Chamber–Specific Molecular Profiling of Doxorubicin-Induced Cardiotoxicity via Integrated Multi-Omics Analysis

Background: Doxorubicin (Dox) induces dose-dependent Dox–induced cardiotoxicity (DIC), but the chamber-specific molecular mechanisms remain unclear, as traditional analyses rely on whole-heart homogenates. Methods: We established rat acute single–dose and chronic cumulative–dose DIC models, and separated left atrium (L–atrium) and left ventricle (L–ventricle) tissues for subsequent detection. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to quantify regional Dox tissue and serum concentrations. Histological HE and Masson’s staining were used to evaluate myocardial structural injury and collagen deposition. Integrated chamber-specific bulk proteomics, phosphoproteomics, and untargeted metabolomics were applied to systematically compare molecular perturbations between the L-atrium and L-ventricle. Two–way factorial statistical decomposition coupled with functional enrichment analysis was subsequently performed to identify both shared and chamber-specific molecular signatures. Results: Histological staining confirmed pronounced obvious myocardial fibrosis in both the L–atrium and L–ventricle under chronic Dox exposure. LC–MS/MS revealed prominent L–atrium–predominant Dox accumulation in the acute model, while inter–chamber drug distribution differences vanished after long–term repeated administration. Proteomic and metabolomic responses exhibited high inter–chamber consistency (r = 0.864 and r = 0.911, respectively). In contrast, the phosphoproteome was the dominant regulatory layer driving chamber divergence. A total of 14 core phosphosites with significant tissue–drug interaction effects were identified as key molecular switches. Conclusions: Our findings reveal preferential atrial Dox accumulation and distinct atrial phosphorylation remodeling following Dox exposure. The identified differential phosphosites serve as promising candidate targets for chamber-specific cardioprotection against DIC, pending further functional validation.