Introduction
Chemotherapy-related cardiovascular complications, in particular, doxorubicin (DOX)-induced cardiotoxicity, are a major quality-of-life compromise that can, in some cases, result in the death among cancer survivors [1]. DOX treatment impairs mitochondrial energetics through multiple pathways, resulting in dysfunctional cardiac metabolism. The hallmarks of cardiac metabolic dysfunction are decreased fatty acid oxidation, the main energy supply for the healthy heart, and increased reliance on other substrates, such as glucose. Recent work has highlighted the loss of mitochondrial pyruvate carriers 1 and 2 (MPCs), which are responsible for importing pyruvate into mitochondria, as correlating with more severe cardiometabolic dysfunction [2, 3]. We hypothesized that exposure to DOX decreases MPC1/2 expression in the mouse heart in a manner that can be imaged by positron emission tomography (PET). We tested this hypothesis in a preclinical murine model of DOX-induced cardiotoxicity using [3-¹¹C]pyruvate PET imaging and a multi-omics approach.

Methods
We established chronic cardiotoxicity in male C57BL/6J mice by administering doxorubicin (cumulative dose 24 mg/kg) intraperitoneally over 2 weeks. Over 16 weeks, the doxorubicin-treated (DOX) mice and age- and sex-matched controls were imaged with [3-¹¹C]pyruvate PET. The dynamic PET acquisitions commenced immediately upon injection and were reconstructed as 12x5s, 12x10s, 8x15s, 10x30s, 10x60s, and 2x300s frames. Tissue transit time (τ) in the heart was determined to enable quantitative comparisons between groups. Stable isotope tracing metabolomics were performed in excised cardiac tissue following the administration of [3-¹³C]pyruvate. Finally, we extracted cardiac tissue samples at PET imaging time points and analyzed the bulk RNA-seq and MPC1/2 expression by Western blot. We used the STRING database and GeneOntology (GO) to identify the most significantly affected biological processes (BP) and KEGG pathways resulting from exposure to DOX.
Results
Mice exposed to DOX exhibited characteristic physiological differences, including decreased body weight and heart-weight-to-tibia-length ratios, compared to control mice. Four weeks after the first dose of DOX, bulk RNA-seq (FDR < 0.001, the absolute log2FC cutoff > 1) analysis showed significantly decreased expression of genes related to pyruvate and fatty acid metabolism, including Mpc1, Mpc2, Pdhb, and Fabp3, in DOX heart tissue. At the protein level, MPC1/2 expression was also significantly decreased at this time point. This translated to a significant decrease in ¹³C-labeled tricarboxylic acid (TCA) cycle intermediates in DOX hearts compared to controls. [3-¹¹C]Pyruvate PET kinetics generally indicated delayed clearance of cardiac PET signal in the mice treated with DOX, resulting in an increased transit time of 423±140 compared to 317±34. Interestingly, cardiac MPC1/2 protein expression gradually recovered in the DOX mice and was not different to the controls 16 weeks after the initial DOX dose. This was reflected in the similar cardiac flux of [3-¹¹C]pyruvate uptake in the two groups, resulting in τ = 364±56 for the DOX mice and 384±63 for the control mice.
Conclusions
Our results suggest that DOX rapidly alters cardiac metabolism through decreased MPC expression and suppression of pyruvate metabolism. Significantly, this biochemical change can be detected in vivo by imaging the cardiac flux of [3-¹¹C]pyruvate by PET. MPC expression gradually recovers after cessation of DOX treatment, and similarly, the transit time of [3-¹¹C]pyruvate in the heart converges between the two groups. These findings highlight the dynamic cardiometabolic reprogramming undergone by mouse hearts exposed to DOX and supports the use of [3-¹¹C]pyruvate PET to detect potentially deleterious changes in MPC expression in cancer patients treated with doxorubicin.

Author