Molecular Imaging in Ischemic Heart Disease
Purpose of Review
The purpose of this paper is to review current and new modalities to image key biological processes in ischemic heart disease and after myocardial infarction non-invasively.
New imaging targets have been developed to detect and quantify myocardial damage after ischemia. Although positron emission tomography (PET) has been leading the development of new probes in the past, continuous improvements of magnetic resonance imaging (MRI) together with the development of new novel MRI contrast agents opens new research avenues including the combination of both PET and MRI to obtain anatomic, functional, and molecular information simultaneously, which is not possible from a single imaging session.
This review summarizes the state of art of non-invasive molecular imaging of the myocardium during ischemia and after myocardial infarction using PET and MRI. We also describe the different contrast agents that have been developed to image the different phases of cardiac healing and the biological processes associated with each of those phases. Importantly, here we focus on imaging of inflammation as it is the key biological process that orchestrates clearance of dead cells, tissue remodeling, cardiac repair, and future outcome. We also focus on clinical translation of some of the novel contrast agents that have been tested in patients and discuss the need for larger, multi-center patient studies to fully validate the applicability of new imaging probes.
KeywordsCardiovascular imaging Inflammation Ischemic heart disease Vascular remodeling Myocardial infarction
Cardiovascular diseases remain the leading cause of morbidity and mortality in western societies with coronary artery disease and associated myocardial infarction (MI) being the most common type of disease in the circulatory system . Myocardial infarction occurs when the coronary arteries are obstructed, limiting the transport of nutrients and oxygen to the cardiomyocytes that form part of the ventricular wall. After the ischemic event, a plethora of molecular and cellular pathways are activated to compensate and resolve the injury to the heart. A proportion of the cardiomyocytes die in response to the coronary obstruction, creating an environment that will stimulate the infiltration of inflammatory phagocytes such as neutrophils immediately after the event and is followed by the influx of inflammatory and reparative monocytes . Inflammation plays a crucial role in cardiac healing, first by removing dead cells and secondly by activating cells that will contribute to the healing response which includes the production of extracellular matrix proteins that will become an integral part of the scar tissue that will replace the dead cardiomyocytes . However, if the inflammatory response is unbalanced (too strong or too weak), the effect on cardiac healing can be deleterious, resulting in ventricular dilation, hypertrophy or thinning of the myocardium among others, a phenomenon known as adverse ventricular remodeling. Patients with adverse ventricular remodeling have a higher chance of developing progressive heart failure and associated poor prognosis [4, 5]. Therefore, the identification of patients undergoing adverse ventricular remodeling is crucial in influencing their therapeutic intervention and ultimately improving their prognosis.
Current clinical imaging techniques are mainly focused on assessing the anatomy and function of the heart. However, the development of new molecular-targeted probes can increase significantly the information obtained from each imaging session, by better visualization and understanding of the molecular pathways involved in myocardial healing and, thus, help clinicians to provide more personalized treatments to their patients. In this review, we focus on the two primary imaging modalities used for cardiac molecular imaging, positron emission tomography (PET) and magnetic resonance imaging (MRI), and we discuss new molecular-targeted probes used to detect different biological processes after myocardial infarction with special focus on inflammation, as it is considered the key biological process in cardiac healing.
Clinical Need for Non-invasive Imaging of Post-MI Remodeling
Optimal myocardial remodeling after ischemia relies on a suitable degree of inflammation and its timely resolution. Inadequate recruitment of granulocytes and monocytes into the infarcted area triggers impaired healing frequently promoting adverse cardiac remodeling, impaired scar stability, and long-term risk of heart failure [4, 6•]. Blood biomarker tests including serum troponin levels, together with clinical symptoms of chest pain, and ST segment changes or T-wave inversion on the electrocardiogram (ECG) constitute the basis for identifying patients suffering from myocardial ischemia . Although increased levels of inflammatory blood biomarkers, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and C reactive protein (CRP), may predict poor prognosis, they do not provide information on the local inflammatory response triggered in the ischemic myocardial segment. Therefore, the ability to directly visualize and assess the healing process and cardiac remodeling may produce useful clinical information to help individualize patient treatment. In this review, we will focus on clinical and experimental non-invasive imaging approaches available for assessing cardiac remodeling after ischemia, with a specific focus on the inflammatory phase and macrophage imaging.
