Advanced Cardiac Organoid Model for Studying Doxorubicin-Induced Cardiotoxicity

Abstract

Cardiac organoids provide an in vitro platform for studying heart disease mechanisms and drug responses. However, a major limitation is the immaturity of cardiomyocytes, restricting their ability to mimic adult cardiac physiology. Additionally, the inadequacy of commonly used extracellular matrices (ECM), which fail to replicate the biochemical and mechanical properties of natural heart tissue, poses significant challenges. Consequently, structural integrity in cardiac organoids is impaired. Moreover, scalability remains an obstacle, as conventional ECM substitutes hinder mass production of organoids for high-throughput toxicology screening. To overcome these challenges, we developed an advanced model promoting fibroblast-driven ECM self-secretion, enabling physiologically relevant tissue architecture and function. Using the ECM-free, mature cardiomyocyte-integrated organoid model, we investigated the cardiotoxicity of doxorubicin, a widely used chemotherapeutic agent known to impair cardiac function. Cardiomyocytes derived from induced pluripotent stem cells were characterized for maturity by immunostaining for cTNT and MYL2 alongside gene expression analysis. Organoids treated with doxorubicin showed reduced size and increased collagen deposition. These structural changes correlated with functional impairments, including decreased contraction rate and disrupted synchronous beating. In 2D culture, exposure to doxorubicin induced fibroblast activation, promoted endothelial-to-mesenchymal transition in endothelial cells, and triggered cytotoxic effects in cardiomyocytes. This study highlights the importance of ECM remodeling in advancing cardiac organoid models and demonstrates its potential for more accurate cardiotoxicity assessment. Addressing these limitations enhances the physiological relevance of cardiac organoid systems for drug safety assessment and cardiac disease modeling.

Keywords: cardiac organoid; cardiotoxicity; chemotherapy; pluripotent stem cells.

Figure 1.

Characterization of iPSC-derived mature cardiomyocytes. (A) Immunofluorescence staining of human iPSC-derived cardiomyocytes using mature cardiomyocyte-specific antibodies (cTnT, MYL2). (B) Gene expression analysis by RT-PCR comparing selected mature cardiomyocyte markers at day 10 (D10) and day 40 (D40). Data presented as mean fold-change (2−ΔΔCt) ± SD (*p < 0.05; n = 3, with 10 cardiac organoids per group). CPT1B: carnitine palmitoyltransferase 1B; SLC25A20: mitochondrial carnitine/acylcarnitine carrier protein; KCNJ2: potassium inwardly rectifying channel subfamily J member 2; SCN5A: sodium voltage-gated channel alpha subunit 5.

Figure 2.

Cardiac organoid formation and characterization. (A) Self-organized cardiac organoids immunostained for fibroblasts (vimentin), endothelial cells (CD31), cardiomyocytes (MYL2), and extracellular matrix component collagen I (COL1A1). (B) Contractility assessment of cardiac organoids at day 10 (D10). Organoids were treated with norepinephrine (0.25, 0.5, and 1 μM) for 20 min, and beating rates (beats per minute, BPM) were quantified through video-based region-of-interest (ROI) analysis (*p < 0.05; n = 10).

Figure 3.

Doxorubicin disrupts cardiac organoid contractility. Cardiac organoids were cultured for 10 days in a 96-well ultralow attachment plate before exposure to doxorubicin for 3 days, followed by a 7-day recovery period. (A) Organoid size quantified by ImageJ analysis (*p < 0.05; n=10). (B) Contractility measured by video analysis; beating rates (BPM) were quantified (*p < 0.05; n=10). (C) Rhythm visualization of representative contraction patterns. (D, E) Western blot analysis of collagen I (COL1A1) protein expression and quantification (*p < 0.05; n=3, 10 organoids per group). (F) Immunofluorescence staining for COL1A1.

Figure 4.

Doxorubicin-induced activation of cardiac fibroblasts. (A) Viability of fibroblasts seeded at 10,000 cells/well assessed by MTT assay following exposure to doxorubicin (0.1 μM) for 1, 2, and 3 days (*p < 0.05; n=8). (B, C) Western blot analysis and quantification of α-smooth muscle actin (α-SMA) expression in fibroblasts exposed to 0.1 μM doxorubicin.

Figure 5.

Doxorubicin induces endothelial cell toxicity and endothelial-to-mesenchymal transition. Endothelial cells were seeded at 10,000 cells/well, exposed to doxorubicin (0, 0.01, 0.1, 1 μM), and assessed for viability by MTT assay at 1, 2 and 3 days post-treatment (*p < 0.05; n=8). (A, B) Western blot analysis and quantification of α-SMA protein expression after 0.1 μM doxorubicin exposure (*p < 0.05; n=3, 10 organoids per group). (C) MTT assay evaluating cell viability after doxorubicin treatment on days 1–3. (D) Representative immunofluorescence staining demonstrating doxorubicin-induced α-SMA changes in endothelial cells.

Figure 6.

Doxorubicin reduces cardiomyocyte viability. Mature cardiomyocytes were seeded in 96-well plates at a density of 10,000 cells per well. Cells were exposed to doxorubicin (0, 0.01, 0.1, 1 μM) starting one day post-seeding, and viability was measured by MTT assay after 3 days (*p < 0.05; n=8).