Abstract Fri076: Endothelial Mitochondria-rich Extracellular Vesicles Protect In Vitro Cardiomyocytes from Doxorubicin Toxicity by Restoring ATP Production, Reducing Oxidative Stress, and Modulating Free Calcium

Doxorubicin (Doxo) is a first-line chemotherapeutic agent that leads to heart failure due to irreversible mitochondrial dysfunction. Endothelial-derived extracellular vesicles (e-EVs) may protect cardiomyocytes from injury, though the role of large mitochondria-rich e-EVs (e-mtEVs) in cardiotoxicity remains unknown. We hypothesized that e-mtEVs uptake by cardiomyocytes may counteract Doxo-induced toxicity. e-mtEVs were isolated via serial centrifugation from human aortic endothelial cell culture media, with JC-1 staining confirming ∼90% contained active mitochondria. H9c2 cardiomyocytes were exposed to 0.3 µM Doxo for 12h and treated with e-mtEVs (~1 EV/cell). At 24h, Seahorse analysis showed a 52% reduction in glycolytic ATP production in Doxo cells (Control Mdn 0.02 [IQR 0.02-0.03] vs Doxo Mdn 0.009 [IQR .003-.010], p=0.046), which normalized after e-mtEVs treatment. Extracellular acidification rate (ECAR) decreased by 46% with Doxo (Control Mdn 0.005 [IQR 0.004-0.006] vs Doxo Mdn 0.003 [IQR 0.002-0.003], p=0.025), but remained at control levels in Doxo+e-mtEVs cells. Cell viability, reduced by 67% in Doxo cells (p≤0.001), improved by 30% with e-mtEVs (p=0.029). Spectrofluorimetric analysis showed increased oxidative stress in Doxo-treated cells, with superoxide (O2–, +82%, Control Mdn 0.180 [IQR 0.139-0.181] vs Doxo Mdn 0.990 [IQR 0.915-1.044], p≤0.0001) and hydrogen peroxide (H2O2, +76%, Control Mdn 0.225 [IQR 0.138-0.362] vs Doxo Mdn 0.933 [IQR 0.815-1.190], p≤0.0001), whereas e-mtEVs significantly reduced O2– (-57%, e-mtEVs Mdn 0.421 [IQR 0.290-0.566], p=0.077) and H2O2 (-40%, e-mtEVs Mdn 0.561 [IQR 0.337-0.708], p=0.021) after Doxo. Intracellular Ca++ levels, measured with Fluo-4 AM and Fura-2 AM, increased after Doxo (+80% bound Ca++, Control Mdn 0.193 [IQR 0.158-0.572] vs Doxo Mdn 0.978 [IQR 0.929-1.110], p=0.026; +61% free Ca++, Control Mdn 0.324 [IQR 0.087-0.494] vs Doxo Mdn 0.825 [IQR 0.697-1.478], p=0.002), but were reduced by 60% with e-mtEVs (Mdn 0.334 [IQR 0.263-0.339], p=0.009) compared to Doxo cells. In conclusion, the uptake of e-mtEVs protects cardiomyocytes from Doxo toxicity, restoring glycolytic ATP production, reducing oxidative stress, and modulating calcium dynamics. These findings suggest that transplanting e-mtEVs could offer a groundbreaking therapeutic approach to combat chemotherapy-induced cardiotoxicity.