Abstract Fri056: Mitochondrial DNA (mtDNA) Serves as a Potential Mediator and Prognostic Indicator of Doxorubicin Induced Cardiotoxicity

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Background: Cardiotoxicity and subsequent cardiac dysfunction are well-documented sequelae of anthracycline-based chemotherapy treatment. Previous studies have established inflammation and activation of the innate immune system as key mediators of doxorubicin (DOX)-induced cardiotoxicity, the most frequently used anthracycline. However, the underlying mechanisms driving inflammatory activation in this pathology remain elusive. Hypothesis: Here, we posit release of circulating cell-free mitochondrial DNA (ccf-mtDNA), a known activator of inflammation in other cardiovascular disease states, increases following DOX treatment, in a manner independent of cell death. We further hypothesize ccf-mtDNA released following DOX treatment activates innate immune cells via Toll-Like Receptor 9 (TLR9) signaling. Finally, we suspected that mtDNA release increased in DOX patients, in a manner dependent on cumulative dose administration. Methods: Human cardiomyocyte AC-16 cells were treated with increasing doses of DOX (vehicle, 0.5μM, 1μM, 1.5μM, and 2μM) for 24 hours. Following treatment, culture media was collected, cleared of cells and debris, and analyzed through quantitative PCR (qPCR) for mtDNA release by quantifying mtDNA-encoded genes such as MT-ATP6, MT-COX1, and MT-CYB. The collected supernatant was also used in conditioned media experiments with human monocyte derived THP-1 cells to assess TLR9 activation using a SEAP reporter assay. Finally, plasma samples (n=9) from patients undergoing DOX treatment were analyzed for ccf-mtDNA content via qPCR over the course of treatment. Results: Our results indicated a dose-dependent increase in ccf-mtDNA release for all genes, with the largest statistically significant differences observed for MT-COX1 at the 1μM (p ≤ 0.01), 1.5μM (p ≤ 0.001), and 2μM (p ≤ 0.0001) doses, as compared to vehicle. Also, ccf-mtDNA levels correlated with activation of TLR9 in THP1 cells, which was statistically significant at 2μM (p ≤ 0.05). Interestingly, plasma samples from patients showed a transient 3-fold increase in ccf-mtDNA levels after initial DOX treatment, followed by a return to basal levels after approximately 2-3 cycles of DOX treatment. Conclusions: In conclusion our data suggest that ccf-mtDNA release may contribute to DOX-induced cardiotoxicity by activating TLR9 signaling, highlighting its potential as a biomarker and therapeutic target.

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