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bioRxiv · 10.1101/2021.04.12.439436

Imatinib mesylate induces necroptotic cell death and impairs autophagic flux in human cardiac progenitor cells

Walmsley, R.·Steele, D. S.·Ellison-Hughes, G. M.·Smith, A. J.
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Abstract

The receptor tyrosine kinase inhibitor imatinib mesylate has improved patient cancer survival rates but has been linked to long-term cardiotoxicity. This study investigated the effects of imatinib on cell viability, apoptosis, autophagy and necroptosis in human cardiac progenitor cells in vitro. After 24 hours, imatinib significantly reduced cell viability (75.9{+/-}2.7% vs._100.0{+/-}0.0%, n=5, p<0.05) at concentrations comparable to peak plasma levels (10 {micro}M). Further investigation showed no increase in caspase 3 or 7 activation. Imatinib also significantly reduced the fluorescence of cells stained with TMRM (74.6{+/-}6.5% vs. 100.0{+/-}0.0%, n=5, p<0.05), consistent with mitochondrial depolarization. Imatinib increased lysosome and autophagosome content relative to the control, as indicated by changes in acridine orange fluorescence (46.0{+/-}5.4% vs. 9.0{+/-}3.0, n=7, p<0.001) and expression of LAMP2 (2.4{+/-}0.3 fold, n=3, p<0.05) after 24 hours treatment. Although imatinib increased the expression of proteins associated with autophagy, it also impaired the autophagic flux, as demonstrated by the proximity ligation assay staining for LAMP2 (lysosome marker) and LC3II (autophagosome marker), with control cells showing 11.3{+/-}2.1 puncta per cell and 48 hours of imatinib treatment reducing the visible puncta to 2.7{+/-}0.7 per cell (n=10, p<0.05). Cell viability was partially recovered by autophagosome inhibition by wortmannin, with a 91.8{+/-}8.2% (n=5, p>0.05) increase in viability after imatinib and wortmannin co-treatment. Imatinib-induced necroptosis was associated with an 8.5{+/-}2.5-fold increase in activation of mixed lineage kinase domain-like pseudokinase. Imatinib-induced toxicity was rescued by RIP1 inhibition relative to the control; 88.6{+/-}3.0% vs. 100.0{+/-}0.0% (n=4, p>0.05). In summary, imatinib applied to human cardiac progenitor cells depolarizes mitochondria and induces cell death through necroptosis, which can be recovered by inhibition of RIP1, with an additional partial role for autophagy in the cell death pathway. These data provide two possible targets for co-therapies to address imatinib-induced long-term cardiotoxicity.

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Walmsley, R., Steele, D. S., Ellison-Hughes, G. M., Smith, A. J.. 2021-04-13. Imatinib mesylate induces necroptotic cell death and impairs autophagic flux in human cardiac progenitor cells. https://doi.org/10.1101/2021.04.12.439436

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