Abstract Body: Intro: Doxorubicin (DOX), a widely used chemotherapy, is often limited by dose-dependent cardiotoxicity. While the clinical impact of doxorubicin-induced cardiotoxicity (DIC) is well known, its precise molecular mechanisms remain poorly defined, and effective cardioprotective strategies are limited. Accumulating evidence, together with our data, suggest endothelial cell (EC) dysfunction and disrupted EC-cardiomyocyte (CM) signaling contribute to DIC. In particular, abnormal EC-CM communication, like neuregulin 1 (NRG1) signaling from ECs and its interaction with ERBB receptors on CMs, appears to play a critical role in DIC pathogenesis.
Hypothesis: Aberrant EC-CM communication, including the NRG1-ERBB axis, plays a critical role in DIC
Aim: To establish vascularized cardiac organoids (vCOs) using a CDH5-GFP reporter hiPSC line to model DIC and investigate EC-CM crosstalk
Methods: We developed a vCO DIC model using human induced pluripotent stem cells (hiPSCs). A CDH5-GFP reporter hiPSC line was used for real-time tracking of EC differentiation and vascular network formation within the organoids. To model DIC, vCOs were treated with DOX, and functional changes in both ECs and CMs were evaluated. We also engineered CDH5 promoter-driven and TNNT2 promoter-driven CRISPRi systems to enable EC- or CM-specific repression of communication pathways in the vCO model.
Results: Spatial transcriptomic analysis of DIC mouse hearts using Xenium revealed EC-CM crosstalk was significantly altered by DOX treatment, including changes in the NRG1-ERBB signaling axis. Our vCOs developed vascular-like networks, exhibited spontaneous beating, and preserved key EC-CM crosstalk features. scRNA-seq confirmed the vCOs were enriched for both ECs and CMs. DOX-treated vCOs showed reduced CM function like decreased calcium amplitude. Angiogenesis and gap junction integrity were also significantly impaired by DOX treatment, indicating EC dysfunction. scRNA-seq also showed that EC-CM communication changes in DOX-treated vCOs were consistent with those observed in the mouse heart dataset, including alterations in the NRG1-ERBB pathway. We are now validating the role of EC-CM crosstalk in DIC using CDH5 promoter-driven NRG1 and TNNT2 promoter-driven ERBB CRISPRi knockdown under DOX treatment.
Conclusions: Using vCOs as a human relevant DIC model, our results support disrupted EC-CM crosstalk, including the NRG1-ERBB axis, may serve as a key mechanism of DIC and a potential therapeutic target.