Abstract Body: INTRODUCTION: Doxorubicin (DOX) is an effective chemotherapeutic agent for various cancers, but its use is restricted due to potentially life-threatening cardiac toxicity. This cardiotoxicity persists long after treatment, necessitating strategies to repair myocardial damage and identify target genes in cardio-oncology. Our previous research indicated that lysosomal dysfunction significantly contributes to DOX-induced myocardial damage. The ubiquitin E3 ligase SMURF1, a crucial enzyme for lysosomal regeneration, may offer protective effects against DOX-induced cardiotoxicity, yet this remains unexplored.
HYPOTHESIS: This study tested the hypothesis that enhancing lysosomal function through upregulation of SMURF1 can decrease DOX-induced cardiomyocyte death.
METHODS: We utilized human AC16 and rat H9c2 cardiomyoblasts to determine if adenoviral treatment, which increases SMURF1 expression, can mitigate DOX-induced death in cardiomyocytes. To evaluate the impact of SMURF1 on cardiotoxicity and lysosomal function, we measured cell death using Propidium Iodide (PI) staining along with lysosomal acidity and enzyme activity via LysoSensor and DQRed BSA staining.
RESULTS: AC16 cardiomyoblasts treated with 1 μM DOX for 16 hours showed a significant increase in cell death (PI staining). However, the upregulation of SMURF1 expression before DOX decreased cell death (control: 3.3% ± 0.3, SMURF1: 3.2% ± 0.2, DOX: 12.2% ± 0.7, DOX+SMURF1: 9.9% ± 0.7; DOX vs DOX+SMURF1 p ≤ 0.05, n = 12). Similar results were obtained with H9c2; LysoSensor staining indicated that DOX increased pH and reduced lysosomal acidity. Additionally, lysosomal proteolysis was worsened. In contrast, the upregulation of SMURF1 enhanced the intensity of LysoSensor staining, while DQRed staining indicated improved lysosomal activity. (Both DOX vs DOX+SMURF1 p ≤ 0.05 n=12)
CONCLUSIONS: Our findings confirm that DOX induces lysosomal injury, contributing to cardiotoxicity. SMURF1 aids in maintaining lysosomal function and alleviates DOX-induced toxic effects. While further validation in chronic DOX cardiotoxicity models is needed, these findings suggest that SMURF1-targeted therapy could mitigate the risks of DOX cardiotoxicity while preserving anticancer effects.