Molecular Imaging of the Heart

Abstract

Multimodality cardiovascular imaging is routinely used to assess cardiac function, structure, and physiological parameters to facilitate the diagnosis, characterization, and phenotyping of numerous cardiovascular diseases (CVD), as well as allows for risk stratification and guidance in medical therapy decision-making. Although useful, these imaging strategies are unable to assess the underlying cellular and molecular processes that modulate pathophysiological changes. Over the last decade, there have been great advancements in imaging instrumentation and technology that have been paralleled by breakthroughs in probe development and image analysis. These advancements have been merged with discoveries in cellular/molecular cardiovascular biology to burgeon the field of cardiovascular molecular imaging. Cardiovascular molecular imaging aims to noninvasively detect and characterize underlying disease processes to facilitate early diagnosis, improve prognostication, and guide targeted therapy across the continuum of CVD. The most-widely used approaches for preclinical and clinical molecular imaging include radiotracers that allow for high-sensitivity in vivo detection and quantification of molecular processes with single photon emission computed tomography and positron emission tomography. This review will describe multimodality molecular imaging instrumentation along with established and novel molecular imaging targets and probes. We will highlight how molecular imaging has provided valuable insights in determining the underlying fundamental biology of a wide variety of CVDs, including: myocardial infarction, cardiac arrhythmias, and nonischemic and ischemic heart failure with reduced and preserved ejection fraction. In addition, the potential of molecular imaging to assist in the characterization and risk stratification of systemic diseases, such as amyloidosis and sarcoidosis will be discussed.

Introduction

Cardiovascular disease (CVD) remains a major healthcare problem in the United States (US) that affects nearly 92.1 million Americans (29). Despite a ~25% decrease in death rates attributable to CVD over the last decade, CVD remains the leading cause of mortality in the US with over 600,000 deaths related to complications of CVD occurring per year (29). The total health cost associated with CVD was estimated at $316.1 billion in the US in 2012 to 2013 (29). Given the high socioeconomic burden of CVD, there has been a shift from treatment of CVD to primary and secondary disease prevention (130). The ability for early detection of disease allows for risk stratification and guided clinical management to curtail or attenuate the development of overt disease. Advancements in basic science and innovations in technology have led to a greater understanding of cellular and molecular processes that underlie and/or modulate changes in cardiac function, structure, and physiology in CVD. Molecular imaging, defined as the visualization, characterization, and noninvasive measurement of biological processes at the molecular and cellular level, has ability to translate advancements in basic science to humans to facilitate early diagnosis, improve prognostication and guide targeted therapy across the spectrum of CVD (343). This comprehensive review describes multimodality molecular imaging instrumentation and established and novel molecular imaging targets and probes, and highlights their application within the myocardium in preclinical models and in patients with CVD. We have focused on the myocardium, given the recent publication of several excellent and comprehensive reviews on similar applications within the coronary and peripheral vasculature (108, 268, 360, 384). Although not discussed, the interested reader is referred to several excellent reviews on nonmolecular, but important, electrophysiological imaging applications (235, 280, 314).

Imaging Systems for Molecular Imaging

Nuclear imaging modalities

Nuclear imaging modalities include, single photon emission computed tomography (SPECT) and positron emission tomography (PET), both of which provide high sensitivity for detection of molecular processes, although have limited resolution. SPECT cameras detect single gamma emissions from the decay of several low-energy radioisotopes that are linked to physiological or molecularly targeted probes. PET cameras detect two antiparallel 511 keV energy photons generated when positrons emitted during radioisotope decay collide with tissue electrons (annihilation) (67). These imaging modalities are widely available and are extensively used to evaluate myocardial perfusion, metabolism, and function. In addition, nuclear imaging systems are the most broadly used imaging modalities for molecular imaging due to their high sensitivity to detect cellular and molecular targets (picomolar level) and a multitude of available molecular imaging probes (343). Common radioisotopes used for SPECT imaging are technetium-99m (^99mTc), thallium-201 (²⁰¹Tl), iodine-123 (¹²³I), and indium-111 (¹¹¹In). Frequently used PET isotopes include, carbon-11 (¹¹C), fluorine-18 (¹⁸F), copper-64 (⁶⁴Cu), gallium-68 (⁶⁸Ga), nitrogen-13 (¹³N), rubidium-82 (⁸²Rb), iodine-124 (¹²⁴I), zirconium-89 (⁸⁹Zr), and oxygen-15 (¹⁵O). Multiple strategies exist that allow for molecular imaging with nuclear imaging systems that include: radiolabeling of antibodies, antagonists/inhibitors, agonists, or peptides that bind to specific molecular targets; radiolabeling of endogenous molecules; radiolabeling analogs of biologically available molecules; and direct delivery of radioisotopes that target endogenous or engineered biological targets.

SPECT and PET have well described advantages and disadvantages (308). For small animal imaging, microSPECT cameras can provide higher spatial resolution (<0.3 mm) than microPET cameras (>1 mm) due to physical factors governing radioactive decay of PET isotopes. On the other hand, traditional clinical SPECT cameras have lower sensitivity to detect molecular/cellular targets and have poorer spatiotemporal resolution than PET cameras. In addition, more established methods for kinetic modeling and absolute quantification of radiotracer uptake exist for PET compared to SPECT cameras. Alternatively, in terms of translatability, SPECT cameras are less expensive, are more widely available, offer the ability for multi-isotope imaging, and are compatible with radioisotopes that do not require complex on-site labeling. In addition, the longer half-life of SPECT isotopes allows for delayed imaging and delivery of radioisotopes from central distributors, which is only possible for very few PET isotopes. The recent development of dedicated cardiac SPECT cameras with cadmium-zinc-telluride (CZT) semi-conductor detectors instead of sodium iodide scintillation detectors have overcome several of the limitations of traditional SPECT cameras and allow for higher temporal resolution, higher spatial resolution, higher detection sensitivity, better energy resolution and the capability for dynamic imaging for kinetic modeling. Combined, these advancements allow for the use of less radiation and improve the quantitation of radiotracers with SPECT (106, 306, 426). Indeed, the use of either system is limited by the need for ionizing radiation and by limited spatial resolution (submillimeter to centimeter) compared to computed tomography (CT) and cardiac magnetic resonance imaging (MRI). While SPECT and PET imaging systems offer higher detection sensitivity of cellular and molecular processes, the relatively low spatial resolution of these systems makes it difficult to interpret focal molecular signals without corresponding anatomical information. To overcome this issue, hybrid imaging systems have emerged that integrate nuclear imaging modalities with CT or MRI systems. In addition to tracer localization, anatomical information generated from CT or MRI also facilitates correction of attenuation, scatter, and partial volume effects, thus improving image quality and accuracy of radiotracer quantification (62, 249, 294). Beyond providing an anatomical map for nuclear images, hybrid imaging systems, such as PET/MRI holds the potential to obtain synergistic, nonredundant data that allows for better characterization of disease pathology as has recently been reviewed elsewhere (307).

Computed tomography

Computed tomography (CT) is a commonly used imaging modality for the evaluation of the cardiovascular system, as it is cost effective, widely available and provides excellent spatial resolution. However, the lower detection sensitivity of CT for cellular and molecular processes compared to PET and SPECT makes CT less suitable for the use of molecular imaging. Clinically used iodine-based contrast agents provide good tissue contrast for anatomic evaluation; however, these agents are rapidly cleared from the blood and show nonspecific tissue distribution, both of which limit their use for targeted imaging. To overcome these issues, novel nanoparticles (1–100 nm diameter) have recently been assembled that incorporate high payloads of iodinated or inorganic contrast agents that may also use specific peptides or antibodies for improved sensitivity to detect molecular/cellular targets, while also improving signal-to-noise (361, 362).

Iodine-based nanoparticles can be assembled as micelles, liposomes, nanoemulsions, and dendrimers (255) (Fig. 1). Micelles are self-assemblies of amphiphilic molecules, consisting of outward pointing hydrophilic head groups and inward pointing hydrophobic tails. The iodinated contrast is attached to the hydrophobic tails allowing for the delivery of high payloads of iodine. Liposomes can also be constructed from amphiphilic molecules or polymers self-assembled into bilayers, allowing for the encapsulation of hydrophilic contrast agents in the core and hydrophobic contrast agents within the bilayer. Nanoemulsions are assembled by surrounding a hydrophobic iodinated contrast-containing core through a mixture of phospholipids and cholesterol. Dendrimers are polymers that are assembled by sequential branched growth steps leading to molecular stability and to the formation of a large number of terminal surface functional groups that can be used for modification.

Figure 1.

Lipid-based nanoparticles for molecular imaging. (A) Schematic representation of amphiphilic lipids. (I) Amphiphiles consist of a hydrophilic head and a hydrophobic tail. (II) Micelle-forming lipids have a relatively large head compared with the hydrophobic part, whereas (III) bilayer-forming lipids usually have two hydrophobic tails. (IV) PEG-lipids are used to improve pharmacokinetic properties and (V) cholesterol is used to stabilize liposomes. (B) Possible lipid aggregates for in vivo use. (I) Micelles can be prepared from micelle-forming lipids and from PEG-lipids. (II) A conventional liposome consists of a phospholipid bilayer. (III) Improved stabilization of liposomes can be achieved by incorporating a small amount of PEG-lipids and cholesterol. (IV) Microemulsions consist of a surfactant (amphiphile) monolayer covering oil. (V) Micelles can contain a hydrophobic nanoparticle. (VI) Bilayer on nanoparticles of silica, mica, glass, or iron oxide. Modified, with permission, from Mulder WJ, NMR Biomed. 2006 (255).

Inorganic contrast materials offer several advantages over iodine-based contrast agents. Contrast generation on a CT image relies on the distinct attenuation coefficients of different tissue types, which is determined by the atomic number (Z) and the electron density of the tissue components. Higher atomic number and higher electron density result in a higher attenuation coefficient. Novel inorganic contrast agents carry high Z elements, such as gold (Z = 79) or bismuth (Z = 83), thus produce higher attenuation on CT (193). Inorganic contrast-based nanoparticles have also been reported to be more ideal for spectral CT imaging than iodinated contrast agents. Spectral CT employs energy-sensitive photon-counting detectors that are used to differentiate photons from different energy levels (326). When a photon collides with these X-ray detectors, it generates a current pulse that is proportional to the energy of the photon. The detected photon then gets allocated to energy bins representing different electron voltage intervals. Importantly, detection of a specific element can be improved by placing the bins at the K-edge of the imaged element, where K-edge represents the characteristic energy needed to eject the innermost and most strongly bound electrons (K-shell electrons). Spectral CT uses the phenomenon that the attenuation of the X-ray photons suddenly increases at the photon energy just above the K-edge. Importantly, the human body absorbs a substantial proportion of the low energy photons, which significantly limits the use of low K-edge elements (such as iodine, 33 keV) for spectral CT. On the other hand, inorganic contrast agents, such as gadolinium (Gd) (50 keV), gold (81 keV), or bismuth (91 keV) have higher K-edge, which makes them more suitable for spectral CT imaging. However, these contrast agents are currently only used in preclinical settings due to the significant concern for human toxicity of the inorganic CT contrast materials.

Molecular magnetic resonance imaging and magnetic resonance spectroscopy

Magnetic resonance imaging (MRI) is capable of providing both anatomical and physiological information of a biological system at high spatial and temporal resolution. Molecular MRI imaging utilizes targeted contrast agents that create contrast by changing the magnetism of the targeted tissue’s surrounding environment. Although offering higher spatial resolution, molecular MRI has substantially lower sensitivity to detect molecular/cellular targets than SPECT or PET. Therefore, similar to CT molecular imaging, high in vivo concentration of the imaged contrast agent, and/or a very high concentration of the imaged molecular target is usually required for successful signal detection.

The most frequently used MRI contrast agents for clinical applications are nonspecific Gd-based contrast agents that increase signal intensity on T1-weighted images by shortening the T1 relaxation time of the surrounding water protons (343). Specific targeting of Gd-based agents has been achieved by conjugating them to protein-based probes, monoclonal antibodies, or antibody fragments (351). Similar to concepts described above for CT, higher detection sensitivity for molecular/cellular targets and higher signal amplification can be accomplished by incorporating large payloads of Gd into nanoparticles such as micelles, liposomes, emulsions or by assembling Gd-based dendrimers (255). As a different strategy, novel Gd-based contrast agents have been constructed to undergo spontaneous oligomerization upon target-enzyme activation, leading to prolonged retention and increased MRI signal (263).

Superparamagnetic iron oxide (SPIO) particles are the most frequently utilized non-Gd-based MRI agents in pre-clinical studies. These particles shorten T2 and T2* relaxation times of the nearby water protons, therefore, creating a signal void on T2 and T2* weighted images. The high relaxivity of these particles amplifies signal intensity, thus they can be used to image sparsely expressed molecular/cellular targets. SPIO particles can be used to image phagocytic cells, since they are nonselectivity taken up by cells of the mononuclear phagocytic system, or they can be engineered to bind to specific targets. SPIO particles are classified based on a size scale that ranges from 3 nm up to 10 μm. The most frequently used particles for molecular MRI imaging are ultrasmall (15–30 nm) SPIO particles (USPIO) (343).

Less frequently used MRI molecular imaging strategies use perfluorocarbon nanoparticles and chemical exchange saturation transfer (CEST) contrast agents. When using perfluorocarbon nanoparticles, an image signal is generated from ¹⁹F instead of from water protons, which results in a lower background signal at the cost of lower target signal intensity (117). CEST contrast agents contain a narrow band of off-resonance protons that exchange with tissue water protons (416). When radiofrequency saturation pulses are applied at the shifted CEST resonance, the CEST contrast agent becomes activated. In response to the activation, the saturated CEST protons exchange with the tissue water protons, which reduces the on-resonance water signal leading to reduced signal intensity.

Cardiac magnetic resonance spectroscopy (MRS) is a non-invasive tool for the assessment of myocardial metabolism, without the need for radiation or intravenous contrast use (161). Using the intrinsic magnetic resonance signals from nuclei, including hydrogen (¹H), carbon (¹³C), fluorine (¹⁹F), sodium (²³Na), and phosphorous (³¹P), MRS provides a comprehensive metabolic assessment of myocardial metabolism. However, MRS is limited by low signal-to-noise, low spatial resolution, and long acquisition times; therefore, it has been mostly used in the research setting. The availability of higher field strength (>3T) magnets and novel hyperpolarization techniques may enhance the clinical applicability of this technique.

Ultrasound

Ultrasound is the most widely used imaging modality for the evaluation of cardiovascular structure and function given its wide availability, noninvasive nature, and cost-effectiveness. However, similar to MRI and CT, ultrasound has lower detection sensitivity for molecular/cellular processes compared to nuclear modalities. To overcome this issue, targeted ultrasound contrast agents have been developed with molecular imaging capability (1, 343). An advantage of ultrasound molecular imaging compared to other systems is that it provides simultaneous anatomic information from traditional 2D echocardiography, which assists in the spatial localization of the observed molecular signals.

The most frequently used ultrasound contrast agents are microbubbles composed of a lipid, albumin, or biopolymer shell filled with perfluoropropane or sulfur hexafluoride gas. As these microbubbles are usually 1–8 μm in diameter, they readily pass through the microcirculation, but remain stable in the intravascular space and produce a detectable signal distinct from tissue background. At sufficiently high acoustic pressures, the bubbles expand and contract (resonate) in nonlinear fashion, emitting signals (harmonics) that are acoustically distinct from tissue backscatter. Certain ultrasound imaging systems are capable of detecting microbubble generated signals in the harmonic frequency range, whereas at the same time suppressing tissue backscatter in the fundamental frequency range. For the purposes of targeted imaging, the surface of the microbubbles is modified to express ligands that bind to the molecular target of interest. As the microbubbles are pure intravascular contrast tracers that remain within the vasculature (176), the molecular target needs to be expressed in the vascular compartment such as on endothelial cells, or on adherent, activated leukocytes or platelets. Consequently, targeted ultrasound imaging is mostly used in the field of vascular imaging. An additional challenge in ultrasound molecular imaging is that the specific signal from the molecularly attached microbubbles (usually only a small fraction of the total injected microbubbles) needs to be separated from the circulating nonattached microbubbles (1). Multiple approaches have been proposed to overcome this challenge. One strategy to assist in qualitative assessment is to delay imaging after the injection (approximately 10 min) to allow for the clearance of the nonattached microbubbles (1). For more quantitative assessment, the destruction-replenishment method can be used. In this method, the delivery of high intensity acoustic signals (mechanical index >0.5) destroys the microbubbles in the target tissue of interest. This allows the determination of the difference in imaging signal measured pre and post microbubble destruction, which corresponds to the molecularly targeted ultrasound imaging signal (1).

Ultrasound molecular imaging mainly occurs in the pre-clinical space, but recent reports showing the safety and feasibility of the first clinical grade targeted ultrasound contrast agent (BR55) in cancer patients may accelerate clinical translation of this molecular imaging modality (348, 421). Specifically, BR55 detects neovascularization in patients with various cancer types, including breast and ovarian cancer, by binding to vascular endothelial growth factor receptor 2 (VEGF-2), which is a receptor that is robustly upregulated during tumor angiogenesis.

Optical imaging

Optical molecular imaging techniques take advantage of the interaction between light and matter. Advantages to using optical imaging over other imaging approaches, include: very high image sensitivity to detect molecular signals (picomolar to femtomolar concentration); a wide variety of targeting platforms, such as peptides, proteins, antibody fragments and nanoparticles and widely available imaging systems, several of which are discussed in the following text (104, 172).

Fluorescence imaging is usually performed by delivering a fluorescent probe that interacts with the desired molecular target or by directly imaging an endogenously protein with fluorescent properties. The imaging signal is generated by excitation of the fluorescent probe/protein at a specific wavelength and detecting the emission signal at a different wavelength by a charge-coupled device (CCD) camera built into either a planar (fluorescence reflectance imaging; FRI) or a tomographic imaging system (fluorescence molecular tomography; FMT) (64). Fluorescent systems offer excellent ex vivo spatial and temporal resolution; however, their application is limited by poor in vivo spatial resolution. A potential strategy to overcome this limitation is the use of hybrid fluorescence imaging by incorporating MRI or CT modalities for anatomic reference. One major concern for fluorescent imaging is the limited penetration of light into deep tissues, which is limited from few millimeters (wavelength <500 nm blue, green) to several centimeters (wavelength >650 nm, red, near-infrared). Therefore, fluorescent imaging has been mainly used with combination of molecular probes that fluoresce in the red to near infrared spectral range. FMT is increasingly used to image cardiovascular targets in mouse models, where the depth of light penetration in tissue is not a significant limiting factor (200). Compared to fluorescence imaging in the visible light and mid-IR range, NIRF imaging allows for deeper tissue penetration and reduced tissue auto-fluorescence, which allows for higher sensitivity detection of molecular signals (172). Recently quantum dots, nanoparticles of 2 to 8 nm diameter with a semiconductor core and shell that possess adjustable optical and electrical properties, have been employed for fluorescent imaging; however, their potential toxicity needs to be investigated before translation to the clinical setting(411). In addition to these signal modal probes, magneto-fluorescent probes have been developed that take advantage of the strengths afforded by both fluorescent imaging (high imaging sensitivity) and MRI (high spatial resolution) (354).

Bioluminescence imaging (BLI) utilizes light generated from an enzyme-substrate pair as an imaging signal and an ultrasensitive cooled CCD camera for signal detection (64). The most frequently used BLI approach is the exogenous expression of a luciferase enzyme, followed by systemic delivery of the substrate (luciferin). The lack of competing background signal allows for high sensitivity (femtomolar concentration) detection of probes. Notably, the low cost and the high throughput capacity make BLI a very attractive modality for tracking stem cell delivery/survival, and for evaluation of therapeutic gene expression and biodistribution in small animal models (442). However, several technical details make BLI less favorable for clinical application, such as the limited penetration of light, requirement for exogenous reporter genes, and the need for delivery of a large amount of potentially immunogenic substrate.

Photoacoustic imaging is based on the optoacoustic effect, in which pulses of laser delivered light are absorbed in the visible and near-infrared wavelengths in tissue and generate an acoustic signal (ultrasound waves) upon absorption that can be detected in vivo with an ultrasound detector. Since ultrasound waves scatter less in tissue than light, this technique gives the advantage of high-resolution optical imaging at deeper tissue depth (several centimeters) compared to conventional optical imaging techniques. The acoustic signal is proportional to the local absorption of light, which is dependent on the absorption coefficient of different biological constituents (e.g., oxyvs. deoxy-hemoglobin), thus allowing for endogenous contrast for optical imaging of certain structures, such as the vasculature (440). In addition, exogenously delivered dye-based contrast agents or bioconjugated nanoparticles that possess significantly greater optical absorption than biological constituents have been used to improve image detection sensitivity of molecular targets in tissues or cells that weakly absorb visible and near-infrared wavelengths (175).

Intravascular based optical approaches, such as optical-coherence tomography (OCT) catheters have also been developed to overcome the limitations of light penetration into the tissue. OCT catheters employ near-infrared coherent light that generates very high resolution (4–16 μm) cross-sectional and volumetric images of the vessel wall at a suitable imaging depth (2–3 mm) based on principles of light scattering. OCT imaging contrast agents enhance light scattering, and a variety of molecularly targeted OCT probes have been developed to assess vascular inflammatory processes in vivo and at a high-resolution using intravascular OCT catheters (177, 234).

Figure 2 provides an overview of the strengths/weaknesses of each imaging modality as it relates to anatomical, physiological, metabolic, and molecular imaging. This figure also identifies which molecular processes can be best evaluated with each specific imaging approach. In addition, Table 1 provides a comprehensive summary of the links between a specific imaging modality and relevant tracers to pathological disease processes discussed in the review.

Figure 2.

