Stage-resolved single-cell profiling identifies GASK1B as an interceptive target for doxorubicin cardiotoxicity
Y Qin, M Mergiotti, J Van Fraeyenhove, M Russo, M Lee, A M A Miranda, S N Barnett, R Toscano-Rivalta, P Chaves Guerrero, M D Schneider, A R Lyon, E Hirsch, R Lombardi, A Ghigo, M NosedaAbstract
Background
Doxorubicin (DOX) is a widely used chemotherapy agent limited by cumulative cardiotoxicity. DOX-induced cardiotoxicity (DIC) typically evolves from an early asymptomatic phase to a late stage with irreversible cardiac damage, ultimately leading to symptomatic heart failure. Despite extensive studies, the underlying cellular and molecular mechanisms of DIC remain unclear, highlighting the need for stage-resolved analyses linking early transcriptional responses to functional outcomes.
Methods
We injected BALB/c mice with saline (VEH) or 4 mg/kg DOX. Two DOX groups received either a single dose with analysis at 3 days (DOX_3D), or weekly injections for 3 weeks with analysis at 6 weeks after the first dose (DOX_6W). We obtained human DIC heart samples from the Cardiac Tissue Biobank at the University of Colorado. We performed single-nucleus RNA sequencing (snRNA-seq) on the mouse and human myocardium, integrated the human data with published datasets, and analysed the data using Seurat and Scanpy. Downstream analyses included drug2cell for DOX target prediction, miloR for differential abundance testing and CellChat for cell–cell interaction analysis. We performed functional validation in zebrafish and mouse DIC models, as well as in human induced pluripotent stem cell derived cardiomyocytes.
Results
We analysed mouse hearts with DOX_3D representing early cardiotoxicity and DOX_6W representing late-stage with overt cardiotoxicity, confirmed by reduced fractional shortening and cardiac atrophy. We identified 8 major cell types that further resolved into 42 distinct cell states. Drug2cell predicted fibroblasts, cardiomyocytes and myeloid cells significantly targeted by DOX; these populations also showed marked abundance shifts in DOX_6W hearts, indicating they are the most DOX-responsive cell types. We defined the cellular and molecular programmes of DIC across disease stages. At the late stage, we identified an inflammatory–fibrotic circuit involving activated fibroblasts and myeloid cells (including Trem2Hi macrophages and conventional dendritic cells type 2) via TGF-β, SPP1 and IL-1β signalling. In parallel, we identified an early-onset cardiomyocyte stress programme with the emergence of a proto-stressed intermediate state, which progressed to enrichment of a stressed state at the late stage. This cardiomyocyte-intrinsic stress programme was marked by GASK1B kinase upregulation, which persisted in advanced human DIC, as shown by snRNA-seq analysis of predominantly end-stage heart failure samples. Cross-species studies in zebrafish and mice demonstrated that GASK1B knockdown preserved cardiac function in DIC, while complementary assays in human cardiomyocytes confirmed protection against DOX-induced cell death and injury.
Conclusion
We define a stage-dependent framework that distinguishes early, potentially reversible cardiomyocyte stress from late-stage cardiac remodelling, and identify GASK1B as a potential therapeutic target in DIC.