Myocardial Remodeling After Ischemia
Coronary artery disease is the consequence of progressive and gradual narrowing of the coronary arteries by the build-up of fatty material within the vessel walls or by the obstruction of the coronary lumen by clot formation as a consequence of acute plaque rupture. A complete obstruction can severely limit the availability of oxygen and nutrients inducing rapid cardiomyocyte damage and death [8, 9, 10]. Within the culprit coronary artery myocardial territory, the subendocardial layer is damaged first, due to effects of cardiac contraction, vascular pressure-dependent compliance, and potential transmural differences in vessel anatomy . If the obstruction persists, the injury will progress along a wave front starting from the subendocardial layer towards the subepicardial layer [12, 13, 14]. Therefore, the time lapse between acute coronary obstruction and revascularization directly determines the extent of myocardium injury and significantly affects long-term cardiac function and prognosis. However, the restoration of blood flow and subsequent reperfusion paradoxically induces injury in the myocardium [15, 16, 17, 18]. Several approaches to minimize myocardial injury inflicted by reperfusion have been tested throughout processes known as pre- and post-conditioning [17, 19].
As mentioned above, the extent and duration of the inflammatory response post-MI may have profound effects on the cardiac remodeling and clinical outcome. High levels of specific inflammatory markers, such as IL-6 and TNF-α, in blood have been correlated with long-term heart failure and poor prognosis . High levels of white blood cells within 24 h after ischemia have shown to be a strong predictor of 30-day mortality and recurrent clinical events . The CANTOS study has demonstrated the importance of the inflammatory marker IL-6 in patients with history of myocardial infarction showing the potential beneficial effects of the modulation of its signaling pathways, thereby reducing cardiovascular event rates independently of lipid lowering [29, 30•]. Similarly, high levels of other inflammatory markers such as TNF-α and IL-10 have been associated with recurrent events and poor prognosis [31, 32]. It is also important to note that a weak inflammatory response after MI can be deleterious, as the inflammatory cells might not be able to clear up the injured area from damaged and dead cells and not be able to produce strong enough signals to activate other cells involved in the healing response and scar formation . Therefore, non-invasive imaging of cardiac remodeling and more specifically inflammation after myocardial infarction can be a useful clinical tool for patient stratification allowing more targeted and individualized therapeutic approaches.
Molecular Imaging of Cardiac Remodeling After Myocardial Infarction
Several imaging approaches can be employed to image the structural, functional, and molecular changes during persistent ischemia and after infarction. Cardiovascular magnetic resonance imaging (CMRI) is a non-ionizing imaging modality which allows the assessment of cardiac function by using cine imaging and anatomical evaluation with bright and black blood imaging. In addition, CMRI allows detailed myocardial tissue characterization using T1 and T2 mapping by exploiting the differences in T1 and T2 relaxation time of the myocardium during health and disease [34, 35]. Following myocardial infarction, the area at risk is characterized by edema, which is representative of the percentage of myocardium affected after the insult. Edematous regions are usually visualized on T2-weighted but can also be detected on T1-weighted images as both are sensitive to changes in water concentration, without the need for contrast administration . Native T1 and T2 mapping are starting to replace T1w and T2w imaging as they can quantify the amount of edema and, thereby, further improve diagnostic accuracy . The detection and quantification of the extent of the area at risk and especially the area of infarction can be improved using contrast agents. Gadolinium-based contrast agents can be used to determine the damaged areas by using early gadolinium enhancement (EGE) for area at risk detection and late gadolinium enhancement (LGE) for direct infarct visualization . As the contrast agent washes out slower from scar tissue compared to the healthy tissue, scar appears bright on heavily T1-weighted inversion recovery prepared LGE images. In addition, myocardial perfusion can be assessed qualitatively and quantitatively using first pass MR perfusion imaging with gadolinium-based contrast agents, as damaged and poorly perfused myocardium will show less gadolinium uptake and, therefore, reduced signal on T1-weighted images [37, 38].
Immediately after myocardial infarction, a hypoxic environment is generated in the territory supplied by the obstructed coronary artery leading to cardiomyocyte damage and death. Cells generally die through a non-programmed or a programmed cell death pathway, which are known as necrosis and apoptosis, respectively. During necrosis, the cell membrane is disrupted and the cell content is released to the extracellular space, leading to the activation of the inflammatory response. However, during apoptosis, cells shrink and form apoptotic bodies exposing specific markers in their membrane that are detected and subsequently phagocytosed by macrophages.