Schematic representation of the relative strengths of each imaging modality as it relates to multiple facets of cardiovascular imaging. The weight of the connecting arrows on the left indicates the relative strengths of each imaging modality as it relates to anatomical (e.g., spatial resolution), physiological (e.g., flow, function), metabolic and molecular imaging. The connecting arrows on the right links each imaging modality to specific biological targets, including: thrombosis, angiogenesis, inflammation, autonomic nervous system function, the renin angiotensin-aldosterone system (RAAS), cell death, extracellular matrix, and tracking of cell and gene therapies. CT, computed tomography; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy.

Table 1.

Links Between Imaging Modalities and Relevant Tracers to Pathological Disease Processes

	Tracer	Mechanism	Myocardial ischemia/MI	Heart failure	PAH	Transplant/transplant rejection	Sarcoidosis	Myocarditis	Amyloidosis	References
PET	18F-FDG	Glucose analog (myocyte and inflammatory cell uptake	X	X	X	x	X	X	x	(2,3,20,28,31,33,40,54,55,103,107,129,134,149,202,215,226,244,274,286,291,304,311,316,325,336,383,408,413,425,431,437)
	11C-palmitate	Fatty acid analog	x							(328)
	18F-FTHA	Fatty acid analog			x					(134)
	11C-acetate	Index of myocardial oxygen consumption	x	X		X				(23,28,47,79)
	18F-annexin V	Binding to phosphatidylserine	x							(257)
	18F-WC-4-116 (isatin analogue)	Binding to caspase-3 and -7	x							(389)
	11C-methionine	Inflammatory cell uptake	X					x		(236,251,386)
	68Ga-pentixafor	Inflammatory cell recruitment by targeting CXCR4	X							(387)
	18F-GE180	Macrophage mitochondrial translocator protein (TSPO)	X							(388)
	64Cu dextran nanoparticles	Inflammatory cell uptake				x				(398)
	18F-fluorothymidine	Inflammatory cell uptake					X			(282)
	18F-galacto-RGD	αvβ3 integrin expression	X							(206,228,335)
	68Ga-PRGD2	αvβ3 integrin expression	X							(366)
	18F-Fluciclatide	αvβ3 and αvβ5 integrin expression	X							(178)
	64Cu-DOTA-VEGF121	VEGF receptor expression	x							(313)
	111In-DTPA-eNGR	CD13 expression	x							(151)
	64Cu-N0TA-TRC105	CD105 expression	x							(289)
	18F-FXIII	Substrate for FXIII, crosslinked to ECM proteins	x							(261)
	68Ga-collagelin	Binding to collagen I and III	x							(405)
	64Cu-NOTA-collagelin	Binding to collagen I and III	x							(189)
	18F-fluorobenzoyl-lisinopril	ACE inhibitor		x						(96)
	11C-KR31173	Angiotensin receptor I blocker	x							(127,156)
	18F-florbetapir	Unknown, binds to myocardial amyloid deposits							X	(102,295)
	Pittsburgh Compound B (11C-PIB)	Thioflavin-T derivative, binds to amyloid deposits							X	(8,213)
	11C-meta-hydroxyephedrine (HED)	Norepinephrine analog	X	X		X				(4,7,23,24,26,27,50,53,57,93,114,124,126,180,225,231,233,305,310,331)
	11C-epinephrine	Norepinephrine analog	x							(322)
	11C-CGP-12177	Nonselective β-antagonist	X	X						(53,180,272,285,359,396)
	11C- CGP-12388	Nonselective β-antagonist		X						(81)
	11C-MQNB	Nonselective muscarinic receptor antagonist	X	X						(208,237)
SPECT	123I-IPPA	Fatty acid analog	x	X						(336,406)
	123I-BMIPP	Fatty acid analog	x	X	X					(95,146,186,192,260,373,434)
	99mTc-annexin V	Binding to phosphatidylserine	X	x		x		x		(30,160,270,299,381)
	111 In-antimyosin Fab antibody	Binding to myosin	X			x				(125,232)
	99mTc-glucarate	Unknown	X							(230)
	111In-GSAO	Binding to hsp90 and filamin-A	x	x						(371)
	99mTc-duramycin	Binding to phosphatidylethanolamine	x							(414)
	99mTc-labeled C2A domain of synaptotagmin I	Binding to phosphatidylserine	x							(445)
	111In-Oxine	In vitro white blood cell labeling	x							(267)
	111In-RP748	Binding to αvβ3 integrin	x							(182,241)
	123I-Gluco-RGD	Binding to αvβ3 integrin	x							(181)
	99mTc-RAFT-RGD	Binding to αvβ3 integrin	x							(98)
	99mTc-maraciclatide	Binding to αvβ3 integrin	x	X						(100,218,221,254)
	111In-DOTA-FXIII	Substrate for FXIII, crosslinked to ECM proteins	x							(261)
	99mTc-Collagelin	Binding to collagen I and III	x							(258)
	111In-antitenascin-C monoclonal ab Fab′	Binding to tenascin C	x					x		(283,323)
	125I-antitenascin-C ab	Binding to tenascin C	x							(375)
	99mTc-RP805	Broad spectrum MMP inhibitor	x							(320,364)
	99mTc-losartan	Angiotensin receptor I blocker	x							(407)
	99mTc-pyrophosphate	Unknown, calcium deposits?	X						X	(39,52,60,61,73,278,433)
	123I-metaiodobenzylguanidine (mIBG)	Norepinephrine analog	X	X		X			X	(6,10,18,22,42,74,113,145,162,171,188,216,233,242,245,259,269,271,279,281,321,327,333,372,374,377,379)
CT	CNA-35 gold containing nanoparticles	Collagen-homing ligand	x							(75)
	AnxCLIO-Cy5.5	Binding to phosphatidylserine via Annexin V	x	x						(352,354)
MRI	Monocrystalline iron oxide nanoparticles with antimyosin Fab	Binding to myosin	x							(417)
	Gd-TO	DNA-binding gadolinium chelate	x							(164)
	SPIO bound C2A domain of synaptotagmin I	Binding to phosphatidylserine	x							(445)
	USPIO	Inflammatory cell uptake	x			x				(184,262,353,428,429)
	Ferumoxytol	Inflammatory cell uptake	X					X		(5,119,363,435)
	MPO-Gd	myeloperoxidase (MPO) activated gadolinium (Gd)	x							(263)
	Gd labeled EP-3533	Collagen I specific binding	x							(150)
	CLIO-VT750	Proteolytic cleavage by MMP-2 and MMP-9	x							(65,265)
US	P-selectin targeted microbubbles	Binding to P-selectin	x							(77,168,185)
	Phosphatidylserine-containing microbubbles	Complement-dependent binding to inflammatory cells	x							(252)
	ICAM-1 antibody conjugated microbubbles	Binding to ICAM-1				x				(419)
Optical	AnxCLIO-Cy5.5	Binding to phosphatidylserine via Annexin V	X	x						(352,354)
	Oregon Green labeled annexin V	Binding to phosphatidylserine	X							(105)
	Dendritic polyglycerol sulfates - 6S-ICG propargyl (NIR dye) conjugate	Binding to P- and L-selectin	x							(382)
	CLIO-Cy5.5	Inflammatory cell uptake	X							(353)
	Dendritic polyglycerol sulfates - 6S-ICG propargyl (NIR dye) conjugate	Binding to P- and L-selectin	x							(382)
	CLIO-VT75	Proteolytic cleavage by MMP-2 and MMP-9	x							(65,265)
	Prosense-680	Proteolytic cleavage by cathepsin B	x							(265)

Tracers and contrast agents that have been used for positron emission tomography (PET), single photon emission tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), and optical imaging applied to preclinical models (x) and patients (X) with the following of cardiovascular diseases: myocardial ischemia/myocardial infarction (MI); Heart failure; pulmonary arterial hypertension (PAH); cardiac transplant/transplant rejection; cardiac sarcoidosis; myocarditis; cardiac amyloidosis. Tracers and contrast agents that target metabolism (green); cell death (brown); inflammation (navy blue); angiogenesis (red); extracellular matrix (black); the renin angiotensin aldosterone system (gold); amyloid (light blue); and the autonomic nervous system (purple) are represented. Tracers and contrast agents that have not been applied to disease models or patients are not listed in this table, but are discussed in the text.

Metabolic Imaging of the Heart

Cardiac contraction and relaxation are energy-consuming processes; thus, the heart requires a high rate of adenosine triphosphate (ATP) turnover to maintain proper function. This is met primarily by mitochondrial oxidative phosphorylation (98%), with a small portion being supplied from substrate level phosphorylation during glycolysis (370). Under normal physiologic conditions at rest, the oxidative metabolism of fatty acids (FAs) provides the majority of mitochondrial ATP (~60%-90%), with the remainder of ATP being supplied from the oxidative metabolism of pyruvate that is generated from either glucose or lactate. Similar to other metabolically active organs, ATP can also be produced by the phosphocreatine (PCr) system and by anaerobic glycolysis, especially under certain stresses, such as hypoxia. In addition to energy provision, cellular metabolism also regulates or participates in numerous other functions, including, but not limited to antioxidant defense, cell signaling, and growth and survival (370). It has long been recognized that numerous cardiac diseases are associated with alterations in cardiac metabolism and that these changes may contribute to disease progression. More recently, it has been recognized that in vivo assessment of cardiac metabolism may help elucidate underlying disease mechanisms and etiology, guide medical therapy and offer information on CVD progression and risk (137).

Radiotracer approaches are the oldest techniques used to assess in vivo cardiac metabolism, with PET being by far the most frequently used cardiac metabolic imaging modality due to its quantitative capabilities and large inventory of available metabolic radiotracers. In PET, two major radiolabeling strategies have been employed for metabolic imaging. The most frequently used method involves the radiolabeling of the analogs of biologically available substrates with fluorine-18 (¹⁸F). Following uptake, these tracers often undergo enzymatic modification resulting in products that are unable to undergo further metabolism and become trapped in the cell. The best example in this category of metabolic imaging is 2-[¹⁸F] fluoro-2-deoxy-D-glucose (¹⁸F-FDG) (301). The alternative approach is to directly radiolabel the naturally occurring substrates, such as various fatty acids (FAs) (1-¹¹C-palmitate or 1-¹¹C-oleate), glucose (1-¹¹C-glucose), and lactate (L-3-¹¹C-lactate) with carbon-11 (¹¹C) in selected locations to reflect specific metabolic pathways (301, 343). Using this approach, the metabolism of the radiolabeled substrate is identical to the unlabeled substrate. With the application of appropriate kinetic models, the myocardial uptake and downstream metabolism of these substrates can be assessed quantitatively. The major disadvantages of this approach include the requirement of an on-site cyclotron and advanced radiochemistry capabilities due to the relatively short half-life of ¹¹C of only 20.4 minutes. This has significantly limited the widespread utilization of 11C-labeled radioisotopes. SPECT imaging has also been used for the evaluation of FA metabolism with radioiodine labeled compounds; however, difficulty in quantifying cellular processes and the relatively poor temporal resolution of SPECT imaging systems has limited the widespread use of this method for metabolic imaging. This limitation may disappear with the recent introduction of solid-state multidetector SPECT systems that provide high detection sensitivity for molecular processes and the capability to perform dynamic imaging.

In addition to nuclear imaging modalities, MRI and MRS have been used to assess components of myocardial metabolism. For example, myocardial oxygen extraction can be measured with MRI and the blood oxygenation level dependent (BOLD) method (see below) (441). Furthermore, MRS can be used to evaluate myocardial substrate metabolism, especially in the preclinical setting. This method allows the assessment of metabolites (e.g., 1-¹³C-pyruvate), high-energy phosphates (e.g., ATP, PCr) and triglyceride (TG) and sodium content (34). Notably, ¹³C MRS offers the ability to assess the metabolic flux of key metabolites through multiple pathways (e.g., tricarboxylic acid cycle (TCA), ß-oxidation). However, the low imaging sensitivity of this technique has precluded its widespread use. To overcome these issues, hyperpolarized ¹³C MRS was developed, which has the ability to increase signal by >10,000 times, but is also limited by technical challenges, such as the need for short-acquisition windows and supra-physiological tracer concentration (133). Lastly, CEST has been used to determine reaction rates and flux of key enzymatic processes of metabolites with exchangeable protons. For example, CEST has been used to determine ATP production rates via the exchange of inorganic phosphate (Pi) during the creatine kinase catalyzed reaction, adenosine diphosphate (ADP) + PCr → ATP + creatine (144). However, long acquisition times have precluded the widespread use of this technique. Figure 3 provides an overview of the key metabolic pathways and molecular imaging targets to assess metabolic events in vivo.

Figure 3.

Schematic representation of cardiac metabolic imaging, including imaging of myocardial perfusion, substrate utilization [glycolysis, β-oxidation, and tricarboxylic acid cycle (TCA) cycle], and high-energy phosphate metabolism: Myocardial perfusion can be quantified with gadolinium (Gd) using first-pass MRI or by using PET (¹⁵O-water, ⁸²Rb, or ¹³NH₄) or SPECT (^99mTc-tetrofosmin, ^99mTc-sestamibi or ²⁰¹Tl) radiotracers. Substrate utilization: Proton-MRS (¹H-MRS) is used for static measurement of the triglyceride (TG) pool. ¹³C-octanoate, 1-¹³C-pyruvate, and 2-¹³C-pyruvate can be used as tracers for hyperpolarized carbon-MRS (¹³C-MRS). ¹³C-octanoate can be found downstream as ¹³C-acetylcarnitine and can be used as an estimate of fatty acid uptake and oxidation. 1-¹³C-pyruvate will be converted to ¹³CO₂ and ¹³C-bicarbonate and can be used to determine the pyruvate dehydrogenase (PDH) fluxes as an estimate of glucose oxidation. By tracing 2-¹³C-pyruvate, the complete TCA cycle can be visualized, as the ¹³C label will be retained in acetyl-CoA and downstream in lactate, acetylcarnitine, citrate, and glutamate. Metabolic trapping of β-methyl-¹¹C-heptadecanoic acid (β-Me-HA) and 2-deoxy-2-¹⁸F-fluoro-D-glucose (FDG-glucose) enables dynamic estimates of fatty acid and glucose uptake by PET, respectively. 3- and 5-Methyl-17-¹⁸F-fluoroheptadecanoic acid (3-MFHA and 5-MFHA), 16-¹⁸F-fluoro-4-thiapalmitic acid (FTP), and 14-¹⁸F-fluoro-6-thiaheptadecanoic acid (FTHA) are used to estimate fatty acid uptake and metabolism (although metabolism and kinetics of these traces are not fully elucidated). The metabolically cleared ¹¹C-palmitate is used for estimation of fatty acid uptake, oxidation, and esterification; ¹¹C-acetate is used for the assessment of TCA activity coupled to oxygen consumption in the electron transport chain (ETC). ¹¹C-glucose, which is fully metabolized, enables kinetic modeling of glucose metabolism. Fatty acid tracers for SPECT are the metabolically trapped ¹²³I-β-methyl-p-iodophenylpentadecanoic acid (BMIPP) and the fully metabolized ¹²³I-iodophenylpentadecanoic acid (IPPA). High-energy phosphate metabolism: In the cytoplasm, adenosine diphosphate (ADP), formed by hydrolysis of adenosine triphosphate (ATP), can be resynthesized by cytoplasmatic creatine kinase (MM-CK) to ATP through hydrolysis of phosphocreatine (PCr). PCr can be quickly resynthesized by mitochondrial creatine kinase (Mi-CK) through hydrolysis of newly formed ATP in the mitochondria. PCr levels are dependent on cellular creatine (Cr) uptake, as creatine is not synthesized in the heart but actively taken up by cardiomyocytes. ¹H-MRS and phosphorus-MRS (³¹P-MRS) are used for measurement of Cr and PCr/ATP ratio, respectively. Modified, with permission, from van de Weijer T, J Appl Physiol (1985). 2018 (400).

Evaluation of fatty acid metabolism

Once taken up by the cell, FAs are either oxidized to acetyl-CoA via ß-oxidation for subsequent entry into the TCA cycle or stored in the form of TG. In oxidative metabolism, the TCA cycle generates the reducing equivalents, nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FADH₂) that participate in electron transfer within the mitochondria membrane for ultimate production of ATP following electron transfer to oxygen. Under fasting conditions, FA oxidation predominates, while FA storage may occur in the post-prandial state, during energy surplus and/or insulin resistance as discussed below. Complete or partial FA oxidation and storage can be measured with PET, SPECT, and MRS.

There are few available tracers for measuring myocardial metabolism with SPECT. One successful SPECT approach for evaluation of oxidative FA metabolism involves the use of 15-(p-iodophenyl)-pentadecanoic acid (¹²³I-IPPA), a medium-sized straight chain fatty acid that contains an aromatic ring at the omega position radiolabeled with radioiodine (197). ¹²³I-IPPA follows similar kinetics to ¹¹C-palmitate and the clearance rates of this tracer correlate directly with ß-oxidation. Unfortunately, conventional SPECT imaging systems does not have the detection sensitivity or temporal resolution to effectively evaluate the rapid kinetics of ¹²³I-IPPA, therefore this SPECT imaging approach was never widely applied. However, as mentioned above, this limitation may be overcome by the introduction of high spatiotemporal resolution solid-state SPECT cameras. The development of branched-chain analogs of IPPA, such as ¹²³I-beta-methyl-P-iodophenylpentadecanoic acid (BMIPP), provides an alternative solution to the challenges of SPECT imaging of ¹²³I-IPPA (109), since the alkyl branching of ¹²³I-BMIPP inhibits β-oxidation, thus increasing radiotracer retention and improving SPECT image quality. Tissue retention of ¹²³I-BMIPP appears to reflect initial activation of extracted FA by acyl-CoA carboxylase and their incorporation into TG. Consequently, static images provide an index of the initial portions of FA metabolism. However, quantification of myocardial substrate use with ¹²³I-BMIPP has been difficult because of the technical limitations of SPECT and incomplete metabolism of ¹²³I-BMIPP relative to endogenous fatty acids (432).

Labeling endogenous FAs with ¹¹C has advantage over nonendogenous analogs of FA, since the kinetics of labeled endogenous FAs is identical to that of unlabeled FAs. Therefore, the uptake, oxidation, and storage of ¹¹C-labeled endogenous FAs can be derived with appropriate kinetic modeling and metabolite correction (301). The most widely used example of this is ¹¹C-palmitate. Several preclinical studies demonstrated that the washout of extracted ¹¹C-palmitate from the myocardium correlates with myocardial oxygen consumption (132, 328, 329). However, the kinetic modeling associated with palmitate is complex and image quality is suboptimal due to rapid degradation. To overcome these issues, several FA analogs have been radiolabeled with ¹⁸F that get “trapped” at a point in ß-oxidation causing accumulation of the radiotracer in the cell which leads to improved image quality (86). However, improved image quality comes at a cost of losing the ability to separate between FA uptake, oxidation, and storage. Several FA analogs have been developed, such as 14-(R,S)-¹⁸F-fluoro-6-thiaheptadecanoic acid (FTHA) (85), 16-¹⁸F-fluoro-4-thia-palmitate (FTP) (86) and trans-9(RS)-¹⁸F-fluoro-3,4(RS,RS) methylenehepta-decanoic acid (FCPHA) (340), each with well described strengths and weaknesses (138, 301). Since oleate has a higher rate of oxidation than palmitate in the myocardium, a 4-thia-substituted analog of oleate, 18-¹⁸F-fluro-4-thia-oleate (FTO) was developed to take advantage of this property (84). Preclinical studies in rats show greater specificity for ß-oxidation and image quality of FTO compared to FTP (84), however the utility of this tracer in patients has not been established.

Recently, 15-(4-(2-¹⁸F-fluroethoxy)phenyl)pentadecanoic acid (¹⁸F7) was synthesized based on the structure of the SPECT tracer, IPPA (397). ¹⁸F7 has in vivo kinetics similar to ¹¹C-palmitate; therefore, may be used to separate between FA uptake, oxidation, and storage with appropriate kinetic modeling. Initial studies with ¹⁸F7 have shown excellent image quality in preclinical models when compared to ¹¹C-palmitate (397). Indeed, the potential clinical utility of this tracer needs to be studied.

The above-mentioned PET techniques and tracers only represent FA metabolism from circulating FA bound to albumin or in free form. However, a significant proportion of FA delivered to the myocardium are in bound form as TG within circulating chylomicrons or very low-density lipoproteins (131). To overcome this issue, oral administration of FA radiotracers to allow for incorporation of FA in chylomicrons has been proposed. This approach has been employed using ¹⁸F-FTHA in both small animals and humans showing successful incorporation of ¹⁸F in chylomicrons and the ability to measure FAs derived from these particles within the myocardium and other organs (203, 204). Although interesting, interpreting radiotracer uptake is difficult due to technical challenges highlighted succinctly and elegantly elsewhere (339).

In addition to circulating FA, intracellular TG also contribute to the overall oxidation of FA within the myocardium, and the proportion of energy provision from these sources appears to be altered in different disease states (48). The regulation of TG turnover within the myocardium is unclear, but two other PET approaches have been established that may allow for elucidation of these mechanisms. The first approach involves radiolabeling the TG pool with ¹¹C-palmitate, and after equilibrium is reached, measuring tracer washout under various pharmacological or biological stimuli (194). However, this method is limited by the need to use high doses of radioactivity and only providing and index of overall TG turnover. The second approach to assess TG turnover combines FA oxidation measures with PET (¹¹C-palmitate) and intracellular lipids quantification with ¹H-MRS to estimate the contribution of plasma and intracellular lipids to myocardial FA oxidation (48). This approach was applied in obese humans and was able to show that a significant proportion of energy provision for myocardial work is derived from the oxidation of intracellular FAs, which could be reduced with novel therapeutics. Although interesting, this method requires further validation.

