Rodolfo A. Medina

PET imaging of cardiac hypoxia: Opportunities and challenges

Myocardial hypoxia is a major factor in the pathology of cardiac ischemia and myocardial infarction. Hypoxia also occurs in microvascular disease and cardiac hypertrophy, and is thought to be a prime determinant of the progression to heart failure, as well as the driving force for compensatory angiogenesis. The non-invasive delineation and quantification of hypoxia in cardiac tissue therefore has the potential to be an invaluable experimental, diagnostic and prognostic biomarker for applications in cardiology. However, at this time there are no validated methodologies sufficiently sensitive or reliable for clinical use. PET imaging provides real-time spatial information on the biodistribution of injected radiolabeled tracer molecules. Its inherent high sensitivity allows quantitative imaging of these tracers, even when injected at sub-pharmacological (≥pM) concentrations, allowing the non-invasive investigation of biological systems without perturbing them. PET is therefore an attractive approach for the delineation and quantification of cardiac hypoxia and ischemia. In this review we discuss the key concepts which must be considered when imaging hypoxia in the heart. We summarize the PET tracers which are currently available, and we look forward to the next generation of hypoxia-specific PET imaging agents currently being developed. We describe their potential advantages and shortcomings compared to existing imaging approaches, and what is needed in terms of validation and characterization before these agents can be exploited clinically.

Cardiac Hypoxia Imaging: Second-Generation Analogues of 64Cu-ATSM

Myocardial hypoxia is an attractive target for diagnostic and prognostic imaging, but current approaches are insufficiently sensitive for clinical use. The PET tracer copper(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) has promise, but its selectivity and sensitivity could be improved by structural modification. We have therefore evaluated a range of 64Cu-ATSM analogs for imaging hypoxic myocardium. Methods: Isolated rat hearts (n = 5/group) were perfused with normoxic buffer for 30 min and then hypoxic buffer for 45 min within a custom-built triple-γ-detector system to quantify radiotracer infusion, hypoxia-dependent cardiac uptake, and washout. A 1-MBq bolus of each candidate tracer (and 18F-fluoromisonidazole for comparative purposes) was injected into the arterial line during normoxia, and during early and late hypoxia, and their hypoxia selectivity and pharmacokinetics were evaluated. The in vivo pharmacokinetics of promising candidates in healthy rats were then assessed by PET imaging and biodistribution. Results: All tested analogs exhibited hypoxia sensitivity within 5 min. Complexes less lipophilic than 64Cu-ATSM provided significant gains in hypoxic-to-normoxic contrast (14:1 for 64Cu-2,3-butanedione bis(thiosemicarbazone) (ATS), 17:1 for 64Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS), 8:1 for 64Cu-ATSM, P < 0.05). Hypoxic first-pass uptake was 78.2% ± 7.2% for 64Cu-ATS and 70.7% ± 14.5% for 64Cu-CTS, compared with 63.9% ± 11.7% for 64Cu-ATSM. Cardiac retention of 18F-fluoromisonidazole increased from 0.44% ± 0.17% during normoxia to 2.24% ± 0.08% during hypoxia. In vivo, normoxic cardiac retention of 64Cu-CTS was significantly lower than that of 64Cu-ATSM and 64Cu-ATS (0.13% ± 0.02% vs. 0.25% ± 0.04% and 0.24% ± 0.03% injected dose, P < 0.05), with retention of all 3 tracers falling to less than 0.7% injected dose within 6 min. 64Cu-CTS also exhibited lower uptake in liver and lung. Conclusion: 64Cu-ATS and 64Cu-CTS exhibit better cardiac hypoxia selectivity and imaging characteristics than the current lead hypoxia tracers, 64Cu-ATSM and 18F-fluoromisonidazole.

64Cu-CTS: A Promising Radiopharmaceutical for the Identification of Low-Grade Cardiac Hypoxia by PET.