One of the first approaches to measure myocardial apoptosis was targeting the intracellular protein myosin using indium-111 (111In)-labeled anti-myosin antibodies in dogs with MI . Clinical validation of targeting the intracellular myosin was performed using technicium-99 (99mTc) labeled anti-myosin antibodies in 30 patients that underwent percutaneous revascularization after myocardial infarction. This approach allowed imaging of the necrotic area, but not the border zones that may or may not have irreversible injury . No follow-up studies have been reported since. Annexin-V is one of the markers expressed in apoptotic cells and has been used as target for non-invasive imaging of apoptosis after MI using MRI. This approach uses Annexin-V-labeled nanoparticles, known as AnxCLIO-Cy5.5, in a murine model of heart failure  and acute ischemia . Other approaches are focused on the development of contrast agents targeting caspases, which are key proteins during the apoptotic process. However, all efforts so far have been focused on imaging apoptosis in cancer , with little to no work in cardiac apoptosis.
Vascular Permeability (Endothelial Damage and Angiogenesis)
Acute ischemia is associated with damage of the endothelial cells that cover the ventricle and, therefore, an increase in vascular permeability is observed. Moreover, during the chronic maturation phase, increased levels of VEGF and basic fibroblast growth factors are released in the injured area, triggering the formation of immature or leaky neovessels contributing also to an increase in permeability in the infarcted area. Intense research efforts have been undertaken to develop contrast agents that can detect and quantify vascular permeability to better understand the cardiac healing process during the acute and chronic phase. Several different imaging targets sensitive to vascular permeability have been tested, including αvβ3, VEGF, VCAM-1, and albumin.
The maturation or chronic phase after ischemia is characterized by the reorganization and production of new extracellular matrix proteins, the development of a fibrotic scar, typically rich in type I collagen fibers to prevent heart dilation and rupture. The presence and extent of myocardial scar carries important diagnostic and prognostic information [52, 53, 54]. LGE MRI is currently considered the standard of reference for scar detection and quantification . The increase in extracellular volume (ECV) in the damaged myocardium together with the delayed wash allows gadolinium to accumulate in the infarcted region, thereby providing exquisite visualization of scar on highly T1-weighted inversion recovery LGE MR images . While LGE MRI can visualize the location and extent of the scar, pre- and post-contrast T1 mapping enables the quantification of ECV non-invasively . ECV has been shown to be an excellent marker for focal and diffuse fibrosis . While LGE MRI and T1 mapping provide important information about scar size and extent of transmurality and the amount of fibrosis, those methods fall short in providing detailed information on the underlying biology. An alternative approach overcoming the above limitation is the use of Gd-based target-specific contrast agents such as EP-3533, which binds to collagen types I–IV and, thus, provides additional information beyond LGE MRI or T1 mapping with non-specific Gd-based agents. EP-3533 has been successfully validated in a murine model of ischemia-reperfusion and demonstrated the ability to detect collagen-rich scar tissue 6 weeks after MI . Other extracellular matrix proteins that are overexpressed after MI include elastin and tropoelastin. Importantly, studies suggest that elastin formation after MI leads to improved ejection fraction and decreased risk of myocardial rupture  and therefore may be an attractive imaging candidate. We and other groups have demonstrated the merits of an elastin-binding contrast agent, known as ESMA, to visualize and quantify the infarcted area . While the area of infarction was similar to that measured with a conventional Gd-based contrast agent, elastin imaging enabled the monitoring of changes in extracellular matrix remodeling over time (higher signal at 3 weeks post-MI) in both the infarct and remote area, which were not seen with standard LGE MRI [59, 60]. In addition, we have evaluated the interplay between elastin remodeling and inflammation after MI using simultaneous 1H and 19F imaging which may be beneficial to predicting future outcomes . To enable the assessment of ECM turnover and fibrosis activity, which may be directly linked to inflammation, we have recently developed a new Gd-based contrast agent which specifically binds to tropoelastin (TESMA), the precursor of elastin. We have successfully demonstrated the feasibility of imaging of tropoelastin in mouse models of atherosclerosis  and abdominal aortic aneurysm and future studies will focus on the added value of TESMA for the detection adverse myocardial remodeling after myocardial infarction.
Finally, altered levels of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, which are enzymes involved on the extracellular matrix remodeling, have been associated with adverse myocardial remodeling and poor prognosis after MI [63, 64, 65]. Non-invasive imaging using radiotracers enabled the quantification and localization of MMP activation in a murine model of myocardial infarction . This is one of the first approaches that enables the measurement of the biological activity of enzymes involved in the remodeling process directly and, thus, could provide important prognostic information and guide treatment decisions.