Although useful for assessment of overall FA delivery, oxidation, or storage, the aforementioned techniques are unable to provide information on the flux of FA and FA metabolites through key metabolic reactions. Therefore, hyperpolarized ¹³C MRS has used for more nuanced insights into FA metabolism (330). Considering that acetate is a short chain FA, [1-¹³C]acetate has been used to assess FA flux through the TCA in preclinical models (15, 118). In addition, key metabolic reactions involving acetate have been explored with [1-¹³C]acetate, such as the production of the central metabolite acetyl-CoA via acetyl-CoA synthetase (179). Furthermore, [1-¹³C] labeled butyrate, a short-chain FA derived from colonic bacteria, has been used to assess multiple enzymatic reactions of ß-oxidation and subsequent flux through the TCA (13).

Evaluation of carbohydrate metabolism

Once taken up by the cell, glucose can either enter glycolysis for breakdown to pyruvate, become stored as glycogen or enter the pentose phosphate pathway. Pyruvate is converted to lactate or to alanine in the cytosol during ischemic conditions or high glycolytic flux, or is converted to acetyl-CoA via the pyruvate dehydrogenase (PDH) complex within the mitochondria for participation in oxidative phosphorylation (370). Generally, oxidative metabolism of glucose is favored, although the anaerobic metabolism can occur during cardiac stress to maintain ATP production (436). Although FA metabolism is favored over glucose under normal conditions, this preference can change in certain CVD states; therefore, the ability to assess in vivo glucose metabolism provides a valuable tool to metabolically phenotype different CVD states. In addition, assessing in vivo myocardial glucose metabolism has been useful in distinguishing viable myocytes from scarred myocardium (see the following text).

PET imaging of ¹⁸F-FDG is by far the most common and widely applied tracer to assess carbohydrate metabolism in the heart, which has been facilitated by the longer half-life of ¹⁸F (110 min) compared to other PET tracers combined with established networks for ¹⁸F-FDG distribution. ¹⁸F-FDG is taken up via glucose transporters, where it is subsequently phosphorylated by hexokinase to ¹⁸F-FDG-phosphate and becomes trapped in the cytosol unable to undergo further metabolism, thereby causing tracer retention and excellent image quality (301). ¹⁸F-FDG uptake can be expressed in relative (static imaging) or absolute terms (dynamic imaging with kinetic modeling), with the need of a lumped constant to correct for kinetic differences between the radiotracer and endogenous glucose (155). However, the limited metabolism of ¹⁸F-FDG precludes the determination of the intracellular metabolic fate of glucose (i.e., anaerobic/aerobic glycolysis vs. glycogen synthesis) (201). Alternatively, myocardial glucose uptake and metabolism can be determined with PET imaging of ¹¹C-glucose. Unlike ¹⁸F-FDG, ¹¹C-glucose is metabolized beyond the hexokinase step, having the same metabolic fate as endogenous glucose (155). However, kinetic modeling of ¹¹C-glucose is more complicated and requires correction for the arterial input function for the production of ¹¹CO₂ and ¹¹C-lactate. The heart has the ability to oxidize circulating and locally produced lactate, which becomes an important source of energy during periods of increased cardiac work (368). The PET tracer, L-3-¹¹C-lactate provides an index of lactate metabolism. The extraction of lactate in the heart using this PET imaging approach correlates with lactate oxidation measured by direct sampling of arterial and coronary sinus blood (154). However, the use of this technique has been limited.

Unlike nuclear imaging, hyperpolarized ¹³C MRS has the ability to probe key reactions in glucose metabolism. Specifically, [1-¹³C]-pyruvate has been used to assess in vivo activity of lactate dehydrogenase ([1-¹³C]-lactate production), alanine aminotransferase ([1-¹³C]-alanine production), or PDH flux (¹³CO₂ and ([¹³C]-bicarbonate production), key reactions that represent redox state of the cytosol, cardiac work and oxidative metabolism of glucose, respectively (12, 138). In addition, the use of [2-¹³C]-pyruvate has been used to assess the real-time flux of [2-¹³C]-pyruvate through the TCA cycle, as the metabolism of this tracer leads to the production of [1-¹³C]-acetyl-CoA (330). Although useful to understanding biology in preclinical applications, the use of hyperpolarized pyruvate has remained largely preclinical, since tracer concentrations needed to detect an adequate signal are supraphysiological. Therefore, [1-¹³C]-lactate, has been proposed as an alternate strategy to assess key reactions in glucose metabolism, since lactate is present in the plasma at a much higher concentration than pyruvate and is readily metabolized in the heart following conversion to pyruvate (95).

Evaluation of myocardial oxygen consumption

As discussed above, the oxidation of FAs and carbohydrates leads to the production of acetyl-CoA for entry into the TCA cycle within the mitochondria. Reducing equivalents produced in the TCA then enter the electron transport chain (ETC) for a series of electron transfers that end in the reduction of oxygen. This collective process creates the electrochemical gradient necessary for the phosphorylation of ADP to ATP, termed oxidative phosphorylation (147).

Myocardial oxygen consumption can be noninvasively evaluated with PET imaging by either ¹⁵O-oxygen or ¹¹C-acetate. PET imaging of ¹⁵O-oxygen provides an index of myocardial oxygen extraction, which when combined with measures of blood flow and arterial oxygen content yields a direct measure of myocardial oxygen consumption (MVO₂) (170). This multitracer approach involves complex compartmental modeling (170). PET imaging of ¹¹C-acetate can also be used for the estimation of overall oxidative metabolism by two basic principles: (i) ¹¹C-acetate is rapidly converted to acetyl-CoA through the TCA cycle, and (ii) the TCA and oxidative phosphorylation are closely coupled (301). Therefore, in combination with kinetic modeling, PET imaging with ¹¹C-acetate can provide an index of MVO₂ but does require correction of blood activity for ¹¹CO₂ production (47, 365).

Recently, MRI BOLD imaging has emerged as a promising alternative imaging modality to assess myocardial oxygen utilization (238). BOLD provides an index of the myocardial extraction of oxygen and takes advantage of differences in magnetism of hemoglobin between its oxygenated (diamagnetic) and deoxygenated (paramagnetic) states. Therefore, increased myocardial extraction of oxygen from oxyhemoglobin causes a signal loss on T2 and T2* weighted images, whereas a decrease in myocardial extraction from oxyhemoglobin will cause a signal increase. The measure of myocardial oxygen extraction derived from BOLD can be combined with separate measures of myocardial blood flow and blood volume from first-pass perfusion imaging to determine MVO₂ (238, 239). MVO₂ derived from this method is comparable to values obtained from ¹¹C-acetate PET imaging. However, several technical difficulties exist with this method that must be overcome prior to its widespread adoption.

Clinical applications

Myocardial ischemia

Myocardial ischemia causes a decrease in the ß-oxidation rate of FA (220). Therefore, to maintain ATP production and cell viability, there is an increase myocardial glucose usage through increased translocation of glucose transporter type (GLUT) 1 and GLUT 4 (Fig. 4) (436). In the ischemic state, glucose metabolism occurs mainly through anaerobic glycolysis as demonstrated by ¹³C-MRS (240). Shi et al. demonstrated this metabolic substrate switch in a pre-clinical model of graded coronary stenoses, demonstrating an initial reduction in ¹²³I-IPPA uptake in ischemic regions and subsequent delayed clearance with a concomitant increase in ¹⁸F-FDG accumulation in the ischemic regions (Fig. 5) (336). Similar findings have been demonstrated in patients post exercise by both an elevation in myocardial ¹⁸F-FDG (91% of patients with angiographic CAD) (149) or a reduction in ¹²³I-BMIPP uptake (96% of patients with ²⁰¹Tl SPECT perfusion defects). (95). Interestingly, when ischemia resolves and oxidative metabolism resumes, abnormalities in substrate metabolism may persist for several hours to days, also known as ischemic memory. This was first demonstrated with PET ¹⁸F-FDG imaging, showing an increase in glucose uptake during the recovery phase following exercise-induced ischemia when perfusion decrements and ECG changes returned to normal (54). Dilsizian et al. also demonstrated this phenomenon with ¹²³I-BMIPP in patients with exercise-induced myocardial ischemia that were imaged up to 30 hours after the ischemic episode (Fig. 6) (95). Considering that perfusion deficits are quickly resolved after an acute ischemic episode, this metabolic ischemic fingerprint may assist in the diagnosis of coronary artery disease (CAD) in patients with acute chest pain. For example, ¹²³I-BMIPP SPECT imaging has been shown to have superior diagnostic sensitivity (74%) over ^99mTc-tetrofosmin perfusion imaging (38%) in identifying ischemic lesions (coronary stenosis or spasm) as the cause of acute chest pain in patients whom also underwent coronary angiography (186). In addition, the diagnostic accuracy of identifying patients with ischemic lesions (coronary stenosis, 82%; coronary spasm, 83%) was highest when both ¹²³I-BMIPP and ^99mTc-tetrofosmin imaging were abnormal. However, nonstandardized imaging protocols, dependence on substrate availability and preference, and the uncertainty regarding the clinical utility, contributed to the reasons why cardiac metabolic imaging is not a clinical standard in the setting of acute ischemic events or following exercise.

Figure 4.

Low-flow ischemia leads to translocation of GLUT-4 and GLUT-1 to the sarcolemma: (I) Immunofluorescence of glucose transporter-4 (GLUT-4) and GLUT-1 in sections from nonischemic (A) and ischemic (C) regions of left ventricle by confocal microscopy. (II) Myocardial extraction (% of arterial) of glucose and lactate in the left anterior descending (LAD) and left circumflex coronary (LCx) regions during the 30 min before ischemia (Baseline) and during the last 30 min of low-flow ischemia (Low Flow). AV extractions were calculated from quadruplicate time points. *P < 0.01 versus both baseline LAD extraction and LCx extraction during low-flow ischemia. Values represent mean ± SEM (n = 9). (III) Sarcolemma and intracellular membrane content of GLUT-4 and GLUT-1 in myocardium from nonischemic and ischemic regions of the left ventricle. Glucose transporter content was quantified by ¹²⁵I-protein A binding (cpm/μg membrane protein) multiplied by yield of membrane fraction. Data for each membrane fraction are expressed as a percentage of total GLUT-4 or GLUT-1 content in sarcolemma and intracellular membrane fractions. Values are mean ± SEM (n = 9). *P < 0.05 versus nonischemic myocardium. Modified, with permission, from Young LH, Circulation. 1997 (436).

Figure 5.

Correlation of the myocardial free fatty-acid analog, p-¹²³I-iodophenylpentadecanoic acid (IPPA) retention with 2-[¹⁸F] fluoro-2-deoxy-D-glucose (¹⁸F-FDG) accumulation during experimental low-flow ischemia: (I) After baseline (BASE) measurements, partial coronary stenosis was created in canines and maintained throughout protocol. Arterial and venous samples were obtained for metabolic measurements, and radiolabeled microspheres were injected at times designated. IPPA was injected 60 min after creation of stenosis. ¹⁸F-FDG was injected 90 min later. (II) Myocardial ¹²³I-IPPA and ¹⁸F-FDG retained activities expressed as nonischemic percentage for all segments (n = 576). Segments were segregated on basis of normalized flows in segments into 20%-flow increments. Numbers within each bar represent number of segments falling into each flow range. (III) (A) Serial short-axis and vertical long (v-long) axis SPECT ¹²³I-IPPA images from a representative dog displayed in standard format. Time after injection is designated on left margin. Note perfusion defect in anteroseptal and anteroapical regions, which normalizes over time. (B) Myocardial ¹²³I-IPPA clearance curves derived from ischemic and nonischemic regions for same dog. (C) Early clearance data for same dog are also displayed as semilogarithmic plot. Early myocardial clearance appears linear on this semilogarithmic plot, suggesting early monoexponential clearance of ¹²³I-IPPA. Delayed myocardial ¹²³I-IPPA clearance is seen in ischemic region. This research was originally published in JNM. Shi CQ, Young LH, Daher E, DiBella EV, Liu YH, Heller EN, Zoghbi S, Wackers FJ, Soufer R, and Sinusas AJ. Correlation of myocardial p-(123)I-iodophenylpentadecanoic acid retention with (18)F-FDG accumulation during experimental low-flow ischemia. J Nucl Med 43: 421–431, 2002. © SNMMI. (336).

Figure 6.

SPECT imaging showing delayed recovery of regional fatty acid metabolism in heart tissue after transient exercise-induced ischemia (“ischemic memory”): Representative stress and rest short-axis thallium tomograms after reinjection (left two panels) demonstrate a reversible inferior defect consistent with exercise-induced myocardial ischemia. Cardiac SPECT of a patient injected with radiolabeled [¹²³I]-β-methyl-p-iodophenyl-pentadecanoic acid (BMIPP), acquired at rest 22 h after exercise-induced ischemia, shows persistent metabolic abnormality in the inferior region despite complete recovery of regional perfusion (center panel). Retention of BMIPP in the heart of a normal adult is shown as a control (right panel). Modified, with permission, from Taegtmeyer H, Nat Clin Pract Cardiovasc Med. 2008 (369).

Hypertension/left ventricular hypertrophy

The metabolic phenotype of left ventricular hypertrophy (LVH) is characterized by a reduction in FA oxidation, secondary to a downregulation of several ß-oxidation enzymes, and an increase in anaerobic glucose metabolism that is accompanied by increases in glucose transporters and glycolytic enzymes (174). This metabolic phenotype has been confirmed in vivo with PET imaging in preclinical models and in humans. In mice, ¹⁸F-FDG PET imaging showed that myocardial glucose uptake increases within 24 hours of pressure overload and continues to rise in parallel to increases in LV size (448). Interestingly, the increase in myocardial ¹⁸F-FDG occurs prior to decrements in systolic function, and inhibition of glucose oxidation with propranolol treatment leads to an attenuation of LV dysfunction (448) (Fig. 7). These data suggest that pressure overload induced cardiac stress leads to an increase in glucose metabolism, and this shift appears to stimulate/accelerate LV hypertrophy through activation of protein synthetic pathways. In hypertensive humans, ¹¹C-palmitate imaging showed a reduction in FA oxidation that was independently predictive of an increased LV mass (r = −0.24, P = 0.018). Specifically, for every unit (nmol/g per minute) decrease in myocardial FA oxidation, there is an associated 0.17 g increase in LV mass (82). Other studies in hypertensive patients with LVH have observed that a decrease in ¹¹C-palmitate PET imaging derived FA oxidation was paralleled by a decrement in myocardial contractile efficiency when compared to controls (83). Therefore, aberrant changes in substrate metabolism which, at least in part, may promote LVH and contribute to reduced contractile efficiency, which at least in part, may contribute to the progression of HF.

Figure 7.

Quantitative PET imaging detects early metabolic remodeling in a mouse model of pressure-overload left ventricular hypertrophy in vivo: Gated end-diastolic transverse ¹⁸F-FDG PET images for sham mice, transverse aortic constriction (TAC) mice, and TAC mice treated with propranolol at baseline, day 1, and day 7 after surgery are shown. All serial scans are from same animals. Images show increase in ¹⁸F-FDG uptake in TAC mice starting at day 1, indicative of metabolic adaptation in pressure-overload left ventricular hypertrophy. This research was originally published in JNM. Zhong M, Alonso CE, Taegtmeyer H, and Kundu BK. Quantitative PET imaging detects early metabolic remodeling in a mouse model of pressure-overload left ventricular hypertrophy in vivo. J Nucl Med 54: 609–615, 2013. © SNMMI. (448).

Heart failure

Ischemic cardiomyopathy

In chronic ischemia, the myocardial preference for glucose metabolism promotes cell survival (129). As a result, myocytes may remain viable despite reductions in myocardial blood flow and contractile function, a state referred to as myocardial hibernation. Hibernating myocardium retains metabolic function, as evidenced by 123I-IPPA (336) or more commonly,¹⁸F-FDG uptake despite a reduction in perfusion, a condition known as flow-metabolism mismatch (Fig. 8) (129). On the other hand, a concomitant decrease in both metabolism and perfusion is associated with scarred, nonviable myocardium. Importantly, viable, but hibernating myocardium has the potential to recover contractile function following revascularization, while scarred myocardium does not (17). Early studies using ¹²³IPPA imaging in patients with chronic myocardial ischemia, showed that the presence of ≥7 ¹²³IPPA -viable segments was the best predictor of functional recovery (≥10% increase in LVEF) following surgical revascularization compared to other clinical variables (72% accuracy, CI [64%-80%]) (406). More recently, ¹⁸F-FDG PET imaging has been shown to be the most diagnostically sensitive (92%) non-invasive viability imaging technique for predicting recovery of segmental contractile function of hibernating myocardium following revascularization (19, 325). It has been suggested that ¹⁸F-FDG PET viability imaging may help guide revascularization decision making in patients with chronic ischemic cardiomyopathies, since revascularization is associated with high perioperative morbidity and mortality in these patients (391, 404). However, evidence to support this hypothesis is controversial, with the largest prospective, randomized trial of 430 ischemic HF patients (LVEF <35%) only showing a nonsignificant trend for a decrease (30%) in the composite endpoint of cardiac death, myocardial infarction, or recurrent hospital stay for cardiac cause within 1 year in the ¹⁸F-FDG imaging guided group (30%) versus standard of care (36%) (relative risk 0.82, 95% CI[0.59–1.14], P = 0.16) (20). On the other hand, sub-analyses of this dataset have shown a significant benefit of ¹⁸F-FDG image guided revascularizations in reducing the composite event rate (19%) versus standard care (41%) (relative risk 0.37, 95% CI [0.17–0.78], P = 0.009) when imaging was performed in an experienced center using standardized protocols (2). Now that PET is more widely available, larger trials that include experienced imaging centers may be needed to reestablish the clinical value of ¹⁸F-FDG viability imaging in guiding revascularization under conditions of chronic ischemia and HF.

Figure 8.

PET scan showing perfusion–metabolism mismatch in hibernating heart tissue as an example of preserved cardiometabolic reserve: (A) ⁸²Rubidium (⁸²Rb) PET in short-axis view shows markedly decreased perfusion in the apical, inferior, inferolateral, and septal regions of the left ventricle at rest, which extends from distal to basal slices. (B) Images acquired under glucose-loaded conditions, labeled with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG), show perfusion-metabolism mismatch pattern (the scintigraphic hallmark of hibernation) in all abnormally perfused myocardial regions at rest. An exception is the anteroseptal region, which demonstrates matched perfusion-metabolism pattern (compatible with scarred myocardium). Modified, with permission, from Taegtmeyer H, Nat Clin Pract Cardiovasc Med. 2008 (369).

Nonischemic dilated cardiomyopathy

Similar to pressure overload hypertrophy, patients with nonischemic dilated cardiomyopathy have a reduced cardiac FA oxidative capacity that is accompanied by an increase in anaerobic glucose metabolism (174). Nuclear imaging with both SPECT and PET has confirmed this metabolic shift in these patients and has provided valuable information on disease severity and prognosis. For example, SPECT imaging of ¹²³I-BMIPP demonstrated deficits in FA oxidation in patients with an idiopathic dilated cardiomyopathy that correlated with other indicators of HF severity, such as left-sided filling pressures (pulmonary capillary wedge pressure: r = 0.68, P < 0.001, LV end diastolic pressure: r = 0.65, P < 0.001), fractional shortening (r = −0.58, P = 0.001), myocyte diameter (r = 0.66, P < 0.001) and percentage area of interstitial fibrosis measured from right heart endomyocardial biopsies (r = 0.69, P < 0.001) (434). In addition, multi-isotope PET imaging in patients with idiopathic dilated cardiomyopathy confirmed a reduction in FA uptake (134 ± 44 vs. 213 ± 49 nmol/g/min, P = 0.003) and oxidation (113 ± 50 vs. 205 ± 49 nmol/g/min, P = 0.001) that occurred concomitantly with an increase in glucose uptake compared to age-matched controls (79). Metabolic imaging has also been useful in elucidating the mechanisms responsible for therapeutic improvements in cardiac function in HF patients. Specifically, improvements in SPECT ¹²³I-BMIPP derived FA oxidation were observed in HF patients, the majority with nonischemic etiology, following 6 months of candesartan (angiotensin II receptor blocker) treatment (373). More recently, a high defect SPECT ¹²³I-BMIPP score (≥4) in nonischemic HF patients with preserved ejection fraction (HFpEF) was prognostic of higher major adverse cardiovascular events (MACE, composite event of cardiac death, AMI, unstable angina or HF hospitalization) during a mean follow-up of 2.5 years compared to patients with low (<4) ¹²³I-BMIPP defect scores (MACE rate 59% vs. 9% respectively, P < 0.001) (146).

Right ventricular imaging in pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a rare, progressive disease, which is associated with increased pressure in the arterial segment of the pulmonary circulation (116). Right ventricular (RV) remodeling, and RV failure are common long-term complications of PAH, with RV failure being the most common cause of death in these patients (116).

Growing evidence suggests that PAH shifts RV metabolism from aerobic oxidation of FAs and carbohydrates to the anaerobic metabolism of glucose (318). Considering that anaerobic glycolysis produces significantly less ATP than oxidative metabolic pathways, there is a marked upregulation of glucose uptake to maintain an adequate energy supply. PET imaging of ¹⁸F-FDG has confirmed this elevation in glucose uptake in both animal models of PAH (304) and in patients with PAH (55, 226, 316). In PAH patients, the LV to RV ¹⁸F-FDG uptake ratio correlated well with pulmonary artery systolic pressure (r = 0.75, P = 0.0085), pulmonary artery mean pressure (r = 0.87, P = 0.001) (40), and RV ejection fraction (r = −0.40, P < 0.001) (244). In addition, higher RV ¹⁸F-FDG uptake (partial volume corrected standardized uptake value [SUV] ≥8.3) in PAH has been associated with worsening clinical status (383). Moreover, improvements in RV function and pressures following therapy have been associated with reductions in RV ¹⁸F-FDG uptake in both preclinical PAH models (103) and in patients with PAH, where the percentage change of the corrected ¹⁸F-FDG RV SUV significantly correlated with the percentage change of the pulmonary vascular resistance (r = 0.78, P < 0.01) (286, 413) (Fig. 9).

