Mitochondrial sORF-Encoded Peptide MODICA Protects the Heart From Doxorubicin-Induced Cardiac Injury by Suppressing VDAC Oligomerization

BACKGROUND: Doxorubicin (DOX) cardiotoxicity increases cardiovascular risk in cancer patients, mainly through mitochondrial damage. However, the underlying mechanisms remain unclear, and whether mitochondrial short open reading frame-encoded peptides can mitigate DOX-induced cardiotoxicity is unknown. METHODS: Five adeno-associated viruses expressing mitochondrial short open reading frame-encoded peptides under the cardiac troponin T promoter, including MODICA (mito-SEP protector against DOX-induced cardiac injury), were screened in a DOX-induced cardiotoxicity mouse model (n=3–5 per group). Male and female mice were randomized to adeno-associated virus-CTRL or adeno-associated virus-MODICA, respectively, combined with saline or DOX treatment. Sample sizes were: males—saline-CTRL (n=4), saline-MODICA (n=4), DOX-CTRL (n=11), DOX-MODICA (n=10); females—saline-CTRL (n=8), saline-MODICA (n=10), DOX-CTRL (n=10), DOX-MODICA (n=13). MODICA-heterozygous mice generated by CRISPR/Cas9 were also included: saline-WT (n=7), saline-heterozygous (n=4), DOX-WT (n=11), DOX-heterozygous (n=8). Echocardiography was performed at baseline and after 2 weeks of DOX treatment; myocardial tissue and serum samples were collected for molecular and histological analyses. RESULTS: The mitochondrial short open reading frame-encoded peptide MODICA was identified through biochemical analysis and functional screening in a DOX-induced cardiac injury model. MODICA localizes to the outer mitochondrial membrane and is significantly downregulated by DOX (1.00±0.08 versus 0.42±0.09; P <0.001). Cardiac-specific overexpression of MODICA via adeno-associated viruses significantly attenuated DOX-induced cardiac injury in both males and females (fractional shortening: males 38.86% versus 51.54%, P <0.001; females 39.81% versus 51.39%, P <0.001, DOX-CTRL versus DOX-MODICA) and was supported by bulk RNA-seq analysis. Conversely, MODICA deficiency exacerbated DOX-induced injury, resulting in reduced fractional shortening (40.37% versus 31.85%, P <0.001; DOX-WT versus DOX-heterozygous) and increased cardiac fibrosis ( P =0.009). Proteomic analyses revealed that MODICA interacts with apoptosis-related voltage-dependent anion channel proteins, inhibiting their DOX-induced oligomerization ( P <0.001) on the outer mitochondrial membrane, thereby reducing mitochondrial permeability, decreasing cardiomyocyte apoptosis and improving calcium handling. CONCLUSIONS: Our study shows that the mitochondrial short open reading frame-encoded peptide MODICA alleviates DOX-induced cardiac dysfunction and may represent a therapeutic target against DOX cardiotoxicity.