René R. Sevag Packard

Cardiac PET perfusion tracers: current status and future directions.

PET myocardial perfusion imaging (MPI) is increasingly being used for noninvasive detection and evaluation of coronary artery disease. However, the widespread use of PET MPI has been limited by the shortcomings of the current PET perfusion tracers. The availability of these tracers is limited by the need for an onsite ((15)O water and (13)N ammonia) or nearby ((13)N ammonia) cyclotron or commitment to costly generators ((82)Rb). Owing to the short half-lives, such as 76 seconds for (82)Rb, 2.06 minutes for (15)O water, and 9.96 minutes for (13)N ammonia, their use in conjunction with treadmill exercise stress testing is either not possible ((82)Rb and (15)O water) or not practical ((13)N ammonia). Furthermore, the long positron range of (82)Rb makes image resolution suboptimal and its low myocardial extraction limits its defect resolution. In recent years, development of an (18)F-labeled PET perfusion tracer has gathered considerable interest. The longer half-life of (18)F (109 minutes) would make the tracer available as a unit dose from regional cyclotrons and allow use in conjunction with treadmill exercise testing. Furthermore, the short positron range of (18)F would result in better image resolution. Flurpiridaz F 18 is by far the most thoroughly studied in animal models and is the only (18)F-based PET MPI radiotracer currently undergoing clinical evaluation. Preclinical and clinical experience with Flurpiridaz F 18 demonstrated a high myocardial extraction fraction, high image and defect resolution, high myocardial uptake, slow myocardial clearance, and high myocardial-to-background contrast that was stable over time-important properties of an ideal PET MPI radiotracer. Preclinical data from other (18)F-labeled myocardial perfusion tracers are encouraging.

Absolute Quantitation of Myocardial Blood Flow in Human Subjects With or Without Myocardial Ischemia Using Dynamic Flurpiridaz F 18 PET

Absolute quantitation of myocardial blood flow (MBF) by PET is an established method of analyzing coronary artery disease (CAD) but subject to the various shortcomings of available radiotracers. Flurpiridaz F 18 is a novel PET radiotracer that exhibits properties of an ideal tracer. Methods: A new absolute perfusion quantitation method with flurpiridaz was developed, taking advantage of the early kinetics and high first-pass extraction by the myocardium of this radiotracer, and the first-in-human measurements of MBF performed in 7 healthy subjects and 8 patients with documented CAD. PET images with time–activity curves were acquired at rest and during adenosine stress. Results: In healthy subjects, regional MBF between coronary artery territories did not differ significantly, leading to a mean global MBF of 0.73 mL/min/g at rest and 2.53 mL/min/g during stress, with a mean global myocardial flow reserve (MFR) of 3.70. CAD vascular territories with <50% stenosis demonstrated a mean MBF of 0.73 at rest and 2.02 during stress, leading to a mean MFR of 2.97. CAD vascular territories with ≥50% stenosis exhibited a mean MBF of 0.86 at rest and 1.43 during stress, leading to a mean MFR of 1.86. Differences in stress MBF and MFR between normal and CAD territories, as well as between <50% and ≥50% stenosis vascular territories, were significant (P < 0.01). Conclusion: Absolute quantitation of MBF in humans with the novel PET radiotracer flurpiridaz is feasible over a wide range of cardiac flow in the presence or absence of stress-inducible myocardial ischemia. The significant decrease in stress MBF and ensuing MFR in CAD territories allows a clear distinction between vascular territories exhibiting stress-inducible myocardial ischemia and those with normal perfusion.

Genetic Architecture of Atherosclerosis in Mice: A Systems Genetics Analysis of Common Inbred Strains

Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis.

4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.

Cardiac Light-Sheet Fluorescent Microscopy for Multi-Scale and Rapid Imaging of Architecture and Function.

Light Sheet Fluorescence Microscopy (LSFM) enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. We hereby demonstrate cardiac LSFM (c-LSFM) imaging to assess the functional architecture of zebrafish embryos with a retrospective cardiac synchronization algorithm for four-dimensional reconstruction (3-D space + time). By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin treatment. By integrating the resolution enhancement technique with c-LSFM to increase the resolving power under a large field-of-view, we demonstrate the use of low power objective to resolve the entire architecture of large-scale neonatal mouse hearts, revealing the helical orientation of individual myocardial fibers. Therefore, our c-LSFM imaging approach provides multi-scale visualization of architecture and function to drive cardiovascular research with translational implication in congenital heart diseases.

Blood flow modulation of vascular dynamics

Purpose of review Blood flow is intimately linked with cardiovascular development, repair and dysfunction. The current review will build on the fluid mechanical principle underlying haemodynamic shear forces, mechanotransduction and metabolic effects. Recent findings Pulsatile flow produces both time (∂&tgr;/∂t) and spatial-varying shear stress (∂&tgr;/∂x) to modulate vascular oxidative stress and inflammatory response with pathophysiological significance to atherosclerosis. The characteristics of haemodynamic shear forces, namely, steady laminar (∂&tgr;/∂t = 0), pulsatile shear stress (PSS: unidirectional forward flow) and oscillatory shear stress (bidirectional with a near net 0 forward flow), modulate mechano-signal transduction to influence metabolic effects on vascular endothelial function. Atheroprotective PSS promotes antioxidant, anti-inflammatory and antithrombotic responses, whereas atherogenic oscillatory shear stress induces nicotinamide adenine dinucleotide phosphate oxidase–JNK signalling to increase mitochondrial superoxide production, protein degradation of manganese superoxide dismutase and post-translational protein modifications of LDL particles in the disturbed flow-exposed regions of vasculature. In the era of tissue regeneration, shear stress has been implicated in reactivation of developmental genes, namely, Wnt and Notch signalling, for vascular development and repair. Summary Blood flow imparts a dynamic continuum from vascular development to repair. Augmentation of PSS confers atheroprotection and reactivation of developmental signalling pathways for regeneration.

