oxygenation, or a coronary vascular steal⁴⁹. An alternative explanation of these findings may be heterogeneity in the etiologies underlying INOCA⁵⁰. The visualization of the tissue oxygenation status and its regional heterogeneity by maps acquired by OS-CMR with breathing maneuvers suggest that this methodology could play an important role in a more direct and comprehensive examination of regional myocardial vascular function in these patients beyond a simplified global measure of perfusion or oxygenation.

Limitations
Some limitations of the methodology remain. From a physiological perspective, utilizing the BOLD effect to make inferences about tissue oxygenation requires the consideration of other variables as the OS-CMR signal intensity is also impacted by blood inflow and blood volume²^,⁵¹. Fortunately though, the effects on the signal intensity are synergistic and physiologically linked (induced coronary vasodilation simultaneously increases blood flow, blood volume, and blood oxygenation). The potential bias of these confounders is, therefore, systematic and unidirectional, with little relevance when assessing microvascular function. Other factors related to the blood (hemoglobin, hematocrit) and field strength have been identified as potential confounding factors in OS-CMR image interpretation and analysis⁵¹^,⁵² and will have to be taken into account if significantly abnormal. To address these factors, novel biomarkers derived from OS-CMR signal intensity response may control for or minimize the confounding effects of hemodilutional state and hematocrit, for example, by normalizing the signal intensity response to the left or right ventricular blood pool of each participant.

Until recently, the evaluation of OS-MR data has required laborious manual annotation, segmentation, and analysis. User-friendly postprocessing tools for the automated or semi-automatic analysis of dynamic OS-CMR data sets are being developed⁵³. Finally, there is a lack of population-based normal values and clinical studies comparing OS-CMR results with invasive measurements of microvascular dysfunction, as well as data on prognosis, cost efficiency, and the impact of its use on clinical outcomes.

Conclusion
Non-invasive monitoring of dynamic regional or global changes in myocardial oxygenation by OS-CMR with vasoactive breathing maneuvers provides unique, clinically meaningful information on coronary vascular function and may play a particularly important role in patients with microvascular dysfunction. Further clinical studies should be performed to investigate its clinical utility in various patient populations.

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Disclosures

MGF is listed as a holder of United States Patent No. 14/419,877: Inducing and measuring myocardial oxygenation changes as a marker for heart disease; United States Patent No. 15/483,712: Measuring oxygenation changes in tissue as a marker for vascular function; United States Patent No 10,653,394: Measuring oxygenation changes in tissue as a marker for vascular function - continuation; and Canadian Patent CA2020/051776: Method and apparatus for determining biomarkers of vascular function utilizing bold CMR images. EH is listed as a holder of International Patent CA2020/051776: Method and apparatus for determining biomarkers of vascular function utilizing bold CMR images.

Acknowledgments

This paper and methodology review was made possible by the entire team of the Courtois CMR Research Group at the McGill University Health Centre. Special thanks to our MRI technologists Maggie Leo and Sylvie Gelineau for the scanning of our participants and feedback on this manuscript.

Materials

Name	Company	Catalog Number	Comments
balanced SSFP MRI sequence	Any		To modify to create the OS-CMR sequence
DICOM/ Imaging Viewer	Any		Best if the viewer has the ability for quantitative measurements (i.e., Area19 prototype software)
Magnetic Resonance Imaging scanner	Any		3 Tesla or 1.5 Tesla
Metronome	Any		Set to 30 breaths per minute. To use if manually communicating breathing maneuver instructions to participants.
Speaker system	Any		To communicate breathing maneuver instrucitons to participants through
Stopwatch	Any		To use if manually communicating breathing maneuver instructions to participants

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