(D-155) Doxorubicin-induced Cardiotoxicity is Propagated by Paracrine Signaling through Exosomal Factors

Introduction:: Cardiovascular disease (CVD) is the leading cause of death in the United States and worldwide. While most of these deaths are the result of chronic diseases of the heart which lead to heart failure after insult, some CVDs can be induced artificially, such as via off-target effects of other therapies. Doxorubicin (DOX), an anthracycline, is a chemotherapeutic that has been commonly used to treat breast cancer, one of the most common types of cancer in the United States, affecting 1 in 8 women. While DOX and DOX cocktails show success in mitigating or otherwise destroying breast cancer and other cancers, over 10% of women showed signs of acute cardiotoxicity immediately following treatment and approximately 2% developed severe cardiotoxicity up to 10 years after the end of treatment, despite known clearance of DOX in less than 48 h. This DOX-induced cardiotoxicity has since been well-established, operating through transcriptional and mitochondrial damage, but the mechanism by which the onset of this cardiotoxicity occurs over time is not well understood. We suggest that initial DOX exposure is able to alter the paracrine signaling behaviors of some myocardial cells, which over time propagate this dysfunction to the surrounding tissue to eventually result in observable cardiotoxic effects. This can be demonstrated by recapitulating the effects of DOX treatment independently utilizing only isolated extracellular vesicles (EVs) from DOX-treated cells.

Materials and Methods:: Stem cell derived cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells were treated with physiological DOX concentration for up to 48 h. After this, EVs were isolated from media by typical exosome ultracentrifugation protocol. Isolated EVs were assessed via nanoparticle tracking analysis (NTA) and western blot to identify population changes as a result of DOX treatment, and were also assessed for presence of DOX cargo, via UV-Vis spectrophotometry, and changes in miRNA profile, via Nanostring assay. The effects of paracrine signaling on CMs and CFs were then assessed via cell assays in 2D and 3D culture conditions, with ECs being excluded from individual assays due to showing little response to DOX exposure. All cell assays were performed in the absence of any DOX exposure, except for when as a positive control. CMs were assessed for beating velocity, rate, and area, as well as calcium handling and beating regularity, to identify arrhythmia-like behaviors. CFs were assessed for survival and epithelial-to-mesenchymal transdifferentiation (EMT). All cells were also assessed for metabolic activity and reactive oxygen species (ROS) generation. Assays were also tested with cell-free DOX media EV isolate, to ensure that media DOX carryover was not driving any observed differences. 3D models were constructed from a combination of gelatin methacrylol (GelMA) and collagen, using a combination of CMs and CFs. Gels were stable and capable of regular beating within 7 days. CMs were kept isolated from DOX, and CFs were exposed to physiological DOX or a null control for 48 h before seeding.

Results, Conclusions, and Discussions:: DOX treatment induced a non-significant increase in EV production in CFs and ECs, and a non-significant decrease in EV production in CMs. In both CFs and CMs, EV size was significantly decreased and CD63 expression was significantly increased. TSG101 expression was also significantly increased in CM EVs. For all EVs, UV-Vis spectrophotometry revealed the DOX-treated cell EVs were indistinguishable from control EVs, with DOX group EVs also lacking characteristic DOX absorbance peak at ~480 nm and emission at 590 nm. Direct treatment of CMs with DOX resulted in significantly decreased beating velocity, but not rate, and dysregulation of calcium handling that resulted in an irregular beating profile, similarly to clinical DOX cardiotoxicity. Treatment of CFs with DOX resulted in a significant increase in both transdifferentiation and cell death in normal culture conditions. Interestingly, in 2D culture EVs from DOX-treated CFs alone were sufficient to induce DOX-like dysregulation of CM beating velocity, calcium handling, and metabolism, while EVs from DOX-treated CMs emulated some of these effects. Treatment with control CM EVs caused no noticeable difference in CM behavior, while treatment with control CF EVs enhanced beating velocity and calcium handling. To further assess these effects, CMs were encapsulated with control or DOX-treated CFs. Gels seeded with control CFs began beating several days before gels seeded with DOX-treated CFs, with DOX-treated CF seeded gels showing decreased beating velocity and calcium handling, similar to the effects observed in 2D culture. These results suggest that DOX-mediated cardiotoxicity can be propagated in the absence of DOX itself, via paracrine signaling from cells that have been exposed to DOX. In particular, this effect may be mediated by differential expression of several exosomal miRNAs. Some miRNAs, such as miR-140-5p, miR-199a, and miR-320a, have already been suggested as individual targets in the induction of DOX-mediated cardiotoxicity, with the miRNAs each able to partially recapitulate the effects of DOX treatment in vitro. We suggest that these miRNAs may be part of a exosome population shift in DOX exposed cells which drive the onset of DOX cardiotoxicity.

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