Figure 9.

Increased ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) accumulation in right ventricular free wall in patients with pulmonary hypertension and the effect of epoprostenol: Representative midventricular transaxial ¹⁸F-FDG positron emission tomography images of a patient with primary pulmonary hypertension before and after the pulmonary vasodilator therapy with epoprostenol for three months. (A) Before the pulmonary vasodilator therapy, the right ventricular (RV) ¹⁸F-FDG accumulation was highly increased and the corrected RV standardized uptake value (SUV) of ¹⁸F-FDG was 13.4. (B) After the therapy, the corrected RV SUV of ¹⁸F-FDG markedly decreased to 7.5. (Lower panels) Correlations between the percentage change of RV SUV of ¹⁸F-FDG corrected for the partial volume effect and the percentage change of the pulmonary vascular resistance and peak-systolic wall stress in the RV free wall. Modified, with permission, from Oikawa M, J Am Coll Cardiol. 2005 (286).

Metabolic imaging has also been useful in elucidating other metabolic derangements in PAH. As described above, PAH is associated with a reduction in the oxidation of FAs. PET imaging of the FA analog, ¹⁸F-FTHA, showed reduced FA uptake in the RV that paralleled a lesser increase in ¹⁸F-FDG uptake in an animal model of PAH (134). In addition, reduced SPECT ¹²³I-BMIPP uptake has been detected in patients with severely hypertrophic RV due to RV overload (192, 260). Taken together, these exciting preliminary studies indicate that metabolic imaging of the RV may serve as a marker of disease severity, facilitate the tracking of therapeutic responses and predict outcomes in PAH patients; however, large studies are needed to confirm this postulation.

Cell Death Imaging

Cell death plays a significant role in the pathogenesis of many diseases that affect the myocardium. Molecular imaging has been used to image two distinct forms of cell death linked to cardiovascular pathology: necrosis (uncontrolled cell death) and apoptosis (programmed cell death).

Cell necrosis imaging agents

In necrosis, disruption of the cell membrane leads to loss of membrane integrity and release of intracellular contents into the extracellular environment. Therefore, many necrosis avid agents target intracellular components that are not normally found in the extracellular environment, such as myosin (¹¹¹In-antimyosin) (232), heat shock protein (hsp) 90 (371), or calcium deposits (^99mTc-pyrophosphate [PYP]) (73, 433), whereas some tracers demonstrate high affinity to necrotic tissue via unclear mechanisms (^99mTc-glucaric acid) (230). More recently, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO), an arsenic compound tagged with ¹¹¹In has been used to home into dithiol-containing intracellular molecules, such as hsp90 and filamin-A (371). In addition, many MRI molecular probes provide a unique opportunity for imaging necrosis. For example, gadoporphyrin is an MRI contrast agent that shows accumulation in necrotic tissue (214), while monocrystalline iron oxide nanoparticles can be coupled to antimyosin antibodies to enhance MR contrast (417). In addition, novel deoxyribonucleic acid (DNA)-binding Gd chelates, such as Gd-TO (164) have been developed to image necrosis via binding to exposed DNA in acutely necrotic cells.

Cell apoptosis imaging agents

Unlike necrosis, apoptosis is a highly regulated process that is characterized by controlled enzymatic degradation and relatively clean removal of the cell (334). Apoptosis is triggered by activation of cytoplasmic caspase-3, which initiates a cascade of intracellular events that leads to the exposure of phospholipids in the cell membrane that normally face the inner cell including, phosphatidylserine (PS) and phosphatidylethanolamine (PE). The exposure of these phospholipids to the outer layer of the cell membrane facilitates recognition of the cell to phagocytic cells. The most commonly used apoptosis molecular imaging tracers have been developed to target PS and caspases. The most well described PS targeted tracer is Annexin-V, which binds to PS with high affinity. Annexin-V has been radiolabeled with both PET (¹⁸F, ⁶⁸Ga, ¹²⁴I) (187, 257) and SPECT (^99mTc,¹²³I) (36, 205) radioisotopes. Annexin-V has been also linked to SPIO nanoparticles and Gd-based contrast agents for higher spatial resolution detection of apoptosis with MRI (158, 352). AnxCLIO-Cy5.5, an Annexin V cross-linked SPIO tagged with Cy5.5 (fluorescein probe), has been demonstrated to have high affinity for apoptotic cardiomyocytes (352, 354). In addition, annexin-V has been linked to Gd-diethylenetriamine pentacetate (DTPA)-coated liposomes (158) and the fluorescent probe Oregon Green (105). Another PS homing ligand, the C2A domain of synaptotagmin I has been used to image apoptosis by labeling with ^99mTc (445) or by conjugation to SPIOs (444). Lastly, PE targeted imaging with ^99mTc-duramycin has been proposed as an alternative to PS-targeted imaging (414).

As phospholipids are exposed to the extracellular environment both during apoptosis and necrosis, the differentiation between necrosis and apoptosis can be difficult. To overcome this issue, isatin sulfonamide analogs, which are potent inhibitors of caspase 3 and 7, have been labeled with ¹⁸F to more specifically assess cellular apoptosis with PET imaging (389). In addition, an MRI technique has been also proposed to image both cardiomyocyte apoptosis and necrosis by employing AnxCLIO-Cy5.5 (apoptosis) and delayed enhancement imaging with Gd-DTPA-NBD (necrosis) (352).

Clinical applications of cell death imaging

Acute myocardial infarction

Cell death is a key component in the pathogenesis of AMI. Within hours of vascular occlusion, cardiomyocytes start to die from necrosis and apoptosis resulting in significant myocellular loss within the injured myocardium. Both necrosis and apoptosis occur mainly in the infarct zone; however, cell death occurs to a lesser degree in the border-zone of the infarct, as well as in the remote myocardium. While necrosis is usually limited to the immediate post-infarct period, apoptosis can continue at a lower level for up to a few months after AMI, which may contribute to LV remodeling and to the development of HF.

Necrosis has been demonstrated in AMI by using ^99mTc-pyrophosphate (73), ¹¹¹In-antimyosin Fab antibody (232), ^99mTc-glucarate (230), and antimyosin antibody conjugated to monocrystalline iron oxide nanoparticles (417). From the applied agents for necrosis imaging, ¹¹¹In-antimyosin Fab antibody appears to be the most diagnostically sensitive tracer for detecting Q-wave (87%-98%) and non-Q-wave (78%-84%) infarctions that also has high specificity in patients with chest pain, but no clinical evidence of infarction (85%-96%) (120, 376). In addition, imaging apoptosis has also gained great interest in assessing the response to novel therapeutics in the setting of AMI, as preclinical studies demonstrated a reduction in infarct size and an attenuation of LV remodeling by modulating apoptosis after AMI (Fig. 10) (121, 381). Radiolabeled and SPIO nanoparticle bound Annexin-V have been used to demonstrate increased abundance of PS in acute coronary syndromes (Fig. 11) (160, 352). However, the observed low target-to-background ratios, along with slow clearance resulting in significant off-target signal, have limited widespread clinical use of Annexin V. To overcome this issue, ^99mTc-duramycin, a smaller agent with better clearance profile, has been applied and tested successfully in large animal models of myocardial ischemia-reperfusion (I/R) injury (414).

Figure 10.

Evaluation of the cardioprotective effects of Fv-HSP72 by Annexin-V based apoptosis imaging in a rabbit ischemia-reperfusion model: the effect of a single intravenous dose of Fv-HSP72 [the heat shock protein-72 (HSP72) coupled to a single-chain variable fragment (Fv) of monoclonal antibody 3E10 (3E10Fv)] was tested in rabbits undergoing left coronary artery occlusion for 40 min followed by 3 h reperfusion. Higher and more extensive uptake of ^99mTc-annexin-V was seen in the control groups compared with the two therapy groups (pre and post ischemia reperfusion) in in vivo sagittal slices of SPECT images obtained 3 h after the injection of ^99mTc-annexin-V (white circle = apex) and in ex vivo images of excised heart (traced with dotted lines, arrows = high uptake area; asterisk = apex). Modified, with permission, from Tanimoto T, J Am Coll Cardiol. 2017 (381).

Figure 11.

Molecular MRI imaging of cardiomyocyte apoptosis with AnxCLIO-Cy5.5 (A, D, G) and simultaneous delayed enhancement (DE) MRI of necrosis with Gadolinium-DTPA-NBD (B, E, H) in a mouse with severe myocardial injury after transient coronary artery ligation (35 min): Images at three slice locations are shown, moving progressively from the midventricular level (A and B) to the left ventricular apex (G and H). (B) At the midventricular level, only a small area in the subendocardium of the lateral wall shows DE (red arrows). (E and H) The extent of DE increases progressively in the more apical slices (red arrows) and is fairly extensive at the apex. Although the accumulation of AnxCLIO-Cy5.5 is fairly transmural, DE of Gd-DTPA-NBD is seen predominantly in the subendocardium. (C, F, and I) Immunohistochemistry for Gd-DTPA-NBD confirming the in vivo MRI findings. (C) Control area in the uninjured septum showing no evidence of DE (magnification × 200). (F) (× 200) and (I) (× 400) Sections from the antero-apical wall of the left ventricle show positive staining for Gd-DTPA-NBD in areas of the subendocardium with significant amounts of cardiomyocyte degeneration. Modified, with permission, from Sosnovik DE, Circ Cardiovasc Imaging. 2009 (352).

Heart failure

Although the level of apoptosis is much lower in HF compared to the ongoing apoptosis in MI, the persistent low-grade loss of cells over an extended period of time can lead to a significant decrease in the number of functioning cardiomyocytes. Importantly, the level of apoptosis has been shown to be associated with the progression and disease severity in HF patients (402). Along these lines, AnxCLIO-Cy5.5 has demonstrated apoptosis in a transgenic model of chronic HF (354). In addition, radiolabeled Annexin V has been used to detect increased level of apoptosis in preclinical models of chemotherapy-induced cardiomyopathy (30) and experimental myocarditis (299).

Transplant rejection

Necrosis and apoptosis are hallmark features of transplant rejection. Current clinical guidelines recommend serial endomyocardial biopsies for the surveillance of transplanted patients to detect transplant rejection. This procedure carries risks for periprocedural complications; therefore, many groups have attempted to diagnose allograft rejection with noninvasive imaging by targeting myocyte necrosis or apoptosis. Along these lines, ¹¹¹In labeled myosin-targeting antibody (125) and ^99mTc labeled Annexin V (270) have been applied to image rejection in preclinical studies and small patient studies, respectively. Interestingly, patients with no histological evidence of rejection had no myocardial uptake of ^99mTc-Annexin V, while patients with histological evidence of rejection had positive myocardial uptake of ^99mTc-Annexin V (270). Despite these promising findings, the full clinical potential of Annexin-V imaging to diagnose allograft rejection in transplant patients has yet to be realized.

Inflammation Imaging

Growing evidence suggests that the immune system is actively involved in the pathogenesis of multiple cardiac pathologies (298). For example, immune mediated injury is a central component in myocarditis, cardiac sarcoidosis, transplant rejection, and also plays an important role in the natural history of MI and the development of HF.

Labeling leukocytes provides a unique opportunity to track active inflammatory cell recruitment to the myocardium. ¹¹¹In-Oxine is an FDA approved, lipid soluble isotope cell-tracker that has been widely used to label leukocytes and track their in vivo distribution by SPECT imaging (267). In addition, the natural phagocytic behavior of monocytes/macrophages make these cells relatively easy targets for cell labeling. For example, iron oxide-based nanoparticles, especially USPIOs, have been extensively used to track the in vivo monocyte/macrophage recruitment to the myocardium following MI with MRI in both preclinical models and in the clinical setting (5, 262, 353, 435). Lastly, iodine and gold containing nanoparticulate contrast agents have been fabricated to detect macrophage recruitment to atherosclerotic plaques in pre-clinical models, but have yet to be translated to larger animal models or humans (32, 68, 166, 167).

Besides direct labeling of inflammatory cells, several strategies have been used to image in vivo inflammatory cell recruitment to the myocardium. Active inflammatory cells show high glycolytic activity to sustain their energy demand (122). As such, ¹⁸F-FDG has been used to assess inflammatory cell activity in the myocardium. However, the cell-specificity of this tracer for inflammatory cells is compromised by myocardial glucose metabolism, particularly in the setting of ischemia (101, 332). Therefore, suppressing myocardial ¹⁸F-FDG uptake is essential to optimize the target-to-background ratio, which can be achieved by (i) administering a fat-enriched, low carbohydrate diet, (ii) prolonged fasting, and/or (iii) the use of intravenous unfractionated heparin shortly before tracer injection (35).

Other strategies to image inflammatory cell recruitment to the myocardium include, ¹⁸F-fluorothymidine (282) and ¹¹C-methionine (251, 386), since both tracers represent inflammatory cell activity, but show low background myocardial uptake. More recently, the PET tracer, ¹⁸F-GE180 has been used to image myocardial inflammation in both small animals and in patients (388).¹⁸F-GE180 targets the 18-kDa mitochondrial translocator protein (TSPO), which is upregulated in activated macrophages. Instead of targeting inflammatory cell activity, the CXCR4 targeted PET tracer, ⁶⁸Ga-pentixafor, has been used to assess myocardial inflammation by targeting leukocyte recruitment (387). The inflammatory monocyte marker CCR2 promotes monocytes recruitment, especially following I/R injury. The ⁶⁴Cu-radiolabeled CCR2 binding peptide (⁶⁴Cu-DOTA-ECL1i) has been successfully used with PET imaging to assess monocyte recruitment following I/R injury in lung transplant models (222) and has also been used to visualize in vivo monocyte recruitment into atherosclerotic plaques (219). However, the application of this tracer to assess in vivo myocardial inflammation following I/R in the heart has yet to be realized.

For imaging inflammatory cell activity with MRI, myeloperoxidase (MPO) activatable Gd-based probes (MPO-Gd) have been used in preclinical models, since activated monocytes and neutrophils release high quantities of this peroxidase enzyme (263). In addition, molecular imaging of inflammation has been performed with myocardial contrast echocardiography in rodent and non-human primate models using microbubbles targeting different endothelial cell inflammatory markers, including the selectins (77, 168, 185, 252) and intercellular adhesion molecule-1 (ICAM-1) (419).

Clinical applications of inflammation imaging

Acute myocardial infarction

Following myocardial infarction, the recruitment of inflammatory cells to the myocardium is essential to ensure adequate wound healing; however, excessive or insufficient inflammatory cell recruitment can exacerbate post-infarct remodeling. The inflammatory cascade following an MI is complex. Within minutes to hours after the ischemic event, neutrophils and monocytes are recruited to the injured myocardium (262, 266, 395). The neutrophil influx quickly falls, but proinflammatory monocytes continue to be recruited to the injured myocardium for several days. During the first 3–5 days after an AMI, pro-inflammatory monocytes (Ly6-C^High [Mouse], CD14^High CD16− and CCR2+ [Human]) are recruited into the myocardium. These cells differentiate into M1-like macrophages and are involved in matrix degradation (via matrix metalloproteases [MMPs] and cathepsins) and the removal of cellular debris (phagocytosis) and apoptotic cells (efferocytosis) (420). After this time period, monocytes with a lesser pro-inflammatory profile (Ly-6C^Low [Mouse], CD14^Low CD16+ [Human]) are recruited to the myocardium, which differentiate into M2-like macrophages and facilitate tissue repair by secreting cytokines that promote angiogenesis and collagen production. In addition, other lymphocytes, such as natural killer (NK) cells, T and B cells get recruited to the myocardium and participate in the resolution of the pro-inflammatory state (159, 349, 451). This biphasic recruitment of monocyte subpopulations was elucidated in a mouse model of MI by employing ¹¹¹In-Oxine labelling of Ly-6C^High and Ly-6C^Low monocyte subtypes (267). Tracking these events are important as higher levels of circulating proinflammatory monocytes have been linked to compromised LV recovery in patients with MI, suggesting that excessive recruitment of proinflammatory monocytes may promote adverse LV remodeling (395).

Preclinical studies demonstrated the feasibility of using USPIO nanoparticles for tracking macrophage recruitment in MI, detected as MRI hypo-enhancement in the infarcted myocardium that correlated well with the number of infiltrating macrophages on ex vivo analysis (262, 353). These experiments were followed by small clinical studies that investigated the use of ferumoxytol, a recently FDA approved USPIO probe, for imaging cardiac inflammation in patients with ST elevation MI (STEMI). These studies demonstrated signal hypoenhancement on T2-weighted images and a signal void on T2*-maps in the infarct area following ferumoxytol administration early post MI (within 7 days) (5, 435). In addition, a decrease in T2* values were also detected in the peri-infarct-zone and the remote myocardium, but to a lesser extent than observed in the infarct area (Fig. 12) (5, 435). Recently the therapeutic potential of ferumoxytol in post-infarct setting has been suggested by a small clinical study, however larger studies are needed to confirm these findings (119).

Figure 12.

Ferumoxytol-enhanced magnetic resonance imaging assessing inflammation after myocardial infarction: examples of myocardial edema and ferumoxytol enhancement in the infarct zone of 3 patients after myocardial infarction (1—anteroseptal, 2—lateral, and 3—inferior). Accumulation of ferumoxytol reduces T2* decay time and creates signal deficits that can be quantified and visualized using T2* MRI. To describe ferumoxytol accumulation, the relaxation rate, R2*, which is the inverse of the mean T2* was calculated for each region of interest, where the higher the value, the greater the ferumoxytol accumulation. The separate columns illustrate late gadolinium enhancement (LGE, first column), ferumoxytol enhancement (R2* map, second and third column) and edema (T2 map, fourth and fifth columns). At early time points (up to 10 days) increased inflammation was detected by ferumoxytol (dark regions on R2* maps) and edema was detected on T2 maps (light region). These changes have improved or have resolved by 3 months. Modified, with permission, from Stirrat CG, Heart. 2017 (363).

Preclinical studies demonstrated increased in vivo ¹⁸F-FDG uptake with PET imaging 7 days following experimental myocardial I/R injury, which correlated well with macrophage infiltration in the infarct region as assessed immunohisto-chemically (31). However, despite the elevated signal, this study showed a significant amount of background myocardial uptake. Other preclinical studies using myocardial glucose suppression strategies for more specific uptake have confirmed findings of elevated ¹⁸F-FDG uptake in the infarct region and the border zone 5 to 7 days after MI that correlated well with postmortem analysis of monocyte and macrophage infiltration (215, 425). Recently, a small sized clinical study (n = 29) has tested the value of 18F-FDG PET in imaging myocardial inflammation post MI and in predicting LV remodeling (Fig. 13) (311). In this study, increased ¹⁸F-FDG uptake 5 days after MI in the infarct zone (expressed as standardized uptake value (SUV) mean) was found to be an independent predictor of adverse LV remodeling (Δ LVEF, P < 0.04; Δ LV end-diastolic volume, P < 0.02; Δ LV end-systolic volume, P < 0.005) at 6 months when accounting for circulating leukocyte levels and MRI determined infarct size (late gadolinium enhancement (LGE)). Interestingly, the peak levels of CCR2⁺ (inflammatory) and CD14^High CD16⁺ (intermediate inflammatory) monocytes were associated with LV ¹⁸F-FDG uptake extent (r = 0.40, P < 0.02 and r = 0.42, P < 0.02, respectively), while CD14^High CD16⁻ (inflammatory) and CD14^Low CD16⁺ (noninflammatory) monocyte subtypes were not (r = 0.31, P = 0.07 and r = 0.02, P = 0.93, respectively).

Figure 13.

Prospective evaluation of ¹⁸F-Fluorodeoxyglucose (¹⁸F-FDG) uptake after acute myocardial infarction by PET/MRI imaging as a prognostic marker of functional outcome: (Left panel) Short- and long-axis views of late gadolinium enhancement (LGE) MRI (left), ¹⁸F-FDG–PET images (middle), and overlay (right) of patients with anterior (A) or inferior (B) myocardial infarction. (Right Panel) (A) Correlation between LGE extent and Δ ejection fraction (EF at follow-up - EF at initial imaging), Δ end diastolic volume (EDV at follow-up – EDV at initial imaging), and Δ end-systolic volume (ESV at follow-up – ESV at initial imaging). (B) Correlation between postischemic ¹⁸F-FDG uptake in the infarct area mean standard uptake value (SUV_mean) and ΔEF, ΔEDV and ΔESV. (C) Segmental analysis of the wall motion recovery at follow-up. Comparison of LGE transmurality and ¹⁸F-FDG uptake in the infarct (SUV_mean). Modified, with permission, from Rischpler C, Circ Cardiovasc Imaging. 2016 (311).

Elevated relative uptake of ¹¹C-methionine has also been demonstrated in the infarct area compared to the remote zone in patients within 2 weeks after reperfusion, with a greater magnitude of tracer uptake occuring when patients are imaged closer to reperfusion (251). Preclinical studies recaptured these early findings and showed the selective uptake of ¹¹C-methionine by polarized M1 macrophages, neutrophils, monocytes and Tand NK cells, but not by M2 macrophages or by B cells (386). This same group also showed that ⁶⁸Ga-pentixafor uptake followed a similar temporal uptake pattern in the infarct region as ¹¹C-methionine post MI in mice, with in vivo tracer uptake correlating well with inflammatory cell infiltration on ex vivo analysis (387). ⁶⁸Ga-pentixafor was translated to a small number of patient’s early post reperfusion (within 5 days) following an AMI. Findings from this study showed, a significant increase in ⁶⁸Ga-pentixafor uptake in infarct (2.2 ± 0.7) versus remote myocardium (1.3 ± 0.4; P = 0.002), but large variability in the magnitude of tracer uptake between patients. More recently, Thackeray et al. (388) demonstrated elevated myocardial TSPO uptake in a mouse model of experimental MI 1 week post-MI, which was predictive of LV remodeling 8 weeks later. In this elegant study, the authors also provided evidence of a parallel increase in TSPO signal in the brain in the setting of AMI in both preclinical models and in patients (Fig. 14).

