Contents

📒 Maillet 2016

Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes1

Sciwheel.

Introduction

  • Doxorubicin-induced cardiotoxicity (DIC): acute atrial and ventricular arrhythmia, as well as chronic cardiomyopathy and heart failure, cumulative dose-dependent
  • topoisomerase-II beta Top2Îē) in the pathogenesis of DIC
  • human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as the testing platform

Results

Generation and characterization of hPSC-derived CMs

  • upregulation of cardiac markers cTnT, NKX2-5 and MYH7, predominantly ventricular-like

Doxorubicin triggers cell death with apoptotic and necrotic features in hPSC-derived CMs

  • measurement of ATP levels => cell viability
  • IC50 of 30.1 ξM. Starting @ 5ΞM
  • Annexin V (early apoptosis) & propidium iodide (late apoptosis/necrosis) staining

Doxorubicin leads to mitochondrial dysfunction and ROS production in hPSC-derived CMs

  • AA = antimycin A (positive control). MitoSOX Red dye
  • significant decrease in ΔÎĻm after 1 h of treatment with 5 ξM of doxorubicin

Doxorubicin induces an increase in intracellular Ca2+ and the formation of double-strand DNA breaks

  • [Ca2+]i level before treatment was relatively low and increased 2.5 fold after treatment with 5 ξM of doxorubicin for 16 h
  • doxorubicin treatment induced the DNA damage signal Îģ-H2AX in hPSC-derived CMs in a dose-dependent manner

Multi-electrode array electrophysiology and response to doxorubicin

  • dose- and time-dependent decrease in beat period and spike amplitude @ as low as 0.25 ξM of doxorubicin
  • doxorubicin treatment resulted in a dose-dependent reduction in the corrected field potential duration (cFPD) ~= QTc interval: earlier repolarization caused by doxorubicin

Effects of doxorubicin on gene expression

  • KEGG pathway analysis & GO analysis
  • structural gene-based cardiomyopathy (remodeling) pathways as being commonly upregulated among the three conditions (Fig. 6d). Conversely, the Wnt signaling pathway was found to be down-regulated in all conditions

TOP2B is essential for doxorubicin-induced cell death in hPSC-derived cardiomyocytes

  • TOP2B KO by CRISPR-Cas9: more resistant to doxorubicin-induced double-strand DNA breaks (DSBs) and cell deaths

Discussion

  • DOX treatment: significant production of ROS, mitochondrial dysfunction and increased [Ca2+]i in hPSC-derived CMs
  • DOX-induced arrhythmias
  • transcriptome profiling: doxorubicin induces a broad program of gene expression associated with cellular remodeling and impaired cardiomyocyte function
  • CMs express TOP2B but not TOP2A, and recently, Top2B was shown to be critical for DIC in a mouse model. Using CRISPR-Cas9, we showed that disruption of TOP2B reduces the sensitivity of hPSC-CMs to doxorubicin-induced DSBs
  • limitations
    1. hPSC-derived CMs are less mature than native adult myocytes
    2. mixture of cells atrial-, ventricular- and nodal-like phenotypes
    3. single-cell models and lack 3D tissue interactions

  1. Maillet A, Tan K, Chai X, et al. Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes. Sci. Rep. 2016;6:25333. doi:10.1038/srep25333. PMC4855185 ↩︎