Abstract
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Objectives: Cardiac muscle satisfies its high-energy requirements by fatty acid oxidation (FAO), that is a promising therapeutic target for heart failure. 18-18F-fluoro-4-thia-oleic acid (FTO) has been introduced as a marker of myocardial FAO. We newly developed a FTO analog 18F-AS3504073-00 aiming to improve tracer characteristics for cardiac imaging. In this study, the subcellular kinetics of uptake and trapping of 18F-AS3504073-00 was examined in freshly isolated cardiomyocytes.
Methods: 18F-AS3504073-00 was prepared by aliphatic radiofluorination in the presence of [18F]TBAF and deprotection under basic conditions using sodium hydroxide solution. All animal experiments were conducted in compliance with the current laws of the Federal Republic of Germany. Murine cardiomyocytes were freshly isolated from healthy mouse according to the previous reports with some modifications (Timothy D. O’Connell et al., www.signaling-gateway.org/reports/v1/CM0005/CM0005.htm). Cells were treated with 100 kBq 18F-AS3504073-00 in DMEM containing 0.5% BSA. Non-specific binding or uptake was examined by incubation with 18F-AS3504073-00 at 4oC. To investigate uptake pathway, cardiomyocytes were treated with 18F-AS3504073-00 for 10 min in the presence or absence of DMSO, 100 μM, or 500 μM sulfo-N-succinimidyl oleate (SSO), a selective inhibitor for fatty acid transporter CD36. For investigating metabolic trapping of 18F-AS3504073-00, cardiomyocytes were treated with 18F-AS3504073-00 for 60 min in the presence or absence of 100 μM etomoxir (ETO), a selective inhibitor for carnitine palmitoyltransferase 1 (CPT1), or 100 μM trimetazidine (TMZ), a partial inhibitor for β-oxidation. After washing with medium, cells were further incubated in medium for 60 min and the radioactivity in supernatant and cells was measured.
Results: Radiochemical yield of 18F-AS3504073-00 was 17.5±3.6%, and radiochemical purity was greater than 99%. 18F-AS3504073-00 was time-dependently taken up into cardiomyocytes, and the uptake was inhibited to 27.7±5.6% of control by the treatment with 500 μM SSO (P<0.01). The non-specific background binding of 18F-AS3504073-00 was 29.9±7.6% of control. These results confirmed highly specific tracer 18F-AS3504073-00 uptake via fatty acid transporter CD36. Furthermore, in the presence of ETO, the uptake of 18F-AS3504073-00 was decreased down to 43.2±13.8% of control (P<0.001), indicating CPT1 dependent tracer transport into mitochondria as a key uptake pathway. Extracellular release of 18F-AS3504073-00 and its metabolites were increased in the presence of ETO or TMZ. These results confirmed that β-oxidation is critical for tracer retention in the cells.
Conclusions: A preliminary in vitro cellular assay confirmed that the newly developed FTO analog 18F-AS3504073-00 was taken up into cardiomyocytes via fatty acid transporter CD36 and metabolically trapped in cardiomyocytes by β-oxidation. The new class of PET tracer 18F-AS3504073-00 is worthy of further pursuit as a marker of altered cardiac metabolic phenotype in heart failure.