Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): This work was supported by the European Union’s Horizon 2020 research and innovation program (ERC Advanced Grant) and the Novo Nordisk Foundation. Background Doxorubicin is a common and highly effective chemotherapeutic. However, its use is limited by cardiotoxic side effects, which occur in 5-23 % of patients treated, and the lack of methods to detect these conditions at early time points. Purpose In the present study, we evaluated if 64CuCu-RGD positron emission tomography/computed tomography (PET/CT) could detect cardiotoxicity in a rat model of doxorubicin induced heart failure. Methods Male Lewis rats were divided into two groups and treated with either a cumulative dose of 15 mg/kg doxorubicin (n = 15) or left untreated (n = 10). Cardiac anatomy and function were assessed using magnetic resonance imaging at baseline and in week eight. 64CuCu-RGD PET/CT scans were performed in week four. All animal experiments were approved by the Danish Animal Experiments Inspectorate (authorization number 2016-15-0201-00920) and study performed in accordance with the ARRIVA guidelines. Results Doxorubicin treatment led to a decline in left ventricular ejection fraction (66.5 ± 0.85 % to 46.67 ± 0.84 %, p < 0.001), as well as an increase in septal and thymic uptake of 64CuCu-RGD (0.46 ± 0.02 vs. 0.29 ± 0.04, <0.001 and 0.87 ± 0.03 vs. 0.40 ± 0.07, p < 0.001, respectively). In addition, doxorubicin altered the cardiac gene expression, led to infiltration of macrophages (0.90 ± 0.12 % vs. 0.30 ± 0.04 %, p <0.001), reduced endothelial content (0.19 ± 0.02 % vs. 0.47 ± 0.07 %, p < 0.001), and increased interstitial fibrosis (13.6 ± 0.86 % vs. 10.5 ± 0.66 %, p = 0.014). Furthermore, concentrations of inflammatory plasma proteins were increased in the doxorubicin group. Conclusion Doxorubicin treatment resulted in the development of cardiotoxicity and heart failure, which could be detected using 64CuCu-RGD PET/CT at early time points. 64CuCu-RGD uptake in the myocardial septum and thymus predicted low left ventricular ejection fraction in week eight, thus it may serve as an early marker of cardiotoxicity.Experimental setup and timeline