Molecular Imaging of Inflammation After Ischemia
Receptor-based targeting of macrophages has also been an important area of research. Myeloperoxidase (MPO) is an inflammatory enzyme produced by neutrophils and macrophages that is highly expressed in acute MI or in culprit atherosclerotic lesions . Bogdanov et al. developed a gadolinium-based contrast agent that targets MPO (MPO-Gd)  which was validated in murine models of ischemia and reperfusion . The use of Gd-MPO allowed them to asses MPO activity in vivo in the infarcted myocardium, which is a direct measurement of the inflammatory response triggered in the heart by an acute MI. They were also able to demonstrate the beneficial effect of an anti-inflammatory therapeutical intervention, using atorvastatin, which resulted in lower levels of MPO activity . Other target-specific contrast agents such as gadolinium immunomicelles targeted to the macrophage scavenger receptor CD206 , gadolinium-loaded LDL-based nanoparticles , and CX3CL1 nanoparticles  have been successfully tested to image and quantify macrophages in atherosclerosis. However, these agents have not been yet tested in the context of MI.
Nuclear medicine research has also been very active and focused on the development of novel radiotracers to target inflammation. 18F-deoxyglucose (FDG) is the best characterized and widely available tracer for imaging inflammation (metabolic activity of cells) in different diseases, such as cancer and cardiovascular diseases, among others . Leukocytes that infiltrate the infarcted myocardium have very high metabolic activity and consume high concentrations of glucose. FDG imaging takes advantage of the metabolic change allowing the visualization and quantification of the inflammatory response at difference stages of the disease. FDG imaging in combination with PET/MRI technology has allowed the visualization of the biphasic nature of monocyte infiltration after an ischemic insult in a murine model of MI and in patients with acute MI . In addition, FDG-PET uptake 5 days after percutaneous coronary intervention had an inverse correlation with myocardial outcome measure by MRI 6–9 months after the ischemic event in patients with cardiovascular disease [90•]. In addition, FDG signal exceeded the scar area measured by MRI, demonstrating that FDG measures the area at risk. Importantly, blood leukocyte counts correlated with both, area at risk measured by FDG-PET and scar size measured by MRI [90•]. However, cardiomyocytes have high metabolic activity and therefore produce a high FDG signal, which can mask inflammatory activity. To overcome this limitation, patients undergo special dietary requirements, including fasting or high fat meals to potentiate fatty acid metabolism and suppress cardiomyocyte glucose uptake . Because this method does not always provide the desirable signal suppression from cardiomyocytes, we need new, more specific imaging radiotracers to detect and quantify inflammation. One alternative is the use of the glucose isomer mannose. Because macrophages express on their membranes the scavenger receptor known as CD206 or mannose receptor, 18F-deoxymannose (FDM) can be used for macrophage imaging. In addition, this receptor is highly expressed on M2-like macrophages opening a new research window not only for macrophage detection, but also for the differentiation between the two major macrophage subsets. FDM has been tested in atherosclerotic rabbits showing that the uptake obtained with FDM is not inferior to the one when using FDG . However, FDM remains to be evaluated in myocardial infarction and in patients with cardiovascular disease.
The somatostatin receptor type 2 (SSTR2) is also highly expressed in activated macrophages, especially in M1-like macrophages, and is routinely used for imaging neuroendocrine tumors. Two imaging agents that are a newer generation of somatostatin analogs have been developed, known as 68Ga-DOTATATE and 68Ga-DOTATOC. 68Ga-DOTATATE has higher affinity to the SSTR2, while 68Ga-DOTATOC has higher affinity to SST5. Both radiotracers have shown different imaging benefits compared to FDG, as it not only presents superior accuracy and sensitivity, but also allows characterization of whole-body SSTR expression . 68Ga-DOTATATE has shown very promising results for the detection of inflammation in atherosclerosis ; however, due to the very quick metabolism and blood clearance, no conclusive results were observed in a murine model of MI . In contrast, with 68Ga-DOTATOC, the elevation of SSTR2 in the infarcted myocardium at 3 and 10 days post-acute MI has been observed in patients .
