Investigation into the mechanism of Nox2 NADPH oxidase-dependent signalling in doxorubicin cardiotoxicity

Abstract

Although the antibiotic, doxorubicin (DOX), is a widely-used and successful first-line treatment for cancers, it can induce cardiotoxicity which is associated with increased production of myocardial reactive oxygen species (ROS). Recent work has suggested that ROS generated specifically from Nox2 NADPH oxidase, contributes to key processes underlying cardiac dysfunction associated with chronic DOX treatment. The aim of this study was therefore (1) to characterise the role of Nox2 NADPH oxidase-derived ROS in DOX-induced cardiotoxicity in a 4 week murine experimental model as the basis for detailed mechanistic studies, (2) identify Nox2-regulated genes and signalling pathways which may play a key role in the development of DOX-induced cardiotoxicity in vivo, and (3) investigate detailed Nox2-dependent signalling mechanisms in DOX-treated cardiomyocytes in vitro. Wild type (WT) and Nox2-/y mice were treated with or without DOX (4mg/kg) and studied after 4 weeks. DOX-induced cardiac contractile dysfunction and cardiomyocyte remodelling was attenuated in Nox2-/y as compared to WT mice. Furthermore, DOX treatment was found to increase Nox2 mRNA expression in WT mice, whilst Nox2 mRNA was not detected in Nox2-/y animals and was not induced by DOX. Inhibitor studies conducted in WT animals confirmed that the observed effects were mediated by ROS. Taken together, these data give further support to our previous data suggesting that ROS specifically-derived from Nox2 NADPH oxidase make a significant contribution DOX-induced cardiac remodelling and provide a solid basis for use of the 4 week experimental model for detailed mechanistic studies. In this regard, a whole-genome gene expression array (Illumina MouseWG-6 v2.0) was performed on ventricular tissue from these animals in order to identify key Nox2-regulated pathways which may mediate protection against DOX cardiotoxicity. Pathway analysis identified 156 differentially expressed genes (P<0.1) and highlighted ‘Cell Death and Survival’ as the network of most significance to the dataset. Further examination using heat maps highlighted cell death and survival functions, with a focus on 5 genes associated with cardiomyocyte apoptosis. Of these genes, peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC1α), and the mitochondrial membrane protein, mitofusin 2 (Mfn2), which is known to be up- vi regulated by PGC1α, were selected as good candidates for further investigation. qPCR confirmed increased expression of Mfn2 mRNA in hearts from WT (1.3-fold) but not Nox2-/y DOX-treated animals, although no changes in PGC1α mRNA were apparent between groups. To further investigate the signalling mechanism by which Mfn2 may mediate the protective effects of Nox2, an in vitro model of DOX-induced apoptosis in HL-1 cardiomyocytes was employed. Initial characterization studies showed that DOX treatment for 24 hours resulted in increased expression of both Nox2 and Mfn2 and superoxide production. Targeted siRNA knockdown of Nox2 and Mfn2 produced significant downregulation of protein expression, and exhibited a protective effect against DOX-induced decreases in cell viability and caspase/viability activity. Furthermore, pharmacological inhibition of NADPH oxidase using the inhibitor, VAS2870, significantly reduced DOX-induced HL-1 cardiomyocyte apoptosis and cytotoxicity. Taken together, the findings of this study add further support to a key role for Nox2 NADPH oxidase-derived ROS in DOX cardiotoxicity, and suggest that apoptotic cell death may represent a central mechanism in this process, which may involve mitochondrial fusion, thereby contributing to the associated contractile dysfunction.

Date of Award	2014
Original language	English
Awarding Institution	Queen's University Belfast