The subtle hypoxia underlying chronic cardiovascular disease is an attractive target for PET imaging, but the lead hypoxia imaging agents 64Cu-2,3-butanedione bis(N4-methylthiosemicarbazone) (ATSM) and 18F-fluoromisonidazole are trapped only at extreme levels of hypoxia and hence are insufficiently sensitive for this purpose. We have therefore sought an analog of 64Cu-ATSM better suited to identify compromised but salvageable myocardium, and we validated it using parallel biomarkers of cardiac energetics comparable to those observed in chronic cardiac ischemic syndromes. Methods: Rat hearts were perfused with aerobic buffer for 20 min, followed by a range of hypoxic buffers (using a computer-controlled gas mixer) for 45 min. Contractility was monitored by intraventricular balloon, energetics by 31P nuclear MR spectroscopy, lactate and creatine kinase release spectrophotometrically, and hypoxia-inducible factor 1-α by Western blotting. Results: We identified a key hypoxia threshold at a 30% buffer O2 saturation that induces a stable and potentially survivable functional and energetic compromise: left ventricular developed pressure was depressed by 20%, and cardiac phosphocreatine was depleted by 65.5% ± 14% (P < 0.05 vs. control), but adenosine triphosphate levels were maintained. Lactate release was elevated (0.21 ± 0.067 mmol/L/min vs. 0.056 ± 0.01 mmol/L/min, P < 0.05) but not maximal (0.46 ± 0.117 mmol/L/min), indicating residual oxidative metabolic capacity. Hypoxia-inducible factor 1-α was elevated but not maximal. At this key threshold, 64Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS) selectively deposited significantly more 64Cu than any other tracer we examined (61.8% ± 9.6% injected dose vs. 29.4% ± 9.5% for 64Cu-ATSM, P < 0.05). Conclusion: The hypoxic threshold that induced survivable metabolic and functional compromise was 30% O2. At this threshold, only 64Cu-CTS delivered a hypoxic-to-normoxic contrast of 3:1, and it therefore warrants in vivo evaluation for imaging chronic cardiac ischemic syndromes.

Demonstration of the retention of 64Cu-ATSM in cardiac myocytes using a novel incubation chamber for screening hypoxia-dependent radiotracers.

ObjectiveWe have designed a low-cost, reusable incubation system that allows cells to be cultured in either plated or suspension culture under complete atmospheric control for radiotracer characterization. We demonstrate its utility here in the first quantification of the hypoxia-dependent accumulation of 64Cu-diacetyl bis(N4-methylthiosemicarbazone) (64Cu-ATSM) in adult rat ventricular myocytes (ARVMs). Materials and methodsARVMs were allowed to adhere overnight in 9 cm culture plates (2×105 cells/dish) or were used in suspension culture, placed inside the chamber and equilibrated with either oxic (95 or 21% O2/5% CO2) or anoxic gas (95% N2/5% CO2). 64Cu-ATSM of 100 kBq was administered, and the cells were incubated for 30 or 60 min. Cells were then harvested, counted and fractionated to determine intracellular 64Cu biodistribution. ResultsAfter 1 h, the average cellular 64Cu retention in plated ARVMs under oxygenated conditions was 23.9±2.5 mBq/cell (95% O2), increasing to 27.3±5.1 under 21% O2 (P<0.05) and to 36.1±3.1 under 0% O2 (P<0.05). When ARVMs were cultured in suspension, normoxic–hypoxic contrast was less marked but still significant [63.2±14.1 vs. 53.4±10.9% mBq/cell after 30 min (P<0.05)]. Sixty percent of tracer accumulated in the cytosol, and, although total cellular retention increased during hypoxia, there was no enrichment in any particular cellular compartment. ConclusionThis apparatus allows the conduction of radiotracer uptake studies in cells under complete atmospheric control, as evidenced by our first demonstration of the hypoxia-dependent uptake of 64Cu-ATSM in ventricular myocytes. It is ideally suited for screening, validating and characterizing novel hypoxia-selective radiotracers.

