Imidazoline Receptor Agonists Alleviate Doxorubicin-Induced Lysosomal Injury and Cardiomyocyte Death

Due to its broad efficacy, Doxorubicin (DOX) is widely recognized as a potent antineoplastic agent. However, its clinical use is severely limited by dose-dependent cardiotoxicity that often causes heart failure. This cardiotoxicity is a critical issue in cancer therapy and has recently been linked to lysosomal dysfunction. Lysosomal membrane permeabilization (LMP) is a central mechanism affecting cellular homeostasis and cell death and is also a target for anticancer activity. We aimed to test the hypothesis that DOX-induced LMP is responsible for cardiomyocyte cell death and, thus, suppression of LMP is a potent therapeutic target for cardioprotection. First, we assessed LMP levels in human cardiomyoblasts AC16 by confocal imaging, using the fluorescent LMP reporter tfGalectin3, and observed that treatment with one µM of DOX for 16 hours enhanced the count of puncta of Galectin3 that accumulated on injured lysosomes (Control 3.17±0.78, DOX-treated 29.33±3.89, p=0.0033, five cell counts), indicating that DOX induces LMP. Further analysis using Lysosensor staining revealed that treatment with DOX markedly reduced lysosomal acidity (relative changes in DOX are 0.7±, while those for Con are normalized to 1.0±, with p = 0.038 n=12). DQRed BSA staining showed that overall lysosomal proteolysis was also reduced in DOX treatment (Relative changes in DOX 0.7± while normalized to that of Con in 1.0±, p = 0.05 n=12), indicating lysosomal dysfunction due to DOX. Western blot analysis revealed that the isolated lysosomal fraction obtained via lysosomal immunoprecipitation (Lyso-IP) showed a reduction in the active form of lysosomal protease Cathepsin D (CTSD) within lysosomes. Conversely, the total expression of CTSD increased following DOX exposure. This indicates lysosomal damage induced by LMP, suggesting leakage of lysosomal proteins from the compromised lysosomal lumen. Interestingly, treatment with 2-(Benzofuran-2-yl)-2-imidazoline hydrochloride (2BFI), an agonist of the imidazoline receptor that has been reported to improve lysosomal membrane permeability, reduced DOX-induced myocyte death. Propidium Iodide (PI) staining showed that treatment with DOX reduced myocyte death (the cell death ratios were recorded as follows: control 5% ±, DOX 40%, and DOX combined with 2BFI 20%, with a p-value ≤ 0.05 when comparing DOX to DOX + 2BFI, n = 12. This study was repeated twice). On the other hand, LMP inducer L-Leucyl-L-Leucine methyl ester (LLOMe) treatment further induced cell death (cell death ratios: control 5% ±, DOX 40%, and DOX combined with LLOMe 60%, with a p-value ≤ 0.05 when comparing DOX to DOX + LLOMe, n = 12. This study was repeated twice). These results suggest that DOX-induced LMP is one of the significant mechanisms mediating myocardial cell death and that suppression of LMP is a novel therapeutic target for myocardial protection. Further studies are warranted to verify the reinforcement of LMP stability and safeguard against DOX-induced cardiotoxicity by the 2BFI intervention. 1R15HL163707-01A1 1R15HL161737-01 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.