Automated Segmentation of Light-Sheet Fluorescent Imaging to Characterize Experimental Doxorubicin-Induced Cardiac Injury and Repair.

This study sought to develop an automated segmentation approach based on histogram analysis of raw axial images acquired by light-sheet fluorescent imaging (LSFI) to establish rapid reconstruction of the 3-D zebrafish cardiac architecture in response to doxorubicin-induced injury and repair. Input images underwent a 4-step automated image segmentation process consisting of stationary noise removal, histogram equalization, adaptive thresholding, and image fusion followed by 3-D reconstruction. We applied this method to 3-month old zebrafish injected intraperitoneally with doxorubicin followed by LSFI at 3, 30, and 60 days post-injection. We observed an initial decrease in myocardial and endocardial cavity volumes at day 3, followed by ventricular remodeling at day 30, and recovery at day 60 (P < 0.05, n = 7–19). Doxorubicin-injected fish developed ventricular diastolic dysfunction and worsening global cardiac function evidenced by elevated E/A ratios and myocardial performance indexes quantified by pulsed-wave Doppler ultrasound at day 30, followed by normalization at day 60 (P < 0.05, n = 9–20). Treatment with the γ-secretase inhibitor, DAPT, to inhibit cleavage and release of Notch Intracellular Domain (NICD) blocked cardiac architectural regeneration and restoration of ventricular function at day 60 (P < 0.05, n = 6–14). Our approach provides a high-throughput model with translational implications for drug discovery and genetic modifiers of chemotherapy-induced cardiomyopathy.

Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics

Currently, there is a limited ability to interactively study developmental cardiac mechanics and physiology. We therefore combined light-sheet fluorescence microscopy (LSFM) with virtual reality (VR) to provide a hybrid platform for 3D architecture and time-dependent cardiac contractile function characterization. By taking advantage of the rapid acquisition, high axial resolution, low phototoxicity, and high fidelity in 3D and 4D (3D spatial + 1D time or spectra), this VR-LSFM hybrid methodology enables interactive visualization and quantification otherwise not available by conventional methods, such as routine optical microscopes. We hereby demonstrate multiscale applicability of VR-LSFM to (a) interrogate skin fibroblasts interacting with a hyaluronic acid-based hydrogel, (b) navigate through the endocardial trabecular network during zebrafish development, and (c) localize gene therapy-mediated potassium channel expression in adult murine hearts. We further combined our batch intensity normalized segmentation algorithm with deformable image registration to interface a VR environment with imaging computation for the analysis of cardiac contraction. Thus, the VR-LSFM hybrid platform demonstrates an efficient and robust framework for creating a user-directed microenvironment in which we uncovered developmental cardiac mechanics and physiology with high spatiotemporal resolution.

Fractional flow reserve by computerized tomography and subsequent coronary revascularization

Aims Fractional flow reserve by computerized tomography (FFR-CT) provides non-invasive functional assessment of the hemodynamic significance of coronary artery stenosis. We determined the FFR-CT values, receiver operator characteristic (ROC) curves, and predictive ability of FFR-CT for actual standard of care guided coronary revascularization. Methods and results Consecutive outpatients who underwent coronary CT angiography (coronary CTA) followed by invasive angiography over a 24-month period from 2012 to 2014 were identified. Studies that fit inclusion criteria (n = 75 patients, mean age 66, 75% males) were sent for FFR-CT analysis, and results stratified by coronary artery calcium (CAC) scores. Coronary CTA studies were re-interpreted in a blinded manner, and baseline FFR-CT values were obtained retrospectively. Therefore, results did not interfere with clinical decision-making. Median FFR-CT values were 0.70 in revascularized (n = 69) and 0.86 in not revascularized (n = 138) coronary arteries (P < 0.001). Using clinically established significance cut-offs of FFR-CT ⩽0.80 and coronary CTA ≥70% stenosis for the prediction of clinical decision-making and subsequent coronary revascularization, the positive predictive values were 74 and 88% and negative predictive values were 96 and 84%, respectively. The area under the curve (AUC) for all studied territories was 0.904 for coronary CTA, 0.920 for FFR-CT, and 0.941 for coronary CTA combined with FFR-CT (P = 0.001). With increasing CAC scores, the AUC decreased for coronary CTA but remained higher for FFR-CT (P < 0.05). Conclusion The addition of FFR-CT provides a complementary role to coronary CTA and increases the ability of a CT-based approach to identify subsequent standard of care guided coronary revascularization.

Technical aspects of acquiring and measuring myocardial blood flow: Method, technique, and QA

Measuring absolute myocardial blood flow (MBF) is becoming a common aid for diagnosing patients suspected to have coronary artery disease. An MBF study, however, requires a scanner with high count rate capability, is more susceptible to artifacts, and is much more technically involved than static imaging, which leads to a greater risk of artifactual results contaminating the final result. This technical note gives the reader an introductory understanding of the method for calculating MBF. It then describes the scanning protocol, potential pitfalls and how to recognize them, and quality control steps that should be taken to avoid basing a clinical decision on possibly inaccurate flow information.