234Cardiotoxicity of chemotherapeutic agents assessed in human embryonic or induced pluripotent stem cell-derived cardiomyocytes

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Abstract

Purpose

The development of new anti-cancer drugs is limited by their damaging effects on cardiac function. Chemotherapies target survival and proliferation pathways both leading to cardiac dysfunction and cell death. Here we evaluate cultured cardiomyocytes differentiated from human pluripotent stem cells as a cardiotoxicity screen to investigate the interaction between the chemotherapeutic agents Doxorubicin and Lapatinib.

Material and Methods

Human embryonic stem cell H7 cell line-derived cardiomyocytes (H7-CM) and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) were plated at 35,000 cells per cm2 or cultured as spontaneously beating clusters in M199 media + 5μg/ml insulin. After incubation with drugs, live cells were stained with TMRM (for mitochondrial membrane potential), Vybrant Caspase 3 and 7 substrate, TOTO-3/BOBO-1 (for cell permeability) and Hoechst 33342 (nuclear structure). Permeabilized cells were stained with anti-caspase3 antibody and anti-α/β-MHC. Plates were scanned with an automated microscope (Cellomics) and a toxicity profile was built from combined cell health/death parameters. Beating rate was recorded from spontaneously beating clusters.

Results

In isolated H7-CM the anthracycline Doxorubicin (DOX) induced apoptosis, nuclear remodelling and cell loss in a concentration-dependent (1-100μM) manner, whereas the ErbB-2 blocker Lapatinib (LAP) did not produce any toxicity. However, the combination of the 2 chemotherapeutic agents worsened the toxicity profile compared to DOX alone (p < 0.001, n=12), consistent with clinical observations. Activation of caspase-3 in H7-clusters was coincident with the dissipation of the mitochondrial membrane potential, while in hiPSC-CM the MMP was preserved during this phase. In H7-clusters exposed to low (1μM) DOX the first signs of contractile dysfunction appeared after 7 to 12 days (p < 0.001, n=14 at d12), whereas arrest in contraction and activation of programmed cell death occurred within 24h when exposed to 10μM DOX.,

Conclusion

We have shown that human pluripotent stem cell-derived cardiomyocytes reproduce the clinical adverse cardiac effects after treatment with anti-cancer drugs, making these cells a relevant system for future drug development. However, our data suggest that the pathways involved may differ between cell lines and culture method.

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