Unveiling doxorubicin-induced cardiotoxicity: into the heart with single-cell resolution

Abstract Background Doxorubicin (DOX), a potent chemotherapeutic agent used to treat a wide range of malignancies, is limited in its clinical use due to dose-dependent cardiotoxicity, which can lead to heart failure and increased mortality among cancer survivors. The underlying molecular basis of DOX-induced cardiotoxicity (DIC) remains ambiguous, as no study has yet conducted an unbiased characterization of the transcriptional landscape of DOX-treated hearts, distinguishing an early but reversible phase of the disease. Aim Identifying early transcriptional changes in DIC could be key to developing new cardioprotective strategies aimed at preventing disease progression. Methods BALB/c mice received either saline or DOX (single-dose or weekly injections for 3 weeks; 4 mg/kg) as previously established (1). Hearts were collected at 3 days (3-day, early) or 6 weeks (6-week, late) post-injection. Nuclei isolated from frozen hearts underwent single-nuclei transcriptomic analysis (snRNAseq). Seurat pipeline along with Drug2cell and CellChat tools were used for downstream analysis. Results Analysis of snRNAseq data from 12 hearts revealed 8 major cell types and 42 distinct cell states, highlighting the high cellular complexity within the cardiac environment. Differential abundance testing revealed enrichment of immune cell subpopulations, including CD8+ T cells (TC2_CD8) in DOX treated groups. Notably, CellChat analysis indicated a direct proinflammatory signaling from fibroblast (FB) and TC2_CD8 to cardiomyocytes (CMs) upon DOX, specifically targeting a CM population identified as CM3_Stressed, characterized by elevated expression of well-known cardiac stress markers (e.g. Nppb, Myh7). ScFate trajectory analysis of CMs uncovered 4 unbiased clusters of gene signatures depicting a progression path from a CM_Basal cell state to CM_Stressed, suggesting that the maladaptive cardiac remodeling driven by DOX underlies profound and specific transcriptional changes, as well as complex cross talk between cardiac subpopulations. Intriguingly, among these gene signatures, we found Golgi associated kinase 1B (Gask1b), which has never been associated to cardiac pathophysiology, as one of the top differentially upregulated genes specifically in CMs of both 3-day and 6-week DOX-treated groups. SnRNAseq dataset of a human cardiotoxic heart confirmed Gask1b overexpression upon anthracycline treatment. Interestingly, preliminary results in our zebrafish model of DIC reveal that Gask1b silencing prevents DOX-induced decline of heart function, identifying Gask1b as a new potential player of DIC. Conclusions We generated a single-nucleus dataset of DOX-treated mouse hearts and identified transcriptional changes driven by DOX in CMs at early and late stages of the disease. Furthermore, we identified Gask1b gene, whose role in cardiac pathophysiology was previously unappreciated, as a new potential marker and determinant of DIC.