Trapped Platelets Activated in Ischemia Initiate Ventricular Fibrillation

Background— We tested the hypothesis that ischemia-induced ventricular fibrillation (VF) is facilitated by platelets, trapped regionally in the ischemic zone and activated to release arrhythmogenic secretome. Methods and Results— In a randomized study in blood-free, buffer-perfused isolated rat hearts, ischemic zone territory (34±1% of left ventricle) was selected so that ischemia evoked VF in only 42% of controls. VF incidence was increased to 91% (P<0.05) by coronary ligation–induced trapping of freshly prepared autologous platelets (infused before and during coronary ligation, with trapping confirmed by 111In-labeled platelet autoradiographic imaging). Trapping of platelet secretome prepared ex vivo, or platelet-sized fluorospheres, did not increase ischemia-induced VF incidence. Secretome alone did, however, evoke VF in 2 sham coronary-ligated hearts. Perfusion did not activate infused platelets in sham coronary-ligated hearts, whereas ligation activated trapped platelets (assessed by thromboxane release). In a separate study, trapping whole-heparinized blood mimicked the ability of trapped platelets to increase VF incidence. This effect was not prevented by >5 days oral pretreatment in vivo with clopidogrel (10 mg/kg per day) or indomethacin (2.4 mg/kg per day). Conclusions— Platelets facilitate VF during acute ischemia independently of their ability to participate in occlusive thrombosis. Moreover, the effect is unresponsive to antiplatelet drugs commonly used. Labile secretome constituents appear to be responsible. This opens a novel avenue for antiarrhythmic drug research.

Assessing radiotracer kinetics in the Langendorff perfused heart

BackgroundThe Langendorff perfused heart is a physiologically relevant and controllable model with potential for assessing the pharmacokinetics of new radiotracers under a range of pathophysiological conditions.. We assess the feasibility of extending the methods validated for in vivo PET data analysis to the characterisation of PET tracer kinetics applied to Langendorff perfused hearts.MethodsMonte Carlo simulations were used to study the accuracy and reproducibility of linear and non-linear spectral analysis (SA/NLSA), the Patlak graphical method and normalised tissue activity (NA). The methods were used to analyse time-activity curves of two widely used PET tracers, [18 F]-FDG and [18 F]-FMISO, acquired ex vivo from Langendorff perfused rat hearts under normoxic and hypoxic conditions.ResultsMonte Carlo simulations showed NLSA to be superior to SA in identifying and quantifying the presence of irreversible trapping component (αo), for low values of αo. The performance of NLSA and SA for high values of trapping was comparable. NLSA was also more precise than SA in determining the absence of trapping over the range of simulated kinetics and SNR. Simulations also suggest that the semi-quantitative method NA is adequate for the evaluation of trapping, and it was found to be more accurate than Patlak. The values of α0 estimated with NLSA from the time series of both [18 F]-FDG and [18 F]-FMISO increased significantly from normoxia to hypoxia in agreement with previous studies. The values of trapping derived using SA increased but not significantly, reflecting the larger error associate with this method. Patlak estimated from the experimental datasets increased from normoxia to hypoxia but was not significant. NA estimated from the [18 F]-FDG data increased from normoxia to hypoxia, but was not significant, whilst NA calculated for [18 F]-FMISO time-activity curves increased significantly.ConclusionsMonte Carlo simulations suggested that spectral-based quantitative analysis methods are adequate for the kinetic characterisation of time-activity curves acquired ex vivo from perfused hearts. The uptake rate Patlak and the index NA also represent a good alternative to the SA and NLSA algorithms when the aim of the kinetic analysis is to measure changes in the amount of tracer trapped in the irreversible compartment in response to external stimuli. For low levels of trapping, NLSA and NA were subject to lower errors than SA and Patlak, respectively.