Figure 14.

Mitochondrial translocator protein (TSPO)-targeted positron emission tomography reveals myocardial inflammation and neuroinflammation in patients after acute myocardial infarction (AMI): (A) tomographic images of the heart display elevated TSPO signal in the hypoperfused infarct region in a representative patient (arrows). Images were acquired 4 to 6 days after reperfusion for first AMI. (B) Parametric brain images (statistical parametric mapping) show regional group difference of TSPO signal between infarct patients and healthy volunteers, superimposed to a magnetic resonance imaging template. Elevation of signal is regionally seen in the frontal and temporal cortex. HLA, horizontal long axis; VLA, vertical long axis. Modified, with permission, from Thackeray JT, J Am Coll Cardiol. 2018 (388).

In addition to PET imaging, MRI and MPO-Gd have been used to detect myocardial inflammation in a mouse model of experimental MI (263). In this study, MPO-Gd uptake and image enhancement peaked 2 days following MI and was strongly related to ex vivo MPO activity in inflammatory cells. Unfortunately, this novel imaging strategy has yet to be applied to larger animal models or humans.

By injecting P-selectin targeting ultrasound contrast microbubbles, a fourfold to fivefold increase in ultrasound signal has been demonstrated in rodent models of myocardial I/R injury within hours after transient ischemia (168, 185). These results have been also confirmed in a non-human primate model of myocardial ischemia (77), but have not yet been investigated in humans. Recently, PS-containing microbubbles have been used to detect regional inflammation via a complement dependent binding to inflammatory leukocytes in a canine model of myocardial I/R injury. In a closed-chest model of MI, this method detected a signal in the risk area that was five-fold higher than control myocardium (Fig. 15) (252). In addition, a dendritic polyglycerol sulfate-based NIRF agent targeted to Pand L-selectin has been successfully used with photo-acoustic imaging for the visualization of inflammation in experimental MI in rodents (382).

Figure 15.

Myocardial contrast echocardiographic (MCE) ischemic memory in dogs with selectin-targeted microbubbles: (A) mean (± SEM) video intensity in the risk and remote areas after selectin-targeted microbubble administration in dogs undergoing ischemia-reperfusion, and in both left anterior descending coronary artery and left circumflex territories together in closed-chest nonischemic controls (n for closed-chest represents region rather than animal number). (B) Example of MCE from a closed-chest control animal. (C-E) Examples of MCE, triphenyltetrazolium chloride staining and risk area by method of intracoronary injection of contrast from a single animal undergoing left circumflex ischemia reperfusion. ANOVA, analysis of variance. Modified, with permission, from Mott B, JACC Cardiovasc Imaging. 2016 (252).

Sarcoidosis

Sarcoidosis is a multisystem inflammatory granulomatous disease of unknown origin. This disease is characterized by the formation of noncaseating granulomas consisting of compact collections of macrophages and epitheloid cells that are surrounded by lymphocytes (346). It is estimated that cardiac involvement can be found in approximately one quarter of sarcoidosis patients (346). Cardiac sarcoidosis can affect the whole heart but generally manifests as focal areas of inflammation. Therefore, nonguided endomyocardial biopsies to diagnose cardiac involvement have low diagnostic yield (20–30%) (346). On the other hand, molecular imaging of the increased metabolic activity associated with inflamed granulomatous tissue provides detailed information regarding the myocardial distribution of sarcoidosis.

Historically ⁶⁷Ga scintigraphy has been used to detect inflammation for the diagnosis of cardiac sarcoidosis; however, this technique has largely been replaced by ¹⁸F-FDG PET imaging secondary to the low diagnostic accuracy of 67Ga imaging and poor image quality (related to the high-energy positron emission) (431). A recent meta-analysis that included a total of 164 patients demonstrated good sensitivity (87%) and specificity (78%) of ¹⁸F-FDG PET for the diagnosis of sarcoidosis (437), and newer data suggests even better diagnostic accuracy of ¹⁸F-FDG PET when combined with MRI. Specifically, Vita et al. report that in patients being evaluated for sarcoidosis, the addition of ¹⁸F-FDG imaging results to MRI findings allowed for the reclassification of 45% of patients, of which 80% were correctly reclassified when compared to the final diagnosis (408). In this regard, hybrid PET/MRI imaging holds great promise for the diagnosis of cardiac sarcoidosis, providing simultaneous, synergistic information on disease distribution (LGE) and disease activity (¹⁸F-FDG) (Fig. 16) (107). In response to these studies, recently published expert consensus statements have incorporated abnormal ¹⁸F-FDG PET findings as a diagnostic criterion for cardiac sarcoidosis (33, 346). Whether ¹⁸F-FDG PET imaging would be able to help guide biopsies in this patient population remains to be evaluated. Recently, ¹⁸F-fluorothymidine has been tested as an alternative tracer for the diagnosis of cardiac sarcoidosis (282). These preliminary findings suggest that ¹⁸F-fluorothymidine can detect cardiac and thoracic involvement in newly diagnosed sarcoidosis patients, however, uptake of ¹⁸F-fluorothymidine in lesions was significantly less than observed with ¹⁸F-FDG PET (SUVmax 4.91 ± 2.20 vs. 9.89 ± 5.07, respectively, P < 0.001).

Figure 16.

Patients with imaging evidence of active cardiac sarcoidosis on hybrid PET/MRI: late gadolinium enhancement (LGE) MRI images on the left with hybrid ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET/MRI images on the right. (A) Subepicardial (near transmural) LGE in the basal anteroseptum extending in to the right ventricular free wall with increased ¹⁸F-FDG uptake localizing to exactly the same region on fused PET/MRI. (B) Subepicardial LGE in the basal anterolateral wall with increased ¹⁸F-FDG uptake colocalizing to exactly that region on PET/MRI. (C) Patchy midwall LGE in the anterolateral wall with matched increased ¹⁸F-FDG uptake on PET/MRI. (D) Multifocal LGE in the lateral wall with matched increased ¹⁸F-FDG uptake on PET/MRI. Modified, with permission, from Dweck MR, JACC Cardiovasc Imaging. 2018 (107).

In addition to facilitating the diagnosis of cardiac sarcoidosis, ¹⁸F-FDG PET imaging has been used to monitor response to immunosuppressive therapies (3, 202) and to predict patient prognosis (37). Sarcoid-mediated focal myocardial inflammation has been associated with corresponding regional impairment in coronary flow (202). In a recent prospective study, immune-suppression that led to a reduction in myocardial ¹⁸F-FDG PET uptake was associated with an improvement in coronary flow reserve, whereas areas of similar or higher ¹⁸F-FDG PET uptake post-therapy were associated with a reduction in coronary flow reserve (r = −0.47; P = 0.048) (202).

Myocarditis

Myocarditis is an inflammatory state affecting the myocardium, which is caused by viral or other types of infection, autoimmune conditions or drug mediated reactions. The inflammation can be present as a focal or global process. Despite the low diagnostic sensitivity (28–63%) (69, 148), endomyocardial biopsy remains the gold standard for the diagnosis of myocarditis. Smaller clinical studies suggest that ¹⁸F-FDG PET may be able to detect active inflammatory lesions in myocarditis (274, 291). Using an integrated PET/MRI system to assess patients with suspected myocarditis, Nensa et al. demonstrated reasonable spatial agreement (κ = 0.73) between ¹⁸F-FDG PET and MRI findings (LGE or myocardial edema on T2 weighted imaging) (274). However, the role of molecular imaging in guiding biopsies to facilitate diagnosis of myocarditis has yet to be tested.

The feasibility of tracking active inflammation with the amino acid tracer, ¹¹C-methionine, has been also demonstrated in a rat model of autoimmune myocarditis (Fig. 17) (236). More recently, a small clinical study investigating 14 patients with suspected myocarditis surprisingly found that the USPIO-enhanced MRI did not provide any additive benefit to the more traditional MRI evaluation of myocarditis such as, LGE and T2 mapping (change in R2* after ferumoxytol in patients with myocarditis vs. healthy volunteers 35.0 ± 15.0 vs. 37.2 ± 9.6 s⁻¹, respectively, P > 0.05) (363). Larger clinical studies are needed to confirm this finding. In addition to myocarditis, ¹⁸F-FDG PET (135, 319) and ¹¹¹In-oxine SPECT imaging of labeled autologous white blood cells (56) have been utilized to assist with the clinical diagnosis of infective endocarditis.

Figure 17.

¹¹C-Methionine PET of myocardial inflammation in a rat model of experimental autoimmune myocarditis (EAM): (A) representative ¹¹C-methionine PET images in a rat model of EAM 30 min after intravenous tracer administration. Strong focal cardiac ¹¹C-methionine uptake was observed in EAM rats but not in control animals. Extracardiac tracer accumulation in thymus (asterisk) and the liver (arrowheads) was noted in both EAM and control rats, whereas respective tracer activities in lungs and blood pool were rather low. (B) Cardiac tracer uptake in EAM rats (black bar) was significantly higher than that in control rats (white bar). (C) Representative short-axis PET images and time-activity curves of dynamic PET imaging in EAM rat. Ten to 20 min after administration, tracer uptake increased in heart together with rapid clearance of blood activity. Cardiac signals remained stable for 30 to 40 min. Gray scale images serve as reference for location of heart. This research was originally published in JNM. Maya Y, Werner RA, Schutz C, Wakabayashi H, Samnick S, Lapa C, Zechmeister C, Jahns R, Jahns V, and Higuchi T. 11C-Methionine PET of Myocardial Inflammation in a Rat Model of Experimental Autoimmune Myocarditis. J Nucl Med 57: 1985–1990, 2016. © SNMMI. (236).

Transplant rejection

Macrophages play an essential role in acute cardiac allograft rejection, and selective depletion of macrophages protects the heart against allograft rejection in rodent models (428). In addition, macrophage infiltration during acute rejection is associated with cardiac fibrosis in the long term (401). By using biopsy specimens obtained from patients with acute allograft rejection, a recent study demonstrated that the lesser inflammatory monocytes (CD16⁺) and M2 macrophages are the predominant subtypes in rejected allografts (401). Given the available tracers to target active inflammatory processes, several preclinical studies have used molecular imaging to target macrophage infiltration of transplanted organs (70, 184, 398, 429). For example, reduction in MRI signal intensity was seen in a heterotopic heart transplantation rat model using dextran coated USPIO nanoparticles, and a good correlation between signal change in MRI and clinical rejection grade was demonstrated (184). In this study, postmortem analysis confirmed USPIO uptake by transplant infiltrating macrophages. In addition, PET imaging after the injection of dextran nanoparticles that incorporate ⁶⁴Cu showed increased signal in hearts undergoing rejection 7 days after heart transplantation in mice (398). Moreover, ultrasound imaging of microbubbles targeted to the endothelial cell inflammatory marker, ICAM-1, has shown higher intensity in rejecting versus nonrejecting rat cardiac transplant myocardium (419). These elegant approaches are promising for the noninvasive detection of allograft rejection, and hold potential for translation of these preclinical findings to large animal models or humans.

Angiogenesis Imaging

Angiogenesis is defined as new capillary formation from pre-existing microvessels (341). In adults, angiogenesis is stimulated by tissue hypoxia via activation of hypoxia-inducible factor (HIF) 1-α. HIF1-α triggers the transcription of numerous angiogenic genes, such as vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR), neuropilin-1, and angiopoietin-2 (341). Besides the transcription of growth factors and their receptors, hypoxia triggers inflammatory cells (e.g., monocytes/macrophages, mast cells and lymphocytes) to secrete soluble proangiogenic and antiangiogenic molecules that regulate this process (264). Inflammatory cells secrete proteolytic enzymes (e.g., MMPs) that facilitate the degradation of the vasculature and surrounding extracellular matrix (ECM). Following degradation, via the secretion of proangiogenic molecules, endothelial or progenitor cells migrate, proliferate, and organize to form new vessels with a lumen. Activated and proliferating endothelial cells express integrins, which are crucial to the angiogenic process, as these integrins facilitate the interaction between endothelial cells and the ECM and promote signal transduction, cell migration, and survival (152). Molecular-targeted imaging of angiogenesis in acute and chronic ischemia has been explored in multiple preclinical models and in some small-scale patient studies with nuclear imaging techniques. Although MRI and ultrasound compatible imaging probes have also been developed for the in vivo detection of angiogenesis, these imaging targets have only been applied in the setting of atherosclerosis, peripheral vascular disease, and in tumor angiogenesis, thus will not be discussed further (21, 99, 412, 423, 424, 443).

αvβ3 integrin-targeted imaging

The most widely applied strategy for molecular imaging of angiogenesis has been with the use of SPECT and PET tracers that target the αvβ3 integrin, which is a cell membrane glycoprotein that is highly expressed on endothelial cells during angiogenesis, but not on normal quiescent endothelial cells (94). For example, the αvβ3 integrin antagonist, ¹¹¹In-RP748 has been used to define angiogenesis with SPECT imaging. This tracer binds to the αvβ3 integrin with high affinity and selectivity (241). Later generation SPECT and PET tracers have employed the arginine-glycine-aspartic acid (RGD) peptide, or its derivatives, for angiogenesis imaging, since this sequence is highly selective for the αvβ3 integrin (98, 181, 206). Commonly used tracers include the SPECT tracer, ^99mTc-NC100692 (^99mTc-maraciclatide), and the PET tracers, ¹⁸F-galacto-RGD, ⁶⁸Ga-PRGD2, and ¹⁸F-Fluciclatide.

Vascular endothelial growth factor and endothelial cell imaging

As discussed above, VEGF is a growth factor that is expressed in response to tissue hypoxia and is able to potently stimulate angiogenesis via binding and activation of its cognate receptors (VEGFR-1, VEGFR-2) (59, 248). Considering the importance of VEGF in angiogenesis, there have been efforts to radiolabel VEGF isoforms or antibodies against specific VEGFRs with both PET and SPECT radioisotopes for the noninvasive assessment of angiogenesis (217, 224, 313). For example, the VEGF isoform, VEGF₁₂₁, has been radiolabeled with ¹¹¹In and ⁶⁴Cu for SPECT and PET imaging of angiogenesis, respectively. Alternatively, other approaches for angiogenesis imaging have used tracers that target membrane proteins of activated endothelial cells (CD13) or endothelial cells of newly formed vessels (CD105) (151, 289).

Clinical applications of angiogenesis imaging

Acute myocardial infarction

Following an acute MI, angiogenesis is stimulated by hypoxia and the inflammatory cell milieu to promote partial restoration of tissue perfusion to the ischemic myocardium, which contributes to ischemic tissue salvage (198). In addition, angiogenesis participates in infarct healing following the proinflammatory phase and has several attenuating effects on postinfarct LV remodeling (173). Given these important roles, molecular-targeted imaging of angiogenesis, mostly with PET and SPECT compatible tracers, has been explored in multiple preclinical models and in some small-scale patient studies to assess the magnitude and spatiotemporal distribution of angiogenesis early post MI and to relate this signal to future LV remodeling.

Early preclinical SPECT imaging studies described the in vivo spatiotemporal distribution of angiogenesis in the infarct and peri-infarct regions following surgically induced nontransmural infarcts using the αvβ3 integrin antagonist, ¹¹¹In-RP748 (182, 241) (Fig. 18). Importantly, the in vivo SPECT signal was associated with αvβ3 expression on endothelial cells in the infarct and peri-infarct regions, confirming the imaging signal was selective for angiogenesis. In addition, the SPECT cyclic RGD tracer, ^99mTc-NC100692 (^99mTc-maraciclatide) has been well described in several pre-clinical models of acute MI, showing good in vivo image quality and focal uptake that is associated with angiogenic specific markers on ex vivo tissue analysis (218, 221). Furthermore, quantitative methods have been establish for in vivo evaluation of the uptake of this tracer that have been validated against the ex vivo gold standard, tissue well counting (218).

Figure 18.

In vivo and ex vivo ¹¹¹In-RP748 and ^99mTc-sestamibi (^99mTc-MIBI) images from dogs with chronic infarction: (A) Serial in vivo ¹¹¹In-RP748 SPECT short axis, vertical long axis (VLA), and horizontal long axis (HLA) images in a dog 3 weeks after left anterior descending coronary artery (LAD) infarction at 20 min and 75 min after injection in standard format. ¹¹¹In-RP748 SPECT images were registered with ^99mTc-MIBI perfusion images (third row). The 75-min ¹¹¹In-RP748 SPECT images were colored red and fused with ^99mTc-MIBI images (green) to better demonstrate localization of ¹¹¹In-RP748 activity within the heart (color fusion, bottom row). Right ventricular (RV) and left ventricular (LV) blood pool activity is seen at 20 min. White arrows indicate region of increased ¹¹¹In-RP748 uptake in anterior wall. This corresponds to the anteroapical ^99mTc-sestamibi perfusion defect (yellow arrows). (B) Sequential ^99mTc-sestamibi (top row) and ¹¹¹In-RP748 in vivo SPECT HLA images at 90 min after injection (middle row) from a dog at 8 h (acute), 1 week, and 3 weeks after LAD infarction. Increased myocardial ¹¹¹In-RP748 uptake is seen in the anteroapical wall at all three time points. Color fusion ^99mTc-MIBI (green) and ¹¹¹In-RP748 (red) images (bottom row) demonstrate ¹¹¹In-RP748 uptake within ^99mTc-MIBI perfusion defect. (C) Ex vivo ^99mTc-sestamibi (left) and ¹¹¹In-RP748 (center) images of myocardial slices from a dog 3 weeks after LAD occlusion, with color fusion image on the right. Short axis slices are in the standard orientation. Yellow arrows indicate anterior location of nontransmural perfusion defect region; white arrows indicate corresponding area of increased ¹¹¹In-RP748 uptake. Reprinted with permission (241).

Similar to SPECT tracers, several RGD-targeted PET tracers have also been evaluated in preclinical studies and in several small clinical trials. ¹⁸F-galacto-RGD has been widely described in preclinical models of acute MI, and greater myocardial uptake of ¹⁸F-galacto-RGD early after an MI (1 week) has been shown to be an independent predictor of attenuated LV remodeling in rodents (335). However, despite widespread preclinical use, ¹⁸F-galacto-RGD imaging has only been assessed in case reports and small studies in patients early post-MI; with 18F-galacto-RGD uptake apparent in only some, but not all, patients (228). Several other PET RGD-based tracers have shown clinical feasibility for tracking angiogenesis in patients early post-MI. For example, ⁶⁸Ga-PRGD2 imaging demonstrated focal uptake in the infarct and border regions that peaked early (~1 week) after an MI and stayed elevated, albeit to a lesser extent, for up to 2.5 months in some patients (366). In addition, ¹⁸F-Fluciclatide, an α_vβ₃ and α_vβ₅ selective radiotracer, has been applied in post-acute ST elevation MI patients and was shown to be retained in the infarct region early post MI (~2 weeks) (Fig. 19). Interestingly, ¹⁸F-Fluciclatide was higher in hypokinetic regions that subsequently displayed functional recovery compared to regions with no change or worsening in function at 9-month follow-up (mean tissue-to-background ratio (TBR_mean): 0.95 ± 0.33 vs. 0.81 ± 0.27, P = 0.002). In predictive models, the magnitude of myocardial ¹⁸F-Fluciclatide uptake was associated with functional recovery (odds ratio: 1.27, 95% CI [1.08–1.50] per 10% increase in ¹⁸F-Fluciclatide TBR_mean, p = 0.003) (178). In line with some animal studies, ¹⁸F-Fluciclatide uptake was higher in patients with subendocardial infarction compared with transmural infarcts.

Figure 19.

Assessment of cardiac αVβ3 integrin expression following acute myocardial infarction in humans by ¹⁸F-Fluciclatide PET imaging: (Left panel) ¹⁸F-Fluciclatide uptake in three patients with recent subendocardial myocardial infarction (MI). (A) Patient 1, 13 days after anterior MI, displaying a short-axis PET image of the left ventricle with crescentic ¹⁸F-fluciclatide uptake that correlates with the interventricular septum and anterior wall on CT angiography (B). The fused PET/CT-angiography image (C) shows this uptake to correspond exactly with the region of late gadolinium enhancement (LGE) on magnetic resonance imaging (MRI) (D). Further delineation of myocardial uptake on PET/CT is clearer in the two-chamber view (E) and on a fused CT/three-dimensional-Patlak image, which shows this uptake to follow a watershed-pattern emerging from the coronary stents present in the left anterior descending coronary artery (F). (G and H) Patient 2, 8 days following anterior MI, displaying focal uptake of ¹⁸F-fluciclatide in the anterior wall and apex in the three-chamber view on PET/CT (G) which corresponds to the region of infarction on LGE MRI imaging (H). (I and J) Patient 3, showing focal uptake of ¹⁸F-fluciclatide in the inferior wall 19 days following MI on PET/CT (I) that again corresponds to the infarction on MRI LGE imaging (J). (Right Panel) ¹⁸F-Fluciclatide uptake in MI is shown. Uptake of ¹⁸F-fluciclatide in (A) patients with acute MI at 2 and 10 weeks, patients with chronic total occlusion (CTO) and healthy control subjects is shown. Uptake was greatest at 2 weeks after MI (B). ¹⁸F-Fluciclatide uptake in the acute MI group was greater in regions of hypokinesis when compared with sites of normal function or akinesis (C). This translated to a higher ¹⁸F-fluciclatide uptake in those regions, which subsequently improved in function on follow-up cardiac magnetic resonance (D). CTO, chronic total occlusion; TBR, tissue-to-background ratio. WMA, wall motion abnormality. Modified, with permission, from Jenkins WS, Heart. 2017 (178).