C-X-C chemokine receptor 4 (CXCR4) is a receptor involved in leukocyte migration and recruitment to injured tissue, such as the infarcted myocardium. 68Ga-pentixafor is a PET radiotracer that has shown promising results for the detection of inflammation in a murine model of MI. Tracer uptake in the infarcted myocardium was proportional to leukocyte infiltration as detected by flow cytometry . In the same study of 12 patients with myocardial infarction, this radiotracer showed heterogeneous uptake at time points between 2 and 8 days post-MI, suggesting a patient specific modulation of the chemokine response [99, 100]. Studies with larger number of patients are now required to better understand the chemokine response provided by the CXCR4 receptor after MI.
Although several strategies have been developed to image macrophages, none of them has had the ability to clearly distinguish between macrophage subsets. Thus, the development of M1 or M2 specific probes remains an interesting challenge on the horizon.
Important advances in imaging the molecular processes associated with the ischemia-induced myocardial injury and resulting remodeling have been made in the last decade. The development of new target-specific contrast agents, with some of them currently being clinically validated, could open a new era in the management of patients with myocardial infarction, thereby allowing better patient stratification and enable more personalized treatments. However, intense research is still needed at both the preclinical and clinical level to develop not only more specific contrast agents, but also to better understand the biological information provided by those reagents. For clinical translation, further validation of these new agents needs to be performed in larger patient cohorts to evaluate the diagnostic and prognostic value. The discovery and development of new agents able to differentiate between the different subsets of leukocytes, monocytes, and macrophages present at different time points in the infarcted myocardium could open new avenues for more personalized treatments, prognosis and outcome studies.
The authors acknowledge financial support from (1) EPSRC (EP/P001009/1 and EP/P007619/1), (2) the British Heart Foundation (RG/12/1/29262), (3) the Centre of Excellence in Medical Engineering funded by the Wellcome Trust and EPSRC (203148/Z/16/Z), and (4) the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.
Compliance with Ethical Standards
Conflict of Interest
All authors declare that they have no conflict of interest.
Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.
Papers of particular interest, published recently, have been highlighted as: • Of importance
- 1.Writing Group Members, et al. Heart disease and stroke statistics-2016 update: a report from the American Heart Association. Circulation. 2016;133(4):e38–60.Google Scholar
- 6.• Thackeray JT, Bengel FM. Molecular imaging of myocardial inflammation with positron emission tomography post-ischemia. A determinant of subsequent remodeling or recovery. JACC Cardiovasc Imaging. 2018;11(9):1340–55. This study provides a good overview of the emerging molecular imaging radiotracers that can be used to study myocardial inflammation following ischemia.PubMedCrossRefGoogle Scholar
- 30.• Ridker PM, et al. Modulation of the interleukin-6 signalling pathway and incidence rates of atherosclerotic events and all-cause mortality: analyses from the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS). Eur Heart J. 2018;39(38):3499–507. This clinical trial provides evidence that anti-inflammatory therapy modulating the interleukin-6 signalling pathway is associated with reduced cardiovascular event rates, independent of lipid lowering therapy.PubMedCrossRefGoogle Scholar
- 37.Knott, K.D., et al., Quantitative myocardial perfusion in coronary artery disease: a perfusion mapping study. J Magn Reson Imaging, 2019.Google Scholar
- 38.Kotecha, T., et al., Automated pixel-wise quantitative myocardial perfusion mapping by CMR to detect obstructive coronary artery disease and coronary microvascular dysfunction: validation against invasive coronary physiology. JACC Cardiovasc Imaging, 2019.Google Scholar
- 61.Ramos IT, et al. Simultaneous assessment of cardiac inflammation and extracellular matrix remodeling after myocardial infarction. Circ Cardiovasc Imaging. 2018;11(11).Google Scholar
- 62.Phinikaridou, A., et al., Tropoelastin: a novel marker for plaque progression and instability. Circ Cardiovasc Imaging, 2018. 11(8).Google Scholar
- 87.Hernández-Gil J, et al. P11 developing new targeted molecular contrast agents for imaging inflammation of vulnerable plaques. Cardiovasc Res. 2018;114(suppl_2):S3–4.Google Scholar
- 90.• Rischpler C, et al. Prospective evaluation of 18F-fluorodeoxyglucose uptake in postischemic myocardium by simultaneous positron emission tomography/magnetic resonance imaging as a prognostic marker of functional outcome. Circ Cardiovasc Imaging. 2016;9(4):e004316 This study demonstrates that the intensity of 18F-FDG uptake in the myocardium after acute myocardial infarction correlates inversely with functional outcome at 6 months, demonstrating that 18F-FDG uptake in infarcted myocardium may serve as a potential novel biomarker of myocardial injury.PubMedPubMedCentralCrossRefGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.