330 - Doxorubicin-Mediated Toxic Effects Are Mediated Via NO/eNOS in a Novel 3D in Vitro Model of the Human Heart

Abstract

Background Nitric oxide (NO●) and endothelial nitric oxide synthase (eNOS) play a dual role in the human heart by both preventing and mediating the toxic effects of chemotherapeutics such as doxorubicin (DOX). Treatment of cancer patients with DOX can result in severe late-onset dilated cardiomyopathy more than one year after completion of treatment. This cardiotoxicity has been linked to DOX-mediated upregulation of eNOS, peroxynitrous acid generation and caspase 3 activation. However, currently available models used to study this signalling pathway have limited potential to identify the cellular and molecular targets of DOX and the mechanism of toxicity in humans. Aims to use a novel in vitro 3D co-culture model, the “cardiac spheroid” (CS), generated from human endothelial cells (ECs), cardiac myocytes (CMs) and fibroblasts (CFs) to recapitulate biochemical and morphological features typical of the human heart and examine the mechanisms of DOX toxicity. Results DOX-mediated dose dependent toxic effects on CSs are shown to be dependent on NO● synthesis. Inhibition of NO● formation using L-NIO reduced DOX-mediated toxic effects in CSs at DOX concentrations detected in DOX-treated patients. Analysis at the cellular level showed that EC-derived NO● plays a protective role, whereas most of the toxic effects of DOX/NO was derived from CFs. Silencing of eNOS with lentiviral NOS3 shRNA particles in ECs confirmed a dual role of NO in mediating DOX-derived toxic effects in CSs., Genetic eNOS inhibition in CFs supported the toxic role played by these cells. Conclusions This study provides the first direct evidence for a dual role of NO. in mediating both toxic and protective DOX-mediated effects in the human heart, and cellular crosstalk responsible for this. Determining the molecular and cellular targets of novel drugs in CSs has the potential to prevent unexpected toxic effects.