VEGF and other endothelial targeted imaging agents have also been applied in preclinical models of MI. Specifically, PET imaging of ⁶⁴Cu-DOTA-VEGF₁₂₁ was efficacious at detecting myocardial angiogenesis early post MI in animal models (313). In addition, ¹¹¹In-DTPA-eNGR (targeting CD13) (151) and ⁶⁴Cu-NOTA-TRC105 (targeting CD105) (289) showed favorable results for imaging angiogenesis in preclinical models of acute MI with SPECT and PET imaging, respectively. Despite promising results, these tracers have yet to be applied in human studies.

Ischemic cardiomyopathy

Therapeutic angiogenesis has been proposed as a treatment strategy to increase blood flow to chronically ischemic tissue in an effort to salvage tissue and prevent infarct expansion (341). Therefore, the efficacy of several novel angiogenic stimulating cell and gene-based therapies have been assessed with angiogenesis imaging in the chronic post-infarct setting in preclinical models and in small clinical trials. For example, SPECT imaging of ^99mTc-NC100692 detected enhanced angiogenesis at 4 weeks following delivery of IGF-1 adeno-associated virus gene therapy into the peri-infarct region at the time of permanent occlusion of the left anterior descending artery (100). In this study, increased in vivo ^99mTc-NC100692 uptake in the IGF-1 treatment group was associated with greater angiogenic markers on ex vivo analysis at 4 and 16 weeks following MI compared to nontreated infarcted mice. Furthermore, the increase in ^99mTc-NC100692 uptake in the treatment group at four weeks was associated with improvement in echocardiography-derived indices of LV function and volumes at 4 weeks that remained significantly improved compared to nontreated infarcted animals at 16 weeks. In addition to preclinical studies, ^99mTc-NC100692 imaging of angiogenesis has been applied in a small pilot study of patients with chronic ischemic HF following intravenous delivery of bone marrow-derived stem cells (BMSC) to assess the relationship between therapeutic angiogenesis and improvements in myocardial function (254). Although the BMSC treatment group had slightly higher ^99mTc-NC100692 myocardial uptake compared to control treated patients, this difference did not reach statistical significance (P = 0.41). Interestingly, these ischemic HF patients demonstrated baseline 99mTc-NC100692 uptake, suggesting ongoing angiogenesis. The future application of PET angiogenesis tracers with higher detection sensitivity is required to assess the therapeutic response of novel celland gene-based therapies delivered to the myocardium of patients with an ischemic cardiomyopathy.

Extracellular Matrix Imaging

Fibrosis, or deposition of components of the ECM, occurs as a compensatory mechanism to cardiac injury or stress. The process and underlying mechanisms mediating cardiac fibrosis in response to acute ischemic injury have been the most well studied and described. Immediately after MI, the abrupt increase in vascular permeability results in extravasation of fibronectin and fibrinogen, which leads to the formation of a fibrin-based provisional matrix that provides a scaffold for inflammatory cell migration. The provisional matrix is lysed after the hyper-acute period. The increased local production of fibronectin, TGF-ß, and changes in mechanical tension initiate the differentiation of fibroblasts into myofibroblasts. The myofibroblasts then promote collagen deposition in the infarcted area to support scar formation in an effort to preserve cardiac geometry. However, scar formation increases the mechanical load experienced by remote myocardium, resulting in reactive fibrosis, additional deleterious remodeling, and eventual progression to HF (123). In addition to postinfarct remodeling, reactive fibrosis occurs in response to pressure (378) and volume overload (43) and in certain cardiomyopathies (11, 380). Given the presence of fibrosis in many CVDs, targeted ECM imaging has been proposed as a tool to assess disease progression, evaluate response to therapies, and improve prognostication. The majority of the studies have applied ECM imaging in the postinfarct setting.

Clinical applications of extracellular matrix imaging

Acute myocardial infarction and postinfarct remodeling

Factor XIII imaging

During scar formation, newly synthesized immature collagen needs to be cross-linked and deposited into the ECM for infarct stability. Factor (F) XIII is a key player in this crosslinking process, as reduced FXIII levels have been associated with infarct rupture in acute MI patients (261). As such, imaging of FXIII with SPECT (¹¹¹In) and PET (¹⁸F) labeled isotopes has been proposed as a method to predict LV remodeling. Along these lines, preclinical data suggest that lower FXIII imaging signal post MI is associated with enhanced LV dilatation (227, 261). However, the clinical utility of this method has yet to be determined.

Collagen imaging

Collagen type I and collagen type III, albeit to a lesser extent, are the major collagen isoforms involved in reactive cardiac fibrosis. As collagen is exclusively located in the extracellular space and increases 10-fold or more during fibrosis formation, nontargeted tracers that are trapped in the extracellular space and do not appreciably enter the intracellular space have been used as surrogates of collagen accumulation associated with cardiac scar/fibrosis. In this context, the MRI extracellular contrast agent, Gd-DOTA, is the most widely applied technique for the noninvasive assessment of infarct size and viability after MI (290, 300). PET tracers with similar distribution characteristics, such as ⁶⁴Cu-DOTA, have also been developed and show good localization and visualization in the infarct region in experimental models (189). However, the nonspecificity of these tracers does not afford the image sensitivity needed to detect diffuse fibrosis often present in pressure/volume overload and certain cardiomyopathies. Therefore, efforts have been made to synthesize collagen specific probes to overcome this issue.

The first approach for collagen specific imaging was the radiolabeling of collagelin (col) with ^99mTc for SPECT imaging. Col is a cyclic 22 amino-acid peptide with high specificity binding capability to collagen I and III (258). Initial preclinical studies showed Col uptake in the infarct region, but image quality was limited by high liver uptake. Therefore, radiolabeled Col analogs were synthesized with ⁶⁸Ga and ⁶⁴Cu for PET imaging in an effort to reduce background uptake, while maintaining collagen specificity (190, 405). For example, the ⁶⁴Cu-labeled Col analog, NOTA-CRPA, demonstrated significantly higher target-to-background ratio compared to ⁶⁴Cu-NOTA-Col and the nonspecific, ⁶⁴Cu-DOTA compound, in a preclinical model of post MI fibrosis (190). However, despite the higher signal-to-noise and in vitro collagen specificity, the in vivo kinetics of ⁶⁴Cu-NOTA-Col did not differ from nonspecific ECM targeted tracers, suggesting a potential lack of collagen specificity during in vivo imaging.

EP-3533, a type I collagen specific peptide that is freely diffusible to the extracellular space has been conjugated to Gd for collagen targeted imaging by MRI (58). In a chronic mouse MI model, Gd labeled EP-3533 showed a significantly longer retention rate in scarred myocardium when compared to commercial Gd agents (Fig. 20) (150). Another collagen-homing ligand, CNA-35, has been attached to paramagnetic/fluorescent micellar nanoparticles and tested for collagen formation in a mouse model of abdominal aortic aneurysm, but has yet to be tested in the heart (195). In addition to MRI compatible agents, CNA-35 has been attached to the surface of gold containing nanoparticles for CT molecular imaging of collagen (75). With the use of this nanoparticle, focal contrast enhancement was detected in explanted hearts 30 days after experimental MI, with the imaging signal correlating well with myocardial scar formation on histology. However, in vivo imaging failed to detect contrast enhancement in the scarred regions, possibly due to noncardiac gated microCT imaging.

Figure 20.

Postinfarction myocardial scarring in mice: molecular MRI imaging with use of a collagen-targeting contrast agent: Midventricular short-axis double inversion-recovery gradient-echo MRI images (A, B, D, and E) and corresponding picrosirius red-stained histologic sections (C and F) of the mouse left ventricle 6 weeks after left anterior descending artery occlusion-reperfusion. Arrows point to area of scarring. (A and D) Standard anatomic MR images acquired by using a double inversion-recovery gradient-echo sequence. (B and E) Midventricular short-axis MR images of the left ventricles at two section locations obtained 40 min after injection of a gadolinium-based collagen-targeting contrast agent EP-3533. The regions of contrast enhancement correlate closely with (C and F) photomicrographs of picrosirius red-stained tissue sections. Modified, with permission, from Helm PA et al. Radiology 2008 (150).

Tenascin-C imaging

Tenascin-C is a matrix glycoprotein that regulates collagen assembly. It is transiently upregulated in the infarct ECM, where it plays important role in fibroblast recruitment. High in vivo tenascin C expression has been demonstrated in rodent models of MI by using SPECT imaging and ¹¹¹In-labeled antitenascin-C monoclonal antibody Fab^′ fragments (283) or ¹²⁵I-labeled antitenascin-C antibodies (375). In these models, tenascin-C expression peaked 3 days post-MI, and was almost undetectable by 7 days following MI. Besides postinfarct ECM imaging, ¹¹¹In-labeled antitenascin-C monoclonal antibody Fab^′ fragments have been used to demonstrate elevated focal in vivo tenascin C expression with SPECT in a rat model of autoimmune myocarditis (323).

Extracellular matrix protease imaging

MMPs are zinc-dependent proteases, which play a key role in the turnover of ECM under normal physiologic conditions (356). The activity of MMPs is highly regulated by tissue inhibitors of MMPs (TIMPs). MMP activity is upregulated in many CVDs, including acute MI. During the inflammatory phase, MMP activation facilitates the infiltration of immune cells into the infarct zone. During the reparative phase, MMPs facilitate removal of cellular debris, help the migration of cardiomyocytes and myofibroblasts and play an essential role in angiogenesis and matrix deposition (80). However, unbalanced MMP activity in the post-infarct setting can lead to cardiac aneurysm formation with subsequent cardiac rupture or may accelerate adverse LV remodeling and the development of HF (303, 355).

The most well characterized MMP imaging agent for the evaluation of post MI MMP activation and subsequent LV remodeling is RP805, which is a ^99mTc labeled broad-spectrum MMP inhibitor (320, 364). In vivo uptake of ^99mTc-RP805 was demonstrated in the ²⁰¹Tl defined infarct zone in both small and large animal models of MI (320, 364). In these models, ^99mTc-RP805 uptake peaked around 1 week after MI and remained elevated for up to 3–4 weeks there-after (Figs. 21 and 22) (320, 364). Of note, a modest uptake of ^99mTc-RP805 was also detected in the infarct border zone and in the remote myocardium, suggesting global LV remodeling. Other broad-spectrum MMP inhibitors have been labeled with PET radioisotopes for imaging MMP activity; however, their use has not been tested in cardiac pathologies (46, 446). In addition, specific fluorescent peptides that are activated by proteolytic cleavage by MMP-2 and MMP-9 have been developed to assess the role of these MMPs in the post-infarct setting with NIRF tissue imaging and fluorescence microscopy (65). In addition, similar fluorescent probes that are activated by other key proteolytic enzymes of post-infarct remodeling, such as cathepsin B (prosense-680), have been tested in rodent models of MI (265).

Figure 21.

Noninvasive targeted imaging of matrix metalloproteinase activation in a murine model of postinfarction remodeling: hybrid micro-SPECT/CT reconstructed short-axis images were acquired without x-ray contrast (A) in a control sham-operated mouse (left) and selected mice at 1 week (middle) and 3 weeks (right) after surgical myocardial infarction (MI), after injection of ²⁰¹Tl (top row, green) and ^99mTc-RP805, (middle row, red). A black-and-white and multicolor fusion image is shown on bottom. Control heart demonstrates normal myocardial perfusion and no focal ^99mTc-RP805 uptake within the heart, although some uptake is seen in chest wall at the thoracotomy site (dashed arrows). All post-MI mice have a large anterolateral ²⁰¹Tl perfusion defect (yellow arrows) and focal uptake of ^99mTc-RP805 in defect area. A dashed circle is drawn around the heart to demonstrate localization of ^99mTc-RP805, within the infarcted area of the heart. Some activity is also seen in the peri-infarct border zone. Additional micro-SPECT/CT images were acquired by use of a higher-resolution SPECT detector after the administration of x-ray contrast, at 1 week (B) and 3 weeks (C) after MI. The contrast agent permitted better definition of the LV myocardium, which is highlighted by white dotted line. Representative short-axis (SA), horizontal long-axis (HLA), and vertical long-axis (VLA) images are shown for two additional mice by use of the same format and color scheme. Focal uptake of ^99mTc-RP805 is seen within the central infarct and peri-infarct regions, which again corresponds to ²⁰¹Tl perfusion defect. Modified, with permission, from Su H, Circulation. 2005 (364).

Figure 22.

Dual-isotope in vivo SPECT/CT imaging reflecting myocardial perfusion (²⁰¹Thallium) and matrix metalloproteinase activity (^99mTc-RP805): In vivo Thallium-201 and ^99mTc-RP805 SPECT/CT images of pigs at 1 week, 2 weeks and 4 weeks post myocardial infarction are shown in transaxial, coronal, and sagittal views. Note the perfusion defect in the lateral wall and the time dependent changes in the intensity of ^99mTc-RP805 retention in the same regions (green arrows). The yellow double arrows point to ^99mTc-RP805 activity in the surgical sternal wound. A known point source (orange arrow) can be used to quantify hotspot uptake. Scale bars: 2 cm. Modified, with permission, from Sahul ZH, Circ Cardiovasc Imaging. 2011 (320)

Molecular Imaging of Thrombus Formation

Thrombosis development in either the venous or the arterial system is a major component of CVD related mortality and morbidity. Thrombosis can affect the heart in both a direct and indirect manner. Thrombotic occlusion can occur in the coronaries following plaque rupture or endothelial erosion, leading to AMI. In addition, thrombus can be formed within the chambers of the heart in cardiovascular pathologies that are associated with blood stagnation in either the ventricles (e.g., ischemic and nonischemic cardiomyopathies) or in the atria (e.g., atrial fibrillation). Moreover, the heart can be indirectly affected by thrombotic complications occurring elsewhere such as pulmonary embolism, stroke, and aortic aneurysms. Detailed information about the molecular imaging of extracardiac thrombus formation can be found in a recently published comprehensive review by Wang et al (415).

Thrombosis, provides an ideal target for molecular imaging, as thrombi contain multiple molecular signatures that are present in high concentration and do not exist in other tissue types. As such, molecular imaging of coagulation factors (e.g., FXIII), fibrin, and activated platelets have been applied to detect thrombus formation.

The main role of FXIII is the cross linking of fibrin. As discussed above, FXIII has been labeled with SPECT (¹¹¹In) and PET (¹⁸F) isotopes (227, 261). In addition to nuclear approaches, dual MRI/fluorescent probes that target FXIII and are either linked to MRI contrast agents (Gd-DTPA and rhodamine) (246) or fluorescently labeled cross-linked SPIO nanoparticles (71) have been developed to assess intravascular thrombosis.

The formation of an insoluble network of fibrin is the final endpoint of the coagulation pathway. Multiple molecular imaging probes have been developed that target fibrin for the imaging of thrombus formation including PET (287, 399), SPECT (287), MRI (358, 410) and CT (191, 292, 293) compatible probes. Of note, the feasibility of thrombosis imaging by a fibrin specific, Gd-based contrast agent (EP-2104R) has been recently demonstrated in patients (410).

Similar to fibrin, activated platelets are highly abundant in thrombi, and provide specific targets for the molecular imaging of thrombosis. Upon activation, platelets express a wide variety of epitopes that can be used as molecular targets. One such target is P-selectin that is only expressed in activated platelets (415). P-selectin has been targeted successfully by USPIOs (367) and by ultrasound contrast microbubbles (78). However, P-selectin is also expressed on activated endothelial cells and macrophages, which limits the use of selectin as a thrombus-imaging target. Therefore, multiple groups have attempted to image the GPIIb/IIIa receptor (integrin αIIbβ3), which is only expressed in activated platelets and platelet precursors and is the most abundant receptor on these cells. RGD analogs, which resemble the GPIIb/IIIa binding site of fibrinogen (natural ligand for GPIIb/IIIa), have been used to target GPIIb/IIIa receptors by ultrasound (312), MRI (196) and nuclear approaches (183, 449). However, RGD analogs are not specific for GPIIb/IIIa and bind to many other integrins including αvβ3 integrin (163). Therefore, other tracers, such as the small molecule PET tracer, ¹⁸F-GP1that binds specifically to GPIIb/IIIa with high affinity have been synthesized (223). Recently, ¹⁸F-GP1 has shown promising results for the detection of small arterial and venous thrombi, thrombotic depositions on damaged endothelial surfaces and small cerebral emboli in non-human primates by in vivo PET imaging (223). In addition, ¹⁸F-GP1 showed rapid blood clearance and a low background after intravenous injection. These results have promoted evaluation of ¹⁸F-GP1 in clinical studies.

Clinical applications

Molecular imaging of intracoronary thrombus formation

The majority of acute coronary syndromes are believed to originate from local thrombosis at sites of ruptured atherosclerotic plaques (143); therefore, early and noninvasive detection of thrombus formation may be beneficial for early diagnosis of acute MI to guide therapeutic decisions. Coronary artery thrombosis has been visualized in preclinical models by fibrin-binding molecular MRI contrast agents (358), by using P-selectin-targeted microbubbles (78) or by GPIIb/IIIa targeting iron oxide particles (409). These studies show feasibility of targeted intracoronary thrombus imaging and hold great potential for the detection of in-stent thrombosis or for the assessment of thrombus burden in acute coronary syndromes. Given the significance of these issues, human studies are highly anticipated.

Molecular imaging of intracardiac thrombus formation

The early detection of intra-cardiac thrombus formation is important as the presence of a thrombus in the heart affects clinical outcomes and changes clinical management by providing rationale for initiating anticoagulation. In addition, the accurate detection or exclusion of atrial thrombi is important before performing cardioversion. A preclinical study used the fibrin targeting, EP-2104R, for the visualization of chronic human thrombi that were surgically implanted in the left atrial appendages of swine (358). After contrast administration, all atrial clots were selectively visualized as white spots on MR images with a high contrast-to-noise ratio. The use of EP-2104R has advanced to human use, and a phase II study including six patients with intra-cardiac thrombus demonstrated that MRI imaging was able to localize contrast at the thrombus after EP-2104R administration (357). This study also confirmed the safety profile of EP-2104R, as administration of this agent was not associated with major adverse events. In addition, another phase II trial including 15 patients with intracardiac thrombus demonstrated similar results with this tracer (410). These preliminary findings need to be validated in larger clinical populations.

Renin Angiotensin Aldosterone System Imaging

In the early stages of HF, activation of the renin angiotensin aldosterone system (RAAS) is one of the key compensatory mechanisms to preserve mean arterial pressure, which is accomplished by sodium retention and vasoconstriction (212). However, chronic activation of the RAAS leads to adverse LV remodeling and the progression of HF (63, 212). Angiotensin II (Ang II), the key mediator of RAAS, is formed by the conversion of Ang I to Ang II by the angiotensin-converting enzyme (ACE). ACE is highly abundant in the lungs, but Ang II can also be produced in the heart by local ACE activity (115, 297). Ang II exerts its biological effects through binding to Ang II receptor 1 (AT₁R) or Ang II receptor 2 (AT₂R), with the most deleterious effects mediated by AT₁R. Overactivation of RAAS stimulates cardiac hypertrophy and fibrosis, promoting LV remodeling and worsening of HF (51, 430). ACE inhibitors and angiotensin receptor blockers (ARBs) are well characterized and interfere with this pathophysiological cascade, resulting in improved survival in patients with HF and reduced EF (72, 439). However, recent reports demonstrated that the treatment benefit exerted by RAAS inhibition is highly variable and depends on certain genetic and baseline clinical factors (136, 284). Therefore, it has been postulated that molecular imaging of tissue RAAS activity may assist in verifying pharmacological efficacy and in guiding the titration of RAAS inhibition on an individual basis (338).

Imaging myocardial angiotensin converting enzyme activity

The angiotensin converting enzyme (ACE) inhibitor, lisinopril, has been labeled for both PET (N-succinimidyl-4-[F-18]-fluorobenzoyl-lisinopril (¹⁸F-FBL)) and SPECT (^99mTc-lisinopril) imaging of RAAS. ^99mTc-lisinopril has been successfully used for the in vivo detection of increased activity of ACE in rats with cardiac-specific overexpression of human ACE (97). Among the available PET radiolabeled ACE inhibitors (¹⁸F-FBL, ¹⁸F-captopril, and ¹¹C-zofenopril), ¹⁸F-FBL shows the least nonspecific binding, highest binding affinity to tissue ACE and the most optimal tracer kinetics for in vivo imaging (338). However, despite promising preclinical and ex vivo results, lisinopril-targeted tracers have yet to be tested in vivo in patients.

Imaging myocardial angiotensin II receptor 1 expression

Several ¹¹C labeled nonpeptide compounds have been evaluated for the in vivo assessment of angiotensin receptors in several preclinical models and in humans (337, 338). Among these tracers, ¹¹C-KR31173 is the most well characterized in the myocardium of animal models and in humans (Fig. 23) (127, 156, 450). Despite the initial positive findings and enthusiasm for this radiotracer, ¹¹C-KR31173 shows significantly higher uptake in the liver compared to the myocardium, which may compromise image interpretation and quantification. In addition, ¹¹C-KR31173 is not derived from a clinical ARBs, therefore translating this tracer to clinical practice may be more difficult when compared to radiolabeled derivatives of currently used ARBs. Along these lines, losartan has been labeled with both PET (9, 139, 140) and SPECT (169) isotopes. However, radiolabeled losartan has yet to be applied in patients with CVD, thus the true clinical potential is currently unknown.

Figure 23.

Positon emission tomography (PET)/computed tomography (CT) of angiotensin II type 1 receptors (AT₁R) in healthy pigs: panel A shows anterior maximum intensity projections (MIP, left), transaxial fusion images (middle) and reangulated PET images (right) (SA, short axis; HL, horizontal long axis; VL, vertical long axis), using AT₁R ligand [¹¹ C]-KR31173 (top row), and myocardial perfusion tracer [¹³N]-ammonia (NH₃) (bottom row). Panel B shows myocardial kinetics of [¹¹C]-KR31173 at baseline, and (panel C) after intravenous AT₁R blockade (representative coronal PET slices on top, time activity curves for arterial blood (pink) and myocardium (cyan) on bottom). Modified, with permission, from Fukushima K, J Am Coll Cardiol. 2012 (127).

Clinical implications

Acute myocardial infarction

Increased RAAS activity in the post-infarct setting is associated with deleterious LV remodeling. Nuclear imaging probes that target ACE and AT₁R have been used to assess the in vivo upregulation of these RAAS components in animal models of post-infarct remodeling. Specifically, SPECT imaging of ^99mTc-losartan demonstrated an upregulation of myocardial AT₁R in the infarct region, but not the border and remote zones, of mice 3 weeks following MI (407). Similarly, other studies have shown an upregulation of AT₁R expression with PET imaging of ¹¹C-KR31173 in the infarct region of the myocardium of both rodent and porcine models 3 to 4 weeks following myocardial I/R injury (127, 156). In contrast to the aforementioned study using ^99mTc-losartan, there was an increase in ¹¹C-KR31173 uptake in the remote regions in infarcted animals, albeit to a lesser extent than the in the infarct zone. Reasons for this discrepancy are unknown but may be due to differences in tracer specificity and image resolution between PET and SPECT cameras. Despite these promising results, in vivo myocardial RAAS activity imaging has yet to be applied in patients in the setting of acute MI.

Ischemic cardiomyopathy

As discussed above, it is believed that chronic activation of the RAAS leads to adverse LV remodeling and the progression of HF (63, 212). Along these lines, ¹⁸F-FBL was used to detect increased ACE expression with PET imaging in explanted hearts from 3 patients with ischemic cardiomyopathy at the time of heart transplantation (96). This study demonstrated specific binding of ¹⁸F-FBL to myocardial ACE and showed the highest radiotracer binding with autoradiography in the peri-infarct area. Specifically, ¹⁸F-FBL binding was 6.3 ± 4.5 photostimulating luminescence units (PSL)/mm² in infarcted, 7.6 ± 4.7 PSL/mm² in peri-infarcted, and 5.0 ± 1.0 PSL/mm² in remote, noninfarcted segments (P < 0.02 vs. peri-infarcted). These data are in line with observations that remodeling occurs in border and remote zone following scar formation due to alternations in mechanical stress experienced by these regions (123) and provides evidence that increased myocardial ACE activity participates, at least in part, in the remodeling process.

Cardiac Amyloidosis Imaging

Amyloidosis is a heterogeneous group of diseases associated with extracellular deposition of insoluble fibrillary proteins that may be confined to a single organ or can affect the entire human body. Cardiac involvement usually results in progressive LV hypertrophy, diastolic dysfunction, and HFpEF and can lead to conduction abnormalities and arrhythmias. Certain types of amyloidosis, such as primary light chain (AL) amyloidosis and transthyretin (ATTR) amyloidosis are more commonly associated with cardiac involvement, whereas other types including secondary (AA) amyloidosis rarely affect the heart. The diagnosis is usually established by endomyocardial biopsy coupled with immunohistochemistry. Determination of the type of amyloidosis is critical as the type determines the clinical course and the available treatment options.

Different radiolabeled phosphate derivatives targeting calcium deposition associated with necrosis have been applied for detecting amyloid deposits since the 1970’s, including: 99mTc-pyrophosphate (PYP), 99mTc-methylene diphosphonate (MDP), and 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid (DPD), with the majority of clinical studies using ^99mTc-PYP (41). More recently novel PET tracers, such as ¹⁸F-florbetapir and the Pittsburgh Compound B (¹¹C-PIB) have been used with promising results for imaging cardiac amyloidosis (8, 102, 213, 295). ¹⁸F-florbetapir binds to amyloid plaque through unknown mechanisms, while ¹¹C-PIB, a derivative of thioflavin-T, is thought to bind to all amyloid fibril types.

Clinical applications

Cardiac amyloid detection and amyloid type differentiation

The ratio of myocardial ^99mTc-PYP mean counts to ventricular cavity mean counts (accumulation rate >41.5%) has been found to be a sensitive (84.6%) and specific way (97.3%) of distinguishing cardiac amyloidosis from other causes of HF (433). Moreover, ^99mTc-PYP readily differentiates ATTR from AL amyloidosis, as ATTR amyloid has been shown to have higher tracer retention, whereas AL amyloid shows no or only minimal retention (heart-to-contralateral ratio: 1.80 ± 0.04 vs.1.21 ± 0.04 respectively, P < 0.0001) (39). This finding has been confirmed by a recent large multicenter study including 229 patients that were being evaluated for cardiac amyloidosis, in which the heart to-contralateral median ratio for ATTR amyloid (median: 1.73 [IQR: 1.53–1.98] was significantly higher than observed in non-ATTR amyloid (median: 1.16 [IQR: 1.02–1.30]; P < 0.001) (60). In addition, this trial demonstrated with ^99mTc-PYP planar imaging, that a heart to contralateral chest ratio of ≥1.6 was associated with worse survival among patients with ATTR cardiac amyloidosis (hazard ratio for death: 7.91 [95% CI: 1.68–37.30], P = 0.01).

Recently, in vitro studies showed that ¹⁸F-florbetapir binds specifically to myocardial amyloid deposits in human tissue from patients with documented AL or ATTR amyloid (295). In addition, a pilot study demonstrated myocardial uptake of ¹⁸F-Florbetapir in all the investigated amyloid patients (n = 5) compared to control subjects that had no evidence of myocardial uptake (n = 9) (102). A small study demonstrated a significant increase in myocardial ¹¹C-PIB retention index in patients with amyloidosis (mean retention index (RI_mean) 0.054 min⁻¹ [range: 0.033–0.134 min⁻¹] compared to healthy volunteers (RI_mean : 0.025 min⁻¹ [range: 0.020–0.031 min⁻¹], P = 0.0007) (8). In this study, amyloid patients had lower myocardial blood flow than healthy volunteers, but notably, the ¹¹C-PIB blood-flow corrected retention index remained highly significantly elevated in amyloid patients (RI_mean : 0.124 min⁻¹ [range: 0.069–0.309 min⁻¹] compared to healthy controls (RI_mean : 0.042 min⁻¹ [range: 0.025–0.052 min⁻¹], P = 0.0007) (Fig. 24) (8). In another small study, the myocardium-to-blood pool ratio of ¹¹C-PIB was significantly higher in patients that showed cardiac amyloidosis on subsequent biopsy (median 3.9 [range 1.7–19.9]) compared to those who did not (1.0 [range 0.8–1.2], P < 0.001) (213). Interestingly, in this study, a lower median myocardium-to-blood pool ¹¹C-PIB ratio was observed in AL amyloid patients who received chemotherapy (2.3 [range 1.7–3.8]) prior to PET imaging compared to patients who did not (10.4 [range 1.7–19.9], P = 0.014). These findings suggest that ¹¹C-PIB PET imaging may be a useful a tool in monitoring response to therapy in patients with cardiac amyloidosis. Taken together, the promising results of these initial studies will need to be verified by larger clinical investigations.

Figure 24.

In vivo visualization of amyloid deposits in the heart with Pittsburgh compound B (¹¹C-PIB) and PET: short-axis images of the Pittsburgh compound B (¹¹C-PIB) retention index and myocardial blood flow (MBF) in (left to right) patients with high, intermediate, and partially increased ¹¹C-PIB retention and a healthy control. Liver is clearly visible in ¹¹C-PIB images of the second patient and healthy control and is just outside PET field of view for other 2 patients. Liver uptake is due to biliary excretion of ¹¹C-PIB and is likely not related to amyloid binding. This research was originally published in JNM. Antoni G, Lubberink M, Estrada S, Axelsson J, Carlson K, Lindsjo L, Kero T, Langstrom B, Granstam SO, Rosengren S, Vedin O, Wassberg C, Wikstrom G, Westermark P, and Sorensen J. In vivo visualization of amyloid deposits in the heart with 11C-PIB and PET. J Nucl Med 54: 213–220, 2013. © SNMMI (8).

Autonomic Nervous System Imaging

The autonomic nervous system (ANS) consists of the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS). The ANS plays a key role in maintaining cardiovascular homeostasis at rest and in response to a variety of stimuli (e.g., exercise and postural changes) by regulating cardiac output, vasoreactivity, and metabolism via the release of neurotransmitters that act directly or indirectly on myocardium and the peripheral vasculature (16, 142, 243). Not surprisingly then, dysregulation or dysfunction in either ANS subdivision due to aging and other environmental stressors contributes to the pathophysiology and progression of many cardiovascular pathologies, such as hypertension, atrial and ventricular arrhythmogenesis, and HF (14, 112, 229, 288). The use of radiotracers that target SNS and PNS pre- and postsynaptic receptors allows for direct, noninvasive evaluation of cardiac ANS innervation. The use of SPECT and PET radioligands that target presynaptic SNS neural activity (e.g., uptake-1 and metabolism) are the most widely applied and clinically relevant for cardiovascular ANS imaging.

Sympathetic nervous system imaging

Following an action potential, postganglionic SNS nerve endings release stored norepinephrine (NE) from secretory vesicles. This NE enters the synaptic cleft for subsequent binding to adrenergic, G-protein coupled receptors on myocytes, with the subtypes α₁, β₁ and β₂ being the predominant isoforms (347). After release, most of the NE in the synaptic space (50–80%) is cleared via the NE reuptake transporter (NET or uptake-1) on presynaptic nerve terminals in an energy dependent manner (142). The majority of this NE is repackaged into secretory vesicles, while a small portion is metabolized (142). The remaining NE in the synaptic space is cleared by postsynaptic cells by the uptake-2 mechanism in an energy-independent fashion, or by diffusion into the vascular space.

Tracers of SNS presynaptic nerve activity

The most widely characterized and clinically used radiotracer for SNS imaging is the FDA approved SPECT analog of NE, ¹²³I-metaiodobenzylguanidine (mIBG) (153, 157, 392). Although SPECT compatible, many studies using 123I-mIBG have applied planar scintigraphy to determine the heart to mediastinal ratio (H/M) has been used as a surrogate for cardiac SNS innervation. The washout ratio from early and late ¹²³I-mIBG images has been used as a surrogate for SNS presynaptic nerve activity. In addition to ¹²³I-mIBG, several PET tracers have been synthesized that also target the uptake-1 mechanism; with the metabolically resistant analog of NE, ¹¹C-meta-hydroxyephedrine (HED), being the most well characterized in animal and human studies (256, 390). Importantly, ¹²³mIBG and ¹¹C-HED have similar, but not identical, myocardial uptake and clearance kinetics, and use of either tracer has advantages and limitations. Specifically, PET has higher spatiotemporal resolution and superior quantitative capabilities compared to planar scintigraphy and SPECT imaging; however, ¹¹C has a short-half life and requires a cyclotron for synthesis; therefore, precludes full kinetic modeling and offsite labeling, respectively. To overcome these challenges, the longer half-life, 18F-N-[3-bromo-4-(3-fluoro-propoxy)-benzyl]-guanadine (¹⁸F-LMI 1195) was synthesized to resemble the structure and uptake kinetics of ¹²³I-mIBG, while taking advantage of PET imaging (438). First-in-human studies showed favorable dosimetry and imaging characteristics of ¹⁸F-LMI1195, with rapid blood clearance, significant myocardial retention, and excellent target to background activity (Fig. 25) (66, 344). The application of this agent is being explored in patients with depressed LV function and HF that are at risk for sudden death. Other commonly used experimental SNS pre-synaptic tracers exist, such as ¹¹C-epinephrine (EPI) and ¹¹C-phenylephrine (PHEN) that offer information on NE storage and metabolism, respectively (88, 390).

Figure 25.

First-in-human study of a novel ¹⁸F-labeled tracer LMI1195 for imaging myocardial sympathetic innervation: Representative series of whole body [¹⁸F]-LMI 1195 coronal images at mid-myocardial level in a healthy human volunteer acquired 5 hours after injection (average, 193.5 ± 37.4 MBq [5.23 ± 1.01 mCi]). Each whole-body image is scaled to maximum value within that image. This research was originally published in JNM. Sinusas AJ, Lazewatsky J, Brunetti J, Heller G, Srivastava A, Liu YH, Sparks R, Puretskiy A, Lin SF, Crane P, Carson RE, and Lee LV. Biodistribution and radiation dosimetry of LMI1195: first-in-human study of a novel ¹⁸F-labeled tracer for imaging myocardial innervation. J Nucl Med 55: 1445–1451, 2014. © SNMMI (344).

Tracers of SNS postsynaptic receptors

Although less commonly used, PET tracers also exist that target SNS postsynaptic receptors in the myocardium, with the nonselective β-antagonists, ¹¹C-CGP-12177 and ¹¹C-CGP-12388, being the most widely used and characterized tracers in preclinical and human studies (110, 250, 276, 277). In addition, a PET compatible α₁-adrenoceptor antagonist, ¹¹C-GB67, also exists and has shown high in vivo binding specificity and favorable retention in the myocardium in preclinical models (207, 296); however, this tracer has yet to be tested in humans.

Parasympathetic nervous system imaging

Imaging of PNS cardiac innervation has been more elusive and challenging than imaging the SNS due to the denser distribution of PNS terminal nerves and receptors in the thinner walled atria compared to the ventricles, coupled with the rapid degradation of acetylcholine (ACh) by acetylcholinesterase (AChE). PNS nerve conduction is mediated by the release of ACh from postganglionic terminal nerve endings following an action potential and nerve depolarization. ACh that is released into the synaptic space subsequently binds to either muscarinic or nicotinic receptors, with the G_i-protein coupled M₂ muscarinic receptor being the most abundantly expressed cholinergic receptor in the myocardium (92, 142). Despite rapid degradation of ACh into its constituent molecules (e.g., choline and acetate) by AChE, there is rapid re-uptake of choline by the terminal nerve endings resulting in continual re-synthesis of ACh.

Tracers of PNS presynaptic nerve activity

Strategies to image PNS presynaptic nerves have included radiolabeling inhibitors of the vesicular ACh transporter and the high-affinity choline transporter since these proteins mediate ACh and choline uptake into synaptic vesicles of cholinergic neurons, respectively (324). However, the results of in vitro and preclinical studies have been largely unsuccessful due to low myocardial uptake that is confounded by high nonspecific binding (87, 128, 302, 447). Despite these disappointing results, recent data in large animals and in healthy human suggest that assessing AChE density as a surrogate of PNS presynaptic nerve density with the radiolabeled AChE antagonist, ¹¹C-donepezil shows favorable characteristics for in vivo PET imaging of cardiac PNS innervation. The clinical utility of ¹¹C-donepezil has yet to be explored but awaits future study.

Tracers of postsynaptic PNS receptors

The density of both muscarinic and nicotinic cholinergic receptors has been successfully assessed with PET imaging. The 11C-labeled, nonselective muscarinic receptor antagonist, methiodide quinuclidinyl benzilate (¹¹C-MQNB), is the most well characterized and clinically used radiotracer for cardiac PNS PET imaging (89–91, 209, 210). Recently, the feasibility of imaging cardiac nicotinic ACh receptors with PET was reported with the use of an ¹⁸F-radiolabeled analogue of the highly selective agonist of the α₄β₂ nicotinic ACh receptor, A-85380 (49). Initial studies showed favorable imaging characteristics of 2-[¹⁸F]-F-A-85380 in healthy volunteers and in patients with neurodegenerative disease (49). A schematic overview of ANS tracers is shown in Figure 26.

Figure 26.

Depiction of postganglionic sympathetic nervous system (SNS) and parasympathetic nervous system (PNS) nerve endings: (Left panel) The synthesis and release of norepinephrine in postganglionic SNS nerve endings and subsequent binding to postsynaptic receptors on cardiomyocytes. The tracers in red depict SNS pre- and postsynaptic radioanalogs. (Right panel) the synthesis and release of acetylcholine in the terminal nerve ending and varicosities of postganglionic PNS nerve endings and subsequent binding to postsynaptic receptors on cardiomyocytes. Tracers in blue depict PNS pre- and postsynaptic radioanalogs. AC, adenylyl cyclase; ACh, acetylcholine; AChE, acetylcholinesterase; ATP, adenosine triphosphate; CAT, choline-acetyl-transferase; COM, catechol-O-methyltransferase; cAMP, cyclic adenosine monophosphate; MAO, monoamine oxidase; mIBG, metaiodobenzylguanidine; MR2, muscarinic receptor 2; NE, norepinephrine; NR, nicotinic receptor; VMAT, vesicular monoamine transporter; ¹⁸F-6F-DA, 6-¹⁸F-fluorodopamine; PHEN, phenylephrine; EPI, epinephrine; HED, hydroxyephedrine; MQNB, (R,S)-N-[¹¹C]-methyl-quinuclidin-3-yl benzilate. Modified, with permission, from Boutagy NE, Curr Cardiol Rep. 2017 (45).

Clinical applications of autonomic imaging

Cardiac transplantation

Postganglionic SNS nerves are surgically transected at the base of the donor heart prior to transplantation, thus the allograft is devoid of SNS innervation, which leads to altered SNS cardiac responsiveness in the recipient. Therefore, reinnervation is an important processes in achieving normal cardiac function (26, 93). Imaging studies with both ¹²³I-mIBG and ¹¹C-HED have been valuable in elucidating the reinnervation process and the physiological consequences of insufficient reinnervation in transplant recipients (27, 113, 331). Observational studies with ¹²³I-mIBG and ¹¹C-HED described a time-dependent reinnervation process that never fully completes, with some patients remaining denervated up to 15 years after surgery (27, 113). Longitudinal studies with ¹¹C-HED indicated numerous factors that influence the reinnervation process, such as donor and recipient age, duration and complexity of surgery (24) and diabetic status (25). In addition, studies with ¹²³I-mIBG indicated that patients who develop coronary artery vasculopathy are less likely to have reinnervated myocardium following transplant (113). Importantly, denervated myocardium, as assessed with ¹¹C-HED, is associated with abnormal basal cardiac metabolism despite normal coronary flow (higher ¹⁸F-FDG uptake; 6.9 ± 6.6 vs. 6.0 ± 6.2 mmol/min/100 g; P = 0.03) compared to reinnervated areas in the same subjects (28); an attenuated myocardial blood flow response to SNS stimulation compared to vascular territories with lesser reinnervation in the same subjects (cold pressor increase in flow in LAD region [46 ± 10%] vs. right coronary artery [16 ± 5%, P = 0.01] and left circumflex artery [23 ± 6%, P = 0.06]) (93); and blunted responses to aerobic exercise (exercise time: 6.1 ± 1.5 minutes, vs. 8.2 ± 1.2, P < 0.01) when compared to HT recipients with greater myocardial reinnervation (26).

Myocardial ischemia and infarction

SNS imaging has been used to characterize the greater susceptibility of sympathetic nerves to ischemia than myocytes (7, 22, 245). Preclinical studies using ¹¹C-HED PET imaging report that progressive chronic ischemia can lead to abnormalities in SNS nerve function prior to decrements in resting myocardial blood flow and in the absence of tissue necrosis (225). In line with preclinical findings, studies in patients with chronic CAD, but without a history of infarction, show defects in both ¹²³I-mIBG and ¹¹C-HED uptake that were typically larger than perfusion defects (50, 145).

Several clinical studies using ¹²³I-mIBG or ¹¹C-HED, combined with perfusion imaging, show that sympathetic denervation extends beyond the infarct area in patients following an acute MI (7, 22, 124, 245). Importantly, areas of denervated myocardium that have normal perfusion post MI, termed autonomic/perfusion mismatch areas, are postulated to be particularly arrhythmogenic (322) (see the following text). Some clinical studies suggest that sympathetic denervation is permanent after an MI (7, 124) and that the severity of SNS denervation post-MI is associated with future LV remodeling (321). In addition, PET imaging of ¹¹C-CGP-12177 has shown global β-adrenergic receptor downregulation in acute MI patients compared to normal controls (109, 285, 359). Interestingly, the global reduction of β-adrenergic receptor density at 1-month post-MI is modestly predictive of an increase in end-diastolic (r = 0.31, P < 0.02) and end-systolic volumes (r = 0.29, P < 0.03) at 6 months (359). It has also been observed that a reduction in β-adrenergic receptor density in the remote, noninfarcted zone following an MI is strongly associated with impaired peak longitudinal strain (r = −0.71, P < 0.001) prior to the development of HF (285).

Despite the multitude of reports on SNS function in ischemia and MI, there has not been thorough investigation of the cardiac PNS in these states. One report by Mazzadi et al. (237) suggests that cardiac muscarinic receptor density, as determined by PET imaging of ¹¹C-MQNB, was significantly upregulated in the remote (67 ± 30 pmol/mL tissue) and border zone (71 ± 30 pmol/mL tissue) compared to the infarct zone (42 ± 21 pmol/mL tissue, P < 0.001) of MI patients and compared to normal myocardium of healthy controls (32 ± 17 pmol/mL tissue, P < 0.001). Of significance, the mean muscarinic receptor density in the remote zone was negatively associated with resting heart rate in MI patients (ρ = −0.745, P = 0.008). Indeed, larger studies are needed to fully elucidate the potential of PNS imaging in patients with acute and chronic ischemic disease.

Heart failure

During the initial stages of HF, there is a compensatory increase in cardiac and peripheral SNS activity in an effort to maintain cardiac output. Centrally, prolonged SNS activation and release of NE overwhelms the uptake-1 mechanism, leading to excessive NE in the synaptic cleft and eventual desensitization/downregulation of this transporter. This is turn exposes the myocardium and postsynaptic adrenergic receptors to greater concentrations of NE, which contributes to desensitization/downregulation of β-adrenergic receptors, cardiac remodeling, and worsening of HF and prognosis (141). Cardiac imaging of the SNS has provided useful information into the underlying disease process of HF, and has been helpful in assessing the severity and prognosis of patients with HF.

The magnitude of cardiac SNS denervation, as assessed with either ¹²³I-mIBG or ¹¹C-HED imaging, is associated with HF severity and cardiac function in patients with HFrEF of ischemic and nonischemic etiologies (23, 53, 233, 242, 269, 310, 327) and in HFpEF patients (4). More importantly, an ever-growing body of literature suggests that the magnitude of cardiac SNS denervation, determined either by ¹²³I-mIBG or ¹¹C-HED imaging, is predictive of worse disease prognosis and independently predicts cardiovascular and all-cause mortality in patients with HFrEF (126, 171, 269, 305) (Fig. 27). Of note, the largest (n = 961), prospective, multi-center trial (ADMIRE-HF) in HF patients with NYHA Class II-III symptoms and reduced EF (<35%) showed that a ¹²³I-mIBG H/M ratio of >1.6 was associated with lower risk of HF progression (hazard ratio [95% CI], 0.49 [0.32–0.77], P < 0.002), arrhythmic deaths (hazard ratio [95% CI], 0.37 [0.16–0.85)] and cardiac deaths (hazard ratio [95% CI], 0.14 [0.03–0.58]) at 2-years follow-up. In addition, the H/M ratio was predictive of time to first cardiac event, even when controlling for NYHA functional class, LVEF, and circulating brain natriuretic peptide in multivariate analyses (171, 271). Furthermore, sub-analyses of the ADMIRE-HF trial showed that the addition of the ¹²³I-mIBG H/M ratio to the Seattle Heart Failure Model provided a 22.7% improvement in the prognostic power of this commonly used algorithm in predicting morality in HF patients (188).

Figure 27.

¹²³I-metaiodobenzylguanidine (¹²³I-mIBG) imaging for prediction of mortality and potentially fatal events in heart failure: representative ¹²³I-mIBG images from patients from the ADMIRE-HFX Study. (A) Image from 37-year-old man with nonischemic cardiomyopathy demonstrated ¹²³I-mIBG heart to mediastinum (H/M) ratio of 1.69. (B) Image from 51-year-old woman with ischemic cardiomyopathy showed H/M ratio of 1.80. Right panel shows 2-year all-cause mortality rates based on 0.1 increments of H/M indicating progressive decline from maximum of 29.4% for H/M < 1.10. There were no deaths among subjects with H/M ≥1.80. This research was originally published in JNM. Narula J, Gerson M, Thomas GS, Cerqueira MD, and Jacobson AF. ¹²³I-mIBG imaging for prediction of mortality and potentially fatal events in heart failure: the ADMIRE-HFX study. Journal of Nuclear Medicine 56: 1011–1018, 2015 (271).

Although there is less robust data than exists for presynaptic SNS imaging, PET imaging of β-adrenergic receptor density with ¹¹C-CGP-12177 and ¹¹C-CGP-12388 has provided valuable insights into cardiac β-adrenergic receptor density in patients with HFrEF. For example, investigators have reported β-adrenergic receptor density downregulation in patients with nonischemic cardiomyopathy with PET imaging and ¹¹C-CGP-12177 (272, 396) or ¹¹C-CGP-12388 (81). In some studies, the reduction in β-adrenergic receptor density occurred concomitantly with an increased ¹²³I-mIBG H/M washout, a noninvasive assessment of pre-synaptic nerve activity. On the other hand, β-adrenergic receptor density derived from ¹¹C-CGP-12177 PET imaging only tended to be reduced globally (B’_max:10.0 ± 6.4 vs. 13.4 ± 4.2 pmol/mL, P = 0.056) in patients with ischemic cardiomyopathy compared to age-matched healthy volunteers, despite significant cardiac SNS denervation (permeability-surface area product: 0.32 ± 0.34 vs. 0.81 ± 0.33, P < 0.0001) as determined by dynamic ¹¹C-HED PET imaging (53). Even less data exists on the role of PET PNS targeted imaging in HF patients. One small study in idiopathic dilated cardiomyopathy patients with a reduced EF showed that muscarinic receptor density, as derived from ¹¹C- MQNB PET imaging, was significantly higher in patients (B’_max: 34.5 ± 8.9 pmol/mL) than in healthy controls (B’_max: 25 ± 7.7 pmol/mL, P < 0.005) (208). Indeed, more studies are needed to fully elucidate the role of postsynaptic SNS and PNS receptor imaging in HF.

Cardiac arrhythmias

In HF patients with NYHA Class II-III symptoms, LVEF ≤35% is the only criterion for eligibility of implantable cardioverter-defibrillator (ICD) therapy for primary prevention of sudden cardiac death (SCD) (111). However, only a small fraction of patients with depressed LVEF develop SCD (418). This is a major issue considering the high cost and patient risk (i.e., infection) associated with implantable electrical devices (350). Altered cardiac adrenergic activity in vulnerable myocardium causes electrical instability and increases the risk of arrhythmias (394). Therefore, cardiac sympathetic innervation imaging has been proposed as a strategy to stratify HF patients for arrhythmogenic risk (393, 394). Multiple studies indicate that patients with HF demonstrating significant cardiac denervation as determined by ¹²³I-mIBG (H/M ratios <1.6) have an independently higher risk of ICD discharges and SCD than patients with higher H/M ratios (171, 259, 279, 333, 377). In some (10, 279), but not all of these studies (18, 42), the extent of autonomic/perfusion mismatch area was also associated with ICD discharges and SCD. Some data suggests that the relationship between denervated myocardium and ICD discharge/SCD was not continuous, in that patients with an intermediate H/M ratio (1.30–1.59) have the highest risk for these events (271, 333). In the large, prospective ADMIRE-HF trial, patients with a H/M ratio <1.6 were more likely to experience arrhythmic events (10.4%) than patients with a H/M ratio ≥1.6 (3.5%) (171). Similar findings have been observed with PET ¹¹C-HED imaging. Specifically, in the prospective prediction of arrhythmic events with positron emission tomography (PAREPET) trial (114), denervated myocardial volume as determined by PET ¹¹C-HED imaging independently predicted time to SCD (arrhythmic death or appropriate ICD discharge) (hazard ratio [95% CI], 1.069 [1.023–1.117], p = 0.003) in patients with an ischemic cardiomyopathy (LVEF <35%, n = 204) (Fig. 28). In this study, the volume of denervated myocardium was also associated with SCD risk, with each 1% increase in the volume of denervated myocardium being associated with a 5.7% increase in risk of SCD. Interestingly, infarct volume and LVEF were not different in patients that developed SCD compared to those who did not.

Figure 28.

Regional myocardial sympathetic denervation assessment by [¹¹C]-meta-hydroxyephedrine (HED) PET in ischemic cardiomyopathy: (Top panel) PET images from two representative subjects from the PAREPET trial comparing resting flow (measured by ¹³N-ammonia [¹³NH₃]), viability (by insulin-stimulated ¹⁸F-fluorodeoxyglucose [¹⁸F-FDG]), and myocardial sympathetic innervation (by ¹¹C-HED) are shown. (Bottom panel) Kaplan-Meier curves show the incidence of sudden cardiac arrest for tertiles of PET-defined myocardial substrates (median follow-up 4.1 years). As continuous variables, the total volume of denervated myocardium, as well as viable denervated myocardium, predicted sudden cardiac arrest. Neither infarct volume nor hibernating myocardium was significant as continuous variables. Modified, with permission, from Fallavollita JA, J Am Coll Cardiol. 2014 (114).

Cardiac resynchronization therapy

Cardiac resynchronization therapy (CRT) has been shown to improve HF symptoms and reduce hospitalizations and life-threatening arrhythmias in some HF patients (273, 317). However, about one-third of patients receiving CRT do not respond to therapy (76). Some evidence suggests that CRT modulates the neurohormonal environment, which in turn, may lead to improvements in LV function (44, 61). Therefore, it has been suggested that cardiac SNS imaging may be used to determine appropriate candidates for this expensive therapy. In several small-scale studies, responders to CRT therapy had improvements in¹²³I-mIBG derived H/M ratios and washout rates (52, 61, 278) and ¹¹C-HED uptake values compared to nonresponders (57, 231). In addition, some studies also showed that higher baseline cardiac innervation, as determined by either ¹²³I-mIBG or ¹¹C-HED, was associated with response to CRT therapy (e.g., an improvement in NYHA class and/or a decrease in LV end-systolic volume ≥15%) (61, 231). These studies suggest that improvements in SNS presynaptic function with CRT may contribute to CRT response and provide some evidence that cardiac SNS adrenergic imaging with either ¹²³I-mIBG or ¹¹C-HED may also help to guide CRT placement.

Autonomic nervous system imaging in other conditions

Chemotherapy induced cardiotoxicity

Doxorubicin is an effective chemotherapy agent that is associated with cardiotoxicity (342). Doxorubicin induced cardiotoxicity is dose dependent and is clinically identified as a reduction in LVEF that may progress to overt HF in some patients (342). In addition to affecting the cardiomyocytes, doxorubicin has toxic effects on the cardiac adrenergic nervous system (216). Several small-scale patient studies have shown that cardiac ¹²³I-mIBG uptake is reduced with doxorubicin chemotherapy, and that this reduction typically occurs prior to reductions in systolic function (216, 372, 374). These findings suggest that cardiac SNS innervation imaging with ¹²³I-mIBG may provide an early index of cardiotoxicity in cancer patients treated with doxorubicin, but these findings need to be corroborated with a larger number of patients.

Cardiac amyloidosis

Amyloidosis frequently leads to abnormalities in the autonomic innervation of the heart, which is especially common in patients diagnosed with hereditary ATTR (hATTR) amyloidosis and to a lesser extent in wild type ATTR and AL amyloidosis (345). The aberration in SNS innervation has been postulated to play a role in the development of conduction disorders and fatal cardiac arrhythmias in these patients. As such, SNS innervation imaging has been applied in amyloidosis patients to elucidate these relationships. Specifically, several studies have reported a markedly reduced ¹²³I-mIBG H/M ratio in hATTR patients (6, 74, 379). Importantly, in patients with hATTR, the ¹²³I-mIBG H/M ratio is a powerful independent predictor of survival (6, 74). Specifically, the reported 5-year mortality is 42% for patients with H/M ratio <1.60 and 7% for patients with H/M ratio ≥1.60 (74). In addition, AL amyloidosis patients show some reduction in the ¹²³I-mIBG H/M ratio, but to a lesser extent than it is seen in ATTR amyloidosis (162). However, unlike the other frequently used amyloid imaging tracer ^99mTc-PYP, ¹²³I-mIBG is unable to differentiate between AL and ATTR amyloid, with significant overlap in ¹²³I-mIBG uptake between these subtypes (281). The relationship between impaired sympathetic innervation and the incidence of life-threatening arrhythmias in cardiac amyloidosis is unclear, and it is currently an area of investigation.

Monitoring Cell and Gene-Based Therapies with Imaging

Cell and gene-based therapies have been proposed as alternative approaches to treat CVDs, especially in conditions that do not improve appreciably with standard medical therapy. Along these lines, patients with ischemic cardiomyopathy have been the target of cell and gene-based therapy trials, with some trials reporting improved LV remodeling and function with treatment and many more reporting no benefit (165, 275). One explanation for the large failure rate of these trials may be due to the inability to noninvasively track the initial engraftment, persistence, and magnitude of these therapies in vivo. The high image sensitivity of nuclear or MRI systems to detect cellular and molecular events offers a unique opportunity to address these limitations. The ability to visualize cellular and gene-based therapies with nuclear or MRI imaging systems relies on two distinct strategies that include (i) direct labeling and (ii) the use of reporter genes.

Direct labeling

The gene itself, or components of the gene delivery system (e.g., viral capsid, viral proteins), have been directly radiolabeled with both gamma (¹¹¹In) and positron emitting (¹²⁴I) isotopes for SPECT and PET imaging, respectively. In addition, stem cells have been directly radiolabeled with techniques that allow for cellular incorporation or uptake of radiotracers for SPECT and PET imaging (38, 199, 315). Direct labeling techniques that allow stem cells to the incorporate or bind to SPIO, paramagnetic ions (e.g., Gd-chelates) or perfluorocarbon particles have also been used to track stem cell delivery in vivo with MRI (315). Although useful for monitoring initial cell/gene delivery to a target tissue, major issues exist with these techniques. For example, direct labeling does not allow for long-term follow-up due to loss of imaging signal strength because of radioactive isotope decay or MRI contrast biodegradation. In addition, some evidence suggests that direct labeling may affect cellular viability. Lastly, and most importantly, direct labeling does not allow for the ability to differentiate viable cells from dead cells or cellular debris (315).

Reporter genes

Reporter gene technology overcomes limitations of direct labeling. Simply, reporter genes are genes for specific proteins that are able to elicit an imaging signal by biochemically interacting with an imageable substrate (probe). In gene therapy applications, the reporter gene is attached to the regulatory sequence of the protein of interest; therefore, the magnitude of imaging signal is related to gene expression of both the report gene and the gene of interest. Similarly, in stem cell applications, reporter genes are introduced into the stem cell line prior to transplantation, thus probe uptake in vivo relates to stem cell number. Considering that only cells with active gene expression will take up an imaging substrate, the imaging signal is also an index of cell viability.

Common nuclear imaging reporter genes are the her-pes simplex virus type 1 thymidine kinase (HSV1-tk), the dopamine type 2 receptor (D₂R), and the human sodium iodide symporter (hNIS). Each of these proteins interact with reporter probes differently. Specifically, expression of the HSV1-tk in the target cell leads to the production of thymidine kinase, which in the presence of its radioisotope substrates, 9-(4–18F-fluoro-3-[hydroxymethyl]butyl)guanine (¹⁸F-FHBG) and 1-(29-deoxy-29-fluoro-b-D-arabinofuranosyl)-5-ethyl-uridine) (¹⁸F-FEAU), causes phosphorylation and trapping of these probes in the cell. Intuitively, radioanalogs of receptor ligands are able to bind to endogenously expressed receptors and these interactions follow receptor-ligand binding kinetics. Lastly, the hNIS is a membrane protein that is able to transport several radioisotopes (e.g., ^99mTcO₄⁻, ¹²³I, ¹⁸F-tetrofluroborate) when expressed in a target cell. Uptake is facilitated by active transport of Na⁺, thus uptake can occur against a concentration gradient and lead to higher radiotracer accumulation and image sensitivity than with the use of enzymes or receptors (38, 315). Alternatively, reporter genes for MRI are endogenous proteins that bind to iron (e.g., transferrin), enzymes that activate paramagnetic ions (β-gal), bioengineered receptors that bind to a variety of MRI contrast agents without eliciting a cell-signaling response, or are artificial peptides that when expressed allow for CEST-MRI (403). Several of these MRI techniques have been used to track stem cell delivery and survival in the myocardium in small animal models of MI; however, these techniques do not appreciably increase MRI image sensitivity for detecting in vivo gene expression and thus have not been used beyond small animal models. On the other hand, nuclear imaging, especially when combined with anatomical CT imaging, offers adequate image sensitivity for detecting in vivo processes, and thus has been more widely used.

Clinical applications of tracking cell and gene-based therapy with imaging

Monitoring gene therapy in the myocardium

The most widely applied reporter genes for tracking gene expression in the myocardium are HSV1-tk and hNIS. For example, the mutant HSV1-tk, HSV1-sr39tk, has been used to track the expression of adenovirus driven VEGF as an angiogenic therapy in the myocardium following experimental MI in rats (427). Delivery of the adenovirus vector in the peri-infarct region following surgical MI induction allowed for the in vivo assessment of HSV1-sr39tk gene expression for up to two weeks with microPET and ¹⁸F-FHBG imaging. Importantly, the in vivo PET imaging signal correlated well with ex vivo VEGF expression and histological angiogenic markers, such as capillary density. Other investigators have compared HSV1-tk, HSV1-sr39tk and hNIS to determine the optimal reporter gene for monitoring cardiac transgene expression in rodents with microPET (247). As discussed above, the ability of hNIS to transport and accumulate radioisotopes in vivo provided higher signal intensity and image contrast for PET imaging at equal adenoviral vector doses encoding for each reporter gene. The use of NIS as a reporter gene for assessing in vivo gene expression in myocardium has also been translated to large animal models. Specifically, the use of canine NIS on AAV9 allowed for the serial evaluation of several novel strategies for delivering gene therapy to the myocardium of adult canines using SPECT/CT (253). These technologies have yet to be applied to monitor gene expression in patients with CVD, but have been used safely and efficaciously in humans with other diseases (309).

Monitoring stem cell engraftment and survival in the myocardium

Reporter gene technology and nuclear imaging has been used to assess initial stem cell engraftment and long-term survival in the myocardium of small and large animals (315). The ability to visualize the fate and survival of induced pluripotent stem cells (iPSCs) and human mesenchymal stem cells immediately following myocardial delivery in several large animal models has been demonstrated with the use of HSV1-tk as a reporter gene and ¹⁸F-FHBG PET/CT imaging (211, 422). In addition, the use of hNIS as a reporter gene facilitated the first demonstration of long-term survival and engraftment of human iPSC delivered into the myocardium following MI in a large animal model. Specifically, the expression of hNIS in hiPSC allowed for serial SPECT/CT ¹²³I imaging of these cells for up to 15 weeks following delivery into porcine myocardium following MI (Fig. 29) (385). Tracking initial delivery and long-term survival has yet to be applied in humans, but merits future study.

Figure 29.

Tracking of human induced pluripotent stem cells in a pig model of myocardial infarction: assessment of cell survival, engraftment and distribution by hybrid SPECT/CT imaging of sodium iodide symporter transgene expression: In vivo SPECT/CT imaging ~1 h following intracoronary injection of ¹²³I demonstrating long-term surviving derivatives of sodium iodide symporter (NIS) positive (^pos) transgenic human induced pluripotent stem cells (NIS^pos-hiPSCs, each site 5 × 10⁷ cells) in pig hearts after 5 days and 15 weeks, respectively. Note, NIS^pos-hiPSCs were only visualized when co-injected with an equal number of mesenchymal stem cells (MSC). Immunohistochemical staining of tissue sections of corresponding areas of the left ventricular wall confirmed the presence of hiPSC derivatives, since delivered NIS^pos-hiPSCs were also transfected to express the reporter protein, venus. Notably, the vast majority of venus positive iPSC derivatives were found to represent endothelial cells integrated into the cardiac vasculature 15 weeks after cell transplantation. Modified, with permission, from Templin C, Circulation 2012 (385).

Conclusions

CVD and associated morbidity carry high socioeconomic burdens nationally and worldwide. Current clinical imaging has focused on assessment of functional, structural, and physiological parameters for detection of CVD. Although essential to clinical medicine, these imaging techniques lack the ability to detect early disease and to interrogate the underlying cellular and molecular mechanisms that contribute to disease pathology. On the other hand, advancements in molecular and cellular biology combined with advances in image instrumentation, targeted probe development, and image reconstruction and processing have led to the application of multimodality cardiovascular molecular imaging. Over the past several decades, molecular imaging has provided valuable insights in determining the underlying fundamental molecular processes and pathophysiology of many CVDs, including: ischemic, hypertrophic, infiltrative, or inflammatory heart disease, and complicating arrhythmias and heart failure (HF). Importantly, these insights have led to the translation of some of these imaging techniques to patients, which have assisted in guiding therapy, in understanding the response to established and novel therapies, and in predicting disease progression and risk stratification. A specific example of this is cardiac SNS presynaptic imaging with ¹²³I-mIBG and ¹¹C-HED, which has provided additional clinical benefit in terms of predicting the risk and cause of mortality in HFrEF patients. Another notable imaging technique that has had a clinical impact is ¹⁸F-FDG imaging for the assessment of hibernating myocardium in patients with ischemic cardiomyopathy. This review has also highlighted the potential for molecular imaging to assist in the characterization and risk prediction in less prevalent diseases, such as amyloidosis, sarcoidosis, and poorly characterized conditions like HFpEF. Indeed, larger trials are needed to elucidate the potential of molecular imaging for these applications. Despite the promising horizons, advancements in imaging technology and tracer development are needed to better characterize disease pathologies with chronic, low-level molecular activation of pathogenic signals that occur in a diffuse pattern, which challenge the detection sensitivity of current in vivo imaging modalities.

Didactic Synopsis.

Major teaching points

• Molecular imaging refers to the visualization, characterization, and noninvasive measurement of biological processes at the molecular and cellular levels in humans and other living systems.

• The nuclear imaging systems, single-photon emission tomography (SPECT) and position emission tomography (PET) are the most commonly used modalities for molecular imaging.

• Nanoparticles, contrast agents, and microbubbles are used to enhance the molecular imaging capability of computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound that have inherently lower sensitivity for detection of these molecular processes.

• Molecular imaging has provided valuable insights in determining the underlying pathophysiology and molecular processes associated with many cardiovascular diseases (CVDs), including ischemic, hypertrophic, infiltrative or inflammatory heart disease, and complicating arrhythmias and heart failure (HF).

• Cardiac molecular imaging is already currently used as a sensitive tool in the diagnosis of certain CVDs, such as cardiac sarcoidosis and amyloidosis.

• Molecular imaging of the heart has the capability to provide early detection of disease, improve the understanding of the mechanisms of disease progression, and allows for risk stratification.